MINI-SYMPOSIUM: BREAST PATHOLOGY
Molecular diagnosis in
breast cancer
Fresia Pareja

Caterina Marchio
Jorge S Reis-Filho
Abstract
Breast cancer is a complex and heterogeneous disease, encompass-
ing a plethora of entities with distinct biological features and clinical
behaviour. The advent of high throughput molecular methods has
allowed a systematic characterization of the genomic landscape of
breast cancer, revealing a profound heterogeneity in this disease.
These methods are having a profound effect on the understanding
of breast cancer. Some have already been incorporated in clinical
practice, such as the prognostic ‘gene signatures’ that allow the
tailoring of therapy in the subgroup of patients with oestrogen receptor
(ER)-positive and HER2-negative breast cancer. In this review, we
discuss the contribution of the main molecular methods in breast can-
cer research and how this information is changing our approaches to
the diagnosis and management of this disease. We also address novel
developments in the diagnosis and management of HER2-positive
breast carcinomas and familial breast cancer.
Keywords in situ hybridization; liquid biopsies; molecular taxonomy;
precision medicine; prediction; prognosis; prognostic signatures
Molecular pathology techniques have had a dramatic effect on
the diagnosis of haematological malignancies and soft tissue
sarcomas. This has led to a paradigm shift in the way entities are
defined: from purely morphological, descriptive classification
systems to a combined histopathological and molecular taxon-
omy. Many haematological malignancies are defined by specific
recurrent chromosomal translocations and/or molecular
aberrations.
The contribution of molecular pathology to the study of most
types of carcinomas has been less profound. With the boom of
high throughput technologies and increasingly coherent data on
the molecular features of epithelial malignancies, molecular
techniques are becoming an integral part of the armamentarium
of surgical pathology laboratories.
Breast cancer has been more extensively studied with mo-
lecular methods than any other epithelial malignancy. Some of
the ‘molecular-era’ breakthroughs have been translated into
Fresia Pareja MD PhD is an Instructor in Pathology at Memorial Sloan
Kettering Cancer Center, New York, NY, USA. Conflicts of interest:
none declared.
Caterina Marchio  MD PhD is Assistant Professor of Pathology at the
University of Turin, Italy. Conflicts of interest: none declared.
Jorge S Reis-Filho MD PhD FRCPath is the Director of Experimental
Pathology at Memorial Sloan Kettering Cancer Center, New York,
NY, USA. Conflicts of interest: none declared.
DIAGNOSTIC HISTOPATHOLOGY 24:2
methods amenable to histopathologists (e.g. immunohisto-
chemistry) and made ‘prime time’ in diagnostic practice (e.g. E-
cadherin immunohistochemistry to differentiate between lobular
neoplasia and low-grade solid ductal carcinoma in situ). Molec-
ular data have also confirmed the concept that breast cancer is a
heterogeneous disease, comprising several histological types
with distinct biological features and different clinical behaviour.
Understanding the molecular features of breast cancer may
potentially provide additional diagnostic, prognostic and pre-
dictive information that may, in the not so distant future, facili-
tate the development of tailored therapy.
The main contribution of molecular methods for under-
standing breast cancer will be addressed, and the way this in-
formation is changing the management of breast cancer will be
contextualized in this review.
Molecular classification of breast cancer
Microarray-based gene expression profiling has solidified the
notion that breast cancer, rather than being a single disease,
represents a group of entities with different molecular alterations
and clinical behaviour. Seminal studies by the Stanford group led
to the classification of breast cancer into four intrinsic subtypes:
luminal A, luminal B, HER2-enriched and basal like.1 Later on,
additional molecular subtypes of breast cancer were identified,
such as the claudin-low2 and the molecular apocrine.3
The ER-positive group comprises the luminal A and luminal B
tumours, which are characterized by the expression of ER, genes
pertaining to the ER pathway, and other transcripts usually found
in luminal epithelial cells. The prognosis of luminal tumours is
largely determined by the expression of proliferation-related
genes. Luminal B cancers display higher levels of genes per-
taining to the proliferation cluster than luminal A tumours, and
have a worse prognosis.4,5 Luminal tumours show intrinsic
heterogeneity. Along these lines, luminal A cancers can be
further stratified into four subgroups, with different copy number
alterations, somatic mutations profiles and clinical outcomes,
including the copy number-high subgroup, which displays high
genomic instability, recurrent TP53 mutations and over-
activation of Aurora kinases, and is associated with a worse
clinical outcome.6 The ER-negative cluster encompasses the
HER2-enriched subgroup, characterized by high levels of
expression of genes pertaining to the HER2 amplicon (17q11);
the basal-like subgroup, characterized by the expression of genes
expressed by basal/myoepithelial cells, such as basal cytoker-
atins; claudin-low tumors, which are enriched for genes related
to cancer stem cells, epithelial to mesenchymal transition and
immune response2; and the molecular apocrine subgroup, which
shows increased androgen signaling and a molecular apocrine
gene expression profile.3 This molecular taxonomy of breast
cancer has important clinical implications, as the different mo-
lecular subtypes display distinct biology, responses to therapy
and clinical outcomes.7
Triple negative breast cancer (TNBC) shows a vast inter-
tumour heterogeneity, and seven molecular subtypes have
been put forward by Lehmann et al.,8 including the basal-like 1
(BL1), basal-like 2 (BL2), mesenchymal (M), mesenchymal stem-
like (MSL), immunomodulatory (IM), luminal androgen receptor
(LAR), and unstable subgroups.8 Nonetheless, it was later
shown, by the same group, that the transcriptomic profiles of the
71 Ó 2018 Elsevier Ltd. All rights reserved.
 MINI-SYMPOSIUM: BREAST PATHOLOGY
IM and the MSL subgroups might not derive from tumour cells,
but rather stem from tumour infiltrating lymphocytes and stro-
mal cells, respectively,9 indicating that the most parsimonious
number of TNBC molecular subtypes is four. The clinical impli-
cation of this classification was confirmed by studies showing
that the different TNBC subgroups significantly differ in terms of
their response to neoadjuvant chemotherapy.9 The rate of path-
ologic complete response (pCR) for BL1 tumours is much higher
than the one for BL2 and LAR tumours.9
An alternative molecular classification of breast cancer, based
on the integrative analysis of copy number alterations and gene
expression, was put forward by the Molecular Taxonomy of
Breast Cancer International Consortium (METABRIC), which
categorized breast cancer into ten integrative clusters
(IntClusts).10 A gene expression method for the classification of
breast cancer into the different IntClusts was later developed,11
and provided an independent validation of the clinical rele-
vance of this classification, as breast cancers corresponding to
the different IntClusts displayed varying responses to neo-
adjuvant chemotherapy and different clinical outcomes.11
Prognostic gene signatures in breast cancer
The identification of patients who will benefit from adjuvant
chemotherapy remains challenging. Multigene prognostic tests
have become useful tools in the determination of the risk of
recurrence and in the decision making of whether or not
chemotherapy should be spared for some patients.12 Whilst first
generation prognostic assays, such as Mammaprint13 and
Oncotype Dx14 have a better predictive power for recurrences
within the first five years, more recent tests, such as Prosigna,15
Endopredict,16 and the Breast Cancer Index (BCI)17 have good
predictive power both for early and late recurrences (Table 1).18
The utility of multigene assays is limited in ER-negative breast
cancer, as most cases are classified as “high-risk” due to their
elevated proliferative rates,5 restricting the prognostic value of
multigene assays to ER-positive disease. Of note, all these five
tests may be performed using formalin-fixed paraffin-embedded
(FFPE) samples, facilitating their widespread use, and whilst
Mammaprint, Oncotype Dx and BCI should be performed by
central laboratories, Prosigna and EndoPredict may be set up in
local pathology laboratories.18
Oncotype Dx is a reverse transcriptase-PCR (RT-PCR) assay
which measures the relative expression of 21 genes, including
16 cancer-related genes and five reference genes, and computes
a recurrence score (RS) from zero to 100, assigning individual
List of commercially available prognostic gene signature assays that are clinically useful in the context of ERD/HER2-
disease
Mammaprint
Method Microarrays
Feasibiity on FFPE samples Yes
Type of assessment Central laboratory
Level I evidence Yes, IA
Information regarding the molecular subtype No
Table 1
DIAGNOSTIC HISTOPATHOLOGY 24:2
patients into the low- (RS < 18), intermediate- (RS 18e30) and
high-risk (RS
31) categories,14 which determines the risk of
distant recurrence at 10 years and the benefit of the addition of
chemotherapy in ER-positive, HER2-negative, node-negative
breast cancer patients treated with tamoxifen. Interestingly, a
recurrence score predicted by the integration of morphologic
and immunohistochemical parameters including histologic
grade, receptor status, tumour size and Ki67 expression can
predict the Oncotype Dx RS with relative accuracy.19 The clin-
ical utility of Oncotype DX was validated by the initial results of
the TAILORx study.20 MammaPrint is a DNA microarray-based
prognostic assay for patients younger than 61 years old with
stage I or II ER-positive node-negative breast cancers.13 This
assay entails the evaluation of the expression of 70 genes,
enabling the stratification of patients into low-risk and high-risk
categories, and its utility was validated by the prospective
randomized phase III MINDACT trial.13 Prosigna is an RT-PCR
based assay which, using the NanoString technology, mea-
sures the expression of 50 classifier genes from the PAM50
molecular classification algorithm and of 5 control genes, and
computes a risk of recurrence (ROR) score, placing patients into
the low-, intermediate- or high-risk categories, depending on
their 10 year-risk of distant recurrence, which correlates with
the intrinsic subtype of the case.15 Its use was approved for the
prediction of distant recurrence-free survival in postmenopausal
women with stage I and stage II ER-positive breast cancer
treated with adjuvant hormone therapy. EndoPredict is an RT-
PCR based assay which calculates a risk score based on the
expression of eight cancer-related genes and three reference
genes, allowing the stratification of patients with early ER-
positive breast cancer treated with adjuvant endocrine therapy
alone, into high-risk and low-risk groups for 10 year-recur-
rence.16 The integration of EndoPredict score with tumour size
and nodal status allows the computing of EPclin, a compre-
hensive risk score, which has been validated in the ABCSG-6
and ABCSG-8 randomyzed phase III trials.16 Lastly, BCI is an
RT-PCR based assay which quantifies the expression ratio of
HOXB13 and IL17BR,21 and integrates it with the molecular
grade index (MGI), which assesses the expression of five genes,
related to tumour grade and proliferative status.17 It was
designed for the identification of patients with early ER-positive,
node-negative breast cancer receiving adjuvant hormone ther-
apy at a high risk of recurrence. Its prognostic utility was vali-
dated in postmenopausal patients with early ER-positive breast
cancer from the Stockholm trial.22
Oncotype Dx Prosigna EndoPredict Breast Cancer Index
qRT-PCR NanoString qRT-PCR qRT-PCR
Yes Yes Yes Yes
Central laboratory Local laboratory Local laboratory Central laboratory
Yes, IA Yes, IB Yes, IB Yes, IB
No Yes No No
72 Ó 2018 Elsevier Ltd. All rights reserved.
 MINI-SYMPOSIUM: BREAST PATHOLOGY
Although these multigene tests show an appropriate agree-
ment at the population level, comparative studies have revealed
discordant risk prediction when individual cases are evaluated
by different assays.23 For instance, about a third of cases classi-
fied as high-risk by Mammaprint fell in the low-risk category
when evaluated by Oncotype Dx.24 Despite these limitations,
these biomarkers are changing the way we manage breast can-
cer. Underscoring their clinical relevance, a multidisciplinary
team of breast cancer experts recommended the incorporation of
the Oncotype Dx score in the staging of patients with ER-positive
HER2-negative breast cancer in the 8th edition of the American
Joint Committee on Cancer (AJCC) staging system.25
In situ hybridization: diagnosis, prognosis and prediction
In situ hybridization (ISH) is a molecular technique that allows
the visualization of genes on a glass slide. This technique is
commonly used in clinical practice, both for diagnostic and
prognostic/predictive purposes. In breast pathology ISH enables
the evaluation of copy number alterations and the detection of
fusion genes. Three methods of ISH have been introduced in
breast pathology: fluorescent in situ hybridization (FISH), chro-
mogenic in situ hybridization (CISH), and silver in situ hybridi-
zation (SISH). They allow for the semi-quantitative assessment of
gains, losses and amplifications (FISH and CISH) directly on
tissue sections, combining molecular genetics with traditional
pathology.
In diagnostic breast pathology, one of the main uses of ISH is
HER2 gene testing, for which FISH, CISH and SISH methods are
available and have been FDA approved. Nevertheless, HER2
testing is not the sole utility of ISH in breast pathology, as these
techniques can also be used for the detection of amplification of
novel promising therapeutic targets (for instance FGFR1),26 and
Diagnostic, prognostic and predictive utility of in situ
hybridization in diagnostic practice at present
Gene Significance
MYB/NFIB Diagnosis of breast adenoid cystic
translocation carcinoma
MYBL1 Diagnosis of breast adenoid cystic
rearrangements carcinoma
(MYBL1-ACTN1 and
MYBL1-NFIB
translocations)
MYB amplification Diagnosis of breast adenoid cystic
carcinoma (identified in a single case at
present)
ETV6/NTRK3 Diagnosis of secretory carcinoma
translocation
HER2 amplification Prediction of response to anti-HER2
agents þ chemotherapy in adjuvant,
neoadjuvant and metastatic settings
FGFR1 amplification Predictive factor for response to FGFR1
tyrosine kinase inhibitors (at present in
the context of clinical trials)
Table 2
DIAGNOSTIC HISTOPATHOLOGY 24:2
for the study of fusion genes in rare histologic types of breast
cancer (Table 2).
A subset of rare breast cancers display distinctive morphol-
ogies and are underpinned by pathognomonic genetic alter-
ations,27 such as fusion genes, including adenoid cystic
carcinoma (AdCC) and secretory carcinoma. ISH techniques are
of great diagnostic utility in this context, especially when dealing
with less differentiated lesions (for instance the solid variant of
AdCC). AdCC, a common salivary gland tumour, may arise in
other organs, including the breast, and is characterized by the
t(6;9)(q22-23;p23-24) translocation, which results in the MYB-
NFIB fusion gene, regardless of its anatomic origin.28 Bona fide
AdCCs lacking the typical MYB-NFIB fusion gene are on re-
cord.29,30 We recently described that MYB-NFIB fusion-negative
AdCCs are characterized by alternative genetic alterations
resulting in activation of the MYB pathway, including MYBL1
rearrangements (MYBL1-ACTN1 and MYBL1-NFIB) and MYB
amplification,30 all of which are detectable by ISH techniques.
Another special histologic type for which ISH can be of diag-
nostic value is breast secretory carcinoma. This is an exceedingly
rare histologic type of breast cancer which harbours the highly
recurrent t(12;15)(p13;q25) translocation, resulting in the ETV6-
NTRK3 fusion gene.31 Whilst other neoplasms outside the breast
harbour this fusion gene, it is pathognomic for secretory carci-
noma in a breast-specific context.32 The correct identification of
secretory carcinomas is of clinical significance,31 because these
tumours are associated with a favourable prognosis.33
HER2 assessment in diagnostic practice
HER2, a member of the epidermal growth factor receptor family,
is an orphan tyrosine kinase receptor overexpressed in about 15
e20% of breast cancers.34,35 Targeting of HER2 is perhaps the
best example of tailored therapy for breast cancer patients as
HER2 is a tumour driver and constitutes an excellent example of
‘oncogene addiction’. Trastuzumab, a humanized monoclonal
anti-HER2 antibody, is offered to breast cancer patients whose
tumours have HER2 3þ immunohistochemical expression or
HER2 gene amplification (Figure 1), in the adjuvant, neoadjuvant
and metastatic settings.36e38 Over the past years novel anti-HER2
therapeutic strategies have been proposed: monoclonal anti-
bodies that are allegedly more effective in blocking HER2 heter-
odimerization (e.g. pertuzumab),39 HER2 tyrosine kinase
inhibitors (e.g. lapatinib),40 and more recently antibody-drug
conjugates, such as trastuzumab emtansine, i.e. T-DM1.41 In
the latter case it is important to note that as the chemotherapeutic
agent is linked to the anti-HER2 antibody, successful delivery of
the chemotherapeutic agent will utterly depend on the real
presence of the HER2 protein, thus highlighting the importance
of providing accurate HER2 testing.
In this respect it should be mentioned that good laboratory
practice preanalytical variables, such as cold ischemia time
together with type and time of fixation, should be strictly moni-
tored, and kit preparations should be employed for immunohis-
tochemical evaluation.42 HER2 testing can be approached by
either performing first immunohistochemistry (IHC) followed by
reflex ISH in IHC-equivocal cases, or doing front-line ISH on each
newly diagnosed breast cancer (Figure 1). Readers should refer to
the latest version of the ASCO/CAP guidelines for the details in the
73 Ó 2018 Elsevier Ltd. All rights reserved.
 MINI-SYMPOSIUM: BREAST PATHOLOGY
IHC testing
SCORE 1+ SCORE 2+
ISH testing
ISH Negative ISH Equivocal
HER2/CEP17<2 HER2/CEP17<2
and HER2 copy and HER2 copy
number <4 number ≥4 but <6
Figure 1 Schematic representation of the decision-making algorithm for HER2 testing according to the ASCO/CAP 2013 guidelines (using a dual-
color probe in situ hybridization) (Ref. Wolff et al., 201343).
interpretation of IHC and ISH results.43 Of note, a new version of
these guidelines is expected to be released in mid 2018.
Over the past years HER2 ISH assessment in breast cancer has
undergone important changes. The 2013 updated ASCO/CAP
guidelines43 recommended the use of the HER2/chromosome 17
centromere (CEP17) ratio cut-off of 2 (i.e. the original criterion
used in the first-generation clinical trials leading to trastuzumab
approval) to define amplification, rather than the 2.2 cut-off
employed since 2007. In addition, since several studies using
different technologies have consistently demonstrated that true
chromosome 17 (Chr17) polysomy is a quite rare event and high
level gains or amplification of the centromeric region of Chr17 are
more frequently encountered in breast cancer,44e47 an algorithm
for dual-color ISH testing was introduced by the 2013 ASCO/CAP
guidelines. This algorithm takes into account the HER2/CEP17
ratio first and the HER2 copy number value as a second level of
evaluation. In such a way, whenever a HER2/CEP17 ratio <2 is
encountered (possibly due to a gain/amplification of the
DIAGNOSTIC HISTOPATHOLOGY 24:2 74
SCORE 3+
ISH Positive
HER2/CEP17>2
or
HER2/CEP17<2
but HER2 copy
number ≥6
centromeric region), the tester is advised to double check the
absolute HER2 copy number to look for HER2 amplification based
on a cut-off of >¼6 mean copy number value. This algorithm has
allowed HER2 gene amplification not to be underestimated in a
subgroup of tumours harbouring high HER2 copy numbers in
conjunction with CEP17 gain or amplification.48
Some authors have proposed the use of additional Chr17
probes in cases harbouring CEP17 abnormalities; nevertheless,
we do not envisage this approach in the clinical setting, as Chr17
is known to show complex rearrangements in breast can-
cer44,49,50: in this respect, any chromosomal region may be
affected by local copy number alterations, thus rendering the use
of alternative probes not an ideal surrogate for Chr17 ploidy. In
addition, this approach has not been used in clinical trials
showing efficacy of anti-HER2 therapy.
Additional patterns that can be challenging at ISH testing are
represented by tumours showing equivocal expression of the
HER2 protein (score 2þ by IHC) and mean HER2 copy numbers
Ó 2018 Elsevier Ltd. All rights reserved.
 MINI-SYMPOSIUM: BREAST PATHOLOGY
in the equivocal range (i.e. HER2/CEP17 ratio <2.0 and mean
HER2 copy numbers
4 and <6), as assessed by ISH (see
Figure 1). These carcinomas are also labelled as “HER2 double-
equivocal” breast cancers and are typically ER-positive and
defined as luminal B-like when using the IHC surrogate proposed
by the St. Gallen consensus, since they usually display relatively
high Ki67 indices.51,52 According to the 2013 ASCO/CAP guide-
lines,43 if a case is finally deemed to be equivocal by both IHC
and ISH, oncologists may consider the option of offering anti-
HER2 therapy by also taking into account patient’s perfor-
mance and wish.43 Nevertheless, it should be noted that at pre-
sent data on the efficacy of response to anti-HER2 therapies are
not available.
Lastly, we bring to the readers’ attention a potential pitfall in
ISH testing related to HER2 re-testing on surgical specimens
following neoadjuvant treatment. Residual carcinomas following
taxane-based chemotherapy may present giant syncytial
multinucleated-looking cells harboring an abnormally high
number of HER2 signals. These cells typically are scattered
within a background of tumour cells with normal HER2 copy
numbers. This phenomenon is most likely due to a polyploidy
status induced by chemotherapy rather than to a focal amplifi-
cation of the HER2 locus, as demonstrated by the fact that in such
cases additional copies of CEP17 as well as of many regions in
other chromosomes are present.53 Caution should be exercised in
this scenario in order not to misinterpret this phenomenon as
heterogeneous amplification of the HER2 locus.
Of note, only a proportion of HER2-positive cases (defined
by IHC or ISH) respond to anti-HER2 tailored therapy; most
initially trastuzumab-sensitive metastatic breast cancer patients
show progression within one year,54 and 15% of patients in the
adjuvant setting eventually develop metastatic disease. In the
neoadjuvant setting, about half of the patients are predicted to
have residual disease following treatment. A significant group
of patients with HER2-positive disease therefore have tumours
that show de novo or acquired resistance to anti-HER2 targeted
agents  chemotherapy. Identification of tumours that are
sensitive to this therapeutic regimen (i.e. better companion di-
agnostics for anti-HER2 regimens) is crucial and HER2 testing
should get beyond a standardized assessment of HER2 expres-
sion or gene copy number status. Nevertheless, after 15 years of
translational research on HER2-positive breast cancer, there are
currently no validated tools for treatment tailoring beyond
HER2 and the evaluation of this biomarker based on IHC and
ISH still remains the cornerstone for prediction of response to
therapy.
Familial breast cancer
Hereditary breast cancer accounts for 10% of breast cancers,
half of which are due to germline mutations in two high-
penetrance genes: BRCA1 and BRCA2 (Table 3).55 Pathogenic
germline mutations in BRCA1 or BRCA2 genes are associated
with a high risk of development of breast and ovarian cancer.56
Inactivation of the wild type allele is found in most breast can-
cers developing in BRCA1/2 germline mutation carriers. Unlike
the pattern of mutations found in TP53, no hotspot regions of
mutations in BRCA1 and BRCA2 genes have been described.
Selection of individuals with familial breast cancer for genetic
DIAGNOSTIC HISTOPATHOLOGY 24:2
testing is difficult. Genetic testing models used are based on
strong family history, young age and other clinical and familial
characteristics, depending on the model adopted. These models
have been shown to have suboptimal specificity, ranging from
25% to 30% depending on the model employed.57
Pioneering work by the Breast Cancer Linkage Con-
sortium58,59 and others has shed light in the characterization of
the pathological features of BRCA1 and BRCA2 cancers. BRCA1
tumours have been shown to have characteristic morphological
features.56 These tumours are more frequently of ductal NST,
medullary or atypical medullary histological morphology, of
histological grade III; and more often display pushing borders,
brisk lymphocytic infiltrate and necrosis when compared to
grade-matched controls and tumours arising in BRCA2 mutation
carriers.56 These features are found particularly in BRCA1 can-
cers developing in patients aged <55 years. Most BRCA1 cancers
lack ER, PR and HER2 expression, and express basal makers (i.e.
CK 5/6, CK 14, CK 17, EGFR and P-cadherin) in >80% of cases.60
The differences between BRCA2 cancers and grade-matched
controls are not so conspicuous. These tumours have been re-
ported in some, but not all, studies to be more often of invasive
lobular, pleomorphic lobular, tubular and cribriform histological
types than sporadic controls.56 BRCA2 cancers seem to be more
often of high histological grade and have pushing borders than
matched controls,56 but are more frequently positive for ER.61 The
morphological features of BRCA2 cancers are of limited help in
identifying patients to be screened for mutations, but histopatho-
logical models to predict BRCA1 germline mutations have been
developed. Farshid et al.57 proposed a system based on ER, PR, and
the morphological features of BRCA1 tumours. It has similar
sensitivity when compared to clinical models, but much higher
specificity (86%) and positive and negative predictive values (61%
and 98%, respectively).57 Recent studies have also highlighted
similarities between tumours arising in BRCA1 mutation carriers
and sporadic basal-like breast cancers,60 and demonstrated that the
BRCA1 pathway is dysfunctional in most sporadic basal-like can-
cers.62 An immunohistochemical predictor of BRCA1 germline
mutation using ER and CK5/6 has been shown to have a sensitivity
of 56%, a specificity of 87%, and positive and negative predictive
values of 28% and 99%, respectively.63 Based on this evidence,
models incorporating the clinical features, family history, histo-
pathological features and immunohistochemical profiles of the tu-
mours would be best suited for the identification of patients with
BRCA mutations. Further evidence in support of the predictive
values of the models described above is required, but these findings
should at least help decide which gene should be tested in a patient
with family history strongly suggestive of familial breast and
ovarian cancer: if the tumour lacks ER expression and is positive for
‘basal’ markers, BRCA1 rather than BRCA2 should be sequenced.
The knowledge of the phenotype and pathological character-
istics of BRCA cancers extends beyond patient and family sur-
veillance given that novel, targeted therapies for patients
harbouring BRCA1 and BRCA2 germline mutations have been
developed. These regimens address one of the defining features of
BRCA cancers: inactivation of BRCA1 or BRCA2 leads to deficiency
in homologous recombination repair of DNA double-strand breaks
and interstrand crosslinks. The rationale for this therapeutic
approach resides on the fact that BRCA1 and BRCA2 tumours may
be more sensitive to crosslinking agents (e.g. platinum salts) than
75 Ó 2018 Elsevier Ltd. All rights reserved.

Syndromes associated with hereditary breast cancer
Syndrome (OMIM)
Hereditary breast cancer and
ovarian cancer syndrome (113705)
Hereditary breast cancer and
ovarian cancer syndrome (600185)
PALB2 mutations (610355)
CHEK2 mutations (LieFraumeni 2
Syndrome?)
Other FANC genes (114480,
607139, 600901, 605882)
Familial-linitis-plastica type gastric
cancer and lobular breast
carcinomas syndrome (192090)
Louis-Bar’ syndrome (208900)
Li-Fraumeni syndrome (151623)
DIAGNOSTIC HISTOPATHOLOGY 24:2
MINI-SYMPOSIUM: BREAST PATHOLOGY
Gene involved and Clinical features Distinctive histological features
cytoband of the hereditary breast cancer
BRCA1 (17q21) Breast cancer, high risk (50e80%) High prevalence of histological
Ovarian cancer, high risk (40e50%) grade 3
Enriched for medullary and
atypical medullary histologic
type (but invasive carcinomas of
no special type are the most
frequently found in this group)
Pushing margins, lymphocytic
infiltrates, central necrosis
Predominantly ER-, PR- and
HER2-negative (>80%)
Association with basal
phenotype
BRCA2 (13q12.3) Breast cancer, high risk (50e70%) Histological features similar to
Ovarian cancer, intermediate risk (10%) those of age- and grade-
Prostate cancer matched controls.
Pancreatic cancer Possible higher prevalence of
Melanoma pushing borders and high
histological grade
More often invasive lobular,
pleomorphic lobular, tubular
and cribriform histological types
than sporadic controls
More frequently ER-positive
PALB2 (16p12.2) High risk of breast cancer development Histological features similar to
(breast-cancer risk for PALB2 mutation carriers those of BRCA2 and sporadic
may overlap with that for BRCA2 mutation controls
carriers)a
CHEK2 (22q12.1) Breast cancer, intermediate risk (w2 fold) Reported association with the
Sarcomas I157T missense mutation and
Brain tumours lobular histotype
CHEK2*1100delC mutation is
more prevalent in ER-positive
cancers
FANCA (16q24.3) Low risk of development of breast cancer
FANCE (6p22-p21)
BRIP1 (17q22)
CDH1 (16q22.1) Gastric cancer Infiltrating lobular carcinoma (E-
Lobular breast cancer cadherin negative)
ATM (11q22.3) Lymphoma No association with distinctive
Cerebellar ataxia histopathological features
Immune deficiency (predominantly infiltrating
Glioma carcinomas) and no similarities
Medulloblastoma with the phenotype of BRCA1
Breast cancer tumours
Disputable association with
poorly differentiated tumours
TP53 (17p13.1) High penetrance for breast cancers
at young age
Risk of soft tissue sarcomas and
osteosarcomas, brain tumours,
leukemia, and adrenocortical
carcinomab
76 Ó 2018 Elsevier Ltd. All rights reserved.
 MINI-SYMPOSIUM: BREAST PATHOLOGY

Table 3 (continued )
Syndrome (OMIM) Gene involved and Clinical features Distinctive histological features
cytoband of the hereditary breast cancer

Cowden syndrome (158350) PTEN (10q23.31) Increased risk of developing neoplasms

Bannayan-Riley-Ruvalcaba
syndrome (153480)
Peutz-Jeghers syndrome (175200)
Lynch cancer family syndrome II
(114400)
a
See Ref. Antoniou et al. (2014)76.
b
See Ref. McBride et al. (2014)83
(breast cancer, thyroid carcinoma, endometrial
carcinoma and others)
Hamartomatous polyps of the gastrointestinal
tract
Mucocutaneous lesions
PTEN (10q23.31) Breast cancer
Meningioma
Follicular cell tumours of the thyroid
STK11 (19p13.3) Melanocytic macules of the lips, buccal Invasive carcinomas of no
mucosa and digits special type and papillary
Multiple gastrointestinal hamartomatous lesions
polyps
Increased risk of various neoplasms (breast,
testis, pancreas, cervix)
MSH2 (2p22-p21) Increased risk of endometrial carcinoma and
MSH3 (5q11-q12), colorectal carcinoma
MSH6 (2P16), High risk of multiple primary malignant
MLH1 (3p21.3), neoplasms (including breast, ovarian,
PMS1 (2q31-q33), gastrointestinal and genitourinary carcinomas,
PMS27 (p22) sarcomas, glioblastoma and leukaemia)

Table 3

to spindle poisons. Preclinical studies have demonstrated that
BRCA cancers have an exquisite sensitivity to PARP enzyme in-
hibitors, agents that block one of the alternative mechanisms of
DNA repair (i.e. base excision repair). In cells deficient in ho-
mologous recombination repair, the inhibition of base excision
repair has been shown to result in dramatic levels of chromosomal
instability, cell cycle arrest and subsequent apoptosis.64
Based on the preclinical data showing a synthetic lethality
interaction between PARP inhibition and BRCA mutation sta-
tus,64,65 a phase 1 clinical trial of olaparib, including patients
with germline BRCA1 or BRCA2 mutations was initiated and
demonstrated a clinical benefit in 63% of germline BRCA mutant
patients.66 Phase 2 trials involving patients with germline BRCA
mutant breast, ovarian, pancreatic, or prostate cancers confirmed
that olaparib offered clinical benefit.67e69 The results of these
trials in familial breast cancer patients have been pivotal to de-
vice more effective therapies for sporadic TNBCs as these tu-
mours have been shown recapitulate several cardinal features of
breast cancers arising in BRCA1 mutation carriers and to have a
dysfunctional BRCA1 pathway.62 The term “BRCAness” has been
coined to describe tumours that have not arisen from a germline
BRCA1 or BRCA2 mutation but nonetheless share certain mo-
lecular features, in particular a homologous recombination
defect, with these hereditary cancers.70
In TNBC, clinical responses to olaparib have been shown to be
somewhat mixed; although patients with BRCA1/2-mutant tu-
mours showed some disease stabilization when treated with
olaparib, there were no sustained responses in either BRCA1/2-
mutant or nonmutant cohorts, suggesting that BRCAness might
be less frequent in TNBCs than in high-grade serous ovarian
carcinomas for instance,71 or that other clinical parameters or
previous treatment effects might be important.70 In the attempt to
detect BRCAness, different DNA-based assays, based on LOH,72
telomeric allelic imbalance,73 and large state transitions74 have
been developed for the assessment of homologous recombination
deficiency. More recently, an assay called “HRDetect” has been
described based on mutational signatures derived from whole
genome sequencing experiments.75 This is an assay which uses a
logistic regression model to identify mutational signatures pre-
dictive of BRCA1/2 deficiency, allowing for the selection of pa-
tients who could benefit from PARP inhibition therapies.75
Besides BRCA1 and BRCA2 there is a vast array of other genes
related to hereditary breast cancer (Table 3). Due to their low
frequency, the risk conferred by these rare variants has not yet
been fully elucidated. Partner and Localizer of BRCA2 (PALB2) is
a tumour suppressor which, akin to BRCA1 and BRCA2, plays a
key role in homologous recombination, and has emerged as an
important hereditary breast cancer gene. The cumulative risk for
the development of breast cancer in patients with PALB2 germ-
line mutations by age 70 is approximately 35%, overlapping with
the one associated to BRCA2.76 In line with its function in ho-
mologous recombination repair, PALB2 deficient cellular models
display an increased susceptibility to PARP inhibitors.77 Our
study of breast cancers in PALB2 germline carriers has shown
that PALB2 biallelic inactivation, which occurs by LOH and
second somatic mutations, confers features of homologous

DIAGNOSTIC HISTOPATHOLOGY 24:2 77 Ó 2018 Elsevier Ltd. All rights reserved.

 MINI-SYMPOSIUM: BREAST PATHOLOGY
recombination deficiency, suggesting that identification of pa-
tients with PALB2 biallelic inactivation may facilitate the selec-
tion of individuals who might benefit from PARP inhibitors and
platinum salts based therapies.78
ATM encodes for the ataxia-telangiectasia mutated protein
and plays a key role in the DNA damage repair response upon
DNA double strand breaks.79 ATM loss-of-function mutations
and the V2424G missense mutation result in an increased sus-
ceptibility to hereditary breast cancer.80 Consistent with its
function in DNA damage response, ATM germline mutations
appear to confer susceptibility to PARP inhibitors in prostate
cancer.81 Surprisingly, we have shown that breast cancers in
ATM germline mutation carriers lack the genetic hallmarks of
homologous recombination deficiency, such as large state tran-
sitions and mutational signature three, suggesting that the
increased susceptibility to PARP inhibitors related to ATM
germline mutations might be due to a mechanism unrelated to
homologous recombination repair.82
Hereditary breast cancer may also be related to other genes
which are not definitely related to genome repair. Mutations in
PTEN, a phosphatase of the PI3K-AKT-mTOR pathway underlie
Cowden syndrome and Bannayan-Riley-Ruvalcaba syndrome,
which fall under the umbrella of PTEN hamartoma syndromes,
and are associated with an elevated risk of breast cancer.84
STK11, a serine threonine kinase which regulates the AMPK
pathway is related to Peutz-Jeghers syndrome.85 Notably, recent
evidence suggests an association between STK11 and homolo-
gous recombination repair, as it has been recently demonstrated
that STK11edeficient cells have increased DNA double strand
breaks, suggesting that STK11 may play a role in DNA repair.86
CDH1 mutations result in hereditary diffuse gastric cancer,87
and the second most common neoplasm in such patients is
breast cancer, in particular invasive lobular carcinoma.88
Intratumour heterogeneity and liquid biopsies
The study of breast cancer using massively parallel sequencing
(MPS) approaches has shed light on the constellation of the genetic
alterations that characterize breast cancer and has provided us
with the opportunity to tailor therapy according to the genomic
landscape of a given tumour. We have learnt that breast cancer is a
heterogeneous disease even at base pair level and that a handful of
genes appear to be highly recurrently mutated in breast cancer:
only TP53, PIK3CA, and GATA3 were found to be consistently
mutated in more than 10% of unselected breast cancers, the
remaining genes being mutated in less than 7.7% of cases, with a
very long list of genes mutated in less than 1% of cases.10 Poten-
tially actionable mutations, such as those affecting HER2 and ESR1
have been found in a relatively low proportion of breast carci-
nomas, accounting for up to 2% of unselected primary tumours.
We have also come to terms with the fact that tumours are
composed of multiple clones with distinct genetic alterations,
which may result in the emergence of resistant cell populations
upon therapeutic pressure.89 Indeed, there is evidence indicating
that metastatic outgrowths might stem from a minor cell sub-
population of the primary tumour.90 Traditional approaches in
which DNA from the bulk of the tumour is sequenced, do not
have the power to detect minor subclones,91 which may be
responsible for progression and resistance to therapy.92 Liquid
DIAGNOSTIC HISTOPATHOLOGY 24:2
biopsies refer to the study of circulating cell-free tumour DNA
(cfDNA) and circulating tumour cells (CTC) and constitute a
novel non-invasive sensitive and specific approach to monitor
tumour burden, as well as the dynamic changes of tumour ge-
nomes in real time.93,94
Liquid biopsies have been shown to have potential utility in
both early and advanced breast cancer. The analysis of ctDNA in
plasma of patients with early breast cancer receiving neo-
adjuvant chemotherapy showed that mutation tracking in ctDNA
could be used to predict relapse with a shorter median lead time
than current standard of care methods.95 Furthermore, targeted
capture MPS of ctDNA detected somatic mutations present in
metastatic foci and absent in the primary tumours, showing that
liquid biopsies might allow a more accurate study of the genomic
landscape of metastatic foci compared than the study of the
primary tumours.95
Liquid biopsies might also have a potential role in the iden-
tification of genetic alterations related to therapeutic resistance in
breast cancer. ESR1 mutations mediate resistance to oestrogen
deprivation,96 and whilst rare in primary breast cancer, they are
found at a higher frequency in metastatic breast cancer, and may
be detected in cfDNA in this setting.97 The identification of ESR1
Y537S and D538G mutations in patients with ER-positive meta-
static breast cancer treated with aromatase inhibitors was asso-
ciated with a reduction in overall survival.98 Assessment of ESR1
mutations by liquid biopsies might have a potential utility in the
triaging of patients to further endocrine therapies as shown by
the study by Fribbens et al.99 In this study, the analysis of ESR1
mutations in archived baseline plasma showed that patients with
ESR1 mutations from the SoFEA trial had a better progression-
free survival following treatment with fulvestrant compared to
exemestrane, whist patients with wild-type ESR1 had similar
outcomes following either treatment.99
Finally, cfDNA analysis was shown to be useful for the
detection of BRCA1/2 somatic reversion mutations in BRCA1 or
BRCA2 germline mutation carriers with metastatic breast cancer
previously treated with platinum and/or PARP inhibitors,100
underscoring the potential role of liquid biopsies in the selec-
tion of patients for PARP inhibition therapies.
Taken together, multiple lines of evidence show that liquid
biopsies might be a powerful technique to circumvent intra-
tumour heterogeneity and guide therapy, both in the early breast
cancer and in the metastatic setting.
Conclusions
Despite the advances of molecular pathology, histopathological
analysis remains a cornerstone of breast cancer management.101
Diligent use of clinicopathological features to define therapy has
positively contributed to the reduction of breast cancer mortality
rates. Incorporation of new technologies has helped improve the
accuracy of prognostic and predictive systems. Biotechnology is
developing at an unprecedented pace, with the emergence of
several techniques that may allow us to address questions that
could not have been answered in the past. To achieve the goals of
individualized therapy, some of these novel methods and/or
their derivatives must be incorporated into clinical practice.
These techniques will inevitable need to undergo the same level
of scrutiny that diagnostic methods have been subjected to. A
78 Ó 2018 Elsevier Ltd. All rights reserved.
 MINI-SYMPOSIUM: BREAST PATHOLOGY
Practice points
C Histopathological and immunophenotypical characteristics of
breast cancers should be recorded systematically.
C Careful monitoring of the preanalytical conditions that may
impact the preservation of specimens should be exercised to
guarantee the reliability of prognostic and predictive marker
assessment.
C Multigene prognostic signatures are useful in the context of ERþ/
HER2- disease to identify patients that may be spared from
adjuvant chemotherapy and may be incorporated in the staging
system.
C Sporadic basal-like carcinomas and tumours arising in BRCA1
mutation carriers have similar morphology, immunohistochem-
istry and molecular features.
C Pathologists should be involved in the discovery and validation of
diagnostic and predictive molecular ancillary methods.
Research directions
C Consolidation of a combined morphological and molecular tax-
onomy for breast cancer.
C Unravelling of the genetic and transcriptomic pathways of the
development and progression of breast cancer.
C Identification of therapeutic targets for specific molecular sub-
groups of breast cancer, particularly the basal-like subgroup.
C Development of more accurate companion diagnostics for pa-
tients with HER2-positive cancers.
C Understanding the potential benefit of anti-HER2 agents in breast
carcinomas showing an equivocal HER2 status at both IHC and
ISH levels.
C Development of methodologies to exploit the full potential of
liquid biopsies in tackling tumor heterogeneity in the era of
precision medicine.
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Acknowledgements
Caterina Marchio is supported in part by a grant of the Italian Ministry
of University, Education and Research (PRIN 2015HAJH8E).

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