Clinical

Cancer
Review Research

Deep Molecular Response in Chronic Myeloid Leukemia: The

New Goal of Therapy?
Francois-Xavier Mahon1 and Gabriel Etienne2

Abstract
Chronic myeloid leukemia (CML) is caused by formation of the BCRABL1 fusion protein. Tyrosine
kinase inhibitors (TKI) that target BCRABL1 are now the standard of care for patients with CML.
Molecular monitoring of residual BCRABL1 mRNA transcripts, typically performed using real-time
quantitative PCR, has improved treatment management, particularly for patients with CML in chronic
phase. Major molecular response (MMR; i.e., a
3-log reduction in BCRABL1 transcript levels) is used
in current treatment guidelines to assess prognosis. Recent evidence suggests that deeper molecular
responses (
4-log reductions in BCRABL1 transcript levels), particularly when attained early during
treatment, may have even better correlation with long-term outcomes, including survival and disease
progression. Furthermore, achieving deep molecular response is a requirement for entering trials
evaluating treatment-free remission (TFR). In this review, we discuss the evolving definition of minimal
residual disease and the various levels of molecular response under evaluation in current clinical studies.
In addition, the available clinical data on achieving MMR and deeper levels of molecular response with
TKI therapy, the prognostic value of deep molecular response, and factors that may predict a patients
ability to achieve and sustain a deep molecular response on TKI therapy are also discussed. Available
data from TFR studies are addressed. We discuss current knowledge of the ideal conditions for
attempting treatment discontinuation, factors predictive of molecular relapse, when TKI therapy should
be restarted, and which therapeutic strategies (when administered in the first-line setting and beyond)
are expected to best enable successful TFR. Clin Cancer Res; 20(2); 31022.
2013 AACR.

Introduction With over a decade of imatinib use as first-line therapy in

Dysregulated protein tyrosine kinase (PTK) activity is
the hallmark of multiple neoplasms (1). Over the past
decade, a broad array of drugs designed to selectively inhibit
PTKs [i.e., tyrosine kinase inhibitors, (TKI)] have emerged as
novel therapies for patients with cancer (2). Perhaps the most
well-known PTK target to date is the BCRABL1 oncoprotein,
which is critical to the pathogenesis of chronic myeloid
leukemia (CML; ref. 3). The successful treatment of patients
with CML with the BCRABL1 TKI imatinib has definitively
validated this therapeutic strategy and established CML as
a model disease for targeted cancer treatment (4).
Authors' Afliations: 1Laboratoire d'He
matologie, Centre Hospitalier Uni-
versitaire de Bordeaux and Laboratoire He
matopoe se Leuce
mique et
Cible The
rapeutique, Biothe

rapies des maladies ge
ne

tiques et cancers,
Inserm U1035, Universite
Bordeaux Se
galen; and 2Centre Re
gional de
Lutte Contre le Cancer de Bordeaux et du Sud-Ouest, Institut Bergonie
,
De
partement d'Oncologie Me
dicale, Bordeaux, France
Corresponding Author: Francois-Xavier Mahon, Laboratoire d'He
mato-
logie, CHU de Bordeaux and Laboratoire He
matopoe se normale et patho-

Victor Se
logique, Universite
galen Bordeaux 2, 146 rue Le

o-Saignat, Inserm
U1035, 33076 Bordeaux Cedex, France. Phone: 33-5-5757-1524; Fax: 33-5-
5693-8883; E-mail: francois-xavier.mahon@u-bordeaux2.fr
doi: 10.1158/1078-0432.CCR-13-1988

2013 American Association for Cancer Research.
patients with CML, surrogate markers that strongly correlate
with prognosis have been identified (5). The validation of
surrogate endpoints for treatment effectiveness, such as
complete hematologic response (CHR) and complete cyto-
genetic response (CCyR), has improved treatment manage-
ment (6, 7). Most patients, particularly those with CML in
chronic phase (CML-CP), will achieve these endpoints on a
BCRABL1 TKI (810). The progress of molecular biology
has presented the opportunity to look beyond CHR and
CCyR and monitor residual disease on a molecular level
(11, 12). Current clinical practice recommendations for
CML advocate monitoring patients for hematologic
response (i.e., blood counts returning to normal values),
cytogenetic response (i.e., disappearance of the Philadel-
phia chromosome), and molecular response (i.e., reduction
in BCRABL1 gene expression; refs. 6 and 7). Molecular
monitoring is typically performed using real-time quanti-
tative PCR (RQ-PCR), a simple technique that can be
performed on peripheral blood samples and is both more
sensitive and more convenient than conventional cytoge-
netics (13). The first level of response evaluated on the
molecular scale, a major molecular response (MMR), cor-
responds to a 3-log reduction in BCRABL1 transcript levels
from a standardized baseline (0.1% BCRABL1 on the
International Scale [BCRABL1IS]; refs. 11 and 14). MMR

310 Clin Cancer Res; 20(2) January 15, 2014


has been found to be associated with improved progression-
free survival (PFS; refs. 15 and 16) and event-free survival
(EFS; refs. 1719), although its prognostic value is still
debated.
The European LeukemiaNet (ELN) defines response to
TKI therapy based on molecular and cytogenetic milestones
achieved at 3, 6, and 12 months, or beyond (6). The ELN
recommends evaluating molecular response using buffy
coat from peripheral blood every 3 months until MMR is
achieved, and then every 3 to 6 months. Cytogenetic
response should be assessed using bone marrow at 3, 6,
and 12 months, and then every 12 months once CCyR is
achieved. Some patients (e.g., patients with monosomy 7,
del[7q] or clonal chromosomal abnormalities) may require
additional long-term bone marrow follow-up.
Until recently, deep molecular responses beyond the level
of MMR have remained largely unexplored, given a lack of
standardized assay techniques (20). Several recent studies
have demonstrated that some patients with such responses
can achieve treatment-free remission (TFR), thereby chal-
lenging the dogma that CML cannot be cured without
allogeneic bone marrow transplantation (21, 22). The
overall goals of therapy in CMLdisease remission,
reduced risk of progression, and improved overall survival
(OS)are clear; however, many questions remain about
the impact of molecular response on achieving these goals.
Recent data, to be discussed herein, suggest that in select
populations, obtaining a deep molecular response should
be considered a primary therapeutic goal.
Log
BCRABL1
reduction in Reduction in BCRABL1
(%IS)
BCRABL1
100
10 1 1:10
1 2 1:100
Figure 1. Levels of molecular
response in CML. MCyR, major 0.1 3 1:1,000
cytogenetic response; MR,
molecular response.
0.01 4 1:10,000
0.0032 4.5 1:32,000
0.001 5 1:100,000
0.0001 6 1:1,000,000
CCR Reviews
www.aacrjournals.org
Deep Molecular Response in Chronic Myeloid Leukemia
The Challenge of Dening Deep Molecular
Response
The development of the International Scale for BCR
ABL1 RQ-PCR assessment has enabled quantification of
molecular response in relation to a standardized baseline
(14, 23). The definition of MMR as BCRABL1IS 0.1%
originates from the International Randomized Study of
Interferon Versus STI571 (IRIS; ref. 15). However, standard-
ization of deeper levels of molecular response has proven
less straightforward and is urgently needed to facilitate
improved interpretation of clinical results. Deeper levels
of molecular response may also be defined according to the
International Scale, wherein MR4 indicates
4.0-log
reduction (BCRABL1IS 0.01%), MR4.5 indicates
4.5-log
reduction (BCRABL1IS 0.0032%), and MR5 indicates

5.0-log reduction (BCRABL1IS  0.001%; Fig. 1; ref. 11).
However, the observation of undetectable BCRABL1 tran-
scripts is inherently linked to the sensitivity of the PCR
method, as well as the control gene, used. An ongoing
European Treatment and Outcome Study (EUTOS) collab-
oration aims to facilitate standardization of deep molecular
response across laboratories by establishing recommenda-
tions for response definitions and quality control (11).
Varying definitions of complete molecular response
(CMR) have been reported. For example, the National
Comprehensive Cancer Network (7) defines CMR as "no
detectable BCRABL1 chimeric mRNA as assessed by RQ-
PCR using the International Scale with a sensitivity of 4.5-
Baseline
Approximates
MCyR
Approximates
CCyR
MMR
MR4
MR4.5
Deep
MR5 molecular
response
MR6 (with currently available technology,
this level of response cannot be assessed)
Level of response
2013 American Association for Cancer Research
Clin Cancer Res; 20(2) January 15, 2014 311
 Mahon and Etienne
log reduction or more from the standardized baseline,"
whereas the ELN (6) recommends using the term "molec-
ularly undetectable leukemia" instead of "CMR" and notes
the importance of specifying control gene copy number
when reporting this level of response. Undetectable mini-
mal residual disease indicates a negative RQ-PCR result and
must be associated with a defined PCR assay sensitivity;
however, leukemic cells may still be present even if RQ-PCR
results are negative (24). Notably, current RQ-PCR meth-
odology is largely optimized for detection of so-called
"typical" BCRABL1 transcripts, or those involving the
major breakpoint cluster region, and may fail to detect
atypical transcripts derived from other breakpoints (25);
thus, assessment of transcript type at baseline is essential to
ensure accurate interpretation of RQ-PCR results.
Deep Molecular Response in Clinical Trials of
CML-CP
Deep molecular responses have been assessed in mul-
tiple ongoing clinical trials of TKIs in patients with newly
diagnosed CML-CP (Table 1). Given the current lack of
standardization of PCR sensitivity, rates of CMR reported
in these studies are not necessarily comparable. Discre-
pancies in rates of deep molecular response may also
result from differences in assay techniques or study
design. For example, PCR may be assessed using blood
or bone marrow, and response may be defined by a PCR
result at a single time point or confirmed by multiple
samples. Other variables include the study population
(e.g., the proportion of high-risk patients), median fol-
low-up at time of analysis, and type and dose of TKI
therapy received. Although several studies have shown
that imatinib can elicit deep molecular responses in some
patients, second-generation TKIs, such as nilotinib and
dasatinib, have demonstrated higher rates of deep molec-
ular response attained within shorter time periods
(Table 1). The combination of TKI therapy with interfer-
on (IFN), which may help drive leukemic stem cells (LSC)
into the cell cycle (26), thereby inducing deep molecular
response, has also been explored. Rates of deep molecular
response were higher with this combination versus ima-
tinib alone in one study using pegylated IFN (26), but
similar in another study using IFN-a (Table 1; ref. 27).
The Clinical Relevance of Deep Molecular
Response
Defining the value of achieving deep molecular
response in patients with CML is an active area of ongoing
research. There is conflicting evidence about whether
achieving an MMR provides additional benefit beyond
a CCyR (20). The leukemic cell burden is similar in
patients with MMR and CCyR (only 1-log difference in
BCRABL1 levels), and this difference may be too small to
impact long-term outcomes such as PFS and OS. Events
like progression or death are quite infrequent with front-
line TKI therapy; therefore, long-term follow-up and large
312 Clin Cancer Res; 20(2) January 15, 2014
cohorts of patients would be required to definitively
demonstrate any added benefit to achieving MMR.
However, deeper molecular responses provide more
separation from CCyR in terms of residual disease bur-
den; therefore, it may be easier to distinguish the unique
benefits of such responses. Patients who achieve deep
molecular response seem less likely to lose MMR, and
several studies have shown that deep molecular responses
correlate with better long-term clinical outcomes, such as
EFS, PFS, and OS, and a low risk of progression and
disease relapse (Table 2; refs. 28 and 29). For example, a
study by Etienne and colleagues (30) found that EFS and
PFS were longer in patients with CMR than those with
CCyR, regardless of MMR status; OS was not significantly
different between these groups. Another study by Falchi
and colleagues (31) showed that patients with undetect-
able BCR-ABL1 levels by 18 or 24 months had 100% rates
of OS, EFS, and transformation-free survival (TFS) at 6
years. In the German CML study IV, life expectancy in
patients with MR4 or MR4.5 was the same as that in an age-
matched population, and only 4 of 792 patients (0.5%)
who achieved MR4 had disease progression (32).
These results provide encouraging evidence that achieve-
ment of deep molecular response is an important clinical
goal and prompt the question: should patients with CCyR,
who do not achieve these deep levels of molecular response,
be switched to an alternate therapy? The ongoing Evaluating
Nilotinib Efficacy and Safety in Clinical TrialsCMR
(ENESTcmr) study aims to address this question by ran-
domizing patients with detectable BCRABL1 transcripts
after
2 years on imatinib to either continue on imatinib or
switch to nilotinib (33). After 12 and 24 months of follow-
up, more patients achieved MR4.5 and undetectable BCR
ABL1 (
4.5-log RQ-PCR assay sensitivity) on nilotinib
compared with continued imatinib (33). Yet perhaps the
most convincing reason to strive for sustained deep molec-
ular response in patients with CML-CP is the possibility that
it may enable eventual achievement of TFR.
Deep Molecular Response and TFR
Reports of imatinib discontinuation in patients without
sustained deep molecular responses have shown high and
rapid rates of disease relapse (3437). In contrast, a small
pilot study of imatinib discontinuation with the stringent
entry criteria of sustained CMR for
2 years on imatinib
showed promising results, with 6 of 12 patients remaining
in molecular remission after a median 18 months of fol-
low-up (38). This proof of concept inspired numerous
clinical studies of imatinib discontinuation in patients with
stable, deep molecular responses on TKI therapy (Table 3).
In all cases, many patients were able to remain relapse-free
off therapy.
Importantly, molecular relapse, the trigger for reintro-
duction of TKI therapy in these studies, was often defined
differently. For example, in the Stop Imatinib (STIM) study,
molecular relapse was defined as positive RQ-PCR results
on 2 consecutive assessments (39, 40), whereas in the STIM
Clinical Cancer Research
 Table 1. Clinical trials of TKI therapy reporting deep molecular responses (MR4 or deeper) occurring in patients with newly diagnosed CML-CP

www.aacrjournals.org
Assay sensitivity Median
Trial Samples, na (IS standardized?) f/u, mo Treatment!MR endpoints assessed
IRIS (15, 24) 1
4.5 logs (established 25 IM 400 mg qd (n 333) ! CMR at 12 mo 4%b
the IS) 81 IM 400 mg qd (n 29c) ! MR4/MR4.5 by 81 mo 70%/52%. MR4.5 at 81 mo 45%
ID-01_151 (66) 1
5 logs (No) 15 IM 400 mg b.i.d. (n 112) ! CMR 28%
de Lavallade
2 consecutive NR (Yes) 38 IM 400 mg qd (n 204d) ! CMR 5%
et al. (67)
RIGHT (68) 1
4.5 logs (Noe) 17f IM 400 mg b.i.d. (n 115) ! CMR by 6, 12, 18 mo 39%, 44%, 55%
Verma 1
4.5 logs (NR) 65 IM 400 mg qd (n 73) or 800 mg qd (n 208) ! CMR 44%
et al. (29)
g
SPIRIT (26) 1 (Yes) 47h IM 400 mg qd (n 159) ! MR4 at 12, 24 mo 14%, 21%. CMR at 24 mo 9%
IM 400 mg qd AraC (n 158) ! MR4 at 12, 24 mo 15%, 26%. CMR at 24 mo 8%
IM 400 mg qd PEG-IFN (n 159) ! MR4 at 12, 24 mo 30%, 38%. CMR at 24 mo 16%
IM 600 mg qd (n 160) ! MR4 at 12, 24 mo 17%, 26%. CMR at 24 mo 8%
CML Study IV (27) 1 NR (Yes) 43 IM 400 mg qd (n 324) ! MR4 by 12, 24, 36 mo 8%, 31%, 46%
48 IM 400 mg qd IFN-a (n 350) ! MR4 by 12, 24, 36 mo 12%, 30%, 41%
28 IM 800 mg qd (n 338) ! MR4 by 12, 24, 36 mo 20%, 43%, 57%
i
ENESTnd (10) 1 (Yes) Min 36 NIL 300 mg b.i.d. (n 282) ! MR4 by 1, 3 y 20%, 50%. MR4.5 by 1, 3 y 11%, 32%
NIL 400 mg b.i.d. (n 281) ! MR4 by 1, 3 y 15%, 44%. MR4.5 by 1, 3 y 7%, 28%
IM 400 mg qd (n 283) ! MR4 by 1, 3 y 6%, 26%. MR4.5 by 1, 3 y 1%, 15%
ENEST1st (69) 1 NR (Yes) 6.5f NIL 300 mg b.i.d. (n 205) ! MR4 by 3, 6 mo 5%, 20%
GIMEMA (70, 71) 1; stable,
4 logs (Yes) 48 NIL 400 mg b.i.d. (n 73) ! MR4 at 12, 24, 36 mo 12%, 27%, 25%
3  4 mo apart 51 NIL 400 mg b.i.d. (n 73) ! Stable MR4 25%
MDACC NIL 1
5 logs (Yes) 17.3 NIL 400 mg b.i.d. (n 51) ! CMR by 6, 12, 30 mo 4%, 11%, 15%
phase II (72)
Nicolini 1 NR (Yes) 13.6 NIL 300 mg b.i.d. PEG-IFN (n 40) ! MR4 at 6, 12, 15 mo 23%, 57%, 80%. MR4.5 at
et al. (73) 6, 12, 15 mo 10%, 21%, 50%. MR5 at 6, 12, 15 mo 10%, 15%, 40%
DASISION (9) 1 NR (Yes) Min 24 DAS 100 mg qd (n 259) ! MR4.5 by 2 y 17%
IM 400 mg qd (n 260) ! MR4.5 by 2 y 8%

(Continued on the following page)

Clin Cancer Res; 20(2) January 15, 2014

Deep Molecular Response in Chronic Myeloid Leukemia

313
 314
Mahon and Etienne

Table 1. Clinical trials of TKI therapy reporting deep molecular responses (MR4 or deeper) occurring in patients with newly diagnosed CML-
CP (Cont'd )

Assay sensitivity Median

Clin Cancer Res; 20(2) January 15, 2014

Trial Samples, na (IS standardized?) f/u, mo Treatment!MR endpoints assessed
S0325 (74) 1 NR (j) 36 DAS 100 mg qd (n 99) ! MR4/MR4.5 at 1 y 27%/21%
IM 400 mg qd (n 91) ! MR4/MR4.5 at 1 y 21%/15%
MDACC DAS 1
5 logs (Yes) 24 DAS 100 mg qd or 50 mg b.i.d. (n 50) ! CMR at 6, 12, 30 mo 0%, 7%, 0%
phase II (75)
BELA (76) 1
4 logs (Yes) 13.8f BOS 500 mg qd (n 250) ! MR4 at 12 mo 12%
IM 400 mg qd (n 252) ! MR4 at 12 mo 3%

Abbreviations: AraC, cytarabine; BELA, Bosutinib Efcacy and Safety in Chronic Myeloid Leukemia; b.i.d., twice daily; BOS, bosutinib; DAS, dasatinib; DASISION, Dasatinib Versus
Imatinib Study in Treatment-Naive CML patients; ENEST1st, Evaluating Nilotinib Efcacy and Safety in Clinical Trials as First-Line Treatment; ENESTnd, Evaluating Nilotinib Efcacy
and Safety in Clinical TrialsNewly Diagnosed Patients; f/u, follow-up; GIMEMA, Gruppo Italiano Malattie Ematologiche dell'Adulto; IM, imatinib; IRIS, International Randomized
Interferon Versus STI571; IS, International Scale; MDACC, MD Anderson Cancer Center; min, minimum; MR, molecular response; NIL, nilotinib; NR, not reported; qd, daily; PEG-IFN,
pegylated interferon a-2a; qd, once daily; RIGHT, Rationale and Insight for Gleevec High-Dose Therapy; SPIRIT, STI571 Prospective Randomized Trial; SWOG, Southwest Oncology
Group.
a
Number of samples required to meet criteria for response.
b
Patients with CCyR only.
c
Subgroup analysis of patients enrolled in the IRIS study in Australia and New Zealand from June 2000 to February 2007.
d
Note: 17 of these patients were also in the IRIS study.
e
Baseline BCRABL1/ABL1 ratio based on prestudy samples from participating laboratory.
f
Median exposure.
g

25,000 copies of ABL were required for a sample to be considered adequate.
h
For all patients; 48 mo for patients alive as of data cutoff.
i

3,000 copies of ABL were required for a sample to be considered adequate.
j
Standardized to SWOG-specic BCRABL1 baseline level.

Clinical Cancer Research

 Table 2. Clinical outcomes of patients with newly diagnosed CML-CP who achieved deep molecular responses with TKI therapy

Outcomes for patients with or without deep molecular

response, %
Median
Trial description N f/u, mo Comparators EFS rate PFS rate OS rate TFS rate
a
Etienne et al. (30): 266 53.2 CCyR MMR CMR 95 98 OS was not NR

www.aacrjournals.org
rst-line IM 400 mg CCyR MMR  CMRa 65 82 different among NR
CCyR  MMR 28 56 the 3 groups NR
Falchi et al. (31): 495 73 No MR at 18 mo 78 (6 y) NR 93 (6 y) 90 (6 y)
rst-line IM (400 mg, n 83; MMR at 18 mo 94 (6 y) NR 98 (6 y) 100 (6 y)
800 mg, n 204), NIL (n 106), MR4 at 18 mo 97 (6 y) NR 97 (6 y) 100 (6 y)
or DAS (n 102) MR4.5 at 18 mo 93 (6 y) NR 95 (6 y) 100 (6 y)
Undetectable BCR-ABLb at 18 mo 100 (6 y) NR 100 (6 y) 100 (6 y)
No MR at 24 mo 80 (6 y) NR 92 (6 y) 90 (6 y)
MMR at 24 mo 90 (6 y) NR 97 (6 y) 100 (6 y)
MR4 at 24 mo 97 (6 y) NR 100 (6 y) 100 (6 y)
MR4.5 at 24 mo 95 (6 y) NR 97 (6 y) 100 (6 y)
Undetectable BCR-ABLb at 24 mo 100 (6 y) NR 100 (6 y) 100 (6 y)
CML study IV (32): rst-line IM 1,525 67.5 After a median duration of MR4 of 3.7 y, only 4 of 792 patients with CMR4 (0.5%) progressed; life expectancy
400 mg, IM 400 mg IFN-a, with MR4 and MR4.5 was identical to that of the age-matched population
IM 400 mg AraC, IM after
IFN-a failure, or IM 800 mg
Kantarjian et al. (28): 276 48 Durable CMRb (
6 mo) NR 100 (5 y) NR NR
retrospective analysis. IM (400 mg, Nondurable CMRb (<6 mo) NR 98 (5 y) NR NR
n 71; 800 mg, n 205)
Verma et al. (29): 281 65 CCyR MMR CMRb at 2 y 100 (5 y) NR NR 100 (5 y)
retrospective analysis. First-line CCyR MMR  CMRb at 2 y 96 (5 y) NR NR 96 (5 y)
IM (400 mg, n 73; 800 mg, CCyR  MMR  CMRb at 2 y 86 (5 y) NR NR 91 (5 y)
n 208)
Press et al. (77): all 90 49 MMR CMRc NR Not reachedd NR NR
patients achieved CCyR on MMR  CMRc NR 44 mod NR NR
IM and had
2 measurements of
BCRABL1 level after achieving
CCyR

Abbreviations: AraC, cytarabine; DAS, dasatinib; f/u, follow-up; IM, imatinib; MR, molecular response; NIL, nilotinib; NR, not reported.
a
CMR was dened as undetectable BCRABL1 transcripts with a sensitivity of
4.5 logs on 2 consecutive analyses
2 mo apart.
b
Minimum sensitivity 4.5 logs.
c
Minimum sensitivity 4 logs.
d
Data shown are median relapse-free survival, dened as progression to accelerated phase/blast crisis, loss of complete hematologic response, or loss of CCyR.

Clin Cancer Res; 20(2) January 15, 2014

Deep Molecular Response in Chronic Myeloid Leukemia

315
 Mahon and Etienne

Table 3. Clinical trials of TKI discontinuation in patients with CML-CP and deep molecular response

Relapse-
free pts, %
Treatment before d/c (median Patients responding
Trial (response required for d/c) Denition of relapse f/u, mo) to TKI after relapse
Trials of IM d/c
STIM (ref. 40; N 100) IM for
36 mo (MR5 for
24 mo) Conrmed loss of MR5 39% (30) 56/61 regained MR5
ALLG CML8/TWISTER IM for
36 mo (MR4.5 for Conrmed loss of 45% (42) 22/22 regained MMRa
(ref. 55; N 40)
24 mo) MR4.5
According to STIM IM for
36 mo (MR4.5 for Loss of MMR 64% (23) 24/24 regained MMRa
(ref. 41; N 66)
24 mo)
Korean study (ref. 78; IM for
36 mo (CMR for
24 mo) Conrmed loss of 81% (15.8) 8/9 regained MMRa
N 48) MMR
Yhim et al. IM for median 56.4 mo; range, Conrmed loss of 28.6% at 7/10 regained CMR
(ref. 54; N 14) 26.282.0 (CMR for
12 mo) CMR 12 mo (23)
Keio STIM (ref. 79; IM for median 98 mo; range, Loss of UMRDb 55.4% at 17/18 regained CMR
N 40) 24126 (UMRDb for
24 mo) 12 mo (15.5)
Takahashi IM for median 45.2 mo; range, Molecular recurrence 56% (22.4) 17/17 regained MMRa
et al. 4.592.7 [UMRD (
4-log after d/c of IM for
retrospective analysis sensitivity) by RQ-PCR,
6 mo
(ref. 80; N 43) RT-PCR, or TMA]
STIM 2c (ref. 57; IM (MR5 for
2 y) Loss of MMR and/or Study Study ongoing
N 200) >1-log increase in ongoing
BCRABL1
Trials of NIL d/c
Nilo post-STIMc NIL for
2 y in patients who Loss of MR5 on 2 Study Study ongoing
(ref. 57; N 70) failed TFR in STIM or STIM 2 consecutive ongoing
(conrmed CMR after 2 y assessments
of NIL)
ENESTFreedomc First-line NIL for
2 y and 1 y NIL Loss of MMR Study Study ongoing
(ref. 57; N 175) on study (MR4.5 for
1 y) ongoing
ENESTopc (ref. 57; TKI therapy for
3 y, including Conrmed loss of MR4 Study Study ongoing
N 117)
2 y of second-line NIL and or any loss of MMR ongoing
1 y NIL on study (MR4.5 for

1 y)
ENESTgoalc (ref. 57; IM for
1 y and NIL on study Conrmed loss of MR4 Study Study ongoing
N 300) (MR4.5 for 1 or 2 y) ongoing
ENESTPathc (ref. 57; IM for
2 y and NIL on study Loss of MR4 Study Study ongoing
N 1,058) (MR4 for 1 or 2 y) ongoing
TIGERc (ref. 57; N 652) First-line NIL PEG-IFN-a vs. Loss of MMR Study Study ongoing
NIL for
2 y on study, then ongoing
PEG-IFN-a vs. NIL
maintenance therapy
(stable MR4)
Trials of DAS d/c
DASFREEd (57) (N 75) DAS for
2 y (MR4.5 for
1 y) Loss of MMR Study Study ongoing
ongoing
Trials of TKI d/c
STOP 2G-TKI (ref. 42; NIL or DAS for
36 mo (UMRD Loss of MMR 58.3% at 13/15 regained MMR
N 34) for
24 mo, with
20,000 12 mo (14) 10/13 regained CMR
ABL1 copies)
EURO-SKId (ref. 57; TKI therapy (rst-line or second- Loss of MMR Study Study ongoing
N 500) line) for
3 y (MR4 for
1 y) ongoing

(Continued on the following page)

316 Clin Cancer Res; 20(2) January 15, 2014 Clinical Cancer Research

Table 3. Clinical trials of TKI discontinuation in patients with CML-CP and deep molecular response (Cont'd )
Treatment before d/c
Trial (response required for d/c)
DESTINYd (ref. 57; IM, NIL, or DAS for
3 y and
N 168) half-standard dose of TKI for
1 y on study (MMR or MR4 for

1 y at therapeutic dose and
1 y at half-standard dose)
NCT00573378c (ref. NIL or IM for
3 y, with stable
57; N 40) dose for
1 y and same
TKI PEG-IFN-a for 2 y on
study (NR)
Abbreviations: ALLG, Australasian Leukaemia and Lymphoma Group; DAS, dasatinib; d/c, discontinuation; f/u, follow-up; IM, imatinib;
MR, molecular response; NIL, nilotinib; NR, not reported; PEG-IFN-a, pegylated interferon-a; RT-PCR, nested reverse transcriptase
PCR; TMA, transcription-mediated amplication; UMRD, undetectable minimal residual disease.
a
Patients achieved this level of response or better.
b
UMRD dened as <100 copies by TMA; thus, loss of UMRD was >100 copies by TMA.
c
Currently recruiting participants.
d
Not yet recruiting participants.
study, molecular relapse was less stringently defined as loss
of MMR at any time (41). In According to STIM, all 24
patients who relapsed regained a response of MMR or better
once imatinib was reintroduced (41); therefore, waiting
until a patient loses MMR to reinitiate TKI therapy does
not seem to be detrimental. This suggests that BCRABL1
positivity after TKI cessation does not necessarily equal
disease relapse (22). However, the optimal molecular
response threshold for triggering restart of TKI therapy
remains to be established.
Data on discontinuation of second-generation TKIs are
limited; however, the ongoing STOP 2G-TKI study has
shown similar results to those of imatinib discontinuation
trials (Table 3; ref. 42). Thus, available results suggest that
stopping TKI therapy in patients with sustained deep molec-
ular response can be safe and associated with prolonged
TFR. Given the preliminary nature of available clinical data
on TFR, both the ELN and the National Comprehensive
Cancer Network currently recommend that patients remain
on TKI therapy indefinitely and that TKI discontinuation
only be attempted in controlled clinical trials (6, 7).
Factors Affecting Achievement of Deep
Molecular Response
Achievement of BCRABL1IS 10% at 3 or 6 months after
initiating first-line imatinib has been shown to be predictive
of long-term molecular response and improved outcomes
(19, 43, 44), whereas delayed cytogenetic and molecular
response on imatinib is associated with increased risk of
progression (45). Landmark studies of second-generation
TKIs have found BCRABL1 levels at 3 months to be
similarly predictive (46, 47). A retrospective analysis of
www.aacrjournals.org
Deep Molecular Response in Chronic Myeloid Leukemia
Relapse-
free pts, %
(median Patients responding
Denition of relapse f/u, mo) to TKI after relapse
Loss of MMR Study Study ongoing
ongoing
NR Study Study ongoing
ongoing
patients treated with second-line nilotinib, dasatinib, or
bosutinib found that BCRABL1IS 10% at 3 months from
start of second-line treatment was associated with signifi-
cantly higher cumulative incidence of MMR and CMR and
improved OS, PFS, and EFS (48). An online database of
patients with CML-CP treated with first-line imatinib in
Japan found that patients with an MMR at 12 months were
significantly more likely than those without to achieve
undetectable BCRABL1 transcripts by 72 months (49).
Another study found a similar association in patients trea-
ted with first-line or second-line imatinib (24).
Other factors that may affect achievement of deep molec-
ular response include risk score, sex, adherence to treat-
ment, and dose intensity. For example, in the Evaluating
Nilotinib Efficacy and Safety in Clinical TrialsNewly Diag-
nosed Patients (ENESTnd) phase III trial of first-line nilo-
tinib versus imatinib, rates of deep molecular response were
lowest among patients with high Sokal score, although
more high-risk patients achieved MR4.5 by 3 years on
nilotinib than imatinib (24%, 27%, and 9% in the nilotinib
300 mg b.i.d., nilotinib 400 mg b.i.d., and imatinib arms,
respectively; ref. 10). Univariate analysis of a study in
patients treated with first-line imatinib showed that females
were more likely than males to achieve stable undetectable
BCRABL1 (with a RQ-PCR sensitivity of
4.5 log) by 8
years (68% vs. 30%, respectively; ref. 50). Multivariate
analysis of a study in Japanese patients found adherence
to standard-dose imatinib to be predictive of CMR achieve-
ment (51). Another study reported adherence to standard-
dose imatinib as the only independent predictor of CMR;
poor adherence and failure to achieve MMR predicted
treatment discontinuation and eventual loss of CCyR
(52, 53).
Clin Cancer Res; 20(2) January 15, 2014 317
 Mahon and Etienne

Factors Affecting Durability of Deep Molecular ment of highly sensitive techniques like ultra-deep
Response Off Treatment sequencing (UDS) and massive parallel sequencing (MPS)
may help clarify these differences. Indeed, both UDS and
The high rates of and rapidity of disease relapse
MPS detected more complex mutation dynamics in TKI-
observed in patients who discontinue TKI therapy with-
resistant patients than conventional Sanger sequencing
out deep molecular response (3437) suggest that the
(59, 60).
main factor influencing relapse risk off therapy is the level
Mathematical modeling of the kinetics of molecular
of residual molecular disease present when TKI therapy is
relapse in patients in the STIM study has led to a hypothesis
stopped. However, even among patients with sustained,
that selective pressure exerted by imatinib treatment results
undetectable disease on TKI therapy, a significant per-
in an increased frequency of LSC clones with slower growth
centage of patients have experienced disease relapse (40),
and differentiation than the predominant clone at baseline
and several studies have evaluated factors potentially
(61). Imatinib therapy may affect the ability of LSCs to
contributing to a patients ability to achieve successful
produce differentiated populations and may affect the cell
TFR.
division of progenitors and differentiated leukemic cells
Studies of imatinib discontinuation have found an
(61). The effects of imatinib on leukemia-initiating cells
association between Sokal score and relapse risk (39,
(LIC) may differ from patient to patient, and these differ-
40, 54, 55), suggesting that patients with high Sokal risk
ences may manifest in varying times to molecular relapse in
may have inherent biologic attributes that drive develop-
patients with deep molecular response who stop imatinib
ment of disease relapse once BCRABL1 inhibition is
treatment (Fig. 2).
relieved. The duration of deep molecular response before
It remains to be established whether the ultimate goal
TKI discontinuation and the overall duration of prior TKI
of treatment for patients with CML-CP will be a "clinical
treatment also seem to be important (39, 40, 56). Based
cure" (i.e., the absence of relapse) or a "biological cure"
on this, the majority of ongoing studies require patients
(i.e., absence of all leukemic cells, including LSCs;
to maintain a deep molecular response for
2 years and
refs. 22, 62, 63). Multiple strategies for specifically tar-
have had
3 years of prior TKI treatment before attempt-
geting the LSC reservoir in patients with CML are under
ing discontinuation. More rapid achievement of deep
investigation, including combination of a BCRABL1 TKI
molecular response may also be predictive of successful
with other targeted agents, such as inhibitors of the
TFR (54, 55).
Hedgehog, Wnt/B-catenin, or Notch signaling pathways,
In the STIM study, the only factors associated with
or immunomodulatory agents, such as IFN (22, 62, 63).
lower risk of relapse were low/intermediate Sokal score
In one study, maintenance therapy with peglyated IFN
and duration of imatinib treatment >5 years (39, 40).
following imatinib discontinuation led to retained or
Another study identified a subgroup of patients with
improved molecular responses in the majority of
increased risk of molecular relapse following imatinib
patients, most of whom did not have deep molecular
discontinuation, characterized by the following: high
responses (64). This may represent a viable strategy for
Sokal score, >24 months to CMR, and <33 months of
patients who wish to stop TKI therapy in the absence of
imatinib after achieving CMR; patients with any of these
deep molecular response; the ongoing TIGER study is
characteristics had a 0% probability of CMR persistence
further investigating this approach (Table 3). Quantifi-
at 1 year, compared with 80% probability in patients
cation of a patients residual LSC reservoir and the effect
without these characteristics (54). Several other ongoing
of such combination treatments on LSCs remains a
TKI discontinuation studies aim to determine other fac-
challenge (22, 65). Given the potential diversity in dis-
tors that may play a role in relapse off treatment (Table
ease burden and relapse risk in patients who discontinue
3). For example, the large, phase III EURO-SKI study
TKI therapy, rigorous long-term follow-up is essential,
(NCT01596114) will explore factors associated with
and discontinuation should only be attempted in clinical
molecular relapse in patients with stable MR4 who stop
trials.
TKI therapy, and STIM 2 (NCT01343173) will evaluate
factors predictive of sustained deep molecular response
after imatinib discontinuation (57). Current data suggest Conclusions
that deep molecular response is heterogeneous, and Molecular monitoring affords a precise, convenient
different patients may have differing levels of residual method for monitoring residual disease burden in
disease burden despite having undetectable disease (22). patients with CML-CP. As TKIs have improved patient
Most, if not all, patients with sustained CMR have outcomes, clinical trial designs have begun to evaluate
persistent BCRABL1-positive cells (based on PCR at a deeper levels of molecular response. Deep molecular
DNA level; ref. 55 and 58); however, this persistence does responses are associated with improved rates of PFS, EFS,
not necessarily lead to relapse after treatment discontinu- and OS, and reduced risk of progression to advanced
ation. Furthermore, the kinetics of relapse after treatment disease. Furthermore, a sustained deep molecular
discontinuation vary, with early and late molecular response is an essential entry criterion for studies of TFR.
relapses observed (39). This discrepancy may reflect the The ability to achieve deep molecular response on ther-
heterogeneity of CML at the stem cell level. The develop- apy, and sustain this response off therapy, may be

318 Clin Cancer Res; 20(2) January 15, 2014 Clinical Cancer Research
 Deep Molecular Response in Chronic Myeloid Leukemia

Survival without molecular relapse

1.0
0.9
0.8
STOP
0.7 Stem cell persistence but
LSCs LICs Imatinib therapy
0.6 undetectable using RQ-PCR
0.5 (5-log sensitivity)
0.4
0.3
0.2
0.1
0.0
0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 54 57 60
Months since discontinuation of imatinib

B
LIC with intrinsically Survival without molecular relapse
faster growth rate 1.0
0.9
0.8
STOP
0.7 Early molecular relapse
LSCs LICs Imatinib therapy
0.6
0.5
0.4
0.3
Late molecular relapse
0.2
0.1
0.0
0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 54 57 60
LIC with intrinsically Months since discontinuation of imatinib
slower growth rate

2013 American Association for Cancer Research

CCR Reviews

Figure 2. Hypothesis for variability in duration of deep molecular response in patients who discontinue imatinib. Imatinib treatment has differing effects on LICs
that introduce variability in times to molecular relapse after imatinib discontinuation. A, in some patients, imatinib treatment may successfully eradicate LICs,
leaving only a small population of quiescent LSCs that is undetectable by conventional RQ-PCR (5-log sensitivity), enabling prolonged TFR. B, in other
patients, populations of LICs with variable growth kinetics remain. Patients with faster-growing residual LICs experience more rapid molecular relapse on
discontinuation of imatinib treatment, whereas patients with slower-growing residual LICs experience later molecular relapse. Inset graphs show the Kaplan
Meier estimate of relapse-free survival in the STIM study (relapse dened as conrmed loss of molecular response
5-log reduction) based on a median
follow-up of 30 months (40).

affected by a variety of factors, including disease char- Authors' Contributions

acteristics, risk score, and adherence to and time on Conception and design: F.-X. Mahon, G. Etienne
Development of methodology: F.-X. Mahon
treatment. As more is learned about the optimal criteria Writing, review, and/or revision of the manuscript: F.-X. Mahon, G. Etienne
for successful TKI discontinuation, patients may have
increased chances of achieving treatment-free disease Acknowledgments
The authors thank K. Miller-Moslin, PhD, and P. Tuttle, PhD (Articulate
control, a first step toward the elusive cure for CML. Science), for medical editorial assistance.

Disclosure of Potential Conicts of Interest Grant Support

F.-X. Mahon has received commercial research grants from Bristol-Myers Financial support for medical editorial assistance was provided by Novar-
Squibb and Novartis Pharma. F.-X. Mahon is a consultant/advisory board tis Pharmaceuticals Corporation.
member of Bristol-Myers Squibb, Novartis Pharma, and Ariad. G. Etienne is a
consultant/advisory board member of Bristol-Myers Squibb, Novartis, Pfizer, Received July 19, 2013; revised October 8, 2013; accepted October 9, 2013;
and Ariad. published OnlineFirst October 22, 2013.

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