Medicine

A DECADE IN
MEDICINE
November 2015
UROLOGY
ENDOCRINOLOGY
CLINICAL ONCOLOGY
NEPHROLOGY
CARDIOLOGY
NEUROLOGY
GASTROENTEROLOGY &
HE
PATOLOGY
RHEUMATOLOGY
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A Decade in Medicine
The articles included in A Decade in Medicine were originally
published online and in the November 2014 or November2015
issues of the eight clinical Nature Reviews journals. To celebrate
the 10th anniversary of the launch of thesejournals, the editors
commissioned international experts to write short essays
highlighting the key papers that made the biggest contribution
to their field in the past decade. Between them, the clinical
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acute coronary syndromes | Successes and future objectives in acute
coronary syndrome
Frans Van de Werf
3
5
arrhythmias | Cardiac fibrillationchallenges and evolving solutions

Stanley Nattel
cardiomyopathies | Cardiomyopathy on the move
Magdi H. Yacoub
6
8
dyslipidaemia | Resurgence of targets and compounds to treat dyslipidaemia

John J. P. Kastelein
heart failure | 10Years of progress in HF researchwhat have we learned?
Henry Krum
10 hypertension | The past decade in hypertensionfacts, hopes, and hypes
Thomas Unger
12 peripheral vascular disease | 10Years of breakthroughs in peripheral
vascular disease
Mark A. Creager
14 valvular disease | Current perspectives on treatment of valvular heart disease
Friedrich W. Mohr
Nature Reviews Clinical Oncology

16 clinical trials | Shifting paradigms in cancer clinical trial design
Daniel J. Sargent and Edward L. Korn
18 targeted therapy | Successes, toxicities and challenges in solid
tumours
Joel W.Neal and George W.Sledge
19 haematological cancer | Advances in biology and therapy
S. Vincent Rajkumar and Philippe Moreau
21 cancer immunotherapy | Entering the mainstream ofcancer
treatment
Steven A. Rosenberg
23 genomics | A decade of discovery in cancer genomics
Kenneth Offit
25 funding in cancer research | National Cancer Institute awards
a work in progress
Tito Fojo and Paraskevi Giannakakou
Nature Reviews Endocrinology

28 bone | Great strides made but still further to go
Ian R. Reid
29 thyroid disease | The endocrinology of thyroid disease from 2005
to2015
P. Reed Larsen
31 type 2 diabetes mellitus | At the centre of things
Guang Ning
33 paediatric endocrinology | New genes, new therapies
Mehul T. Dattani
35 reproductive endocrinology | Understanding reproductive
endocrine disorders
Ursula B. Kaiser
53 polycystic kidney disease | Slowing progression of autosomal

dominant polycystic kidney disease
Robert W. Schrier
54 renal transplantation | A spectrum of advances in renal
transplantation
Bruce Kaplan
Nature Reviews Neurology

57 dementia | A decade of discovery and disappointment
indementia research
John R. Hodges
58 multiple sclerosis | New drugs and personalized medicine
formultiple sclerosis
Paul M. Matthews
60 epilepsy | Edging toward breakthroughs inepilepsy diagnostics
andcare
Daniel H. Lowenstein
62 movement disorders | Tracking the pathogenesis ofmovement
disorders
Oksana Suchowersky
63 stroke | Progress in acute ischaemic stroke treatment
andprevention
Jose G. Romano and Ralph L. Sacco
65 migraine | Incredible progress for an era ofbetter migraine care
Peter J. Goadsby
67 cns infections | Major advances against a moving target
of CNS infections
Lisa F. P. Ng and Tom Solomon
Nature Reviews Rheumatology
Nature Reviews Gastroenterology & Hepatology
69 translational rheumatology | Tenyears after: rheumatology

research from bench to bedside
Nunzio Bottini and Gary S.Firestein
37 hcv | Hepatitis C therapya fast andcompetitive race

Stefan Zeuzem
71 paediatric rheumatology | A field on the move

Seza Ozen
38 hepatocellular carcinoma | HCCsubtypes, stratification

andsorafenib
Gregory J. Gores
72 technology | Technological advances transforming rheumatology

William H. Robinson and Rong Mao
40 gut microbiota | The gut microbiota era marches on

Francisco Guarner
42 fgids | Functional gastrointestinal disordersa paradigm shift
Nicholas J. Talley
43 pancreatic diseases | Advances in understanding andcare
ofpancreatic diseases
Randall E. Brand
45 ibd | IBDgenes, bacteria and new therapeutic strategies
Jean-Frederic Colombel
74 clinical rheumatology | 10years of therapeutic advances in the

rheumatic diseases
John D.Isaacs
Nature Reviews Urology

77 bladder cancer | International progress: from cytology to genomics
James C. Costello and Dan Theodorescu
79 imaging | A decade in image-guided prostate biopsy
Baris Turkbey and Peter L. Choyke
Nature Reviews Nephrology
81 urinary incontinence | Advances in female urology andvoiding

dysfunction
Marisa M. Clifton and Howard B. Goldman
48 glomerular disease | The glomerulus reveals some secrets

Agnes B. Fogo
82 kidney cancer | Discoveries, therapies andopportunities

W. Marston Linehan and Christopher J. Ricketts
50 genetics of kidney diseases | Genetic dissection of kidney

disorders
Friedhelm Hildebrandt
84 sexual dysfunction | Post-RP erectile dysfunctiontherapies

forthe next decade
Emmanuel Weyne and Maarten Albersen
51 acute kidney injury | Acute kidney injurya decade ofprogress

Rinaldo Bellomo
86 prostate cancer | A decade of progress in detection and treatment

Behfar Ehdaie and Peter T. Scardino
CARDIOLOGY
DECADE IN REVIEWACUTE CORONARY SYNDROMES
Successes and future objectives in acute

coronary syndrome
Frans Van de Werf
The past decade has seen considerable advances in the treatment of acute coronary syndromes (ACS),
particularly in the search for improved antithrombotic therapies. Despite these successes, however, renewed
efforts are needed to improve long-term outcomes after ACS by reducing recurrent ischaemic events and
lowering the risk of bleedingcomplications.
Van de Werf, F. Nat. Rev. Cardiol. 11, 624625 (2014); published online 2 September 2014; doi:10.1038/nrcardio.2014.129
By 10years ago, aspirin and clopidogrel

therapy for 1year was already the standard
treatment for all patients with acute coronary syndrome (ACS), despite the benefit
of this dual antiplatelet therapy never
having been formally studied in patients
with ST-segment elevation myocardial
infarction (STEMI). Without a doubt, the
majority of clinical studies in ACS performed in the past decade have focused on
attempts to find more efficacious and safer
antithrombotic treatments than aspirin
and clopidogrel dual antiplatelet therapy.
The most successful trial in this regard
was PLATO,1 which showed significantly
improved outcomes (including recurrent
myocardial infarction, stent thrombosis,
death from vascular causes, and even allcause mortality) with ticagrelor, a new
oral, reversible, direct-actingP2Ypurinoreceptor12 (P2Y12) antagonist, when compared with clopidogrel; the study included
patients with either STEMI or non-STEMI
(NSTEMI). Pleiotropic actions, such as
increased adenosine plasma concentrations,
were suggested to have contributed to the
beneficial effect of ticagrelor. The overall
excess spontaneous bleeding rate with this
new, more powerful agent was small, but
the trend towards an increased risk of intra
cranial haemorrhage observed in this study
was a concern.
One of the possible reasons why newer
P2Y12 antagonists, such as cangrelor, pra
sugrel, and ticagrelor, are superior to
clopid ogrel in large clinical trials is the
existence of genetic variants of hepatic
cytochromeP450 (CYP) enzymes responsible for converting clopidogrel to its active
metabolite. Several studies have shown that
carriers of an allele that resulted in reduced

CYP function had lower levels of the active
metabolite and higher rates of major cardio
vascular events. As such, carriers of CYP
loss-of-function variants who are receiving
clopidogrel are likely to be especially at risk
of stent thrombosis. The concerns about
interindividual variability in the antiplatelet
effect of clopidogrel have led to a number
of studies aimed at monitoring platelet
function and adjusting treatment accordingly. Surprisingly, in one of the largest
randomized studies, which included 2,440
patients scheduled for coronary stenting
(of whom 657 had NSTEMI), no improvement in clinical outcomes at 1year was
observed with platelet-function monitoring and treatment adjustments as compared
with standard treatment without monitoring.2 Genetic testing and platelet-function
monitoring have not become routine tests
in patients taking clopidogrel.
recurrent ischaemic
eventsand long-term mortality
remain high
Bivalirudin has replaced heparin in many

hospitals, especially for patients undergoing primary percutaneous coronary intervention (PCI), owing mainly to the results
of the HORIZONSAMI study.3 This trial
showed significantly reduced bleeding rates
and decreased 30day mortalitywith bivalirudin alone when compared withheparin
plus a glycoprotein (GP)IIb/IIIa (integrinIIb3) antagonist.3 However, the benefit
of bivalirudin for primary PCI was challenged by the results of a single-centre
trial (HEATPPCI4),in which GPIIb/IIIa

antagonists were used only for bailout
(bivalirudin versus heparin): fewer ischaemic events occurred with heparin than with
bivalirudin and, surprisingly (in contrast to
all previous trials), no reduction in bleeding
complications was found with bivalirudin.
Whether these data should change practice
and whether a new, large multicentre trial is
needed is currently a matter of hot debate.
Despite the progress made with anti
thrombotic therapy and revascularization
in the early phase of an ACS, recurrent
ischaemic events and long-term mortality remain high. One explanation could be
that dual antiplatelet therapy consisting of
aspirin and clopidogrel does not provide
sufficient long-term protection against
recurrent thrombotic events. In two large
studies, 5,6 an additional antithrombotic
agent was tested in combination with
aspirin and c lopidogrel: vorapaxar, an
antagonist of the thrombin receptor PAR1
(protease-activated receptor1), was used
in the TRA2P trial, 5 and rivaroxaban, a
direct oral inhibitor of factorXa, was used
in ATLAS2.6 Although increased bleeding
rates were observed in both studies (and
the TRA2P study even had to be stopped
prematurely in the subgroup of patients
with a history of stroke, owing to an excess
rate of intracranial haemorrhage), selected
populations did benefit from adding a
third antithrombotic drug. In the group
of >17,000 patients with a previous myocardial infarction, addition of vorapaxar
reduced the risk of cardiovascular death,
myoc ardial infarction, or strokeat the
cost of an increased risk of moderate or
severe bleeding, but without a significant
NOVEMBER 2015 | 1
A DECADE IN MEDICINE
CARDIOLOGY
PLATO1 and subgroups
of TRA 2P5 or ATLAS-26
Haemorrhagic risk
No option
Standard
antithrombotic
treatment
Range of possible results

of a simplified antithrombotic regimen
Risk of thrombosis
Figure 1 | The balance of risks in treatment

ofacute coronary syndrome. Patients with
acute coronary syndrome receive therapies
aimed at reducing the risk of thrombosis,
butantithrombotic treatments carry an
increased risk of bleeding complications.
New therapeutic approaches should minimize
the possibility of thrombotic events while
keeping haemorrhagic risk at acceptable
levels, or even reduce the risk when
compared with current treatment.
increase in intracranial haemorrhage.5 In

ATLAS2,6 low-dose rivaroxaban (2.5mg
twice daily) also significantly reduced the
risk of the composite end point of cardio
vascular death, myocardial infarction,
and stroke and, surprisingly, also reduced
rates of stent thrombosis and death from
any cause. Whether these agents are also
beneficial and safe when, for instance,
ticagrelor or prasugrel is used in combination with aspirin, is unknown. Similarly,
whether aspirin can be dropped if a new
P2Y12 antagonist is given in combination
with vorapaxar or low-dose rivaroxaban
remains to be determined. New studies of
simplified antithrombotic regimens that
are underway or in the planning phase
should take into account the possible
benefits and risks of new antithrombotic
combinations(Figure1).
use of increasingly powerful

antithrombotic agents has raised
concern about bleeding
A number of trials to study the optimal

timing of angiography and revascularization
in patients with NSTEMI yielded discordant results. In the TIMACS trial,7 >3,000
patients with ACS were randomly assigned
to either routine early intervention or
delayed intervention (either <24h or >36h
after treatment assignment, respectively).
In the total population, a nonsignificant
2 | NOVEMBER 2015
15% reduction in the composite end point

of death, myocardial infarction, or stroke
was observed when comparing early versus
delayed treatment. However, in line with
other studies, early intervention in highrisk patients led to a significant 35% reduction in the risk of death, new myocardial
infarction, or stroke when compared with
delayedintervention.7
The use of increasingly powerful anti
thrombotic agents has raised concern
about bleeding. Major bleeding complications are indeed common, and most occur
at the vascular access site. In the RIVAL
trial,8 >7,000 patients with ACS, including almost 2,000 patients with STEMI,
were randomly allocated to either radial
or femoral access. Overall, a lower rate of
local vascular complications was observed
with the radial approach than with femoral
access. Remarkably, patients with STEMI
had better clinical outcomes (including reduced mortality) when treated via
radial access, suggesting that this approach
might be particularly preferable in patients
withSTEMI.8
Data from all registries show that, in
patients with STEMI, door-to-balloon
times have declined significantly over the
past 10years. The implementation of emergency medical systems based on a network
of hospitals with various levels of technology connected by an efficient ambulance
system has reduced pre-PCI delays in many
places. 9 Howe ver, in a large US study of
almost 100,000 patients with STEMI, inhospital mortality remained unchanged
despite significant improvement in doorto-balloon times, indicating that other
components of the total ischaemic time
need to be targeted.9 Administration of a
fibrinolytic agent to patients who cannot
undergo timely PCI is a strategy that might
be incorporated into prehospital care. In
the STREAM trial 10 of ~2,000 patients
presenting with early STEMI, prehospital
administration of tenecteplase (half the
normal dose in elderly individuals) to
patients who could not undergo PCI within
1h resulted in rates of the composite end
point of death, shock, congestive heart
failure, or recurrent infarction at 30days
that were similar to those in patients receiving standard primary PCI. Emergency
coronary angiography on arrival in the
PCI hospital could be avoided in almost
two-thirds of patients treated with tenecte
plase.10 This strategy needs to be further
explored in patients with long transport
times, especially elderly patients.
In summary, 30day mortality after

ACS has decreased remarkably in all age
categories in the past decade, thanks to
improved antithrombotic treatment and
early revascularization. As a consequence,
profound changes in the epidemiology of
ACS have also taken place: more patients
with ACS survive the initial event and go
on to develop recurrent ischaemic events,
heart failure, atrial fibrillation, or noncardiac diseases (such as cancer), and are
dying at an older age. In the future, more
attention will have to be paid to the longterm care of patients after ACS, of which
antithrombotic agents will remain an
importantcomponent.
Department of Cardiovascular Sciences,
University of Leuven, Herestraat 49,
B3000Leuven, Belgium.
frans.vandewerf@med.kuleuven.be
Acknowledgements
I thank all my colleagues on both sides of the
Atlantic with whom I was able to collaborate in
performing important clinical trials that have
improved the treatment of patients with acute
coronary syndrome.
Competing interests
F.V.d.W. declares that he has received research
grants and fees for participating in advisory boards,
data and safety monitoring boards, and speaking
activities from AstraZeneca, Boehringer Ingelheim,
Merck, and The Medicines Company.
1.
Wallentin, L. etal. Ticagrelor versus clopidogrel

in patients with acute coronary syndromes.
N.Engl. J. Med. 361, 10451057 (2009).
2. Collet, J.P. etal. Bedside monitoring to adjust
antiplatelet therapy for coronary stenting.
N.Engl. J. Med. 367, 21002109 (2012).
3. Stone, G.W. etal. Bivalirudin during primary PCI
in acute myocardial infarction. N. Engl. J. Med.
358, 22182230 (2008).
4. Shahzad, A. etal. Unfractionated heparin
versus bivalirudin in primary percutaneous
coronary intervention (HEAT-PPCI): an openlabel, single centre, randomised controlled trial.
Lancet http://dx.doi.org/10.1016/S01406736(14)60924-7.
5. Morrow, D.A. etal. Vorapaxar in the secondary
prevention of atherothrombotic events. N. Engl.
J. Med. 366, 14041413 (2012).
6. Mega, J.L. etal. Rivaroxaban in patients with a
recent acute coronary syndrome. N. Engl. J. Med.
366, 919 (2012).
7. Mehta, S.R. etal. Early versus delayed invasive
intervention in acute coronary syndromes.
N.Engl. J. Med. 360, 21652175 (2009).
8. Jolly, S.S. etal. Radial versus femoral access
for coronary angiography and intervention in
patients with acute coronary syndromes (RIVAL):
a randomised, parallel group, multicentre trial.
Lancet 377, 14091420 (2011).
9. Menees, D.S. etal. Doortoballoon time and
mortality among patients undergoing primary
PCI. N. Engl. J. Med. 369, 901909 (2013).
10. Armstrong, P.W. etal. Fibrinolysis or primary
PCI in STsegment elevation myocardial
infarction. N. Engl. J. Med. 368, 13791387
(2013).
www.nature.com/reviews
CARDIOLOGY
DECADE IN REVIEWARRHYTHMIAS
Cardiac fibrillationchallenges
and evolving solutions
Stanley Nattel
Cardiac rhythm disorders, or arrhythmias, are major sources of

morbidity and mortality, and have been challenging to treat because
classic pharmacological therapies are often ineffective and sometimes
dangerous. In the past decade, groundbreaking developments have
revolutionized the management of arrhythmias and prepared the
groundwork for new advances in the future.
Nattel, S. Nat. Rev. Cardiol. 11, 626627 (2014); published online 9 September 2014;
doi:10.1038/nrcardio.2014.133
At the onset of the 2000s, effective and

definitive therapies were available for most
types of cardiac arrhythmia. However, two
common and important rhythm disorders
remained major therapeutic challenges
sudden cardiac death (SCD) and atrial
fibrillation (AF). SCD typically occurs
owing to a chaotic, disorganized rhythm,
ventricular fibrillation, which renders
effective heart pumping impossible and
causes death if not effectively managed
within minutes of onset (Figure1). AF
resembles ventricular fibrillation in being a
totally disorganized rhythm, but AF affects
the atria, which are not essential for cardiac
pumping (Figure1). AF causes problems
by firing the heart at an inappropriately
rapid rate, leading to a range of symptoms
and, in extreme cases, heart muscle weakening that leads to heart failure. In addition, the ineffect ive pumpi ng action of
the atria allows blood clots to form in the
stagnant atrial blood pool. These clots can
propagate to the brain and cause strokes.
Consequently, AF is a major cause of
strokes, particularly in the elderly. Work
during the past decade has greatly improved
our ability to prevent and manage both SCD
and AF, and enabled further progress in
thefuture.
Towards the end of the 20thcentury, the
use of implantable devices to detect and
rapidly terminate ventricular tachyarrhythmias that might otherwise lead to SCD had
become widespread. However, their effectiveness in reducing mortality was unclear.
Despite preventing SCD, such devices might
not actually reduce mortality, particularly
if SCD constitutes a small portion of the
overall mortality, and if device-related
complications can themselves increase the
likelihood of death. An important milestone
was a study by Bardy and colleagues, who
compared the effects of amiodarone (the

most effective antiarrhythmic drug) with
an implantable cardioverterdefibrillator
(ICD), or placebo in patients with systolic heart failure, who are known to be
at high risk of SCD.1 Amiodarone did not
reduce all-cause mortality during a median
45.5month follow-up period compared
with placebo; however, ICD-therapy significantly reduced all-cause mortality (by 23%;
P=0.007).1 These findings established ICDs
as a potentially life-saving intervention in
patients at increased risk of SCD.
Whereas some patients at increased risk
of SCD are easily identified, the majority of
these arrhythmias occur in individuals
without known high-risk factors. More
work is, therefore, needed to understand
the factors leading to SCD and thereby
effectively identify at-risk individuals.
An important contribution was provided
by Hassaguerre etal. who showed that
a specif ic electrocardiographic variant

(known as early repolarization) is much
more common in individuals resuscitated
after SCD than in a matched control population.2 This study led to extensive clinical
and basic science investigations into the
underlying mechanisms and clinical importance of early repolarization. The early
repolarization pattern is a common electrocardiographic variant in otherwise normal
individuals, particularly young, physicallyactive men, so distinguishing between the
common benign early-repolarization pat
tern and early repolarization associated
with increased SCD risk is an important
challenge. The combination of electro
cardiographic Jwaves (an elevated junction between the end of the QRS and the
onset of the ST-segment) and a horizontal
or descending ST-segment is associated
with increased risk of SCD. Howe ver, no
early repolarization pattern alone (in the
absence of malignant arrhythmias) is presently sufficient to warrant prophylactic
therapy (such as an ICD) to prevent SCD.
A further major advance in understanding the pathophysiology of SCD was the
demonstration by Itzhaki and colleagues
that skin fibroblasts from patients with
an inherited SCD syndrome can be transformed into induced pluripotent stem cells,
and then differentiated into cardiomyocytes that reproduce the cardiac electrical
dysfunction underlying SCD risk. 3 This
work establishes a novel technique to aid
the understanding of SCD pathophysio
logy in individual patients and might ultimately lead to patient-specific medical and
biological therapies.
Ventricular fibrillation
Implanted
defibrillator
prevents death1
Atrial fibrillation
Early
repolarization:
a novel cause2
Rate control as good as

rhythm control in heart failure4
Lenient versus strict

rate control5
Prevention
by risk-factor
reduction10
Drug therapy
complications in
high-risk patients6
Novel
anticoagulants
prevent stroke9
Ablation as
initial therapy7
LA
AVN
RA
RV
LV
IPSCs for pathophysiology

and therapy3
Mechanism-directed
ablation8
Figure 1 | Major advances in arrhythmia research during the past decade. The rate of impulse
transmission through the AVN determines the ventricular response rate during atrial fibrillation,
and is the target of rate-control therapy. Abbreviations: AVN, atrioventricular node; IPSC, induced
pluripotent stem cell; LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle.
NOVEMBER 2015 | 3
CARDIOLOGY
Two potential approaches to managing AF exist: leaving the patient in AF, but
controlling the ventricular response rate
(known as rate control); and keeping the
patient out of AF, generally with the use of
antiarrhythmic drugs (known as rhythm
control). AF is a particularly important
risk factor for death and complications
in patients with heart failure, so rhythm
control would be expected to be of maximal
value in these individuals. This hypothesis
was tested in a prospective, randomized
trial by Roy etal. who found that an effective rhythm-control strategy had no bene
ficial effects on cardiac function, survival,
or physical function in patients with heart
failure and AF.4
In the past decade, the

management and understanding
of cardiac arrhythmias have been
greatly advanced
Despite the value of rate control in controlling adverse consequences of AF, the
parameters characterizing optimum rate
control are largely unknown. VanGelder
and colleag ues compared a lenient
rate-control strate gy (resting heart rate
<110bpm) with a strict strategy (resting
heart rate <80bpm; exercise heart rate
<110bpm). 5 The lenient strategy was as
effective in preventing adverse effects and
easier to achieve than strict rate control.
A further assessment of pharmacological
management of patients handled by rate
control was performed by Connolly and
colleagues.6 Based on previous work suggesting that the antiarrhythmia drug
dronedarone reduces cardiac mortality
and stroke rate in patients with AF, and
that this effect might be independent of
maintaining sinus rhythm, these investigators administered dronedarone or placebo
to patients with permanent AF and markers
of increased risk. Instead of improving
outcomes, dronedarone increased rates
of heart failure, stroke, and cardiovascular death. 6 This study reinforced concerns about the risks of antiarrhythmic
agents in patients with AF at high risk of
cardiovascular complications.
Given the concerns about antiarrhythmic drugs and continual improvements
in technology, ablation procedures have
assumed an increasingly important role in
the treatment of AF over the past 10years.
Although cardiac ablations have generally
4 | NOVEMBER 2015
been reserved for patients who did not

respond to at least one antiarrhythmic
drug, ablation as first-line therapy in AF
has been given increasing consideration.
Cosedis Nielsen etal. compared antiarrhythmic drugs with radiofrequency
ablation as an initial therapy for paroxysmal AF.7 Ablation reduced the recurrence
rate of symptomatic AF (from 16% to 7%),
but the primary end point (AF burden) was
not significantly affected.7 This result suggests that antiarrhythmic drugs still have
a role in the management of AF. However,
encouraging developments in ablat ion
technology have been made. Many failures of ablation techniques are suspected
to be owing to procedures that do not
target patient-specific pathophysiology.
Narayan etal. have developed a method
using intra-atrial basket-catheter arrays
and complex mathematical algorithms to
identify and target patient-specific mechanisms of AF. 8 Comparing their method
with conventional ablation approaches in
a multicentre, prospective, randomized
trial, the investigators noted substantial
improvements in AF prevention: patients
undergoing mechanism-directed ablation
showed much greater maintenance of sinus
rhythm compared with those undergoing
standard procedures (77.8% versus 38.5%;
P=0.001).8
Stroke is the most important complication of AF. Effective orally administered anticoagulation drugs can prevent
AF-related stroke, but until the approval
of dabigatran by the European Medicines
Agency in 2008, the only medications
available for this indication were vitaminK
antagonists, which have a very narrow toxicto-therapeutic wind ow. A number of
direct-acting oral anticoagulants, including
thethrombin-inhibitor dabigatran, and the
factorXa antagonists apixaban, edoxaban,
and rivaroxaban, were developed in the past
decade. The first to be studied, dabigatran,
was demonstrated in a large, multicentre,
placebo-controlled trial to be at least as
effective and safer than warfarin for stroke
prevention in patients with AF. 9 Similar
findings were subsequently published for
the other direct-acting oral anticoagulants,
which have had a major and growing effect
on AF management.
Ultimately, the best approach to AF management would be to prevent the arrhythmia from occurring in the first place. An
exciting proof-of-principle was provided
by Abed etal. who compared aggressive
weight-m anagement intervention with
lifestyle advice (control group) in a randomized study of patients with AF who were
overweight.10 In addition to a substantial
(19%) reduction in body mass, the intervention group showed significant reductions
compared with the control group in scores
for AF frequency (3.4 versus 0.7; P<0.001),
duration (5.0 versus 0.8; P<0.001), and
symptoms (8.4 versus 1.7; P<0.001).10 This
study will motivate further investigation
of factors and mechanisms that promote
AF,as well as public-health measures forAF
prevention.
In the past decade, the management and
understanding of cardiac arrhythmias have
been greatly advanced. The groundwork
has now been laid for important further
developments in the near future.
Department of Medicine and Research
Center, Montreal Heart Institute and
Universit de Montral, 5000 Belanger Street
East, Montreal, QCH1T 1C8, Canada.
stanley.nattel@icm-mhi.org
Acknowledgements
S.N. is funded by the Canadian Institutes of Health
Research (6957 and 44365) and the Heart and
Stroke Foundation of Canada.
Competing interests
S.N. declares no competing interests.
1.
Bardy, G.H. etal. Amiodarone or an

implantable cardioverter-defibrillator for
congestive heart failure. N. Engl. J. Med. 352,
225237 (2005).
2. Hassaguerre, M. etal. Sudden cardiac arrest
associated with early repolarization. N. Engl. J.
Med. 358, 20162023 (2008).
3. Itzhaki, I. etal. Modelling the long QT syndrome
with induced pluripotent stem cells. Nature
471, 225229 (2011).
4. Roy, D. etal. Rhythm control versus rate control
for atrial fibrillation and heart failure N. Engl. J.
Med. 358, 26672677 (2008).
5. Van Gelder, I.C. etal. Lenient versus strict rate
control in patients with atrial fibrillation. N. Engl.
J. Med. 362, 13631373 (2010).
6. Connolly, S.J. etal. Dronedarone in high-risk
permanent atrial fibrillation. N. Engl. J. Med.
365, 22682276 (2011).
7. Cosedis Nielsen, J. etal. Radiofrequency
ablation as initial therapy in paroxysmal atrial
fibrillation. N. Engl. J. Med. 367, 15871595
(2012).
8. Narayan, S.M. etal. Treatment of atrial
fibrillation by the ablation of localized sources:
CONFIRM (Conventional Ablation for Atrial
Fibrillation With or Without Focal Impulse and
Rotor Modulation) trial. J. Am. Coll. Cardiol. 60,
628636 (2012).
9. Connolly, S.J. etal. Dabigatran versus warfarin
in patients with atrial fibrillation. N. Engl. J. Med.
361, 11391151 (2009).
10. Abed, H.S. etal. Effect of weight reduction
and cardiometabolic risk factor management
on symptom burden and severity in
patients with atrial fibrillation: a randomized
clinical trial. JAMA 310, 20502060
(2013).
CARDIOLOGY
further stimulate studies to determine the

worldwide distribution of d
isease-causing
mutations, their possible determinants,
and importantly, the underlying biological
mechanismsresponsible.
Inheritance of DCM or HCM can be
autosomal dominant, Xlinked, and occasionally, autosomal recessive. However, the
incidence of these diseases in the population is grossly underestimated, has low
penetrance, and low expressivity (that is,
low severity within a single family). The

development and availability of large genetic
databases, which include phenotypicallydefined populations of different ethnic
origin, such as the 1000 Genomes Project
and the Exome Sequencing Project, has
enabled researchers to identify a discrepancy between the prevalence of potential
disease-causing variants and the estimated
prevalence of the disease.3,4 One explanation for this discrepancy might be that the
cardiomyopathy phenotype is affected by
modifier genes (protective or otherwise),
epigenetics, or environmental factors that
have yet to be identified. 5 The increasing use of next-generation sequencing has
enabled simultaneous, more efficient, and
cost-effective detection of mutations in a
large number of genes.2 This development,
and formal population studies to determine
the true prevalence of the disease in different
populations, might provide some answers to
these seemingly complex issues.
Myocardial interstitial and focal fibrosis are hallmarks of HCM pathology that
DECADE IN REVIEWCARDIOMYOPATHIES
Cardiomyopathy on the move

Magdi H. Yacoub
Since Wallace Brigden first used the term cardiomyopathy in 1952,

this group of diseases has continued to attract the interest of clinicians,
researchers, and importantly, patients. The past decade has seen a
substantial accumulation of knowledge relating to various cardiomyopathies,
which has partially lifted the mystery surrounding thistopic.
Yacoub, M.H. Nat. Rev. Cardiol. 11, 628629 (2014); published online 30 September 2014;
doi:10.1038/nrcardio.2014.157
The current definition of cardiomyopathy,

suggested by the ESC and now widely
accepted worldwide, is a collection of myocardial disorders in which the heart muscle is
structurally and functionally abnormal in the
absence of coronary artery disease, hypertension, valvular or congenital heart disease
sufficient to cause the observed myocardial
abnormality. 1 This definition excludes
channelopathies, which do not lead to
structural changes in the myocardium. The
phenotypic classification proposed by John
Goodwin in 1961 of hypertrophic cardio
myopathy (HCM), dilated cardiomyopathy
(DCM), and restrictive cardiomyopathy has
withstood the test of time, with the addition
of arrhythmogenic, noncompaction, and
Takotsubo (also known as acute broken heart
syndrome) cardiomyopathies. Although
clinically useful, this classification does not
consider the cause or molecular mechanisms
responsible for the disease.
In the past decade, the realization that a
familial link for DCM is more frequent than
originally thought led to the discovery of a
large number of mutations in genes encoding proteins involved in various functions of
the myocardium, particularly the sarcomere
(Figure1). One example is thegene that
encodes titin (TTN), which has an important role in cardiac function, but the sheer
length of the gene and molecule it encodes
(the titin protein is ~1m long and spans
half a sarcomere) has inhibited attempts to
sequence it completely. Next-generation
sequencing approaches have been used to
identify mutations in the gene that result
in a truncated molecule in 14% of patients
with DCM, which increased to 27% when
parallel-capture next-generation sequencing was used.2 These findings have substantially increased the number (up to
70%) of patients with DCM who have a
known genetic mutation, and established
TTN as the gene most-commonly associated with DCM. This development will
HCM
DCM
ARVC
LV noncompaction
cardiomyopathy
Sarcomere
Desmosome
TTN
TPM1
TNNT2
JAG1
DES
DSP
TNNC1
TNNI3
NOTCH1
NICD
MYBPC3
ACTC1
DSG2
DSC2
Sarcolemma
MYL2/3
MYH6
MYH7
MIB1
Cytoplasm
PKP2
JUP
NICD
TFs
Figure 1 | Genetic mechanisms of cardiomyopathies. a | Mutations in TTN and other genes

encoding sarcomeric proteins have been shown to cause HCM (yellow stars). b | Similarly,
mutations in genes encoding either sarcomeric or desmosomal proteins have been linked with
DCM (pink stars). c | Mutations in genes encoding desmosomal proteins have also been
implicated in the pathogenesis of ARVC (green stars). d | Mutations involved in the NOTCH1
signalling pathway are thought to underlie LV noncompaction cardiomyopathy (blue stars).
Abbreviations: ARVC, arrhythmogenic right ventricular cardiomyopathy; DCM, dilated
cardiomyopathy; HCM, hypertrophic cardiomyopathy; LV, left ventricular; NICD1, NOTCH1
intracellular domain; TF, transcription factor. Permission for inset panels from partsac obtained
from NPG Hershberger, R.E. etal. Nat. Rev. Cardiol. 10, 531547 (2013).
NOVEMBER 2015 | 5
CARDIOLOGY
contribute to the impaired diastolic and systolic function observed in patients with this
disease. The exact molecular mechanisms
responsible for interstitial fibrosis remain
unknown, with a unifying hypothesis invoking developmental biology being proposed.6
Investigators using comprehensive transcriptional RNA analysis of cardiomyocytes and
nonmyocytes from two animal models of
HCM identified several changes in expression of profibrotic genes in nonmyocytes,
which preceded myocardial hypertrophy.7
The investigators then prevented pathological myocardial remodelling with allelespecific silencing of mutant transcripts to
target profibrotic molecules. This study
has important mechanistic and therapeutic
implications with regard to preventing or
reversing fibrosis in HCM.
Studies designed to improve our understanding of how genetic mutations are
translated into a clinical phenotype have
employed a wide spectrum of tools in the
past decade. These have included molecular modelling,8 structural and molecular
biology, biochemistry and pharmacology, as
well as biophysics. Coppini and colleagues
used patch clamping to compare the electro
mechanical properties of 26 myectomy
samples from patients with HCM with
control tissue.9 The HCM specimens had
abnormalities in the late sodium current,
which were corrected by administration of
ranolazine, an inhibitor of the late sodium
channels.9 Aside from its value in understanding the pathophysiology of the disease,
this study has stimulated the development
of a clinical trial using ranolazine for the
treatment of HCM.9
The initial discovery that mutations in
genes encoding sarcomeric proteins, such
as myosin heavy chains, can cause DCM
and HCM (Figure1) has raised the hope
that such diseases might be prevented or
cured with strategies to counteract the
molecular abnormality. Progress towards
this goal is slowly being made. For example,
in 2013, a study involving allele-specific,
gene-mediated RNA interference in the
cardiomyocytes of a mouse model of HCM
resulted in partial silencing of the mutated
molecule.10 This approach was sufficient to
prevent the mice from developing the HCM
phenotype. The discovery of genetic cell
engineering, without the use of viral vectors,
might provide effective ways of using such
strategies in humans with cardiomyo
pathy, in the future. The past 10years have
seen a rapidly accelerating rate of research
into the mechanisms and treatment of
6 | NOVEMBER 2015
cardiomyopathy, which has markedly

enhanced our understanding and management of the disease, and is poised to deliver
much more in the coming decade.
Qatar Cardiovascular Research Center,
POBox5825, Doha, Qatar.
m.yacoub@imperial.ac.uk
Competing interests
The author declares no competing interests.
1.
2.
3.
4.
Cecchi, F., Tomberli, B. & Olivotto, I. Clinical and

molecular classification of cardiomyopathies.
Glob. Cardiol. Sci. Pract. 2012, 4 (2012).
Herman, D.S. etal. Truncations of titin causing
dilated cardiomyopathy. N. Engl. J. Med. 366,
619628 (2012).
Golbus, J.R. etal. Population-based variation in
cardiomyopathy genes. Circ. Cardiovasc. Genet.
5, 391399 (2012).
Pan, S. etal. Cardiac structure and sarcomeric
genes associated with cardiomyopathy exhibit
marked intolerance of genetic variation.

Circ.Cardiovasc. Genet. 5, 602610 (2012).
5. Kalozoumi, G., Tzimas, C. & Sanoudou, D.
Theexpanding role of epigenetics. Glob. Cardiol.
Sci. Pract. 2012, 7 (2012).
6. Olivotto, I. etal. Developmental origins of
hypertrophic cardiomyopathy phenotypes:
aunifying hypothesis. Nat. Rev. Cardiol. 6,
317321 (2009).
7. Teekakirikul, P. etal. Cardiac fibrosis in mice
with hypertrophic cardiomyopathy is mediated
by non-myocyte proliferation and requires Tgf.
J. Clin. Invest. 120, 35203529 (2010).
8. Gaendrarao, P. etal. Molecular modeling of
disease causing mutations in domainC1
ofcMyBPC. PLoS ONE 8, e59206 (2013).
9. Coppini, R. etal. Late sodium current inhibition
reverses electromechanical dysfunction in
human hypertrophic cardiomyopathy. Circulation
127, 575584 (2013).
10. Jiang, J. etal. Allele-specific silencing of
mutantMyh6 transcripts in mice suppresses
hypertrophic cardiomyopathy. Science 342,
111114 (2013).
DECADE IN REVIEWDYSLIPIDAEMIA
Resurgence of targets and

compounds to treat dyslipidaemia
John J. P. Kastelein
Over the past decade, we have witnessed the unparalleled success of statins
to treat dyslipidaemia. Target identification by Mendelian randomization,
human monoclonal antibodies, gene therapy, RNA-based targets, and
atherogenic lipoproteins other than LDL cholesterol have fuelled intense
development efforts that might bear fruit in the very nearfuture.
Kastelein, J.J.P. Nat. Rev. Cardiol. 11, 629631 (2014); published online 9 September 2014;
doi:10.1038/nrcardio.2014.132
When Terje Pedersen first presented the

results of the Scandinavian Simvastatin
Survival Study at the AHA Scientific
Sessions on 16November1994 in Dallas,
TX, USA, he probably did not realize that
the study would initiate a global revolution in statin therapy. Today, statins are
the most widely prescribed class of drugs,
and although the results of this study were
presented 20years ago, no other drug has
since been shown to have any additional
benefit for patients with dyslipidaemia.
Inhibitors of phospholipases, cholesteryl
ester transfer protein (CETP), and cholesterol absorption, as well as agonists of
peroxisome proliferator-activated receptor,
thyroxin receptor, and nicotinic acid have
either been toxic or not provided a beneficial
outcome for patients. However, not all discoveries in the past decade for the therapeutic control of dyslipidaemia can be claimed
by statins. In 2003, Boileau and colleagues
mapped a locus associated with familial
hypercholesterolaemia in the gene encoding proprotein convertase subtilisin/kexin

type9 (PCSK9). As with the Scandinavian
Simvastatin Survival Study, Boileau etal.
could not have foreseen that their discovery would develop into the currently most
exciting class of lipid-lowering drugs
monoclonal antibodies against circulating
PCSK9. These two discoveries have led to an
unprecedented number of developments in
the field of dyslipidaemia in the past decade.
In 2004, the authors of two papers first
established the clinical importance of statins
in the treatment of cardiovascular disease,
which turned the hypothesis that lower
LDL cholesterol is better into a core principle. In the PROVEIT study,1 Cannon and
co-workers convincingly demonstrated that
80mg of atorvastatin was superior to 40mg
of pravastatin in outcome parameters including death, myocardial infarction, unstable
angina, and revascularization (a 16%
risk reduction in favour or atorvastat in;
CARDIOLOGY
1986
450
2013
First
Synvinolin
study
400
350
LDL-C levels (mg/dl)
300
RUTHERFORD
250
ENHANCE
ASAP
200
RADIANCE
150
100
50
th
va
er
ap
y
4
At
0
or
m
va
g
Si 80
m
m
va
Si
g
m
va
Si 40
m
m
80
g
va
m
g + 80
To
m
Ez
rc
g
et
e
10
60
Ev
m
m
g
o
g
42
+
st
0
at
m
in
g
+
st
at
in
Si
(apoCIII), apolipoprotein(a) [apo(a)], and

PCSK9, and which have all reached phaseI
and further clinical studies. These developments are just the beginning, and many
more will come inthe next decade. In the
second paper, which garnered little attention when first published, Stroes and colleagues performed the first proof-of-concept
study of gene therapy for dyslipidaemia.
Alipogene tiparvovec increases the removal
of postprandial chylomicrons, the particles
known to cause the acute and potentially
lethal haemorrhagic pancreatitis associated with the chylomicronaemia syndrome.
Alipogene tiparvovec (commercially known
as Glybera; UniQure, Netherlands) became
the first gene therapy product to be approved
for any indication in the Westernworld.5
Many developments since 1994 have
revolv ed around the biology, pathology, and therapeutic lowering of LDLcholesterol levels. Only after a number of
international consortia performed large
Mendelian randomization studies was the
link made between CAD and apo(a), triglyceride-rich lipoproteins, and remnant
cholesterol.6,7 These observations are semi
nal for our understanding of atherogenesis
and initiated an international effort to identify novel strategies to decrease circulating levels of these lipoproteins. Moreover,
the results of these Mendelian randomization studies, such as those performed
by Nordestgaard 6 and Kathiresan, 7 have
questioned the relationship between HDL
cholesterol and CAD, which might explain
the disappointing results of strategies that
raise HDL-cholesterol levels. By contrast,
Mendelian randomization and intervention studies have both validated the role of
lowering LDL-cholesterol levels to treat dyslipidaemia. Consequently, the discoveries by
investigators that CETP, triglyceride-rich
lipoproteins, and apo(a) are indeed involved
in atherogenesis have led to a search for
compounds to safely lower the levels of these
proteins, as well as those of apoCIII and, of
course, PCSK9. Mendelian randomization
studies are now an integral approach in the
development of drugs to treat dyslipidaemia.
Of all the developments over the past
decade, a paper by Stein and colleagues in
2012 deserves the title of game changer. In
this study, a monoclonal antibody against
PCSK9 (alirocumab), reduced the relative
LDL-cholesterol level by >60% in both
volunt eers with normal lipid levels and
patients with familial hypercholesterolaemia.8 The excellent safety profile of this
drug class has been a relief for clinicians,
No
P=0.005), when administered shortly after

an acute coronary syndrome (ACS).1 This
study transformed care for patients with
ACS; 80mg of atorvastatin straight after
an ACS episode has now become routine
clinical practice in coronary care units in
most countries. This strategy has saved
innumerable lives of patients with coronary
artery disease (CAD) and protected them
against recurrent events.
In the TNT study,2 also published in 2004,
LaRosa and colleagues demonstrated that
80mg was superior to 10mg of atorvastatin
for treating patients with stable CAD. Highdose atorvastatin reduced the primary end
point (occurrence of a major cardiovascular
event) by 22% compared with the lower dose
of the drug (P<0.001).2 This study has also
enabled many secondary analyses that aid
our understanding of renal function during
statin therapy, the clinical safety of low LDLcholesterol levels, and that a reduction in
adverse clinical outcomes is independent
of almost any baseline patient characteristic, including LDL-cholesterol level itself.
These statin principles were extended into
the realm of primary prevention in 2008 in
the JUPITER trial.3 Ridker etal. selected
patients with an elevated Creactive protein
level (measured by high-sensitivity assay),
but who were free from CAD, and demonstrated that a daily dose of 20mg rosuvastatin robustly reduced the incidence of major
adverse cardiovascular events by 44% compared with placebo (P<0.00001).3 Again,
efficacy was observed in all subgroups,
including men and women, the elderly and
young, tobacco smokers and nonsmokers,
and those with or without metabolic syndrome. More importantly, the JUPITER
trial3 results highlighted the link between
inflammation, dyslipidaemia, and atherosclerotic vascular disease, which has led to
the current development of selective antiinflammatory strategies in outcome trials
by the same research group.
In two papers published in 2006 and
2008, respectively, investigators applied
advanced molecular biology techniques
to the study of lipid control.4,5 In the first
study, mRNA inhibition by subcutaneously administered small oligonucleotides
against apolipoproteinB100 (apoB100)
lowered the levels of all atherogenic lipoproteins in human volunteers with mild
dyslipidaemia.4 These results have led to
the development of small inhibitory RNAs,
mRNA inhibitors, and locked nucleic acid
inhibitors that target angiopoietin-related
protein3, apoB100, apolipoproteinCIII
Therapy
Figure 1 | LDL-cholesterol levels achieved by

lipid-lowering therapies developed during the
past 3decades in patients with familial
hypercholesterolaemia. Dosing frequencies are
per day unless otherwise stated. Abbreviations:
Atorva, atorvastatixn; Evo, evolocumab; Eze,
ezetimibe; LDLC, LDL-cholesterol; Simva,
simvastatin; Torcet, torcetrapib.
especially given the previous toxicityrelated failures of other compounds. Simi

lar results were obtained in studies with
bococizumab and evolocumab, and all three
monoclonal antibodies targeted against
PCSK9 are already in advanced, phaseIII
outcome trials. Only 9years have passed
between Boileau and colleagues initial
PCSK9 discovery and Stein and co-workers
2012 paper. This rapid development has
been made possible by recombinant DNA
and antibody technology. Compared with
the >25years between cloning of the lipoprotein lipase (LPL) gene and the LPL gene
therapy trial by our own group, the development of PCSK9-based technologies has
been a remarkable achievement.
However, for which patients is the discovery of PCSK9 monoclonal antibodies
most beneficial in the short term? To have
personally witnessed the ever-decreasing
LDL-cholesterol levels that different therapies have achieved in patients with familial
hypercholesterolaemia has been immensely
gratifying. Between the first simvastatin
trial in 1986 and the RUTHERFORD
study of evolocumab just last year, a steady
NOVEMBER 2015 | 7
CARDIOLOGY
decrease in LDL-cholesterol levels from
9.6mmol/l (371mg/dl) to ~1.7mmol/l
(65mg/dl) has been achieved (Figure1).
The discovery that monoclonal antibodies
against PCSK9 can lower LDL-cholesterol
levels to such an extent has essentially
cured familial hypercholesterolaemia; in
fact, the LDL-cholesterol levels achieved
in these patients are now lower than in the
general population. Who would have had
the temerity to predict that in 2003?
The final discovery that I would like to
highlight was reported in two papers in
which triglyceride-rich lipoprotein and
remnant cholesterol levels were causally
linked to apoCIII and CAD. 9,10 ApoC
III was first hypothesized to reduce trigly
ceride levels and contribute to the risk of
CAD, and on that basis a mRNA inhibitor
ofapoCIII was developed. These two papers
strengthen the association between apoCIII,
triglyceride metabolism, and CAD risk.9,10
The fact that the apoCIII inhibitor is already
in clinical development is, therefore, timely
and fortuitous. With all of these results
comes the hope that novel small-molecule
compounds, monoclonal antibodies, and
RNA technology will transform dyslipid
aemia treatment in the coming decade, for
a second time since 1994. We might finally
be able to eliminate dyslipidaemia and subsequent atherosclerotic vascular disease for
our patients in the very near future.
Department of Vascular Medicine,
AcademicMedical Center, University
ofAmsterdam, Meibergdreef 9,
1105AZAmsterdam, Netherlands.
j.j.kastelein@amc.uva.nl
Competing interests
J.J.P.K. declares that he has acted as a consultant
and received honouraria from the following
companies: Aegerion, Amgen, AstraZeneca,
Atheronova, Boehringer Ingelheim, Catabasis,
Cerenis, CSL Behring, Dezima Pharmaceuticals,
EliLilly, Esperion, Genzyme, Isis, Merck, Novartis,
Omthera, Pronova, Regeneron, Sanofi, The Medicines
Company, UniQure, and VascularBiogenics.
1.
2.
3.
4.
Cannon, C.P. etal. Intensive versus moderate

lipid lowering with statins after acute coronary
syndromes. N. Engl. J. Med. 350, 14951504
(2004).
LaRosa, J.C. etal. Intensive lipid lowering with
atorvastatin in patients with stable coronary
disease. N. Engl. J. Med. 352, 14251435
(2005).
Ridker, P.M. etal. Rosuvastatin to prevent
vascular events in men and women with
elevated Creactive protein. N. Engl. J. Med.
359, 21952207 (2008).
Kastelein, J.J.P. etal. Potent reduction of
apolipoproteinB and low-density lipoprotein
cholesterol by short-term administration of
anantisense inhibitor of apolipoproteinB.
Circulation 114, 17291735 (2006).
8 | NOVEMBER 2015
5.
6.
7.
Stroes, E.S. etal. Intramuscular administration

of AAV1-lipoprotein lipase S447X lowers
triglycerides in lipoprotein lipase-deficient
patients. Arterioscler. Thromb. Vasc. Biol. 28,
23032304 (2008).
Kamstrup, P.R., Tybjrg-Hansen, A.,
Steffensen, R. & Nordestgaard, B.G.
Genetically elevated lipoprotein(a) and
increased risk of myocardial infarction.
JAMA301, 23312339 (2009).
Do, R. etal. Common variants associated with
plasma triglycerides and risk for coronary artery
disease. Nat. Genet. 45, 13451352 (2013).
8.
Stein, E.A. etal. Effect of a monoclonal

antibodies to PCSK9 on LDL cholesterol.
N.Engl. J. Med. 366, 11081118 (2012).
9. Jrgensen, A.B., Frikke-Schmidt, R.,
Nordestgaard, B.G. & Tybjrg-Hansen, A.
Lossof-function mutations in APOC3 and risk of
ischemic vascular disease. N. Engl. J. Med. 371,
3241 (2014).
10. The TG and HDL Group of the Exome
Sequencing Project, National, Heart, Lung, and
Blood Institute. Loss-of-function mutations in
APOC3, triglycerides, and coronary heart
disease. N. Engl. J. Med. 371, 2231 (2014).
DECADE IN REVIEWHEART FAILURE
10Years of progress in HF
researchwhat have we learned?
Henry Krum
In this Decade in Review article, I highlight the top 10 advances in heart

failure (HF) over the past decade, including new pharmacological therapies
and expanded indications for devices in HF with reduced ejection fraction.
The poor progress in acute HF and HF with preservedejection fraction is
emphasised. Biomarkers and devices that help prevent, detect, and guide
treatment represent the future of HFmanagement.
Krum, H. Nat. Rev. Cardiol. 11, 631633 (2014); published online 2 September 2014;
doi:10.1038/nrcardio.2014.127
One of the great advances in cardiovascular

therapy during the past 20years has been
the use of cardiac resynchronization therapy
(CRT) in patients with heart failure (HF)
with reduced ejection fraction (HFrEF).
Several studies have indicated major out
come benefits in patients with HFrEF and
advanced NYHA classIIIIV symptoms.
Investigators in MADITCRT1 tested the
hypothesis that mildly symptomatic patients
with HFrEF would also benefit from CRT.
In a cohort of >1,000 patients, the primary
end point (HF events or death) was reduced
to 17.2% with CRT versus 25.3% without
CRT, driven primarily by a reduction in HF
events.1 These findings were supported by
a subsequent trial, in which HF events and
mortality were reduced by CRT. CRT is now
guideline-recommended for all patients
with HFrEF and a prolonged QRS duration
(>150ms).
Major drug therapy trials directed towards
novel targets have also been conducted in
the past decade (Figure 1). Pharmacological
inhibition of neprilysin can augment the
physiological effects of endogenous natri
uretic peptides, which have beneficial
vasodilatory, anti-fibrotic, and natriuretic
actions. Dual therapy with angiotensinconverting enzyme (ACE) inhibitors was
shown to be unsuccessful because of study

design flaws and off-target effects; however,
combination with an angiotensin-receptor
blocker (ARB) seems to overcome this risk
and might offer clinical benefits beyond
ARB monotherapy. The researchers in the
PARADIGMHF study 2 aimed to determine whether the ARBneprilysin inhibitor
LCZ696 would be superior to the goldstandard ACE-inhibitor enalapril in patients
with HFrEF. Patients received either the
ARBneprilysin inhibitor LCZ696 (200mg
twice daily) or enalapril (10mg twice daily),
with the primary end point of cardiovascular
death or HF hospitalization (n=8,442).
Although the final results have not yet been
presented, the study was terminated pre
maturely for overwhelming efficacy benefit.
Given the positive outcome, and assuming
that the drug has an acceptable safety profile,
LCZ696 should supplant ACE inhibitors as
standard therapy for thesepatients.
The clinical benefit of blockers for HF
might be partly attributable to their capacity to lower heart rate. If channel-blocking
agents, which are direct sinus node inhibitors, were used to investigate this heart-ratelowering hypothesis in the SHIfT study 3
(n=6,558; 89% receiving background
blocker therapy), in which the If channel
CARDIOLOGY
blocker ivabradine was compared with
placebo. The primary end point (cardiovascular death or HF hospitalization) was
reached by 24% of patients receiving ivabradine compared with 29% of patients taking
placebo, driven primarily by reduced hospitalization for HF. The benefits of adding
ivabradine were diminished with increasing
dose of background blockers, possibly
because the additive effect of ivabradine on
lowering the heart-rate was abrogated by
full-dose blocker therapy. Ivabradine is
now approved as an adjunct to background
ACE inhibitor, blocker, and mineralo
corticoid-receptor antagonists (MRAs) in
symptomatic patients with HFrEF and a
sinus rhythm heart rate >70bpm.
MRAs increase survival in patients with
advanced HFrEF and in those who have
experienced a myocardial infarction and
have HF. However, until the EMPHASISHF
trial,4 patients with mild symptoms of HFrEF
had not been formally studied. Researchers
in EMPHASISHF4 evaluated the efficacy of
eplerenone compared with placebo in 2,737
patients in NYHA classII. The trial was terminated prematurely (median follow-up
21months) because only 18.3% of patients
receiving eplerenone reached the primary
end point (cardiovascular death or hospitalization for HF) compared with 25.9% in the
placebo group.4 Importantly, these benefits
were additional to background ACE inhibitor and blocker therapy. MRAs have, therefore, become mandated therapy for patients
with mild HFrEF.
In contrast to HFrEF, pharmacological
therapy has so far failed to reduce major
adverse events in patients with HF and preserved ejection fraction (HFpEF). Given the
established benefit of MRAs in patients with
HFrEF, researchers in the TOPCAT study 5
evaluated the efficacy of spironolactone
(1545mg per day) versus placebo in 3,445
patients with an ejection fraction >45% in
addition to one other HF indicator, in an
attempt to recruit a more homogenous population with true HFpEF. The occurrence
of the primary end point (cardiovascular
death, cardiac arrest, or HF hospitalization)
was not significantly different between the
two groups. Patients who entered the study
because of an elevated plasma Btype natri
uretic peptide (BNP) concentration exhibited
a stand-alone benefit on the primary end
point with spironolactone. This result suggests that patients who fulfilled the criteria of
HF hospitalization rather than elevated BNP
might not have had true HFpEF, and in turn
did not benefit from MRA therapy. Further
Novel drug therapies

Improved understanding
of pathophysiology
Expanding indications
for HF therapeutic devices
Advances in HF
HF preventive strategies
Gene-based therapies
Plasma biomarker and
implantable device-based
tracking of patient clinic status
Figure 1 | Key advances in the past 10years of HF research.
efforts to recruit a more homogenous patient

population and establish the existence of
true HFpEF before study entry are required
to evaluate the role of MRAs definitively in
thissetting.
Improving HF outcomes by treating comorbidities has become a concept
of great therapeutic interest. Two inter
related strategies involve the correction of
HFrelated anaemia and iron deficiency.
Erythropoietin-stimulating agents (ESAs)
are frequently used to treat anaemia, but have
not reduced major cardiovascular events in
HF patients. By contrast, correction of iron
deficiency has produced more encouraging
results. Investigators in the FAIRHF study 6
enrolled 459 patients with HFrEF and iron
deficiency, based on either serum ferritin or
transferrin saturation levels. Patients were
randomly allocated to either intravenous
ferric carboxymaltose or saline. Fifty percent
of the patients who received ferric carboxymaltose showed improvement according to
the Patient Global Assessment, compared
with only 28% of those receiving placebo.
However, an adequately-sized trial to assess
the effect of intravenous iron on morbidity
and mortality has not yet been performed in
patients with HFrEF who are iron-deficient.
Treatment of acute HF has not changed for
40years. Numerous proposed agents have
demonstrated favourable effects on natri
uresis and diuresis, but at the expense of worsened renal function and lack of benefit on
prognosis. Relaxin is an endogenous peptide
with diuretic, natriuretic, vasodilatory, and
anti-fibrotic properties. Investigators in
the RELAXAHF study 7 evaluated 1,061
patients with acute HF randomly allocated to
serelaxin (a recombinant form of relaxin) or
placebo. Serelaxin improved one of two dyspnoea end points, but had no effect on cardiovascular/renal death or hospital readmission
at 60days in patients with acute HF. However,
fewer deaths occurred at 180days with serelaxin, and evidence existed of reduced inhospital worsening of HF. On the basis of
these mixed findings, regulatory authorities

did not approve the agent; however, the findings were sufficiently compelling to warrant
a definitive mortality trial, RELAXII, which
is currentlyongoing.
Gene-based approaches for HF management are beginning to emerge, after much
promise. Insertion of the sarcoplasmic reticu
lum Ca2+-ATPase (SERCA2a) gene into the
failing heart has been considered a potential
therapeutic strategy on the basis of its pivotal
role in myocardial contractile function. The
CUPID study 8 involved 39 patients with HF
who received intracoronary adeno-associated
virus type1/SERCA2a or placebo. Initial
findings suggested that gene transfer might
improve relevant surrogate end points and
reduce cardiovascular events. The CUPID2
trial, with an increased cohort of patients,
iscurrently ongoing.
The discovery of biomarkers that indicate the likelihood of risk factors trans
itioning to left ventricular (LV) dysfunction
might change the future of HF diagnosis.
Investigators in the STOPHF study 9 trialled
a BNP screening programme in which highrisk asymptomatic patients (n=1374) were
randomly assigned to receive care based on
knowledge of the plasma BNP level, or usual
care. Using this approach, 5.3% of the BNP
group developed LV dysfunction (with or
without HF) compared with 8.7% receiving
usual care, over a 4.2year period. Additional
biomarkers, including some specific to the
LV dysfunction transition phase (such as
ST2, galectin3), have been proposed and
are currently under assessment.
Tracking of patient clinical status has
underg one substantial technological
advance in the past decade. In an individual
patient meta-analysis of over 2,000 individuals with HFpEF or HFrEF, BNP-guided HF
treatment was shown to significantly reduce
all-cause mortality and HF hospitalization
in those <70years of age.10 Newly available tools for tracking patient clinical status
remotely, such as pulmonary pressure
NOVEMBER 2015 | 9
CARDIOLOGY
measurement using the CardioMEMS HF
system (St.Jude Medical, USA), can complement biomarker-guided therapy, permitting earlier intervention in, for example, the
acute decompensation setting.
Substantial advances have been made in
HF treatment over the past decade. Prog
nosis is improving for patients with HFrEF,
with novel pharmacological strategies, new
devices and interventions, as well as celland gene-based therapies currently being
developed (Figure 1). However, successful strategies in both acute HF and HFpEF
remain elusive. Personalized medicine will
be a major research focus in the future,
integrating neurohormonal, proteomic,
metabolomic, and genomic information into
therapeutic decision-making.
Centre of Cardiovascular Research
&Education in Therapeutics, School of
PublicHealth & Preventive Medicine,
MonashUniversity, 99 Commercial Road,
Melbourne, VIC 3004, Australia.
henry.krum@monash.edu
Competing interests
H.K. declares that he has received research grants
from Bayer, Medtronic, Novartis, Pfizer, and Servier.
1.
Moss, A.J. etal. Cardiac-resynchronization

therapy for the prevention of heart failure events.
N.Engl. J.Med. 361, 13291338 (2009).
2. McMurray, J.J. etal. Baseline characteristics and
treatment of patients in Prospective comparison
of ARNI with ACEI to Determine Impact on
Global Mortality and morbidity in Heart Failure
trial (PARADIGM-HF). Eur. J.Heart Fail. 16,
817825 (2014).
3. Swedberg, K. etal. Ivabradine and outcomes
inchronic heart failure (SHIFT): a randomised
placebo-controlled study. Lancet 376, 875885
(2010).
4. Zannad, F. etal. Eplerenone in patients with
systolic heart failure and mild symptoms.
N.Engl. J.Med. 364, 1121 (2011).
5. Pitt, B. etal. Spironolactone for heart failure
with preserved ejection fraction. N.Engl. J.Med.
370, 13831392 (2014).
6. Anker, S.D. etal. Ferric carboxymaltose in
patients with heart failure and iron deficiency.
N. Engl. J. Med. 361, 24362448 (2009).
7. Teerlink, J.R. etal. Serelaxin, recombinant
human relaxin2, for treatment of acute heart
failure (RELAX-AHF): a randomised, placebocontrolled trial. Lancet 381, 2939 (2013).
8. Jessup, M. etal. Calcium upregulation by
percutaneous administration of gene therapy
incardiac disease (CUPID): a phase2 trial
ofintracoronary gene therapy of sarcoplasmic
reticulum Ca2+-ATPase in patients with
advanced heart failure. Circulation 124,
304313 (2011).
9. Ledwidge, M. etal. Natriuretic peptide-based
screening and collaborative care for heart
failure: the STOP-HF randomized trial. JAMA
310, 6674 (2013).
10. Troughton, R.W. etal. Effect of Btype
natriuretic peptide-guided treatment of chronic
heart failure on total mortality and
hospitalization: an individual patient metaanalysis. Eur. Heart J. 35, 15591567 (2014).
10 | NOVEMBER 2015
DECADE IN REVIEWHYPERTENSION
The past decade in hypertension

facts, hopes, and hypes
Thomas Unger
Hypertension research in the past decade has been a mixture of hope

and hype. In the absence of new drug developments, clinical intervention
procedures such as renal nerve ablation and baroreflex activation therapy
have dominated the research, but the results have not yet fulfilled the
great expectations.
Unger, T. Nat. Rev. Cardiol. 11, 633635 (2014); published online 2 September 2014;
doi:10.1038/nrcardio.2014.131
A simple, brief, catheter-based procedure to

ablate sympathetic nerves can be done safely
without long-term complications and could
result in persistent reductions of blood pressure.1 Such was the hope 5years ago after the
publication of a study involving 45 patients
by an international team ofresearchers who
investigated the efficacy of catheter-based
renal sympathetic denervation for resistant
hypertension.1 Indeed, initial findings suggested that resistant hypertension, which
is refractory to pharmacological therapy,
could be treated with an interventional
approach. This study fuelled an outburst
of studies on renal sympathetic nerve ablations for the treatment of hypertension.
However, much to the disappointment of
cardiologists worldwide, findings from the
SYMPLICITYHTN3 trial2 released early in
2014 demonstrated no significant differences
in systolic blood pressure between the renaldenervation group and the sham-controlled
group after 6months (Figure 1). Now that
the hype surrounding renal sympathetic
denervation has subsided, we must determine
whether this procedure can offer permanent
benefit to patients with hypertension and,
most importantly, which treatment-resistant
subgroups (age, ethnicity, sex, duration
of hypertension, state of the sympathetic
nervous system) are eligible for denervation.
Immunization against angiotensinII to
reduce blood pressure represented another
promising strategy to treat hypertension
6years ago. The efficacy of this strategy
was evaluated in a phaseIIa trial involving 72 patients.3 After a 14week treatment
period, a significant reduction in ambulatory blood pressure was observed in patients
who received a higher dose of the vaccine,
compared with placebo. However, these
findings could not be reproduced in further
clinical studies. Nevertheless, vaccinations
against a population-wide disease such as
hypertension might still be effective if an

appropriate target can be identified, and the
associated immunological problems can
beovercome.
A third nonpharmacological approach for
the treatment of hypertension has emerged
for patients with resistant hypertension.
Baroreflex activation therapy (BAT) involves
the implantation of a device that permanently
stimulates the baroreceptor reflex, which is
usually attenuated in patients with hypertension. Although BAT for lowering blood
pressure is no longer a novel concept, it
has regained attention because of marked
improvements in device technology. In a randomized trial published in 2011, 265 patients
with resistant hypertension were assigned to
either groupA (BAT applied permanently for
up to 12months) or groupB (BAT deferred
until after 6months).4 No differences were
apparent between the two groups after
6months, but an ancillary analysis revealed
a significantly higher percentage of patients
(42% versus 24%, P<0.005) who achieved
systolic blood pressure 140mmHg in
groupA compared with groupB.4 A treatment regimen involving surgical implant
ation of a device is unlikely to become
standard therapy for all hypertensive patients,
but this strategy might be suitable for selected
patients who do not respond sufficiently to
anti-hypertensive drug therapy.
In the past 2decades, the development
of drugs for the treatment of hypertension and other cardiovascular diseases has
been largely dominated by inhibitors of
the reninangiotensin system, reflected by
many comparative drug studies with large
patient cohorts. A prime example is the
ONTARGET trial,5 in which the efficacy
of the angiotensin receptor blocker (ARB),
telmisartan, was compared with the goldstandard angiotensin-converting-enzyme
(ACE) inhibitor, ramipril, in patients with
CARDIOLOGY
Baroreflex
activation therapy
Immunisation
against
angiotensin II
Reduction in
blood pressure
Renal
denervation
RAS inhibitors
Drug intervention
Nondrug intervention
Figure 1 | The hits and misses in the development of blood-pressure lowering therapies
duringthe past decade. The ARB telmisartan was shown to be as effective as the goldstandardACE-inhibitor ramipril in the ONTARGET trial. However, nondrug interventions were
lesssuccessful. Renal denervation failed to reduce systolic blood pressure in patients with
resistant hypertension in the SYMPLICITY HTN3 trial. Furthermore, initial findings that
immunisation against angiotensionII reduced ambulatory blood pressure could not be
reproduced in subsequent clinical studies. Finally, baroreflex activation therapy did not
significantly reduce systolic blood pressure after 12months in a randomized controlled trial,
butsignificantly increased the number of patients who achieved systolic blood pressure of
140mmHg. Abbreviations: ACE, angiotensin-converting-enzyme; ARB, angiotensin receptor
blocker; RAS,reninangiotensin system.
coronary artery disease or diabetes mel

litus, in addition to other cardiovascular
risk factors, including hypertension. After
a mean follow-up period of 5years, telmi
sartan was noninferior to ramipril in terms of
the composite primary end point of cardio
vascular death, myocardial infarction, stroke,
or hospitalization for heart failure, and was
better tolerated than the ACE inhibitor.
A combination of the two drugs did not
provide further benefit, but in fact increased
the risk of hypotension, syncope, renal dysfunction, and hyperkalaemia. In the parallel
TRANSCEND trial6 involving 5,926 patients
who were unable to tolerate ACE inhibitors,
telmisartan was superior to placebo treatment only when hospitalization for heart
failure was removed from the combined
primary end point.
At first glance, these two trials do not
seem to have conveyed any new knowledge; however, on further inspection, many
lessons can be gained from them. Firstly,
the conventional strategy of clinical trials
in cardiovascular disease (that is, to extract
marginally significant benefits of one drug
versus another) will be questioned because
the gain in clinical evidence is disproportionate to the time and financial expenditure of
such a trial. In the future, mega-trials on

therapeutic regimens will have to be replaced
by smaller, better defined, and more individualized clinical studies. Secondly, the
long-debated efficacy of ACE inhibitors
versus ARBs in treating cardiovascular
disease can now be considered settled; each
is noninferior to the other, but ARBs are
generally better tolerated. Thirdly, in the
TRANSCEND trial,6 a combination of two
different classes of reninangiotensin inhibitors was not beneficial for patients at high
cardiovascular risk. For these reasons, the
ONTARGETTRANSCEND trial5,6 was a
hallmark study of the past decade, and will
probably be the last mega-trial of this kind
in cardiovascular therapy.
Concerning the pathophysiology of
hypertension, two seminal publications have
aroused much interest in the past decade. An
article describing hydrogen sulphide (H2S)
as a physiological vasorelaxant was published in 2008.7 Genetic deletion of the H2Sgenerating enzyme cystathionine lyase
markedly reduced H2S levels in serum and
tissue. Mutant mice lacking this enzyme
showed pronounced hypertension, together
with diminished endothelium-dependent
vascular relaxation. These results strongly
indicate a physiological role for H2S as a

vasodilator and blood-pressure regulator.
In 2011, a paper examining the association
between angiotensinII-induced hyper
tension and vascular injury, and its relationship with Tregulatory lymphocytes was
published.8 The investigators convincingly
showed that Tregulatory lymphocytes suppressed angiotensinII-mediated vascular
injury through their anti-inflammatory
actions,8 adding support to the hypothesis
that immune mechanisms modulate the
various physiological and pathophysio
logical actions of angiotensinII. This notion
is consistent with the pathological concept
that undercurrents, such as immune mechanisms, inflammation, proliferation, and
fibrosis, are important in disease processes,
forming a link between various pathologies
(such as those involving the kidneys, heart,
and blood vessels).9
Finally, we should not forget genetics.
The findings from a study published in 2009
offered an early insight into the genetic basis
of hypertension, and the identified loci might
be new targets for the reduction of blood
pressure.10 After decades of heavy spending
and international collaborative endeavours
to unveil the complex genetic background
underlying hypertension, we might be
moving away from the general one-size-fitsall approach and towards more personalized
methods of diagnosis and treatment.
At present, the future of hypertension
research is somewhat diffuse. The golden
age of hypertension research appears to have
come to a temporary end, as most of the
large pharmaceutical drug companies have
stopped developing new anti-hypertensive
therapies. Despite this, new areas of research
are emerging; scientists are beginning to view
hypertension as a pathological interface in
the development of disorders involving the
blood vessels, heart, and kidneys, and will
develop strategies to target common pathological undercurrents such as inflammation,
fibrosis, and proliferative processes.
CARIM School for Cardiovascular Diseases,
Maastricht University, PO Box 616, 6200 MD
Maastricht, Netherlands.
t.unger@maastrichtuniversity.nl
Competing interests
1.
2.
Krum, H. etal. Catheter-based renal sympathetic

denervation for resistant hypertension:
amulticentre safety and proofofprinciple
cohortstudy. Lancet 373, 12751281 (2009).
Bhatt, D.L. etal. A controlled trial of renal
denervation for resistant hypertension. N. Engl.
J. Med. 370, 13931401 (2014).
NOVEMBER 2015 | 11
CARDIOLOGY
3.
4.
5.
6.
Tissot, A.C. etal. Effect of immunisation

against angiotensin II with CYT006-AngQb on
ambulatory blood pressure: a double-blind,
randomised, placebo-controlled phaseIIa
study. Lancet 371, 821827 (2008).
Bisognano, J.D. etal. Baroreflex activation
therapy lowers blood pressure in patients with
resistant hypertension: results from the doubleblind, randomized, placebo-controlled Rheos
Pivotal Trial. J. Am. Coll. Cardiol. 58, 765773
(2011).
Yusuf, S. etal. Telmisartan, ramipril, or both
inpatients at high risk for vascular events.
N.Engl. J. Med. 358, 15471559 (2008).
Yusuf, S. etal. Effects of the angiotensin-receptor
blocker telmisartan on cardiovascular events in
high-risk patients intolerant to angiotensin-
converting enzyme inhibitors: arandomised

controlled trial. Lancet 372, 11741183 (2008).
7. Yang, G. etal. H2S as a physiologic
vasorelaxant: hypertension in mice with
deletion of cystathionine gamma-lyase. Science
322, 587590 (2008).
8. Barhoumi, T. etal. T regulatory lymphocytes
prevent angiotensin IIinduced hypertension
and vascular injury. Hypertension 57, 469476
(2011).
9. Hess, K., Marx, N. & Lehrke, M. Cardiovascular
disease and diabetes: the vulnerable patient.
Eur. Heart J. Suppl. 14, B4B13 (2012).
10. Newton-Cheh, C. etal. Genome-wide
association study identifies eight loci
associated with blood pressure. Nat. Genet. 41,
666676 (2009).
DECADE IN REVIEWPERIPHERAL VASCULAR DISEASE
10Years of breakthroughs
in peripheral vascular disease
Mark A. Creager
Clinical trials published during the past decade have had substantial
effects on the treatment of peripheral vascular diseases. In this article,
Idiscuss ten important trials that have influenced treatment for common
vascular disorders, including peripheral artery disease, abdominal aortic
aneurysm, renal artery disease, extracranial carotid artery disease, and
venous thromboembolism.
Creager, M. A. Nat. Rev. Cardiol. 11, 635636 (2014); published online 30 September 2014;
doi:10.1038/nrcardio.2014.153
Patients with peripheral artery disease

(PAD) are at increased risk of myocardial
infarction, stroke, and cardiovascular death.
Antiplatelet therapy, including aspirin, is
recommended for patients with PAD. Two
prospective studies were designed to determine whether aspirin can prevent cardio
vascular events in asymptomatic patients
with PAD. Investigators in the Aspirin
for Asymptomatic Atherosclerosis Trial1
recruited 28,980 individuals free from
clinical cardiovascular disease, of whom
3,350 were found to have PAD on the basis
of an anklebrachial index (ABI) 0.95.
Participants received either aspirin (100mg
daily) or placebo, and were followed up for
a mean of 8.2years. The primary end point
of fatal or nonfatal coronary events, stroke,
or revascularization was not significantly
different between the aspirin and placebo
groups (HR1.03, 95%CI 0.841.27).1 In
the POPADAD trial,2 investigators examined the efficacy of aspirin in reducing
cardiovascular events in 1,276 patients with
asymptomatic PAD (defined as an ABI
0.99) and either type1 or type2 diabetes
mellitus. Patients received aspirin (100mg
12 | NOVEMBER 2015
daily) or placebo, and were followed up for

a median of 6.7years. No significant difference in the primary composite end point
of fatal coronary heart disease or stroke,
nonfatal myocardial infarction, nonfatal
stroke, or above ankle amputation for critical limb ischaemia was observed in patients
treated with or without aspirin (HR0.98,
95%CI 0.761.26).2 These findings reject the
notion that aspirin therapy is effective for all
patients with PAD, and led to modifications
in ACCF/AHA guideline recommendations
regarding the use of antiplatelet therapy in
patients with asymptomatic PAD.
Although both exercise training and
endovascular revascularization improve
symptoms of intermittent claudication in
patients with PAD, the merits of each treatment have been controversial. The CLEVER
study 3 involved 111 patients with PAD and
aortoiliac artery stenosis randomly allocated to receive optimal medical therapy
alone, or in combination with supervised
exercise training or stent revascularization.
After 6months, the change in peak walking
time was greater in the exercise group than
in the stenting group (5.84.6min versus
3.74.9min; P=0.04), and both interventions were superior to medical therapy alone
(1.21.6min; P<0.001 and P<0.02, respectively).3 This study confirms the efficacy of
exercise training for patients with claudi
cation and aortoiliac artery disease, but
also supports stenting for patients unable
or unwilling to participate in an exercise
training programme.
Repair of large abdominal aortic aneurysms (AAAs) reduces the risk of aortic
rupture and aneurysm-related mortality.
The efficacy of endovascular repair versus
open surgical repair was evaluated in
345 patients with an AAA 5.0cm in the
DREAM trial.4 The primary end point, a
composite of 30day operative mortality
and severe complications, was numerically
greater in the open surgical group than in
the endovascular repair group (risk ratio
[RR]2.1, 95%CI 0.95.4, P=0.1), and
operative mortality alone trended higher
in the open surgical group (RR3.9, 95%CI
0.932.9, P=0.1).4 The incidence of severe
complications was higher after surgical
repair (RR3.9, 95%CI 1.39.1, P<0.01). The
efficacy of endovascular repair versus open
repair of AAAs 5.5cm was also assessed
in 1,082 patients in the EVAR trial1.5 Allcause mortality was not significantly different between the two groups, although
aneurysm-related mortality was significantly reduced after endovascular repair
(HR0.55, 95%CI 0.310.96, P=0.04). 5
Postoperative complications and the need
for reintervention occurred more frequently
in the endovascular group. Together, these
studies support endovascular repair of
AAAs as an alternative to surgical repair,
but highlight the importance of long-term
surveillance for endograft complications.
The efficacy of endovascular repair of
AAAs among patients deemed unfit for
open surgical repair was evaluated in the
EVAR trial2.6 Patients with an AAA 5.5cm
(n=3,338) were randomly allocated to endovascular repair or no intervention and followed up for up to 4years. Neither all-cause
nor aneurysm-related mortality differed
significantly between the two groups.6 The
30day operative mortality in the endo
vascular repair group was 9%, indicating the
high-risk nature of the study participants.6
These observations raise an important note
of caution for endovascular repair of AAAs
in patients unfit for open surgery, and suggest
that attention should be instead focused on
improving overall medicalfitness.
The role of renal artery stenting for the
treatment of renal artery stenosis has been
CARDIOLOGY
controversial. Investigators in the CORAL
study 7 compared the efficacy of stenting
plus standard medical therapy to medical
therapy alone in 947 patients with severe
renal artery stenosis and systolic hyper
tension or chronic kidney disease. Standard
medical therapy included an angiotensin
receptor blocker, with or without a diuretic,
a calcium channel blocker, and a statin. After
up to 5years of follow-up, no significant difference was found between the two groups
in the primary composite end point of death
from cardiovascular or renal causes, myocardial infarction, stroke, hospitalization for
congestive heart failure, progressive renal
insufficiency, or renal replacement therapy.7
Furthermore, no significant difference
existed in all-cause mortality. Systolic blood
pressure decreased by 15.625.8mmHg
in the medical therapy only group and by
16.621.2 mmHg in the stented group,
and in a longitudinal analysis, was modestly less in patients from the stented group
(2.3mmHg; 95% CI 0.24.4mmHg,
P=0.03).7 The observation that stenting
was no more effective than evidence-based
medical therapy to reduce cardiovascular
or renal events should diminish referral
for stenting in the majority of patients with
renal artery stenosis. However, percutaneous
balloon angioplasty and stenting should still
be considered in patients with hypertension
associated with renal artery fibromuscular
dysplasia, and those with bilateral renal
artery steno sis andepis odic pulmonary
oedema, respectively, because these groups
were not included in the CORALstudy.
Both carotid artery endarterectomy and
carotid artery stenting are used to treat
extracranial carotid artery stenosis, but
considerable debate exists about the relative
efficacy and safety of the two procedures.
Researchers in the CREST trial8 compared
2,502 patients with carotid artery stenosis
who underwent either stenting or endarter
ectomy, and found no significant difference
in the primary end point of stroke, myo
cardial infarction, or all-cause mortality
during the periprocedural period or any
ipsilateral stroke within 4years (HR1.11,
95%CI 0.811.51, P=0.51). There was no
difference between treatment groups in
the 4 year rate of ipsilateral stroke in either
symptomatic or asymptomatic patients.
The perip rocedural rate of stroke was
higher in the stented group (4.1% versus
2.3%; P=0.01), whereas the periprocedural
rate of myocardial infarction was higher
in the endarterectomy group (2.3% versus
1.1%; P=0.03).8 No significant difference
was observed in periprocedural mortality.

These findings indicate that carotid artery
endartere ctomy and stenting are equally
effective for treating patients with carotid
artery stenosis. Stenting, therefore, can be
considered as a therapeutic alternative to
carotid endarterectomy. Importantly, carotid
revascularization was not compared with
optimal medical therapy in the CREST trial.
Whether medical therapy is the preferred
approach, particularly in asymptom atic
patients with carotid s tenosis, remains to
bedetermined.
These findings reject the

notion that aspirin is effective for
all patients with PAD...
Anticoagulation therapy is indicated in

patients with venous thromboembolism
(VTE) to reduce the recurrence of deep vein
thrombosis (DVT) and pulmonary embolism. Several clinical trials have examined
the efficacy of novel oral anticoagulants,
which work by inhibiting thrombin or
factorXa. Investigators in the RECOVER
trial,9 a noninferiority study, compared the
efficacy and safety of dabigatran, a direct
thrombin inhibitor, with warfarin in 1,274
patients with acute DVT or pulmonary
embolism. Patients were initially treated
with a parenteral anticoagulant for at least
5days, and subsequently received either
dabigatran or warfarin. There was no difference between the two groups in the
primary composite outcome of the incidence of recurrent symptomatic venous
thromboembolism or related deaths at
6months (HR1.10, 95%CI 0.651.84),
and the difference in risk was 0.4% (95%CI
0.8% to 1.5%; P<0.001 for noninferiority). There was no significant difference
in major bleeding events between groups,
although the incidence of major or clinically
relevant nonmajor bleeding was reduced
with dabigatran.9 In the EINSTEIN-DVT
trial,10 the efficacy and safety of rivaroxaban, an oral factorXa inhibitor, were compared with those of a vitaminK antagonist
in 3,449 patients with acute symptomatic
DVT. Patients received either rivaroxaban,
or enoxaparin followed by a vitaminK
antagonist. Rivaroxaban was noninferior
to vitaminK antagonism for recurrent VTE
(HR0.68, 95%CI 0.441.04, P<0.001 for
noninferiority).10 No significant difference
was observed in the primary safety outcome
of major bleeding or clinically relevant nonmajor bleeding. Taken together, these two
studies indicate that these novel oral anticoagulant drugs are as effective and safe as
warfarin (or similar vitaminK antagonists),
have similar or improved safety profile, and
can thus be a safe alternative to warfarin for
the management of acute VTE.
Over the past decade, evidence from
clinical trials has guided the management of
peripheral atherosclerotic vascular diseases
and venous thrombosis. The next 10years
of research is anticipated to uncover novel
treatments to reduce the risk of peripheral
atherosclerotic events, inhibit the growth
of aortic aneurysms, improve symptomsof
peripheral artery disease, and improve the
outcomes of patients with venous thrombosis
and pulmonary embolism.
Cardiovascular Division, Brigham and
WomensHospital, 75 Francis Street, Boston,
MA 02115, USA.
mcreager@partners.org
Competing interests
M.A.C. declares that he has received consulting fees
from AstraZeneca and Novartis.
1.
Fowkes, F.G. etal. Aspirin for prevention of

cardiovascular events in a general population
screened for a low ankle brachial index:
a randomized controlled trial. JAMA 303,
841848 (2010).
2. Belch, J. etal. The prevention of progression of
arterial disease and diabetes (POPADAD) trial:
factorial randomised placebo controlled trial
ofaspirin and antioxidants in patients with
diabetes and asymptomatic peripheral arterial
disease. BMJ 337, a1840 (2008).
3. Murphy, T.P. etal. Supervised exercise versus
primary stenting for claudication resulting
from aortoiliac peripheral artery disease:
six-month outcomes from the claudication:
exercise versus endoluminal revascularization
(CLEVER) study. Circulation 125, 130139
(2012).
4. Prinssen, M. etal. A randomized trial comparing
conventional and endovascular repair of
abdominal aortic aneurysms. N. Engl. J. Med.
351, 16071618 (2004).
5. EVAR trial participants. Endovascular aneurysm
repair versus open repair in patients with
abdominal aortic aneurysm (EVAR trial1):
randomised controlled trial. Lancet 365,
21792186 (2005).
6. EVAR trial participants. Endovascular aneurysm
repair and outcome in patients unfit for open
repair of abdominal aortic aneurysm (EVAR
trial2): randomised controlled trial. Lancet
365, 21872192 (2005).
7. Cooper, C.J. etal. Stenting and medical therapy
for atherosclerotic renal-artery stenosis.
N.Engl. J. Med. 370, 1322 (2014).
8. Brott, T.G. etal. Stenting versus endarterectomy
for treatment of carotid-artery stenosis. N. Engl.
J. Med. 363, 1123 (2010).
9. Schulman, S. etal. Dabigatran versus
warfarin in the treatment of acute venous
thromboembolism. N. Engl. J. Med. 361,
23422352 (2009).
10. EINSTEIN Investigators. Oral rivaroxaban
forsymptomatic venous thromboembolism.
N.Engl. J. Med. 363, 24992510 (2010).
NOVEMBER 2015 | 13
CARDIOLOGY
technical challenges after the launch of these
prostheses. Meanwhile, with the availability
of a multitude of improvised valve prostheses and hardware used for implantation, as
well as the growing experience of cardiac
surgeons and interventional cardiologists,
TAVI has become a routine proc edure
and a good alternative for inoperable and
highrisk patients (Figure1).
Similarly in mitral valve surgery, attempts
to minimize patient trauma have led to
interesting progress in minimally-invasive
surgery. Both mitral valve replacement and
reconstruction can be performed safely
using minimally-invasive or even robotassisted techniques in centres with sufficient
experience. Repair for mitral regurgitation
has become an indicator of quality in many
centres. We have learned that reconstruction
is feasible and desirable in most patients with
degenerative disease. However, the results of
reconstruction for ischaemic mitral regurgitation are mixed, particularly in the long
term. 3 As a result of the poor prognosis
in these patients owing to the combination of progressive heart failure and mitral
DECADE IN REVIEWVALVULAR DISEASE
Current perspectives on treatment

of valvular heart disease
Friedrich W. Mohr
Tremendous advances in the understanding and treatment of structural

heart valve disease have been made in the past decade, including
widespread utilization of minimally-invasive surgical procedures and
the invention and evolution of numerous interventional techniques.
These innovations will become the norm in therapy for valvular disease
inthefuture.
Mohr, F.W. Nat. Rev. Cardiol. 11, 637638 (2014); published online 23 September 2014;
doi:10.1038/nrcardio.2014.138
14 | NOVEMBER 2015
AlainCribier,2 and its establishment as an

effective treatment in old, morbid, and virtually inoperable patients. The benefits of
avoiding a sternotomy, extracorporeal circulation, cardioplegic arrest, and (with a
transfemoral approach) even general anaesthesia, have given this technique a boost,
despite higher rates of residual aortic regurgitation, pacemaker implantations, and early
20,000
TAVI
AVR allograft
AVR biological
AVR mechanical
18,000
16,000
14,000
Total number of procedures
12,000
10,000
8,000
6,000
4,000
2,000
12
13
20
11
20
10
20
09
20
08
20
07
20
06
20
05
20
04
20
03
20
02
20
01
20
00
20
99
20
98
19
97
19
96
19
95
19
19
94
19
Valve repair and replacement continue to

be better treatment options than conservative management for patients with structural heart valve disease. The past decade
has seen major advances in the approaches
used to perform these procedures, especially with the advent of minimally-invasive
surgical and interventional techniques.
Some of these techniques are still in their
infancy, whereas others are being routinely
used globally, particularly in high-volume
centres. These developments have enabled
heart specialists to treat an increasingly
wide spectrum of patients, especially those
who would otherwise have been considered inoperable. Simultaneous progress in
imaging has improved delineation of the
anatomy and pathology of heart valves.
One of the most remarkable accomplishments has been the establishment of the
Heart Team concept, which involves experi
enced cardiologists and cardiac surgeons
collaborating to devise unbiased treatment
strategies for patients.
In the surgical treatment of aortic valve
disease, two new trends have been observed
in the past 10years. Firstly, a major shift
worldwide has occurred towards increased
use of biological valve prostheses, which
has been boosted by the availability of
newer-generation bioprostheses with excellent haemodynamic characteristics and
long-term durability (Figure1).1 Secondly,
minimally-invasive procedures through
small incisions have been shown to have
the same safety and efficacy as those performed conventionally through a standard
median sternotomy. The development of
sutureless implants has further facilitated
theseapproaches.
One of the most memorable developments was the first transcatheter aortic
valve implantation (TAVI) in 2002 by
Year
Figure 1 | Development of isolated AVR in Germany from 1994 to 2013. The graph shows
therise in the total number of AVRs over this period, the increasing use of biological valves,
andtherapid growth in TAVI since 2008. Abbreviations: AVR, aortic valve replacement; TAVI,
transcatheter aortic valve implantation.
CARDIOLOGY
regurgitation, the idea of interventional
treatment seems rational. Several devices
that imitate particular steps of the surgical
repair technique have been developed. The
most widely used device, the MitraClip
(Evalve, USA), mimics the edge-to-edge
leaflet repair (Alfieris stitch). The device has
been shown to relieve symptoms,4 but the
long-term results of this technique have yet
to be evaluated. The next logical step is true
catheter-based mitral valve replacement,
and first-in-human experience has already
been reported. Even though the anatomical
challenges of mitral valve replacement are
much more difficult to overcome than those
with aortic valve implantation, the sheer
number of high-risk patients with mitral
valve disease seems to justify the effort of
perfecting these devices.
The tricuspid valve, which has long
been neglected, has received due attention
during the past decade. For many years,
minimal or mild tricuspid valve incompetence was widely accepted to resolve, or at
least not worsen, after treatment of other
valves. However, several studies published
in the past 10years have revealed improved
long-term outcomes after concomitant tri
cuspid valve repair, which has resulted in a
rising trend for tricuspid valve reconstruction for enlarged tricuspid annuli, even if
regurgitation is only minimal.5
Another development that has influenced
decision-making in valve surgery is the
concept of implanting a transcatheter valve
inside a previously implanted degenerated
bioprosthesis or ring after valve repair. 6
These valve-in-valve or valve-in-ring procedures have not only helped to prevent
high-risk patients from having toundergo
complex reoperations, but have also resulted
in an increased use of bioprostheses during
the primary operation, often in younger

patientsa trend that will only rise in
thefuture.
The valve-in-valve concept has also been
applied to the pulmonary valve with the
availability of the MelodyTranscatheter
Pulmonary Valve (Medtronic, USA), which
was the first transcatheter valve available
on the market. This technology has been a
blessing for children and young adults, who
would otherwise have to undergo multiple
pulmonary valve reoperations.7
During the past decade, continued
efforts have been made to minimize surgical trauma without compromising clinical
outcomes, and to make minimally-invasive
valve surgery a routine and low-risk oper
ation. Simultaneously, the development of
catheter-based valve implantation has complemented the therapeutic arsenal, thereby
expanding the indications to patients who
were formerly deemed inoperable. The rapid
development of TAVI systems and, more
importantly, the strong push to spread this
technology by the medical industry, interventional cardiologists, and some surgeons
has triggered competition between TAVI
and conventional surgical valve replacement. With only two randomized studies to
compare TAVI and conventional surgery in
high-risk patients having been published to
date,8,9 further research and trials need to be
conducted in the near future, to determine
the most suitable therapy for each patient.
Unfortunately, TAVI is already being offered
to intermediate-risk and low-risk patients
in the absence of scientific evidence. Care
needs to be exercised in advising patients
to undergo these procedures, at least until
long-term outcomes are known. Therefore,
the importance of large, independent
studies and surveys, such as the German
Aortic Valve Registry (GARY),10 which will

provide 5year follow-up data on all aortic
valve interventions performed in Germany,
cannot be overemphasized. Nonetheless,
the fast progress in valve repair and replacement technologies over the past decade is
extremely encouraging, and raises hopes for
further advances in the future.
Herzzentrum Leipzig GmbHUniversitaetsklinik,
Struempellstrasse39, 04289 Leipzig, Germany.
friedrich.mohr@medizin.uni-leipzig.de
Competing interests
1.
Bavaria, J.E. etal. The St Jude Medical Trifecta

aortic pericardial valve: results from a global,
multicenter, prospective clinical study. J. Thorac.
Cardiovasc. Surg. 147, 590597 (2014).
2. Cribier, A. etal. Percutaneous transcatheter
implantation of an aortic valve prosthesis for
calcific aortic stenosis: first human case
description. Circulation 106, 30063008 (2002).
3. Acker, M.A. etal. Mitral-valve repair versus
replacement for severe ischemic mitral
regurgitation. N. Engl. J. Med. 370, 2332
(2014).
4. Glower, D.D. etal. Percutaneous mitral valve
repair for mitral regurgitation in high-risk
patients: results of the EVERESTII study.
J. Am. Coll. Cardiol. 64, 172181 (2014).
5. Navia, J.L. etal. Moderate tricuspid
regurgitation with left-sided degenerative
heart valve disease: to repair or not to repair?
Ann. Thorac. Surg. 93, 5967 (2012).
6. Dvir, D. etal. Transcatheter aortic valve
implantation in failed bioprosthetic surgical
valves. JAMA 312, 162170 (2014).
7. Momenah, T.S. etal. Extended application of
percutaneous pulmonary valve implantation.
J.Am. Coll. Cardiol. 53, 18591863 (2009).
8. Smith, C.R. etal. Transcatheter versus surgical
aortic-valve replacement in high-risk patients.
N. Engl. J. Med. 364, 21872198 (2011).
9. Adams, D.H. etal. Transcatheter aortic-valve
replacement with a self-expanding prosthesis.
N. Engl. J. Med. 370, 17901798 (2014).
10. Mohr, F.W. etal. The German Aortic Valve
Registry: 1year results from 13680 patients
with aortic valve disease. Eur. J. Cardiothorac.
Surg. http://dx.doi.org/10.1093/ejcts/ezu290.
NOVEMBER 2015 | 15
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CLINICAL ONCOLOGY
DECADE IN REVIEWCLINICAL TRIALS
Shifting paradigms in cancer clinical trial design

Daniel J. Sargent and Edward L. Korn
Over the past decade, there have been profound shifts in clinical trial design. PhaseII randomized studies,
phaseII/III and other adaptive designs, early surrogate end points, and prospective biomarker-based patient
selection have all increased in popularity. We discuss these shifts in clinical trial designs that have increased
efficiency in identifying which patients will benefit from specific treatments.
The past decade has witnessed an expansion in knowledge and understanding of

the biological basis of cancer, as a result
of mega-projects such as the Human
Genome Project and The Cancer Genome
Atlas, as well as continued scientific pursuit
by thousands of individual researchers. This
knowledge has enabled the development
and advancement of rationally designed
targeted therapies in oncology, often (but
not always) accompanied by a predictive
biomarker to identify the patients who
have the greatest likelihood of benefit. This
paradigm of targeted therapy in potentially
biomarker-defined populations has required
evolution of the methods used to clinically
evaluate new therapies. For instance, when
a therapy demonstrates a profound and
unambiguous treatment benefit (such as
anaplastic lymphoma kinase [ALK] inhibition in ALK-positive metastatic non-smallcell lung cancer), performing a randomized
trial versus a standard therapy might be
unnecessary.1 Unfortunately, most effective therapeutic agents are unlikely to show
such a dramatic benefit, and randomized
trials will continue to be the fundamental
approach to de veloping evidence-based
treatmentparadigms.
a paradigm shift in
the design of phaseII trials
hasoccurred
Over the past decade, much work has

been conducted to improve randomized
clinical trial designs. For example, a paradigm shift in the design of phaseII trials has
occurred. Before 2004, randomized phaseII
trials were relatively rare, and mainly used
a pick the winner design to identify the
iStock/Thinkstock
Sargent, D. J. & Korn, E. L. Nat. Rev. Clin. Oncol. 11, 625626 (2014); published online 7 October 2014; doi:10.1038/nrclinonc.2014.167
most-promising of several experimental

regimens, which would subsequently be
evaluated in a controlled trial; rather than
randomized phaseII studies, the activity
of a novel regimen was typically evaluated in a single-arm trial with a tumour
response rate end point. Rubinstein etal.2
proposed expanding the role of randomization in phaseII trials to explicitly include
a control arm, with the intention of generating more-robust data to justify the large
resource expenditure of a phaseIII trial.
Randomization is particularly critical in the
setting of newly characterized, biomarkerdefined patient groups for which the natural
history of disease is poorly understood; for
new therapies with activity that might be
best assessed using a time-related end point,
such as progression-free survival (PFS); and
when novel drugs are being combined with
standard treatments known to have activity.
It has been suggested that randomization
in phaseII trials should, in most circumstances, now become the expectation as
opposed to the exception.3
PhaseII/III trials attempt to expedite

drug development by combining previously distinct trials into a single protocol.
In the past decade, these trials have become
both increasingly attractive and feasible.
Key design issues face phaseII/III trials:
firstly, the choice of phaseII end point; secondly, whether to temporarily halt accrual
to enable maturation of the phaseII data
before making the decision about proceeding to phaseIII; and thirdly, the choice of
phaseII efficacy cut-off that informs this
decision.4 Ideally, the phaseII end point
should be an intermediate end point (for
example, PFS) available earlier than the
definitive phaseIII end point (for example,
overall survival). The stringency of the
efficacy cut-off determines the chance
of proceeding to an ultimately negative
phaseIII trial versus missing a potentially
positive phaseIII trial by stopping investigation of a therapy too early based on the
phaseIIresults.
The search for, and use of, predictive
biomarkers (that is, those that identify
the patients most likely to benefit from a
specific therapy) has become a dominant
theme in oncology drug development. In
the context of a single biomarker (such
as a specific gene mutation in a tumour),
the major trial design choice is whether to
restrict eligibility to the biomarker-positive
population (enrichment design), or to randomize all patients (or all patients who
have completed biomarker evaluation) and
then evaluate the treatment effect within
the biomarker-positive and biomarkernegative subgroups separately. 5 The use
of an enrichment design potentially offers
greater trial efficiency, but at the cost of
not knowing how well the treatment would
have worked in the biomarker-n egative
NOVEMBER 2015 | 16
CLINICAL ONCOLOGY
subgroup, and whether the cost and inconvenience of routine use of the biomarker
to select patients for treatment is justified. Thus, the choice of trial design (and
analysis strategy) should be dictated by the
strength of the pretrial evidence that any
treatment benefit would be restricted to
the biomarker-positive group. Continuing
improvements in biomarker assay technologies (for example, smaller sample requirements, the use of formalin-fixed instead
of frozen tissue, and assessments of blood
or molecular-imaging biomarkers) enable
more patients to be included in trials
incorporating biomarkerevaluations.
the next decade will be

faced with the realization
that every tumour is a
raretumour
A number of trial designs have been

proposed for the situation when multiple
biomarkers are potentially associated with
therapeutic efficacy. Examples of explora
tory trials in which the treatment is not (at
least initially) restricted by the biomarkers
are available. The phaseII BATTLE trial6
randomly assigned patients with metastatic non-small-cell lung cancer to four
treatments and subsequently evaluated
outcomes among five biomarker-defined
subgroups for each of these treatments. The
ongoing ISPY2 trial is randomly assigning
patients with breast cancer between multiple investigational therapies after initial
stratification by standard FDA-approved
biomarkers, with efficacy then estimated
within patient groups with predefined biomarker profiles accompanied by post-hoc
analyses of a variety of additional biomarkers in an attempt to simultaneously speed
up biomarker and therapeutic discovery.
These trials demonstrate that obtaining
data on multiple biomarkers for patients
being treated with multiple treatments
during a trial is possible. Signal-finding
trials, in which the treatment is specifi
cally directed by multiple biomarkers, are
also being undertaken. For example, the
phaseII National Cancer Institute MATCH
trial will assign patients with disease progression after at least one standard therapy
for advanced and/or metastatic disease to
potentially any of 2025 targeted agents
depending on the specific genomic mutations detected in thepatients tumours (ifan
actionable mutation is present).8
17 | NOVEMBER 2015
Definitive trials with multiple biomarkers, in which patients are assigned to different randomized phaseIII trials depending
on their biomarker profiles, are also being
performed. For example, the Lung Cancer
Master Protocol (Lung-MAP) trial is
screening patients with advanced-stage
squamous cell lung cancer and assigning
them to four different randomized trials
of targeted agents depending upon the
genomic alterations in their tumours, or a
fifth randomized trial of an immunotherapy (all versus appropriate conventional
chemotherapy). 8 Similar to many other
biomarker-based trials, Lung-MAP is
designed as a phaseII/III trial. The ability
to have a uniform screening platform to
assign patients to different trials enhances
efficiency and enables more patients to be
included than if each trial screened separately for its associated genomic alteration
(with patients without that alteration not
being included in any trial). Biomarkerbased trials are often adaptive, owing to the
ability to use the accumulating trial data to
make study modifications. Adaptive trials
are not new, as methods for formal interim
analyses date to the 1970s. The past decade,
however, has seen a rapid expansion of
the types and goals of adaption, including
defining subpopulations, adding or dropping arms on the fly, altering the randomization ratio based on accumulating data,
and many other options. Adaptive trials
offer the possibility of obtaining clinical
answers more quickly.
Efficient clinical trial design, and in particular the ability to conduct phaseII/III
or other adaptive trials, requires end points
that are rapidly accessible. Pursuit of such
early, or surrogate, end points is a major
current research focus. In oncology, the
vigorous debate between PFS and overall
survival as a phase III trial end point
continues unabated, with the questions
including, but not limited to, whether PFS
is a valid surrogate for overall survival or
quality of life. The decision on which end
point to select is specific to the trial and
situation. For phaseII trials, an intermediate end point need not be a validated surro
gate for the definitive end point because
the phaseIII evaluation will be performed
eventually using the definitive end point.
The meta-analytical approach, which
requires accurate prediction of within-trial
results for the true end point based on the
surrogate, has in the past decade been established as the preferred method of surrogate
end point validation for assessing whether
new therapies provide a group-level advantage (as opposed to predicting how well an
individual patient will respond).9
Oncology trial design in the next decade
will be faced with the ever-increasing subclassification of tumours with the realization that every tumour is a rare tumour.
One potential strategy, in addition to the
strategies mentioned earlier, is to raise
thebar and demand greater efficacy from
new treatments.10 This strategy might be
possible owing to predictive biomarkers
and targeted therapies. Continued advances
in clinical trial design and analysis will be
required to meet these challenges and
to ensure rapid translation of biological
knowledge into clinical practice.
Department of Health Science Research,
MayoClinic, 200 First Street SW, Rochester,
MN 5590, USA (D.J.S.). Biometric Research
Branch, Division of Cancer Treatment and
Diagnosis, National Cancer Institute,
9609Medical Center Drive, Room 5W112,
Bethesda, MD 208929735, USA (E.L.K.).
Correspondence to: D.J.S.
sargent.daniel@mayo.edu
Competing interests
The authors declare no competing interests.
1.
Sherman, R.E., Li, J., Shapley, S., Robb, M. &

Woodcock, J. Expediting drug developmentthe
FDAs new breakthrough therapy designation.
N.Engl. J.Med. 369, 18771880 (2013).
2. Rubinstein, L.V. etal. Design issues of
randomized phaseII trials and a proposal
forphaseII screening trials. J.Clin. Oncol. 23,
71997206 (2005).
3. Seymour, L. etal. The design of phaseII clinical
trials testing cancer therapeutics: consensus
recommendations from the clinical trial design
task force of the National Cancer Institute
Investigational Drug Steering Committee.
Clin.Cancer Res. 16, 17641769 (2010).
4. Korn, E.L., Freidlin, B., Abrams, J.S. &
Halabi,S. Design issues in randomized
phaseII/III trials. J.Clin. Oncol. 30, 667671
(2012).
5. Sargent, D.J., Conley, B.A., Allegra, C. &
Collette, L. Clinical trial designs for predictive
marker validation in cancer treatment trials.
J.Clin. Oncol. 23, 20202027 (2005).
6. Kim, E.S. etal. The BATTLE trial: personalizing
therapy for lung cancer. Cancer Discov. 1,
4453 (2011).
7. Barker, A.D. etal. ISPY 2: an adaptive breast
cancer trial design in the setting of neoadjuvant
chemotherapy. Clin. Pharmacol. Ther. 86,
97100 (2009).
8. Abrams, J. etal. National Cancer Institutes
precision medicine initiatives for the new
National Clinical Trials Network. Am. Soc. Clin.
Oncol. Educ. Book 2014, 7176 (2014).
9. Burzykowski, T., Molenberghs, G. & Buyse, M.
(eds) The Evaluation of Surrogate Endpoints
(Springer, 2005).
10. Ellis, L.M. etal. American Society of Clinical
Oncology perspective: raising the bar for
clinical trials by defining clinically meaningful
outcomes. J.Clin. Oncol. 32, 12771280
(2014).
CLINICAL ONCOLOGY
DECADE IN REVIEWTARGETED THERAPY
Successes, toxicities and

challenges in solid tumours
The rise of targeted therapy for solid tumours over the past decade
has yielded a cornucopia of novel agents across an array of cancers.
Amidst multiple acclaimed successes, targeted therapies are associated
with considerable toxicity, and durable responses are often thwarted by
genomic chaos driving the evolution of resistant clones; key examples
ofsuccesses in solid tumours are highlighted herein.
Neal, J. W. & Sledge, G. W. Nat. Rev. Clin. Oncol. 11, 627628 (2014); published online 7 October 2014;
doi:10.1038/nrclinonc.2014.171
The past decade has seen a major transition in drug therapy for cancer, with a shift
from broad-spectrum cytotoxic agents to
highly targeted therapies. In the past few
years, FDA approvals of targeted therapies
for solid tumours have increased substantially (Supplementary Table1). Encouraged
by the success of the first FDA-approved
small molecule tyrosine kinase inhibitor (TKI), imatinib, for the treatment of
chronic myeloid leukaemia, oncologists
and patients hoped that targeted therapies
would prove to be highly effective in most
cancers and associated with less toxicity than
the cytotoxic chemotherapeutic agents that
preceded them. With a decade of clinical
research behind us, spurred on by several
signal triumphs such as HER2-directed
therapy in breast cancer, EGFR and ALK
inhibitor therapy in non-small-cell lung
cancer (NSCLC), antiangiogenictherapy
in kidney cancer, and BRAF-targeted
therapy in metastatic melanoma, a more
nuanced understanding of targeted therapy
hasemerged.
A rapid expansion in the number of
antibody and small-molecule therapies
occurred over the past decade. These agents
were tested broadly for therapeutic efficacy,
often resulting in refinement of the biological mechanism of action after signals of
clinical benefit were observed. For example,
cetuximab, a chimeric monoclonal antibody that targets the extracellular portion
of EGFR, improved survival in colorectal
cancer (CRC), initially in an unselected
patient population; however, subsequent
analysis of tumour specimens from large
trials showed that the tumours with KRAS
mutations were insensitive to cetuximab.1
At the same time, a complementary strategy was developed to inhibit the EGFR
targeting the ATP-binding domain with
small-molecule TKIs. One such drug, erloti

nib, was granted initial FDA approval in
the second-line treatment of unselected
NSCLC in 2004 (Supplementary Table1).
Interestingly, in early clinical trials, a subgroup of patients experienced dramatic
tumour shrinkage, leading to the identification of tumour-specific, sensitizing EGFR
mutations that strongly predicted response
to erlotinib. Subsequently, the indications for
erlotinib therapy were expanded to include
first-line treatmentin lieu of chemotherapy
for EGFR-mutant NSCLC;2 nevertheless,
as in many other tumour types, responses
to this targeted therapy are not durable,
with a simple secondary point mutation in
EGFR occurring as the dominant mechanism of acquired resistance in around half
of these cancers. Importantly, the identi
fication of such mutations also points
to potential th erapeutic approaches to
overcomeresistance.
The past decade has seen a

major transition in drug therapy
for cancer
The BRAF V600E mutation in melanoma

is another case in point; although relatively
common (approximately 50% of all melanomas harbour this mutation), and readily
targetable with oral small-molecule TKIs in
the metastatic setting,3 in general, tumour
responses are of relatively brief duration and
are associated with only a modest improvement in overall survival. Indeed, the rapid
emergence of drug resistance in this highly
mutated type of cancer is common, multifactorial, and occurs at an early time point
after treatment. Preclinical analyses have
suggested that targeting multiple drivers
of tumour growth simultaneously will be
Thinkstock/iStock
Joel W.Neal and George W.Sledge
required to maximize cell killing and, thus,

thwart the emergence of resistance. For
instance, in the case of melanoma, combining direct BRAF inhibition with blockade
of the downstream kinase MEK has been
shown to improve the progression-free
survival of patients with metastatic dis
ease (and received FDA approval in 2014;
Supplementary Table1), but this approach
is associated with increased adverse events
and fiscaltoxicity.4
In NSCLC, the identification of an
uncommon tumour gene rearrangement,
EML4ALK, led to clinical testing and rapid
approvalin only 4yearsof crizotinib,
a drug designed as a hepatocyte growth
factor receptor (also known as MET) inhibitor that has off-target activity against ALK.5
Repeat biopsies of tumours have revealed a
variety of secondary resistance mutations in
ALK, which could be overcome with newer-
generation TKIs targeting this receptor. One
such second-generation ALK inhibitor, ceritinib, received accelerated approval by the
FDA in 2011 based on the demonstration
of promising efficacy in phaseI/II trials.6
Other approaches to overcoming resistance
have been introduced in the past decade,
and include the use of the initial targeted
agent as a delivery vehicle for nontargeted
drugs. Trastuzumab emtansine, a conjugate of the anti-HER2 monoclonal antibody
trastuzumab and the cytotoxic maytansine
derivative mertansine (TDM1), was the first
molecule using this strategy to be approved
by the FDA (Supplementary Table1), and
has proven activity in trastuzumab-resistant
HER2positive breastcancer.7
A new hope for targeted therapy is in
preventing the development of disease
recurrence following curative treatment.
HER2-targeting with trastuzumab began
in the 1990s, but it was not until 2005 that
a series of adjuvant trials of this agent in
patients with breast cancer demonstrated a
dramatic reduction in the rates of recurrence
in HER2-positive early stage disease; with
NOVEMBER 2015 | 18
CLINICAL ONCOLOGY
further follow-up, these reductions translated into durable improvements in overall
survival for this once-lethal breast cancer
subtype.8 Other attempts at translating therapeutic benefit in the metastatic disease to
the adjuvant setting have been less successful,
with no benefit observed for the anti-EGFR
antibody cetuximab or anti-VEGF antibody
bevacizumab following complete resection
of CRC. However, in patients with imatinibsensitive gastrointestinal stromal tumours,
both disease-free survival and overall survival were improved with imatinib treatment
after surgery.9 Intriguingly, prolonged 3year
treatment is superior to 1year regimens,
suggesting that targeted therapies might
exert a cytostatic rather than cytotoxic effect
against micrometastatic disease.9
Not all targeted therapies have yielded
such clear insight into the molecular basis
of tumour sensitivity and resistance. Anti
angiogenic therapy with bevacizumab
found an initial role in the treatment of CRC
and NSCLC, but extensive searches in both
normal and tumour tissues for biomarkers
of therapeutic benefit have not identified
a straightforward predictive marker of
response. By contrast, renal-cell cancers,
which are generally chemoresistant, are sensitive to a variety of antiangiogenic treatment
strategies, including bevacizumab and smallmolecule VEGF-receptor inhibitors such as
sorafenib, sunitinib, pazopanib, and axitinib.
Of these, sunitinib more than doubled the
median progression-free survival duration
in patients, compared with the historical
standard treatment of IFN.10
With regard to toxicity, many targeted
therap ies are arguably associated with
important adverse events, although these
toxicities tend to be mechanistic in nature
(for example, skin and gastrointestinal toxi
city for EGFR inhibitors and hypertension
for VEGF inhibitors) and nonmyeloablative.
Indeed, targeted therapies are generally
associated with increased incidence of highgrade adverse events, treatment discontinuation, and treatment-related fatalities
compared with the control interventions in
registration trials. Targeted therapies should
not, therefore, be expected to be less toxic
than the nontargeted chemotherapeutics
that preceded them.
For tumour types with known effective
targeted therapies, the challenges over the
upcoming decade will be in both reactively
and proactively overcoming resistance to the
existing agents with next-generation drugs,
and through combinat orial therapeutic
approaches. Additionally, use of targeted
19 | NOVEMBER 2015
therapies in the adjuvant setting might delay

or prevent disease recurrence. In the next
decade, studies might also reveal additional
targetable tumour-drug dyads, but much of
the low-hanging fruit has probably already
been identified; thus, in light of the limitations of the current approaches, targeted
therapy might not be a panacea for the
majority of patients with cancer. Indeed,
genomic analyses point to a landscape dominated by orphan diseases, with untargetable
or rare driver mutations being exceptionally
common across a broad spectrum of human
cancers, and with many cancers having
multiple drivers. For patients with these
cancers, hope lies in improving access to
approved and investigational targeted drugs
with plausible efficacy in their cancer, and in
new avenues of anticancer research, such as
immunotherapy, that might synergize with
chemotherapy and targeted therapy.
Division of Oncology, Stanford University
School of Medicine, 875 Blake Wilbur Drive,
Stanford, CA 943055826, USA (J.W.N., G.W.S.).
Correspondence to: J.W.N.
jwneal@stanford.edu
Competing interests
J.W.N. has received grants for research support
from, ArQule, Boehringer-Ingelheim, Merck, and
Roche/Genentech. G.W.S. has received grants for
research support from Roche/Genentech and
honouraria for speaking from Genentech, is a
consultant for Symphogen, and is a member of
theBoard of Directors for Syndax Pharmaceuticals.
1.
Karapetis, C.S. etal. KRas mutations and

benefit from cetuximab in advanced colorectal
cancer. N.Engl. J.Med. 359, 17571765
(2008).
2. Rosell, R. etal. Erlotinib versus standard
chemotherapy as first-line treatment for
European patients with advanced EGFR
mutation-positive nonsmallcell lung cancer
(EURTAC): a multicentre, open-label,
randomised phase3 trial. Lancet Oncol. 13,
239246 (2012).
3. Chapman, P.B. etal. Improved survival with
vemurafenib in melanoma with BRAF V600E
mutation. N.Engl. J.Med. 364, 25072516
(2011).
4. Flaherty, K.T. etal. Combined BRAF and MEK
inhibition in melanoma with BRAF V600
mutations. N.Engl. J.Med. 367, 16941703
(2012).
5. Kwak, E.L. etal. Anaplastic lymphoma kinase
inhibition in nonsmallcell lung cancer. N.Engl.
J.Med. 363, 16931703 (2010).
6. Shaw, A.T. etal. Ceritinib in ALK-rearranged
nonsmallcell lung cancer. N.Engl. J.Med. 370,
11891197 (2014).
7. Verma, S. etal. Trastuzumab emtansine for
HER2-positive advanced breast cancer. N.Engl.
J.Med. 367, 17831791 (2012).
8. Romond, E.H. etal. Trastuzumab plus adjuvant
chemotherapy for operable HER2-positive
breast cancer. N.Engl. J.Med. 353,
16731684 (2005).
9. Joensuu, H. etal. One vs three years of
adjuvant imatinib for operable gastrointestinal
stromal tumor: a randomized trial. JAMA 307,
12651272 (2012).
10. Motzer, R.J. etal. Sunitinib versus interferon
alfa in metastatic renal-cell carcinoma. N. Engl.
J. Med. 356, 115124 (2007).
Supplementary information is linked to the online
version of the paper at www.nature.com/nrclinonc.
DECADE IN REVIEWHAEMATOLOGICAL CANCER
Advances in biology and therapy

S. Vincent Rajkumar and Philippe Moreau
It has been a decade of remarkable progress in the field of

haematological malignancies with the rapid translation of basic science
discoveries into effective targeted therapies. We discuss the most exciting
advances in this field, many of which have already produced meaningful
improvements in survival and quality of life of patients.
Rajkumar, S. V. & Moreau, P. Nat. Rev. Clin. Oncol. 11, 628630 (2014); published online 14 October 2014;
In the 1990s, the median survival of multiple myeloma was 23years. Nowadays, the
median survival estimates are approximately
710years. In the early 2000s, the advent of
bortezomib and lenalidomide changed the
treatment of multiple myeloma in a radical
way. While bortezomib was developed with
a clear mechanistic goal in mind, lenalidomide had a serendipitous discovery that
promulgated its use in the treatment setting.
Bortezomib is a first-in-class proteasome
inhibitor and functions by blocking the cellular protein catabolism via the ubiquitinproteasome dependent pathway. Multiple
myeloma has been the first disease in which
inhibition of the proteasome was used as a
therapeutic target for the treatment of cancer.
The drug was initially tested in patients with
relapsed and refractory disease; however,
bortezomib has now become one of the
major options for the treatment of newly
diagnosed multiplemyeloma.1
CLINICAL ONCOLOGY
clinical success with these

agents provides new hope for
patients
In patients with non-Hodgkin lymphoma, two key areas that were of great
therapeutic frustration saw important
advances. First, the need to prolong time
to relapse in patients with follicular lymphoma using an approach that does not
compromise quality of life was addressed.
A large randomized trial of 1,018patients
undergoing maintena nce therapy with
rituximab administered every 2months
for 2years significantly prolonged PFS and
time to next chemotherapy; 3year PFS was
75% with rituximab maintenance versus
58% in the observation arm (P<0.0001).3
Nevertheless, relapse in patients with
indolent lymphomas is usually inevitable,
and eventually the disease is refractory to
available classes of agents. Thus, the second
area of interest was to identify active drugs
that are directed against novel targets to
help overcome this issue in patients with
20
04
06
5
200
20
200
200
2008
2012
3
01
10
20
201
2014
NP
G
Lenalidomide is an analogue of thalido

mide that is more active and less toxic
than the parent drug. The original finding
that lenalidomide was remarkably effective in patients with relapsed myeloma
preceded the identification of its cellular
target, cereblon, by several years. Cereblon
is a component of E3 ubiquitin ligase, a
key enzyme in the ubiquitinproteasome
protein degradation pathway. The binding
of lenalidomide to cereblon enhances its
E3 ubiquitin ligase activity, which leads to
selective ubiquitination of two transcription factors, lkaros and Aiolos. This causes
the degradation of these proteins by the
proteasome catalytic complex, and leads to
myeloma cell death.
As with bortezomib, the use of lenalidomide has now moved rapidly to the frontline
setting. In a recent landmark randomized
trial, long-term therapy administered until
disease progression with lenalidomide
plus low-dose dexamethasone improved
progression-free survival (PFS) as well as
overall survival of patients with newly diagnosed multiple myeloma.2 In addition to
their direct role in prolonging the survival
of patients with myeloma, these drugs have
spawned the development of several agents
with a similar mechanism of action, including the proteasome inhibitors carfilzomib,
ixazomib, marizomib, oprozomib and the
lenalidomide analogue pomalidomide.
relapsed and/or refractory disease. The

phosphatidylinositol 3kinase (PI3K) path
way has an important role in cell proliferation and survival. PI3K is particularly
active in Bcell cancers, including indolent
and non-Hodgkin lymphoma, and thus represents an attractive new target. In a phaseI
trial in patients with relapsed indolent lymphoma, idelalisiba specific inhibitor of
PI3Kshowed a striking response rate of
57% (71 out of 125 patients), including 6%
complete responses.4 These results identify
a new therapeutic target in Bcell malignancies, and led to the FDA regulatory approval
of idelalisib in the USA.
Further advances are sure to follow as we
improve our understanding of the precise
pathogenetic mechanisms involved in such
diseases, and identify druggable targets. For
example, although translocations involving
MYC and the immunoglobulin genes have
been recognized for decades as the cyto
genetic signature of Burkitt lymphoma, this
has not been translated into specific targeted
therapy. Of note, an interesting study has
reported new insights that might provide
new targets for Burkitt lymphoma. Using
high-throughput RNA sequencing and
RNA interference screening, Schmitz etal.5
identified several new molecular abnormalities in the majority of patients with sporadic
Burkitt lymphoma, including mutations of
the transcription factor TCF3 (E2A) or its
negative regulator ID3, as well as mutations
of CCND3, many of which can be targeted
for therapeutic benefit.
The treatment of Hodgkin lymphoma
has long represented one of the greatest
successes in oncology; however, a subset of
patients develop refractory or relapsed dis
ease. These refractory or relapsed patients
usually fail to respond to multi-agent
combination chemotherapy and stem-cell
transplantation; therefore, new targets
are needed for their treatment. The CD30
antigen is overexpressed in tumour cells
in patients with Hodgkin lymphoma and
anaplastic large-cell lymphoma. However,
previous attempts to target CD30 using
monoclonal antibodies were not successful.
Brentuximab vedotin is an antibody conjugate in which monomethyl auristatinE, an
antitubulin agent, is attached to a CD30
monoclonal antibody. In a phaseI trial,
treatment with brentuximab vedotin res
ulted in 13 complete responders among
45patients with relapsed and/or refractory
Hodgkin lymphoma or anaplastic largecell lymphoma, a remarkable result that
provides hope for these patients.6
In patients with chronic lymphocytic leukaemia (CLL), the main advance is ibrutinib. CLL, similar to indolent non-Hodgkin
lymphoma and multiple myeloma, is
characterized by almost inevitable relapse.
With each relapse, remissions are harder
to achieve and less durable. In fact, until
a definitive cure is found, the developments of new agents will continue to be
paramount. The hope for patients in these
situations lies in the continued identification of such treatments. Ibrutinib is an
oral inhibitor of Bruton tyrosine kinase
(BTK), an enzyme critical for CLL cell
survival. In a phaseI/II trial of 85 patients
with advanced-stage CLL, approximately
70% responded to therapy, and the PFS
rate at 2years was over 75%. 7 Ibrutinib
is also active in mantle-cell lymphoma,
Waldenstrom ma croglobulinaemia, and
other lymphomas.
Another major practice-changing discovery in the field of haematological
malignancies came from a randomized
trial of all-trans retinoic acid (ATRA) plus
arsenic trioxide versus ATRA plus chemotherapy in patients with acute promyelocytic leukaemia. 8 The trial was designed
to test whether a regimen without any
NOVEMBER 2015 | 20
CLINICAL ONCOLOGY
standard chemotherapy drugs could effectively treat this disease. Complete remissions were achieved in all 77 patients with
the ATRAarsenic trioxide combination
versus 75 (out of 79) patients in the ATRA
chemotherapy arm. The 2year event-free
survival rates were 97% and 86%, respectively. Overall survival was superior with
ATRAarsenic trioxide; specifically the 2
year overall survival was 99% in the ATRA
arsenic trioxide arm versus 91% in the
ATRAchemotherapy arm (P=0.02).
Chronic myeloid disorders have also had
their share of success in the past 10years.
The first drug ever to prolong overall survival in myelodysplastic syndrome (MDS)
was reported in 2009. In a randomized trial,
azacitidine was found to improve overall
survival compared with conventional care
selected by investigators before randomization: 24.5months versus 15.0months,
respectively (P=0.0001).9
One of the biggest advances of the past
decade in haematology, was the finding
that mutations in Janus kinase 2 (JAK2) are
associated with almost all casesofpoly
cythemia vera. The BCRABL translocation,
known as the Philadelphia translocation,
separates chronic myelogenous leukaemia
from other myeloproliferative disorders;
the ability to target this translocation using
imatinib rocked the field in 2001. However,
until the discovery of the JAK2 mutation,
the pathogenetic basis of the remaining myeloproliferative disorders was not
known. The JAK2 mutation is a valine-tophenylalanine substitution at amino acid
position 617 (JAK2 V617F) that results in
the constitutive activation of JAK2. The
JAK2 V617F substitution induced polycythemia in a mouse model, and has been
reported in polycythemia vera, but not
secondary polycythemia.10 It is, however,
not specific for polycythemia, and has been
detected in other myeloproliferative dis
orders, such as essential thrombocythemia
and myelofibrosis. These findings led the
way for the development of JAK2 inhibitors,
and clinical success with these agents provides new hope for patients with a variety of
myeloid disorders.
The advances discussed above have
resulted in meaningful clinical benefit to
patients affected by different lymphoid and
myeloid malignancies. The improved understanding of the molecular pathogenesis
gained in the past 10 years will certainly
translate into a major expansion of targeted therapy for numerous haematological
cancers in the next decade.
21 | NOVEMBER 2015
Division of Haematology, Mayo Clinic, 200 First

Street SW, Rochester, MN 55905, USA (S.V.R.).
Department of Haematology, University Hospital
Htel-Dieu, 44093 Nantes, France (P.M.).
Correspondence to: S.V.R.
rajkumar.vincent@mayo.edu
Competing interests
P.M. has served on advisory boards and received
honouraria from Celgene, Janssen and Millennium.
S.V.R. declares no competing interests.
1.
2.
3.
San Miguel, J.F. et al. Bortezomib plus

melphalan and prednisone for initial treatment
of multiple myeloma. N.Engl. J.Med. 359,
906917 (2008).
Benboubker, L. etal. Lenalidomide and
dexamethasone in transplant-ineligible patients
with myeloma. N.Engl. J.Med. 371, 906917
(2014).
Salles, G. et al. Rituximab maintenance for
2years in patients with high tumour burden
follicular lymphoma responding to rituximab plus
chemotherapy (PRIMA): a phase 3, randomised
4.
Gopal, A.K. et al. PI3K Inhibition by idelalisib

in patients with relapsed indolent lymphoma.
N.Engl. J.Med. 370, 10081018 (2014).
5. Schmitz, R. et al. Burkitt lymphoma
pathogenesis and therapeutic targets from
structural and functional genomics. Nature
490, 11620 (2012).
6. Younes, A. et al. Brentuximab vedotin (SGN-35)
for relapsed CD30-positive lymphomas. N.Engl.
J.Med. 363, 18121821 9(2010).
7. Byrd, J.C. et al. Targeting BTK with ibrutinib in
relapsed chronic lymphocytic leukemia. N.Engl.
J.Med. 369, 3242 (2013).
8. Lo-Coco, F. et al. Retinoic acid and arsenic
trioxide for acute promyelocytic leukemia.
N.Engl. J.Med. 369, 111121 (2013).
9. Fenaux, P. et al. Efficacy of azacitidine
compared with that of conventional care
regimens in the treatment of higher-risk
myelodysplastic syndromes: a randomised,
open-label, phase III study. Lancet Oncol. 10,
223232 (2009).
10. James, C. etal. A unique clonal JAK2 mutation
leading to constitutive signalling causes
polycythaemia vera. Nature 434, 11441148
(2005).
DECADE IN REVIEWCANCER IMMUNOTHERAPY
Entering the mainstream

ofcancer treatment
Steven A. Rosenberg
By November 2004, when the first issue of Nature Reviews Clinical

Oncology was published, cancer immunotherapy had been successfully
applied to the treatment of selected human cancers; however, dramatic
progress in the following decade has moved immunotherapy from the
sidelines of cancer treatment into the mainstream of modern oncology.
Rosenberg, S. A. Nat. Rev. Clin. Oncol. 11, 630632 (2014); published online 14 October 2014;
Administration of IL2, the first immuno

therapy capable of mediating complete,
durable cancer responses was originally
described in 1985 and approved by the
FDA for the treatment of patients with renal
cancer and melanoma in 1992 and 1998,
respectively. In 2003, the first evidence
thatthe administration of an antibody
targeting the inhibitory Tcell costimulatory molecule CTLA4, (often referred to
as a checkpoint modulator) could mediate
cancer regression in patients with metastatic
melanoma was published. Despite extensive
efforts to develop therapeutic cancer vaccines, prior to 2004 none had been shown
to be effective.
The first effective adoptive cell-transfer
(ACT) immunotherapy for cancer was
reported in 1988 and showed that cultured
tumour infiltrating lymphocytes (TIL) isolated from resected metastatic melanomas
and administered to the autologous patients
could mediate objective cancer regressions (Figure1). The effectiveness of this

Tcell therapy was dramatically improved
in 2002 with the discovery that lympho
depletion prior to Tcell administration
could improve the antitumour activity of
the transferred cells. Thus by 2004, there
were clear indications that either direct
stimulation of Tcells, blockade of checkpoint modulators or the administration of
antitumour Tcells could mediate regression
of invasive, vascularized cancers inhumans.
In the subsequent decade, increasing information about the importance of
cell-surface inhibitory molecules present
on lymphocytes led to striking advances
in immunotherapy, and antibodies that
could block these inhibitory receptors
were administered to patients. The first
rand omized protocol to demonstrate a
survival benefit for a cancer immunotherapy was published by Hodi etal.1 in 2010.
CLINICAL ONCOLOGY
Inthis study, patients with metastatic melanoma were randomly assigned to receive
a peptide vaccine alone or in combination
with anti-CTLA4 antibody (ipilimumab)
therapy.1 This trial was controversial, as it
was already known that ipilimumab could
mediate objective (and sometimes complete) responses in patients with melanoma,
whereas peptide vaccines alone had virtually no evidence of efficacy, and no crossover between cohorts was allowed. In the
vaccine plus ipilimumab arm, 35 of 540
patients (6.5%) experienced an objective
response compared with two of 136 patients
(1.5%) treated with peptide alone.1 Of note,
the patients who received ipilimumab
showed an increase in median survival from
6.5months to 10months. 1 These results
ultimately led to the approval of ipilimumab
for the treatment of patients with metastatic
melanoma by the FDA in March 2011. The
clinical value of ipilimumab in other cancer
types (with the possible exception of renal
cancer) is currentlyunknown.
has moved immunotherapy

from the sidelines of cancer
treatment into the mainstream
ofmodern oncology
Clinical utility of the blockade of a

second inhibitory lymphocyte receptor
programmed cell death 1 (PD1) was anticipated in 2010 and firmly demonstrated in
2012 in a successive trial that used this agent
in 296 patients with different cancer types.2
Among 236 evaluable patients, objective
response rates were seen in 18%, 28% and
27% of patients with non-small-cell lung
cancer, melanoma and renal cell cancer,
respectively.2 No objective responses were
observed in patients with colorectal or
prostate cancers.2 Response rates seemed
to be increased when PDL1, the ligand for
PD1, was expressed on the cancer cells,
and the use of a blocking antibody against
PDL1 resulted in responses in nine of 52
(17%) patients with melanoma, two of 17
(12%) with renal cancer, five of 49 (10%)
with non-small-cell lung cancer and one of
17 (6%) patients with ovarian cancer.3 The
impressive responses seen with pembrolizumab, an anti-PD1 antibody, in multicentre phaseII studies led to approval of
this agent by the FDA in September 2014
for the treatment of patients with metastatic
melanoma refractory to ipilimumab and to
a BRAF inhibitor, if the tumour was BRAF
Cell infusion
plus IL-2
Preconditioning:
chemotherapy
TIL isolation
Tumour
Figure 1 | Adoptive cell transfer immunotherapy. Cells are obtained from a resected tumour
(autologous TILs) or using peripheral lymphocytes genetically engineered to express antitumour
T-cell receptors, expanded in vitro and infused to patients after they have received a preparative
lymphodepleting regimen. Abbreviation: TIL, tumour-infiltrating lymphocyte.
V600-mutation-positive. Evaluation of the

blockade of other inhibitory receptors such
as TIM3 or LAG3 is ongoing.
Very little progress has been made in the
past 10years concerning the application
of therapeutic cancer vaccines. The only
positive randomized trial of a therapeutic
vaccine was published by Kantoff etal.4 in
July2010. Patients with hormone-refractory
prostate cancer received the administration
of autologous peripheral blood mononuclear
cells activated exvivo with a recombinant
protein consisting of prostatic acid phosphatase fused to granulocyte-macrophage
colony stimulating factor (sipuleucelT
vaccine) or placebo. 4 Median survival
improved from 21.7months in the placebo
cohort to 25.8months in the vaccine group.4
Such increase in median survival was surprising as only one of the 341 patients
(0.3%) treated with the vaccine had an
objective response, and only 2.6% of patients
had a decrease in serum prostate-specific
antigen (PSA) levels;4 the time to disease
progression was similar in the two groups
(14.4weeks compared to 14.6weeks).4 On
the basis of these results, the sipuleucelT
vaccine was approved bythe FDA in 2010.
So far, this modest clinical result remains
the only trial to show a convincing benefit
of an active im munization approach to
cancertreatment.
The past 10years have seen considerable progress in the development of ACT
immunot herapies using both naturally
occurring and genetically engineered
lymphocytes. Long-term follow-up studies
showing that ACT immunotherapy using
autologous TIL could mediate durable
complete (and likely curative) responses in
patients with refractory metastatic melanoma was published in 2011.5 In a series of
three sequential trials that used increasing

lymphodepleting regimens, objective cancer
regressions were seen in 52 of 93 patients
(56%), including 20 patients (22%) with
complete regressions. 19 of the 20 patients
had ongoing complete responses beyond
5years, with the longest ongoing responses
persisting beyond 10years.5 These results
represented the highest reported response
rates of any clinical trials in patients with
metastatic melanoma. Furthermore, Bollard
etal.6 reported responses in patients with
EpsteinBarr virus (EBV)-related Hodgkin
or non-Hodgkin lymphomas receiving
autologous cytotoxic Tcells that target the
EBV latent membrane proteins LMP2 or
LMP1. Specifically, 13 of 21 patients with
measurable relapsed or resistant lymphomas exhibited clinical responses, including
11 completeresponses.6
The difficulty in identifying cells with
invitro antitumour activity against many
cancer types led to extensive efforts to
genetically engineer Tcells for use in ACT
therapy. Retroviruses encoding either conventional Tcell receptors (TCR) or chimeric antigen receptors (CAR; composed
of a single chain antibody fused to intracellular signalling chains) are capable of
inserting genes into the genome of human
lymphocytes with efficiencies exceeding
80%. The first successful use of genetically
engineered lymphocytes to mediate cancer
regression was reported in 2006. Morgan
etal.7 showed that retroviral transduction
of autologous lymphocytes with the gene
encoding a TCR against the melanocyte/
melanoma differentiation antigen, MART1,
mediated objective cancer regressions in
two of 15 (13%) patients with metastatic
melanoma. Objective cancer regressions
were later reported in 30% of 20patients
NOVEMBER 2015 | 22
CLINICAL ONCOLOGY
The past 10years have

seen considerable progress
in the development of ACT
immunotherapies
receiving gene-modified cells targeting the

MART1 antigen and in 19% of 16 patients
who received autologous cells transduced
with a mouse TCR that recognized the
gp100 antigen, demonstrating for the first
time that adoptive transfer of genetically
modified cells could mediate cancer regression. In 2008, Pule etal.8 reported that EBVspecific autologous Tcells transduced with a
CAR targeting the GD2 molecule mediated
objective tumour regressions in two of eight
patients with measurable neuroblastoma.
In 2010, the first report of the successful
use of CAR-transduced Tcells targeting
the CD19 molecule in patients with Bcell
lymphomas was reported.9 A heavily pretreated patient with advanced lymphoma
exhibited a dramatic response after receiving two cycles of autologous anti-CD19
CAR-transduced T cells and remained
progression-free 5years later.9 Additional
reports confirmed the ability of these antiCD19 CAR-transduced Tcells to mediate
the regression of both indolent as well as
aggressive large B-cell lymphomas, and
acute and chronic lymphoblastic leukaemias.
These early studies using gene-modified
cells have greatly expanded the reach of ACT
therapy to different tumourtypes.
The greatest obstacle to the further
development of cancer immunotherapy
is the identification of target molecules
expressed on cancer cells and not on essential human tissues. Immunogenic mutations
expressed in individual cancers could be the
ideal immunotherapy target. In 2014, Tran
etal.10 reported a general technique for the
identification of lymphocytes capable of
targeting the rare but unique immunogenic
mutations present in individual epithelial
cancers and used this ACT approach to
mediate objective regression in a patient
with metastatic cholangiocarcinoma. The
intrinsic ability of the immune system to
specifically target the unique mutations
expressed by common cancers is likely to
be the key to further progress in the field of
cancer immunotherapy.
National Cancer Institute, 10 Center Drive
MSC1201, Bethesda, MD 20892, USA.
sar@nih.gov
Competing interests
23 | NOVEMBER 2015
1.
2.
3.
4.
5.
6.
Hodi, F.S. etal. Improved survival with

ipilimumab in patients with metastatic
melanoma. N.Engl. J.Med. 363, 711723
(2010).
Topalian, S.L. etal. Safety, activity, and immune
correlates of antiPD1 antibody incancer.
N.Engl. J.Med. 366, 24432454 (2012).
Brahmer, J.R. etal. Safety and activity of
antiPDL1 antibody in patients with advanced
cancer. N.Engl. J.Med. 366, 24552465
(2012).
Kantoff, P.W. etal. SipuleucelT immunotherapy
for castration-resistant prostate cancer. N.Engl.
J.Med. 363, 422 (2010).
Rosenberg, S.A. etal. Durable complete
responses in heavily pretreated patients
withmetastatic melanoma using Tcell transfer
immunotherapy. Clin. Cancer Res. 17,
45504557 (2011).
Bollard, C.M. etal. Sustained complete
responses in patients with lymphoma receiving
autologous cytotoxic T lymphocytes targeting

EpsteinBarr virus latent membrane proteins.
J.Clin. Oncol. 32, 798808 (2012).
7. Morgan, R.A. etal. Cancer regression in
patients after transfer of genetically
engineered lymphocytes. Science 314,
126129 (2013).
8. Pule, M.A. etal. Virus-specific Tcells
engineered to coexpress tumor-specific
receptors: persistence and antitumor activity
inindividuals with neuroblastoma. Nat. Med.
14, 12641270 (2008).
9. Kochenderfer, J.N. etal. Eradication of
Blineage cells and regression of lymphoma
ina patient treated with autologous Tcells
genetically engineered to recognize CD19.
Blood 116, 40994102 (2010).
10. Tran, E. etal. Cancer immunotherapy based
onmutation-specific CD4+ Tcells in a patient
with epithelial cancer. Science 344, 641645
(2014).
DECADE IN REVIEWGENOMICS
A decade of discovery in cancer

genomics
Kenneth Offit
Over the past decade, genetic testing for rare inherited mutations, such
as BRCA1 and BRCA2 mutations, has been successfully incorporated
into clinical practice. Next-generation sequencing of cancer-susceptibility
genes and entire tumour genomes has transformed cancer care and
prevention. The discoveries of new cancer syndromes have raised exciting
opportunities and potential liabilities for cancer-care providers seeking to
incorporate genomic approaches into preventive oncology practice.
Offit, K. Nat. Rev. Clin. Oncol. 11, 632624 (2014); published online 7 October 2014;
The past decade has witnessed the incorporation of genetic testing for cancersusceptibility syndromes into the evidence-
based practice of oncology, and the emergence of next-generation genome scans
for cancer-risk loci. Herein, I discuss a
series of seminal papers published over
the past decade that described new cancer
syndromes, but also raised new challenges
related to informed consent, incidental
findings, and the management of genetic
variants of unknown significance or
unproven clinicalactionability.
In the 1980s and 1990s, rare but highlypenetrant cancer-predisposition genes were
identified by studying cancer-prone families
that demonstrate Mendelian inheritance of
cancer susceptibility. These studies implicated genes, such as BRCA1 and BRCA2, the
DNA-mismatch-repair genes (relevant for
colon cancer), TP53 in LiFraumeni syndrome, and APC in familial adenomatous
polyposis. The genetic basis of these and
other syndromes had a powerful impact
on the practice of preventive oncology.

The incorporation of genetic testing for
BRCA mutations in breast cancer marked
one of the first applications of personalized genomics in medicine, and enabled
targeted cancer screening, prevention
and, in some cases, the ability to personal
ize therapies according to the patients
genetic lansdcape.1 The translation of BRCA
testing to clinical practice was highlighted
by Domchek and colleagues2 who showed
that preventive surgery of the ovaries over
a 34-year period decreased mortality in a
cohort of 2,482 women with BRCA1 or
BRCA2 mutations; compared with women
who did not have salpingo-oophorectomy,
women who underwent this procedure had
a 60% decrease in all-cause death rates,
driven by lower mortality associated with
both breast and ovarian cancer. 2 In this
study, the subset of women found to have
occult microscopic ovarian cancer at the
time of preventive surgery were excluded
from analysis.2 During subsequent years,
CLINICAL ONCOLOGY
risk-reducing ovarian surgery, along
withbreast MRI, the option of prophy
lactic breast surgery and hormonal chemoprevention, became standard practice in
preventive oncology.1
oncologists will soon be

screening the inherited genomes
of all patients with cancer
In the past decade it had become obvious

that highly penetrant cancer genes (such
as BRCA1/2 and MSH2) did not account
for the bulk of familial risk of the common
hereditary cancers. A debate ensued regarding whether there were many common lowrisk genetic variants or undiscovered rare
high-risk variants, which would explain the
missing heritability of cancer. Apivotal
paper tested the common variant hypothesis using the emerging technology of gene
chips to assess hundreds of thousands of
single nucleotide polymorphisms (SNPs).3
In a two-stage design, 227,876SNPs were
assessed in 4,398 breast-cancer cases
and 4,316controls, identifying 30SNPs
of interest that were further analys ed in
21,860cases and 22,578controls.3 The SNP
that emerged as the best hit, which was
proximal to the gene FGFR2, had a relative
risk of around 1.2times the baseline risk,
compared with BRCA1 that elevated risk of
early onset breast cancer by up to 40fold.3
Subsequent genome-wide association
studies of other cancer types identified hundreds of hits near potentially causal genes,
which were all statistically significant, but
none of a magnitude to influence preventive management in the clinic.4 A possi
ble exception to this lack of clinical utility
emerged from studies we performed as part
of an international consortium investigating
modifiers of risk in the carriers of BRCA
mutations. In studies involving tens of thousands of BRCA1/2 mutation carriers worldwide, panels of risk-associated SNPs could
partition breast cancer risk from 20%up to
100%in BRCA-mutation carriers.5 These
findings will likely mark the first application of SNP-based risk profiling to inform
clinical management of individuals with
hereditary risk of a common cancer.
Over the second half of the past decade,
a shift to identifying rare genomic variants was made possible by the emergence
of next-generation sequencing (NGS)
approaches. NGS involves a series of repeating sequencing reactions, performed and
detected automatically, with the production
of thousands to millions of simultaneous

sequence reads. An immediate and obvious
application of NGS was to sequence several
genes at the same time. A technological
tourde force prefigured the current era
in cancer panel testing. Using targeted
capture and massively parallel genomic
sequencing, a group at the University of
Washington screened 21candidate genes in
360women with ovarian cancer.6 Strikingly,
24%of these women carried germline lossof-function mutations in genes such as
BRCA1, BRCA2, BARD1, BRIP1, CHEK2,
MRE11A, MSH6, NBN, PALB2, RAD50,
RAD51C, and TP53.6 Fuelled by this techno
logical innovation, plus the equally impactful loss of patent protection for BRCA1
and BRCA2 sequence analysis, a plethora
of commercial cancer panels flooded the
oncologymarketplace.
At the same time, NGS technologies were
rapidly applied to studying unexplained
familial cancer clusters. Over the past
5years, whole-exome sequencing (WES)
and whole-genome sequencing (WGS) has
resulted in a renaissance in the discovery
of new syndromes of cancer susceptibility
(Box1). One of the early applications of this
technology came from a group at the Johns
Hopkins University, who applied WES of
20,661coding genes in a single case of familial pancreatic cancer.7 Of 15,461germline
variants not found in the reference human
genome, a deletion of four base pairs within
the PALB2 gene was discovered and tested
as a pancreatic-cancer-susceptibility gene.7
Despite this early report, we and others have
failed to confirm PALB2 as a major factor in
hereditary breastpancreas-cancer families;
however, PALB2 maintained its status as a
rare breast-cancer-susceptibility gene.
Another example of a new syndrome with
a striking phenotype was described by Testa
and colleagues in 2011,8 on the basis of their
observation of gene clustering of mesotheliomas and melanomas. Using exome
sequencing strategies, germline mutations were discovered in the gene encoding BRCA1associated protein1(BAP1) in
two families with multiple cases of meso
thelioma, and in some cases of uveal melanoma.8 These findings built on the earlier
observation of inherited germline BAP1
mutations in uveal and cutaneous melanocytic tumours. Remarkably, this syndrome
was extended by other groups to include
renal-cell cancers in rare families.
In some cases the new familial cancer
types studied were not rare. For example,
we studied families with acute lymphoblastic
Box 1 | Cancer susceptibility syndromes*

Familial pancreatic cancer
PALB2 identified by exome sequencing; ATM
identified by exome sequencing and WGS
Familial ovarian cancer
BRIP1 identified by WGS
Familial pheochromocytoma
MAX identified through exome sequencing
Acute myelogenous leukaemia
(withEmberger syndrome)
GATA2 identified by exome sequencing
Familial Hodgkin lymphoma
NPAT identified by exome sequencing
Familial preB-cell acute lymphoblastic
leukaemia
PAX5 identified by exome sequencing
Familial melanoma
MITF identified by WGS; TERT identified
bytargeted sequencing
Familial mesothelioma, melanoma
andrenal-cell cancer
BAP1 identified through exome and targeted
sequencing
Hereditary mixed polyposis syndrome
(HMPS)
GREM1 identified by targeted sequencing
Colorectal adenomas and colon cancer
POLE and POLD1 identified by WGS
Familial breast cancer
XRCC2 and FAN1 identified by exome
sequencing; PPM1D (mosaic) by targeted
sequencing
*Discovered recently by NGS.4 Abbreviations: NGS,
nextgeneration sequencing; WGS, whole-genome
sequencing.
leukaemia, the most-common malignancy

of childhood, and identified a mutation in
a lymphoid-associated transcription factor,
PAX5, in two such families,9 with a third
Israeli family more recently reported to
harbour the same mutation. These new
cancer syndromes have redefined our notion
of inherited cancer (Box1).
Despite these advances over the past
decade, clinical interventions for these syndromes remain relatively rudimentary, and
the ethical implications of these discoveries remain daunting. Risk reduction for
the adult-cancer syndromes includes organ
removal surgeries.1 True genetic prevention using assisted reproductive techno
logies is an option oncologists should
remember to discuss with their younger
patients, or patients families, taking into
account ethical or religious considerations.
A broader ethical debate has emerged
regarding the extent to which incidental, or
secondary genetic findings, termed the incidentalome, should be disclosed to patients.
Particularly challenging for oncologists
NOVEMBER 2015 | 24
CLINICAL ONCOLOGY
are the unexpected results of NGS analysis
of tumour and normal pairs, which might
include identification of genetic predispositions to noncancer-related diseases, such as
cardiac or neurological diseases.10 A vigorous discussion is in progress regarding the
potential obligations of physicians to inform
individuals of incidental genetic findings.
At the same time there have been recent
calls for population-based screeningfor
example, BRCA testing of all 30yearold
women worldwide. Although such requests
by laboratory-based scientists have the best
intentions, they overlook a more-pressing
clinical reality: oncologists will soon be
screening the inherited genomes of all
patients with cancer. In both scenarios,
population testing of healthy individuals
and tumournormal screening in patients
with tumours, we must recognize what
has been learnt over the past decade: not
all individuals wish to know all genomic
information; risks might reflect both popu
lation heterogeneity and differences in
penetrance; and not all genomic information is clinically actionable. Oncology has
become the ground zero for a tectonic shift
in paradigms regarding personalized medicine, both for targeted treatment as well as
prevention based on genomic profiles.
Department of Medicine, Clinical Genetics
Service, Program in Cancer Biology and
Genetics, Sloan-Kettering Institute, Memorial
Sloan Kettering Cancer Center, Box192,
1275York Avenue, New York, NY 10021, USA.
Department of Medicine and Public Health,
Weill Cornell Medical College,
CornellUniversity, 445 East 69th Street,
NewYork, NY10021, USA.
offitk@mskcc.org
Acknowledgements
K.O. would like to acknowledge funding support from
The Robert and Kate Niehaus Clinical Cancer
Genetics Research Initiative, and The Breast Cancer
Research Foundation.
Competing interests
1.
2.
3.
4.
Couch, F.J., Nathanson, K.L. & Offit, K.

Twodecades after BRCA: setting paradigms
inpersonalized cancer care and prevention.
Science 343, 14661470 (2014).
Domchek, S.M. etal. Association of riskreducing surgery in BRCA1 or BRCA2 mutation
carriers with cancer risk and mortality. JAMA
304, 967975 (2010).
Easton, D.F. etal. Genome-wide association
study identifies novel breast cancer
susceptibility loci. Nature 447, 10871093
(2007).
Stadler, K., Schrader, K.A., Vijai, J.,
Robson,M.E. & Offit, K. Cancer genomics
andinherited risk. J.Clin. Oncol. 32, 687698
(2014).
25 | NOVEMBER 2015
5.
6.
7.
Gaudet, M.M. etal. Identification of a BRCA2specific modifier locus at 6p24 related to

breast cancer risk. PLoS Genet. 9, e1003173
(2013).
Walsh, T. etal. Mutations in 12 genes for
inherited ovarian, fallopian tube, and peritoneal
carcinoma identified by massively parallel
sequencing. Proc. Natl Acad. Sci. USA 108,
1803218037 (2011).
Jones, S. etal. Exomic sequencing identifies
PALB2 as a pancreatic cancer susceptibility
gene. Science 324, 217 (2009).
8.
Testa, J.R. etal. Germline BAP1

mutationspredispose to malignant
mesothelioma. Nat.Genet. 43, 10221025
(2011).
9. Shah, S. etal. A recurrent germline PAX5
mutation confers susceptibility to preBcell
acute lymphoblastic leukemia. Nat. Genet. 45,
12261231 (2013).
10. Bombard, Y., Robson, M. & Offit, K.
Revealingthe incidentalome when targeting
thetumor genome. JAMA 310, 795796
(2013).
DECADE IN REVIEWFUNDING IN CANCER RESEARCH
National Cancer Institute awards

a work in progress
Tito Fojo and Paraskevi Giannakakou
Over the past decade, funding for cancer research by the

USgovernmentand othershas stagnated, while the demand for
investment has grown because of the increasing cancer incidence
worldwide. We discuss how National Cancer Institute funding efforts
havedeveloped during this period, and the contemporary and future
impact of these measures on cancer research in the USA.
Fojo, T. & Giannakakou, P. Nat. Rev. Clin. Oncol. 11, 634636 (2014); published online 14 October 2014;
The USA has been at the forefront of

cancer research with regard to spending,
and this investment has benefited not only
US citizens, but also the wider global community. Although dwarfed by the money
invested by pharmaceutical companies and
a diverse group of foundations, govern
ment expenditure remains critical, and
is unencumbered by bias. The National
Cancer Institute (NCI) was established as
the principal agency for cancer research
inthe USA in 1937, and with their mandate
further expanded to include coordination of
the National Cancer Program in 1971, the
NCI has been responsible for the management of the grants programme that allocates research funds to investigators and
cooperative groups. For most of the past
decade, the NCI has had essentially flat
budgets, requiring it to curtail many efforts
and reduce the percentage of grants funded
to numbers that hover close to single digits.
Nobody believes that all grants deserve
support, however, there is no doubt that
the current allocation leaves many valuable
ideas unfunded, and a much higher percentage of grant funding would be optimal.
Indeed, given cancers increasing impact on
mortality statistics worldwide, the measures
taken by the NCI during the past 10years to
maximize the value of their budgets are of
clinical research has

nevertheless been severely
affected by the flat NCI budget
heightened importance; herein, we provide

our personal views, and not the views of the
NCI, of these measures.
The NCI portfolio is heavily weighted
to supporting investigator-initiated projects, usually focusing on the basic science
of cancer rather than clinical trials. The
wisdom of this model can be debated, but
few can deny that support for clinical trials
has suffered greatly during the ongoing
period of fiscal austerity.1 This latter view,
held widely by clinical researchers, was ratified in a 2010 Institute of Medicine (IOM)
report,2 which concluded that clinical trials
for cancer in the USA were approaching a
state of crisis, especially those conducted
under the auspices of the NCI Cooperative
Group Program; since that report, the
outlook has worsened. The IOM recommended 12major changes,2 many of which
the NCI has initiated as part of a comprehensive overhaul. An important change
was consolidation of nine Cooperative
Groups into four, with the immediate goal
of improving efficacy and avoiding duplication of effort, and a long-term goal of
hopefully enabling the conduct of studies

in patients with specific molecular tumour
subtypes who might be more difficult to
accrue in smaller groups. This programme,
now referred to as the National Clinical
Trials Network (NCTN), had an overall
budget of US$151 million in the 2014 fiscal
year, representing only 3.1% of the total
NCI budget and the same amount provided for the Cooperative Group awards
in each of the 2012 and 2013 fiscal years.3
Although the distribution of funds to
various components of the NCTN is different to the allocation under the former
Cooperative Group Program, clinical
research has nevertheless been severely
affected by the flat NCI budget. Amajor
consequence is low reimbursem ents to
particip ating institutions for patients
enrolled in NCI-sponsored clinical trials,
which has remained largely unchanged for
nearly a decade and stands at US$2000
per patient. This level of reimbursement
is increasingly inadequate, considering the
expenditures of US$10,000 per patient, a
reality recognized by pharmaceutical companies that now reimburse organisations
conducting clinical trials US$10,00040,000
per patient.4 The shortfall in reimbursement means participating institutions
must find other revenue sources to support
NCI-funded trials, discouraging involvement and preventi ng investigators from
conducting the correlative studies that are
the essence of cutting-edge clinical trials.
The shortfall is also largely responsible for
the increasing reliance on pharmaceuticalcompany sponsored trials, a trend that
some argue should be embraced, as those
with the most to gain (financially) should
cover thecosts. However, most investigators
would counter that industry involvement
affects the choice of trials conducted and
the relevance of the questions asked, as
profits remain an important consideration
for publicly heldcorporations.
Although the NCI predominantly funds
scientific research, valid metrics to assess
the success of any of such programmes (not
their applicants) are needed. Some might
argue that publications represent an indirect
measure of scientific output and success, but
few agree, as this metric also has its flaws.5
Although assessments of established NCI
programmes are also lacking, efforts have
been made to develop effective methodologies and quantitative indicators to ascertain the success or failure of new initiatives.
Two examples include the novel Physical
ScienceOncology Centers (PSOC) and
the Provocative Questions programme.

At its launch in 2009, the PSOC funded
12 centres throughout the USA, with over
250 investigators dedicated to building
transdisciplinary teams and infrastructure
to conduct transformative research. An
emphasis on engaging individuals with
backgrounds in the physical sciences and
limited previous involvement in cancer
studies was an important goal of the PSOC.
Initial assessments revealed rapid scientific
progress, such as the application of concepts based on physical science principles in
clinical trials.6 Further evidence indicated a
clear increase in crossdisciplinary collabor
ations and publications in post-award years
among the PSOC primary investigators.
Although encouraging prog ress is being
made, sufficient time to reach a level of
common un derstandingespecially for
interdiscip linary pr ogrammeswillbe
crucial before a final assessment can
bemade. Similarly, a combination of subjective and objective assessments will be used
to measure the success of the Provocative
Questions programme in cancer research,
an equally novel initiative that launched
in 2012 and will be evaluated in the
comingyears.7
Initial assessments revealed

rapid scientific progress
Progress has been more difficult as the

NCI has tried to think outside of the box
in order to achieve much-needed breakthroughs. Economists typically argue that
taking risks is critical to any investment
strategy and that a minimum of 510% of
a portfolio should be held in alternatives,
which generally have low correlation with
the majority of the portfolio and provide
welcome diversification. One could argue
the NCI should take the same approach:
part of the budget should be devoted to such
high-risk/high-payoff alternative investment strategies. However, pursuing such a
strategy has been difficult.
Although most agree that current NCI
funding mechanisms are far from perfect,
there seems to be a reluctance to pursue
alternatives. This reluctance is emphasized by the fact that peer-reviewed NIH
funding currently represents one of the
most important metrics for academic
career development. Perhaps scientists
are more risk-averse; how else could one
explain the skepticism and hesitation
expressed by so many when the Provocative
iStock/Thinkstock
CLINICAL ONCOLOGY
Questions initiat ive was launched, 8 or

conc erns regarding the well-validated
PSOC programme that represent only
a very small fraction of the entire NCI
budget (0.3% and 0.6%, respectively)? In
launching the Provocative Questions programme, Varmus and Harlow 7 noted that
pa rticularly during periods of fiscal
austeritygrantapplic ants and reviewers tend to favour grants converging on
similar, popular and conservative ideas,
thus narrowing research portfolios; efforts
such as those taken by the NCI in the latter
half of the past decade are attempting to
challenge such thinking. Similarly, the NCI
Outstanding Investigator grants, 7year
people grants of up to US$600,000 a year
(plus another 50% for overhead costs) that
will replace project-based grants, has been
launched with the hope of unbridling innovation. The amount of expenditure will not
be trivial, as the NCI expects to fund 50
additional Outstanding Investigator Awards
per year for 7years, by which time the programme will total US$317million per year,
or about 16% of the US$2billion that the
NCI spends on research grants currently. To
be sure, these people grants are not uniformly endorsedgiven concerns that they
might be unfair to younger investigators,
favouring established investigators based
on past track record and not based on the
scientific rigour of proposed research, as
well as the fact that their long duration will
result in less money turning over each year
and, therefore, fewer awards;9 however, if
properly executed, this grants programme
might promote much needed outside of the
box thinking and research.
Continued funding is critical to enable
substantive progress in the search for thera
pies that can prolong the lives of patients
with cancer. The stakes are high and the
need for progress essential. The NCI efforts
in this direction over the past decade
remain a work in progress.
NOVEMBER 2015 | 26
CLINICAL ONCOLOGY
Medical Oncology, Center for Cancer Research,
National Cancer Institute, Building 10,
Room12N226, 9000 Rockville Pike, Bethesda,
MD 20892, USA (T.F.). Department of Medicine,
Division of Hematology and Medical Oncology,
Weill Cornell Medical College/Weill Cornell
Cancer Center, 520East70thStreet, Starr 341,
New York, NY10021, USA (P.G.).
Correspondence to: T.F.
fojot@mail.nih.gov
2.
3.
Competing interests
1.
American Society of Clinical Oncology.

TheFunding Crisis in Cancer Clinical Trials:
AFocus on Cooperative Groups [online],
27 | NOVEMBER 2015
4.
http://www.asco.org/advocacy/funding-crisiscancer-clinical-trials-focus-cooperative-groups
(2014).
Institute of Medicine (US) Committee on Cancer
Clinical Trials and the NCI Cooperative Group
Program (eds Nass, S.J., Moses, H.L.
&Mendelsohn, J.). A National Cancer Clinical
Trials System for the 21st Century: Reinvigorating
The NCI Cooperative Group Program
(TheNational Academies Press, 2010).
National Cancer Institute at the National
Institutes of Health. Clinical Trials Programs and
Initiatives: An Overview of NCIs National Clinical
Trials Network [online], http://www.cancer.gov/
clinicaltrials/nctn (2014).
Seow, H.Y. et al. Funding oncology clinical
trials: are cooperative group trials sustainable?
J. Clin. Oncol. 30, 14561461 (2012).
5.
6.
7.
8.
9.
Begley, C.G. & Ellis, L.M. Drug

development:raise standards for preclinical
cancer research. Nature 483, 531533
(2012).
Basner, J.E. et al. Measuring the evolution
andoutput of cross-disciplinary collaborations
within the NCI Physical SciencesOncology
Centers Network. Res. Eval. 22, 285297
(2013).
Varmus, H. & Harlow, E. Science funding:
Provocative questions in cancer research.
Nature 481, 436437 (2012).
Benowitz, S. Provocative Questions initiative
tofund innovative cancers research. J. Natl
Cancer Inst. 104, 970972 (2012).
Kaiser, J. Funding. NIH institute considers
broad shift to people awards. Science 345,
366367 (2014).
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ENDOCRINOLOGY
DECADE IN REVIEWBONE
Great strides made but still further to go

Ian R. Reid
Our understanding of bone biology and the subsequent development of therapies to treat bone diseases have
both expanded greatly in the past 30years. This article reviews some of the key advances made in these fields
during the past decade.
28 | NOVEMBER 2015
The past decade has seen several major

advances in therapies for osteoporosis. First,
the phaseIII trial for zoledronate resulted
in the availability of an annual intravenous
infusion for the management of osteo
porosis. Zoledronate might have slightly
greater efficacy than oral bisphosphonates
in the prevention of vertebral fractures, but
the principal benefit of zoledronate over oral
bisphosphonates is one of convenience. This
benefit increases the likelihood of treatment
adherence, as patients only need to remem
ber to take their medication once a year.
The study by Lyles and colleagues3 broke
new ground by demonstrating the effective
ness of zoledronate within 3months of a
fracture, and also the intriguing finding of
lower mortality in those allocated to active
treatment. This observation is consistent, to
some extent, with the growing body of data,
in which osteoporotic fractures are associ
ated with increased mortality; however, the
mechanism by which mortality is reduced
remains uncertain.
A similar study with denosumab
showed comparable efficacy
for this monoclonal anti
body directed against
the osteoclastogenic
protein, RANKL.4
Denosumab,
which is admin
istered as asub
cutaneous injection
every 6months, does not
cause an acute phase
response and is safe in
patients with impaired
renal function. As
denosum ab has
a rapid offset
ofaction, strict
c ompl i anc e
is important;
however, this
Er
ax
ion
/iS
to
ck
/T
hin
ks
to
ck
The recognition that osteocytes have a

central role in bone biology has been amajor
development in the past decade. Elegant
studies in rodents have demonstrated the
influence of skeletal loading on osteocytic
production of the osteoblast inhibitory
protein, sclerostin. A seminal article by
Robling etal. concluded that modulation of
sclerostin levels appears to be a finely tuned
mechanism by which osteocytes coordinate
regional and local osteogenesis in response
to increased mechanical stimulation.1 This
finding complements the established role of
the osteocyte as a source of receptor activa
tor of nuclear factorB ligand (RANKL),
and enables these cells to directly influence
both bone formation and bone resorption.
Consistent with their key regulatory func
tions, sclerostin and RANKL are both now
targets for new drug development.
The influence of osteocytes is not just
limited to bone, as they also have a central
role in phosphate and vitaminD metab
olism via production of fibroblast growth
factor23 (FGF23). This protein reduces
renal phosphate reabsorption by downregu
lating the expression of sodiumphosphate
cotransporters in the apical membrane of
the proximal tubule. FGF23 also reduces
expression of 25-hydroxyvitamin D 3
1-hydroxylase and increases expression of
1,25-dihydroxyvitaminD3 24-hydroxylase,
which reduces levels of 1,25-dihyroxy
vitaminD3. Serum levels of both phosphate
and 1,25-dihyroxyvitaminD increase levels of
FGF23. The FGF23 receptor is a heteromeric
complex of a canonical FGF receptor and
Klotho.2 Although levels of FGF23 increase
in renal failure and are associated with worse
outcomes for these patients, whether FGF23
has a causative role in these consequences is
unknown. These discoveries have provided
insights into a range of hypophosphataemic
and hyperphosphat aemic disorders, but
many details remain to be elucidated.
property is theoretically advantageous if

patients develop adverse effects resulting
from low bone turnover. Like zoledronate,
denosumab produces a sustained reduc
tion in fracture rates over long-term followup. Denosumab and zoledronate are now
also widely used in managing the skeletal
consequences ofcancer.
In 2014, monoclonal antibodies directed
against the osteoblast inhibitor, sclerostin,
were reported to transiently increase
bone formation and transiently decrease
bone resorption, together with dramatic,
sustained increases in BMD at the hip and
spine.5 The antifracture efficacy of one of
these antibodies, romosozumab, is currently
beingassessed.
Although new agents have been devel
oped, the efficacy and safety of some existing
interventions have been questioned. Metaanalyses have demonstrated an increased
risk of cardiov ascular events with
calcium supplements.6 These risks
are sufficiently common to out
weigh the small beneficial effect
of calcium supplements on frac
tures. When combined with the
established adverse effects of calcium
(such as gastrointestinal symp
toms and kidney stones), calcium
supplements are unlikely to
produce a net benefit
in most patients. A
similar scrutiny of
vitaminD supplem ents
indicates a lack of effect on BMD
and fractures in nonosteomalacic
populations. Furthermore, high doses of
vitaminD supplements might, paradoxically,
increase fractures and falls.
The accumulation of bisphosphonates
in bone has caused concern that their
chronic use could result in skeletal toxicity.
Osteonecrosis of the jaw and atypical femoral
fractures (AFF) might be manifestations of
ENDOCRINOLOGY
this toxicity. Although osteonecrosis of the
jaw is mainly a problem in oncology prac
tice, spontaneous fractures of the femoral
shaft have been reported by many osteo
porosis clinics and seem to have an associ
ation with long-term oral bisphosphonate
use, as demonstrated by Schilcher and col
leagues.7 In this study, AFFs were shown to
be transverse stress fractures originating in
the lateral femoral cortex, whereas classic,
spiral, comminuted, femoral fractures are
no more common in bisphosphonate users
then in other individuals with osteoporosis.7
Risk of AFFs declines rapidly following dis
continuation of bisphosphonate use,7 which
suggests that drug holidays should be part of
long-term osteoporosis management.
In the FLEX study 8a drug holiday
trialwomen already on alendronate for
5years were randomly reassigned to either
continued therapy or placebo. The results
from FLEX showed that halving the conven
tional dose of alendronate did not reduce
its effects on BMD. Additionally, FLEX
demonstrated stable nonvertebral fracture
rates in patients who continued therapy
or crossed over to placebo, but suggested
a benefit from continued intervention in
those whose femoral neck BMD was still
in the osteoporotic range. A similar study
with zoledronate reached similar conclu
sions. Together, these studies now guide the
long-term use of thesedrugs.
In the decades since osteoporosis treat
ment became a reality, the definition of
osteoporosis and which patients should be
treated, has evolved. In the past 10years,
cost-effectiveness has become an important
part of making treatment decisions, and
requires quantification of absolute fracture
risk to target interventions optimally. The
development of fracture risk estimators has
ensued, and has dramatically changed clinical
practice, with FRAX9 and the Garvan calcu
lators being freely available via the internet.
Standard practice is now for treatment to be
provided on the basis of absolute fracture
risk, which is a convenient way of integrat
ing a range of clinical risk factors, including
BMD. The various calculators use slightly dif
ferent selections of risk factors, and comple
ment one another in the assessments they
provide. The Garvan calculator is strongly
influenced by fall history, a risk factor often
ignored by endocrinologists. All calculators
recognize the importance of age in calcu
lating fracture risk, and have probably led
to more widespread intervention in elderly
patients and a more conservative approach
to individuals in the first 12decades after
menopause. Notably, these calculators do not

include certain risk factors, such as calcium
intake and exercise, that have been heavily
emphasized in the past, but which proved
to be of little utility in estimating absolute
fracture risk.
Although much less common than osteo
porosis, Pagets disease can be a source of sub
stantial morbidity and, at the beginning of the
last decade, remained a significant therapeu
tic challenge. Intravenous zoledronate infu
sions have revolutionized the management
ofPagets disease, normalizing serum levelsof
alkaline phosphatase in ~90% of patients.10
These biochemical changes are associated
with improvements in quality of life. Perhaps
more importantly, the biochemical and
quality of life improvements are sustained
in most patients without re-dosing; <1% of
patients relapse over >6years of follow-up
after a single dose of the drug. Zoledronate
has now become the first-line therapy for
this condition and is effectively curative in
the majority of patients.
The past decade has seen many exciting
developments, with new therapeutic and
diagnostic techniques resulting directly
from advances in our understanding of bone
biology. If the coming decade is as productive
as the last, we will be in a much stronger posi
tion to deal with the advancing tide of bone
disease, which increasing longevity is forcing
upon us.
Faculty of Medical and Health Sciences,
University of Auckland, Private Bag 92019,
Auckland, New Zealand.
i.reid@auckland.ac.nz
doi:10.1038/nrendo.2015.143
Published online 25 August 2015
Acknowledgements
I.R.R. thanks J. Cornish, N. Sims and T. J. Martin for
advice during the preparation of this manuscript.
I.R.R. is supported by the Health Research Council
ofNew Zealand.
Competing interests
I.R.R. has received research funding from Amgen,
Merck and Novartis, and has received honoraria
fromAmgen, Lilly, Merck, Novartis and Sanofi.
1.
Robling, A.G. etal. Mechanical stimulation

ofbone invivo reduces osteocyte expression
ofSost/sclerostin. J.Biol. Chem. 283,
58665875 (2008).
2. Urakawa, I. etal. Klotho converts canonical
FGFreceptor into a specific receptor for FGF23.
Nature 444, 770774 (2006).
3. Lyles, K.W. etal. Zoledronic acid and clinical
fractures and mortality after hip fracture.
N.Engl. J.Med. 357, 1799809 (2007).
4. Cummings, S.R. etal. Denosumab for prevention
of fractures in postmenopausal women with
osteoporosis. N.Engl. J.Med. 361,756765
(2009).
5. McClung, M.R. etal. Romosozumab in
postmenopausal women with low bone
mineraldensity. N.Engl. J.Med. 370, 412420
(2014).
6. Bolland, M.J., Grey, A., Avenell, A., Gamble, G.D.
& Reid, I.R. Calcium supplements with or without
vitaminD and risk of cardiovascular events:
reanalysis of the Womens Health Initiative
limited access dataset and meta-analysis.
BMJ342, d2040 (2011).
7. Schilcher, J., Michaelsson, K. & Aspenberg, P.
Bisphosphonate use and atypical fractures
ofthefemoral shaft. N.Engl. J.Med. 364,
17281737 (2011).
8. Black, D.M. etal. Effects of continuing or
stopping alendronate after 5years of
treatment:the Fracture Intervention Trial
Long-term Extension (FLEX): a randomized trial.
JAMA 296, 29272938 (2006).
9. Kanis, J.A., Johnell, O., Oden, A., Johansson, H.
& McCloskey, E. FRAX and the assessment
offracture probability in men and women from
the UK. Osteoporos. Int. 19, 385397 (2008).
10. Reid, I.R. etal. Comparison of a single infusionof
zoledronic acid with risedronate for Pagets
disease. N.Engl. J.Med. 353, 898908 (2005).
DECADE IN REVIEWTHYROID DISEASE
The endocrinology of thyroid

disease from 2005 to 2015
P. Reed Larsen
The past decade has seen exciting progress in the field of thyroid
disease, especially in the evaluation of thyroid nodules, the genomic
characterization of carcinomas and the treatment of carcinomas
after surgery. An improved understanding of hyperthyroidism during
pregnancy, as well as the causes of low T3 syndrome and consumptive
hypothyroidism have also been achieved.
As the importance of fine needle aspiration
for evaluating thyroid nodules for potential
malignancy became apparent, a standard
ized approach to cytologic classification of
this material was necessary. After several

years of preliminary collaborative studies, the
National Cancer Institute hosted a conference
of cytopathologists and endocrinologists. The
NOVEMBER 2015 | 29
ENDOCRINOLOGY
result of this meeting was the development of
the Bethesda System for thyroid cytopathol
ogy.1 Six diagnostic categories were defined
and the cancer risk of each estimated from
preliminary studies. These categories were:
I, nondiagnostic (risk 14%); II, benign (risk
03%); III, atypical or follicular lesions of
undetermined significance (risk 515%);
IV, diagnostic of or suspicious for follicular
neoplasm (risk1530%); V, suspicious for
malignancy (risk 6075%); and VI, malig
nant (risk 9799%). The Bethesda System has
been widely adopted. The main limitation of
this system is that 4060% of patients with an
indeterminate classification (that is, catego
ries III and IV) who undergo surgery actually
have benign lesions, which emphasizes the
need for more-specific preoperative criteria
to reduce the large numbers of unnecessary
diagnostic surgical procedures.
Molecular tools are now being developed
for this purpose. One approach is to rule out
the expression of mutant genes known to be
associated with malignancy while identifying
others that are associated with atypical cytol
ogy using the Gene Expression Classifier
(GEC).2 Of 121 surgical samples classified
as suspicious by GEC, 44% were malignant.
Follow up of 174 patients with tumours classi
fied as benign revealed that only one in 11
patients who underwent surgery had a malig
nancy. Further ultrasonographic and/or sur
gical evaluations did not suggest malignancy
in any other patients in this category. These
findings indicate an improved specificity
ofthe GEC; however, the number of patients
in the studies wassmall.
A second approach that might identify
which patients with papillary thyroid carci
nomas (PTCs) have a high risk of recurrent
disease is to screen nodules for a Val600Glu
mutation in BRAF (BRAFV600E); tumours with
these mutations have a twofold higher recur
rence rate than lesions with wild-type BRAF.
However, this screening would not eliminate
all PTCs as only 5060% of these tumours
express this mutation.3
In a recent landmark paper from the
The Cancer Genome Atlas consortium, in
which genomic and tumour exome, RNAsequencing and microRNA analyses of 496
patients has been performed, ~96% of the
causal mutations in PTCs were identified.
Overall, the main mutations associated with
tumours were in BRAF (62%), NRAS, HRAS
and KRAS (13%) and mutations in genes
encoding transmembrane receptors, mainly
RET (10%).4 Tumours expressing BRAFV600E
and those with BRAF and RET fusion had
high levels of mitogen-activating protein
30 | NOVEMBER 2015
kinase (MAPK) signalling that was associated

with marked suppression of genes required
for thyroid hormone synthesis. Bycontrast,
tumours without the BRAFV600E mutation
and with mutations in the RAS genes did
not. In general, the expression profiles of
PTCs can be categorized as either BRAFV600Elike, poorly differentiated and signalling
through MAPK, or RAS-like and signal
ling via both phosphatidylinositol3kinase
and MAPK, and are well differentiated.
BRAFV600E mutations were present in 78%
of tall cell variant PTCs, whereas follicu
lar variants were RAS-like. Also correlat
ing with aggressive behaviour of tumours
in BRAF-like lesions were levels of miR21
expression and mutations in TERT, which
can explain the heterogeneity of prognoses
of BRAFV600E-expressingtumours.3,5
...the next decade will

see widespread application
ofgenomic analyses
The categorization of tumours into BRAFlike and RAS-like groups is consistent with
results of an important clinical study in
which short-term treatment with the MAPK
antagonist selumetinib was more successful
in increasing iodine uptake totherapeutically
effective levels inlesionswith a mutation in
NRAS thanin lesions with a mutated form
of BRAF.5 Although preliminary, this study
provides a proof-of-principle that MAPK
inhibition can redifferentiate metastatic
PTC lesions. These results also highlight the
importance of molecular characterization
of metastatic, radioiodine-resistant PTCs to
permit individualization of therapy.
Two important publications that address
therapeutic strategies after surgery in
patients with low-risk PTC lesions have
compared remnant ablation strategies and
radioiodine dosages.6,7 In prospective, ran
domized twobytwo comparisons that
involved 1,105 low-risk patients, increas
ing TSH using recombinant TSH was
found to be as effective in ablating residual
thyroid tissue and reducing levels of thyro
globulin as thyroid hormone withdrawal
for 34weeks in patients receiving a single
dose of either 1.1GBq (30mCi) or 3.7GBq
(100mCi) radioiodine.6,7 No difference was
seen in the success rate of the two radio
iodine doses. Infact, in many patients, the
target reduction in serum thyroglobulin
to 2g/l was achieved by surgery alone,
which demonstrates that many patients with
pT1T3 stage PTCs might not require any
radioiodine therapy after surgery. Follow-up

studies were performed 69months after
therapy, but results after longer intervals will
be important, as will studies comparing no
radioisotope with the 1.1Gbq dose. Perhaps
these studies will also include a molecular
characterization of the lesions.
Another key advance in the past 10years
relates to the treatment of pregnant patients
who have thyrotoxicosis. Treatment with
methimazole or carbimazole during the
first trimester of pregnancy is well known
to be associated with a risk of birth defects,
including aplasia cutis and choanal or
oesophageal atresia. Consequently, current
guidelines suggest that, when possible,
propylthiouracil (PTU) should be substi
tuted for these agents during the first tri
mester. In a 2014 nationwide register study
of >800,000 children born in Denmark, 11
of 564 infants exposed to PTU during early
gestation had increased risks of kidney
cyst, hydronephrosis or pre-auricular or
bronchial sinus abnormalities (hazard
ratio increased by threefold to fivefold).8
Although these lesions are not as serious as
those occurring with methimazole or car
bimazole, most required surgical remedi
ation. When antithyroid drugs cannot be
avoided during the first trimester, patients
receiving PTU should be advised of thisrisk.
Consumptive hypothyroidism was first
recognized in infants with large hepatic
haema ngiomas expressing type3 iodo
thyronine deiodinase (DIO3); abnormally
high levels of DIO3 results in thyroid
hormone being inactivated more rapidly
than can be replaced by the healthy thyroid.
This condition has now been identified
in adults with gastroi ntestinal stromal
tumours, many of whom also express DIO3.9
The requirement for thyroid hormone sup
plementation might be further exacerbated
in these patients (and others with different
malignancies) by tyrosine kinase inhibitor
therapy (for example, imatinib or suni
tinib), which can induce DIO3 expression
in thetumour.
For many decades, it has been known that
almost all patients with moderate to severe
illnesses develop abnormal thyroid function
tests, almost universally a reduced T3 and an
increased reverse T3 (rT3) in serum. Other
studies have now revealed that a major cause
of these abnormalities is the generation of
intracellular reactive oxygen species by the
cytokine IL6, which depletes the cytosolic
thiol cofactors required for activation of T4
to T3 in the liver, kidney and skeletal muscle
mediated by type1 and type2 iodothyronine
ENDOCRINOLOGY
deiodinase (DIO1 and DIO2, respectively).10
These deiodinases are the source of 80%
of the T3 produced in iodine-sufficient
humans. Unexpectedly, IL6 might also
induce DIO3 expression and this T3 and T4
inactivating deiodinase, unlike DIO1 and
DIO2, has comes into contact with extra
cellular thiols. Consequently, critically ill
patients are likely to have some degree of
consumptive hypothyroidism during illness,
which further reduces concentrations of
serum T3.
Hopefully, the next decade will see wide
spread application of genomic analyses
of all indeterminate fine needle aspira
tion samples, thus reducing the numbers
of unnecessary diagnostic surgeries. Ialso
anticipate the development of more potent
and specific MAPK inhibitors for thera
peutic redifferentiation of metastatic,
radioiodine-resistant PTCs.
Brigham and Womens Hospital, Division of
Endocrinology, Diabetes and Hypertension,
77Avenue Louis Pasteur, HIM 641, Boston,
MA02115, USA.
plarsen@partners.org
doi:10.1038/nrendo.2015.169
Published online 6 October 2015
Competing interests
1.
Cibas, E.S. & Ali, S.Z. The Bethesda system for

reporting thyroid cytopathology. Thyroid 19,
11591165 (2009).
2. Alexander, E.K. etal. Multicentre clinical
experience with the Afirma gene expression
classifier. Clin. Endocrinol. Metab. 99, 1925
(2014).
3. Xing, M. etal. Association between BRAF V600E
mutation and recurrence of papillary thyroid
cancer. Clin. Oncol. 33, 4250
(2015).
4. Ho, A.L. etal. Selumetinib-enhanced
radioiodine uptake in advanced thyroid
cancer.N. Engl. J. Med. 368, 623632 (2013).
5. Cancer Genome Atlas Research Network.
Integrated genomic characterization of papillary
thyroid carcinoma. Cell 159, 676690(2014).
6. Schlumberger, M. etal. Strategies of radioiodine
ablation in patients with low-risk thyroid cancer.
Tumeurs de la Thyrode Refractaires Network for
the Essai Stimulation Ablation Equivalence Trial.
N. Engl. J. Med. 366, 16631673 (2012).
7. Mallick, U. etal. Ablation with low-dose
radioiodine and thyrotropin alfa in thyroid
cancer. N. Engl. J. Med. 366, 16741685
(2012).
8. Maynard, M.A. etal. Thyroid hormone
inactivation in gastrointestinal stromal
tumors.N. Engl. J. Med. 37, 13271334 (2014).
9. Andersen, S.L., Olsen, J., Wu, C.S.
&Laurberg,P. Severity of birth defects after
propylthiouracil exposure in early pregnancy.
Thyroid 24, 15331540 (2014).
10. Wajner, S. M. etal. IL6 promotes nonthyroidal
illness syndrome by blocking thyroxine
activation while promoting thyroid hormone
inactivation in human cells. J. Clin. Invest. 121,
18341845 (2011).
DECADE IN REVIEWTYPE2 DIABETES MELLITUS
At the centre of things

Guang Ning
Over the past decade, the growing epidemic of type2 diabetes mellitus
(T2DM) and its associated complications has presented both challenges
and opportunities. Progress has been made in incretin-based therapies,
bariatric surgeries and inhibiting renal glucose reabsorption; however,
long-term safety and efficacy studies are required. Advances in the
prevention of macrovascular complications of T2DM from ongoing
clinicaltrials are expected soon.
In the past 10years, major advances have
been made in research into type2 diabetes
mellitus (T2DM). Epidemiological studies
have revealed an increasing worldwide
prevalence of T2DM and related mortality.
Two surveys published in the past 3years on
the prevalence of T2DM and the associated
disease burden, which revealed the worrying
situation we face today, might change global
and national health policies in the future.1,2
The Global Burden of Disease Study (GBD)
20131 provides the latest and most com
prehensive assessment of causes of death,
identifying 240 specific causes of death in
188countries; diabetes (all forms) was iden
tified as one of the major causes of reduced
life expectancy globally. In 2013, mortality
from diabetes reached 1.3million worldwide,
which is almost double the mortality in 1990;
age-standardized death rates increased signi
ficantly from 19.8per100,000 individuals
in 1990 to 21.6 per 100,000 individuals in
2013. The significant increase in mortality
was undoubtedly connected with the con
siderably increased prevalence of diabetes
worldwide, especially inAsia.
A general population-based survey of
98,658 adults conducted in China in 20102
highlights the extent of the problem in Asia.1
This survey, which used the American
Diabetes Association 2010 criteria, estimated
the prevalence of diabetes to be 11.6% and
that of prediabetes to be 50.1%, making
China the country with the highest preva
lence of diabetes in Asia and the largest
absolute disease burden for diabetes in the
world. Furthermore, poor awareness, treat
ment and disease control among patients
with diabetes was revealed, which highlights
an urgent need for a multifaceted approach
in primary diabetesprevention.
Advances in our understanding of the
genetics of T2DM in the past decade can
largely be attributed to the substantial pro
gress made by international collaborations
using genome-wide association studies
(GWAS). A 2014 study reported the largest
(so far) transethnic, GWAS meta-analysis

from four major ethnic groups.3 This study
identified seven novel T2DM susceptibility
loci, which had modest effects on T2DM sus
ceptibility but were conserved across ethnic
groups, adding to the existing 69established
T2DM susceptibility loci.3 Improvements in
the fine-mapping resolution of complex trait
loci were also demonstrated by the trans
ethnic meta-analysis. Combining GWAS
findings from diverse ancestry groups could
facilitate discovery of common variants
forT2DM.
Epidemiological studies have

revealed an increasing worldwide
prevalence of T2DM and related
mortality
Developments in the treatment of T2DM

rely on understanding the complex patho
physiology of the disease and, in the past
decade, breakthroughs in the genetic hetero
geneity of T2DM have revolutionized clini
cal practice. The first success is undoubtedly
incretin-based therapies; the multitarget
modes of action of these therapies make
them ideal for metabolic control. LEAD3
Mono,4 the first long-term randomized trial
evaluating the safety and efficacy of lira
glutide (a glucagon-like peptide1 analogue)
compared with glimepiride (a sulphonyl
urea), is a good example of this promising
strategy. Inthis 52-week, phaseIII trial
involving 746patients, monotherapy with
liraglutide signif icantly reduced HbA 1c
levels compared with glimepiride in a dosedependent manner.4 Although both agents
stimulated insulin secretion, liraglutide
provided more stable long-term control
of HbA1c levels, fewer hypoglycaemic epi
sodes, more weight loss and greater insulin
sensitivity thanliraglutide.
Another success is the use of bariatric
surgery to treat patients with T2DM, which
markedly improves glucose metabolism
NOVEMBER 2015 | 31
ENDOCRINOLOGY
32 | NOVEMBER 2015
High cholesterol levels
High blood pressure
Smoking
Type 2
diabetes mellitus
Sedentary lifestyle
High-fat diet
NPG
and remission in patients with T2DM.

However, the antidiabetic actions of bari
atric surgery are complex; decreasing the
size of the stomach and altering levels of gut
hormones are not the only consequences,
with alterations in bile acid signalling also
being important.5 Following vertical sleeve
gastrectomy, diet-induced obese mice with
targeted deletion of Nr1h4 (encoding the
bile acid receptor; also known as farnesoid
Xactivated receptor) failed to maintain sus
tained weight loss and had worse glycaemic
control than similarly operated wide-type
littermates. 5 Functional bile acid recep
tors are thus essential for the therapeutic
effects of vertical sleeve gastrectomy and
represent a possible new therapeutic target
for T2DM drug development, which offers
a less invasive mode of metabolic control
thansurgery.
The gut microbiota has become a promi
nent target in controlling human health and
many studies have been conducted on its
interaction with T2DM. Qin and colleagues
performed a two-stage metagenome-wide
association study to identify changes in gut
microbiota (using deep shotgun sequen
cing of faecal samples) in 345 patients
with T2DM.6 The researchers identified
>52,484T2DM-associated genetic markers
and established the concept of the meta
genomic linkage group to enable taxonomic
species-level analyses. This study 6 identifies
T2DM-related dysbiosis (associated with
a decline in butyrate-producing bacteria
and an increase in levels of opportunistic
species of pathogenic bacteria in the gut),
which might be a future therapeutic target
for treating T2DM.
Inhibiting renal glucose reabsorption
independent of the traditional glucose low
ering approaches related to cell function
or insulin sensitivity is another approach
to treat T2DM that has emerged in the past
decade. Bailey etal.7 evaluated the safety and
efficacy of dapagliflozin, a highly selective
inhibitor of sodium/glucose cotransporter2
(SGLT2), in a multicentre, phaseIII trial of
546 adult patients with T2DM who had inad
equate glycaemic control with metformin.7
After 24weeks of treatment, mean HbA1c
levels decreased significantly in each dapagli
flozin treatment group compared with
placebo groups. Dapagliflozin treatment
did not significantly increase symptoms of
hypoglycaemia but did result in significant
decreases in bodyweight in each dapagli
flozin group.7 An increase in genital infec
tions were reported in this study, which raises
concerns about the safety ofthistherapy.
Overweight
Nature Reviews | Endocrinology
Other advances in areas related to the

pathophysiology of T2DM have occurred
in the past 10years, including those in
brown adipose tissue (BAT), pancreatic
cell dedifferentiation, inflammation and
proopiomelanocortin (POMC) neuronal
regulation of glucose homeostasis, all of
which might be realistic targets for treating
T2DM in the future. Among these investi
gations, one paper on BAT aroused much
interest, as it was the first large retrospec
tive study to identify physiologically relevant
amounts of BAT in adult individuals using
combined PETCT.8 BAT was detected in
5.4% of the patients (n=1,972). In addition
to significant associations with age, sex and
blocker use, BMI was inversely correlated
with the amount of BAT, especially in elderly
patients, which indicates that BAT could
have a role in protecting against obesity and
metabolicabnormalities.
T2DM-related dysbiosis
might be a future therapeutic
target for treating T2DM
When it comes to evidence-based infor

mation regarding the long-term prevention
of complications related to T2DM, two hall
mark studies in the past decade stand out:
the ADVANCE study 9 and a 10-year, postinterventional follow-up of the UKPDS
study. 10 ADVANCE represented a series
oflong-term studies comparing the effect of
intensive versus standard glycaemic control
on macrovascular outcomes in patients with
T2DM who were at high risk of vascular
events.9 This study provided support for the
well-established benefits of intensive glycae
mic control on microvascluar complications
after a median of 5years of treatment in
11,140 patients with T2DM. Unfortunately,
no significant reduction in macrovascular
outcomes was found in the intensive group
compared with the control group. The
10-year follow-up of the UKPDS study
evaluated the long-term effects of intensive
glycaemic control in the early course of the

disease in patients with T2DM.10 Although
between-group differences in HbA1c levels
had disappeared by the 1year follow-up,
long-term reductions in myocardial infarc
tion and all-cause mortality were observed
inthe intensive treatment group compared
with the conventional therapy group. The
inconsistent findings in cardiovascular out
comes from the two studies raise concern
about which strategy is best for managing
glycaemic control in T2DM. Anticipating
macrovasuclar complications before they
develop in the early stages of T2DM instead
of at advanced stages, integrative care for
multiple vascular risk factors and inten
sive glycaemic control are imperative for
long-term intervention in these patients.
In the past decade, although epidemio
logical studies revealed the challenges of
controlling T2DM, progress in genetics,
pathogenesis and evidence-based inter
ventional studies raises the hope that
the harmful outcomes of T2DM can be
reduced. Even so, additional efforts are
needed to prevent and treat T2DM and its
associated complications. Future research
breakthroughs will require the integration
of methodological innovation and clinical
practice in every facet of T2DM.
Shanghai National Research Center for
Endocrine and Metabolic Diseases,
RuijinHospital, Shanghai Jiao Tong University
School of Medicine, 197 Ruijin 2nd Road,
Shanghai 200025, PR China.
gning@sibs.ac.cn
doi:10.1038/nrendo.2015.147
Published online 25 August 2015
Acknowledgements
The authors work is supported by the National
BasicResearch Program of China (2015CB553601),
the National Science and Technology Pillar
Programof China (2013BAI09B13) and by grants
from the National Natural Science Foundation of
China (grantnumbers 81390350, 81321001
and81261120564).
Competing interests
ENDOCRINOLOGY
1.
2.
3.
4.
GBD 2013 Mortality and Causes of

DeathCollaborators. Global, regional, and
national age-sex specific all-cause andcausespecific mortality for 240 causes ofdeath,
19902013: a systematic analysis forthe
Global Burden ofDisease Study 2013.
Lancet385, 117171 (2015).
Xu, Y. etal. Prevalence and control of
diabetesinChinese adults. JAMA 310,
948959 (2013).
DIAbetes Genetics Replication And
Meta-analysis (DIAGRAM) Consortium etal.
Genome-wide trans-ancestry meta-analysis
provides insight into the genetic architecture
oftype2 diabetes susceptibility. Nat. Genet.
46,234244 (2014).
Garber, A. etal. Liraglutide versus
glimepiridemonotherapy for type2
diabetes(LEAD3 Mono): a randomised,
52-week, phaseIII, double-blind, paralleltreatment trial. Lancet 373, 473481
(2009).
5.
Ryan, K.K. etal. FXR is a molecular target

fortheeffects of vertical sleeve gastrectomy.
Nature 509, 183188 (2014).
6. Qin, J. etal. A metagenome-wide association
study of gut microbiota in type2 diabetes.
Nature490, 5560 (2012).
7. Bailey, C.J., Gross, J.L., Pieters, A.,
Bastien,A.&List, J.F. Effect of dapagliflozin
inpatientswith type2 diabetes who have
inadequate glycaemic control with metformin:
arandomised, double-blind, placebo-controlled
trial. Lancet 375, 22232233 (2010).
8. Cypess, A.M. etal. Identification and importance
of brown adipose tissue in adulthumans. N.Engl.
J.Med. 360, 15091517 (2009).
9. ADVANCE Collaborative Group etal. Intensive
blood glucose control and vascular outcomes
inpatients with type2 diabetes. N.Engl. J.Med.
358, 25602572 (2008).
10. Holman, R.R. etal. 10-year follow-up of
intensive glucose control in type2 diabetes.
N.Engl. J.Med. 359, 15771589 (2008).
DECADE IN REVIEWPAEDIATRIC ENDOCRINOLOGY
New genes, new therapies

Mehul T. Dattani
The past 10years have seen substantial advances in many areas

of paediatric endocrinology. Major progress has been made in our
understanding of the aetiology of many disorders with the advent of
next-generation sequencing. Furthermore, the introduction of novel
therapies has revolutionized clinical management.
Whether the increased incidence of obesity,
in both adults and children, is entirely due
to lifestyle or if a genetic element also pre
disposes an individual to increased weight
gain has been an ongoing debate. However,
in 2007, Frayling etal. performed a genomewide association study comparing patients
with type2 diabetes mellitus (T2DM) and
control individuals,1 and identified single
nucleotide polymorphisms (SNP) in FTO
that were strongly associated with T2DM.
FTO encodes a nucleic acid demethylase
that affects food intake and might also
affect energy balance and nutrient sensing.
One SNP (rs9939609) was also studied in
other European cohorts (19,424 adults of
white European descent and 10,172 chil
dren of white European descent); impor
tantly theassociation of this genetic variant
withthe risk of being overweight or obese
was found to be almost exclusively attribut
able to changes in fat mass.1 Homozygosity
for the rs9939609 risk allele conferred a
1.67-fold increased odds ratio ofdeveloping
obesity and an increase inweight of ~3kg.
Despite birthweight being unaffected in
childrenwith rs9939609, the variant was
associated with being overweight or obese
by the age of 7years.
In 2013, Pearce etal. identified muta

tions in KSR2 in 2.1% of 2,101 patients with
severe early-onset obesity (aged <10years),
compared with only 1.0% of patients in the
control arm.2 In this study of individuals
with severe obesity, 44 of 45 patients were
heterozygous for this KSR2 variant, and
one patient was homozygous for two vari

ants. The phenotype included childhood,
but not adult, hyperphagia, significantly
reduced basal metabolic rate, increased
respiratory quotient, reduced sleeping heart
rate and insulin insensitivity with acantho
sis nigricans. Human KSR2 mutations are
also associated with impaired glucose and
fatty acid oxidation in a mouse C2C12 cell
line.2 Importantly, the impaired fatty acid
oxidation was completely rescued by met
formin. Although rare loss-of-function
KSR2 variants were highly enriched in
patients with early-onset obesity, they
inconsistently cosegregate with severe
obesity, which indicates that other genes or
environmental factors can regulate weight
in theseindividuals.
In another ground-breaking study, a
dominant-negative mutation (p.E403X)
in the thyroid hormone receptor gene
(THRA) was identified by whole-exome
sequencing in a single child with evidence of
hypothyroidism.3 This patient had growth
failure with short limbs, neurodevelop
mental delay, hypotonia, excess weight in
relation to height and chronic severe consti
pation. Although circulating concentrations
of thyroid hormone were near-normal, the
condition seems to be associated with a sub
normal ratio of thyroxine to triiodothyro
nine. Skeletal development improved with
thyroxine treatment, as did height growth,
which suggests that thyroxine treatment
might be of some benefit. Other mutations
have also been identified that clearly define
the role of thyroid hormone receptor in
bone, brain and the gastrointestinal tract;
Table 1 | Advances in clinical paediatric endocrinology

Gene or disease
Mutation phenotype
ormedication
Relevance
FTO
~3kg weight gain, association

from age 7years
First obesity gene identified by GWASaffects

weight in childhood as well as adults1
KSR2
Hyperphagic early-onset
obesity, insulin insensitivity
Mutations affect appetite, energy intake and

energy expenditure. Affects glucose and fatty
acid oxidation2
THRA
Obesity, severe chronic

constipation, growth failure,
neurocognitive delay
First human mutation in THRA. Aids our

understanding of bone and gastrointestinal
phenotypes in common forms of hypothyroidism3
MKRN3
Central precocious puberty
Commonest genetic cause of early puberty.

Provides new insights into regulation of puberty5
GPR101
Pituitary gigantism
Novel gene implicated in pituitary gigantism with

early onset of excess growth hormone secretion6
Hypophosphatasia
Bone-targeted, recombinant
human TNSALP
First treatment for hypophosphatasia. Improves

life expectancy and bone mineralization7
Type1
diabetesmellitus
Islet transplantation using

Edmonton protocol
Improves blood glucose regulation and reduces

number of severe hypoglycaemic episodes8,9
Abbreviation: GWAS, genome-wide association study.
NOVEMBER 2015 | 33
ENDOCRINOLOGY
these observations are pertinent to under
standing the pathophysiology of the con
genital or acquired hypothyroidism seen in
both children and adults.
Our understanding of puberty-related
disorders has also dramatically increased,
and at least 26 genes have been associated
with hypogonadotropic hypogonadism.4
The discovery of heterozygous mutations
in MKRN3 in five of 15 pedigrees with
central precocious puberty revealed a
novel gene that might regulate puberty in
humans.5 MKRN3 is an imprinted, pater
nally expressed gene located within the
PraderWilli syndrome region on chro
mosome 15q1113. MKRN3 is strongly
expressed in the arcuate nucleus before
puberty and expression decreases immedi
ately before puberty, which suggests that
the gene product inhibits the initiation
ofpuberty.
...next-generation sequencing
has enhanced our understanding
of...paediatric endocrine
disorders...
Until 2014, only AIP mutations had been

identified as a common cause of pitui
tary gigantism, a rare condition caused
byexcess secretion of growth hormone by
a pituitary adenoma. However, Trivellin
etal. identified heritable microduplica
tions on chromosome Xq26.3 in 13 patients
(nine female) with early childhood-onset
pituitary gigantism, Xlinked acrogigan
tism.6 One of the three genes in this region
(GPR101) encodes an orphan Gprotein
coupled receptor, which is probably respon
sible for the pituitary gigant ism pheno
type. Expression of GPR101 was greatly
increased in the pituitaries of patients with
the duplication; GPR101 mutations have
also been detected in 11 of 248 patients
with sporadic acromegaly, which sup
ports its role in the aetiology of pituitary
gigantism and/oracromegaly.6
The identification of genetic muta
tions by next-generation sequencing
has enhanced our understanding of the
aetiology and pathogenesis of paediatric
endocrine disorders, and can aid genetic
counselling for patients and their fami
lies (Table1). However, most importantly,
they will help to direct research into novel
therapies for rare geneticdisorders.
The clinical management of childhood
endocrine disorders has also advanced
in the past 10years (Table1). Whyte and
34 | NOVEMBER 2015
colleagues described a major therapeutic

advance for the management of hypophos
phatasia.7 This disorder is characterized by
the extracellular accumulation of inorganic
pyrophosphate that leads to rickets and is
associated with mutations in TNSALP,
which encodes the tissue-nonspecific
isoenzyme of alkaline phosphatase.
Children with hypophosphat asia have
chest deformities that result in respiratory
failure, persistent bone diseasewith poor
mineralization and hypercalcaemia with
nephrocalcinosis. Whyte et al. used a
recombinant human TNSALP (asfotase
alfa), which prevents hypophosphatasia
in Tnsalp null mutant mice, in 11 patients
aged <3years. In nine individuals, rickets
was cured at 6months, with an improve
ment in motorfunction,neurocognition
and pulmonary function. Skeletal abnor
malities also dramatically improved and
in one patient who had very little bone
visible on radiographs at baseline, some
remineralization was noted at 48weeks. At
week48, six of nine patients were breathing
spontaneously, compared with only one at
the beginning of the study.
Finally, one cannot summarize the past
decades research highlights in paediatric
endocrinology without mentioning the
international trial of the Edmonton pro
tocol for islet transplantation in type1
diab etes mellitus (T1DM).8 In this study,
Shapiro etal. used islet transplantation
in 36 individ uals with unstable T1DM
(including severe recurrent hypoglycae
mia). Of these patients, 16(44%) achieved
insulin independence, 10 (28%)had partial
function and a further 10 (28%) had
complete graft loss 1year after the final
transplant. All patients with residual islet
function were completely protected from
severe hypoglycaemic episodes. However,
only five individuals were insulin inde
pendent at 2years. Importantly, serious
adverse effects were either procedurerelated or immunos uppression-related.
However, a recent study suggested that
improved control (HbA1c levels <7%) and
freedom from severe hypoglycaemia can
be maintained in 60% of patients 5years
after transplant, with up to 26% remaining
insulinindependent.9
Although the best hope for a T1DM cure
is alternative sources of insulin-secreting
cells, in the meantime continued improve
ment in T1DM control with closed-loop
insulin delivery using insulin pumps and
continuous glucose monitoring is being
made, as shown in a phaseII randomized
crossover trial by Hovorka and colleagues.10

In this study of young patients with T1DM,
a closed-loop system using real time sub
cutaneous glucose monitoring reduced
the incidence of nocturnal hypoglycaemic
events compared with standard therapy
using an insulin pump and non-real time
continuous glucose monitoring.
Nature Reviews Endocrinology is to be
congratulated on its 10th anniversary; we
look forward to the next 10years, which
will herald the advent of novel genomic
technologies with their inevitable impact
on clinicalpractice.
Genetics and Epigenetics in Health and
Disease, Genetics and Genomic Medicine
Programme, UCL Institute of Child Health,
30Guilford Street, London WC1N 1EH, UK.
m.dattani@ucl.ac.uk
doi:10.1038/nrendo.2015.154
Published online 8 September 2015
Acknowledgements
M.T.D. is funded by the Great Ormond Street
Hospital Childrens Charity.
Competing interests
M.T.D. declares no competing interests.
1.
Frayling, T.M. etal. A common variant in the

FTO gene is associated with body mass index
and predisposes to childhood and adult
obesity. Science 316, 889894 (2007).
2. Pearce, L.R. etal. KSR2 mutations are
associated with obesity, insulin resistance,
andimpaired cellular fuel oxidation. Cell
155,765777 (2013).
3. Bochukova, E. etal. A mutation in the thyroid
hormone receptor gene. N.Engl. J.Med.
366,243249 (2012).
4. Boehm, U. etal. Expert consensus
document:European Consensus Statement
oncongenitalhypogonadotrophic
hypogonadismpathogenesis, diagnosis,
andtreatment. Nat. Rev. Endocrinol. 11,
547564 (2015).
5. Abreu, A.P. etal. Central precocious puberty
caused by mutations in the imprinted gene
MKRN3. N.Engl. J.Med. 368, 24672475
(2013).
6. Trivellin, G. etal. Gigantism and acromegaly
dueto Xq26 microduplications and GPR101
mutation. N.Engl. J.Med. 371, 23632374
(2014).
7. Whyte, M.P. etal. Enzyme-replacement therapy
in life-threatening hypophosphatasia. N.Engl.
J.Med. 366, 904913 (2012).
8. Shapiro, A.M. etal. International trial of the
Edmonton protocol for islet transplantation.
N.Engl. J.Med. 355, 13181330 (2006).
9. Lablance, S. etal. Five-year metabolic,
functional, and safety results of patients with
type 1 diabetes transplanted with allogenic
islets within the Swiss-French GRAGIL network.
Diabetes Care http://dx.doi.org/10.2337/
dc15-0094.
10. Hovorka, R. etal. Manual closed loop insulin
delivery in children and adolescents with
type1 diabetes: a phase2 randomized
crossover trial. Lancet 375, 743751 (2010).
ENDOCRINOLOGY
DECADE IN REVIEWREPRODUCTIVE ENDOCRINOLOGY
Understanding reproductive
endocrine disorders
Ursula B. Kaiser
The past decade has witnessed incredible advances in the field of

reproductive endocrinology. The use of new genetic and genomic tools
has had a particular impact, leading to advances in our understanding,
diagnosis and treatment of reproductive endocrine disorders, particularly
those related to the neuroendocrine control of reproduction and
ovarianbiology.
In the 10 years since Nature Reviews
Endocrinology was first published, many
important advances in the field of repro
ductive endocrinology have occurred.
During this decade, incredible advances
have been made in science and medicine
in general as a result of new tools (such as
genetics, epigenetics and big data) that are
available in discovery research and can be
applied to human biology and medicine.
These advances have made the past decade
an exciting time to be involved in transla
tional research in reproductive biology.
As the many advances in this field over
the past decade could not all be included
in this article, I have limited the scope to
advances involving, directly or indirectly,
hormones, neuroendocrine factors and
paracrinefactors.
I will start at the top of the reproductive
axis, with advances in our understanding of
the neuroendocrine control of reproduction
(Figure1). Hypothalamic gonadotropinreleasing hormone (GnRH) is at the top
of the axis and has effects in the pituitary
gland, where peripheral and central signals
are integ rated to control the output of
gonadotropins, luteinizing hormone (LH)
and follicle-stimulating hormone (FSH).
In turn, LH and FSH act on the gonads to
stimulate gametogenesis and the produc
tion of gonadal sex steroids and peptides,
including estrogens, progesterone and tes
tosterone, as well as inhibins. Although
GnRH has historically been considered to
be at the apex of the reproductive axis, the
past decade has led to important advances in
our understanding of upstream regulators of
GnRH, heralded by two key publications.1,2
Two groups independently reported the
identification of mutations in GPR54 (now
better known as KISS1R), which encodes a
Gprotein-coupled receptor, in patients with
hypogonadotropic hypogonadism, a dis
order characterized by the absence of spon
taneous pubertal maturation, infertility and
low levels of sex steroid hormones in associ

ation with inappropriately low-normal levels
of LH andFSH.
These two landmark papers reinvigorated
the field of reproductive neuroendocrinol
ogy. Subsequently, a multitude of studies
in animal models, as well as in humans,
determined that the ligand for this receptor,
kisspeptin, is a potent stimulator of GnRH
secretion, is produced in specific hypotha
lamic neuronal populations and directly acti
vates kisspeptin receptors in GnRH neurons.
These findings confirm that kisspeptin is an
upstream regulator of GnRH secretion. Four
key roles of kisspeptin have been demon
strated by the >1,400 papers that have been
published on this topic over the past decade.
Firstly, the kisspeptin system is a gatekeeper
of puberty and GnRH secretion. Secondly,
kisspeptin is important for regulation of
male and female fertility. Thirdly, kisspeptinsecreting neurons are important mediators of
Neurokinin B
Kisspeptin
GnRH
both negative and positive feedback of estro

gen, thereby providing new insights into the
regulation of ovulation and the menstrual
or estrous cycle. Fourthly, kisspeptin is an
important component of the link between
metabolism and reproduction, as well as of
other disruptors of reproductive control such
asstress.
The identification of this new regulator
of GnRH secretion using human genomics
ushered in a new era of genetic discover
ies in the control of puberty and reproduc
tion, notably leading to the identification
of loss-of-function mutations in the genes
encoding neurokinin B (NKB) and its
receptor, theneurokinin3 receptor (NK3R),
in patients with hypogonadotropic hypo
gonadism.3 This discovery took us further up
the neuronal ladder of GnRH control, with
demonstrations that both NKB and NK3R
are expressed in kisspeptin neurons and that
this system regulates kisspeptin secretion to
control GnRH release. These discoveries are
already affecting clinical care, with intense
pharmaceutical interest in the development
of kisspeptin agonists and antagonists and
a clinical study demonstrating the poten
tial utility of kisspeptin to effectively and
safely trigger oocyte maturation in women
undergoing invitro fertilization, with a pos
sible reduction in the risk of ovarian hyper
stimulation syndrome.4 In the past few years,
the application of genomic tools to study
patients with central precocious puberty
(CPP; characterized by premature activa
tion of GnRH secretion and early onset of
?
?
MKRN3
?
Letrozole
LH and FSH
Estrogen
AMH
DENND1A
Figure 1 | Advances in reproductive endocrinology related to hypothalamic

and ovarian
control of
Nature Reviews
| Endocrinology
reproduction. NeurokininB, kisspeptin and MKRN3 have been identified as regulators of GnRH
release in both male and female individuals that modulate the timing of puberty, fertility and
feedback effects of estrogen and other sex steroids. Letrozole, an aromatase inhibitor, improves
fertility induction in women with PCOS. DENND1A is involved in the pathogenesis of PCOS and
might be a new target for treatment of PCOS. AMH is now recognized as a biomarker of ovarian
reserve for fertility induction therapy. Abbreviations: AMH, anti-Mllerian hormone; FSH, folliclestimulating hormone; GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone; PCOS,
polycystic ovary syndrome.
NOVEMBER 2015 | 35
ENDOCRINOLOGY
puberty) has led to the identification of
mutations in MKRN3 as a prevalent cause
of CPP, confirming the genetic basis of this
disorder and identifying the first inhibitor of
GnRH secretion in humans.5 This discovery
has already had clinical implications in the
diagnosis of CPP, enabling genetic counsel
ling of family members and early treatment
of thisdisorder.5
These advances in our understanding
of the neuroendocrine control of repro
duction could improve the safety of fertil
ity induction in patients at risk of ovarian
hyperstimulation syndrome, such as those
with polycystic ovary syndrome (PCOS).
However, additional advances in the treat
ment of these patients have also been made
in the past decade. The historical mainstay of
treatment for women with PCOS has been an
estrogenprogestin contraceptive and clomi
phene citrate when fertility is desired. PCOS
is also associated with metabolic distur
bances such as obesity and insulin resistance.
Therapy with the insulin sensitizer, metfor
min, ameliorates the metabolic disturbances
seen in PCOS; however, despite reports of
improved ovulation rates, metformin did not
increase the rate of live births.6 Aromatase
inhibitors (such as letrozole) have been pro
posed as a method to induce ovulation in
women with PCOS (Figure1). These drugs
would also have stimulatory effects on the
hypothalamicpituitarygonadal axis and
might be better than clomiphene citrate, pro
viding more physiologic hormonal stimu
lation of the endometrium, a lower rate of
multiple pregnancies and a more favourable
adverse effect profile. Indeed, a double-blind,
multicentre, randomized trial demonstrated
superiority of letrozole over clomiphene
citrate for fertility induction in women with
PCOS.7 Genes associated with PCOS, most
notably DENND1A, have been identified in
genome-wide association studies and could
open up opportunities for the development
36 | NOVEMBER 2015
of additional targets for the treatment of this

disorder (Figure1).8
The importance of anti-Mllerian hor
mone (AMH) in female reproduction has
been revealed over the past decade. AMH
was initially known as a male hormone
because of its role in male sexual differenti
ation; it is produced in the testes and causes
regression of the Mllerian ducts during
male fetal sexual differentiation. AMH
was subsequently shown to be produced in
granulosa cells of the ovary and identified
as an inhibitor of primordial follicle recruit
ment. Now recognized as a biomarker of
ovarian reserve, serum levels of AMH are
being used in the clinic as reliable predic
tors of responsiveness to fertility induction
therapy,9 ofovarian damage and effects on
the ovarian reserve after chemotherapy or
radiotherapy and of age at the start of meno
pause.10 Thus, AMH is now recognized as an
important female hormone that is crucial
for maintaining the right tempo of folliculo
genesis, and with important diagnostic
implications that enable a personalized
approach to fertility induction therapy.
As we reflect on the many advances
in reproductive endocrinology that have
occurred in the past decade, we likewise can
look forward to the discoveries of the next
decade; for example, the further develop
ment and application of selective progester
one receptor modulators, elucidation of the
effects of endocrine disruptors on oogenesis
and spermatogenesis and the translation of
epigenetic discoveries into clinical care. It
is with great anticipation and excitement
that we can look ahead to the next wave of
discoveries and advances to improve our
understanding of the most fundamental of
biologic processesreproduction and the
preservation of our species.
Brigham and Womens Hospital, 221 Longwood
Avenue, Boston, MA 02115, USA.
ukaiser@bwh.harvard.edu
doi:10.1038/nrendo.2015.179
Acknowledgements
I would like to acknowledge my many colleagues who
contributed ideas and suggestions for inclusion in
this review, including L. Halvorson, W. Kuohung,
E.Norwitz, H. Taylor and L. Heckert, among others.
Iwould also like to acknowledge the support of the
NIH (grant numbers: R01 HD019938, R01 HD
082314, U54 HD028138 and K12 HD051959).
Competing interests
1.
de Roux, N. etal. Hypogonadotropic

hypogonadism due to loss of function of the
KiSS1-derived peptide receptor GPR54.
Proc.Natl Acad. Sci. USA 100, 1097210976
(2003).
2. Seminara, S.B. etal. The GPR54 gene as a
regulator of puberty. N. Engl. J. Med. 349,
15891592 (2003).
3. Topaloglu, A.K. etal. TAC3 and TACR3 mutations
in familial hypogonadotropic hypogonadism
reveal a key role for neurokininB in the central
control of reproduction. Nat.Genet. 41,
354358 (2009).
4. Abbara, A. etal. Efficacy of kisspeptin54 to
trigger oocyte maturation in women with high
risk of ovarian hyperstimulation syndrome
(OHSS) during invitro fertilization (IVF) therapy.
J.Clin. Endocrinol. Metab. 100, 33223331
(2015).
5. Abreu, A.P. etal. Central precocious puberty
caused by mutations in the imprinted gene
MKRN3. N. Engl. J. Med. 368, 24672475
(2013).
6. Legro, R.S. etal. Clomiphene, metformin,
orboth for infertility in the polycystic ovary
syndrome. N. Engl. J. Med. 356, 551566
(2007).
7. Legro, R.S. etal. Letrozole versus clomiphene
for infertility in the polycystic ovary syndrome.
N.Engl. J. Med. 371, 119129 (2014).
8. McAllister, J.M. etal. Overexpression of a
DENND1A isoform produces a polycystic ovary
syndrome theca phenotype. Proc. Natl Acad.
Sci.USA 111, E1519E1527 (2014).
9. Nardo, L.G. etal. Circulating basal antiMllerian hormone levels as predictor of ovarian
response in women undergoing ovarian
stimulation for invitro fertilization. Fertil. Steril.
92, 15861593 (2009).
10. Broer, S.L. etal. Anti-Mllerian hormone
predicts menopause: a long-term follow-up
study in normoovulatory women. J. Clin.
Endocrinol. Metab. 96, 25322539 (2011).
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GASTROENTEROLOGY & HEPATOLOGY

DECADE IN REVIEWHCV
Hepatitis C therapya fast andcompetitive race

Stefan Zeuzem
The past 10years have witnessed incredible developments in the treatment of hepatitis C. From an era in
whichthe standard of care was PEG-IFN and ribavirin back in 2004, various interferon-free regimens with
directacting antivirals are now available or will be soon. Here, major milestones in the hepatitisC treatment
revolution are outlined.
Zeuzem, S. Nat. Rev. Gastroenterol. Hepatol. 11, 644645 (2014); published online 16 September 2014; doi:10.1038/nrgastro.2014.164
Between 2004 and 2014 one of the fastest

revolutions in medicine occurredthe development of tolerable, safe and efficacious treatments for the global disease of hepatitisC.
According to the WHO, >170million people
are infected with HCV, which causes a slowly
fibrosing hepatitis that can subsequently
lead to cirrhosis and its sequelae. Moreover,
HCV is one of the most carcinogenic viruses;
the incidence of hepatocellular carcinoma
(HCC) in patients with HCV-associated liver
cirrhosis ranges between 2% and 5%.1
a race similar to Formula1

in terms of speed and competition
started
Major basic scientific breakthroughs were

published before 2004, including the development of an invitro replicon system, the crystallized structure of the NS3/4A protease and
numerous papers elucidating the biological
life cycle of HCV (reviewed by Lange etal.1).
Antiviral treatment of the disease at this
time, however, was still based on PEG-IFN
and ribavirin, which is poorly tolerable and
can cause substantial adverse effects. The
development and successful clinical application of the first specific and potent directacting antiviral agent (DAA), the NS3/4A
protease inhibitor ciluprevir, marked the first
major milestone of the past decade.2
Subsequent years were characterized by the
clinical development of the first-generation
NS3/4A protease inhibitors telaprevir and
boceprevir, with the publication of phaseII
and III data in 2009 and 2011, respectively.
Owing to cardiotoxicity, the ciluprevir
programme was not pursued. Additional
NS3/4A protease inhibitors and DAAs of
other drug classesmainly nucleoside
37 | NOVEMBER 2015
and non-nucleoside RNA-polymerase

inhibitors and NS5A inhibitorswere
developed and clinically investigated in
combination with PEG-IFN and ribavirin.
Intermittently, patients with chronic hepatitisC were enrolled into hundreds of clinical
trials exploring several dozen DAAs. The
majority of these compounds failed either
because ofinsufficient antiviral activity or
inappropriate safety, or both.
In the middle of the past decade, two
major questions dominated the field. In
2009, Ge etal.3 reported that a genetic variation in IL28B predicts HCV clearance in
patients infected with genotype1 treated with
PEG-IFN and ribavirin. The potential role of
this genetic polymorphism in patients receiving triple therapy, comprising PEG-IFN,
ribavirin and a DAA, was heavily debated.
Step by step, data showed that the predictive role of the IL28B polymorphism in triple
therapy is attenuated in previously untreated
patients and almost extinguished in previous
nonresponders to PEG-IFN and ribavirin.
The discussion around the second question was initially very emotionalthat is,
can HCV be eradicated by interferon-free
regimens using only DAAs? Given that the
majority of leading hepatologists was convinced that interferon is required for HCV
eradication, pharmaceutical comp anies
and regulatory agencies were hesitant to
initiate the first all-oral interferon-free
treatmentregimen.
Consequently, it was neither in the USA
nor in Europe, but in New Zealand that
the first such trial took place. A total of 40
previously untreated patients with HCV
genotype2 or 3 infection were randomly
assigned to four groups; all four groups
received sofosbuvir (at a dose of 400mg once
daily) plus ribavirin for 12weeks. Three of
these groups also received PEG-IFN2a

for 4, 8 or 12weeks. Of the 40 patients, all
30 (100%) who received sofosbuvir plus
ribavirin for 12weeks and interferon for
4, 8, or 12weeks and all 10 (100%) who
received sofosbuvir plus ribavirin without
interferon achieved a sustained virologic
response (SVR).4 When the data were first
presented by ProfessorGane from Auckland
at the Annual Meeting of the American
Association for the Study of Liver Diseases
(AASLD) in November 2011 the audience
was stunned and unable for some minutes
after the presentation to ask questions and
start a scientific discussion.
The possibility of eradicating HCV with
out interferon was confirmed in early 2012
by the combination of the NS3/4A protease
inhibitor asunaprevir and the NS5A inhibitor daclatasvir in patients infected with
genotype1; however, major differences in
SVR rates between HCV-1a and HCV-1b
were observed, indicating differences in
the genetic barrier of HCV genotypes
orsubtypes.5
Thus, from 2011 onwards a race similar to
Formula1 in terms of speed and competition
started in the development of interferon-
free, all-oral combination therapies for
hepatitisC. Generally, two distinct
strategies have been pursued:
regimens either with or
without a nucleoside
polymerase inhibitor. Apotent and
NPG

safe nucleoside polymerase inhibitor has
resembled the Holy Grail of hepatitisC
treatment. Sofosbuvir (invented by the
small biotech company Pharmasset, which
was acquired by Gilead [Foster City, CA,
USA] for >US$11 billion in 2012) fulfilled all
major criteria including potent antiviral and
pangenotypic activity, a high barrier to resistance, a good safety and tolerability profile,
and only minor issues with drugdrug
interactions. Sofosbuvir has been success
fully combined in a step-up process with
NS3/4A protease inhibitors, NS5A inhibitors, and even non-nucleoside polymerase
inhibitors plus or minus ribavirin. A fixed
drug combination consisting of sofosbuvir
and the NS5A inhibitor ledipasvir has completed phaseIIItrials in treatment-naive and
treatment-experienced patients.6,7 SVR rates
>95% were achieved and this combination
will be approved in 2014 both in the USA and
Europe. Other nucleoside polymerase inhibitors have failed owing to either insufficient
antiviral activity (for example, valopicitabine
and mericitabine) or major safety issues
(balapiravir and BMS986094). Yet others
are still in very early clinical development
(ACH3422, IDX21437 andIDX21459).
The second strategy is to combine all drug
classes except for nucleoside polymerase
inhibitors in a step-down processthat is,
NS3/4A protease inhibitors, NS5A inhibitors, non-nucleoside polymeraseinhibitors
and ribavirin. The combination of ritonavirboosted ABT450, with ombitasvir, dasa
buvir and ribavirin achieved SVR rates
>95% both in treatment-naive and treatment-experiencedpatients with chronic
hepatitis C. 8,9 Additional studies have
demonstrated that ribavirin is dispensable
from this regimen in patients infected with
subtype HCV-1b. Furthermore, high SVR
rates have been reported with a two-drug
combination of second-generation NS3/4A
protease and NS5A inhibitors. Ironically,
the company that developed the first potent
and specific DAA against hepatitisC2 and
initiated one of the first large all-oral combi
nation treatment studies10 announced in
June 2014 that they have withdrawn from
further development of this regimen owing
to suboptimal efficacy and tolerability in the
fierce competitiveenvironment.
Generally, both strategies might ultimately
lead to two-drug combinations without ribavirin with treatment durations of 812weeks.
The addition of a third compound might
enable a further decrease in treatment duration, at least in patients without liver cirrhosis. With improved antiviral potency,
broad genotypic activity, and high barriers to

resistance of nucleoside polymerase inhibitors, NS3/4A inhibitors, NS5A inhibitors and
even potentially non-nucleoside polymerase
inhibitors, the development of simple combination therapies suitable for most (standard)
patients is on the horizon. This development
would be one of the first examples in modern
medicine in which a strongly personalized
treatment approach might be overcome by a
simplified regimen.
Taken together, various very potent, safe,
and tolerable all-oral regimens are already
or will soon be approved in many countries.
But will the drugs be accessible to patients?
The price of these treatments might be prohibitive even in the Western world. Amajor
debate has started in the USA about the
US$1,000 price tag for a 400mg tablet of
sofosbuvir. The discussion is very emotional
and frequently not based on the issues that
matter: the price per cure; the cost of total
management including adverse effects; and
the reduced progression rate toward cirrhosis, liver failure and HCC (that is, the
overall impact on morbidity and mortality).
Fortunately, drug companies such as Gilead
have already made agreements with governments of developing countries to provide
anti-HCV drugs at low prices.
The next big question in the field to come
is whether this infectious disease could be
(globally) eradicated exclusively by prevention and antiviral therapy without an available vaccine. The answer to this exciting
question, however, will take many more
years to come.
Department of Medicine, J. W. Goethe

University Hospital, Theodor-Stern-Kai 7,
60590 Frankfurt am Main, Germany.
zeuzem@em.uni-frankfurt.de
Competing interests
The author acts as a consultant for Abbvie,
Boehringer Ingelheim, Bristol-Myers Squibb, Gilead,
Idenix, Janssen, Merck, Novartis, Roche, Santaris
and Vertex. He is a member of the speakers bureau
for Abbvie, Bristol-Myers Squibb, Gilead, Janssen,
Merck and Roche.
1.
Lange, C.M., Jacobson, I.M., Rice, C.M.

&Zeuzem, S. Emerging therapies for the
treatment of hepatitis C. EMBO Mol. Med. 6,
415 (2014).
2. Lamarre, D. et al. An NS3 protease inhibitor
with antiviral effects in humans infected with
hepatitis C virus. Nature 426, 186189
(2003).
3. Ge, D. et al. Genetic variation in IL28B predicts
hepatitis C treatment-induced viral clearance.
Nature 461, 399401 (2009).
4. Gane, E.J. et al. Nucleotide polymerase
inhibitor sofosbuvir plus ribavirin for
hepatitisC. N. Engl. J. Med. 368, 3444
(2013).
5. Lok, A.S. et al. Preliminary study of two antiviral
agents for hepatitis C genotype1. N. Engl. J.Med.
366, 216224 (2012).
6. Afdhal, N. et al. Ledipasvir and sofosbuvir for
untreated HCV genotype1 infection. N. Engl.
J.Med. 370, 18891898 (2014).
7. Afdhal, N. et al. Ledipasvir and sofosbuvir for
previously treated HCV genotype1 infection.
N.Engl. J. Med. 370, 14831493 (2014).
8. Feld, J.J. et al. Treatment of HCV with
ABT450/r-ombitasvir and dasabuvir with
ribavirin. N. Engl. J. Med. 370, 15941603
(2014).
9. Zeuzem, S. et al. Retreatment of HCV with
ABT450/r-ombitasvir and dasabuvir with
ribavirin. N. Engl. J. Med. 370, 16041614
(2014).
10. Zeuzem, S. Faldaprevir and deleobuvir for HCV
genotype1 infection. N. Engl. J. Med. 369,
630639 (2013).
DECADE IN REVIEWHEPATOCELLULAR CARCINOMA
HCCsubtypes, stratification
andsorafenib
Gregory J. Gores
The past 10years have represented a whirlwind of activity with regard

to information on the risk factors, aetiopathogenesis, diagnosis and
treatment of hepatocellular carcinoma. I will describe what I consider
tobe the major advances from both a tumour biology perspective and
aclinical perspective over the past 10years.
Gores, G. J. Nat. Rev. Gastroenterol. Hepatol. 11, 645647 (2014); published online 23 September 2014;
doi:10.1038/nrgastro.2014.157
We are beginning to obtain insights into

the initiating steps of hepatocellular carcinoma (HCC) and to understand the tumour
biology of this lethal malignancy. We have
learnt that polymorphisms in the EGF gene
(encoding epidermal growth factor), which

are associated with alterations in expression, increase the risk of HCC development
in patients with cirrhosis.1 The association
between EFG expression and HCC risk has
NOVEMBER 2015 | 38

Early diagnosis and
chemoprevention
3
Immune
therapies
2
Genetic
stratification
Figure 1 | Future strategies for HCC diagnosis and treatment. Mayo Clinic.
resulted in ongoing clinical studies using

EGF receptor inhibitors for chemoprevention of HCC in high-risk patients. Despite
the tremendous heterogeneity of the genetic
aberrations in advanced HCC, it is likely that
high-frequency somatic mutations in the
telomerase reverse-transcriptase promoter
drive early oncogenesis of this neoplasm.2 In
2008, an important observation was made
by examining gene expression in fixed tissue
samples from patients with HCC largely due
to HCV infection.3 A reproducible genetic
expression signature from nonmalignant
liver tissue in patients with HCC was identi
fied, which predicted late development of
recurrent HCC following hepatic resection.
The nonmalignant genetic signature supports the concept that there is a hepatic field
defect (the concept that cancer results from
tissue alterations throughout the liver as
opposed to somatic mutations within a small
group of cells) in patients who develop HCC.
Although molecular profiling has yet
tobe useful in selecting treatment strategies for HCC, it has become increasingly
useful for prognostication of patients with
HCC. An HCC fivegene score, identified
in 2013, was rigorously validated and associated with survival of patients after liver
resection for HCC.4 The fivegene score
was based on a combination expression
profile of the following genes: HN1, RAN,
RAMP3, KRT19 and TAF9. This profile
was identified empirically and the genes are
not functionally related. Disease-specific
survival was predicted by combining data
on microvascular invasion, the Barcelona
Clinic Liver Cancer classification and the
fivegene score. Thus, for the first time,
39 | NOVEMBER 2015
we have a potential biomarker to stratify

patients for various therapeutic decisions. In
2014, work suggested that KRT19 expression
can be used to identify a subset of patients
who have HCC with a poor prognosis. 5
KRT19 is a cholangiocyte marker, but can
be expressed by a subset of HCC cells either
due to cell differentiation, cell plasticity, or
a common progenitor cell of origin between
hepatocytes and cholangiocytes.5 Indeed, it
has been suggested that the subset of carcinomas that are KRT19 positive should be
considered as a separate subtype of HCC
and perhaps be managed differently.5 The
genetics involved in the pathogenesis of
HCC and those biomarkers that portend a
bad prognosis are now starting to be understood. Stratification of patients with HCC
will ultimately be very important as we
begin to design personalized therapies.
A prominent paper in terms of advances
in the clinical management of HCC of the
past decade was the 2006 study demonstrating that patients infected with HBV
and with elevated serum HBV DNA levels
were at high risk of developing HCC.6 Subse
quently, a variety of studies have confirmed
this observation, demonstrating that reduction of HBV DNA levels reduces the risk of
HCC in this patient population; this finding
provides additional impetus for treating
selected patients who have active HBV
infection with antiviral agents. Radiographic
imaging has greatly improved over the past
decade and it is possible to identify increasingly smaller nodules, which might or might
not be early liver cell cancers. Arigorous
study was published in 2008 indicating
that noninvasive techniques can be used
to identify patients with HCC between

1020mm in diameter, 7 substantiating
the existing practice guidelines for early
diagnosis of HCC by noninvasive criteria.
The response to locoregional therapy has
been assumed to relate to overall survival.
However, this concept remained more of a
supposition than it did an evidence-based
conclusion. Experience from an established
treatment centre showed that the radiographic response to locoregional therapy
does predict patient survival.8 Memon and
colleagues 2011 study gave further credence
for the use of locoregional therapy as treatment for patients with HCC and in monitoring the radiographic response to determine
further locoregional treatment over time.
Liver transplantation for patients with
HCC also remains an area of active interest. However, most issues relate to organ
shortage and how the stewardship of this
rare resource can be counterbalanced by
the potential benefit to the patient. A major
analysis was put forward in 2009 by the original author of the Milan criteria identifying a
subgroup of patients who might do well with
liver transplantation but who have cancers
beyond the Milan criteria.9 A more precise
estimation of survival was suggested by using
the up-to-seven criteria. The total tumour
diameter of up to 7cm in size (for example,
seven small tumours each 1cm or a single
lesion of 7cm) would be predicted to identify patients who would benefit substantially
from livertransplantation.
The manuscript that would probably
end up on everybodys list for advances in
the past decade was the results of the 2008
study demonstrating a survival benefit for
patients treated with sorafenib in advanced
HCC.10 The paper by Llovet etal. was a landmark in so many different ways and from so
many different perspectives. First, the data
illustrated that the sole focus on tumour
response rates might not tell the entire story.
Only a few percent of the patients had classic
response rates (diminution in tumour size).
Rather, therapy worked by delaying progression of the disease and so time-to-event has
become a major end point in oncological
studies. Second, sorafenib was the first systemic therapy to conclusively demonstrate a
survival benefit for HCC in a randomized,
placebo-controlled trial. Finally, no other
chemotherapeutic agent has yet matched
the results obtained with sorafenib. Thus,
sorafenib continues to stand alone as a therapeutic agent for HCC and we have yet to
understand precisely how and why certain
patients benefit from sorafenib therapy.
Division of Gastroenterology and Hepatology,

Mayo College of Medicine, Mayo Clinic, 200
First Street SW, Rochester, MN 55905, USA.
gores.gregory@mayo.edu
Acknowledgements
This work was supported by National Institute
ofHealth Grant DK59427 and the Mayo Clinic.
Competing interests
1.
Tanabe, K.K. et al. Epidermal growth factor

gene functional polymorphism and the risk
ofhepatocellular carcinoma in patients with
cirrhosis. JAMA 299, 5360 (2008).
2. Nault, J.C. et al. High frequency of telomerase
reverse-transcriptase promoter somatic
mutations in hepatocellular carcinoma and
preneoplastic lesions. Nat. Commun. 4, 2218
(2013).
3. Hoshida, Y. et al. Gene expression in fixed
tissues and outcome in hepatocellular
carcinoma. N. Engl. J. Med. 359, 19952004
(2008).
4. Nault, J.C. et al. A hepatocellular carcinoma
5-gene score associated with survival of
patients after liver resection. Gastroenterology
145, 176187 (2013).
5. Govaere, O. et al. Keratin 19: a key role player
in the invasion of human hepatocellular
carcinomas. Gut 63, 674685 (2014).
6. Chen, C.J. et al. Risk of hepatocellular
carcinoma across a biological gradient of
serum hepatitis B virus DNA level. JAMA 295,
6573 (2006).
7. Forner, A. et al. Diagnosis of hepatic nodules
20mm or smaller in cirrhosis: prospective
validation of the noninvasive diagnostic criteria
for hepatocellular carcinoma. Hepatology 47,
97104 (2008).
8. Memon, K. et al. Radiographic response
tolocoregional therapy in hepatocellular
carcinoma predicts patient survival times.
Gastroenterology 141, 526535 (2011).
9. Mazzaferro, V. et al. Predicting survival after
liver transplantation in patients with
hepatocellular carcinoma beyond the Milan
criteria: a retrospective, exploratory analysis.
Lancet Oncol. 10, 3543 (2009).
10. Llovet, J.M. et al. Sorafenib in advanced
hepatocellular carcinoma. N. Engl. J. Med. 359,
378390 (2008).
DECADE IN REVIEWGUT MICROBIOTA
The gut microbiota era marches on

Francisco Guarner
Research on the gut microbial communities harboured in the human gut

is progressing rapidly owing to the availability of novel and reliable tools
for analysis. Dysfunction of the gut microbiota affects human biological
fitness at multiple levels, and understanding of these effects needs to be
improved to benefit human health.
Guarner, F. Nat. Rev. Gastroenterol. Hepatol. 11, 647649 (2014); published online 9 September 2014;
doi:10.1038/nrgastro.2014.156
Knowledge on the microbial communities

that naturally inhabit the human gut has
previously had little effect on clinical medicine. The fight against infectious diseases is
perhaps the greatest challenge of modern
medicine, and it is not surprising that
handbooks typically refer to the gut microbiota as a reservoir of germs with potential
pathogenicity when barrier function fails to
keep them away. In contrast to this assertion, Louis Pasteur, a principal promoter of
the germ theory of disease, wrote in 1885
that animals would not be able to survive
when totally deprived of common micro
organisms. Experiments using germ-free
facilities were successfully developed during
the 1950s and 1960s and demonstrated that
germ-free animals are difficult to breed,
have high nutritional requirements in terms
of food variety and quantity, anddo not
develop normal body anatomyandphysio
logy. Microbial colonization of animals
was shown to be critical for normal growth
and development. As such, it is hard to
understand why these observations were
ignored by medicine for decades, but a
quick overview of leading articles in journals of most, if not all, medical disciplines
makes it easy to predict that understanding
the structure and function of the human
symbiont communities might become the
first great breakthrough of twenty-first
centurymedicine.
This expectation is supported by the
availability of novel tools for analysis
of the gut microbiota. Cultureindependent approaches are
now being used to investigate
microbial ecosystems, combining molecular sequencing
of nucleic acids (DNA, RNA)
with powerful bioinformatics for taxonomic identification and comparative
analysis of large datasets.
Before these approaches, it
was admitted that 6080% of the microbes

observed by microscopic examination of
a faecal specimen were not recoverable by
culture.1 The gold standard for molecular
identification of microbial species is the
phylogenetic analysis of genes highly conserved in all bacteria and archaea: the ribosomal RNA (rRNA) genes. In particular,
the small subunit rRNA gene (16S rRNA)
has become the standard in research into
prokaryotes for determination of phylo
genetic relationships, assessment of diversity in the environment, and detection and
quantificationof specific populations.
In 1999, Jel Dores laboratory in Paris,
France, examined a single faecal sample of
an adult individual by cloning and sequencing 16S rRNA genes, and reported that
only 24% of the sequences could be identified in gene databases, whilst the majority of sequences (76%) would belong to
species unknown at that time.1 6years later,
Eckburg etal.2 cloned and sequenced 16S
rRNA genes extracted from colonic mucosal
tissue and faecal samples from three healthy
adults. Atotal of 13,355 rRNA genesequen
ces were obtained, and again a majority
of the bacterial sequences corresponded
to uncultivated species (80%) and novel
microorganisms (62%). They found 150
300 bacterial phylotypes per participant,
and 5200 clones per phylotype. The term
NP
G
Looking back is easylooking forward

is more challenging, but more intriguing
(Figure1). We are beginning to understand
the heterogeneity of HCC, which clinicians
should be able to use to stratify patients for
clinical trials. I believe that approaches targeting specific subtypes of HCC will prove
to be important, such as different therapies
for those who are negative or positive for
KRT19 expression. I also believe that we
need further studies using immunological therapies for the treatment of HCC and
more attention should be paid to therapies
focused on the tumour microenvironment.
Hopefully in another 10years I will be asked
to write a similar article as this one, which I
would hope will highlight more impressive
therapeutic advances.
NOVEMBER 2015 | 40

phylotype is commonly used instead of
species as this method of taxonomic classification is not based on phenotypic characteristics of the microbe but on phylogenetic
analysis of 16S rRNA sequences (cut-off
values of 98% identity are arbitrarily used to
delimit species). Around 3million aligned
and annotated 16S rRNA sequences are now
available as part of the Ribosomal Database
Project. This figure is a dramatic increase
from the 79,000 and 200,000 av ailable
sequences in 1999 and2004, respectively.
The molecular approach is not limited
to 16S rRNA sequencing. The decreasing
cost and increasing speed of DNA sequencing (next-generation sequencers skip the
cloning step), coupled with advances in
computational analyses of large datasets,
have made it feasible to analyse entire
genomes with reasonable coverage. The
resulting information describes the collective genetic content of the community from
which functional and metabolic networks
can be inferred. Importantly, whole-genome
sequencing provides information about
nonbacterial members in the community,
including viruses, yeasts and protists. The
MetaHIT project addressed this approach.
Full metagenomic analysis of faecal samples
from a cohort of European adults (n=124)
identified a total of 3.3 million non
redundant microbial genes.3 The majority
(99%) of the identifiable genes were bac
terial, with a small proportion of virus-like
genes (bacteriophages and viruses), isolated
DNA (plasmids) and yeasts. Each individual
carries an average of 600,000 nonredundant
microbial genes in the gastrointestinal tract,
and around 300,000 microbial genes are
common in the sense that they are present
in about 50% of individuals. By merging
data from1,267 faecal samples from individuals from Europe, North America and
China, 4 the catalogue of the human gut
microbiome now, as of 2014, incorporates
up to 10million nonredundant genes and,
interestingly, the core number of genes
present in >50% of thestudy participants
remained below 300,000, as in the original
MetaHITcatalogue.
Bacteroides, Faecalibacterium and Bifido
bacterium are the most abundant genera, but
their relative proportion was highly variable
across individuals in the cohort.5 Network
analysis of genus abundance across different
individuals suggested that the gut microbial
ecosystem conforms to well-balanced host
microbial symbiotic states driven by groups
of co-occurring genera. Published in 2011,
a cluster analysis of gut microbial sequences
41 | NOVEMBER 2015
from American, European and Japanese

individuals identified three balanced states
or models, which were designated as enterotypes.5 Each of the three enterotypes is identifiable by the variation in the levels of one
of three genera: Bacteroides (enterotype1),
Prevotella (enterot ype 2) and Rumino
coccus (enterotype3). The discreteness of
these enterotypes is debated; some datasets
support the existence of these categories,
whereas others do not. Nonetheless, the
inverse relationship between Prevotella and
Bacteroides has been reproduced in studies
comparing the gut microbiota of residents
of agrarian societies with that of residents
of industrialized societies. Moreover, a study
exploring the associations between diet and
gut microbiota composition, based on food
frequency questionnaires collected over long
periods, indicates that diet affects the proportions of Prevotella versus Bacteroides in
US populations.6 Thus, the presence of stable
gut microbial communities can be linked to
long-term dietary patterns.6
understandinghuman
symbiont communities
might become the first great
breakthrough of twenty-first
century medicine
A number of studies have associated

increased microbial richness, at either the
taxonomic or gene level, with diets high
in fruits, vegetables and fibre.7,8 Moreover,
diets high in fruits, vegetables and fibre
were linked to reduced levels of frailty in
elderly individuals (mean age 78years, age
range 64102years),7 whereas low microbial
richness has been associated with obesity,9
insulin resistance, 8 dyslipidaemia, 8 lowgrade inflammation8 and IBD.3 A proportion of Europeans (23%) exhibit microbial
gene counts below the previously established median of 600,000.8 Individuals with
low microbial gene counts (below 480,000)
are characterized by more marked overall
adiposity, insulin resistance, leptin resistance, dyslipidaemia and a more pronounced
inflammatory phenotype when compared
with individuals with high gene counts.8
These metabolic parameters are slightly
altered even in otherwise healthy individuals with low microbial gene counts. Obesity
is associated with a gut microbiota that is
highly efficient for harvesting energy from
the diet.9 In addition, the subset of obese
individuals with low gene counts gain more
weight over time and have a propensity

towards severe forms of obesity than individ
uals with high gene counts.8 Low gene richness therefore seems to be a risk factor for
the development of metabolic-syndromerelated complications (type2 diabetes,
hepatic and cardiovascular pathologies) as
well as inflammatory bowel conditions. The
effectiveness of faecal microbiota transplantation in specific disease states (colitis associated with Clostridium difficile infection,
type2 diabetes) is proof of principle that
defects of the gut microbial ecosystem might
be the origin of certain disorders.
Diet can alter the functional metabolism
of the gut microbiome. Food ingredients are
potential substrates for microbial metabolism, which then could produce substances
or molecules that affect host physiology.
For instance, indigestible carbohydrates
in the diet are fermented by the gut micro
biota to produce short-chain fatty acids with
a number of beneficial functions for the
host. Characterizing metabolic interactions
between host and microbes is an important
challenge for understanding human physio
logy and predicting risk of noncommunicable
diseases. An example of this approach is the
discovery that gut microbes can contribute
to the development of atheros clerosis by
producing metabolites of dietary carnitine or
lecithin.10 Catabolism of these compounds by
the gut microbiota results in the formation
of trimethylamine, which is metabolized by
the liver into t rimethylamine-N-oxide. This
small molecule is strongly associated with
an increased risk of adverse cardiovascular
events in humans.10
The gut microbiota is unequivocally integrated into the regulation of multiple host
pathways, giving rise to interactive host
microbiota metabolic and immunological
inflammatory axes. Knowledge of the
structure and functions of the microbial
communities is needed for understanding
human physiology and optimizing strat
egies to combat disease so that the importance of the gut microbiota is not ignored in
the next 10years.
Digestive System Research Unit, University
Hospital Vall dHebron, CIBEREHD, Passeig Vall
dHebron 119129, BarcelonaE08035, Spain.
fguarner@telefonica.net
Competing interests
1.
Suau, A. etal. Direct analysis of genes

encoding 16S rRNA from complex communities
reveals many novel molecular species within
the human gut. Appl. Environ. Microbiol. 65,
47994807 (1999).

2.
3.
4.
5.
6.
Eckburg, P.B. etal. Diversity of the human

intestinal microbial flora. Science 308,
16351638 (2005).
Qin, J. etal. A human gut microbial gene
catalogue established by metagenomic
sequencing. Nature 464, 5965 (2010).
Li, J. etal. An integrated catalog of reference
genes in the human gut microbiome.
Nat.Biotechnol. 32, 834841 (2014).
Arumugam, M. etal. Enterotypes of the human
gut microbiome. Nature 473, 174180 (2011).
Wu, G.D. etal. Linking long-term dietary
patterns with gut microbial enterotypes.
Science 334, 105108 (2011).
7.
Claesson, M.J. etal. Gut microbiota

composition correlates with diet and
healthinthe elderly. Nature 488, 178184
(2012).
8. Le Chatelier, E. etal. Richness of human gut
microbiome correlates with metabolic markers.
Nature 500, 541546 (2013).
9. Turnbaugh, P.J. etal. A core gut microbiome
inobese and lean twins. Nature 457, 480484
(2009).
10. Tang, W.H. etal. Intestinal microbial
metabolism of phosphatidylcholine and
cardiovascular risk. N.Engl. J.Med. 368,
15751584 (2013).
DECADE IN REVIEWFGIDS
Functional gastrointestinal
disordersa paradigm shift
Nicholas J. Talley
In the past decade we have witnessed an explosion in the quantity and

quality of research in the functional gastrointestinal disorders. I discuss
10 top original research papers that, unless recent, have been highly
cited, published in a high-impact journal and have probably shifted
thinking in the field.
Talley, N.J. Nat. Rev. Gastroenterol. Hepatol. 11, 649650 (2014); published online 23 September 2014;
doi:10.1038/nrgastro.2014.163
Post-dysenteric IBS was described by

Chaudhary and Truelove in 1962the initial
clue that IBS might be an organic gut disease.
Although important work has further characterized post-infectious IBS, Mearin and
colleagues prospective study1 from 2005
identified convincingly an increased incidence of dyspepsia, IBS andan overlap syndrome (dyspepsiaIBS) 6and 12months
after a salmonella outbreak; patients with
more severe gastroenteritis were at increased
risk of developing one of these syndromes.1
The overlap of functional gastrointestinal
disorders (FGIDs) has remained mysterious but these key data suggest that infection
might explain it. Salmonella spp. induces
small-intestinal inflammation; a testable
hypothesis is that perhaps the site of inflammation accounts for the symptom phenotype,
for example proximal inflammation might
be associated with dyspepsia, distalinflammation with IBS and more-extensive
inflammation with the overlapsyndrome.
How might inflammation induce the
symptoms of FGIDs? And what about
themysterious extraintestinal symptoms
these patients often report? Liebregts etal.2
tested whether the release of proinflammatory cytokines in IBS is linked to symptoms
and psychiatric comorbidity. They found
that a number of cytokines were increased
in IBS, including TNF and IL6, and that

lipopolysaccharide-induced TNF levels correlated with levels of anxiety.2 These data
further challenge the concept that IBS is a
functional disease (a term that means no
organic, metabolic or biochemical abnormalities), and suggest that gut-to-brain pathways
might be one driver ofpsychological symptoms in IBS. Could psychological distress in
IBS be relieved or even cured by a gut-based
intervention that downregulates the cytokine
response? Meanwhile, the observation that
increased numbers of mast cells are present
in IBS dates back to the 1990s, but it has
been unclear what role mast cells have in the
generation of pain and other symptoms, if
any. In elegant experiments utilizing colonic
biopsy samples from patients with IBS,
Barbara etal.,3 in 2004, observed a substantial increase in the number of degranulating
mast cells releasing histamine and, strikingly, mast cells observed in close proximity
to nerves correlated with abdominal painin
IBS. The role of blocking mast cells, and
inparticular histamine, in IBS is now an area
of activeinterest.
Intense interest in the role of the gut
microbiota in gastrointestinal and nongastrointestinal disease continues, and
the hypothesis that the colonic and small
intestinal flora is altered in IBS is being
actively explored. The study by RajilicStojanovic etal.4 is a technically excellent

example of the current state of the art, demonstrating that although stool microbiota
composition is highly individual, it can be
used to discriminate IBS from health, with
reduced numbers of Bifidobacteriaceae,
Actinobacteria and Bacteroidetes but
increased levels of Firmicutes in patients
with IBS compared with healthy individuals. Problems remain, including limited
sampling techniques, lack of an ability to
culture most bacteria, and the fact that major
differences have been observed in different
studies. Regardless, bacterial manipulation
might be one of the keys to curing FGIDs.
If bacteria are important in symptom generation in IBSfor example by increased
gas production that distends the lumen and
induces bloatingwould starving them
work? In their landmark paper from 2008,
Shepherd and colleagues5 tested dietary fructose and fructan restriction then rechallenge
in patients with IBS; 70% of patients receiving fructose, 77% receiving fructans and
79% receiving a mixture reported symptom
breakthrough versus 14% of patients ingesting glucose. Other data now support the
benefit of a low-FODMAP diet in IBS, which
is being increasingly adopted although large
randomized blinded trials are needed.
No drug has been shown to change the
natural history of IBS (symptoms usually
rapidly reoccur) until the past decade. If
the gut microbiota is important in IBS,
manipulating it should change the natural
history of the condition at least temporarily.
In 2011, Pimentel etal.6 tested rifaximin, a
minimally absorbed antibiotic, for 2weeks
in patients with diarrhoea-predominant and
mixed IBS with 10weeks follow-up. Overall,
global IBS symptoms and, most notably,
bloating improved although the therapeutic gain was modest (~10% of patients).
The striking observation was that symptom
improvement persisted during follow-up.6 It
is unknown why only a subgroup responded
and whether a more targeted attack on the
most relevant bacterial group would yield
better results. The response is known not to
be sustained long term and this factor is also
unexplained; is this because of microbial or
possibly mucosal factors? Probiotics have
also shown modest symptom benefits in IBS
(including a Bifidobacteria infantis strain)
but optimal probiotic therapy remains to
be defined and benefits to date are small
although this area has enormous potential.7
At last a molecular defect in IBS has been
uncovered by Coates etal.8 and these results,
NOVEMBER 2015 | 42
Gutbrain axis
(IBS)
Duodenal
eosinophilia
(functional
dyspepsia)
Intestinal
inflammation
(IBS)
Changes in
gut microbiota
(IBS)
Figure 1 | Functional gut disorders. Many

patients have objective gut pathology and
agut-to-brain syndrome that explains their
gastrointestinal as well as extraintestinal
symptoms (e.g. anxiety, fatigue and
sleepdisturbance).
although initially controversial, have been

confirmed. Serotonin (5-HT) is released from
enterochromaffin cells and mediates peristalsis and reflex responses. In this important
paper, defects in serotonin signalling (including serotonin reuptake) in IBS and ulcerative
colitis were observed (although numbers of
enterochromaffin cells were only reduced in
colitis). Gut-specific molecular alterations
inserotonin might explain constipation
(insufficient 5HT), diarrhoea (excess 5HT)
and mixed IBS (5HT switching between
excess and insufficient levels).
Finally, two key papers indicate a paradigm shift in adult functional dyspepsia.
The Kalixanda study 9 was a landmark
endoscopic population-based study from
northern Sweden that investigated whether
duodenal eosinophilia characterises pure
functional dyspepsia (no overlap of IBS was
permitted). Patients with increased numbers
of duodenal eosinophils had a nearly 12-fold
increased risk of functional dyspepsia, and
the pathology was linked to early satiety, a
finding subsequently confirmed by multiple
studies. A Belgian study 10 from 2014 not only
confirmed duodenal eosinophilia in functional dyspepsia but also showed increased
numbers of mast cells (arguably attributable to the majority of the study population
having both dyspepsia and IBS) and more
43 | NOVEMBER 2015
importantly impaired duodenal permeability.

Increased antigen presentation (from food
or bacteria) might be key in driving a subtle
upper intestinal inflammatory response in
predisposed patients, leading to duodeno
gastric reflex responses and early satiety, a
condition we label as functional dyspepsia.
Our concepts have changed over the
past decade and the short-comings of a
symptom-b ased only classification of
FGIDs are becoming increasingly apparent
(Figure1). IBS is disappearing as we identify more and more subgroups (and recognize organic diseases such as coeliac disease,
microscopic colitis or bile-salt-induced diarrhoea as clinically indistinguishable from
IBS). Accumulating data suggest people
might have either a gut-to-brain or brain-togut syndrome, both with identical gut symptoms. One could make the case it is now time
to drop the arguably pejorative term functional; on the basis of the latest information,
many patients have gut pathology. Identifying
serum and stool biomarkers to objectively
diagnose IBS and functional dyspepsia now
seems plausible. Prediction is dangerous but
I will stick my neck out: a purely symptombased classification of the FGIDs will be
extinct in the next two decades as knowledge
in this areaexplodes.
Faculty of Health and Medicine, University of
Newcastle, HMRI Building Lot 1 Kookaburra
Circuit, New Lambton Heights, NSW 2035,
Australia.
nicholas.talley@newcastle.edu.au
Competing interests
1.
Mearin, F. et al. Dyspepsia and irritable bowel

syndrome after a Salmonella gastroenteritis
outbreak: one-year follow-up cohort study.
2. Liebregts, T. etal. Immune activation in patients
with irritable bowel syndrome. Gastroenterology
132, 913920 (2007).
3. Barbara, G. etal. Activated mast cells in
proximity to colonic nerves correlate with
abdominal pain in irritable bowel syndrome.
4. Rajilic-Stojanovic, M. et al. Global and deep
molecular analysis of microbiota signatures in
fecal samples from patients with irritable bowel
syndrome. Gastroenterology 141, 17921801
(2011).
5. Shepherd, S.J., Parker, F.C., Muir, J.G. &
Gibson,P.R. Dietary triggers of abdominal
symptoms in patients with irritable bowel
syndrome: randomized placebo-controlled
evidence. Clin. Gastroenterol. Hepatol. 6,
765771 (2008).
6. Pimentel, M. etal. Rifaximin therapy for
patients with irritable bowel syndrome without
constipation. N. Engl. J. Med. 364, 2232
(2011).
7. OMahony, L. etal. Lactobacillus and
bifidobacterium in irritable bowel syndrome:
symptom responses and relationship to
cytokine profiles. Gastroenterology 128,
541551 (2005).
8. Coates, M.D. etal. Molecular defects in
mucosal serotonin content and decreased
serotonin reuptake transporter in ulcerative
colitis and irritable bowel syndrome.
9. Talley, N.J. etal. Non-ulcer dyspepsia and
duodenal eosinophilia: an adult endoscopic
population-based case-control study. Clin.
Gastroenterol. Hepatol. 5, 11751183 (2007).
10. Vanheel, H. etal. Impaired duodenal mucosal
integrity and low-grade inflammation in
functional dyspepsia. Gut 63, 262271 (2014).
DECADE IN REVIEWPANCREATIC DISEASES
Advances in understanding
andcare of pancreatic diseases
Randall E. Brand
Clinical and basic science discoveries over the past decade have led to
considerable improvements in our understanding and care of pancreatic
diseases. Findings reported in 10 key papers highlight results that have
already substantially altered the care of patients with acute pancreatitis,
chronic pancreatitis and pancreatic cancer (or soon will).
Brand, R.E. Nat. Rev. Gastroenterol. Hepatol. 11, 650652 (2014); published online 16 September 2014;
doi:10.1038/nrgastro.2014.159
Multicentre collaborations have enabled

investigators to perform well-powered, highquality studies that have led to major changes
in our approach to the care of patients with
acute pancreatitis, chronic pancreatitis or
pancreatic adenocarcinoma. Researchers
have further improved the design of studies

through the use of standardized definitions
of diseases and their complications. Over the
past decade, findings have enabled us to move
toward individualizing preventive and treatment strategies on the basis of our growing

understanding of the molecular and genetic
mechanisms of pancreatic disorders, as
shown in Figure1 (Timeline). In my opinion,
the following 10 studies published over the
past decade best highlight these advances.
Several well-designed prospective, randomized trials by the Dutch Pancreatitis
Study Group have made notable contributions to the management of patients with
acute pancreatitis. In 2010, one of their trials
changed the approach for treating patients
with necrotizing pancreatitis with infected
necrotic tissue, a major cause of death for
patients with acute pancreatitis.1 Their res
ults demonstrated that a minimally invasive
step-up approach (percutaneous drainage
and, if required, a minimally invasive retroperitoneal necrosectomy) had a lower rate
of major short-term and long-term complications than the traditional approach of
opennecrosectomy.
Patients with severe necrotizing pancreatitis have also benefited from research on
the use of prophylactic antibiotics to prevent
polymicrobial pancreatic and peripancreatic
infections. In 2007, results from an inter
national multicentre randomized, prospective, double-blind trial comparing the use of
meropenem to placebo in patients with noninfected necrotizing pancreatitis showed no
statistically significant difference between the
two treatment groups.2 This finding began
the reversal of routine use of broad-spectrum
antibiotic prophylaxis.
In the USA, a large multicentre trial confirmed the effectiveness of a strategy to
prevent endoscopic retrograde cholangiopancreatography (ERCP)-induced pancreatitis.3 Prior to the publication of this study,
no pharmacological prophylaxis was broadly
accepted for the prevention of this potentially life-threatening complication of ERCP.
Results from this randomized, placebo-
controlled, double-blind study established
the benefits of 100mg rectal indomethacin in
reducing the incidence and severity of pancreatitis after ERCP and led to its routine use
in selected patients at high risk of developing
the condition.
The continued identification of both
environmental and genetic risk factors for
pancreatic diseases will enable us to focus preventive strategies in a more directed manner
than previously. For example, one randomized controlled trial demonstrated that
an intervention to help patients reducetheir
alcohol consumption given during their
first hospitalization for alcohol-related
acute pancreatitis and then repeated every
6months at an outpatient gastrointestinal
Adjuvant chemotherapy
shown to improve
survival in patients with
pancreatic cancer
High alcohol intake shown

not to be the only cause
of chronic pancreatitis
FOLFIRINOX improves
survival in patients
with pancreatic cancer
GWAS explains sex
differences in risk of
alcohol-related chronic
pancreatitis
Repeated interventions
reduce recurrence of
acute pancreatitis
2004
2006
Core signalling
pathways in pancreatic
cancer revealed
Routine use of broad-spectrum
antibiotic prophylaxis in necrotizing
pancreatitis begins to decline
2008
2010
Minimally invasive
step-up approach
for treating necrotizing
pancreatitis
2012
2014
First accepted
treatment to prevent
pancreatitis
induced by ERCP
Potential for early detection

of pancreatic cancer
Figure 1 | Timeline of the advances in our understanding of pancreatic diseases and

improvements in patient care over the past decade. Abbreviations: ERCP, endoscopic retrograde
cholangiopancreatography; FOLFIRINOX, folinic acid, fluorouracil, irinotecan and oxaliplatin;
GWAS, genome-wide association study.
clinic reduced the incidence of recurrent

acute pancreatitis by two-thirds compared
with receiving the intervention only during
the initialhospitalization.4
Indeed, several papers over the past decade
have elucidated the relationship between
alcohol and chronic pancreatitis. Examining
data from 540 patients with chronic pancreatitis who were diagnosed according to strict
entry criteria and 695 related and unrelated
control individuals, the North American
Pancreatic Study Group (20 secondary or
tertiary pancreatic care centres) clarified the
relationship between chronic pancreatitis
and both alcohol and smoking consumption.5 The risk of chronic pancreatitis as a
result of alcohol consumption was dosedependent and occurred only in patients
who drank at least five alcoholic drinks
per day. Cigarette smoking was found to
be an independent risk factor. These results
changed the dogma that chronic pancreatitis was mainly attributable to alcohol consumption, as only a minority of patients with
chronic pancreatitis in the study cohort were
heavy drinkers (38% men and 11% women)
and one-quarter had never consumed alcoholic drinks. Results from an international
genome-wide association study that were
reported in 2012 might explain the longrecognized sex differences observed in the
incidence of alcohol-related chronic pancreatitis.6 This study identified an Xlinked
risk allele, CLDN2 (encodes claudin2),
which conferred the greatest risk of developing alcohol-related chronic pancreatitis. As
men only have one copy of the gene (hemi
zygous frequency of 0.26) whereas women
have two copies (homozygous frequency of
0.07), this finding might partially account
for observed sex differences in the incidence

ofalcoholrelated chronic pancreatitis.
The prognosis for patients with pancreatic
adenocarcinoma has remained poor over
the past decade; however, incremental progress has been made in the treatment of this
disease. In 2004, results from a multicentre,
randomized trial by the European Study
Group for Pancreatic Cancer that compared
the use of adjuvant chemoradiation therapy
with or without chemotherapy, chemotherapy alone, or no treatment led to a complete
reassessment of the clinical utility of radiation
therapy.7 By 2011, results from amulticentre
trial from France had shown a considerable survival advantage for a combination
chemotherapy regimen consisting of folinic
acid, fluorouracil, irinotecan and oxaliplatin
(FOLFIRINOX) compared with single-agent
gemcitabine.8 In addition to broadening the
treatment options for patients with metastatic
pancreatic adenocarcinoma who can tolerate
the increased toxicity with FOLFIRINOX,
this study opens the door to new therapeutic
approaches beyond gemcitabine.
With regard to more-personalized therapies, our growing knowledge of the genetic
alterations involved in carcinogenesis has
led to the development of therapies directed
against specific biological targets. One comprehensive genetic analysis of 24 advanced
pancreatic adenocarcinomas showed the
diversity and core pathway changes in these
pancreatic tumours, which had an average of
63 genetic alterations in the proteins involved
in 12 core cellular signalling pathways and
processes.9 These results indicate the importance of moving beyond single-agent therapies to target multiple signalling pathways
and processes. A subsequent study, which
NOVEMBER 2015 | 44

sequenced the genomes of pancreatic adenocarcinoma metastases from seven individuals
obtained during rapid autopsies, compared
the somatic mutations identified in the
metastases with both the primary tumour
and anatomically distinct metastases to
determine the clonal relationships between
the primary and metastatic cancers.10 These
data were incorporated into a mathematical model to estimate the time from initial
tumour initiation to development of metasta
ses, which pointed to an ~15-year window
during which neoplastic cells remain contained within the pancreas. This finding
suggests that the prognosis of patients with
pancreatic adenocarcinoma could be vastly
improved with early detection.
These studies highlight the substantial progress achieved over the past decade in caring
for patients with pancreatic diseases, with
many new findings that can be translated into
clinical practice over the next decade.
Division of Gastroenterology, Hepatology
andNutrition, Department of Internal
Medicine, University of Pittsburgh Medical
Center, 5200 Centre Avenue, Suite 409,
Pittsburgh, PA 15232, USA.
reb53@pitt.edu
Competing interests
1.
van Santvoort, H.C. etal. A step-up approach

or open necrosectomy for necrotizing
pancreatitis. N.Engl. J.Med. 362, 14911502
(2010).
2. Dellinger, E.P. etal. Early antibiotic treatment
for severe acute necrotizing pancreatitis:
arandomized, double-blind, placebo-controlled
study. Ann. Surg. 245, 674683 (2007).
3. Elmunzer, B.J. etal. A randomized trial of rectal
indomethacin to prevent post-ERCP
pancreatitis. N.Engl. J.Med. 366, 14141422
(2012).
4. Nordback, I. etal. The recurrence of acute
alcohol-associated pancreatitis can be
reduced: a randomized controlled trial.
5. Yadav, D. etal. Alcohol consumption, cigarette
smoking, and the risk of recurrent acute and
chronic pancreatitis. Arch. Intern. Med. 169,
10351045 (2009).
6. Whitcomb, D.C. etal. Common genetic variants
in the CLDN2 and PRSS1-PRSS2 loci alter risk
for alcohol-related and sporadic pancreatitis.
Nat. Genet. 44, 13491354 (2012).
7. Neoptolemos, J.P. etal. A randomized trial of
chemoradiotherapy and chemotherapy after
resection of pancreatic cancer. N.Engl. J.Med.
350, 12001210 (2004).
8. Conroy, T. etal. FOLFIRINOX versus gemcitabine
for metastatic pancreatic cancer. N.Engl. J.Med.
364, 18171825 (2011).
9. Jones, S. etal. Core signaling pathways in
human pancreatic cancers revealed by global
genomic analyses. Science 321, 18011806
(2008).
10. Yachida, S. etal. Distant metastasis occurs
late during the genetic evolution of pancreatic
cancer. Nature 467, 11141117 (2010).
45 | NOVEMBER 2015
DECADE IN REVIEWIBD
IBDgenes, bacteria and new

therapeutic strategies
Jean-Frederic Colombel
IBD is known to be associated with an abnormal response to an

unbalanced gut microbiota in genetically predisposed individuals.
Thetherapeutic goal now is to control progression of the disease.
Giventheheterogeneity of IBD, the two universes of basic and
clinicalscience must work in parallel to realize the hope of
personalizedtherapy.
Colombel, J.-F. Nat. Rev. Gastroenterol. Hepatol. 11, 652654 (2014); published online 14 October 2014;
doi:10.1038/nrgastro.2014.170
In the past decade we have witnessed

major advances both in basic and clinical
aspects of IBD. Landmark discoveries in the
fieldsof genetics, immunology and microbiology have provided us with an improved
understanding of the biological and
genetic variations associated with disease.
An increased knowledge of the natural
history of the disease, disease predictive
factors and disease assessment has led us
to demand better therapeutic outcomes, to
develop innovative therapeutic strategies
and to incorporate new end points in daily
clinicalpractice.
The treatment paradigm has

fundamentally shifted in IBD in
the past 10years
Genetic studies culminated in a metaanalysis of Crohns disease and ulcerative

colitis genome-wide association studies
by Jostins etal.1 in 2012. 163 loci associated with IBD were identified.1 Most loci
contribute to both diseases and many of
them are also implicated in other immunemediated disorders, such as ankylosing
spondylitis and psoriasis. It is worth mentioning that the loci identified to date represent ~20% of the potential heritable risk
of developing IBD. The missing heritability
component has led to the conclusion that
some forms of IBDespecially the familial
formscould be due to the profound effect
of rare variants. For example, a familial
form of early-onset Crohns disease has been
identified as a monogenic disorder resulting from homozygous mutations in IL10R
(encoding the interleukin 10 receptor).2
Another major contribution from the
field of genetics was the discovery of
the prominent Crohns disease risk gene

ATG16L1, which encodes an adaptor
protein central to autophagosome formation. This finding introduced the idea
of defective autophagy as a fundamental
disease mechanism, which had not previously been considered by mucosal immuno
logists or gastroenterologists. Patients
homozygous for the ATG16L1 allele have
marked structural alterations in their secretory apparatus along with transcriptional
abnormalities in Paneth cells.3 Autophagy
serves as a compensatory mechanism for
endoplasmic reticulum stress; when endoplasmic reticulum stress in the intestinal
epithelium intersects with an autophagy
defect, a discontinuous, transmural inflam
mation with fissuring lesions spontan
eously develops that mimics Crohns
disease.4 Autophagy also interacts with the
unfolded protein response, a process that
arises from endoplasmic reticulum stress
and manifests in Paneth cells. Deletion of
XPB1, which encodes a transcription factor
important in the unfolded protein response,
resultsin depletion of Paneth cell numbers
along with spontaneous small intestinal
inflammation that mimics human IBD with
crypt abscesses, neutrophil infiltration and
ulcerations. That these three closely connected pathwaysmicrobial signalling
(via NOD2), autophagy and endoplasmic reticulum stressaffect Paneth cell
function clearly implicates altered bacterial handling as having a primary role in
Crohnsdisease.4
The past decade was also marked by the
emergence of the complex and critical role
of the intestinal microbiome in health and
disease. In 2004, Rakoff-Nahoum etal. 5
were amongst the first to demonstrate the
profound role that signals from the gut
microbiota have in determining intestinal

homeostasis, including protection from
injury. Conversely, bacteria-driven innate
colitis, associated with increased production of IL17 and IFN in the colon, is
mediated by IL23-dependent innate lymphoid cellsin mice. 6 The key role of the
IL23 pathway inchronic inflammation
has been established by the association of
IL23R alleles with IBD, psoriasis and ankylosing spondylitis.1 The pathogenic and protective effects of the gut microbiota imply
that an unbalanced microbial community
(dysbiosis) is associated with a dysregulated
immune response. Compared with healthy
individuals as controls, patients with IBD
have fewer bacteria with anti-inflammatory properties and/or more bacteria with
proinflammatory properties. An increased
relative abundance in Enterobacteriaceae,
mostly Escherichia coli, has been observed
in patients with Crohns disease and on
the basis of its pathog enic traits a new
group of E.coli, adherent-invasive E.coli,
has been associated with ileal Crohns
disease.7 By contrast, members of the phyla
Bacteroidetes and Firmicutes are reduced
in patients with Crohns disease; among
Firmicutes, numbers of Faecalibacterium
prausnitzii (which have anti-inflammatory
properties) are reducedin patients with
Crohns disease. Reduced counts of F.praus
nitzii is associated with increased risk of
post-resection recurrence of ileal Crohns
disease. The first study in new-onset
Crohns disease confirmed the presence of
dysbiosis in mucosal samples, with marked
interindividual variation.7 Strikingly, the
strongest factor associated with dysbiosis
was previous exposure to antibiotics, which
is consistent with epidemiological observations that antecedent antibiotic use is a risk
factor for developing Crohns disease.
The past decade was also

marked by the emergence of the
complex and critical role of the
intestinal microbiome
The treatment paradigm has fundamentally shifted in IBD in the past 10years.
Major drivers for this evolution exist: the
recognition that IBD is not an intermittent
but rather a progressive disease, leading to
bowel damage and disability in the long
term; availability of disease-modifying IBD
drugs that are able to halt disease progression; evidence that therapeutic decisions
should be based on objective markers such
Early disease
Late disease
Combination therapy
+ tight control
Combination therapy
+ tight control
Deep remission
Stabilization
De-escalation
Treat-to-target
Figure 1 | A treat-to-target strategy could be applied to all patients with IBD. In early disease, for
patients as defined by disease duration of <18months, no complications and no previous use
of disease-modifying IBD drugs, the target is deep remission with minimal damage whereas in
late disease the target is to stop disease progression. In both cases, combination therapy is the
most effective and requires tight control of therapeutic efficacy and safety. In early disease and
in certain circumstances, that is when the target has been reached, de-escalation therapy could
be considered.
as endoscopy or biomarkers; the demonstration that combination therapy using

an anti-TNF and an immunosuppressor
is the most effective therapeutic strategy;8
and, finally, recognition that treatment
early in the disease course is associated
with improved efficacy of drugs and better
outcomes, such as reduced risk of sur
gery. Figure1 illustrates a treat-to-target
approach that uses the most effective combination therapy in patients with objective
inflammation and tight control of drug
levels and antibodies. The treat-to-target
approach aims either at full control of the
disease as defined by absence of symptoms, normalization of levels of biomarkers (C-reactive protein) and endoscopic
healing (deep remission) in early disease
or stabilization of bowel damage in patients
with long-standing disease. Nonetheless,
many clinical challenges and gaps in our
knowledge remain, particularly as the
current clinical approach is mainly based on
retrospective data. Prospective studies are
needed to validate the concept of treating
beyond symptoms; the ideal target is still
uncertain and could still evolve to become
more stringent (involving histological or
molecular healing) or perhaps more relaxed
than current approaches. Despite close
follow-up and monitoring, many patients
become refractory to the available drugs. In
addition, many promising drugs have failed
to achieve their goals. The notable exception are drugs targeting integrins, with the
first agent, vedolizumab, being approved
in 2013, for which long-term efficacy is
particularly remarkable in patients with

ulcerativecolitis.9
So what does the future hold? The
hope is that more clinical applications
will translate from bench to bedside. For
instance, a huge need exists to be able to
predict disease outcome. One successful example is the study from 2011 by Lee
etal., 10 which reported the first robust
and replicated, biomarker-based method
for predicting severity of Crohns disease
and ulcerative colitis. By assessing the
transcriptome of CD8+ peripheral blood
Tcells, the authors could predict the need
for therapeutic escalation with high specificity and sensitivity. This approach could
be used to identify patients at diagnosis
who might benefit most from early (and
costly) introduction of potent immunotherapies. Finally, although most current
treatments in IBD work by dampening
the immune system, with unavoidable
adverse effects, there is great hope that
re-equilibrating the gut microbiota for
benef icial effects will be possible. For
example, specific components of the diet
have been shown to rapidly and reproducibly alter the human gut microbiota.
Whether this modulation can ultimately
lead to long-term remission and even prevention of disease will be a central question
moving forward.
Department of Medicine, Icahn School
ofMedicine at Mount Sinai, New York,
1428Madison Avenue, NY 10029, USA
jean-frederic.colombel@mssm.edu
NOVEMBER 2015 | 46

Competing interests
J.-F.C. has served as consultant, advisory board
member or speaker for Abbvie, ABScience, Amgen,
BristolMyers Squibb, Celltrion, Danone, Ferring,
Genentech, Giuliani SPA, Given Imaging, Janssen,
Immune Pharmaceuticals, Merck, Millenium
Pharmaceuticals, Nutrition Science Partners, Pfizer,
Prometheus Laboratories, Protagonist Therapeutics,
Receptos, Sanofi, Schering Plough, Second Genome,
Takeda, Teva Pharmaceuticals, UCB Pharma, Vertex
and Dr August Wolff.
2.
3.
4.
5.
1.
Jostins, L. etal. Host-microbe interactions

haveshaped the genetic architecture of
inflammatory bowel disease. Nature 491,
119124 (2012).
47 | NOVEMBER 2015
Glocker, E.O. etal. Inflammatory bowel disease

and mutations affecting the interleukin-10
receptor. N. Engl. J. Med. 361, 20332045
(2009).
Cadwell, K. etal. A key role for autophagy
andthe autophagy gene Atg16l1 in mouse
andhuman intestinal Paneth cells. Nature 456,
259263 (2008).
Adolph, T.E. etal. Paneth cells as a site of
origin for intestinal inflammation. Nature 503,
272276 (2013).
Rakoff-Nahoum, S., Paglino, J.,
EslamiVarzaneh,F., Edberg, S. & Medzhitov, R.
Recognition of commensal microflora by
tolllikereceptors is required for intestinal
homeostasis. Cell 118, 229241 (2004).
6.
Buonocore, S. etal. Innate lymphoid cells drive

interleukin23dependent innate intestinal
pathology. Nature 464, 13711375 (2010).
7. Gevers, D. etal. The treatment-naive
microbiome in new-onset Crohns disease.
CellHost Microbe 15, 382392 (2014).
8. Colombel, J.F. etal. Infliximab, azathioprine,
orcombination therapy for Crohns disease.
N.Engl. J. Med. 362, 13831395 (2010).
9. Feagan, B.G. etal. Vedolizumab as induction
and maintenance therapy for ulcerative colitis.
N. Engl. J. Med. 369, 699710 (2013).
10. Lee, J.C. etal. Gene expression profiling of
CD8+ T cells predicts prognosis in patients
with Crohn disease and ulcerative colitis.
J. Clin. Invest. 121, 41704179 (2011).
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Happy birthday Nature Reviews Gastroenterology & Hepatology!

November 2014 marked the 10th anniversary of the launch of Nature Reviews Gastroenterology
& Hepatology. Tocelebrate this milestone, we present an anniversary issue containing specially
commissioned articles that highlight progress in the field of gastroenterology and hepatology over
the past decade. In addition, our anniversary infographic provides a snapshot of 10 years of the
journal in numbers.
nature.com/nrgastro10
NEPHROLOGY
DECADE IN REVIEWGLOMERULAR DISEASE
The glomerulus reveals some secrets

Agnes B. Fogo
In the past decade, major advances have been made in defining the antigens and pathogenesis of immune
complex diseases such as membranous nephropathy and IgA nephropathy. Probing of rare genetic diseases has
revealed new pathways of injury in proteinuric conditions, including channel abnormalities in the podocyte and
complement dysregulation underlying proliferative glomerular lesions.
Novel approaches to the assessment of
human renal biopsy samples have led to
new insights into several common glo
merular diseases. In primary membranous
nephropathy (MN), the most common
target antigen, phospholipase A2 receptor
(PLA2R), has been identified.1,2 Moreover,
mutations in transient receptor cation
potential channel6 (TRPC6) were dis
covered to cause familial focal segmental
glomerulosclerosis (FSGS),3 expanding the
paradigm beyond previous data showing
that genetic forms of this disease are caused
by mutations in structural proteins of the
podocyte. The markedly increased inci
dence of some chronic kidney diseases
(CKDs) in African Americans was linked
to risk allele variants of APOL1, which
encodes apolipoproteinL1. 4 The patho
genesis of IgA nephropathy (IgAN) was
elucidated, implicating antibody binding
to an abnormally glycosylated region of
IgA1the main IgA subtype present in
the circulation.5 An u
nbiased classification
approach to the varying biopsy appearances
of IgAN has become a model for explor
ing the significance of diverse patterns of
injury. 6 Aberrations in complement C3
regulation are now recognized to underlie
many proliferative glomerular injuries.7,8
Rapidly expanding translational research
is aimed at directing these new insights
into the molecular pathogenesis of disease
towards individualized therapies.
A major goal in nephrology has been to
identify the target antigen in primary MN.
Animal models were developed but the
antigens (for example megalin in Heymann
nephritis) were not present in human MN.
Using an elegant and laborious approach,
Beck etal. collected normal glomeruli from
non-transplanted deceased donor kidneys
and screened protein extracts with serum
48 | NOVEMBER 2015
samples from patients with primary or sec

ondary MN.1 They identified the antigen
PLA2R, which is normally expressed on
podocytes, using additional techniques,
including mass spectrometry and elution
of IgG from the deposits. About 70% of
patients with clinically defined primary
MN had deposits containing PLA2R and
circulating antib odies to this receptor.
Importantly, patients with MN secondary to
lupus erythematosus did not show PLA2R
positivity, and only a very minor proportion
of patients with cancer-associated MN had
PLA2R-positive deposits or serum PLA2R
antibodies. Using a similar approach,
another antigen, thrombospondin type1
domain-containing 7A, was subsequently
identified in approximately 10% of PLA2Rnegative patients with primary MN.2 These
studies indicate that extensive screening
and mass spectrometry of large numbers
of samples will likely identify additional
pathogenic antigens in patients without a
clinically evident secondary cause of MN.
Studies are currently underway to define
the utility of assessing serum levels versus
biopsy demonstration of PLA2R in delineat
ing specific causality and tracking responses
to intervention. Understanding inheri
ted (for example HLA-DQ1-associated
risks9) or acquired (for example drug or
infection-associated) mechanisms under
lying autoantibody formation will yield
additional insights and may provide targets
for early detection or even p
revention in
susceptiblepopulations.
Mass spectrometry was also crucial in
clarifying other types of proteins deposited
in glomeruli. For example, patients with
non-AL, non-AA amyloid can now be cor
rectly classified according to amyloid type,
identifying rare familial and other forms of
amyloid that require differing therapeutic
approaches. This technique has transformed

the approach to characterizing unusual
deposits within the kidney, i ncluding those
in dense deposit disease (DDD).10
Primary FSGS is a common glomerulo
pathy caused by an as yet not definitively
identified circulating factor or factors.
However, patients with familial FSGS may
offer insights into possible pathogenic
pathways that may be perturbed in the
more common sporadic FSGS. Most cases
of familial FSGS are linked to mutations in
structural proteins of the podocyte and slit
diaphragms or in components that anchor
podocytes to the glomerular basement
membrane, such as nephrin, podocin and
actinin4. Recently, a missense mutation
in the cation channel TRPC6 was discov
ered in a kindred with familial adult-onset
FSGS.3 This mutation alters calcium sig
nalling, increasing the response to ago
nists such as angiotensinII. It is possible
that TRPC6 abnormalities also occur in
sporadic FSGS or other proteinu ric dis
eases. Thus, increased TRPC6 activity and
resulting high calcium influx may con
tribute to podocyte injury and play a role
Immunofluorescence image showing granular capillary wall

staining for PLA2R in primary membranous nephropathy.
NEPHROLOGY
in proteinuria and progressive sclerosis.
Pharmacologic targeting of a channel is an
attractive and attainable goal; manipulating
TRPC6 expression and activity thus offers
promise as a therapy for numerous pro
teinuric diseases characterized by podocyte
dysfunction and loss.
Genetic approaches have also shed
light on non-familial kidney diseases.
Compared with Caucasians, African
Americans have a 45-fold increased risk
of some CKDs, including hypertensionassociated CKD, HIV-associated nephro
pathy and FSGS. Genome-wide association
studies found linkage of this excess risk
to the APOL1 allele variants G1 and G2.4
These variants, unlike the G0 variant
common in Caucasians, are trypanolytic
for Trypanosoma brucei rhodesiense, sug
gesting possible evolutionary pressures
underlying their high prevalence in popu
lations of African ancestry. Although the
mechanisms for increased disease risk
have not been elucidated, ongoing studies
postulate an effect on podocytes and/or
innateimmunity.
Mass spectrometry ... has

transformed the approach to
characterizing unusual deposits
within the kidney...
IgAN has a highly variable clinical course

and a range of morphological expres
sions. Aworking group formed by the
International IgA Nephropathy Network
and the Renal Pathology Society undertook
a novel approach to classification of this
disease.6 In a non-biased method, all patho
logical lesions present within a cohort of
archival IgAN biopsy samples were defined
to determine the variables that could be
reproduc ibly scored. The six pathologic
variables thus defined were then assessed
for correlation with clinical outcomes.
Four variables correlated independently
with increasing loss of glomerular filtration
rate: mesangial hypercellularity, endocapil
lary hypercellularity, segmental sclerosis
and significant tubular atrophy/interstitial
fibrosis (>25%). These variables were
used to define the so-called MEST score.
Subsequent studies validated and refined
this scoring approach, which could form
the basis for patient stratification, analysis
of therapeutic responses and assessment of
progression risk, as well as possibly provide
insights into the pathogenesis of various
lesions. Similar to the approach commonly
used in oncology, patients with similar stage

and severity of disease may be grouped
together in clinical trials.
The pathogenesis of IgAN has also been
elucidated. The hinge region of normal
IgA1 has numerous serine and threonine
residues with Oglycosylation. However,
in patients with IgAN, abnormally glyco
sylated IgA1 with deficient galactose is
bound by anti-glycan antibodies, includ
ing IgG, which may have a role in comple
ment activation and a subsequent cascade
of injury.5 This abnormal IgA1 is not effi
ciently cleared by receptors in the liver,
which may enhance its deposition within
the mesangium. These insights provide a
basis for investigation of events leading
to the generation of glycan-specific anti
bodies, not only as disease markers, but
also as a pathogenic m echanism and
therapeutictarget.
Membranoproliferative glomerulo
nephritis (MPGN) is characterized by
mesangial and subendothelial deposits,
double contours of glomerular basement
membranes due to reactions to these
deposits, endoc apillary hypercellularity
and increased matrix. MPGN was pre
viously thought to represent immune
complex diseases of various types, desig
nated as MPGNI, MPGNII (now known
as DDD) and MPGNIII. However, some
patients with an MPGN pattern of injury
have isolated C3 deposits.7 Mass spectrom
etry studies demonstrated that deposits
in DDD contain soluble components
of the terminal complement complex.
Aberrations in complement regulatory
proteins were identified in a group of
patients with dominant C3 deposits or
C3 deposits only, including heterozygous
mutations in factorH and CD46 (key reg
ulators of C3 activation) andexpression
of an autoantibody that inhibits C3 con
vertase, C3 nephritic factor.7 Subsequent
studies in animal models and in patients
led to a new paradigm for approaching
these C3 dominant glomerulonephritides,
now called C3 glomerulopathy.8 Patients
show a spectrum of lesions from DDD to
deposits of complement components that
overlap with the ultrastructural appearance
of immune complexes. Various genetic or
acquired alterations in complement regula
tion have been recognized, including famil
ial forms with mutations in complement
factor Hrelated protein5. The therapeutic
approach in these patients may be control
ling complement dysregulation, rather than
searching for underlying lupus, collagen
vascular diseases, chronic infections or

other triggers ofMPGN.
The field of nephrologyand specifi
cally glomerular diseasesis catching up
with the state-of-the-art in oncology, in
which specific mutation profiles and aber
rant signalling pathways form the basis for
personalized therapy. We are at the dawn
of a new beginning in molecular under
standing of the pathogenesis of some of the
common diseases afflicting the glomerulus.
These key observations form the basis for
rapid progress in the development of new
insights into the pathogenesis of disease
and new targets for intervention.
Department of Pathology, Immunology and
Microbiology, Vanderbilt University Medical
Center, C3310 MCN, 1161 21st Avenue,
Nashville, TN 37232, USA.
agnes.fogo@vanderbilt.edu
doi:10.1038/nrneph.2015.149
Competing interests
1.
Beck, L.H. Jr etal. Mtype phospholipase

A2receptor as target antigen in idiopathic
membranous nephropathy. N. Engl. J. Med. 361,
1121 (2009).
2. Tomas, N.M. etal. Thrombospondin type1
domain-containing 7A in idiopathic membranous
nephropathy. N. Engl. J. Med. 371, 22772287
(2014).
3. Winn, M.P. etal. A mutation in the TRPC6
cation channel causes familial focal
segmentalglomerulosclerosis. Science 308,
18011804 (2005).
4. Genovese, G. etal. Association of
trypanolyticApoL1 variants with kidney
diseasein African Americans. Science 329,
841845 (2010).
5. Suzuki, H. etal. Aberrantly glycosylated
IgA1inIgA nephropathy patients is
recognizedby IgGantibodies with restricted
heterogeneity. J.Clin. Invest. 119, 16681677
(2009).
6. Working Group of the International IgA
Nephropathy Network and the Renal Pathology
Society. The Oxford classification of IgA
nephropathy: pathology definitions,
correlations, and reproducibility. Kidney Int. 76,
546556 (2009).
7. Servais, A. etal. Primary glomerulonephritis
with isolated C3 deposits: a new entity which
shares common genetic risk factors with
haemolytic uraemic syndrome. J. Med. Genet.
44, 193199 (2007).
8. Pickering, M.C. etal. C3 glomerulopathy:
consensus report. Kidney Int. 84, 10791089
(2013).
9. Stanescu, H.C. etal. Risk HLA-DQA1 and
PLA2R1 alleles in idiopathic membranous
nephropathy. N. Engl. J. Med. 364, 616626
(2011).
10. Sethi, S. etal. Laser microdissection and mass
spectrometry-based proteomics aids the
diagnosis and typing of renal amyloidosis.
Kidney Int. 82, 226234 (2012).
NOVEMBER 2015 | 49
NEPHROLOGY
DECADE IN REVIEWGENETICS OF KIDNEY DISEASES
Genetic dissection of kidney

disorders
Friedhelm Hildebrandt
Advances in genome sequencing and genetic manipulation techniques

over the last decade have helped identify numerous single-gene causes
of early-onset kidney diseases and risk alleles for complex, polygenic
traits. Subsequent studies regarding the underlying disease mechanisms
will help lead to personal genetic diagnoses and unique therapeutic
interventions in the future.
50 | NOVEMBER 2015
from c oenzyme Q 10 supplementation.

A high percentage of monogenic causa
tion has also been detected in individuals
manifesting with early-onset CKD due
to other causes, such as cystic kidney dis
eases (>50%), urinary stone disease (21%),
congenital anomalies of the kidneys and
urinary tract (17%), and nephritis (15%).
Overall, monogenic mutations in ~250
recessive or dominant genes are estimated to
account for ~25% of all cases of early-onset
(<25years)CKD.
Cystic kidney diseases, also known as
renal ciliopathies, are caused by a dysfunc
tion of the primary cilia and centrosomes.
Recent discoveries of mutations in the
centrosomal protein CEP1644 and FAN15
have implicated DNA damage response
signalling in the pathogenesis of renal cilio
pathies. This finding led to the proposal
that accumulation of DNA damage with
consecutive premature cellular senescence
could contribute markedly to the develop
ment of renal fibrosis, which is central to all
forms of CKD independent of the primary
cause. Loss-of-function mutations in reces
sive monogenic kidney disease genes can
be readily modelled and characterized
in animals, such as mice or zebrafish, by
SilverV/iStock/Thinkstock
The advent of next-generation sequencing

techniques over the past decade has led to the
discovery that a surprisingly high propor
tion of cases of early-onset chronic kidney
disease (CKD) are caused by mutations in
a single gene (that is, they are monogenic).
Single-gene mutations in such genes have a
very strong effect on disease causation and
are, therefore, considered to exert full pene
trance on genetic causality. This paradigm
was exemplified in a study that performed
a mutation analysis of the 27known causa
tive genes of steroid-resistant nephrotic syn
drome (SRNS) in an international cohort of
1,780 patients with disease that manifested
before 25years of age.1 Strikingly, ~30%
patients were carriers of a causative muta
tion in one of the studied genes that could
fully explain the disease expression. The frac
tion of families in whom a single-gene cause
of SRNS was identified correlated inversely
with age of disease onset, with a single-gene
cause identified in 70% of affected newborns
and 12% of youngadults.
Further investigations into the 27causa
tive genes of SRNS have revealed that their
related gene products are part of protein
protein interaction complexes that con
verge onto specific pathways critical for
glomerular podocyte function. For example,
analysis of mutations in ARHGDIA dem
onstrated that proper regulation by this
gene of the small GTPases Rho, Rac, and
Cdc42, is essential for the integrity of the
glomerular filter.2 This finding could open
inroads into developing Rho and/or Rac
inhibitors and activators to treat SRNS,
for which no efficient treatment currently
exists. Other opportunities for therapeutic
development have also been generated from
mutation analysis in novel disease-causing
genes. Mutations in COQ63 and ADCK4,
which regulate coenzyme Q 10 biosynth
esis, may cause SRNS, and patients who
carry these specific variants could benefit
knockdown and/or knockout approaches.

The first successful treatment approaches
for renal ciliopathies were demonstrated
in mouse models, as exemplified in a study
that addressed impaired cell cycle dysregu
lation as a potential cause of cystogenesis.
Administration of a cyclin-dependent kinase
inhibitor, roscovitine, in mouse models of
juvenile and congenital polyc ystic kidney
disease efficiently arrested cystic disease.
The mechanism of action revealed effective
cell-cycle arrest, transcriptional inhibition,
and attenuation ofapoptosis.6
Studies performed over the past decade
have also focused on determining the con
tribution of single gene causes of adult-onset
CKD. Researchers found that the NPHS2
(podocin) gene variant R229Q, which had
been considered a variant of unknown sig
nificance for many years, is in fact diseasecausing when expressed in a compound
heterozygous state with one of a few other
distinct NPHS2 mutations.7 The combina
tion of heterozygous mutations on both
NPHS2 alleles was shown to result in adult
onset of SRNS. Mild recessive genetic vari
ants are most likely to cause late-onset forms
of disease and, therefore, might explain a
considerable proportion of adult-onsetCKD.
Whereas monogenic mutations are
usually discovered by whole exome sequen
cing, variants that underlie polygenic dis
orders are usually detected by genome-wide
association studies (GWAS). Polygenic dis
orders are more common than are mono
genic disorders, exert weak causa lity (or
risk) on the disease phenotype, usually
manifest later in life, and are often modified
by environmental effects. Risk alleles usually
account for only a small proportion of the
variance of the disease phenotype, and the
mechanistic assignment of an associated
marker allele to loss of gene function is
difficult to ascertain.
NEPHROLOGY
An important discovery attained by GWAS
was the detection of a single nucleotide poly
morphism, rs12917707, located near the
UMOD gene, which if mutated, causes auto
somal dominant medullary cystic kidney
disease type2.8 This finding was later con
firmed in a large worldwide cohort. Another
breakthrough GWAS study, published at the
turn of the decade, reported an association
between APOL1 variants and CKD. Two
variants were found to be strongly associ
ated with an increased risk of focal segmen
tal glomerulosclerosis (FSGS) and CKD
in African Americans.9 More than half of
African Americans in the USA (compared
with 4% of European Americans) carry both
recessive APOL1 risk alleles, increasing the
risk of developing FSGS by 530 fold. These
data likely explain the known racial dispari
ties in non-diabetic nephropathy seen in
African Americans.10 The two APOL1 risk
alleles always occur on different homolo
gous chromosomes and thereby behave like
recessivemutations.
As this article has exemplified, techno
logical approaches to probe and dissect the
human genome have rapidly progressed over
the past 10years, and the cost of perform
ing such investigations has decreased. The
ability to perform GWAS in large, worldwide
cohorts of patients with complex, polygenic
kidney disorders is enabling researchers to
identify new risk alleles for investigation
and to help determine disease causality and
prevalence in various populations. In the near
future we may be able to perform functional
studies of the many genetic variants of reces
sive and dominant causative genes for CKD,
for which there is currently not enough
evidence to indicate a deleterious effect.
Genetic manipulation techniques have also
advanced, enabling scientists to readily and
accurately model gene variants and muta
tions invitro and invivo to ascertain gene
function. The identification of single-gene
mutations in patients with kidney disorders,
by methods such as whole exome sequencing,
has revolutionized our practice of diagnos
ing and understanding disease pathogenesis.
Continued discovery of novel disease-causing
genes will enable the provision of an unequiv
ocal molecular genetic diagnosis to patients,
generate new insights into disease mechan
isms, and have consequences for personal
ized treatment and prevention of CKD in the
future. The functional characterization of
deleterious variants in known kidney disease
genes will be one of the most important and
rewarding contributions to understanding
renal pathogenesis in the coming decade.
Boston Childrens Hospital, Division of

Nephrology, 300 Longwood Avenue,
Enders561, Boston, MA 02115, USA.
friedhelm.hildebrandt@childrens.harvard.edu
doi:10.1038/nrneph.2015.148
Acknowledgements
F.H. has received grant support from the NIH
(grantnumbers DK068306, DK076683, DK88767),
and from the Howard Hughes Medical Institute.
Competing interests
1.
2.
3.
Sadowski, C.E. etal. A single-gene cause in

29.5% of cases of steroid-resistant nephrotic
syndrome. J. Am. Soc. Nephrol. 26, 12791289
(2014).
Gee, H.Y. etal. ARHGDIA mutations cause
nephrotic syndrome via defective RHO GTPase
signaling. J. Clin. Invest. 123, 32433253
(2013).
Heeringa, S.F. etal. COQ6 mutations in human
patients produce nephrotic syndrome with
sensorineural deafness. J. Clin. Invest. 121,

20132024 (2011).
4. Chaki, M. etal. Exome capture reveals
ZNF423and CEP164 mutations, linking renal
ciliopathies to DNA damage response signaling.
Cell 150, 533548 (2012).
5. Zhou, W. etal. FAN1 mutations cause
karyomegalic interstitial nephritis, linking chronic
kidney failure to defective DNA damage repair.
Nat. Genet. 44, 910915 (2012).
6. Bukanov, N.O. et al. Long-lasting arrest of murine
polycystic kidney disease with CDK inhibitor
roscovitine. Nature 444, 949952 (2006).
7. Tory, K. etal. Mutation-dependent recessive
inheritance of NPHS2-associated steroidresistant nephrotic syndrome. Nat. Genet. 46,
299304 (2014).
8. Kottgen, A. etal. Multiple loci associated with
indices of renal function and chronic kidney
disease. Nat. Genet. 41, 712717 (2009).
9. Genovese, G. etal. Association of trypanolytic
ApoL1 variants with kidney disease in African
Americans. Science 329, 841845 (2010).
10. Freedman, B.I. etal. The apolipoprotein L1
(APOL1) gene and nondiabetic nephropathy
inAfrican Americans. J. Am. Soc. Nephrol. 21,
14221426 (2010).
DECADE IN REVIEWACUTE KIDNEY INJURY
Acute kidney injurya decade

ofprogress
Rinaldo Bellomo
The past decade has seen developments in several aspects of acute

kidney injury (AKI), including the discovery of an array of biomarkers,
assessment of the optimal dose intensity for renal replacement therapy,
and the impact of fluid administration. Furthermore, AKI has emerged as
an important risk factor for chronic kidney disease.
Clinical research dedicated to understand
ing, diagnosing, and treating acute kidney
injury (AKI)a term that, by consensus, has
now substituted the previously used acute
renal failurehas increased markedly over
the past 10years. The number of research
papers on this topic has doubled during the
past decade, and during this period several
trends have evolved. One key development
has been the discovery and testing of bio
markers for early AKI. Heralded by the first
publication of the predictive value of neutro
phil gelatinase associated lipocalin (NGAL)
in paediatric cardiac surgery,1 biomarker
discovery and testing for the early indica
tion and prognosis of AKI has undergone a
remarkable degree of exploration. More than
a dozen candidate biomarkers with variable
predictive value have now been identified in
urine and blood, with typical values for the
area under the receiver operating character
istic (AUROC) curve ranging between 0.65
and 0.85 (Figure1). NGAL has been the most
investigated biomarker so far, with mixed
results and some serious concerns regard

ing assay reproducibility together with the
growing understanding that NGAL probably
represents a family of molecules of different
origin and with different epitopes. The most
recent and more systematically studied bio
marker discovery relates to the so-called cell
cycle arrest biomarkers, which have now been
assessed in several large cohorts and have
achieved consistent AUROC values of ~0.8
(Figure1).2 Although now FDA approved, the
value of cell cycle arrest biomarkers in clinical
practice remains unclear and is hampered by
the absence of specific linked therapies.
Irrespective of the possibility of early diag
nosis, some patients develop severe AKI and
are deemed to require renal replacement
therapy (RRT), raising the issue of optimal
dose intensity. After some initial small and
single institution studies that produced
promising results, two large multicentre trials
(one in the USA3 and the other in Australia
and New Zealand4) tested whether increasing
the intensity of RRT from an effluent flow of
NOVEMBER 2015 | 51
NEPHROLOGY
u TIMP-2
IGFBP-7
u TIMP-2
u IGFBP-7
Biomarker
u NGAL
p Cys C
u KIM-1
p NGAL
u IL-18
u pi-GST
u L-FABP
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

AUROC
Nature Reviews | Nephrology
2025ml/kg per hour in continuous modes

or from intermittent dialysis, to 3540ml/kg
per hour or daily dialysis would increase
survival. Both studies were identical in dem
onstrating a lack of effect on survival, thus
establishing a worldwide standard of care in
relation to RRT dose.
The epidemic of chronic kidney disease
(CKD), chronic heart failure, acute decom
pensated heart failure, and AKI prompted
consideration of the complex interactions
between cardiac and renal function, kidney
heart cross talk, and the clinical syndromes
that can ensue to be conceptualized as cardio
renal syndromes. The initial definition,
description, and proposed mechanisms by
which acute disease of each organ could affect
the other has spawned important reinvesti
gations and greater understanding of applied
pharmacology in this field.5 Importantly, and
together with other studies, the concept of
cardiorenal syndrome has shifted the focus
of management in patients with AKI from
the administration of fluids to rescue the
failing kidney, to a greater understanding
and concern that fluid administration might
have little beneficial effect on renal function
and could impose a substantial contribution
to positive fluid balance. A positive fluid
balance might, in turn, cause renal oedema,
delay renal recovery, injure vital organs, and
increase mortalityan effect seen most
strikingly in children with severe AKI.6
Faced with such varied practice patterns,
the utility of several definitions of AKI, and
uncertainty with regard to how best to manage
patients with developing or established AKI,
a large group of experts were funded to
develop a detailed framework of reference
for the definition, diagnosis, prevention,
and treatment of AKI. The Kidney Disease:
Improving Global Outcomes in Acute Kidney
Injury working group delivered an analysis
of all literature in this field and generated a
global definition for this syndrome, which
52 | NOVEMBER 2015
Figure 1 | Histogram summarizing the

estimated AUROC for 12 biomarkers used to
predict early AKI. The predictive value of each
biomarker for AKI stage 2 or 3 (according to
the Kidney Disease: Improving Global
Outcomes in Acute Kidney Injury working group
guidelines) was assessed within 12h of urine
or blood sampling. Data obtained from the
Sapphire study2 under the Creative Commons
agreement. Kashani, K. et al. Crit. Care 17,
R25 (2013). Abbreviations: AKI,acute kidney
injury; AUROC, area under receiver operating
characteristic; p, plasma; u, urinary.
represents the most comprehensive body of

references and information associated with
this c ondition to date.7
Simultaneously, and published in the same
year, intensive care investigators tackled the
possible effect of fluid choice on renal out
comes in critically ill patients. Two large
multicentre double-blind randomized trials
compared the most commonly administered
artificial colloid solution in the world for fluid
resuscitation (hydroxyethyl starch [HES])
and compared it with saline. In a study of
patients with severe sepsis, the Scandinavian
Critical Care Trials Group found that fluid
resuscitation with HES increased mortality
and the incidence of AKI, including severe
AKI requiring RRT.8 Concordant with such
findings, the Australian and New Zealand
Intensive Care Society Clinical Trials Group
investigated a broad cohort of all patients in
intensive care treated with fluid bolus therapy
and also found that the administration of
HES increased the need for RRT.9 These
studies have established beyond doubt that
HES is a nephrotoxin and has led to a major
decrease in its use in developed countries.
Finally, the impact of AKI on the long-term
outcome of patients both from a general and
renal point of view has become a focusof
active investigation after a seminal study
of ~4,000 patients identified a clear associ
ation (a threefold increase in risk) between
an episode of AKI requiring dialysis and the
subsequent need for chronic dialysis despite
initial recovery.10 Other studies have since
confirmed this association. Whether these
findings relate to the possibility that patients
who develop AKI have characteristics that
make them more likely to develop CKD
or represent a specific deleterious effect of
AKI on renal function is unclear. These data
have confirmed, however, that patients with
AKI require specific medical attention and
represent a new at-risk population for CKD.
In summary, we have made notable strides
in understanding the pathology and improv
ing outcomes in AKI over the past decade.
We have identified new biomarkers that could

prove useful in the future, defined a standard
dose for RRT, recognized the adverse conse
quences of overzealous fluid therapy on the
kidney as well as the patient, defined the need
to develop focused interventions to modulate
the cross-effect of simultaneous acute losses
of kidney function and cardiac function,
developed a set of practice guidelines that
provide a baseline reference point definition
and approach to AKI management for future
reference, clearly demonstrated the nephro
toxiciy of HES and, finally, have shown that
AKI is a major risk factor for CKD. Despite
these advances that have undoubtedly
decreased patient harm, we remain deeply
ignorant of the molecular mechanisms
responsible for the development of AKI in
humans and have not yet devised a single spe
cific and effective therapy to prevent, treat, or
attenuate AKI. Much remains to be done in
this field before notable improvements can be
made to patient outcomes.
Australian and New Zealand Intensive Care
Research Centre (ANZICRC), Monash
University, Commercial Road, Melbourne,
VIC3181, Australia.
rinaldo.bellomo@austin.org.au
doi:10.1038/nrneph.2015.147
Competing interests
R.B. has received grants from Baxter and honoraria
from Gambro and B. Braun.
1.
Mishra, J. etal. Neutrophil gelatinaseassociated lipocalin (NGAL) as a biomarker for

acute renal injury after cardiac surgery. Lancet
365, 12311238 (2005).
2. Kashani, K. etal. Discovery and validation of
cell cycle arrest biomarkers in human acute
kidney injury. Crit. Care 17, R25 (2013).
3. VA/NIH Acute Renal Failure Trial Network.
Intensity of renal support in critically ill patients
with acute kidney injury. N.Engl. J.Med. 359,
720 (2005).
4. The RENAL Replacement Therapy Study
Investigators. Intensity of continuous renal
replacement therapy in critically ill patients.
N.Engl. J.Med. 361, 16271638 (2009).
5. Ronco, C. etal. Cardiorenal syndrome.
J.Am. Coll. Cardiol. 52, 15271539 (2008).
6. Sutherland, S.M. etal. Fluid overload and
mortality in children receiving continuous
renalreplacement therapy: the prospective
pediatric continuous renal replacement therapy
registry. Am. J.Kidney Dis. 55, 316325 (2010).
7. KDIGO working group. KDIGO clinical practice
guidelines for acute kidney injury. Kidney Int.
Suppl. 2, 1136 (2012).
8. Perner, A. etal. Hydroxyethyl starch 130/0.42
versus Ringers acetate in severe sepsis.
N.Engl. J.Med. 367, 124134 (2012).
9. Myburgh, J.A. etal. Hydroxyethyl starch or
saline for fluid resuscitation in intensive care.
N.Engl. J.Med. 367, 19011911 (2012).
10. Wald. R. etal. Chronic dialysis and death
among survivors of acute kidney injury requiring
dialysis. JAMA 302, 11791185 (2009).
NEPHROLOGY
DECADE IN REVIEWPOLYCYSTIC KIDNEY DISEASE
our institution receive pravastatin in addi

tion to an angiotensin-converting-enzyme
(ACE)inhibitor.
Despite advances in understanding of
the mechanisms of cyst proliferation in
ADPKD, the development of targeted
therapies has proven difficult. The major
ity of clinical advancement in ADPKD
over the past 10years has primarily been
in determining the optimal management
of hypertension and cardiovascular risk
in affected patients. Hypertensive patients
with ADPKD show increased activation of
the reninangiotensinaldosterone system
(RAAS) compared to patients with essen
tial hypertension of the same age, renal
function and level of blood pressure. The
various potential pathways whereby stimu
lation of the RAAS can cause hypertension
and worsen kidney function include cyst
proliferation and expansion, increased sym
pathetic and endothelin activity, oxidant
injury and fibrosis (Figure1). At the start of
the 21st century, no clear consensus existed
with regard to the most appropriate type of
antihypertensive therapy for patients with
ADPKD. Randomized prospective studies
of ACE inhibitors in this patient population
were lacking, although experimental and
clinical data suggested that ACE inhibition
might be an effective treatment option. In
2002, a 7year study in hypertensive patients
Slowing progression of autosomal

dominant polycystic kidney disease
Robert W. Schrier
Autosomal dominant polycystic kidney disease is a challenging disorder

to diagnose and treat effectively. Promising research over the past
decade has, however, provided novel interventions, modifications to
clinical practice and new areas to investigate with the aim of identifying
approaches to slow disease progression.
Autosomal dominant polycystic kidney
disease (ADPKD) can present in children
at an early age. The disease is character
ized by tubular cystic expansion, kidney
enlargement and vasculature compres
sion of the parenchyma. Understanding
of the pathogenesis of ADPKD has rapidly
advanced since the identification of caus
ative mutations in PKD1 and PKD2 in
the mid-1990s. Approximately 8085%
of patients with ADPKD are carriers of a
PKD1 mutation, with truncation mutations
in this gene known to result in faster disease
progression than mutations in PKD2. The
rapid development of sophisticated tech
niques to manipulate the genome over the
past 10years has further advanced under
standing of the molecular and pathogenic
consequences of perturbed PKD1 func
tion in ADPKD. Using the Cre-lox system,
researchers have shown that inactivation
of Pkd1 in mice during renal development
causes rapid cyst growth, whereas inacti
vation in adulthood causes comparatively
slower cyst growth. Such studies indicate
that the type of mutation as well as the
degree and timing of inactivation of PKD1
and PKD2 might contribute to the severity
of ADPKD in humans.1
Several other risk factors for ADPKD
progression exist, such as early onset hyper
tension and frequent gross haematuria.
ADPKD progression seems to occur faster
in males than in females, which might be
associated with earlier onset of more severe
hypertension in men. Among women with
ADPKD, those with a history of three or
more pregnancies exhibit more rapid loss
of kidney function than those with fewer
previous pregnancies. Overt proteinuria in
patients with ADPKD also predicts a faster
loss of kidney function. The loss of kidney
function observed in these patients strongly
correlates with rapid growth in kidney
volume.2 Research over the past decade has,
therefore, focused on identifying effective
means by which this growth can be slowed

in affected children and young adults. Early
experimental models showed that lovastatin
could decrease the severity of ADPKD by
slowing the increase in kidney volume,3
leading to the proposal that statin admin
istration could decrease the rate of kidney
volume growth in patients with ADPKD.
In 2014, a double blind, randomized trial in
paediatric patients with ADPKD and normal
kidney function found that pravastatin sig
nificantly decreased the percentage change
in height-corrected total kidney volume
over a 3year period compared to placebo
(23%3% versus 31%3%; P=0.02).4 The
mechanisms by which this statin exerts
its effect remain to be defined, but based
on these data, patients aged 822years at
Cyst compression of renal vasculature

Renin
Angiotensin II
Increased
growth factors
Angiogenesis
Cyst proliferation
and expansion
Transforming
growth factor beta
Oxidant-mediated
endothelial damage
Endothelin
Sympathetic
activity
Systemic vascular
resistance
Aldosterone
Sodium retention
Renal fibrosis
Hypertension and renal disease
Figure 1 | Pathogenic role of the reninangiotensinaldosterone system in ADPKD. AngiotensinII

Nature
Reviews
| Nephrology
predominantly acts to increase blood pressure through vasoconstriction,
but is
also known
to
increase cell proliferation, angiogenesis, oxidant injury and fibrosis, which can exacerbate the
renal component of ADPKD. Potential indirect effects of angiotensin II during hypertension include
stimulation of the sympathetic nervous system, endothelin and aldosterone with sodium
retention. Angiotensin II also stimulates reactive oxygen species, which could account, at least
inpart, for the observed endothelial dysfunction in ADPKD. Abbreviation: ADPKD, autosomal
dominant polycystic kidney disease. Reproduced with permission from American Society of
Nephrology Schrier, R. W. et al. J. Am. Soc. Nephrol. 20, 18881893 (2009) and modified with
permission from Remedica Medical Education and Publishing.
NOVEMBER 2015 | 53
NEPHROLOGY
with ADPKD and left ventricular hyper
trophy compared the effects of lowering
blood pressure with RAAS blockade versus
a calcium channel blocker.5 Left ventricu
lar mass index (LVMI) decreased in both
cohorts, but the decrement was greater in
patients administered a RAAS blocker.
The results of a 5year randomized study
later showed that ACE inhibition in chil
dren and young adults with ADPKD and
blood pressures within the upper quartile
of the normal range could prevent decreases
in creatinine clearance and increases in
LVMI.6 ACE inhibition, however, did not
affect kidney volume growth. Nevertheless,
because of enhanced RAAS activation,
we have now entered an era in which
ACE inhibition is a fairly standard firstline treatment for hypertensive patients
withADPKD.
Data from the HALT-PKD double-blind,
placebo-controlled, randomized trial were
reported in 2014.7 This trial assessed the
effects of a standard (120/70mmHg to
130/80mmHg) versus a low (95/60mmHg
to 110/75mmHg) blood pressure target,
and of the ACE inhibitor lisinopril plus
either telmisartan (an angiotensin-receptor
blocker) or placebo, on total kidney volume
in patients aged 1549years with ADPKD
and estimated glomerular filtration rate
>60ml/min/1.73m 2. No difference was
found in the annual percentage change in
total kidney volume associated with dual
versus single RAAS blockade; however, the
annual percentage change in total kidney
volume was significantly lower in the low
blood pressure target group compared to the
standard blood pressure target group (5.6%
versus 6.6%, P=0.006). Moreover, LVMI
decreased to a greater extent in the former
than in the latter group (1.17g/m2 per year
versus 0.57g/m2 per year, P <0.001) as did
urinary albumin excretion (3.77% versus
2.43%, P<0.001).
Improved diagnosis of ADPKD has
also markedly shaped clinical practice
over the past 10years. For example, 2009
sawthe development of unified criteria for
ultras onographic diagnosis of ADPKD,
with at-risk patients aged 3039 years
now requiring a minimum of three renal
cysts for diagnosis. Assessments such as
intracranial screening for aneurysm and
positron-emission and computed tomo
graphy to identify infected renal or liver
cysts are also now routinely performed.8
Improved understanding of the patho
genic mechanisms of cyst formation has
led to the investigation of new treatments
54 | NOVEMBER 2015
for ADPKD, such as the vasopressin

V2-receptor antagonist tolvaptan, which
has not received FDA approval.9 New con
cepts regarding the pathogenic mechanisms
of cyst formation have also been proposed,
such as the identification of human CD133+
progenitor cells and other specialized cells
that can promote renal cyst formation in
mouse models.10 Finally, the results of the
HALT-PKD study have helped to clarify
blood-pressure targets and antihyper
tensive regimens for patients with ADPKD.7
These combined, cross-disciplinary efforts
will enable optimal treatment and diagno
sis of patients with ADPKD as well as the
identification of novel approaches to slow
diseaseprogression.
Division of Renal Diseases and Hypertension,
University of Colorado Denver, 12700 East 19th
Avenue, Campus Box C281, Aurora, CO80045,
USA.
robert.schrier@ucdenver.edu
doi:10.1038/nrneph.2015.164
Competing interests
1.
Lantinga-van Leeuwen, I.S. et al. Lowering of

Pkd1 is sufficient to cause polycystic kidney
disease. Hum. Mol. Genet. 13, 30693077
(2004).
2.
Cadnapaphornchai, M.A. et al. Prospective

change in renal volume and function in children
with ADPKD. Clin. J. Am. Soc. Nephrol. 4,
820829 (2009).
3. Gile, R.D. et al. Effect of lovastatin on the
development of polycystic kidney disease in the
Han:SPRD rat. Am. J. Kidney Dis. 26, 501507
(1995).
4. Cadnapaphornchai, M. et al. Effect of
pravastatin on total kidney volume, left
ventricular mass index, and microalbuminuria in
pediatric autosomal dominant polycystic kidney
disease. Clin. J. Am. Soc. Nephrol. 9, 889896
(2014).
5. Schrier, R.W. et al. Cardiac and renal effects of
standard versus rigorous blood pressure control
in autosomal-dominant polycystic kidney
disease: Results of a seven-year prospective
randomized study. J. Am. Soc. Nephrol. 13,
17331739 (2002).
6. Cadnapaphornchai, M. et al. Increased left
ventricular mass in children with autosomal
dominant polycystic kidney disease. Kidney Int.
74, 11921196 (2008).
7. Schrier, R.W. et al. Blood pressure in early
autosomal dominant polycystic kidney disease.
N. Engl. J. Med. 371, 22552266 (2014).
8. Schrier, R.W. et al. Repeat imaging for
intracranial aneurysms in patients with
N. Engl. J. Med. 22, 23612375 (2013).
9. Torres, V.E. et al. Tolvaptan in patients with
N. Engl. J. Med. 367, 24072418 (2012).
10. Kurbegovic, A. & Trudel, M. Progressive
development of polycystic kidney disease in the
mouse model expressing Pkd1 extracellular
domain. Hum. Mol. Genet. 22, 23612375
(2013).
DECADE IN REVIEWRENAL TRANSPLANTATION
A spectrum of advances in renal

transplantation
Bruce Kaplan
Although the first 50years of renal transplantation were marked by great

advances in immunosuppressive therapies, the past decade has been
marked by an unprecedented increase in technology. This progress has
spurred investigators to challenge old paradigms and investigate how
best to utilize these technologies to further improve patient care.
The establishment of immunological
toleranced efined as the absence of a
detectable immune response against afunc
tional graft in the absence of immuno
suppressionremains a major but as yet
unachieved goal in transplantation. In 2008
Kawai etal. reported on a series of five
patients who received bone marrow condi
tioning with a non-myeloablative regimen
before transplantation, followed by donorderived bone marrow post-transplantation
in the hope of producing a mixed chimeric
tolerogenic state.1 These patients were then
weaned off immunosuppressive therapy.
Two of the first three patients developed

antibody-mediated rejection and a decision
was made to add rituximab therapy to the
pre-transplantation regimen; however, both
subsequently transplanted patients exhib
ited signs of emerging HLA antibodies and
graft damage. This study was meticulously
performed and monitored, and should serve
as a template for careful assessment of the
efficacy of any future attempt to induce tol
erance. Interestingly, analysis of data beyond
1year post-transplantation showed evidence
that mixed chimerism is not necessary to
maintain a tolerogenic state and instead
NEPHROLOGY
Box1 | Advances in renal transplantation in the past decade
Only a few clinical studies of tolerance in transplantation have been performed to date,
butmechanistic insights into tolerance are increasing
The new US kidney allocation system aims to pair the best kidneys to patients with the
longest expected survival
Transplantation of patients with HIV infection has become increasingly common
Great progress has been made in molecular diagnosis of graft rejection, but the findings
require robust validation
Biomarker development has been slow and meticulously performed studies have not been
universally adopted by the transplantation community
The co-stimulatory blocker belatacept is the first biologic agent approved for maintenance
therapy for the prevention of graft rejection, but adoption for newly transplanted patients has
been slow
The pathology of graft loss is coming under greater scrutiny and new paradigms of loss of
allograft function are emerging
suggested that tolerance is more dependent

on the emergence of T regulatory cells. This
study can only be called a partial success in
terms of outcomes, but did reveal tremen
dous mechanistic insights and the long road
that lies ahead in order to fully understand
how best to induce tolerance.
Allocation of kidneys for transplantation
has always been a daunting task that requires
concepts of equity to be balanced with
approaches to achieve greatest utility. After
many years of public debate a new kidney
allocation system was approved by the Board
of Directors of the Organ Procurement and
Transplantation Network and implemented
in 2014. The goal of the new kidney alloca
tion system is to provide greater access to
available organs through national matching
of highly sensitized patients, while using the
Kidney Donor Risk Index (KDRI) as a metric
of kidney quality to match the best organs
with recipients who are expected to survive
longest (as assessed using the Estimated
Post Transplant Survival score). The aim of
this revised system is to increase transplant
outcomes and maximize organ supply by
allocating kidneys with long survival poten
tial to recipients expected to live longest.2
The results of this new system are as yet
unknown, but many in the transplantation
community have raised questions about the
benefits of transplanting so-called compat
ible kidneys into highly sensitized patients
without mandatory testing for HLA-DQ and
HLA-DP alleles. Questions have also been
raised as to how well the KDRI discriminates
kidneys that are not at the extremes of the
scoring metric.
The advent of highly accurate genomics
platforms along with advances in bioinfor
matics analytics has ushered in a number of
genomics based studies to help understand
the underlying mechanisms of graft injury
as well as potentially serve as diagnostic and
prognostic tools in transplantation. Although

this goal has not yet been met, data from two
studies have indicated the great potential of
genomic analyses. In one study, researchers
from Edmonton interrogated kidneys biop
sied for cause, and found a set of molecular
classifiers of tissue injury that preliminarily
showed high correlation with graft loss.3 This
paradigm shift away from diagnostic cat
egories on the basis of histologic findings to
molecular injury-based identifiers could lead
to more accurate prognostic testing as well as
change our perception of graft loss. Taking
this finding a step further, researchers from
the Scripps Research Institute delineated
potential noninvasive molecular classifiers
in whole blood that could predict both acute
rejection and degree of individual immuno
suppression.4 Both of these studies were ele
gantly conceived and analysed; however, as
with all new technologies careful and robust
validation of their f indings arenecessary.
Early noninvasive detection of rejection
has been a goal in the setting of kidney trans
plantation for many years. To date no bio
marker has been comprehensively validated
that meets all the criteria of a fully predictive
diagnostic test. Hricik and colleagues per
formed a beautifully designed and executed
multi-centre study to explore the potential
of immunologic biomarkers as predictors of
acute rejection.5 They found that presence
of the chemokine CXCL9 in the urine had
excellent negative predictive value in deter
mining patients at low risk of acute rejection.
Unfortunately as acute rejection has become
a relatively rare phenomenon the search for a
biomarker with high positive predictive value
will be challenging.
One of the barriers to transplantation
has been sensitization of potential trans
plant recipients to a wide array of HLA
antigens. These highly sensitized individu
als often wait years to receive a transplant
or never find a compatible donor. Over the

past decade a number of centres have per
formed desensitization protocols with excel
lent success rates (at least in the short and
intermediate term of 15years). However,
the outcomes of desensitization are still not
as good those with a fully compatible donor.
Montgomery and colleagues reported 211
patients who underwent desensitization
and were subsequently transplanted.6 These
patients had a significant survival advantage
over those who remained on the transplant
waiting list. Although graft survival in the
desensitized patients was not equivalent
to graft survival associated with transplan
tation of an organ from a compatible live
donor, patients who had been desensi
tized still accrued great benefit. As the
Scientific Registry of Transplant Recipients
(SRTR)which evaluates transplanta
tion outcomes across the USAdoes not
adjust expected survival for desensitized
patients, many centres that perform desen
sitization find themselves at risk of out
comes being rated as below expected and
spurring an extensive audit and large fine
from the Centres for Medicare & Medicaid
Services. Whether the SRTR should adjust
for d
esensitization is open for debate, but
without risk adjustment, transplant pro
grammes have a powerful d isincentive
towards performingdesensitization.
...elaborate donation chains

require elegant and quite complex
algorithms
Another potential partial solution to

helping highly sensitized patients receive a
transplant is through paired kidney dona
tion, where donors incompatible with their
intended donors choose to donate to another
individual they are compatible with, while
their intended recipient receives an organ
from a second compatible donor.7 Simple
two-way swaps require tremendous coordi
nation of logistics, while more elaborate
donation chains require elegant and quite
complex algorithms. It is a great testament
to the transplantation community that such
algorithms have been not only developed,
but that extended altruistic donation chains
have been performed. Despite such inno
vative approaches, however, many highly
sensitized patients still cannot receive a
compatible transplant.
The development of effective antiretroviral
therapies has dramatically improved the life
expectancy of patients with HIV infection;
NOVEMBER 2015 | 55
NEPHROLOGY
however, many HIV infected individuals
develop end-stage renal disease (ESRD). It
had long been held that the immunosuppres
sion necessitated by transplantation would
be dangerous to these individuals and not
provide an adequate risk-to-benefit profile.
In 2010 Stock etal. reported on an NIH
sponsored multi-centre trial of kidney trans
plantation in HIV patients with ESRD. 150
patients were enrolled with 3year patient
and graft survival of 88% and 73.7%, respec
tively.8 Although these results are slightly
inferior to transplantation outcomes in recip
ients without HIV infection, this study pro
vided hope to individuals with HIV infection
and resulted in the formation of guidelines to
help improve results in the future.
Belatacept is the first biologic agent to
be approved by the FDA for maintenance
therapy for the prevention of acute rejec
tion.9 This drug offers the potential promise
of avoiding toxicities associated with calci
neurin inhibition, while maintaining
adequate immunosuppression. Early trials
showed improved glomerular filtration
rates, but a potential signal of a greater risk
of Ebstein Barr-related lymphoma. The
role ofbelatacept in kidney transplantation
remains to bedefined.
Although the prevention of acute rejec
tion has been the subject of numerous
inquiries, the aetiology of graft loss has only
been a major focus over the past decade.
56 | NOVEMBER 2015
ELZoghby and colleagues looked at the

pathology of patients who suffered graft
loss and defined three major aetiologies:10
antibody-associated glomerular lesions,
interstitial fibrosis with tubular atrophy and
recurrent glomerular disease. Interestingly
calcineurin inhibitor toxicity was not a cause
of late graft loss. This study has revised our
thinking about approaches to prolong graft
survival and further studies in this area are
eagerly awaited.
The papers highlighted here are a subjec
tive list and are in no way meant to be all
inclusive. The advances in many areas of
transplantation, including tolerance induc
tion, allocation policy, molecular diagnostics,
desensitization protocols and new immuno
suppressive agents are illustrative of the
major advances that have been made in this
field over the past decade (Box1). These
advances will hopefully pave the way to
more individualized approaches to the care
of transplant recipients. As analytic skills
catch up with technological advances, earlier
and more accurate diagnostics will become
more commonplace. Further approaches
to decrease the toxic immunosuppressive
burden will also improve the quality of life of
transplant recipients over the coming decade.
Arizona State University, 550North 3rd Street,
Phoenix, AZ 85004, USA.
bruce.kaplan@asu.edu
doi:10.1038/nrneph.2015.159
Competing interests
1.
Kawai, T. et al. HLA-mismatched renal

transplantation without maintenance
immunosuppression. N. Engl. J. Med. 358,
353361 (2008).
2. Friedewald, J.J. et al. The kidney allocation
system. Surg. Clin. North Am. 93, 13951406
(2013).
3. Einecke, G. et al. A molecular classifier for
predicting future graft loss in late kidney
transplant biopsies. J Clin. Invest. 120,
18621872 (2010).
4. Kurian, S.M. et al. Molecular classifiers for
acute kidney transplant rejection in peripheral
blood by whole genome gene expression
profiling. Am. J. Transplant. 14, 11641172
(2014).
5. Hricik, D. E. et al. Multicenter validation of
urinary CXCL9 as a risk-stratifying biomarker for
kidney transplant injury. Am. J. Transplant. 13,
26342644 (2013).
6. Montgomery, R.A. et al. Desensitization in
HLA-incompatible kidney recipients and
survival. N.Engl. J. Med. 365, 318326
(2011).
7. Rees, M.A. et al. A nonsimultaneous,
extended, altruistic-donor chain. N. Engl. J. Med.
360, 10961101 (2009).
8. Stock, P.G. et al. Outcomes of kidney
transplantation in HIV-infected recipients.
N.Engl. J. Med. 363, 20042014 (2010).
9. Vincenti, F. et al. Costimulation blockade with
belatacept in renal transplantation. N. Engl. J.
Med. 353, 770781 (2005).
10. El-zoghby, Z.M. et al. Identifying specific
causes of kidney allograft loss. Am. J.
Transplant. 9, 527535 (2009).
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NEUROLOGY
DECADE IN REVIEWDEMENTIA
A decade of discovery and disappointment

indementia research
John R. Hodges
In my personal view, the past decade in dementia research has been marked by remarkable discoveries in
the field of frontotemporal dementia, accompanied by steady scientific consolidation tinged with therapeutic
disappointments related to Alzheimer disease.
In 2011, the last big piece

of the FTD genetic puzzle fell
intoplace
Perhaps an even more impor tant

iscoveryin the same year as the GRN
d
genewas that ubiquitinated TAR DNAbinding protein 43 (TDP43) is the fundamental pathological protein in most cases of
tau-negative, ubiquitin-positive FTD.2 This
protein was found in both sporadic and
familial FTD, as well as in the vast majority of cases of amyotrophic lateral sclerosis
(ALS). These observations opened the way
to understanding the pathophysiology of
FTD, and to further genetic discoveries.
In 2011, the last big piece of the FTD
genetic puzzle fell into place with the simultaneous discovery, by two large research
groups, of a unique hexanucleotide repeat
expansion in the C9orf72 gene on chromosome 9.3,4 This expansion was found in a
57 | NOVEMBER 2015
high proportion of familial FTD and ALS

casesespecially kindreds manifesting
both disordersas well as in a significant
minority of seemingly sporadic behavioural
variant FTD (bvFTD) cases.
The volume of research published since
2011 related to the basic science of C9orf72
and the clinical characterization of patients
with the C9orf72 syndrome has been
astounding. Interestingly, some patients with
the gene expansion have a protracted history
with prominent psychiatric symptoms and
little in the way of brain atrophy, all of which
makes diagnosis challenging.
The fact that some patients with symptoms of bvFTD show minimal progression
over many years has contributed to the
refinement of the diagnostic criteria for
bvFTD, with internationally agreed criteria
for possible, probable and definite bvFTD.5
It is already clear that the probable category
is robust, and that the vast majority of such
cases have one of the pathological forms of
FTD. The utility of the possible level is still
a topic of active research, and the prevalence
of pathological FTD among patients who do
not have one of the accepted clinical forms of
FTD remains open to question. Nevertheless,
the new criteria, which can be applied consistently by researchers around the world,
represent a substantial step forward.
The other parallel development in the
FTD field has been the clear delineation of
three forms of primary progressive aphasia.6
One of the three types, semantic dementia,
stands out as a unique clinicopathological
entity with a highly characteristic clinical
presentation, a signature pattern of asymmetric anterior temporal lobe atrophy on
MRI, and underlying TDP43 pathology.
Nonfluent aphasia has presented more of
a problem, in that the clinical features vary,
there is no signature MRI appearance, and
a proportion of cases have underlying AD

pathology at postmortem. A breakthrough
was the realization that the nonfluent cases
could be split into those with agrammatism
and/or apraxia of speech in association
withinferior frontal atrophy, and thosewith
word-finding difficulty, phonological speech
errors and markedly reduced verbal span, in
association with atrophy around the angular
gyrus. This latter form was termed l ogopenic
progressive aphasia(LPA).
It has become clear that the majority of
individuals with LPA have AD pathology,
which is rare in those with true nonfluent
progressive aphasia, who typically have
FTD-tau pathology. This observation suggests that the clinical presentation of AD
is quite varied, and raises questions about
the received wisdom that AD always begins
with tangle formation in the entorhinal
cortex, spreading gradually to involve the
hippocampus and lateral cortical regions.
An important discovery that unifies
FTD and AD is that the tau pathology
found in AD, and in some cases of FTD,
spreads within the brain by a prion-like
cell-to-cell process. This phenomenon was
elegantly demonstrated using extracts from
a transgenic mouse that expressed mutant
(Pro301Ser) human tau, which were injected
NPG
A decade ago, it had already been known for

some time that up to 30% of patients with
frontotemporal dementia (FTD) have a
strong family history of early-onset dementia, but the genetic basis of the majority of
cases was unknown. This situation began to
change with the discovery of the progranulin
(GRN) gene in 2006.1 GRN was the second
major FTD-associated gene to be identified and, like the microtubule-associated
protein tau (MAPT) gene, which was first
linked to FTD a decade earlier, it is located
on chromosome 17. Mutations in GRN are
associated with tau-negative, ubiquitin-
positive intraneuronal deposits, and they
turned out to be approximately as common
as the MAPT gene mutations, but their discovery still left many apparently familial
cases of FTDunexplained.
NEUROLOGY
into the brains of mice expressing wild-type
human tau.7 This intervention induced the
assembly of the wild-type tau into filaments,
and a progressive spread of pathology from
the site of injection to neighbouring brain
regions. A similar mechanism of prion-like
transmission has now been demonstrated
for a range of tauopathies, and is implicated in other neurodegenerative disorders
associated with the deposition of abnormal
protein aggregates, such as synuclein in
Parkinsondisease.
In the AD field, the Dominantly Inherited
Alzheimer Network (DIAN) has brought
together an amazing range of techniques in a
cohort of at-risk individuals recruited across
three continents.8 This impressive effort led
to the finding that subtle changes, such
as a decline in cerebrospinal fluid (CSF)
amyloid (A) 42, occur 25years before
expected AD symptom onset. In addition,
A deposition can be shown on Pittsburgh B
compound PET (PiBPET) 15years before
symptom onset, when tau concentrations
begin to rise in the CSF and subtle brain
atrophy can be detected.
These findings raise fundamental questions about the possible effectiveness of
disease-modifying treatments for AD, and
perhaps go some way towards explaining
the therapeutic disappointments of the past
decade. Hopes were high for the first A42
(AN1792, Elan Pharmaceuticals) immunization trial, which was, sadly, stopped due
to the occurrence of severe encephalitis in
a few treated individuals. A group of eight
treated patients from the trial underwent
neuropathological examination postmortem.
Seven patients exhibited virtually complete
amyloid plaque removal, showing that the
immunotherapy was successful in achieving
the aim of plaque removal, yet their disease
continued inexorably.9 Later analyses concluded that there was no difference in survival, or time to severe dementia, between
treated and untreated individuals, suggesting that such therapies need to be initiated
much earlier in the disease course, or that
other approaches, perhaps targeted at tau
pathology, are required. Trials of secretase
inhibitors have also proven a disappointment
and, consequently, effective treatments for
AD remain beyond ourreach.
Another highly successful application
of PiBPET, in combination with metabolic 18F-FDGPET and quantitative MRI,
has been to map the relationship between
amyloid deposition, hypometabolism and
atrophy early in the course of AD, and
their effects on medial brain structures that
constitute the default network.10 This work

has brought together a body of knowledge on
the brain systems that are active during quiet
contemplation and memory retrieval, so as to
understand the patterns of c ognitive deficits
typically found in mild cognitive impairment, the prelude to full-blown dementia
inAD.
In conclusion, the past 10years have seen
remarkable discoveries in FTD, tempered by
more-modest, incremental progress in AD
research. We can only hope that the next
decade will be the decade of AD, and that we
will see a giant step forward for mankind in
the treatment of this most p
ernicious killer.
Neuroscience Research Australia (NeuRA) and
University of New South Wales, Barker Street,
Randwick, Sydney, NSW 2013, Australia.
j.hodges@neura.edu.au
doi:10.1038/nrneurol.2015.191
Published online 6 October 2015;
corrected online 4 November 2015
Competing interests
1.
Baker, M. etal. Mutations in progranulin cause

tau-negative frontotemporal dementia linked to
chromosome 17. Nature 442, 916919 (2006).
2.
Neumann, M. etal. Ubiquitinated TDP43

infrontotemporal lobar degeneration and
amyotrophic lateral sclerosis. Science 314,
130133 (2006).
3. Renton, A.E. etal. A hexanucleotide repeat
expansion in C9ORF72 is the cause of
chromosome 9p21-linked ALSFTD. Neuron 72,
257268 (2011).
4. DeJesus-Hernandez, M. etal. Expanded
GGGGCC hexanucleotide repeat in noncoding
region of C9ORF72 causes chromosome
9p-linked FTD and ALS. Neuron 72, 245256
(2011).
5. Rascovsky, K. etal. Sensitivity of revised
diagnostic criteria for the behavioural variant
offrontotemporal dementia. Brain 134,
24562477 (2011).
6. Gorno-Tempini, M.L. etal. Classification of
primary progressive aphasia and its variants.
Neurology 76, 10061014 (2011).
7. Clavaguera, F. etal. Transmission and
spreading of tauopathy in transgenic mouse
brain. Nat. Cell Biol. 11, 909913 (2009).
8. Bateman, R.J. etal. Clinical and biomarker
changes in dominantly inherited Alzheimers
disease. N.Engl. J.Med. 367, 795804
(2012).
9. Holmes, C. etal. Long-term effects of A42
immunisation in Alzheimers disease: follow-up
of a randomised, placebo-controlled phaseI
trial. Lancet 372, 216223 (2008).
10. Buckner, R.L. etal. Molecular, structural, and
functional characterization of Alzheimers
disease: evidence for a relationship between
default activity, amyloid, and memory.
J.Neurosci. 25, 77097717 (2005).
DECADE IN REVIEWMULTIPLE SCLEROSIS
New drugs and personalized

medicine for multiple sclerosis
Paul M. Matthews
The past decade of multiple sclerosis research has been marked by

important advances in understanding the disease, a dramatic increase
in the range of treatment options and a new spirit of data sharingin
research for patient benefit. This progress has made personalized
medicine in multiple sclerosis a realistic possibility.
Clinical research into multiple sclerosis (MS)
has evolved substantially in the past 10years.
The clinical effectiveness of medicines for
relapsingremitting MS has been established,
and optimism is growing that the challenge
posed by progressive disease can be met. My
personal highlights from the decade include
new evidence for a role of B-cell modulation in the treatment of MS, advances in the
understanding of how immune cells traffic
between the peripheral compartment and the
CNS, and the first steps towards evidencebased practice of personalized medicine
forMS.
A conceptual breakthrough with immedi
ate clinical relevance came in 2008 with
clinical evidence for a central role of Bcells in

the immune response in MS. A phaseII trial
showed that a single course of rituximab
a monoclonal antibody that selectively targets
and depletes CD20+ Bcellswas highly effective for suppression of new acute inflammatory activity in the disease.1 The mechanisms
by which these Bcell-directed therapies
modulate the Tcell-associated disease processes still need to be elucidated, but similarly
high efficacy has been observed with other
anti-CD20 monoclonal antibodies that are
now in clinical development. These antibodies promise a new class of treatments for the
relapsingremitting disease that have high
efficacy and fewer serious adverse events.
NOVEMBER 2015 | 58
NEUROLOGY
These developments emphasize important interactions between immune system
effectors in peripheral and CNS compartments, although how CNS antigens are
presented for activation of an adaptive
immune response has been poorly understood. A potentially major breakthrough
came recently in the form of a detailed
anatomical characterization of the way in
which Tcells associate with the meninges.
This characterization identified functional
lymphatic vessels that line the dural sinuses
and express molecular markers of lymphatic endothelial cells.2 These vessels are
capable of carrying fluid and immune cells
from the cerebrospinal fluid and are connected to the deep cervical lymph nodes.
Together, these characteristics suggest that
a functional lymphatic system is associated
with the meninges. The existence of such
a system in the CNS raises the possibility that u
nexplored mechanisms underlie
neuroinflammatory diseases. For example,
pathology that prevents activated adaptive immune cells from exiting the brain
could contribute to disease severity. This
discovery will drive new directions for MS
research in the coming years.
One of the biggest clinical

impacts has been the
expansion in the range of
approved medicines
The past decade has finally seen genetics come of age as an important tool for the
molecular analysis of disease mechanisms
in MS. The collaborative development of an
MS genetic database of unprecedented size
has enabled the International MS Genetics
Consortium to make major discoveries
about susceptibility factors. A genomewide association study that included almost
10,000 people of European descent with MS
identified specific HLADRB1 risk alleles
and confirmed a role for HLAA allelic
variants in protecting against MS.3 Such
genetic studies have revealed that immunologically relevant genes (particularly those
involved in Thelper cell differentiation)
are substantially over-represented among
those associated with susceptibility to MS,
offering further support to the hypothesis
of a primary immunopathogenesis. Work in
the next decade will address how the interactions of these genes with environmental
factors (such as sunlight and vitamin D)
contribute to variation in the severity of
thedisease.
59 | NOVEMBER 2015
Nevertheless, defining the molecular

mechanisms of MS remains a challenge as
long as the disease is mainly diagnosed on
the basis of characteristic features that exist
in an appropriate context, rather than by the
presence of aetiologically specific features
or biomarkers. The discovery that neuromyelitis optica (NMO, also known as Devic
disease) usually has a distinct, antibodymediated aetio-pathogenesis and is not
simply a manifestation of MS, as previously
thought, thus represented an important
step forward.4 Key to this discovery was the
identification of a serum IgG autoantibody
(AQP4IgG) against the membrane protein
aquaporin4, which is a component of the
dystroglycan protein complex in astrocytic foot processes at the bloodbrain
barrier. Evidence suggests that AQP4IgG
is a specific marker of NMO and a causal
agent. Diagnostic testing for AQP4IgG has
enabled identification of a broader spectrum of NMO phenotypes5 and led to the
recognition that the condition of patients
with NMO can deteriorate if treated with
some disease-modifying drugs, making
early accurate diagnosis important in their
clinical management.
One of the biggest clinical impacts of
MS research over the past decade has been
the remarkable expansion in the range of
approved medicines. Six newtreatments have
been approved for the t reatment of relapsing
remitting MS since 2005, including the first
orally administered treatments: fingolimod, dimethyl fumarate and teriflunimide.
US and EU approval of alemtuzumaban
antiCD52 monoclonal antibody that leads
to immune reconstitution after induction
of panlymphocytopeniafor treatment of
MS heralded a new generation of treatments
that are highly effective over long periods,
although alemtuzumab is also associated
with a high rate of serious autoimmunity
related adverse events (for example, thyroid
disease and immune thrombocytopenia).6
The past decade has also seen the first
important steps towards treatments that
limit or halt the worsening of disability in
people with progressive disease. An academically led phaseII trial of high-dose
simvastatin for secondary progressive MS
reached pre-defined clinical and MRI endpoints,7 highlighting the opportunities for
repurposing low-cost drugs. Results of the
RENEW trial suggested that remyelination
promoted by the anti-LINGO antibody
BIIB033 has the potential for neuroprotection, as treatment slowed retinal neuro
degeneration and improved visual function
after acute optic neuritis.8 The next 10years

might see the first treatment become
available for this neglected patientgroup.
The wide variation in convenience,
efficacy and risk of treatments for MS is
fostering a focus on evidence-based personalization of treatment to optimize the
benefit-to-risk ratio for individual patients.
Research into this approach includes the
development of algorithms that can classify
patients according to their risk of disease
progression and enable treatment decisions to be based on subclinical changes and
responses to treatment.
The past 10years of clinical

research in MS have consolidated
a therapeutic success story for
neurology
The biggest development in personalized

treatment for MS in the past decade was
wide implementation of a risk-based model
for personalization of natalizumab treatment. This quantitative model differentiates
patients according to their serum anti-JC
virus antibody status, the length of time for
which they have been using natalizumab and
their history of immunosuppressive therapy.9
As a result, a monitoring programme that
involves regular measurements of serum
anti-JC antibody levels, as well as MRI surveillance and rapid MRI evaluation of new
symptoms that are atypical of MS, is now
recommended in all countries in which
natalizumab is used. The near future could
bring insight into the autoimmune mech
anisms that underlie adverse responses to
alemtuzumab, enabling markers of prospective risk to be identified and increasing
confidence in our u
nderstanding of how the
drug can best be used.
Clinical effectiveness research will be key
in accurately defining the balance between
risk and benefit of treatment for individual
patients. The UK Risk Sharing Scheme
(which ensures access to disease-modifying
treatments for MS through the National
Health Service if patients meet specific
diagnostic criteria and ensures a universal
approach to their follow-up for evaluating
the impact of treatment) has demonstrated
a practical way to couple the results of reallife clinical effectiveness research with
the introduction of new drugs to a health
system. A 2015 report from this large-scale,
pragmatic observational programme confirmed the short-term effectiveness of the
original first-line disease-modifying agents
NEUROLOGY
in a population with relapsingremitting
MS, and showed that this effectiveness
is maintained.10 Global collaborations to
address, at low cost, additional important
questions about patient natural history,
medicine effectiveness and adverse events
are being pioneered by the MSBASE
Consortium and similar organizations
through online registries.
The past 10years of clinical research
in MS have consolidated a therapeutic
success story for neurology. MS is the
first common, chronic neurodegenerative
disease for which treatments that modify
the course of the disease have been successfully developed. The next 10years are likely
to deliver the new treatments indicated specifically for progressive disease and extension of treatment paradigms developed
for MS to the increasing range of other
chronic brain disorders associated with
neuroinflammatorypathology.
Division of Brain Sciences, Department of
Medicine, Hammersmith Hospital, DuCane
Road, London WC12 0NN.
p.matthews@imperial.ac.uk
doi:10.1038/nrneurol.2015.200
Acknowledgements
The author gratefully acknowledges personal
support from the Edmond Safra Foundation and
LilySafra, the Imperial College Healthcare Trust
Biomedical Research Centre, and as an NIHR
SeniorInvestigator.
Competing interests
The author has received funding for his research
from GlaxoSmithKline and Biogen. The author has
received speakers fees or honoraria paid to his
institution from Adelphi Communications, Biogen,
IXICO, GlaxoSmithKline and Novartis.
1.
2.
3.
4.
5.
6.
Hauser, S.L. etal. Bcell depletion with rituximab

in relapsingremitting multiple sclerosis. N. Engl.
J. Med. 358, 676688 (2008).
Louveau, A. etal. Structural and functional
features of central nervous system lymphatic
vessels. Nature 523, 337341 (2015).
International Multiple Sclerosis Genetics
Consortium etal. Genetic risk and a primary
role for cell-mediated immune mechanisms
inmultiple sclerosis. Nature 476, 214219
(2011).
Lennon, V.A., Kryzer, T.J., Pittock, S.J.,
Verkman, A.S. & Hinson, S.R. IgG marker
ofoptic-spinal multiple sclerosis binds to the
aquaporin4 water channel. J. Exp. Med. 202,
473477 (2005).
Tobin, W.O., Weinshenker, B.G. &
Lucchinetti,C.F. Longitudinally extensive
transverse myelitis. Curr. Opin. Neurol. 27,
279289 (2014).
Coles, A.J. etal. Alemtuzumab for patients
withrelapsing multiple sclerosis after
disease-modifying therapy: a randomised
7.
8.
controlled phase3 trial. Lancet 380,

18291839 (2012).
Chataway, J. etal. Effect of high-dose
simvastatin on brain atrophy and disability
insecondary progressive multiple sclerosis
(MSSTAT): a randomised, placebo-controlled,
phase2 trial. Lancet 383, 22132221 (2014).
Multiple Sclerosis Discovery Forum [online]
http://www.msdiscovery.org/researchresources/drug-pipeline/230-biib033 (2015).
9.
Bloomgren, G. etal. Risk of natalizumabassociated progressive multifocal

leukoencephalopathy. N. Engl. J. Med. 366,
18701880 (2012).
10. Palace, J. etal. Effectiveness and costeffectiveness of interferon beta and glatiramer
acetate in the UK Multiple Sclerosis Risk
Sharing Scheme at 6years: a clinical cohort
study with natural history comparator. Lancet
Neurol. 14, 497505 (2015).
DECADE IN REVIEWEPILEPSY
Edging toward breakthroughs

inepilepsy diagnostics and care
Daniel H. Lowenstein
The past decade has yielded a host of important conceptual advances in

epilepsy, along with some promising findings related to diagnostics and
therapeutics. We are on an upswing where precise identification of the
cause of a patients seizure disorder can be matched to therapy that has
a high likelihood of success.
Epilepsy is a disease characterized by recurrent seizures, which are transient signs or
symptoms of abnormal, excessive or synchronous neuronal activity in the brain.
Although the pace of translational discoveries in epilepsy has not yet matched some
of the dramatic achievements seen in fields
such as oncology and infectious disease, a
number of important advances have been
made in the past decade. These advances
have revised our thinking about some core
concepts related to the pathophysiological
mechanisms of epilepsy. Genetic diagnoses
are increasingly available and provide an
opportunity for patient-tailored treatment.
Moreover, on-demand neurostimulation
devices have become available for patients,
and other promising novel treatment
strategies are on the horizon.
The conceptual advances cover the gamut
from the literal definition of epilepsy and
the range of symptoms associated with the
disorder, to the fundamental characteristics
of neuronal network dysfunction that constitute a seizure. Prompted by the problems
created by the prevailing notion that the
diagnosis of epilepsy requires the patient to
have at least two unprovoked seizures, a task
force of the International League Against
Epilepsy recently proposed that the diagnosis can also be established when a patient
has one seizure in the setting of other
factors that make recurrence likely, such as
a family history of epilepsy, or epileptiform
abnormalities seen on EEG.1 This modification to the diagnostic criteria of epilepsy
will have a major impact on direct patient

care in terms of making diagnostic and
treatment decisions, as well as on clinical
research, including epidemiological studies.
we are at the dawn of

precision medicine in epilepsy
Another profound recent innovation in

thinking about epilepsy relates to the range of
behavioural and pathophysiological abnormalities associated with the disorder.2 By far
the dominant conceptualization of epilepsy
has been that it is a brain disease charac
terized by recurrent seizures. However,
this view fails to recognize the existence of
numerous comorbidities in many patients,
including problems in cognition (such as
memory loss, cognitive slowing and autism)
and psychiatric diseases (such as depression,
anxiety and certain personality disorders).
Thus, it seems highly likely that many forms
of epilepsy are complex neuronal network
abnormalities that vary in their timescale
and triggers, with seizures being but one of a
variety of behavioural disturbances.
Our understanding of the basic electrophysiological properties of a seizure has also
evolved in recent years. Until recently, the
canonical view was that a seizure arose from
the hyperexcitability and hypersynchronization of a small network of neurons, with
gradual spread depending on the degree to
which surround inhibition is maintained.
However, recent studies using single microelectrodes demonstrated that a seizure
NOVEMBER 2015 | 60
NEUROLOGY
NPG
begins with micro-seizures emanating

asynchronously from small neuronal clusters, which then coalesce and constitute
the macroscale hypersynchronization that
characterizes most seizures.3 These findings
have important implications regarding our
understanding of the stochastic processes
that seem to govern the transition of a cortical region from the interictal to the ictal
state and, thus, our ability to predict the
occurrence of a seizure.
The past 56years have seen some clear
breakthroughs in our understanding of the
genetic basis of epilepsy. These advances
follow something of a dark age between
2001 and 2009, when some epilepsy-related
genes were discovered among families with
rare epilepsy syndromes that followed a
Mendelian inheritance pattern, but over 100
genome-wide association studies largely produced negative or non-reproducible results.
Arguably the most important discovery,
based on the global collaboration of several
research groups, was the recognition that
denovo mutations are the explanation for a
substantial proportion of patients with socalled epileptic encephalopathiessevere
forms of treatment-resistant epilepsy that
arise early in life and are often associated
with profound developmental delay.4 These
findings have led to expanded use of clinical genetic testing in the routine evaluation
of patients with these syndromes and, as a
consequence, a marked improvement in the
ability to establish a definitive diagnosis and
provide accurate genetic counselling.
The causes of the most common forms
of epilepsygeneralized epilepsy and
nonlesional focal epilepsy, both of which
are thought to have a strong genetic
componentcontinue for the most part to
elude us despite intensive research (although
major improvements in brain imaging have
reduced the number of patients previously
diagnosed as nonlesional). However, two
areas of study have shed light on the genetic
architecture in this group of patients. First,
61 | NOVEMBER 2015
copy number variants, which have been

identified as the basis for a variety of rare
epilepsy syndromes, were shown to account
for at least a few percent of the common
epilepsies.5 Second, a recent large metaanalysis of genome-wide association studies
of this patient population found only three
loci that reached genome-wide significance, providing further evidence that the
common epilepsies are likely to be explained
by the combined influence of numerous
commonvariants of small effect, or multiple
rare variants.6 To adequately assess both
possibilities, further progress will require
the application of next-generation sequen
cing to the analysis of genomes from tens of
thousands ofpatients.
Another major diagnostic advance has
been the recognition that a paraneoplastic
syndrome could be the basis of epilepsy of
particularly explosive and malignant onset
in patients who were previously healthy.7
Interestingly, in some cases, antibodies are
generated against targets that are also implicated in genetic epilepsies. Thus, evaluation
in patients with this presentation increasingly relies on assays for autoantibodies
directed against CNS autoantigens such as
potassium channels or glutamate receptors,
as well as a search for an underlying cancer.
Despite the introduction of well over a
dozen new antiepileptic drugs (AEDs) since
1970, there is little evidence that therapeutic
efficacy has changed. AEDs continue to fail
in approximately 30% of patients, although
some of the new medications arguably offer
better options in terms of ease of use and
adverse effect profile. The past 10years
have, however, seen a definite improvement
in understanding the management of epilepsy in women, as important studies have
demonstrated the effects of specific AEDs
on pregnancy outcome, safety of breast
milk, and the development ofosteoporosis.
The past decade has also seen the introduction of new and promising alternatives to the traditional medical and
surgical approaches for treating epilepsy.
Novel proof-of-principle experiments using
animal models have shown that closed-loop
systems incorporating EEG detection and
optogenetic control of neuronal activity can
rapidly abort seizures at onset, and implantation of inhibitory neuron precursors
can reverse the process of epileptogenesis,
even in the adult animal.8,9 Pivotal clinical
trials have recently led to the approval of
new neurostimulation devices for patients,
including a closed-loop system that detects
incipient seizure activity with intracranial
electrodes, which then deliver focal

stimulation to terminate the seizures.
Finally, the genetic diagnoses described
previously are beginning to provide guidance for treatment decisions. Although the
findings should be considered preliminary,
there are now examples of patients with
defined, causative mutations who respond
to specific drugs prescribed on the basis of
invitro assays of pharmacoresponsiveness.
This step towards individualized treatment
together with the recognition that some of
the underlying mechanisms of epilepto
genesis can be mapped onto distinct cellular pathways (such as mTOR, and pathways
related to synaptic machinery), has led to
the suggestion that we are at the dawn of
precision medicine in epilepsy.10
Department of Neurology, University of
California, San Francisco, 513 Parnassus
Avenue, Room S115, San Francisco,
CA94143, USA.
lowenstein@ucsf.edu
doi:10.1038/nrneurol.2015.193
Acknowledgements
D.H.L. has received research grants from the NIH
(U01NS077274 and U01NS077276).
Competing interests
1.
Fisher, R.S. etal. ILAE official report:

apractical clinical definition of epilepsy.
Epilepsia 55, 475482 (2014).
2. Jensen, F.E. Epilepsy as a spectrum disorder:
implications from novel clinical and basic
neuroscience. Epilepsia 52, 16 (2011).
3. Truccolo, W. etal. Single-neuron dynamics
inhuman focal epilepsy. Nat. Neurosci. 14,
635641 (2011).
4. Epi4K Consortium & Epilepsy Phenome/
Genome Project. Denovo mutations in epileptic
encephalopathies. Nature 501, 217221
(2013).
5. Helbig, I. etal. 15q13.3 microdeletions
increase risk of idiopathic generalized epilepsy.
Nat. Genet. 41, 160162 (2009).
6. International League Against Epilepsy
Consortium on Complex Epilepsies. Genetic
determinants of common epilepsies: a metaanalysis of genome-wide association studies.
Lancet Neurol. 13, 893903 (2014).
7. Seeck, M., Zacharia, A. & Rossetti, A.O.
Autoimmune epilepsy [French]. Rev. Med.
Suisse 6, 925929 (2010).
8. Paz, J.T. etal. Closed-loop optogenetic control of
thalamus as a tool for interrupting seizures after
cortical injury. Nat. Neurosci. 16, 6470 (2013).
9. Hunt, R.F., Girskis, K.M., Rubenstein, J.L.,
Alvarez-Buylla, A. & Baraban, S.C. GABA
progenitors grafted into the adult epileptic
brain control seizures and abnormal behaviour.
Nat. Neurosci. 16, 692697 (2013).
10. EpiPM Consortium. A road map for precision
medicine in the epilepsies. Lancet Neurol.
http://dx.doi.org/10.1016/S14744422(15)00199-4.
NEUROLOGY
DECADE IN REVIEWMOVEMENT DISORDERS
Tracking the pathogenesis

ofmovement disorders
Oksana Suchowersky
Since 2005, we have made substantial progress in understanding

the pathophysiology and natural history of movement disorders such
as Parkinson disease and Huntington disease. However, diseasemodifying therapies for these conditions have proved elusive and,
asaconsequence, treatments remain largely symptomatic.
Over the past few decades, the increasing
recognition of movement disorders as a subspeciality has led to the development of clinical programmes dedicated to diagnosis and
management of disorders such as Parkinson
disease (PD), Huntington disease (HD),
dystonia, tics, and tremors. The formation of multinational collaborative research
groups, such as the Parkinson Study Group
and the Huntington Study Group, has further
enhanced awareness and fuelled expansionof research. Despite improved understanding of the pathophysiology, aetiology
and evolution of these disorders, however,
treatment remains largelysymptomatic.
recently recognized
autoimmune factors result in
paroxysmal and nonparoxysmal
movement disorders
PD is one of the most common neuro

degenerative disorders of ageing, affecting 1
in 100 individuals over the age of 55years.
Diagnosis remains clinical, based on wellrecognized motor symptoms and defined
inclusion and exclusion criteria.1 However,
the past 10years have seen increased recognition of the fact that Lewy body formation and synuclein deposition in
neurons predate the development of motor
symptoms by several decades. At the presymptomatic stage of PD, pathology can
be seen in the anterior olfactory nucleus,
dorsal motor nucleus of the glossopharyn
geal and vagal nerves, and gastrointestinal
nerves. The pathology subsequently spreads
to lower brainstem nuclei before involvement of the substantia nigra. Thus, olfactory dysfunction, anxiety, depression, REM
sleep behaviour disorder and constipation
have been identified as prodromal symptoms of PD, and the development of motor
symptoms indicates moderate disease
(Braakstage3).2
With disease progression, nonmotor

symptoms (NMS) that result from involvement of nondopaminergic neurotransmitter
systems become increasingly evident, resulting in substantial disability and poor quality
of life. As highlighted by the PRIAMO study
published in 2009,3 over 98% of patients with
PD report NMS; the number and severity
of symptoms increase with disease duration. Autonomic dysfunction (constipation,
bladder urgency and postural hypotension),
sleep disturbances and pain require prompt
recognition, as many of these symptoms can
be treated. Over 50% of individuals with PD
develop apathy, fatigue, depression and/or
anxiety;4 cognitive dysfunction and dementia are present in the majority of patients
at the advanced stages. The development
of validated rating scales for both motor
symptoms and NMS has led to improved
monitoring of symptoms. However, reliable
biomarkers to accurately measure disease
progression in PD are still unavailable.
Although descriptions of PD date back
5,000years, its aetiology remains elusive.
Various environmental factors, including
traumatic brain injury, pesticide exposure,
decreased coffee intake and lack of cigar
ette smoking, have been associated with an
increased risk of developing PD. Over the
past decade, however, an important role of
genetic factors, even in sporadic cases, has
become increasingly evident.5 Mutations
in over 10 autosomal recessive and auto
somal dominant genes are now known to
have a major role in at least 10% of affected
families.5 Whole-exome and whole-genome
studies, as well as carefully designed
familystudies, are increasing the number of
identified genetic susceptibility loci. These
discoveries have provided valuable insights
into the pathogenesis of PD, including
common molecular pathways that involve
protein misfolding and aggregation, and
have also enabled the development of new
animalmodels.5
Since 1978, a variety of neuroprotective

or disease-modifying treatments have been
studied in an attempt to slow disease progression in PD. The largest and most recent
study, published in 2015, was NET-PD, a
double-blind, placebo-controlled NIHfunded trial of creatine in 1,741 individuals.6
Like previous trials of potential diseasemodifying treatments in PD, the NET-PD
trial was unsuccessful, perhaps owing to the
fact that the pathology in the study cohort
had progressed to a point at which benefit
was precluded. Intervention in individuals
identified as being at risk of PD could lead to
improved strategies for disease modification
in the future.
HD is an autosomal dominant, slowly
progressive neurodegenerative disorder
characterized by chorea and psychiatric
and cognitive changes. In contrast to PD,
the cause is well known: an expanded CAG
triplet repeat in the huntingtin (HTT) gene
on chromosome4. The disease typically
manifests in midlife, although the age of
onset ranges from early childhood to late
life, depending on the size of the triplet
repeat. Rate of progression also depends
on the repeat size. As in PD, pathological
changes predate symptom onset, with neuro
nal deposition of the abnormal huntingtin
protein identified in the fetal brain.
new insights have been

gained into the premanifest and
early symptomatic stages of PD
and HD
The multinational TRACKHD study

was initiated to determine the natural
history of HD in the premanifest and early
symptomatic stages, and the 36-month data
were reported in 2013.7 Identification of
asymptomatic carriers of the HTT expansion allows long-term evaluation in order
to objectively identify disease onset and
progression, and develop biom arkers.
TRACK-HD showed that atrophy of the
basal ganglia, cortex and white matter, and
loss of structural connectivity between the
basal ganglia and cortex, can be seen on
serial imaging years before symptom onset.
Disease-associated proteins identified in
the cerebrospinal fluid could serve as biomarkers of disease progression, as well as
facilitating the exploration of potential
disease-modifying therapies. Advances in
genetic therapies to silence mutant HTT
gene and mRNA activity, involving zinc
finger proteins, antisense oligonucleotides
NOVEMBER 2015 | 62
NEUROLOGY
and mRNA interference techniques, have
proved to be effective in animal models, and
could lead to successful disease modification
in the nearfuture.8
With respect to other hyperkinetic dis
orders, genotyping has led to improved
classification, as well as an appreciation of
genetic and phenotypic heterogeneity. For
example, mutations in the SLC2A1 gene
that result in GLUT1 deficiency syndrome
can lead to paroxysmal movement disorders
in addition to the classically recognized syndrome of developmental delay and seizures.9
The triplet repeat expansion associated
with spinocerebellar ataxia type2 can cause
ataxia, levodopa-responsive parkinsonism
or amyotrophic lateral sclerosis. A number
of recently recognized autoimmune factors,
such as antiN-methyld-aspartate receptor
antibodies, can result in paroxysmal and
nonparoxysmal movement disorders.10
In summary, over the past decade, the
diagnosis of movement disorders has
improved and expanded with the identification of genetic and environmental factors,
leading to the development of new classifications, validated rating scales, and biomarkers
to measure disease progression. In particular, new insights have been gained into the
premanifest and early symptomatic stages
of PD and HD, which should facilitate the
discovery of strategies for early intervention
in these diseases. Animal models have provided a better understanding of the pathophysiology of movement disorders, and are
enabling the development of new therapeutic approaches. We are optimistic that these
advances will lead to better treatments and
disease modification in the near future.
University of Alberta, 7112Q Clinical Sciences
Building, 1135083 Avenue, Edmonton,
ABT6G 2G3, Canada.
oksana.suchowersky@albertahealthservices.ca
doi:10.1038/nrneurol.2015.201
Competing interests
The author has received research grants from
AbbVie and Biotie. She has received honoraria
fromAbbVie, Allergan and Merz.
1.
2.
3.
Suchowersky, O. etal. Practice parameter:

diagnosis and prognosis of new onset
Parkinson disease (an evidence-based review).
Neurology 66, 968975 (2006).
Tolosa, E., Gaig, C., Santamaria, J. & Compta, Y.
Diagnosis and the premotor phase of Parkinson
disease. Neurology 72 (Suppl. 2), S12S20
(2009).
Barone, P. etal. The PRIAMO study:
amulticenter assessment of nonmotor
symptoms and their impact on quality of life
inParkinsons disease. Mov. Disord. 24,
16411649 (2009).
63 | NOVEMBER 2015
4.
5.
6.
7.
den Brok, M.G. etal. Apathy in Parkinsons

disease: a systematic review and metaanalysis. Mov. Disord. 30, 759769 (2015).
Van der Brug, M.P., Singleton, A, Gasser, T. &
Lewis, P.A. Parkinsons disease: from human
genetics to clinical trials. Sci. Transl. Med. 7,
305ps20 (2015).
Writing Group for the NINDS Exploratory Trials in
Parkinson Disease (NET-PD) Investigators etal.
Effect of creatine monohydrate on clinical
progression in patients with Parkinson disease:
a randomized clinical trial. JAMA 313, 584593
(2015).
Tabrizi, S.J. etal. Predictors of phenotypic
progression and disease onset in premanifest
and early-stage Huntingtons disease in the
TRACK-HD study: analysis of 36-month
observational data. Lancet Neurol. 12,

637649 (2013).
8. Stanek, L.M. etal. Silencing mutant huntingtin
by adeno-associated virus-mediated RNA
interference ameliorates disease
manifestations in the YAC128 mouse model
ofHuntingtons disease. Hum. Gene Ther. 25,
461474 (2014).
9. Leen, W.G. etal. Glucose transporter1
deficiency syndrome: the expanding clinical and
genetic spectrum of a treatable disorder. Brain
133, 655670 (2010).
10. Bigi, S., Hldio, M., Twilt, M., Dalmau, J. &
Benseler, S.M. The growing spectrum of
antibody-associated inflammatory brain
diseases in children. Neurol. Neuroimmunol.
Neuroinflamm. 2, e92 (2015).
DECADE IN REVIEWSTROKE
Progress in acute ischaemic

stroke treatment and prevention
Jose G. Romano and Ralph L. Sacco
Recent decades have seen a dramatic reduction in age-adjusted strokerelated mortality, presumably owing to better control of vascular risk
factors, use of antithrombotic agents and improvements in acute stroke
care. Here, we highlight a few developments in stroke prevention and
acute care that have particularly influenced the care of patients.
Treatment of acute ischaemic stroke has
advanced at a remarkable pace in the past
10years. One important advance was the
broadening of the therapeutic window for
intravenous recombinant tissue plasminogen activator (rtPA) in acute ischaemic
stroke. Two important trials expanded
the original eligibility criteria (initiating treatment within 3h from symptom
onset) for intravenous thrombolytic agents.
The European Cooperative Acute Stroke
StudyIII evaluated the efficacy of thrombolysis conducted 34.5h after the onset
of symptoms.1 Afavourable outcome of no
disability (modified Rankin Scale [mRS]
score 01) was achieved in 52.4% of patients
who were treated with rtPA, in comparison
with 45.2% in the placebo arm of the study,
yielding a significant odds ratio of 1.34 for
the primary outcome of no disability.1 The
third International Stroke Trial demonstrated that individuals older than 80years
derived benefits similar to those seen in
younger patients.2 Recent meta-analysis of
individual patient data from 9 randomized
controlled trials of intravenous rtPA demonstrated better outcomes for patients treated
within 4.5h of symptom onset than for those
given no rtTPA, irrespective of stroke severity and age. The benefit was greater with
earlier thrombolysis (for mRS 01, OR 1.75

at <3h versus OR 1.26 at >3 to <4.5h from
symptomonset).3
Despite these expanded indications,
thrombolytics are of benefit for only a minority of patients with stroke, largely because
of the delay in recognizing symptoms and
the short therapeutic time window. Early
enthusiasm for endovascular recanalization was dampened by three trials published
in2013 that failed to show improvements in
clinical outcomes, highlighting the need for
earlier recanalization, more-effective devices
and appropriate patient selection. Five randomized controlled trials that incorporated
these lessons and were published in 2015
showed substantial benefits of mechanical
endovascular recanalization (MER). These
studies enrolled patients with anterior circulation strokes and large vessel occlusion
(most of whom were treated with intra
venous rtPA) and employed new-generation
stent retrievers. Meta-analysis of the 2015
trials showed that MER after intravenous
rtPA was associated with an increased likelihood of excellent outcomes at 90days (mRS
01 OR 2.59, 95% CI 1.923.48) and functional outcomes (mRS 02 OR 2.48, 95% CI
1.953.15), with no difference in mortality
or symptomatic intracerebralhaemorrhage.4
NEUROLOGY
400
Time to reperfusion (mins)
MR RESCUE*
360
Revascat
IMS3
320
280
SWIFT PRIME
ESCAPE
240
Extend IA
200
0
0
10
20
30
40
50
60
70
80
Percentage of patients with mRS score 2
Figure 1 | Association between time to

recanalization and clinical
in trials
Nature outcomes
Reviews | Neurology
ofmechanical endovascular recanalization.
Across six trials, the mean time from
symptom onset to recanalization* therapy
correlated with the likelihood of low disability
(mRS score2; little or no disability) at
90days. *In the MR RESCUE study, the time
range used was from the latest time point at
which the individual was known to be well to
the groin puncture. Abbreviation: mRS,
modified Rankin Scale.
The key differences between the new

endovascular trials and earlier studies
were a major decrease in the time to
vessel recanalization and tissue perfusion
(Figure1), use of next-generation devices,
and improved selection of patients with
large vessel occlusion.5 Other imaging selection criteria have been applied in individual
trials, including estimation of the infarct
core by diffusion MRI or Alberta Stroke
Program Early CT Score (ASPECTS),
assessment of perfusion mismatch with
CT or MRI techniques, and detection of
adequate collaterals by multiphase CT
angiography. Whether implementation of
these imaging modalities results in better
outcomes or extension of the r ecanalization
window is being investigated.
In light of the current data, the American
Heart Association/American Stroke
Association has updated the guidelines
for acute stroke management, providing a
strong recommendation (Class I, level of
evidenceA) for MER with stent retrievers for patients aged 18years who were
previously independent, were treated with
intravenous rtPA within 4.5hours of stroke
onset, had an NIH Stroke Scale score >6, had
internal carotid or middle cerebral artery
M1 segment occlusion and had anASPECTS
>6, as long as endovascular treatment can
be initiated within 6h ofsymptom onset.6
To realize the benefits of intravenous and
endovascular therapy for acute ischaemic
stroke, reductions in the time to recanal

ization are required; these reductions could
be achieved through improvements to
processes, including prehospital transport
policies and in-hospital workflows, facilitated by data-driven quality performance
improvementprogrammes.
In contrast to the recent success of endovascular devices in acute stroke treatment,
endovascular approaches for stroke prevention have been less successful. Trials
have not demonstrated superiority of
patent foramen ovale closure or stenting of
recently symptomatic intracranial athero
sclerotic disease over usual care, thus
emphasizing the importance of medical
management. Moreover, gaps in the evidence persist regarding the choice between
extracranial carotid artery stenting, carotid
endarterectomy, and medical management for s ymptomatic andasymptomatic
carotidstenosis.
Medical prevention of stroke that results
from atrial fibrillation has advanced dramatically. For many decades, warfarin was
the only effective strategy in this context,
but was under-utilized because warfarin
has a narrow therapeutic range and multiple interactions with medications and foods
making frequent testing and dose adjustment necessary. New non-vitaminK oral
anticoagulants (NOAC) do not require dose
adjustment or monitoring. Currently available agents for non-valvular atrial fibrillation include a direct thrombin inhibitor
(dabigatran) and three factorXa inhibitors
(rivaroxaban, apixaban and edoxaban).
Multiple large international randomized
trials of these drugs have confirmed their
prespecified primary hypotheses. Metaanalysis has confirmed that the available
NOACs reduce stroke or systemic embolism
by 19% when compared with warfarin (RR
0.81, 95% CI 0.730.91) and reduce major
haemorrhagic events by 14% (RR 0.86, 95%
CI 0.731.00).7 The benefits are similar for
those with and without previous cerebral
ischaemic events. Based on the available
evidence, NOACs are recommended in
guidelines for patients with non-valvular
atrial fibrillation who are at risk of stroke.8
Ongoing studies are attempting to develop
ways to rapidly detect and reverse the
anticoagulant effects of NOACs.
Approximately 30% of ischaemic strokes
are classified as cryptogenic, and are usually
treated with antiplatelet agents. Prolonged
electrocardiographic monitoring reveals
atrial fibrillation in a proportion of cryptogenic strokes, though the diagnostic yield
varies with the thoroughness of causative

evaluation and the frequency and modality of monitoring. The Cryptogenic Stroke
and Underlying Atrial Fibrillation study
addressed these concerns; patients with
cryptogenic cerebral ischaemic events
within 90days of extensive diagnostic
testing were randomly assigned to receive
an insertable cardiac monitor or a conventional Holter monitor.9 Among the 441
enrolled patients, the rate of atrial fibrillation detection with continuous monitoring was 8.9%, 12.4% and 30% at 6, 12
and 36months, respectively, compared
with 1.4%, 2% and 3% with conventional
follow-up. Atrial fibrillation was defined
as lasting >30s, but 92.3% of patients had
at least one episode of longer than 6min.
These findings support the recommendation of extended cardiac rhythm monitoring for appropriately selected patients with
cryptogenic stroke, because such monitoring could indicate the need for a change
in therapy. Despite extensive evaluations,
including prolonged cardiac monitoring,
some patients with cryptogenic ischaemic
stroke could be reclassified as having an
embolic stroke of undetermined source.
Ongoing trials are investigating the use of
NOACs versus aspirin for secondary stroke
prevention in this subgroup.
In the coming decade,

research and health policy for
stroke should focus on promoting
healthy lifestyles
Despite a reduction in age-adjusted mortality in recent decades, the global burden of

stroke prevalence and disability is increasing, with a disproportional effect on lowincome countries and minorities.10 With
an ageing population and growing prevalences of obesity, physical inactivity and
diabetes mellitus, the prevalence of stroke
is expected to increase, at a substantial cost
to society. In the coming decade, research
and health policy for stroke should focus
on promoting healthy lifestyles, improving
the control of hypertension and diabetes,
expanding access to health care, enhan
cing systems of care to provide timely
and effective care, exploring better rehab
ilitative measures, and reducing health
disparities. The many successes of the last
decade for stroke prevention and treatment
should provide a solid foundation for continued progress, but many challenges are
stillahead.
NOVEMBER 2015 | 64
NEUROLOGY
University of Miami, Miller School of Medicine,
1120NW 14th Street, Miami, FL 33136, USA
(J.G.R., R.L.S.).
Correspondence to: R.L.S.
rsacco@med.miami.edu
doi:10.1038/nrneurol.2015.199
Acknowledgements
J.G.R. and R.L.S. have received research salary
support from the NIH/NINDS (1R01NS29993,
R01NS40807 and and 1R01NS084288).
Competing interests
J.G.R. has received research support for MaRISS
Study and honoraria from Genentech for a steering
committee role, and honoraria and stock from Vycor
NovaVision for a scientific advisory board role.
R.L.S. has consulted for Boehringer Ingelheim as
co-chair of the RESPECT-ESUS trial with dabigatran
versus aspirin for secondary stroke prevention.
1.
Hacke, W. etal. Thrombolysis with alteplase 3

to 4.5 hours after acute ischemic stroke.
N.Engl. J.Med. 359, 13171329 (2008).
2. IST3 collaborative group. etal. The benefits
andharms of intravenous thrombolysis with
recombinant tissue plasminogen activator
within6h of acute ischaemic stroke (the third
international stroke trial [IST3]): a randomized
3. Emberson, J. etal. Effect of treatment delay,
age, and stroke severity on the effects of
intravenous thrombolysis with alteplase for
acute ischaemic stroke: a meta-analysis of
individual patient data from randomised trials.
Lancet 384, 19291935 (2014).
4. Falk-Delgado, A., Kuntze Sderqvist, .,
Fransn, J. & Falk-Delgado, A. Improved clinical
outcome 3months after endovascular
treatment, including thrombectomy, in patients
with acute ischemic stroke: a meta-analysis.
J.Neurointerv. Surg. http://dx.doi.org/
10.1136/neurintsurg-2015-011835.
5. Prabhakaran, S., Ruff, I. & Bernstein, R.A.
Acute stroke intervention: a systematic review.
JAMA 313, 14511462 (2015).
6. Powers, W.J. etal. 2015 AHA/ASA focused
update of the 2013 guidelines for the early
management of patients with acute ischemic
stroke regarding endovascular treatment:
aguideline for healthcare professionals from
the American Heart Association/American
Stroke Association. Stroke http://dx.doi.org/
10.1161/STR.0000000000000074.
7. Ruff, C.T. etal. Comparison of the efficacy and
safety of new oral anticoagulants with warfarin
in patients with atrial fibrillation: a metaanalysis of randomised trials. Lancet 383,
955962 (2014).
8. Kirchhof, P. etal. Atrial fibrillation guidelines
across the Atlantic: a comparison of the
currentrecommendations of the European
Society of Cardiology/European Heart
RhythmAssociation/European Association of
Cardiothoracic Surgeons, the American College
of Cardiology Foundation/American Heart
Association/Heart Rhythm Society, and the
Canadian Cardiovascular Society. Eur. Heart J.
34, 14711474 (2013).
9. Sanna, T. etal. Cryptogenic stroke and
underlying atrial fibrillation. N.Engl. J.Med.
370, 24782486 (2014).
10. Feigin, V.L. etal. Global and regional burden of
stroke during 19902010: findings from the
Global Burden of Disease Study 2010. Lancet
383, 245254 (2014).
65 | NOVEMBER 2015
DECADE IN REVIEWMIGRAINE
Incredible progress for an era

ofbetter migraine care
Peter J. Goadsby
In the past 10years, the realization that migraine is a brain disorder

rather than a vascular disorder has facilitated development of various
treatments, ranging from innovative immunopharmaceuticals through
to neurostimulation. Many clinical trials have been successful, and
such considerable progress holds promise for the coming decade of
migrainetreatment.
Migraine has been the sixth most common
cause of disability in the world since 1990.
In the same period, medication overuse
headachewhich seems only to occur in
patients with migrainehas emerged from
below 25th to rank 18th. By contrast, progress
has been made with many of the other top
causes: anxiety disorders, iron-deficiency
anaemia and diarrhoeal diseases, to name
just a few.1 This burden of migraine poses
a challenge, but increasing recognition of
the condition offers opportunities. In the
past decade, laboratory and translational
neurobiology has pushed towards a better
understanding of migraine and new therapies. Here, I will highlight the therapies that
are emerging from this work.
An important theme of research in the
past decade has been the steady accumulation of data that point to migraine as a brain
disorder rather than a vascular disorder.
Imaging studies in humans have focused
this discussion further; for example, one
beautifully executed study used magnetic
resonance angiography to produce clearly
negative results and further demonstrate
that no important vascular change occurs
in migraine.2 Moreover, investigation of the
premonitory phase of migraine also indicates brain targets and emphasizes the need
to understand the pathogenesis of attacks in
order to develop new therapies.3
As the biology of migraine has been
unravelled, further questions with clinical implications have arisen. For example,
a carefully conducted study showed that a
light challenge did not trigger a migraine
in any participants who thought their
migraines could be triggered by light.4 This
observation raises the question of whether
the fact that patients notice light in the
premonitory phase contributes to their
attribution of light as a trigger. If so, the
possibility exists that similar phenomena
explain some aspects of migraine triggered
by food, sleep or even stress, as each could

be the result of alterations in brain function, possibly subcortical.5 Such questions
illustrate the point that a greater understanding of the pathogenesisof migraine
would facilitate the development of therapy
and enable clinicians to provide better basic
managementadvice.
An understanding of migraine as a brain
disorder has freed the development of
medicine from the shackles and concerns
of the cranial vessels. As a result, such development in the past decade has been driven
towards targets other than vasoconstrictors
(Supplementary Table1). The most widely
explored candidates have been treatments
that target the calcitonin gene-related
peptide (CGRP) pathway.
The CGRP pathway was first suggested as
a target by translational studies conducted
at the end of the 1980s6,7 that pointed away
from involvement of inflammatory targets.
Studies conducted within the past decade
in which CGRP has been targeted are providing substantial advances in knowledge
that even extend beyond migraine. First,
the general principle that a migraine medicine can either target an acute attack or
prevent attack onset seems to be completely
incorrect: targeting the CGRP pathway is
effective in both contexts (Supplementary
Table1). This finding suggests that further
interrogation of the biology of migraine,
for example further investigation of the
trigeminovascular system, could yield
additional targets for therapeutics that
provide both attack prevention and acute
alleviationwonderfully unorthodox, and
certainly encouraging. Second, targeting
the CGRP pathway drives home the fact
(borne out by the effect of aspirin and the
5HT1F receptor agonist lasmiditan) that
non-vasoconstrictors work: human studies
have shown that CGRP receptor antagonists
are notvasoconstrictors.8
NEUROLOGY
Furthermore, development of the CGRP
monoclonal antibody is the first example of
immunopharmacology in which antibodies
target a molecular pathway instead of modi
fying immune pathways. Antibodies are
highly specific and can target systems that
were previously inaccessible to therapeutics, making antibody therapy a p
romising
strategy for the treatment of migraine.
Millennia after migraine was

first reported, clinicians will be
able to suggest migraine-specific
preventative treatments...
The past 10years have also taught us

lessons about other potential targets for
migraine therapy. A decade ago, nitric
oxide seemed to be a promising target, yet
still eludes. Intravenous administration of
the nonspecific neuronal nitric oxide synthase (NOS) inhibitor 546C88 was effective.
However, tests of inducible NOS as a target
in both acute attack and prevention failed.
These results, together with failures of four
substancePneurokinin1 receptor antagonists, two neurogenic plasma protein extra
vasation antagonists and a TRPV1 receptor
antagonist (Supplementary Table1), indicate
that the role of inflammatory mechanisms
in migraine must be reconsidered and that
therapeutic targeting of these mechanisms
needs rethinking.
Similarly, orexinergic targets seemed
attractive 10years ago on the basis of basic
research, but have now been shown to be
ineffective, at least when both orexin receptors are targeted (Supplementary Table1).
Testing of these targets is incomplete, as
proper targeting of just one or other orexin
receptor might have anti-migraine effects.
Certainly, dual receptor antagonists are
impractical because their sleep-inducing
effects preclude adequate target coverage during the day. Candesartan, which
was developed as an angiotensin receptor antagonist, has been established over
the past decade as a migraine preventative.
However, a negative study with telmisartan
(Supplementary Table1), another angiotensin receptor antagonist, suggests that the
benefit of candesartan is the result of more
than its action on the angiotensin receptor.
Glutamatergic mechanisms are expected
to be involved in migraine, and their
manipulation could offer a neuronal target
(Supplementary Table1). As in other brain
disorders, development of medicines
that target glutamate receptors without
problematic adverse effects has not been

easy. Experimental and clinical studies9 have
demonstrated that the Nmethyldaspartate
receptor is a plausible target in the management of migraine aura, yet the practical use
will be limited unless a way around adverse
side effects is determined. Studies of the
AMPA and kainate (iGluR5) receptors
suggest that targeting the kainate receptor
will be the best way forward (Supplementary
Table1). Targeting of the mGluR5 receptor has already been established as a
next-generation approach.
With the knowledge that migraine is a
brain disorder, neuromodulation seems
an attractive option, and has been tried
with several approaches in the past decade.
Invasive approaches, such as occipital nerve
stimulation, initially seemed promising,
but have been beset by problems with study
design and device suitability. No positive,
well-designed, placebo-controlled study
has yet been completed (Supplementary
Table1), but newer devices and betterdesigned studies could lead to a future role
for invasive techniques.
Noninvasive approaches are particularly
attractive because they carry a low risk, so
little is lost if they do not work. Transcranial
magnetic stimulation and transcutaneous
supraorbital nerve stimulation are now used
in many expert centres in Europe and the
USA. Positive studies have been published
(Supplementary Table1), although the data
are limited. Similarly, noninvasive vagal nerve
stimulation has been studied but is yetto find
its place. Clinical experience suggests to me
that devices, when they are effective, are
truly neuromodulatory but require time and
diligent application over several months to
produce effects that can be excellent.
The future is undoubtedly bright for the
development of new treatments for migraine.
In addition to targets that have already been
tested in clinical studies, one can see other
targets emerging, such as the mGluR5 receptor, pituitary adenylate-cyclase activating
peptide, acid sensing ion channels, neuro
nal nitric oxide synthesis inhibitors and
individual orexin receptors (Supplementary
Table1), to drive the next decade forward.
Millennia after migraine was first reported,
clinicians will be able to suggest migrainespecific preventative treatments to patients
with migraine. Furthermore, some of the
therapies developed to date could be bene
ficial for cluster headache and medication
overuse headache. Experimental medicine
offers a clear pathway from basic biology to
the clinic. One hopes that, as next-generation
genetics evolves,10 the upcoming decade will

see the development of personalized medicine that will see migraine lose its position
among the top of causes of global disability.
Basic and Clinical Neurosciences, Institute of
Psychiatry, Psychology and Neuroscience, and
Kings Clinical Research Facility, Kings College
London, Wellcome Foundation Building, Kings
College Hospital, London SE5 9PJ, UK.
peter.goadsby@kcl.ac.uk
doi:10.1038/nrneurol.2015.203
Competing interests
The author has received grants and personal fees
from Allergan, Amgen and eNeura, and personal
feesfrom Ajinomoto Pharmaceuticals, Akita
Biomedical, Alder Biopharmaceuticals, Autonomic
Technologies, Avanir Pharmaceuticals, Cipla,
CoLucidPharmaceuticals, Dr Reddys Laboratories,
EMKinetics, Ethicon, Heptares, Lilly, NEJM Journal
Watch, Pfizer, Promius Pharma, Teva, UpToDate, Wells
Fargo, W. L. Gore & Associates and Zosano Pharma.
He has also received personal fees for medicolegal
work for individuals. He also has a patent pending for
magnetic stimulation for headache.
1.
Global Burden of Disease Study 2013

Collaborators. Global, regional, and national
incidence, prevalence, and years lived with
disability for 301 acute and chronic diseases
and injuries in 188 countries, 19902013:
asystematic analysis for the Global Burden of
Disease Study 2013. Lancet 386, 743800
(2015).
2. Amin, F.M. etal. Magnetic resonance
angiography of intracranial and extracranial
arteries in patients with spontaneous migraine
without aura: a cross-sectional study. Lancet
Neurol. 12, 454461 (2013).
3. Maniyar, F.H., Sprenger, T., Monteith, T.,
Schankin, C. & Goadsby, P.J. Brain activations
in the premonitory phase of nitroglycerintriggered migraine attacks. Brain 137,
232241 (2014).
4. Hougaard, A., Amin, F., Hauge, A.W., Ashina, M.
& Olesen, J. Provocation of migraine with aura
using natural trigger factors. Neurology 80,
428431 (2013).
5. Akerman, S., Holland, P. & Goadsby, P.J.
Diencephalic and brainstem mechanisms in
migraine. Nat. Rev. Neurosci. 12, 570584
(2011).
6. Goadsby, P.J., Edvinsson, L. & Ekman, R.
Vasoactive peptide release in the extracerebral
circulation of humans during migraine
headache. Ann. Neurol. 28, 183187 (1990).
7. Goadsby, P.J., Edvinsson, L. & Ekman, R.
Release of vasoactive peptides in the
extracerebral circulation of humans and the
catduring activation of the trigeminovascular
system. Ann. Neurol. 23, 193196 (1988).
8. Ho, T.W., Edvinsson, L. & Goadsby, P.J. CGRP
and its receptors provide new insights into
migraine pathophysiology. Nat. Rev. Neurol. 6,
573582 (2010).
9. Ayata, C., Jin, H., Kudo, C., Dalkara, T. &
Moskowitz, M.A. Suppression of cortical
spreading depression in migraine prophylaxis.
Ann. Neurol. 59, 652661 (2006).
10. Tolner, E.A. etal. From migraine genes to
mechanisms. Pain 156 (Suppl. 1), S64S74
(2015).
version of the paper at www.nature.com/nrneurol.
NOVEMBER 2015 | 66
DECADE IN REVIEWCNS INFECTIONS
Major advances against a moving

target of CNS infections
Lisa F. P. Ng and Tom Solomon
CNS infections have severe manifestations, often leading to high

mortality, but the CNS is usually not the primary target of pathogens,
leaving a window of opportunity to prevent neuroinvasion. We must
prioritize development of therapies to prevent neurological sequelae
thatcause long-lasting morbidity and disease burden on society.
The Nobel Prize in Physiology or Medicine
in 2015 was awarded jointly for discoveries
concerning novel therapies against malaria
and infections caused by roundworm parasites, highlighting the global importance of
infectious disease. The past decade has seen
some considerable advances in the diagnosis and treatment of CNS infections,
despite some major challenges, such as
the emergence of novel pathogens, the
spread of existing pathogens to new geographical
areas as a result of travel
and climate change, and
more-severe outbreaks
caused by existing pathogens becoming more
virulent, and increasing
numbers of immuno
compromised people. As
organizers of the Gordon Research
Conferences on Infect ions of the
Nervous System in 2013 and 2015, we
have had the opportunity to closely follow
the advances made in the prevention and
treatment of CNS infections. Despite these
advances, many unmet challenges remain,
including the rise of resistance to the anti
malarial drug artemisin in patients with
severe cerebral malaria, which has increased
morbidity and mortality, re-emergence of
arbovirus-induced CNS disease, limited
treatment options for cryptococcal meningitis, and failure of the Japanese encephalitis
vaccine, to name a few.
In the USA and developed European
countries, viral encephalitis, bacterial
meningitis and Lyme neuroborreliosis
have beenstudied fervently. Less attention
has been given to potentially fatal nervous
system invasion by neurotropic viruses,
parasites or mycobacteria; encephalitides caused by these pathogens are more
common in developing than developed
countries, and are an important health issue
in resource-poor regions, because many of
67 | NOVEMBER 2015
the survivors are left with disability that

reduces health-related quality of life and
causes a great socioeconomicburden.
The epidemiological changes that have
occurred in recent years have placed greater
importance on recently emerged
pathogens, such as the Nipah and
Chikungunya viruses acrossAsia,
and West Nile virus across
the Americas. 1 Meanwhile,
enterovirus71, has continued to cause mass outbreaks of hand, foot and
mouth disease across Asia,
which is associated with
neurological complications that can manifest as meningitis or
severe brainstem encephalitis.2
These neurological complications can, via hitherto unknown
mechanisms, lead to cardiovascular collapse and death.2 Enterovirus71
has even been described by some as the
new polio, because it belongs to the same
enterovirus groupIV as poliovirus, and can
cause acute flaccid paralysis that mimics
poliomyelitis. No antiviral agent or vaccine
against enterovirus71 is currently available.
The arthropod-borne Chikungunya virus,
which was virtually unknown a decade ago,
also causes mass outbreaks of febrile disease.
Chikungunya infections are characterized
by fever, headache, rashes, and debilitating
arthralgia that can continue for years after
infection. Atypical manifestations have
also been described, and include neurological symptoms that range from simple
and complex febrile seizures to meningeal
syndrome,acute encephalopathy, diplopia,
aphasia, acutedisseminated encephalomyelitis, and encephalitis.3 After its first
reported appearance in 1952, the virus
re-emerged in the Indian Ocean islands
in 20052006, and several outbreaks of
Chikungunya infection have occurred since
late 2013, particularly in the Caribbean

islands and the Americas, but also in
several countries in Africa and Southern
Europe. Increased global travel, evolution
of the virus and adaptation of mosquito
vectors have all been suggested as contributing factors to the spread of the virus to
new geographical areas.3 No vaccination or
treatment for Chikungunya virus exists, and
avoiding mosquito bites is c urrently the only
way to prevent thedisease.
Many common infections, including the
big threeHIV, tuberculosis and malaria
have important neurological manifestations. Outbreaks of new influenza strains,
such as H5N1, have also highlighted the
potential of such viruses to cause neurological disease.4 In recent years, the number of
immunocompromised individuals who are
particularly susceptible to CNS infection
has increased owing to the HIV epidemic
and increased use of immunosuppressive
treatments, for example to induce immune
tolerance after organ transplantation or to
treat autoimmune disorders. Neurologists
should be particularly aware of the risk of
CNS infection in patients who are immunocompromised, because many neurological
disorders are treated with immunosuppressive therapies. Perhaps the most prominent
example of such disorders is multiple sclerosis (MS), for which several new drugs have
been approved in the past decade. Similarly,
progressive multifocal leukoencephalo
pathy caused by JC virus was once rare, but
its prevalence has increased with that of
HIV, and in the past 10years, the use of new
immunosuppressive monoclonal antibodies,
such as natalizumab for MS, has become
another important trigger for thedisease.5
During the same period, immune-
mediated encephalitis, such as antiNmethyl-daspartate receptor encephalitis,
has been established as a cause of encephalitis that is as important as are infections.6
Occasionally, some infections of the
nervous system, such as herpes simplex
virus, can also trigger autoimmune
encephalitis.
Improved technologies and diagnostics, such as multiplex PCR,
MALDI-TOF (matrix-assisted
laser desorption/ionization
t ime-of-f light)
NPG
NEUROLOGY
NEUROLOGY
mass spectometry, better immunoassays
and metagenomic next-generation sequen
cing, have improved the speed and accuracy
of discovering pathogens and identifying
immune system correlates of protection from
infectious disease. Despite these advances,
the proportion of patients with CNS infections who are misdiagnosed remains
stubbornlyhigh.6
Treatment of some brain infections,
including acute bacterial meningitis and
tuberculous meningitis, has improved over
the past decade owing to a better understanding of the clinical features and the ways
in which they contribute to poor outcomes.7
Moreover, the mechanism of corticosteroids
in the treatment of some ofthese conditions
has been better defined: the benefit of dexamethasone has been demonstrated in adults
with acute bacterial meningitis in most developed countries, as has its ability to reduce
the incidence and severity of hearing loss in
pneumococcal disease. However, the drug
is often of no benefit to adults or children
in developing, tropical countries, perhaps
because patients in these countries are more
likely to have untreated HIV.8 In Vietnamese
adults with tuberculous meningitis, dexamethasone reduced mortality and is now
used for most patients with this condition.
In the past 10years, no major advances
have been made in the treatment of encephalitis beyond the use of corticosteroids. In
mouse studies, monoclonal antibodies have
shown promise for the treatment of some
viral encephalitides, such as those caused
by West Nile virus, Chikungunya virus and
Japanese encephalitis virus.9 RNA interference has been proposed as another treatment strategy, but the only encephalitides
in which it has been studied to date are
Japanese encephalitis, tick-borne encephalitis andencephalitis caused by alphaviruses,
so it is a long way from clinical applications.
The Holy Grail against CNS infections
effective neuroprotective treatment
remains elusive. Therapeutic hypothermia,
which has proved useful in hypoxic brain
injury after cardiac arrest, was found to be
of no benefit in bacterial meningitis. Novel
cocktails of antivirals and potentially neuroprotective drugs initially looked promising,
but have, in most cases, failed to work.
Vaccination programmes have brought
some of the biggest successes in the prevention of CNS infections. Advances include the
development of new vaccines for pneumococcal meningitis andJapanese encephalitis,10 and, in the case of Japaneseencephalitis,
improved administration methods and
dosage of existing vaccines. By the time
of the next Gordon Research Conference
on Infections of the Nervous System in
2017, these developments should have had
considerable impact.
Singapore Immunology Network, Agency for
Science, Technology and Research (A*STAR),
8A Biomedical Grove, #0406 Immunos,
Biopolis, Singapore 138648, Singapore
(L.F.P.N.). Institute of Infection and Global
Health, University of Liverpool, Ronald Ross
Building, 8 West Derby Street, Liverpool
L697BE, UK (T.S.).
Correspondence to: L.F.P.N.
lisa_ng@immunol.a-star.edu.sg
doi:10.1038/nrneurol.2015.202
Competing interests
1.
Jones, K.E. etal. Global trends in emerging

infectious diseases. Nature 451, 990993
(2008).
2. Chang, L.Y. etal. Neurodevelopment and
cognition in children after enterovirus71
infection. N.Engl. J.Med. 356, 12261234
(2007).
3. Weaver, S.C. & Lecuit, M. Chikungunya virus
infections. N.Eng. J.Med. 372, 12311239
(2015).
4. de Jong, M.D. etal. Fatal outcome of human
influenza A (H5N1) is associated with high
viralload and hypercytokinemia. Nat. Med. 12,
12031207 (2006).
5. Kleinschmidt-DeMasters, B.K. & Tyler, K.L.
Progressive multifocal leukoencephalopathy
complicating treatment with natalizumab
andinterferon beta1a for multiple sclerosis.
N.Eng. J.Med. 353, 369374 (2005).
6. Granerod, J. etal. Causes of encephalitis
anddifferences in their clinical presentations
inEngland: a multicentre, population-based
prospective study. Lancet Infect. Dis. 10,
835844 (2010).
7. Weisfelt, M., van de Beek, D., Spanjaard, L.,
Reitsma, J.B. & de Gans, J. Clinical features,
complications, and outcome in adults with
pneumococcal meningitis: a prospective case
series. Lancet Neurol. 5, 123129 (2006).
8. van de Beek, D. etal. Adjunctive dexamethasone
in bacterial meningitis: a meta-analysis of
individual patient data. Lancet Neurol. 9,
254263 (2010).
9. Oliphant, T. etal. Development of a humanized
monoclonal antibody with therapeutic potential
against West Nile virus. Nat. Med. 11, 522530
(2005).
10. Tauber, E. etal. Safety and immunogenicity
ofaVerocellderived, inactivated Japanese
encephalitis vaccine: a non-inferiority, phaseIII,
randomised controlled trial. Lancet 370,
18471853 (2007).
NOVEMBER 2015 | 68
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RHEUMATOLOGY
DECADE IN REVIEWTRANSLATIONAL RHEUMATOLOGY
Tenyears after: rheumatology research from

bench to bedside
Nunzio Bottini and Gary S.Firestein
Defining key advances in any medical discipline can be challenging, but is especially so in rheumatologya
rapidly advancing field so broad that it defies traditional classifications. Here, we approach the Sisyphean
taskofsummarizing the translational advances in rheumatology in the past decade within several broad
categories ofbasic research.
Translational studies in rheumatology from
the past decade are especially prominent
inthe field of cytokine biology. For instance,
a bench-to-bedside and back loop was
spanned by the quest to understand
IL23IL17 pathobiology. Since IL17producing Thelper (TH)17 cells were first
characterized in 2005 and IL23 defined as
a critical cytokine for their differentiation,
abundant data implicate this pathway in
rheumatic diseases. For example, enthesisresident Tcells that produce IL17 and
IL22 in response to IL23 stimulation
mediate inflammation and new bone formation in a preclinical model of spondylo
arthropathy (SpA).1 Clinical trials of agents
targeting this cytokine axis in SpA have
been spectacular: the anti-IL12/IL23 antibody ustekinumab received FDA approval
for psoriasis in2009 and for psoriatic arthritis (PsA) in 2013, changing the therapeutic
landscape of the former. Theanti-IL17
antib ody secukinumab is also effective
in psoriasis and PsA. Despite high hopes
for IL23IL17 blockade in rheumatoid arthritis (RA), these agents unfortunately showed only modest efficacy in this
setting(Figure1).
IL1 inhibition lost its lustre owing
to the disappointing results in RA, but
enthusiasm re-emerged in the past decade
withthe discovery of a critical role for
IL1 in the inflammas ome and auto
inflammatory diseases. Ultimately, three
anti-IL1 agents demonstrated remarkable
efficacy in cryopyrin-associated periodic
syndromes (CAPS) and were approved for
this use in the USA. The inflammasome and
endogenous danger signals also participate
in crystal-induced diseases, such as gout,
for which anti-IL1 agents are effective.
A newly identified anti-inflammatory

member of the IL1 family, IL37, might
also have therapeutic potential as a target
in autoinflammatorydiseases.
Interferons continue to attract considerable attention in rheumatology, building on
the 2003 observation that leukocytes from
patients with systemic lupus erythematosus
(SLE) have a typeI interferon (IFN) trans
criptome signature. In the past decade,
multiple translational studies focused on
validating typeI IFN as a drug target for SLE
and other autoimmune diseases. However,
modest efficacy of the typeI IFN-blocking
antibody rontalizumab reported in 2015,
as well as experience with B-cell-targeted
approaches, exemplified the persistent
IL-23
IL-17
IL-6
difficulty of approaching a complex disease

such as SLE with traditional clinical
trialdesigns.
Antibodies against two classical cytokine
targets that were the object of translational
efforts have entered clinical development:
targeting of granulocyte-macrophage
colony-stimulating factor (GM-CSF), first
described in the context of RA >25years
ago, has now been validated using blocking antibodies in RA. An open-label trial
showed that the transforming growth
factor (TGF-)-blocking antibody fresolimumab in systemic sclerosis effectively
modulated key predictive fibrosis biomarkers.2 At the preclinical level, IL27, an
IL12 family member highly expressed in
IL-1
B-cell
survival
B-cell
depletion
?
SpA
PsA
Psoriasis
JAK
RA
Autoinflammatory
disease
Systemic
sclerosis
SLE
GM-CSF
PAD
TGF-
Type I IFN
ANCA+
vasculitis
Other kinases
(e.g. BTK, PI3K)
Robust clinical efficacy
Modest clinical efficacy
Preclinical or biomarker data
Figure 1 | Key therapeutic targets for rheumatic diseases explored

in theReviews
past decade.
Nature
| Rheumatology
Targetsfor which agents exist with robust or modest clinical efficacy are presented, as well
astargets for which preliminary or preclinical data support agents currently under development.
Bcell depletion as a therapeutic strategy in SLE is still uncertain. Colours represent cytokines
(blue), cell-targeted therapy (green), enzymes (orange) and diseases (purple). Abbreviations:
ANCA, anti-neutrophil cytoplasmic antibody; GMCSF, granulocyte-macrophage colony-stimulating
factor; IFN, interferon; JAK, Janus-activated kinase; PAD, peptidylarginase deiminase;
PsA,psoriatic arthritis; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus;
SpA,spondyloarthropathy; TGF, transforming growth factor .
NOVEMBER 2015 | 69
RHEUMATOLOGY
the RA synovium, was effective in models
of experimental arthritis, and might have
potential for translation.
In addition to strategies aimed at blocking individual cytokines, approaches that
target cell lineages implicated in patho
genesis have progressed in the last decade.
Studies demonstrating the critical role of
antibodies and Bcells in anti-neutrophil
cytoplasmic antib ody (ANCA)-positive
vasculitis paved the way for FDA approval
of rituximab in2011. Bcells also received
much attention in SLE research: the TNF
superfamily member Bcell stimulating
factors BAFF (TNF ligand superfamily
member13B) and APRIL (TNF ligand
superfamily member13), as well as their
receptors, have been explored as possible
therapeutic targets. The anti-BAFF anti
body belimumab received FDA approval
for SLE in 2011, although its benef its
are limited and utilization in the clinic is
currentlymodest.
Neutrophils have also emerged as critical players in SLE and RA. A seminal
2011 report showed that neutrophils from
patients with SLE extrude portions of their
nuclei and cytoplasm (neutrophil extra
cellular traps, or NETs) in a process called
NETosis. 3 This mechanism, now recognized as a critical pathway for presentation
of altered antigens and self-antigens, is a
key mediator of innate immunopathology in SLE. Importantly, neutrophils are
also a source of citrullinated proteins and
enzymes that convert arginine to citrulline
(peptidylarginine deiminases, or PADs) in
RA, and enzyme-activating antibodies specific for neutrophil PAD might contribute
to RA pathogenesis.4 Interestin targeting
fibroblast-like synoviocytes ininflammatory arthritis was re-ignited this past
decade by the identification of the transmembrane molecule cadherin11 as an
important regulator of cartilage damage
and synovial intimal lining formation.5
The success of therapies based on tyro
sine kinase inhibitors in oncology led to
the notion that similar approaches might
be benef icial in inflammation. Building
upon pioneering studies showing that tyro
sine protein kinase JAK (Janus kinase)3deficient individuals exhibit severe
immunod eficiency, the JAK inhibitor
tofacitinib was developed and approved
for the treatment of RA in 2012. Biopsy
studies showed that tofacitinib is effective
in blocking synovial JAK1STAT3 and
JAKSTAT1 signaling downstream of IL6
and typeI IFN.6
70 | NOVEMBER 2015
However, results in the signaling research

realm have been mixed. For example, fostamatinib (a small-molecule inhibitor of
spleen tyrosine kinase SYK) and multiple
mitogen-activated protein kinase (MAPK)
p38 inhibitors were only modestly effective in RA; back-to-bench studies later
revealed that p38 inhibition increases
activation of other MAPKs and suppresses
anti-inflammatory cytokines like IL10.
Despite these setbacks, kinases remain
high-profile targets in rheumatology: the
tyrosine kinase BTK, a regulator of Bcell
function and innate immunity, and PI3K
(phosphatidylinositol 4,5-bisphosphate
3kinase catalytic subunit isoform, which
regulates both Bcell and synoviocyte functions), are among the kinases currently
under research.
The impressive progress in the genetics
of rheumatic diseases in the past decade
is in part due to the success of genomewide association studies (GWAS). GWAS
in ankylosing spondylitis and RA have
identified clear associations with IL23
and CD40, respectively,7,8 leading to their
explora tion as drug targets. However,
limitations of GWAS have emerged as
well: despite large meta-analyses that
can detect increasing numbers of loci
with ever-smaller effects on riskover
a hundred locihave been identified in
RA to date8GWAS have been unable to
capture most of the risk and severity influences on complex rheumatic diseases.
New epig enetic strategies developed
almost entirely in the past decade might
help fill the gaps left by GWAS. Genomewide epigenetic studies have already been
conducted in blood cells from patients
with SLE, RA or other autoi mmune diseases, in synoviocytesfrom patients with
RA and chondrocytes from patients with
osteoarthritis, with promisingresults.
The study of mucosal microbial populations and how microbial dysbiosis affects
disease is emerging as a new and important
field in rheumatology. High prevalence of
Prevotella copri was identified in the gastro
intestinal microbiome of patients with
early RA,9 and other studies showed that
PADs produced by the gingival pathogen
Porphyromonas gingivalis could contribute
to protein citrullination.
Looking into the future, translational
studies will hopefully provide clues on the
best ways to select patients for personalized approaches to therapy. Current biomarker research is increasingly driven by
knowledge of disease mechanisms: one
possible example was the identification of

CXC motif chemokine4 (CXCL4, also
known as platelet factor4) as a biomarker
in systemic sclerosis in 2014.10 New technology of the past decade, including the
advent of next-generation sequencing,
will enable integrative analyses of multiple
omics platforms and capture a degree
of complexity previously unattainable.
These developments should pave the way
for new algorithms to identify individual
versions of SLE, RA and other rheumatic
diseasesadvances that will hopefully
occur in the next 10years.
La Jolla Institute for Allergy and Immunology,
9420 Athena Circle, La Jolla, CA 92037, USA
(N.B.). UCSan Diego School of Medicine,
9500Gilman Drive, La Jolla, CA 92093, USA
(N.B., G.S.F.).
Correspondence to:
nunzio@lji.org
gfirestein@ucsd.edu
doi:10.1038/nrrheum.2015.126
Competing interests
1.
Sherlock, J.P. etal. IL23 induces

spondyloarthropathy by acting on ROR-t+
CD3+CD4CD8 entheseal resident Tcells.
Nat.Med. 18, 10691076 (2012).
2. Rice, L.M. etal. Fresolimumab treatment
decreases biomarkers and improves
clinicalsymptoms in systemic sclerosis
patients. J.Clin. Invest. 125, 27952807
(2015).
3. Villanueva, E. etal. Netting neutrophils induce
endothelial damage, infiltrate tissues, and
expose immunostimulatory molecules in
systemic lupus erythematosus. J. Immunol.
187, 538552 (2011).
4. Darrah, E. etal. Erosive rheumatoid arthritis
isassociated with antibodies that activate
PAD4 by increasing calcium sensitivity.
Sci.Transl. Med. 5, 186ra65 (2013).
5. Lee, D.M. etal. Cadherin11 in synovial lining
formation and pathology in arthritis. Science
315, 10061010 (2007).
6. Boyle, D.L. etal. The JAK inhibitor tofacitinib
suppresses synovial JAK1STAT signalling
inrheumatoid arthritis. Ann. Rheum. Dis. 74,
13111316 (2015).
7. Australo-Anglo-American Spondyloarthritis
Consortium. etal. Genome-wide association
study of ankylosing spondylitis identifies
non-MHC susceptibility loci. Nat. Genet. 42,
123127 (2010).
8. Okada, Y. etal. Genetics of rheumatoid
arthritis contributes to biology and drug
discovery. Nature 506, 376381 (2014).
9. Scher, J.U. etal. Expansion of intestinal
Prevotella copri correlates with enhanced
susceptibility to arthritis. eLife 2, e01202
(2013).
10. van Bon, L. etal. Proteome-wide analysis
andCXCL4 as a biomarker in systemic
sclerosis. N.Engl. J. Med. 370, 433443
(2014).
RHEUMATOLOGY
DECADE IN REVIEWPAEDIATRIC RHEUMATOLOGY
A field on the move

Seza Ozen
The study of rheumatic diseases that affect children has thrived in

the past 10years. A look at several important advances in this area
illustrates how organized collaborations and advanced technologies
are contributing to the understanding and, ultimately, to improving the
treatment of these disorders.
In this past decade, research in paediatric
rheumatology has been actively revealing
the pathogenesis of the diseases particular
to this specialty. One major contribution of
paediatric research has been the definition
ofmonogenic diseases that have the same
phenotype as corresponding complex diseases. A prominent genetic predisposition
in relevant pathways is expected to lead to
disease manifestation early in life and, thus,
such diseases often present in young children
with affected relatives.1,2 Understanding the
function of these critical genes is hoped to
offer insight into the pathogenesis of these
complex diseases as well. A leading example
is the identification of deficiency of adenosine
deaminase2 (ADA2), in which mutations in a
single gene, CECR1, lead to a disease similar
to polyarteritis nodosa.1 As ADA2 is critical
for macrophage activation and endothelial
cell development, the defect in CECR1 results
in widespread vasculitis with a contribution
from inflammatory macrophages.1 In many
patients, the vessels of the central nervous
system are affected, leading to stroke, whereas
others might have milder symptoms. For
further details of related publications please
refer to Supplementary Box1 online. Another
genetic mutation that mimics a complex
disease is the LACC1 gene, which causes a
form of systemic JIA (sJIA).2
the spectrum of known

autoinflammatory diseases has
enlarged considerably
Gene expression profiling has enabled

the discovery of pathogenetic biomarker
signatures in paediatric rheumatic diseases.
Focusing initially on paediatric systemic
lupus erythematosus (SLE), Virginia Pascuals
group showed that patients with this disease
almost universally have an interferon signature.3 By contrast, the interferon signature is
present in only about half of adult patients
with SLE, a difference that could be due
tothe severity of the disease in children or to
other factors. The same group also identified

transcriptional biomarkers of disease activity
in SLE and, by analysing the transcriptional
pattern of cells from patients with sJIA,
showed that IL1 has a leading role in the
latter disease. In fact, many clinical and laboratory features of sJIA can be explained by
increased IL1 production,3 and these studies
have already paved the way for targeted
therapy for sJIA, with anti-IL1agents.
sJIA has many features typical of an auto
inflammatory syndrome. In fact, most IL1mediated human diseases have been linked to
abnormal activation of this cytokine by the
inflammasome. The IL1-related diseases are
now grouped as autoinflammatory diseases.
Most autoinflammatory diseases are monogenic, affect the innate immune system and
typically present in childhood. Given this
early presentation, it is not surprising that
paediatricians have made major contributions to this field. The leading project in this
area has been the establishment of the multinational, multidisciplinary Eurofever registry,
which has enabled the collection of immense
amounts of information about the clinical features and demographics of autoinflammatory
diseases.4 This large dataset will, it is hoped,
serve as a basis for future development of
diagnostic criteria and t reatment protocols.
In the past 15years, and with paediatricians
leading the field, the spectrum of known autoinflammatory diseases has enlarged considerably. The newly recognized interferon-related
diseases have led us to reconsider the initial
IL1-centred concept of autoinflammation.
The term interferonopathies was intro
duced in 2011 to encompass diseases such as
AicardiGoutires syndrome and spondylo
enchondrodysplasias, and soon included the
PSMB8-related autoinflammatory diseases,
including CANDLE (chronic atypical neutro
philic dermatosis with lipodystrophy and
elevated temperature) syndrome.5 The latest
discovery in this growing field was SAVI
(STING-associated vasculopathy with onset
in infancy) syndrome (see Supplementary
Box1 online). Interferonopathies include
at least 12 monogenic diseases in which the

innate immune response leads to interferon
production and related autoinflammatory
features, with possible overlap with features
of autoimmunity in some cases.5 This new
perception of interferonopathies highlights
the need for different approaches to treatment, and challenges our understanding of
the intersection of the innate and adaptive
immune systems.
A number of genome-wide association
studies (GWAS) have been completed in
the most common paediatric rheumatic
disease, JIA. A 2013 GWAS genotyped
2,816 patients with JIA and 13,056 controls
using the Immunochip array.6 Patients with
oligoarticular and rheumatoid factor (RF)negative polyarticular JIA were studied as
these forms of the disease are specific to
children and include evidence of disorders
in the adaptive immune system. This GWAS
had certain advantages over previously published ones: it included a large international
cohort and the Immunochip array provided
comprehensive coverage of single nucleotide
polymorphisms (SNPs) in regions implicated
in autoimmune diseases. This study confirmed the association with the three previously known JIA risk loci (the HLA region,
PTPN22 and PTPN2) and identified 14 new
loci associated with risk of JIA.6 Notably, the
set of SNPs included on the Immunochip
explained and estimated 18% of JIA suscepti
bility. Thus other regions must exist that
harbour additional JIA risk loci which will
probably be the subject of future research.
Studies of treatments for JIA

have flourished with international
collaboration
Research in JIA has also concentrated

on understanding the cell biology in the
synovium. For instance, the work of two
European research groups focuses on the
autoimmune inflammatory environment of
the rheumatoid joint and the role of regulatory T (TREG) cells in defining the course of
arthritis (see Supplementary Box1 online).
In a comprehensive Review on the subject,
one of the main mechanisms of local autoimmune inflammation is suggested to be
interference with Tcell regulation by inflammatory mediators, inducing Tcell plasticity
and causing unstable TREG cells to convert to
pathogenic effector Tcells.7 Infact, a skewed
Tcell commitment occurs in the synovial fluid and, upon interaction with CD4
Tcells, local monocytes specifically induce
NOVEMBER 2015 | 71
RHEUMATOLOGY
T helper type 17 (TH17) responses, which
are highly inflammatory. The inflammatory
milieu of affected joints has also been shown
to render effector Tcells resistant to suppression. The authors of the Review suggest
that these issues should be considered in the
development of targetedtherapies.7
Studies of treatments for JIA have flourished with international collaboration. In
other rheumatic diseases, however, multi
centre treatment studies in children are
lacking, owing mainly to ethical considerations and a lack of tools that enable the
use of validated classification criteria (often
used as diagnostic criteria in practice). In
the past decade, paediatricians have succeeded in developing, by use of sophisticated approaches, classifications to guide
physicians in diagnosis and to form a basis
for future collaborative studies in two groups
of diseases: juvenile scleroderma8 and the
common childhood forms of vasculitis.9
The Ankara 2006 criteria for IgA vasculitis (HenochSchnlein purpura), granulomatous polyangiitis (formerly known as
Wegener granulomat osis), polyarteritis
nodosa and Takayasu arteritis (which
were endorsed by EULAR, the Paediatric
Rheumatology European Society [PReS] and
the Paediatric Rheumatology International
Trials Organisation [PRINTO]), and the
scleroderma criteria, were validated using
online paediatric registries that included a
large cohort of international patients.
Another important contribution by paediatricians was in defining S100 proteins,
which are released by activated phagocytes
and function as proinflammatory alarmins, as biomarkers of inflammation.10 These
proteins have been implicated in a number
of inflammatory conditions. More importantly, however, they have been shown to
be useful biomarkers for the risk-adapted
stratification of patients with JIA in decisions regarding cessation of therapy; indeed,
levels of S100 proteins have been shown to
correlate well with risk of relapse in a randomized controlled drug trial, and others are
tofollow.10
This overview of developments in paediatric rheumatology in the past decade has
been assigned a limited space and thus many
important developments and papers could
not be cited. However, this selection is hoped
to reflect some of the leading projects in the
field. Paediatric rheumatology is surely progressing in the quest to better understanding
its diseases. The relevant research will enable
us to better manage our young patients, who
have a long life-expectancy.
72 | NOVEMBER 2015
Department of Pediatric Rheumatology,

Hacettepe University, Sihhiye, 06100 Ankara,
Turkey.
sezaozen@hacettepe.edu.tr
doi:10.1038/nrrheum.2015.130
Acknowledgements
The author apologizes to colleagues whose work
could not be cited because of space constraints.
Competing interests
The author declares that she has acted as a
consultant for Novartis and received speaker
honoraria from Roche and Sobi.
1.
2.
3.
4.
Zhou, Q. etal. Early-onset stroke and

vasculopathy associated with mutations
inADA2. N.Engl. J.Med. 370, 911920 (2014).
Wakil, S.M. etal. Association of amutation
inLACC1 with a monogenic form of systemic
juvenile idiopathic arthritis. ArthritisRheumatol.
67, 288295 (2015).
Pascual, V. etal. How the study of children
withrheumatic diseases identified
interferon-and interleukin1 as novel
therapeutic targets.Immunol. Rev. 223, 3959
(2008).
Toplak, N. etal. An international registry on
autoinflammatory diseases: the Eurofever
experience. Ann. Rheum. Dis. 71, 11771182
(2012).
5.
Crow, Y.J. & Manel, N. Aicardi-Goutires

syndrome and the typeI interferonopathies.
Nat. Rev. Immunol. 15, 429440 (2015).
6. Hinks, A. etal. Dense genotyping of
immune-related disease regions identifies
14new susceptibility loci for juvenile
idiopathicarthritis. Nat. Genet. 45, 664669
(2013).
7. Wehrens, E.J., Prakken, B.J. & van Wijk, F.
Tcells out of controlimpaired immune
regulation in the inflamed joint. Nat. Rev.
Rheumatol. 9, 3442 (2013).
8. Zulian, F. etal. The Pediatric
RheumatologyEuropean Society/American
College of Rheumatology/European League
Against Rheumatism provisional classification
criteriafor juvenile systemic sclerosis.
ArthritisRheum. 57, 203212 (2007).
9. Ozen, S. etal. EULAR/PRINTO/PRES criteria
forHenoch-Schnlein purpura, childhood
polyarteritis nodosa, childhood Wegener
granulomatosis and childhood Takayasu
arteritis: Ankara 2008. Part II: final
classification criteria. Ann. Rheum. Dis. 69,
798806 (2010).
10. Rothmund, F.Validation of relapse risk
biomarkers for routine use in patients with
juvenile idiopathic arthritis. Arthritis Care Res.
(Hoboken) 66, 949955 (2014).
version of the paper at www.nature.com/nrrheum.
DECADE IN REVIEWTECHNOLOGY
Technological advances
transforming rheumatology
William H. Robinson and Rong Mao
Technological advances over the past decade have revolutionized many

areas of rheumatology, ranging from diagnosis, prognosis and therapeutic
development to the mechanistic understanding of rheumatic diseases.
This overview highlights key technological innovations and discusses
the major impact that these developments are having on research and
clinical practice.
The past decade has been an exciting time
of technological breakthroughs driving tremendous progress in rheumatology. Many
of these innovations are high-throughput
approaches that enable robust proteomic,
genomic and epigenetic analyses ranging
from the organism level to the single-cell
level.1 Here, we describe the key innovations (examples of which are described in
Box1and illustrated in Supplementary
Figure1 online) and discuss how they have
shaped our understanding of rheumatic diseases as well as our ability to diagnose and
treat thesedisorders.
Historically, many major advancesin the
research and clinical practice of rheumatology were fuelled by technological innovations.
For instance, the advent of MRI introduced a

noninvasive method for visualizing bone and
soft tissues in three dimensions that enables
improved diagnosis. The development of flow
cytometry greatly enhanced our ability to
distinguish between and characterize distinct
cell populations in tissue samples. Molecular
cloning coupled with expression profiling
using DNA microarrays has been pivotalin
identifying key molecules and pathways
inthe pathogenesis of rheumatic diseases, and
thus in uncovering novel therapeutic targets.
Likewise, the past 10years have brought us
a new raft of technological advancesboth
incremental and disruptivethat are enabling
us to interrogate and manage rheumatic
diseases with increasing sophistication.
RHEUMATOLOGY
Proteomics is one notable area in which
great progress has been made over the past
decade, to far-reaching effect. Innovations
in proteomics, including advances in
mass spectrometry and the emergence of
protein-array technologies, have revolutionized our ability to identify proteins and
post-translational modifications associated
with disease. Indeed, mass spectrometry
analyses of proteins in cartilage, synovial
membrane, bone, synovial fluid, plasma and
serum, as well as other tissues and bodily
fluids, have uncovered molecules associated
with pathological changes in osteoarthritis,
rheumatoid arthritis (RA), systemic lupus
erythematosus (SLE) and other rheumatic
diseases. Furthermore, array-based multi
plex profiling of autoantibodies and cyto
kines has deepened our understanding
of pre-disease and early disease states by
enabling the characterization of autoimmu
nity prior to the onset of clinically apparent
symptoms.2 Several of the proteomic pro
files gleaned with these technologies have
potential for use as actionable biomarkers
in predictive medicine. Most rheumatic
diseases are heterogeneous and only certain
subsets of patients respond to any given
therapy. Thus, proteomic profiles and other
biomarkers associated with specific disease
states or with drug responsiveness could
identify individuals at high risk of developing the disease, who can then be enrolled
in primary prevention trials or treated with
preventative therapies. Proteomic profiles
and other biomarkers could also serve as
pharmacodynamic biomarkers to rapidly
assess patients responses to therapy. These
proteomic technologies are ushering in a
new era in biomarker discovery and have the
potential to revolutionize the diagnosis and
treatment of rheumaticdiseases.
Large-scale sequencing is another
technological tour de force that is transforming rheumatology. The advent of highthroughput DNA sequencing has made
possible sequencing of the genome to identify both common and rare genetic variants
associated with rheumatic diseases. This
method can also be applied to sequencing
the expressed genome, which includes thousands of gene transcripts that reflect activation and repression of pathways involved in
rheumatic diseases. Such large-scale DNA
sequencing can now digitally define the
functional antibody and Tcell receptor
(TCR) repertoires in autoimmune rheumatic
diseases.1,3,4 Inaddition, deep sequencing of
transcriptomes via RNA-seq measures the
levels of transcripts and their isoforms, and
Box 1 | Advanced technologies in rheumatology*

CyTOF mass cytometry measures the binding of multiple antibodies (each tagged with a
distinct heavy-metal isotope) to cells1
Single-cell antibody heavy and light chain sequencing enables bioinformatic generation
of phylogenetic trees, which reveal clonal antibody families and guide the selection of
antibodies for expression and characterization3
Single-cell TCR sequencing coupled with amplification of functional genes characteristic
ofT-cell subsets provides insight into the specificity and function of TCRs4
ATAC-seq analyses the epigenome of cells derived from an individual6
iPOP combines genomic, transcriptomic, proteomic, metabolomic, and autoantibody
profiles from an individual to reveal medical risks and dynamic molecular changes in health
anddisease10
*See also Supplementary Figure1 online. Abbreviations: ATAC-seq, transposase-accessible chromatin using
sequencing; CyTOF, cytometry by time-of-flight; iPOP, integrative personal omics profile; TCR, T-cell receptor.
can now even be done with single-cell resolution.5 Furthermore, epigenetic technologies,
such as ATAC-seq (assay for transposaseaccessible chromatin using sequencing),6 can
probe disease-associated changes in DNA
methylation and histone modification. The
scale and efficiency of sequencing that can
now be achieved with these approaches is
facilitating unprecedented progress in both
basic and translational research of rheumatic diseases, and will most likely transform clinical care in the future. However, a
major challenge that these technologies bring
is the need to store, retrieve and analyse
the terabytes and petabytes of data that are
beinggenerated.
The past 10years have also seen the
development of mass cytometry (known
as cytometry by time-of-flight [CyTOF])
and considerable advances in flow cytometry, enhancing our ability to analyse cellu
lar markers and signalling pathways in
rheumatic diseases.7 Mass cytometry uses
heavy metals instead of fluorophores to
label cells, thereby enabling measurement
of >40 parameters per cell without spillover
between fluorescence spectra. Meanwhile,
flow cytometry has been improved by the
introduction of new staining reagents,
laser-excitable fluorescent compounds,
coupling methods and dyes that can be
used to monitor cell replication and physio
logical changes inside cells. As a result of
these advances, CyTOF and polychromatic
flow cytometry have enabled researchers to
delve deeper than ever into the complexity
of disease-relevant cell types and molecules,
monitor their dynamics, andunravel their
normal function as well as their contribution
to rheumatic diseases.
Imaging techniques such as MRI and
ultrasonography, although not new, have
only recently begun to be incorporated
into clinical trials and routine practice in
rheumatology.8 MRI and ultrasonography
are much better than conventional radio

graphy at assessing soft-tissue abnormalities and detecting bone erosions,8 and are
increasingly being used as noninvasive tools
for detecting subclinical inflammation and
progression of joint damage in arthritides.
The growing use of these techniques in
clinical practice and research is transforming diagnostic imaging. By enabling serial
assessment of synovitis, interval imaging
via MRI or ultrasonography yields dynamic
biomarkers that are useful for monitoring
the progression of disease or its response
totherapy.
Last but not least, advances in stem-cell
technologies are offering new opportunities
in tissue engineering and regenerative rheumatology. Recent work on the small mol
ecule kartogenin has shown that it is possible
to direct the differentiation of mesenchymal
stem cells into chondroc ytes and thereby
repair damaged cartilage. 9 Moreover,
induced pluripotent stem cells (iPSCs), first
described in 2006, have emerged as a promising cell source for both drug screening and
cell-replacement therapy.9 Patient-specific
iPSCs are well-suited to autologous stem
cell transplantation, because they elicit only
minimal immune reactions and have the
potential to be used to regenerate tissues,
such as cartilage. Meanwhile, disease modelling using patient-specific iPSCs continues
to expand our knowledge of pathophysiology
and treatment.
In conclusion, technological innovations
in the past decade have opened up new
horizons in our efforts to understand and
treat rheumatic diseases. Moving forward,
it is anticipated that high-throughput
approaches, especially those allowing for
single-cell resolution, will move to the mainstream of rheumatology research and form
the basis for next-generation diagnostics.
The impact of high-throughput sequencing
and other data-rich technologies will be
NOVEMBER 2015 | 73
RHEUMATOLOGY
maximized with the development of more
powerful databases, analytical methods
and analysis pipelines that can handle
the vast amount of data being generated.
Development of new biomarkers will bring
forth an era of predictive medicine, in which
individuals in pre-disease states can be
identified so that primary prevention can
be instituted and in which therapies can be
selected for use in the patient populations
most likely to benefit. Given the hetero
geneity of most rheumatic diseases, the
diverse molecular pathways mediating their
pathogenesis and the multifaceted roles that
these pathways have in normal and pathological states, advances in treatment are likely
to require approaches that integrate genomic,
transcriptomic, proteomic, metabolomic and
autoantibody profiles, such as the recently
described integrative personal omics profile,
oriPOP.10
Division of Immunology and Rheumatology,
CCSR 4135, 269 Campus Drive, Stanford,
CA94305, USA (W.H.R., R.M.).
Correspondence to: W.H.R.
wrobins@stanford.edu
doi:10.1038/nrrheum.2015.137
Acknowledgements
The authors work is supported by grants from the
NIH NHLBI Proteomics Center (N01-HV00242), NIH
NIAMS (R01 AR063676), NIH NIAID (U01 AI101981,
U01 AI057229, U19 AI110491), NIH NCI (R33
CA183659), and NIH NIAMS/NIAID/FNIH AMP
Program (UH2 AR067681), and by the Brennan
Family, the Northern California Chapter of the
Arthritis Foundation (NCCAF) Center of Excellence,
and the Bill and Melinda Gates Foundation.
Competing interests
W.H.R. declares that he is a member of the Board
ofDirectors, consultant for, and owner of equity in
Atreca, Inc. R.M. declares no competing interests.
1.
Maecker, H.T. etal. New tools for classification

and monitoring of autoimmune diseases.
Nat.Rev. Rheumatol. 8, 317328 (2012).
2. Sokolove, J., Lindstrom, T.M. & Robinson,W.H.
Development and deployment of antigen arrays
for investigation of Bcell fine specificity in
autoimmune disease. Front. Biosci. (Elite Ed.)
4,320330 (2012).
3. Robinson, W.H. Sequencing the functional
antibody repertoirediagnostic and
therapeutic discovery. Nat. Rev. Rheumatol. 11,
171182 (2015).
4. Han, A., Glanville, J., Hansmann, L.
&Davis,M.M. Linking Tcell receptor
sequence to functional phenotype at the
single-cell level. Nat. Biotechnol. 32, 684692
(2014).
5. Macosko, E.Z. etal. Highly parallel genomewide expression profiling of individual cells
using nanoliter droplets. Cell 161, 12021214
(2015).
6. Buenrostro, J.D., Giresi, P.G., Zaba, L.C.,
Chang, H.Y. & Greenleaf, W.J. Transposition
ofnative chromatin for fast and sensitive
epigenomic profiling of open chromatin,
DNA-binding proteins and nucleosome
position. Nat.Methods 10, 12131218
(2013).
7. Bendall, S.C., Nolan, G.P., Roederer, M.
&Chattopadhyay, P.K. A deep profilers guide
tocytometry. Trends Immunol. 33, 323332
(2012).
8. Filippucci, E., Di Geso, L. & Grassi, W.
Progressin imaging in rheumatology. Nat. Rev.
Rheumatol. 10, 628634 (2014).
9. Diekman, B.O. & Guilak, F. Stem cell-based
therapies for osteoarthritis: challenges and
opportunities. Curr. Opin. Rheumatol. 25,
119126 (2013).
10. Chen, R. etal. Personal omics profiling reveals
dynamic molecular and medical phenotypes.
Cell 148, 12931307 (2012).
DECADE IN REVIEWCLINICAL RHEUMATOLOGY
10years of therapeutic advances

in the rheumatic diseases
John D.Isaacs
In the past 10years, the rheumatology community has seen an

explosioninthe number of new therapies licensed for use across the
rheumatic diseases, many with outstanding clinical success. Here, thedrugs
and strategies that constitute landmarks in the management ofrheumatic
diseases are highlighted.
The rheumatoid arthritis (RA) investigational
space has remained active in the past decade
(Box1). The major advance in the treatment of this disease has been the successful
development of orally bioavailable smallmolecule inhibitors of signalling proteins.
74 | NOVEMBER 2015
Tofacitinib, the first of this class of drugs to

be licensed for use in RA, inhibits the Janusassociated kinase (JAK) signalling pathways
downstream of various cytokine and growthfactor receptors.1 Tofacitinib is moderately
nonselective and blocks, in descending order
of potency, JAK3, JAK1 and JAK2. In phase3

trials, tofacitinib had a safety and efficacy
profile similar to that of biologic DMARDs.
Several other, more-selective JAK inhibitors
are in development, although it remains to
be seen how these compare with tofacitinib.
Regardless of the results,a treatment that
offers biologic efficacy in apill will surely be
popular among patients withRA.
The need for novel therapies has been
more acute for patients with psoriatic
arthritis (PsA). During most of the past
decade, TNF blockade was the only alternative available after DMARD failure, but
new oral and biologic therapies have now
been licensed. Apremilast, an oral phospho
diesterase4 inhibitor that indirectly reduces
proinflammatory cytokine production,
is now used before biologic therapies for
patients with an inadequate response to
DMARDs.2 The monoclonal antibody (mAb)
ustekinumab blocks the p40 chain common
to the cytokines IL12 and IL23, which drive
T helper (TH)1 and TH17 immune responses,
respectively.3 Both the IL12 and IL23 path
ways have been linked to PsA in genomewide association studies. As the efficacy of
ustekinumab seems similar to that of TNF
blockade,3 this therapy provides an additional, potent option for patients with PsA
refractory to DMARDs. Apremilast seems
less effective than ustekinumab and TNF
blockade but is well-tolerated, andblood
monitoring is not required; therefore, apremilast is also a useful option for patients
withPsA.
Belimumab is the first new therapy to
be licensed for use in systemic lupus erythematosus (SLE) in >50years.4 This mAb
blocks Blymphocyte stimulator (BLyS,
also known as BAFF), a growth factor for
Bcells. Whilst its development was proble
matic and included a failed phase2 clinical
trial, the endpoints in two phase3 studies
of belimumab were achieved, albeit with
only modest improvements over placebo.
Important lessons were learned during this
process, including the need for new clinical
trial outcome criteria. Retrospective analysis
of the phase2 trial also identified an associ
ation between response to therapy and circulating autoantibodies at baseline. Belimumab
is currently licensed as an addition to conventional therapy in patients with active,
seropositiveSLE.
The belimumab trials illustrated the
complexities of clinical trial design in SLE,
perhaps explaining why trials of rituximab
(a Bcell depleting agent) did not demon
strate efficacy in ameliorating this condition.
RHEUMATOLOGY
Rituximab has, however, gained a foothold
in the management of anti-neutrophil cyto
plasmic antibody (ANCA)-associated vas
culitis (AAV), in which it proved noninferior
to daily oral cyclophosphamide in achieving
glucocorticoid-free remission in patients
with newly-diagnosed or relapsing disease.5
In a separate study, rituximab was shown
to be superior to azathioprine in sustaining
remission after treatment with cyclophos
phamide and glucocorticoids. Whilst gluco
corticoids remain an important component
of therapy, many patients with AAV now have
an alternative to traditional cytotoxic and
immunosuppressive medications.
Systemic sclerosis remains one of the most
refractory connective tissue diseases, with
no current treatments that slow its progression. A major aspect of the pathology of this
disease is arterial vasoconstriction, media
ted by endothelin1. Whilst endothelin1
blockade has been available to treat pulmon
ary hypertension for some time, only more
recently has the endothelin-receptor antag
onist bosentan been licensed to treat severe
digital ulcers secondary to poor digital
circulation.6 These ulcers are a particularly
painful and debilitating symptom of systemic sclerosis, and endothelin1 inhibition
has provided new hope for patients with this
unrelentingcondition.
In this past decade, good news for children with juvenile idiopathic arthritis (JIA)
emerged as IL6-receptor blockade with
tocilizumab was shown to be highly effective in children with active systemic or
polyarticular-course JIA. Its effectiveness
was particularly welcome in systemic JIA,
previously a condition with limited therapeutic optionsa situation that resulted
in dependency on growth-inhibiting
glucocorticoids.7
Important advances in therapies for
more common conditions also occurred
in this period. TNF ligand superfamily
member 11, also known as RANK ligand
(RANKL), is an important differentiation,
activation and survival factor of osteoclasts.
Denosumab,an anti-RANKL mAb, has been
developed as anantiresorptive therapy for
postmenopausal women, as well as men,
with osteop orosis, reducing the risk of
both vertebral and nonvertebral fractures.8
Itslong half-life means only twice-yearly sub
cutaneous injections are needed for efficacy,
which will improve adherence considerably
compared with bisphosphanates. Pegloticase
(pegylated uricase), a new treatment for
patients with gout who cannot tolerate, or
have an inadequate response to, standard
treatments, is highly effective in decreasing

uric acid levels, reducing flares and resolving
tophi.9 Clinically important improvements
in quality-of-life were documented within
6months of starting pegloticase treatment,
but anaphylaxis is common (incidence of
~7%), as are infusion reactions associated
with fortnightly dosing.9
Ankylosing spondylitis (AS) remains a
disease with few treatment options, which
currently include NSAIDs, physiotherapy and
TNF blockade. Ustekinumab trials in patients
with PsA suggested benefits for spinal inflammation, and the biology of AS strongly implicates the IL23IL17 axis. Inthis context,
secukinumab, an anti-IL17 mAb, was shown
to rapidly improve the signs and symptoms of
AS, with positive changes assessed in parallel
by use of imaging techniques.10 Intriguingly,
response was associated with polymorphisms
in ERAP1 and, possibly, IL23R. Several mAbs
have been developed that target the IL17
pathway and, notwithstanding any surprises,
patients with AS can look forward to a first
new treatment option since TNF blockade
(none of these are currently licensed for the
treatment of AS).
By reviewing these therapeutic advances,
it is satisfying to see that the majority are
targeted therapies aimed at pivotal patho
genetic pathways. What lies ahead for the
next decade? In RA, a continuing trend
towards earlier treatment is clear, and trials
are underway in pre-RAfocused on
individuals with autoantibodies but without
joint inflammation symptoms. The T
wordtoleranceis increasingly mentioned
and, in several diseases, cell-targeted strate
gies are being developed as potential toler
ogenic therapies. Increasingly sophisticated
methods can be used to purify and expand
Tregulatory (TREG) cells, and mesenchymal
stem cell and tolerogenic dendritic-cell trials
are being reported in the literature. Methods
to generate TREG cells invivo, such as low-dose
IL2 therapy, also seem promising.
Several technologies are likely to contribute to the development of rheumatic disease
therapeutics over the coming decade. In
terms of antibody engineering, we might see
the application of smaller fragments, such as
nanobodies, and of bispecific antibodies. For
example, bispecific agents that target both
TNF and IL17 are currently being studied
in RA. Furthermore, genetic manipulation
and epigenetic modification are already
being applied in nonrheumatic diseases (an
oral antisense oligonucleotide, mongersen,
which targets SMAD7 mRNA and leads to
a subsequent increase in the production of
Box 1 | New drugs for rheumatic diseases

Antibodies
Ustekinumab (PsA)
Belimumab (SLE)
Rituximab (AAV)
Tocilizumab (JIA)
Denosumab (osteoporosis)
Enzyme
Pegloticase (gout)
Small molecule (orally bioavailable) drugs
Tofacitinib (RA)
Apremilast (PsA)
Bosentan (SSc)
Abbreviations: AAV, anti-neutrophil cytoplasmic
antibody (ANCA)-associated vasculitis; JIA, juvenile
idiopathic arthritis; PsA, psoriatic arthritis;
RA,rheumatoid arthritis; SLE,systemic lupus
erythematosus; SSc, systemic sclerosis.
transforming growth factor, improved

symptoms and signs in a phase2 Crohn
disease trial). We can expect similar technol
ogies to be tested in rheumatic diseases, notwithstanding the challenges of extraintestinal
delivery. There is also much interest in the
role of the microbiome in human disease, and
the capacity to modify immune and inflammatory processes by manipulation of the gut
flora is appealing, particularly in the context
of treating early and preclinical conditions.
If therapies become cheaper, they should
become more widely available and be used,
for example, for patients with earlier and lessactive diseasethe first biosimilar infliximab
has now been approved for use in rheumatic
disease, and the next decade will see many
more biosimilars reach the market as patents
for currently licensed biologic agents expire.
Another area of intensive investment is stratified medicine, or prescribing according to a
patients genetic and biologic characteristics.
This approach is likely to pay dividends over
the next decade.
Despite the numerous advances in clinical
management of rheumatic diseases, many
challenges lie ahead for the next decade, not
least the need for disease-modifying drugs
for the most common rheumatic disease,
osteoarthritis. Nonetheless, the new techno
logical and strategic approaches discussed
here, including new drug classes and more
personalized treatment approaches, have the
potential to bring new solutions and algorithms to rheumatology careconsequently,
the way we assess, investigate and treat
patients in 10 years time could look very
different fromtoday.
Newcastle University, Institute of Cellular
Medicine, William Leech Building, Framlington
Place, Newcastle-Upon-Tyne NE24HH, UK.
john.isaacs@ncl.ac.uk
NOVEMBER 2015 | 75
RHEUMATOLOGY
doi:10.31038/nrrheum.2015.138
2.
Acknowledgements
Work in the authors laboratory is supported by
theNational Institute for Health Research (NIHR)
Newcastle Biomedical Research Centre, based
atNewcastle Hospitals NHS Foundation Trust
andNewcastle University, UK. The views expressed
are those of the author and not necessarily those
ofthe NHS, the NIHR or the Department of Health.
Theauthor is grateful to T. Aspray and P. Conaghan
for their insights whilst preparing thismanuscript.
Competing interests
The author has been a member of advisory
boardsfor Celltrion, Hospira, Janssen, Novartis,
Pfizer andRoche.
1.
Norman, P. Selective JAK inhibitors in

development for rheumatoid arthritis.
76 | NOVEMBER 2015
3.
4.
5.
6.
ExpertOpin. Investig. Drugs 23, 10671077

(2014).
Kavanaugh, A. etal. Treatment of psoriatic
arthritis in a phase3 randomised, placebocontrolled trial with apremilast, an oral
phosphodiesterase4 inhibitor. Ann. Rheum. Dis.
73, 10201026 (2014).
McInnes, I.B. etal. Efficacy and safety of
ustekinumab in patients with active psoriatic
arthritis: 1year results of the phase 3,
multicentre, double-blind, placebo-controlled
PSUMMIT 1 trial. Lancet 382, 780789 (2013).
Stohl, W. & Hilbert, D.M. The discovery and
development of belimumab: the anti-BLySlupusconnection. Nat. Biotechnol. 30, 6977
(2012).
Stone, J.H. etal. Rituximab versus
cyclophosphamide for ANCA-associated
vasculitis. N. Engl. J. Med. 363, 221232 (2010).
Matucci-Cerinic, M. etal. Bosentan treatment of
digital ulcers related to systemicsclerosis:
results from the RAPIDS-2randomised,

double-blind, placebo-controlled trial.
Ann.Rheum. Dis. 70, 3238 (2011).
7. De Benedetti, F. etal. Randomized trial of
tocilizumab in systemic juvenile idiopathic
arthritis. N. Engl. J. Med. 367, 23852395
(2012).
8. Cummings, S.R. etal. Denosumab for
prevention of fractures in postmenopausal
women with osteoporosis. N. Engl. J. Med. 361,
756765 (2009).
9. Sundy, J.S. etal. Efficacy and tolerability of
pegloticase for the treatment of chronic gout
inpatients refractory to conventional treatment.
Two randomized controlled trials. JAMA 306,
711720 (2011).
10. Baeten, D. etal. Antiinterleukin17A
monoclonalantibody secukinumabin treatment
of ankylosing spondylitis: a randomised, doubleblind, placebo-controlled trial. Lancet 382,
17051713 (2013).
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UROLOGY
DECADE IN REVIEWBLADDER CANCER
International progress: from cytology to genomics

James C. Costello and Dan Theodorescu
The past decade has seen many notable contributions to bladder cancer research. Here, we highlight the
international efforts in the field, with findings from Europe, USA and China, as well as papers resulting
from international cooperation. We anticipate the next 10years will see even greater collaborative and
internationalefforts.
Costello, J. C. & Theodorescu, D. Nat. Rev. Urol. 11, 609610 (2014); published online 2 September 2014; doi:10.1038/nrurol.2014.236
gene expression profiling

has been used to develop
cellular response models to
drugtreatment
Pogonici/iStock/Thinkstock
The early 2000s saw great changes in bladder

cancer research, with several very important
contributions that helped shape our molecu
lar understanding of the disease and patient
treatment strategies. Dyrskjt andcollea
gues were the first to connect tumour stage
with genome-scale molecular patterns of
the disease using microarrays.1 Dyrskjt
etal.1 showed that gene expression patterns
sampled from patient tumours clustered
into three major groups (stages): Ta, T1 and
T24. They were additionally able to use the
gene expression patterns to predict whether
a tumour would recur or not, providing
insights into the molecular mechanisms
that stratify aggressive muscleinvasive and
non-muscle-invasivetumours.
In addition to stratifying patients by
tumour stage and predicting recurrence,
gene expression profiling has been used to
develop cellular response models to drug
treatment that have been used to predict
how an untreated cell, or patient, will
respond. In a seminal 2007 paper on drug
sensitivity prediction, the NCI60 panel of
cancer cell lines was used to predict how
bladder cancer cells would respond to drug
treatment.2 The coexpression extrapola
tion (COXEN) method demonstrated
77 | NOVEMBER 2015
that drug sensitivities could be accurately

predicted using information derived from
other cancer cell lines. This and additional
retrospective studies showed that COXEN
could be successfully applied to patients.
The results from this study, and those of
Dyrskjt and colleagues,1 provided early
support for the notion of personalized or
precision medicine. Indeed, a clinical trial
(Southwest Oncology Group 1314) is cur
rently underway to validate COXEN pro
spectively in assigning chemotherapy to
bladder cancerpatients.
Urine cytology is particularly insensitive
at detecting low-grade tumours. However,
the finding that bladder tumours could be
genetically stratified, coupled with known
FGFR3 mutations that are frequently
associated with low-grade disease, led to
the hypothesis that FGFR3 could serve as
a urinary biomarker of recurrent disease.
vanRhijn etal.3 reported that combined
microsatellite and FGFR3 mutation analy
sis could detect urothelial cell carcinoma
(UCC) in voided urine. FGFR3 mutations
were found in 44% of urothelial tumours
(n=59), but were absent in 15 G3 tumours.
The sensitivity of microsatellites to detect
cancer in voided urine was lower for
tumours harbouring FGFR3 mutations
(15 of 21 tumours; 71%) than for FGFR3
wild-type UCC (29 of 32 tumours; 91%).
By including the FGFR3 mutation, the
sensitivity of molecular cytology increased
from 71% to 89% and was superior to the
sensitivity of morphological cytology (25%)
for every clinical subdivision. This paper

highlighted the potential power of molecu
lar biology as an adjunct to cystoscopy and
cytology to inform follow-up care.
Minimally invasive surgery has devel
oped into a standard outpatient pro
cedure over the past decade in most areas
of surgery. In urology, it has considerably
changed how we manage men with prostate
cancer, primarily with the advent of robotic
technologies. In 2003, the first worldwide
attempt to perform a robot-assisted laparo
scopic radical cystectomy and completely
intra-abdominal formation of an ortho
topic neobladder (Hautmann neobladder)
was described by Beeken etal.4 Impressively,
operating time was 8.5h and blood loss was
only 200ml. Several other studies have
reported the use of the robot-assisted cys
tectomy, with intracorporeal and extra
corporeal formation of the diversion. Most
recently, several trials have reported com
parisons between open and laparoscopic
approaches. Recent studies have indicated
that robotic cystectomy is carried out in
<15% of patients.
In the late 1980s, the formulation and
early trials of methotrexate, vinblastine,
doxorubicin and cisplatin (MVAC) chemo
therapy represented an important advance
in the treatment of metastatic bladder
cancer. Despite subsequent larger trials
showing durable complete responses were
difficult to achieve, this regimen remained
the standard of care. Further developing
MVAC, a Southwest Oncology Group trial
comparing neoadjuvant chemotherapy
plus cystectomy (n=154) with cystectomy
alone (n=153) for locally advanced bladder
cancer showed the median survival among
patients was 77months and 46months,
respectively.5 Importantly, in both groups,
UROLOGY
improved survival was associated with
the absence of residual cancer in the cys
tectomy specimen. It was also noted that
fewer patients in the combination group
had residual disease than patients in the
cystectomy group (15% versus 38%). The
toxicity of MVAC had previously been
noted in its use in the metastatic setting,
motivating the development of alternative
less-toxic regimens. In 2005, a trial com
paring the survival of patients with locally
advanced or metastatic UCC treated with
MVAC (n=202) to gemcitabine and cispla
tin (n=203) was reported.6 Overall survival
was similar in both arms, with a median
survival of 1415months. The 5year over
all survival rates were 13.0% and 15.3% for
the two regimens, respectively, but were
not statistically different. However, the
lower toxicity of the gemcitabine and cis
platin regimen had a remarkable impact on
practicemost patients are now given this
treatment rather than MVAC.
next-generation sequencing
has been a boon to all cancer
researchand bladder cancer is
no exception
Nomograms for prediction of clinical out

comes after primary therapy gained great
popularity over the past decade in a variety
of diseases, especially in cancer. These
tools are particularly useful for patient
counselling and guidance with respect to
follow-up intensity and adjuvant therapy.
Moving into 2006, a report was published
that described a tool of great clinical
utility in this regard.7 This work was the
result of an international collaboration
that generated a bladder cancer database
from 12centres of excellence and com
prising 9,000 patients treated with radical
cystectomy and pelvic lymphadenectomy.
Aprognostic nomogram was constructed
to predict 5year progression-free prob
ability; its predictive accuracy was found to
be significantly better than standard TNM
or standard pathological subgroupings.
However, its use in clinical practice is, thus
far, unreported.
In the mid-1980s, the presence and
extent of the androgen receptor (AR) in
urothelial bladder cancer tissue was recog
nized to be higher than in normal bladder
mucosa, potentially leading to the gender
disparities in bladder cancer incidence. In
2007, investig ators used animal models
to show that AR signals promote bladder
cancer developm ent and progression. 8

They showed that 92% of wild-type male
and 42% of wild-type female mice treated
with NbutylN-(4-hydroxybutyl)nitrosa
mine (BBN) eventually developed bladder
cancer, whereas none of the male or female
ARknockout mice did. Treatment with
BBN induced bladder cancer in 25% of
ARknockout mice supplemented with
dihydrotestosterone and in 50% of castrated
wild-type male mice. These findings, and
that of other groups using human bladder
cancer cell lines, showed that androgens and
the AR are important promoters of bladder
tumour formation and metastasis. Whether
this evidence that gender differences in
bladder cancer incidence might be due
to AR will result in a change in treatment
protocol is, as yet, unknown.
Mutations in cancer driver genes occur
at high rates; for example, in bladder cancer
TP53 is altered at a frequency of roughly
50%. Traditional single-gene cancer drivers
tend not to exceed 50% of the population,
which is why the study by Killela and col
leagues, which showed TERT promoter
mutations occur at a rate of 66% in bladder
cancer and >80% in glioblastoma, was
so surprising.9 These findings have been
subsequently verified and although the
implications of this discovery will not be
evident for years to come, we have new
insights in the mechanism of bladder
cancer development with the potential for
therapeuticintervention.
Indeed, the advancement of genom
ics technologies into next-generation
sequencing has been a boon to all cancer
researchand bladder cancer is no excep
tion. Although several previous studies
had looked at whole-genome or wholeexome sequencing, the work by Guoetal.10
reported in 2013 was revolutionarybecause
it sampled the genome sequences of 99
Chinese patients with bladder cancer. This
approach enabled comprehensive cata
loguing of genomic alterations. Previously
identified bladder cancer driver mutations
were verified, and many additional candi
date driver genes were identified, including
STAG2, ESPL1 and a group of chromatin
modifying genes. The volume of genomic
information is expanding rapidly, with
efforts such as The Cancer Genome Atlas
and the International Cancer Genome
Consortium producing petabytes of data.
We anticipate that these data will fuel much
of bladder cancer research in the future.
With advances in diagnosis, surgery,
molecular biology and medical therapy,
the management of patients with bladder

cancer has evolved markedly over the past
decade. The convergence of new knowledge
on the genomic landscape of bladder cancer
coupled with new targeted and immuno
therapy approaches promises to change
bladder cancer clinical practice even more
dramatically in the next 10years.
Department of Pharmacology, University
ofColorado, Anshutz Medical Campus,
12801East 17th Avenue, Aurora, CO 80045,
USA (J.C.C.). University of Colorado
Comprehensive Cancer Center, MS F434,
13001 East 17th Place, Aurora, CO 80045, USA
(D.T.).
Correspondence to: D.T.
dan.theodorescu@ucdenver.edu
Competing interests
Acknowledgements
D.T. is supported in part by NIH grants CA075115
and CA104106. The funders had no role in study
design, data collection and analysis, decision
topublish or preparation of the manuscript.
1.
Dyrskjt, L. etal. Identifying distinct classes

ofbladder carcinoma using microarrays.
Nat.Genet. 33, 9096 (2003).
2. Lee, J.K. et al. A strategy for predicting the
chemosensitivity of human cancers and its
application to drug discovery. Proc. Natl Acad.
Sci. USA 104, 1308613091 (2007).
3. van Rhijn, B.W. et al. Combined microsatellite
and FGFR3 mutation analysis enables a highly
sensitive detection of urothelial cell carcinoma
in voided urine. Clin. Cancer Res. 9, 257263
(2003).
4. Beecken, W.D. et al. Robotic-assisted
laparoscopic radical cystectomy and intraabdominal formation of an orthotopic ileal
neobladder. Eur. Urol. 44, 337339 (2003).
5. Grossman, H.B. et al. Neoadjuvant
chemotherapy plus cystectomy compared
withcystectomy alone for locally advanced
bladder cancer. N. Engl. J. Med. 349, 859866
(2003).
6. von der Maase, H. et al. Long-term survival
results of a randomized trial comparing
gemcitabine plus cisplatin, with methotrexate,
vinblastine, doxorubicin, plus cisplatin in
patients with bladder cancer. J. Clin. Oncol. 23,
46024608 (2005).
7. International Bladder Cancer Nomogram etal.
Postoperative nomogram predicting risk of
recurrence after radical cystectomy for
bladdercancer. J. Clin. Oncol. 24, 39673972
(2006).
8. Miyamoto, H. et al. Promotion of bladder
cancer development and progression by
androgen receptor signals. J. Natl Cancer Inst.
99, 558568 (2007).
9. Killela, P.J. et al. TERT promoter mutations
occur frequently in gliomas and a subset
oftumors derived from cells with low rates of
selfrenewal. Proc. Natl Acad. Sci. USA 110,
60216026 (2013).
10. Guo, G. et al. Whole-genome and whole-exome
sequencing of bladder cancer identifies
frequent alterations in genes involved in sister
chromatid cohesion and segregation.
Nat.Genet. 45, 14591463 (2013).
NOVEMBER 2015 | 78
UROLOGY
DECADE IN REVIEWIMAGING
A decade in image-guided
prostate biopsy
Baris Turkbey and Peter L. Choyke
iStock/Thinkstock
The prostate is still largely assessed by random biopsy, but developments

in prostate MRI and fusion with transrectal ultrasonography (TRUS) have
made targeted biopsy of the prostate a reality. MRI/TRUS techniques
promise to address the issues of overdiagnosis and underdiagnosis
inprostate cancer.
Turkbey, B. & Choyke, P. L. Nat. Rev. Urol. 11, 611612 (2014); published online 14 October 2014;
doi:10.1038/nrurol.2014.273
Unlike biopsies of the breast, thyroid

and colon, which are guided into lesions
of radiological concern, the prostate is
still largely assessed by random biopsy.
Transrectal ultrasonography (TRUS) has
proven to be rather poor at detecting pros
tate cancers, so biopsies undertaken using
this method are essentially blind. In the
meantime, multip le technical improve
ments have occurred in prostate MRI, which
has emerged as the imaging technique of
choice for prostate cancer. Specifically,
multiparametric MRI (mpMRI) offers high
sensitivity through a combination of highresolution anatomical T2-weighted (T2W)
MRI and functional pulse sequences,
such as diffusion-weighted MRI (dwMRI),
dynamic-contrast-enhanced MRI (DCE
MRI) and MR spectroscopy imaging
(MRSI). Dramatic technological improve
ments in dwMRI have meant that, over the
past decade, this technique has been more
heavily relied upon than the others.
The wider availability of high field
strength, 3T magnets and new coil designs
have substantially improved mpMRI tech
niques. These methods have proven to be
the most accurate yet for identifying local
ized prostate cancer. However, if mpMRI
simply detected lesions but did not enable
biopsy of those lesions, it would not be
clinically relevant. Thus, from the begin
ning, attempts were made to biopsy lesions
within the MR scanner. Initial efforts
focused on in-bore MRI guided biopsies;
the main advantage of this approach is
that it enables precise lesion sampling. For
instance, Hoeks etal.1 performed in-bore
MRI guided biopsies in 265 patients
with elevated PSA and previous negative
TRUS-guided biopsies. Prostate cancer
was detected in 41% of patients and the
majority of the detected cancers (87%)
were clinically significant. Roethke etal.2
79 | NOVEMBER 2015
investigated the tumour detection rate

of in-bore MRI-guided biopsy technique
in 100 patients with previous negative
TRUS-guided biopsy with a tumour detec
tion rate of 52.0%; 80.8% of the detected
prostate tumours were clinically signifi
cant. However, in-bore MRI-guided biop
sies have considerable limitations, such
as discomfort related to patient position,
increased costs related to long procedure
duration and the requirement for special
nonmagnetic equipment. Another problem
is that there is insufficient MR capacity and
expertise to handle the number of patients
requiring guided biopsy. This approach has
not proved popular with urologists either,
as the biopsy is performed in the radiology
department and interferes with normal
workflow. For these reasons, attempts
have been made to perform the biopsy
outside the MRI suite while retaining the
information affordedby MRI.
the concept of MRI/TRUS

fusion has taken hold
The first attempt to transfer MRI infor

mation to TRUS-guided biopsy was cog
nitive fusion. In cognitive fusion guidance
(CFG), the operator first determines the
location ofthe MRI-positive lesions and
then guides the needle to that location
under real-time TRUS using a best guess
approach. The main advantage of this tech
nique is that it does not need additional
equipment; however, this method depends
strongly on the experience and training
of the operator and, therefore, results in
inconsistent outcomes. The transverse
plane on MRI and the transverse plane on
an axial TRUS are often different, and excel
lent handeye coordination is required to
account for this discrepancy. Also, CFG
does not enable documentation of biopsy

locations for repeat biopsies and active
surveillance. Nevertheless, the impact of the
CFG technique has been positive, Haffner
etal.3 reported that CFG-targeted biopsies
had a sensitivity and specificity of 95% and
100%, respectively, compared with sensi
tivity and specificity values for extended
systematic biopsies of 95% and 83%, respec
tively. CFGbiopsies also detected 16%
more high-grade tumours and produced
longer mean cancer core lengths (5.56mm
compared with 4.70mm [P=0.002]) than
extended systematic biopsies. However,
whether this method can be effective on a
broad scale remains to be proven.
The success of CFG prompted interest
in new technologies, such as MRI/TRUS
fusion, to help guide biopsies based on
MRI findings. MRI/TRUS fusion is a col
lection of technologies that operate under
the same principle. First the MRI image is
obtained, the prostate is segmented from
the remainder of the pelvis and the lesions
are identified. Next the patient is seen in the
ultrasonography suite, where a 3D image is
obtained, to which the segmented prostate
MRI is electronically fused. The TRUS
and the MRI are combined so that as the
TRUS probe is moved or rotated the cor
responding MRI moves and rotates in the
same way, which allows the operator to use
an MRI obtained at a different time during
the TRUS-guided biopsy. MRI/TRUSfusion-guided biopsy is rapidly developing
and several commercial instruments are
alreadyavailable.
In the original implementation, MRI and
TRUS images were linked together using
passive electromagnetic tracking sensors
that transmit the position of the TRUS
probe, allowing the operator to see both
MR and TRUS images moving in real-time.
Initial results reported by Pinto etal.4 dem
onstrated that more cancers per core were
UROLOGY
detected than standard 12-core TRUSguided biopsy alone. Rastinehad etal. 5
reported an overall cancer detection rate
of 62.9%, and 14.3% of cancers were only
detected using MRI/TRUS-fusion biopsy.
Also, approximately 23% of cancers deemed
clinically insignificant by 12-core biopsy
were found to be clinically significant by
MRI/TRUS-fusionbiopsy.
Developments in mpMRI have

been rapid and impressive over
the past 10years
Replacing the freehand electromagnetic

trackers with a mechanical arm that
holds the TRUS probe in place is another
approach. Once the MRI and TRUS images
are fused, the needle and probe positions are
tracked by angle-sensing encoders embed
ded in the joints of the mechanical arm.
Using this device, Wysock etal.6 prospec
tively compared targeted biopsy outcomes
between mechanical arm MRI/TRUSfusion biopsies and CGB. Mechanical arm
MRI/TRUS fusion resulted in a 32.0%
detection rate compared with 20.3% for
CGB for clinically significant cancers, and
similar results were observed by Sonn etal.7
Combining MRI and TRUS images using
spatial features alone, without GPS or
mechanical arm tracking, enables the TRUS
probe to be used freehand. Rud etal.8 evalu
ated accuracy of this method and reported a
52% tumour detection rate. Delongchamps
etal.9 compared the detection rate of CGB
with this form of MRI/TRUS biopsy guid
ance and found the latter to significantly
improve detection rates over that of system
atic 12-core TRUS-guided biopsy. Targeted
biopsies decreased the number of cores
needed and the detection of microscopic
cancer, and increased the detection of highgrade cancer. However, the accuracy of this
method has been questioned.
Transperineal biopsies, which are more
common in Europe, can also be guided
by MRI using a platform that includes a
TRUS probe mounted on a stepper fixed
to the operating table. The probe move
ments are tracked by two encoders and the
biopsy needles are placed through a grid
mounted to the mechanical stepper, similar
to a brachytherapy seed placement setup.
Kuru etal.10 evaluated this platform in 347
patients, demonstrating that 58% hadpros
tate cancer and 73.5% of biopsy-proven
prostate cancer was clinically significant.
The tumour detection rate was 82.6% and

72% oftumours were Gleason score 7.
Overall, the use of targeted cores detected
significantly more cancer than systematic
biopsies (30% compared with 8.2%).Thus,
the concept of MRI/TRUS fusion has
taken hold and multiple companies have
jumped into the market to address the
need for equipment. No direct comparison
of differe nt MRI/TRUS methods exists,
but it is clear that all of the methods are
superior to the current standard of care.
This technology is likely to continue to
evolve and replace traditional blind biop
sies. However, whilst MRI-guided biopsies
are unquestionably superior to unguided
biopsies, they raise concerns over cost, as
the MRI and MRI/TRUS-fusion instru
ments are expensive. Some costs can be
reduced by eliminating the endorectal
coil and it is also unclear whether con
trast enhancement will be needed in the
future. Eliminating these should reduce
the cost of MRI/TRUS-fusion techniques.
As the technology evolves, market forces
should reduce the cost of fusion equipment
further, and better clinical guidelines for
the selection of candidates for biopsy will
emerge, reducing the number of patients
requiring thistechnology.
Over the past decade there has been a
distinct change in philosophy regarding
the nature of prostate cancer. Previously,
prostate cancer was thought to be a multi
focal disease that required random samp
ling to fully identify the extent of disease.
However, experience has shown that many
cancers are incidental and random biop
sies can miss significant disease. Thus, the
concept that there are dominant, clinically
significant cancers and multiple inconse
quential tumours, and that treatment
should be determined by the former, is
being increasingly accepted. The 12-core
random TRUS-guided biopsy is still the
standard of care, but there is growing
awareness of its limitations and the value
of image-guided biopsy. Serum PSA screen
ing significantly increased prostate cancer
diagnosis rates; however, it brought chal
lenges of overdiagnosis without addressing
the persistent problem of underdiagnosis.
Developments in mpMRI have been rapid
and impressive over the past 10years and
have enabled better detection and staging
of prostate cancer. Using mpMRI in biopsy
guidance for prostate cancer diagnosis has
the potential to reduce both overdiagnosis
and underdiagnosis. As genomic assess
ment of prostate cancer becomes more
important, there will be a premium on

obtaining better samples and longer cores
of cancer, which can only be provided
with imaging guidance. Nonetheless, the
ultimate acceptance of MRI/TRUS-fusion
biopsy awaits the results of large-scale
multicentre, randomized studies, which are
needed to convince clinics to pay for this
new technology.
The Molecular Imaging Program, National
Cancer Institute, National Institutes of Health,
10Center Drive, Building 10, Room B3B69,
Bethesda, MD 20892, USA (B.T., P.L.C.).
Correspondence to P.L.C.
pchoyke@mail.nih.gov
Competing interests
1.
Hoeks, C.M. etal. Three-Tesla magnetic

resonance-guided prostate biopsy in men
withincreased prostate-specific antigen
andrepeated, negative, random, systematic,
transrectal ultrasound biopsies: detection
ofclinically significant prostate cancers.
Eur.Urol. 62, 902909 (2012).
2. Roethke, M. etal. MRI-guided prostate biopsy
detects clinically significant cancer: analysis
ofa cohort of 100 patients after previous
negative TRUS biopsy. World J. Urol. 30,
213218 (2012).
3. Haffner, J. etal. Role of magnetic resonance
imaging before initial biopsy: comparison of
magnetic resonance imaging-targeted and
systematic biopsy for significant prostate
cancer detection. BJU Int. 108, E171E178
(2011).
4. Pinto, P.A. etal. Magnetic resonance imaging/
ultrasound fusion guided prostate biopsy
improves cancer detection following
transrectal ultrasound biopsy and correlates
with multiparametric magnetic resonance
imaging. J.Urol. 186, 12811285 (2011).
5. Rastinehad, A.R. etal. Improving Detection
ofClinically Significant Prostate Cancer:
Magnetic Resonance Imaging/Transrectal
Ultrasound Fusion Guided Prostate Biopsy.
J.Urol. 191, 17491754 (2014).
6. Wysock, J.S. etal. A Prospective, Blinded
Comparison of Magnetic Resonance (MR)
Imaging-Ultrasound Fusion and Visual
Estimation in the Performance of MRtargeted
Prostate Biopsy: The PROFUS Trial. Eur. Urol.
66, 343351 (2014).
7. Sonn, G.A. etal. Value of targeted prostate
biopsy using magnetic resonance-ultrasound
fusion in men with prior negative biopsy and
elevated prostate-specific antigen. Eur. Urol.
65, 809815 (2014).
8. Rud, E., Baco, E. & Eggesb, H.B. MRI and
ultrasound-guided prostate biopsy using soft
image fusion. Anticancer Res. 32, 33833389
(2012).
9. Delongchamps, N.B. etal. Prebiopsy
magneticresonance imaging and prostate
cancer detection: comparison of random
andtargeted biopsies. J. Urol. 189, 493499
(2013).
10. Kuru, T.H. etal. Critical evaluation of magnetic
resonance imaging targeted, transrectal
ultrasound guided transperineal fusion biopsy
for detection of prostate cancer. J. Urol. 190,
13801386 (2013).
NOVEMBER 2015 | 80
UROLOGY
DECADE IN REVIEWURINARY INCONTINENCE
Advances in female urology

andvoiding dysfunction
Marisa M. Clifton and Howard B. Goldman
Over the past decade, management of common urological problems has

metamorphosed in the field of female urology and voiding dysfunction;
from treatment of routine stress urinary incontinence (SUI) with synthetic
sling placement, to management of refractory overactive bladder (OAB)
with neuromodulation.
Clifton, M. M. & Goldman, H. B. Nat. Rev. Urol. 11, 613614 (2014); published online 14 October 2014;
doi:10.1038/nrurol.2014.279
For most of the past decade, urodynamic

studies (UDS) were a routine part of the
evaluation for a patient with stress urinary
incontinence (SUI). UDS is an invasive
and relatively expensive procedure that can
cause anxiety in patients and, until recently,
its value in improving treatment outcomes
was unknown. However, in 2012, Nager
etal.1 published a multicentre, random
ized noninferiority trial evaluating the
need for UDS in patients undergoing mid
urethral sling surgery. In this study, women
with uncomplicated SUI were randomized
to undergo office evaluation and UDS or
office evaluation only. Patients had a history
of SUI symptoms for 3months or more, a
score on the Medical, Epidemiological and
Social Aspects of Aging (MESA) inventory
for SUI that was greater than their score for
urinary urgency incontinence (UUI), evi
dence of urethral mobility on examination
and a positive stress test during physical
examination. Patients with previous anti-
incontinence surgery, a history of pelvic
radiation, pelvic surgery within the past
3months and women with pelvic organ pro
lapse of stage III or greater were excluded
from the study. The primary outcome of
treatment success at 12months was deter
mined by a Urogenital Distress Inventory
(UDI) reduction of 70% and a Patient
Global Impression of Improvement (PGII)
Box 1 | Developments in female urology
Urodynamic studies (UDS) are no longer
required in straightforward stress urinary
incontinence (SUI) evaluation
Synthetic midurethral sling placement is
now the standard treatment for SUI
OnabotulinumtoxinA is now a standard
treatment for overactive bladder (OAB)
Percutaneous tibial nerve stimulation
(PTNS) and sacral neuromodulation are
now options for the treatment of OAB
81 | NOVEMBER 2015
of much better. A total of 630 women were

randomized equally into the two study arms.
Successful treatment was observed in 76.9%
of patients who underwent UDS and office
evaluation, compared with 77.2% of patients
who only received office evaluation, showing
that UDS has no real effect on patient out
comes. Eliminating the need to put patients
with straightforward SUI through this
medical examination should expedite
patient care, decrease morbidity, reduce
costs and improve the patientexperience.
the past decade has been

an exciting one for the field
of female urology and voiding
dysfunction
Throughout the previous decade, the gold

standard for the treatment of SUI involved a
retropubic suspension or autologous pubo
vaginal slingboth of which are relatively
invasive, typically involve general or regional
anaesthesia and often require overnight
hospitalization. Tension-free vaginal tape
(TVT)a synthetic retropubic midurethral
slingwas introduced in the late 1990s, and
was widely adopted by urologists over the
decade between 20042014, owing to excel
lent 5year outcome results and minimal
invasiveness compared with retropubic sus
pensions or autologous pubovaginal slings.2
Nilsson etal.3 recently published the 17year
follow-up period results of a multicentre pro
spective TVT study. Atotal of 78% of women
initially included in the study were evalu
ated, with over 90% of these patients objec
tively continent on follow-up after 17years,
and 87% of patients subjectively cured or
significantly improved according to PGII,
Incontinence Impact Questionnaire-short
form (IIQ7), the UDI-short form (UDI6),
and Urinary Incontinence Severity Score
(UISS). This prospective study provides

evidence for the durability of the TVT sling
as well as excellent patient outcomes with
minimal complications. The synthetic retro
pubic midurethral sling is now considered a
gold standard in the management of female
SUI.4 Overall, with the advent of the synthetic
retropubic midurethral sling and the transob
turator midurethral sling (which has accrued
data demonstrating excellent results for most
patients)5 the treatment of SUI has become
less invasive, more predictable and more
acceptable to patients and physicians alike.
One of the most important advances to
have garnered attention over the past few
years has been in the treatment of over
active bladder (OAB). Previously, OAB has
been difficult to treat, and limited thera
peutic options have been available; but now
onabotulinumtoxinA is considered the
standard treatment for patients with either
refractory idiopathic OAB or neurogenic
detrusor overactivity (NDO).6 Recently, Nitti
etal.7 performed a multicentre, random
ized, placebo-controlled trial to evaluate
onabotulinumtoxinA for the treatment of
patients with idiopathic OAB and UUI.
Patients were 18years old and experienced
3 UUI episodes over a 3day period. They
were randomized to receive either 100U
of intravesical onabotulinumtoxinA or
placebo. Primary end points were a change
in the mean number of UUI episodes per
day and the proportion of patients with a
positive treatment response on the treat
ment benefit scale. Secondary end points
were improvement in OAB symptoms, such
as a decrease in the average frequency of
micturition and urgency episodes as well as
scores on health-related quality of life (QOL)
questionnaires. This study demonstrated that
onabotulinumtoxinA therapy decreased
the mean daily frequency of UUI episodes
experienced by patients by 2.65 compared
with a decrease in patients receiving placebo
of only 0.87, which was statistically signifi
cant (P<0.001). Additionally, 60.8% of
patients receiving onabotulinumtoxinA
had a positive response according to QOL
questionnaire results, compared with only
a 29.2% positive response rate in patients
who received placebo (P<0.001). These
patients are generally difficult to treat and
have limited treatment options available to
them; onabotulinumtoxinA therapy has been
shown to improve QOL for these patients,
giving them a viable treatmentoption.
The efficacy of onabotulinumtoxinA in
patients with NDO has also been demon
strated by Schurch etal. 8 usingamulti
UROLOGY
c entre, double-blind, randomized, placebo-
controlled trial. The patients included were
18years old with urinary incontinence
proven by UDS, caused by NDO. Patients
had the option to continue their previously
prescribed anticholinergic therapy while
they were on the study, and all patients
performed intermittent catheterization.
The selected patients were randomized
1:1:1 to receive a single dose of 200U or
300U of onabotulinumtoxinA, or placebo.
Schurch and colleagues8 observed a signifi
cant decrease in incontinence episodes in
both treatment arms (P0.05), but not in
the placebo group. Additionally, patients
who received either 200U or 300U of ona
botulinumtoxinA had improved bladder
function on UDS, including in factors such
as maximum cystometric capacity, reflex
detrusor volume and maximum detrusor
pressure as well as improvements in quality
of life scores. This study did not demonstrate
a significant difference in effect between
the different onabotulinumtoxinA doses,
owing to its small sample size; however, it
did provide significant clinical evidence that
onabotulinumtoxinA decreases signs and
symptoms of urinary incontinence in patients
with NDO and, consequently, fundamentally
changed the management of these patients.
Neuromodulation therapy has gained
support in the literature and has become a
treatment option for patients with medi
cally refractory OAB. Percutaneous tibial
nerve stimulation (PTNS) is an example
of a neuromodulation treatment and is a
simple minimally invasive treatment for
OAB, which can be performed in an office
environment. Peters etal.9 provided signifi
cant support for the use of PTNS in a multi
centre, double-blind, randomized controlled
trial of 220 patients who were 18years old
and had OAB symptoms. These patients
were randomized to 12weeks of treatment
with PTNS or a sham procedure. Participants
who underwent PTNS showed significant
improvement in bladder symptoms, with
54.5% reporting a moderate, or greater than
moderate improvement, compared with
20.9% of patients who underwent the sham
procedure (P<0.001). This study supported
the safe and effective use of PTNS in the
treatment of OAB and it is now considered a
feasible option in the treatment of medically
refractory OAB.6 Sacral neuromodulation
has also become an option in the treatment
of medically refractory OAB over the past
10years.6 vanKerrebroeck etal.10 performed
a 5-year prospective, multicentre trial to
evaluate the use of sacral neuromodulation
therapy for urinary voiding dysfunction.

Atotal of 129 patients 18years old who
had UUI, urinary frequency and/or uri
nary retention were included in the study.
In patients with UUI, the mean number of
leakage episodes per day had decreased from
9.6 to 4.0 at the 5-year follow-up point. In
patients with urgency frequency, the mean
number of voids per day decreased from 19
to 15 and the mean voided volume increased
from 92ml to 165ml. This prospective trial
demonstrated the safety and efficacy of sacral
neuromodulation in patients with refractory
OAB and has improved treatmentoutcomes.
Undoubtedly, the past decade has been
an exciting one for the field of female uro
logy and voiding dysfunction. Significant
advances have been made in the evaluation
and management of patients with SUI and
a host of new treatment options for OAB
have been established (Box 1). Apreviously
difficult-to-treat population now has excel
lent minimally invasive options for treat
ment of their complex problems. Hopefully,
the field will continue to evolve and develop
both better diagnostic and treatmentoptions.
Center for Female Pelvic Medicine and
Reconstructive Surgery, Glickman Urologic
andKidney Institute, The Cleveland Clinic,
LernerCollege of Medicine, Glickman Tower
(QBuilding), 9500 Euclid Avenue, Cleveland,
OH44195, USA (M.M.C., H.B.G.).
Correspondence to: H.B.G.
goldmah@ccf.org
Competing interests
H.B.G. is a consultant and speaker for Allergan,
Medtronic and Uroplasty and a speaker for Astellas,
M.M.C. declares no competing interests.
1.
Nager, C.W. etal. A randomized trial of

urodynamic testing before stress-incontinence
surgery. N. Engl. J. Med. 366, 19871997
(2012).
2. Nilsson, C.G., Kuuva, N., Falconer, C.,
Retzapour, M. & Ulmsten, U. Longer-term
results of the tension-free vaginal tape (TVT)
procedure for surgical treatment of female
stress urinary incontinence. Int. Urogynecol. J.
Pelvic Floor Dysfunct. 12, 58 (2001).
3. Nilsson, C.G., Palva, K., Aarnio, R., Morcos, E.
& Falconer, C. Seventeen years follow-up of the
tension-free vaginal tape procedure for female
stress urinary incontinence. Int. Urogynecol. J.
Pelvic Floor Dysfunct. 24, 12651269 (2013).
4. Nager, C., Tulikangas, P., Miller, D. &
Goldman,H. Position statement on mesh
midurethral slings for stress urinary
incontinence. Female Pelvic Med. Reconstr.
Surg. 3, 123125 (2014).
5. Richter, H.E. et al. Retropubic versus
transobturator midurethral slings for stress
incontinence. N. Engl. J. Med. 362, 20662076
(2010).
6. Gormley, E.A. etal. Diagnosis and treatment of
overactive bladder (non-neurogenic) in adults:
AUA/SUFU guideline. J. Urol. 188, 24552463
(2012).
7. Nitti, V.W. etal. Onabotulinum toxin A for the
treatment of patients with overactive bladder
and urinary incontinence: results of a phase3,
randomized, placebo controlled trial. J. Urol.
189, 21862193 (2013).
8. Schurch, B. etal. Botulinum toxin type A is a
safe and effective treatment for neurogenic
urinary incontinence: results of a single
treatment, randomized, placebo controlled
6month study. J. Urol. 174, 196200 (2005).
9. Peters, K.M. etal. Randomized trial of
percutaneous tibial nerve stimulation versus
sham efficacy in the treatment of overactive
bladder syndrome: results from the SUmiT Trial.
J. Urol. 183, 438443 (2010).
10. van Kerrebroeck, P.E. etal. Results of sacral
neuromodulation therapy for urinary voiding
dysfunction: outcomes of a prospective,
worldwide clinical study. J. Urol. 178,
20292034 (2007).
DECADE IN REVIEWKIDNEY CANCER
Discoveries, therapies
andopportunities
W. Marston Linehan and Christopher J. Ricketts
Several advances in kidney cancer have occurred over the past decade,
including the discovery of mutations in chromatin remodelling genes and
genomic heterogeneity in clear cell renal cell carcinoma (ccRCC). Altered
metabolic patterns in ccRCC and papillary RCC have become apparent,
andnew drugs for ccRCC have been approved.
Linehan, W. M. & Ricketts, C. J. Nat. Rev. Urol. 11, 614616 (2014); published online 7 October 2014;
doi:10.1038/nrurol.2014.262
Between 2004 and 2014, remarkable

improvements in our understanding of
the genetic basis of renal cell carcinoma
(RCC) and in the treatment of patients
with advanced kidney cancer have been
realized. These developments include the

approval by the FDA of seven agents target
ing the VHL/HIF pathway for patients with
advanced-stage RCC, and the identifica
tion of gene mutations in kidney tumours.
NOVEMBER 2015 | 82
UROLOGY
These advances built on the progress of the
previous decade, which had seen the dis
covery of the VHL gene, the identification
of mutations of the VHL gene in clear cell
RCC (ccRCC) and the delineation of the
VHL pathway. Mutations in MET and their
involvement in hereditary type1 papillary
kidney cancer were also identified, and
FLCN gene mutations were shown to be
the primary cause for inherited chromo
phobe kidney cancer associated with
BirtHoggDub syndrome. Mutations in
the FH gene, which encodes a Krebs cycle
enzyme, were also identified as the cause
of most cases of hereditary type2 papillary
kidney cancer associated with hereditary
leiomyomatosis and renal cell carcinoma
(HLRCC). The understanding that kidney
cancer is made up of a number of different
types of cancer, each having different histo
logy, clinical course and genetic causes, has
aided progress in treating this disease.
understanding that kidney

cancer is made up of a number
of different types of cancer
aidedprogress
At the start of this decade, only IL2 was

approved for the treatment of patients with
advanced-stage kidney cancer. The deline
ation of the VHL/HIF pathway and its role in
RCC in the 1990s provided the foundation
for the development of targeted therapeutic
agents for patients with thisdisease. Since
then, several randomized, double-blind
trials have identified effective agents for
the treatment of kidney cancer, targeting
vascular endothelialgrowthfactor(VEGF)
and platelet-derived growth factor(PDGF)
pathways. The first targeted agent against
the VEGF and PDGF receptors, sorafenib,
was approved for patients with advancedstage kidney cancer in 2005. Patients
treated with sorafenib were shown to have
a median progression-free survival (PFS)
of 5.5months, compared with 2.8months
for those given placebo. In 2006, suni
tinib, which also targets the VEGF and
PDGF receptors, was approved. Sunitinib
therapy was associated with an 11-month
PFS compared with 5months for patients
treated with IFN-, a nonspecific immune
modulator that has been used for a number
of years in patients with advanced-stage
kidney cancer.
The mTOR pathway is an important
kidney cancer pathway that ind irectly
83 | NOVEMBER 2015
influences VEGF and PDGF expression by

affecting HIF translation. In 2007, temsiro
limus, an agent that targets mTOR, was
approved after patients with poor prognos
tic factors experienced a PFS of 10.9months
on the drug compared with 7.3months for
those treated with IFN-. Three agents were
approved in 2009: the first, everolimus,
also targets the mTOR pathway and was
approved for patients with RCC who had
previously received sunitinib or sorafenib
therapy. Those treated with everolimus
demonstrated PFS of 4.0months compared
with 1.9months for placebo. The second
drug, bevacizumab (a monoclonal anti
body against VEGFA) was approved for
use in conjunction with IFN- in patients
with advanced-stage RCC. Treatment with
bevacizumab and IFN- increased PFS
to a median of 10.2months, compared
with 5.4months in patients treated with
IFN- alone. Finally, pazopanib, a multi
kinase inhibitor targeting the VEGF and
PDGF receptors, was also approved to treat
patients with advanced-stage RCC. Pazo
panib therapy was found to be associated
with a PFS of 9.2months compared with
4.2months for patients given a placebo.
In January 2012, axitinib, another multi
kinase inhibitor, was the seventh targeted
therapeutic agent this decade approved
by the FDA for the treatment of patients
with kidney cancer. Patients treated with
axitinib were found to have a median PFS
of 6.7months compared with 4.7months
for patient receiving sorafenib.1
These accomplishments provided physi
cians with a range of targeted agents to treat
patients for the first time. Although the
response rates for treatment with agents
such as sunitinib or pazopanib are reported
to be approximately 35%, and there have
been improvements in PFS and overall sur
vival, few treatments result in a complete
response; most patients eventually progress
and many die of their cancer. This eventual
ity could be because targeting the VHL/HIF
pathway with currently available therapies is
inadequate, or because there are other genes
associated with the initiation or progres
sion of kidney cancer that are not currently
beingtargeted.
Exciting new insights into the genetic
basis of ccRCC came from reports identify
ing mutations in several chromatin remod
elling, histone modifying and SWItch/
SNF nucleosome remodelling complex
genes.2,3 When The Cancer Genome Atlas
Research Network performed a compre
hensive molecular characterization of
ccRCC genomic
heterogeneity
VHL/
Sorafenib
Sunitinib
Bevacizumab
Pazopanib
Axitinib
VHL/
TSC2/
Everolimus
Temsirolimus
VHL/
PTEN/
SETD2/
Everolimus
Temsirolimus
VHL/
BAP1/
VHL/
SETD2/
SMARCA4/
???
???
???
???
Figure 1 | Genomic heterogeneity in different

regions of ccRCC. The truncal VHL gene is
mutated in all regions, and can be targeted
with sunitinib, sorafinib, bevacizumab,
pazopanib or axitinib. The branch mutations,
in select regions of the tumour, can occur in
genes for which potential targeted therapies
do or do not exist, such as SETD2, BAP1 or
SMARCA4. Themost effective strategy could
involve combination therapy targeting the
VHL/HIF pathway, or a global strategy
targeting chromatin remodelling gene
pathways. Abbreviation: ccRCC, clear cell
renal cell carcinoma.
genetic changes associated with ccRCC, 19

significantly mutated genes were identified.
After VHL, the most commonly mutated
genes were the chromosome3 chromatin
remodelling genesPBRM1, SETD2 and
BAP1. Mutations of BAP1 were found to be
correlated with poor survival. Furthermore,
mutations were identified in KDM5C, TP53
and the PI3K/Akt pathway genes (PTEN,
MTOR and PIK3CA). It was also demon
strated that the PI3K/Akt pathway has an
important role in tumour progression.
Integrative analysis showed that high-grade,
high-stage, low-survival ccRCC tumours
demonstrated evidence of a Warburg-like
metabolic shift, involving downregula
tion of genes involved in the Krebs cycle,
decreased AMPK and increased fatty acid
synthesis.4 These findings, which are con
sistent with the isotopomer spectral analysis
of VHL/ ccRCC and FH/ type2 papillary
RCC, reveal that a dependence on reductive
glutamine metabolism for lipid biosynthe
sis is important in kidney cancer and could
provide the foundation for the develop
ment of novel forms of therapy targeting
the metabolic basis of RCC.5,6
To further understand the genetic basis
of ccRCC, Gerlinger and colleagues 7
reported the use of multiregion exome
sequencing (M-seq) to study the genomic
architecture and evolution of ccRCC, and
found extensive genomic heterogeneity.
UROLOGY
Truncal mutations of the VHL gene were
found in every segment of each sample,
whereas driver mutations in the chro
matin remodelling genes SETD2, BAP1
and KDM5C, as well as TP53 and genes
in the PI3K/mTOR pathway (MTOR,
PIK3CA, PTEN and TSC2), were found
only in some segments of the tumours;
these were labeled branch mutations. Of all
the driver mutations, 75% were subclonal,
and intratumoral heterogeneity increased
with the number of biopsy specimens analy
sed.7 These findings raise profound ques
tions about the most effective way to detect
driver mutations in ccRCC, and which
gene pathways to target. Should Mseq be
performed on primary tumours and/or
metastatic sites to identify common driver
mutations that are associated with the initia
tion and progression of kidney cancer? Do
both the truncal and branching driver gene
pathways need to be targeted, singularly or
together, to develop an effective form of
therapy for ccRCC? These critical questions
need to be answered in the next decade in
kidney cancer research.
Although a number of novel therapies
for patients with advanced-stage ccRCC
have been developed over the past decade,
we still have no effective treatment for
patients with advanced-stage papillary
kidney cancer. Significant insight into
papill ary kidney cancer has come from
studies of the hereditary form of type2
papillary renal carcinoma found in patients
with HLRCC, caused by mutation of the
FH gene. FHdeficient kidney cancer
cells, which exhibit impaired oxidative
phosphorylation, undergo a Warburg-like
metabolic shift to aerobic glycolysis with
decreased AMPK and increased mTOR and
HIF-1 levels. 8 Studies by Adam etal. 9
and Ooi etal.10 showed upregulation of the
antioxidant signalling pathways in both
FHdeficient kidney cancer and sporadic
(non-inherited) papillary kidney cancer.
Elevated fumarate in FHdeficient RCC
targets and inactivates the electrophile
sensor KEAP1, which results in NRF2 accu
mulation and upregulation of antioxidant
response element-controlled genes, such
as NQO and the GST family genes. These
genes are critical for the survival of cancers,
which are characterized by high levels of
oxidative stress, such as hereditary and
sporadic type2 papillary kidney cancer.9,10
This pathway provides a number of oppor
tunities for the development of targeted
therapeutic approaches for patients with
advanced-stage papillaryRCC.
Overall, this past decade has seen a

numb er of historic advances towards the
understanding and treatment of RCC,
with the FDA approving seven drugs tar
getingthe VHL/HIF pathway for patients
with advanced-stage kidney cancer. Figure1
illustrates the extensive genomic hetero
geneity of ccRCC, which poses a signifi
cant therapeutic challenge; intense efforts
are underway to translate discoveries into
new therapeutic approaches for patients.
The finding of a Warburg-like metabolic
shift and the dependence of the KEAP1/
NRF2 antioxidant pathway in both clear
cell and papillary RCC provides promis
ing opportunities for the development of
therapies ta rgeting the me tabolic basis
ofkidneycancer.
Urologic Oncology Branch, National Cancer
Institute, 10 Center Drive, MSC 1107,
CRCRoom1W5940, Bethesda,
MD208921107, USA (W.M.L., C.J.R.).
Correspondence to: W.M.L.
wml@nih.gov
Competing interests
1.
Jonasch, E. & Motzer, R.J. Ten years of progress

in renal cell carcinoma. J. Natl Compr. Canc.
Netw. 10, 690693 (2012).
2.
Dalgliesh, G.L. etal. Systematic sequencing of

renal carcinoma reveals inactivation of histone
modifying genes. Nature 463, 360363
(2010).
3. Varela, I. etal. Exome sequencing identifies
frequent mutation of the SWI/SNF complex
gene PBRM1 in renal carcinoma. Nature 469,
539542 (2011).
4. The Cancer Genome Atlas Research Network.
Comprehensive Molecular Characterization of
Clear Cell Renal Cell Carcinoma. Nature 499,
4349 (2013).
5. Metallo, C.M. etal. Reductive glutamine
metabolism by IDH1 mediates lipogenesis
under hypoxia. Nature 481, 380384
(2011).
6. Mullen, A.R. etal. Reductive carboxylation
supports growth in tumour cells with
defectivemitochondria. Nature 481, 385388
(2011).
7. Gerlinger, M. etal. Genomic architecture and
evolution of clear cell renal cell carcinomas
defined by multiregion sequencing. Nat. Genet.
46, 225233 (2014).
8. Tong, W.H. etal. The glycolytic shift in
fumaratehydratasedeficient kidney cancer
lowers AMPK levels, increases anabolic
propensities and lowers cellular iron levels.
Cancer Cell 20, 315327 (2011).
9. Adam, J. etal. Renal cyst formation in Fh1deficient mice is independent of the Hif/Phd
pathway: roles for fumarate in KEAP1
succination and Nrf2 signaling. Cancer Cell 20,
524537 (2011).
10. Ooi, A. etal. An antioxidant response
phenotype shared between hereditary and
sporadic type2 papillary renal cell carcinoma.
Cancer Cell 20, 511523 (2011).
DECADE IN REVIEWSEXUAL DYSFUNCTION
Post-RP erectile dysfunction

therapies for the next decade
Emmanuel Weyne and Maarten Albersen
Erectile dysfunction remains a frequent sequela of radical prostatectomy,

owing to injury to the cavernous nerves that innervate the penile erectile
tissue. Over the last decade, many strategies have been proposed to
minimize the duration of denervation and prevent irreversible structural
changes from occurring in the corpora cavernosa.
Weyne, E. & Albersen, M. Nat. Rev. Urol. 11, 616618 (2014); published online 30 September 2014;
doi:10.1038/nrurol.2014.274
Erectile dysfunction (ED) after radical pros

tatectomy remains a major health concern
for patients, urologists and practitioners in
sexual medicine. The presence and persis
tence of ED after radical prostatectomy has
a substantial negative impact on the quality
of daily life for men who have undergone
the procedure. Short-term oncological
results are excellent for radical prostatec
tomy, but the preservation and rehabilita
tion of sexual function remains a challenge.
Potency rates reported after nerve-sparing
radical prostatectomy vary greatly between

14% and 86%, mainly owing to reporting
factors (such as the definition of potency
used in each study), surgeon experience and
patient preoperative characteristics.1
Over the past decade, researchers have
investigated many different strategies to
reduce the time that the corpora caver
nosa lack innervation and limit irreversible
structural changescaused by apoptosis
of smooth muscle cells and fibrosis of the
vascular bedfrom occurring.
NOVEMBER 2015 | 84
UROLOGY
In 1999 the concept of nerve graft inter
position was established 2 and was sub
sequently adopted during the past decade,
mainly in tertiary referral centres. This
technique is especially useful in men at
increased risk of extracapsular extension of
their tumour, who undergo resection of one
or both cavernous nerves resulting in a wide
gap between the nerve ends, which mini
mizes the potential for full nerve regenera
tion. Preclinical rodent studies and initial
reports with sural grafts in observational
prospective studies in humans were promis
ing, reporting high potency rates after uni
lateral nerve reconstruction.3 However, a
2009 large-scale randomized phaseII trial,
which compared patients with unilateral
nerve-sparing radical prostatectomy with
or without unilateral sural nerve graft inter
position, did not show significant differ
ences in recovery of erectile function 2years
after surgery.4 These disappointing results
raised questions regarding the added benefit
of nerve grafting in the setting of unilateral
nerve-sparing radical prostatectomy, and
greatly decreased the general popularity of
the procedure. The use of Schwann-cellseeded artificial grafts, addition of neuro
trophic agents and combination use with
robotic procedures, which provide superior
visualisation and high manoeuvrability, are
future directions in this field.
All the postoperative strategies developed
over the past decade share the common aim
of penile rehabilitation: maintenance of
erectile function by activating the normal
physiological process of erection, with
the ultimate goal of resumption of medi
cally unassisted sexual activity. The loss of
spontaneous and nocturnal erections after
radical prostatectomy mean that the penis
is rendered in chronic state of hypoxia,
causing structural changes to the erectile
tissue. Increasing cavernosal perfusion
would increase penile oxygenation, protect
Image courtesy of Maarten Albersen
85 | NOVEMBER 2015
endothelial function and limit muscular

apoptosis. After the initial introduction
of the concept with intracavernosal injec
tion therapy, PDE5-inhibitors (PDE5Is)
have become the most commonly used
form of penile rehabilitation, owing to
their favourable mode of administration
and ability to increase penile oxygenation.
Preclinical studies in rodent models have
shown evidence of a benefit of PDE5Is in
rehabilitating erectile function. However,
clinical evidence remains limited to one
small randomized trial (76 patients),
in which the sildenafil group (50mg or
100mg) had a significantly higher propor
tion of responders (27%) than the placebo
group (4%) after wash-out.5 The conclu
sions drawn from this study are, however,
impaired by the small number of patients,
high withdrawal rate, the lower placebo
response than generally reported for nervesparing surgery, as well as a nonvalidated
primary study outcome (responses to ques
tion 3 and 4 of the International Index of
Erectile Function [IIEF] questionnaire and
a response to the question were erections
good enough for satisfactory sexual activ
ity?, not overall IIEF-EF score). Conversely,
a larger randomized and well-performed
trial found no difference in potency rates
between groups taking 10mg vardena
fil nightly plus on-demand placebo, ondemand vardenafil plus nightly placebo, or
nightly placebo plus on-demand placebo
for 9months with a subsequent 2month
wash-out period.6 Furthermore, in the latest
REACTT trial,7 tadalafila PDE5I with an
extended half-lifefailed to show an erec
tile function benefit compared with placebo
after the wash-out interval of 6weeks. Penile
rehabilitation with PDE5Is has a sound
conceptual basis, but hard clinical evidence
for its use is still lacking. Starting penile
rehabilitation earlyimmediately after, or
even before, radical prostatectomyand
risk stratification to select the patients
whom could most benefit from the treat
ment, are important considerations looking
to the future of penile rehabilitation.
The absence of conclusive clinical evi
dence in favour of PDE5Is for penile rehabi
litation has, more recently, shifted the focus
towards the initiator of the pathophysio
logical cascade: the nerve injury itself. One
strategy has been the use of immunophilin
proteins and ligands. Immunophilins
such as FK506 (tacrolimus), rapamycin,
and cyclosporin are multifunctional pro
teins with roles in immunoregulation, and
have long histories in the prevention of
allograft transplant rejection. The rationale is

that these agents could temper the neuro
inflammatory reaction occurring after
nerve injury, and they have shown much
promise in preclinical studies. However,
although a 2008 study showed that tacro
limus was highly effective in maintaining
erectile function in a bilateral cavernous
nerve crush model in rats,8 a clinical trial
(NCT00106392) failed to show a differ
ence in recovery of erections 24months
after bilateral nerve-sparing radical pros
tatectomy between men who received
either tacrolimus or a placebo adminis
tered from 4 to 10days before and 6months
aftersurgery.
ADSC migrate to the major

pelvic ganglion (MPG), from which
arise the axons that innervate
thepenis
A similar strategyto enhance neuro

regeneration with neuromodulatory ther
apyis based on the instrinsic ability of the
peripheral nervous system to regenerate
after injury. However, endogenous regener
ation is limited and does not usually result
in full recovery. Neuronal survival is regu
lated by different families of endogenous
neurotrophins among which brain-derived
nerve growth factor (BDNF) is one of the
most known. In 2006, Bella etal.9 elegantly
showed that BDNF is able to increase caver
nous nerve neurite outgrowth invitro and
improve recovery of erectile function after
cavernous nerve crush trauma. These
results looked promising, but BDNF, like
most of these neurotrophin peptides, is not
suited for oral administration. Howe ver,
neurotrophic factors couldhypothetically
be applied in slow-releasing hydrogels
placed in the proximity of the NVB at the
end of the radical prostatectomy procedure,
and this concept is something that could be
investigated in the future.
Mesenchymal stem cells (MSC) have been
shown to be a successful treatment option
in clinical trials for various cardiovascular,
inflammatory and degenerative diseases.
Of the various MSCs, adipose-derived stem
cells (ADSC) show the greatest potential
as a treatment option for ED after radical
prostatectomy. ADSC are easy to isolate and
can be administered as a stromal vascular
fraction during the same surgical session as
the prostatectomy procedure. After admin
istration and cavernous nerve injury (CNI),
ADSC migrate to the major pelvic ganglion
UROLOGY
Many men remain

undertreated with the current
armoury of therapies that are
available in clinical practice
1.
(MPG), from which arise the axons that

innervate the penis10 and can, therefore, be
administered systemically by intravenous
injection. At the site of injury, they act as
a local factory of molecules that promote
neuroregeneration and temper the inflam
matory reaction after nerve injury. In this
way, injection of ADSCs has been shown to
significantly improve erectile function after
CNI in a rat model.10 Multiple clinical trials
are currently enrolling patients, including
one at Johns Hopkins Medical Institutions
(NCT01983709), which will investigate the
safety of systemically administered allo
genic bone-marrow-derived stem cells in
men with prostate cancer and the effect
on erectile recovery after radical prosta
tectomy as a secondary end point. We look
forward to the results of this trial, which
have the potential to stimulate the wide
spread use of stem cells as a new treatment
for CNIassociated ED.
ED remains a frequent concern after
radical prostatectomy, owing to injury to
the cavernous nerves. Many men remain
undertreated with the current armoury of
therapies that are available in clinical prac
tice. Perioperative tools to facilitate NVB
identification and cavernous nerve recon
struction using sural nerve grafts have not
achieved the improvements in erectile func
tion recovery that they seemed to promise
in early studies, and PDE5Is remain the
most commonly used treatment, despite
the paucity of clinical evidence in their
favour. Preclinical studies carried out
over the last decade have shown exciting
potential for stem cell and neurotropic
factor therapy to boost the endogenous
neuroregenerative response after CNI and
we eagerly await their implementation
in clinical trials. As we look to the future,
these therapies represent an auspicious new
avenue for penilerehabilitation.
3.
Laboratory for Experimental Urology,

Department of Development and
Regeneration, KU Leuven and University
Hospitals, Campus Gasthuisberg, O&N 1,
Herestraat 49, Box802, 3000 Leuven, Belgium
(E.W., M.A.).
Correspondence to: M.A.
maarten.albersen@uzleuven.be
Competing interests
2.
4.
5.
Boorjian, S.A. S. etal. A critical analysis of

thelong-term impact of radical prostatectomy on
cancer control and function outcomes. Eur.Urol.
61, 664675 (2012).
Kim, E.D. etal. Interposition of sural nerve
restores function of cavernous nerves resected
during radical prostatectomy. J. Urol. 161,
188192 (1999).
Namiki, S. etal. Impact of unilateral sural nerve
graft on recovery of potency and continence
following radical prostatectomy: 3year
longitudinal study. J. Urol. 178, 212216 (2007).
Davis, J.W. etal. Randomized PhaseII trial
evaluation of erectile function after attempted
unilateral cavernous nerve-sparing retropubic
radical prostatectomy with versus without
unilateral sural nerve grafting for clinically
localized prostate cancer. Eur. Urol. 55,
11351144 (2009).
Padma-Nathan, H. etal. Randomized, doubleblind, placebo-controlled study of postoperative
nightly sildenafil citrate for the prevention of
erectile dysfunction after bilateral nerve-sparing
radical prostatectomy. Int. J. Impot. Res. 20,
479486 (2008).
6.
Montorsi, F. etal. Effect of nightly versus ondemand vardenafil on recovery of erectile

function in men following bilateral nerve-sparing
radical prostatectomy. Eur. Urol. 54, 924931
(2008).
7. Montorsi, F. etal. Effects of tadalafil treatment
on erectile function recovery following bilateral
nerve-sparing radical prostatectomy: a
randomised placebo-controlled study (REACTT).
Eur. Urol. 65, 587596 (2014).
8. Sezen, S.F., Lagoda, G. & Burnett, A.L. Role of
immunophilins in recovery of erectile function
after cavernous nerve injury. J. Sex. Med. 6,
340346 (2009).
9. Bella, A.J. etal. Brain-derived neurotrophic
factor (BDNF) acts primarily via the jak/stat
pathway to promote neurite growth in the major
pelvic ganglion of the rat: part I. J. Sex. Med. 3,
815820 (2006).
10. Fandel, T.M. etal. recruitment of
intracavernously injected adipose-derived
stemcells to the major pelvic ganglion
improveserectile function in a rat model of
cavernous nerve injury. Eur. Urol. 61, 201210
(2011).
DECADE IN REVIEWPROSTATE CANCER
A decade of progress in detection

and treatment
Behfar Ehdaie and Peter T. Scardino
A fundamental shift in the understanding, detection and treatment of

prostate cancer has occurred over the past 10years, especially in the
use of screening, active surveillance, novel therapies and radical surgery.
These discoveries have changed how clinicians and patients approach
prostate cancer.
Ehdaie, B. & Scardino, P. T. Nat. Rev. Urol. 11, 618620 (2014); published online 14 October 2014;
doi:10.1038/nrurol.2014.284
The past decade has witnessed fundamen

tal shifts in our understanding of prostate
cancer and in the detection and treatment
of all stages of this common disease. The
greatest changes in the approach to pros
tate cancer have been in attitudes to screen
ing, the role of patient-reported outcomes
in documenting the effects of therapy
on quality of life (QOL), the move from
radical treatment to active surveillance for
low-risk prostate cancer, the expanding
role of surgery for high-risk cancers and
the move towards multimodality treatment
for advanced cancers. Despite this progress,
prostate cancer remains the second most
lethal cancer among men, and it will con
tinue to challenge our best judgement and
therapeutic acumen as we work to further
reduce morbidity and mortality from this
disease in the coming decades.
PSA has been widely used for the early
detection of prostate cancer in clinical prac
tice since its discovery. However, its value in
population-wide screening remains contro

versial and the results of large randomized
trials have failed to resolve the debate.
The US-based Prostate Lung Colorectal
and Ovarian Cancer screening trial found
no reduction in prostate-cancer-specific
mortality (PCSM) over a median of
10years, but the study was compromised
by pretesting for PSA in 40% of the subjects
and by contamination of the control recruits
(some 70% of the unscreened control
cohort received a PSA test during the study
period), meaning that the study was only
able to report that intense sc reeningis
no better than opportunistic screening
for reducing mortality. 1 In contrast, the
European Randomized Study of Screening
for Prostate Cancer reported a statistically
significant relative reduction of 21% in
PCSM at a follow-up duration of 13years,
equivalent to one prostate cancer death
averted per 781 men invited for screening,
or one per 27 additional prostate cancers
NOVEMBER 2015 | 86
detected.2 Despite this substantial reduction

in PCSM with PSA screening, in 2012 the
US Preventive Services Task Force recom
mended against PSA screening, concluding
that no net benefit is gained from the prac
tice and that the potential harms outweigh
the benefits. Nevertheless, the longest trial
reported to datethe Gteborg random
ized population-based prostate cancer
screening trialshowed a 44% reduction
in PCSM at a follow-up period of 14years,
with screening of 293 men and diagno
sis of 12 cancers preventing one death.3
The power of PSA testing was confirmed
by studies investigating the association
between PSA levels at mid-life, defined
asbetween 4560years, and the lifetime
risk of prostate cancer metastases and death,
enabling decisions to be made regarding
further screening and treatment options.4
These studies have also shown that men
with PSA levels below the reported median
concentration (1.1ng/ml) at 60years have
little chance of dying from prostate cancer
and can, therefore, be excluded from further
screening. This evidence provides weight to
the argument for the value of PSA testing
torisk-adjust screening strategies.
Despite this progress, prostate

cancer remains the second most
lethal cancer among men
The harms of early detection as a result of

PSA screening arise mostly from the con
sequences of unnecessary radical therapy
for low-risk cancers. Patient-reported out
comes provide objective tools for quantify
ing the adverse effects of therapy and its
impact on QOL. In a landmark longitudinal
observational study of the long-term effects
of surgery and radiotherapy on QOL, most
men were shown to experience significant
short-term reductions in urinary and sexual
function after surgery, with less severe
reductions in urinary and sexualfunc
tion, but greater impairment of bowel
function, after radiation. Although these
functional deficits can improve in some
men over time, long-term adverse effects
especially in sexual functionpersist in
many.5 For this reason, active surveillance
for men with low-risk cancer is becoming
more common. Men followed in active
surveillance programmes have little risk of
dying from prostate cancer within 10years,
and many cancers that do progress respond
to delayed treatment with surgery or radia
tion. Nevertheless, longer follow-up periods
87 | NOVEMBER 2015
could reveal greater risks of progression for

men on active surveillance than for those
given radical treatment. Furthermore, active
surveillance requires frequent biopsies,
which are invasive and carry a risk of infec
tion and bleeding, and the criteria under
which men are assessed for delayed inter
vention are poorly defined. However, there
is a growing consensus that men with lowrisk cancer do not usually need immediate
definitive therapy (assuming that no highrisk or intermediate-risk cancer is found on
careful evaluation) and can be put on active
surveillance programmes and men with
intermediate-risk and high-risk cancers
are less likely to develop metastases or to
die of cancer if they are treated with radical
prostatectomy or radiation therapy.
Prediction tools and nomograms have
also been improved and are now widely
available to assist physicians and patients
inthe shared decision-making process
about the treatment options available
for localized prostate cancer. Together
with more accurate biopsy strategies and
improved imaging with MRI, these tools
provide more accurate risk assessments
than were previously available and a greater
confidence that active surveillance is safe
for men with lowrisk prostate cancer.
The ability to make better medical deci
sions in collaboration with patients and
their families will continue to improve
in the next decade, as we standardize the
evaluation of candidates for active surveil
lance. The most promising tools for better
characterization of cancer include advanced
imaging technologies such as multipara
metric MRI, molecular profiling toolsfor
example Prolaris(Myriad Genetics,USA)
and OncotypeDX(Genomic Health, USA)
and tests for circulating biomarkers,
such as the 4Kscore(OPKO Diagnostics,
USA) and the Prostate Health Index (phi).
Improved risk assessment will help to more
precisely define eligibility for active surveil
lance programmes, and the characteristics
that indicate that delayed intervention is an
appropriate treatment option.
During most of the past century, open
radical prostatectomy (ORP) has been the
standard surgical approach in men with
localized prostate cancer. However, over
the past decade, robot-assisted laparoscopic
prostatectomy (RALP) has been widely
adopted, despite an absence of formal evalu
ation documenting superior outcomes. In
fact, population-based observational studies
and single-institution case series continue
to show comparable outcomes for both
iStock/Thinkstock
UROLOGY
approaches, except that patients receiv

ing RALP have slightly shorter hospital
stays (for example 2days for RALP versus
3days for ORP) and fewer blood trans
fusions.6 According to administrative data
RALP does not improve complication rates
or readmission rates compared with ORP.
The skill level of the surgeon, indepen
dent of the technology employed, clearly
has the greatest impact on outcomes. The
positive association of surgical experience
with improved outcomes has been mapped
in learning curves charting surgeon experi
ence with biochemical-recurrence rates and
patientreported functional outcomes.7
Landmark clinical trials have been com
plemented by breakthroughs in translational
research, improving our understanding of
the molecular and genetic factors that drive
prostate cancer. The fusion between trans
membrane protease serine 2 (TMPRSS2)
and ERG, a member of the erythroblast
transformation-specific transcription factor
family, was reported by Tomlins etal. 8
working under Chinnaiyan, and is found in
approximately 50% of primary and meta
static prostate cancers. Although the clini
cal relevance of this fusion gene remains
debatable, its high prevalence provides an
important pathway for investigating new
diagnostic and prognostic markers, as well
as potential targets for novel therapeu
tics. Acomprehensive integrated genomic
profile of prostate cancer found alterations
in key molecular pathways in a surprisingly
large number of primary and metastatic
tumours, and the degree of copy-number
alterations was shown to be associated with
prognosis, independent of Gleason grade.9
Perhaps the single greatest advance in
prostate cancer biology during the past
decade was the finding by the Sawyers
group10 that upregulation of the androgen
receptor was both necessary and sufficient
for the castration-resistant phenotype. This
discovery drove the development of novel
UROLOGY
antiandrogens (for example abiraterone
and enzalutamide), which have markedly
improved the prognosis of patients with
advanced prostate cancer some 75years
after Huggins first identified the effects of
androgen deprivation on prostate cancer,
and after numerous studies of various regi
mens of ADT failed to improve survival in
these patients.
In the past decade, the FDA has approved
six new therapeutic agents that prolong
overall survival in men with castration-
resistant prostate cancer, including drugs
that target androgen-dependent and
androgen-i ndependent pathways, bone
metastas es, and cell cycle regulators.
Evaluation of these therapies is occurring
in multimodality clinical trials, raising the
hope that we might one day develop thera
peutic programmes that can cure many
more men with advanced prostate cancer.
Memorial Sloan Kettering Cancer Center,

1275York Avenue, New York, NY 10065, USA
(B.E., P.T.S.).
Correspondence to: P.T.S.
scardinp@mskcc.org
Competing interests
P.T.S. is a paid scientific advisor to OPKO
Diagnostics, which has licensed the 4KScorefrom
P.T.S. and colleagues for commercial development.
B.E. declares no competing interests.
1.
2.
3.
Andriole, G. L. etal. Mortality results from

arandomized prostate-cancer screening
trial.N.Engl. J.Med. 360, 13101319
(2009).
Schrder, F. H. etal. Screening and prostate
cancer mortality: results of the European
Randomised Study of Screening for Prostate
Cancer (ERSPC) at 13years of follow-up.
Lancet http://dx.doi.org/10.1016/
S0140-6736(14)60525-0.
Hugosson, J. etal. Mortality results from the
Gteborg randomized population-based
prostate-cancer screening trial. Lancet Oncol.
11, 725732 (2010).
4.
Vickers, A. J. etal. Strategy for detection of

prostate cancer based on relation between
prostate specific antigen at age 4055 and
long term risk of metastasis: case-control
study. BMJ 2013, 346357 (2013).
5. Sanda, M. G. etal. Quality of life and
satisfaction with outcome among prostatecancer survivors. N.Engl. J.Med. 358,
12501261 (2008).
6. Gandaglia, G. etal. Comparative effectiveness
of robot-assisted and open radical
prostatectomy in the postdissemination era.
J.Clin. Oncol. 32, 14191426 (2014).
7. Vickers, A. etal. Cancer control and functional
outcomes after radical prostatectomy as
markers of surgical quality: analysis of
heterogeneity between surgeons at a single
cancer center. Eur. Urol. 59, 317322 (2011).
8. Tomlins, S. A. etal. Recurrent fusion of
TMPRSS2 and ETS transcription factor genes in
prostate cancer. Science 310, 644648 (2005).
9. Taylor, B. S. etal. Integrative genomic profiling
of human prostate cancer. Cancer Cell 18,
1122 (2010).
10. Chen, C. D. etal. Molecular determinants of
resistance to antiandrogen therapy. Nat. Med.
10, 3339 (2004).
NOVEMBER 2015 | 88
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A DECADE IN

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From mechanisms to management

design: laura marshall

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