Leukemia & Lymphoma

ISSN: 1042-8194 (Print) 1029-2403 (Online) Journal homepage: http://www.tandfonline.com/loi/ilal20

Characteristics of BCR-ABL kinase domain

mutations in chronic myeloid leukemia from India:
not just missense mutations but insertions and
deletions are also associated with TKI resistance

Nikhil Patkar, Kiran Ghodke, Swapnali Joshi, Shruti Chaudhary, Russel

Mascerhenas, Sona Dusseja, Shashikant Mahadik, Sheetal Gaware, Prashant
Tembhare, Sumeet Gujral, Sharayu Kabre, Pratibha Kadam-Amare, Hasmukh
Jain, Uma Dangi, Bhausaheb Bagal, Navin Khattry, Manju Sengar, Brijesh
Arora, Gaurav Narula, Shripad Banavali, Hari Menon & Papagudi Ganesan
Subramanian

To cite this article: Nikhil Patkar, Kiran Ghodke, Swapnali Joshi, Shruti Chaudhary, Russel
Mascerhenas, Sona Dusseja, Shashikant Mahadik, Sheetal Gaware, Prashant Tembhare,
Sumeet Gujral, Sharayu Kabre, Pratibha Kadam-Amare, Hasmukh Jain, Uma Dangi, Bhausaheb
Bagal, Navin Khattry, Manju Sengar, Brijesh Arora, Gaurav Narula, Shripad Banavali, Hari
Menon & Papagudi Ganesan Subramanian (2016): Characteristics of BCR-ABL kinase
domain mutations in chronic myeloid leukemia from India: not just missense mutations but
insertions and deletions are also associated with TKI resistance, Leukemia & Lymphoma, DOI:
10.3109/10428194.2016.1157868

To link to this article: http://dx.doi.org/10.3109/10428194.2016.1157868

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 LEUKEMIA & LYMPHOMA, 2016
http://dx.doi.org/10.3109/10428194.2016.1157868

ORIGINAL ARTICLE: RESEARCH

Characteristics of BCR-ABL kinase domain mutations in chronic myeloid

leukemia from India: not just missense mutations but insertions and
deletions are also associated with TKI resistance
Nikhil Patkara, Kiran Ghodkea, Swapnali Joshia, Shruti Chaudharya, Russel Mascerhenasa, Sona Dussejaa,
Shashikant Mahadika, Sheetal Gawarea, Prashant Tembharea, Sumeet Gujrala, Sharayu Kabreb,
Pratibha Kadam-Amareb, Hasmukh Jainc, Uma Dangic, Bhausaheb Bagalc, Navin Khattryc, Manju Sengarc,
Brijesh Arorad, Gaurav Narulad, Shripad Banavalid, Hari Menonc and Papagudi Ganesan Subramaniana
a
Hematopathology Laboratory, Tata Memorial Centre, Ernest Borges Marg, Parel, Mumbai, Maharashtra, India; bDepartment of Cancer
Cytogenetics, Tata Memorial Centre, Ernest Borges Marg, Parel, Mumbai, Maharashtra, India; cAdult Haemato-lymphoid Disease
Management Group, Tata Memorial Centre, Ernest Borges Marg, Parel, Mumbai, Maharashtra, India; dPediatric Haemato-lymphoid
Disease Management Group, Tata Memorial Centre, Ernest Borges Marg, Parel, Mumbai, Maharashtra, India
Downloaded by [New York University] at 06:22 22 March 2016

ABSTRACT ARTICLE HISTORY

We document the characteristics of BCR-ABL kinase domain mutations (KDM) in the largest study Received 16 September 2015
from India comprising of 385 patients and demonstrate that more than half (51.9%) of these Revised 12 January 2016
patients have detectable abnormalities in the KD both in adult and in pediatric chronic myeloge- Accepted 14 February 2016
nous leukemia (CML). These comprise singly occurring missense mutations (25.5%), polyclonal/
KEYWORDS
compound point mutations (4.9%), and insertions/deletions (29.6%). Missense mutations were CML India; BCR-ABL Kinase
most commonly seen in the imatinib-binding region followed by the P-loop. The commonest domain mutations; imatinib
mutation in our dataset was T315I. Other common missense mutations were Y253H, M244V, and resistance
F317L. A high prevalence of BCR-ABL exon7 deletion (p.R362fs*) was also seen (25.5% of the
entire cohort), whereas the 35bpintron-derived insertion/truncation mutation detected in 12
patients. In the pediatric age group, 58.8% of patients harbored missense mutations, polyclonal/
compound mutations as well as insertions and deletions. We detected 11 novel mutations (seven
missense mutations and four insertions/deletions).

Introduction kinetics, modulation of other kinase-signaling path-

Development of a specific targeted therapy in the
form of imatinib mesylate (IM), a tyrosine kinase inhibi-
tor (TKI) was a breakthrough in the treatment of
chronic myeloid leukemia (CML). As a result of this
innovative treatment, CML is a paradigm of molecular
medicine.[1,2] TKI therapy for CML is very effective
with rapid responses; nearly, 90% of patients achieve
hematologic remission and 60–80% achieve a com-
plete cytogenetic remission (CCyR). In the landmark
IRIS trial, after six years, nearly 60% of patients treated
with IM have remained in CCyR.[3–5] However, it was
soon realized that almost 30% of patients with CML
have treatment failure or relapse after responding ini-
tially to IM.[6,7] There are a myriad of factors that often
interact and contribute to IM resistance, such as treat-
ment compliance, bioavailability of the drug, pharma-
codynamic covariates, genomic instability resulting in
BCR-ABL kinase domain (KD) mutations, BCR-ABL gene
amplification, BCR-ABL overexpression, altered TKI efflux
ways, and rarely BCR-ABL mutations outside of the kin-
ase domain.[3,6,8] Other causes of resistance typically
seen in CML in advanced phase (accelerated phase/
blast crisis) include chromosomal changes manifested
by an abnormal karyotype. Among these diverse mech-
anisms, kinase domain mutations (KDM) are the com-
monest cause of resistance and have been studied in
detail. KDM are detected in 30–60% of patients with
TKI resistance (depending upon the methodology
used) and with increasing frequencies in accelerated
phase and blast crisis.[9] The detection of a kinase
domain mutation in a patient of CML is an undesirable
event as it may necessitate modification of treatment,
including dose escalation, switching to second or third
generation TKIs or allogeneic bone marrow transplant-
ation depending upon the nature of the mutation.[10]
Numerous studies have demonstrated that KDM-posi-
tive patients have faster progression to accelerated/
blast phase, shorter survival and loss of cytogenetic

CONTACT PG Subramanian pgs_mani@yahoo.com 727, Hematopathology Laboratory, 7th Floor, Annexe Building, Tata Memorial Hospital, Mumbai,
400012, India
ß 2016 Taylor & Francis
 2 N. PATKAR ET AL.
responses and eventual loss of hematological
response.[11–13]
In this manuscript, we describe the spectrum of
BCR-ABL kinase domain mutations (KDM) seen in our
hospital in a relatively short span of two years.
Additionally, we also document KDM analysis of 17
pediatric patients of CML with suspected TKI resistance.
The latter is a relatively rare subset of CML and there
is a paucity of literature describing KDM in this group.
This descriptive series is the largest dataset of BCR-ABL
KDM from India. We describe 11 novel mutations and
document that not just point mutations but also inser-
tions and deletions are associated with resistance to
TKI therapy.
Methods
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Patients
Tata Memorial Center (TMC) has a large volume of
patients with CML. Since 2004, we have enrolled 3117
patients on the Glivec International Patient Assistance
Program (GIPAP). In 2013, 2014 and 2015 the hospital
registered 344, 445, and 393 new patients of CML,
respectively. Since August 2013 when the molecular
hematology laboratory was established, we have tested
996, 3039, and 3725 samples per year for BCR-ABL quan-
titation. Most patients are called for monitoring of BCR-
ABL real-time polymerase chain reaction (PCR) values
once a year. A total of 385 patients were accrued over a
26-month period from May 2013 to July 2015. These
patients had been following up in Tata Memorial Center
for a period ranging from 0–5783 days (median ¼ 1340
days). Patient samples were referred to the hemato
pathology Laboratory, Tata Memorial Center, with a
suspicion of resistance to TKI therapy as per the discre-
tion of the treating physician. Patient records were
assessed on the electronic medical records and/or test
referral form. Response definitions were as per the
European Leukemia-Net criteria.[5,14,15]
RNA extraction and cDNA synthesis
RNA was sourced from a peripheral blood (PB) sample
using a QIAamp RNA Blood Mini Kit (Qiagen,
Germany). Complementary DNA (1 lg) was synthesized
from RNA using a ‘High-capacity cDNA reverse tran-
scription kit’ (Applied Biosystems, CA).
Sequencing of the BCR-ABL kinase domain
We have employed a nested PCR approach to first
amplify the BCR-ABL product followed by another PCR
to amplify the kinase domain as published by the
Hammersmith group.[16] The first round primers amp-
lify the e13a2 or e14a2 fusion product (from exon 13
of BCR gene to exon 10 of ABL1 gene yielding a
1600 bp amplicon) by second round/nested PCR using
primers that amplified exons 4 to 10 of the ABL1 gene
to produce an863 bp amplicon. This product was sub-
jected to Sanger sequencing on an ABI3500 genetic
analyzer using four overlapping primers to ensure that
every base substitution was confirmed at least
twice.[16] These sequences were compared with the
ABL1 wild-type reference sequence (HUMABLA NCBI:
M14752).The PolyPhen – 2 database was used to pre-
dict the damage caused at a protein level for novel
missense mutations.[17] As a part of the proficiency
testing, the laboratory has participated in UK NEQAS
module for BCR-ABL kinase domain mutation, from
2013 till date.
Results
Patients
Blood samples of 385 patients, including 17 children
(<18 years) were submitted to the laboratory for BCR-
ABL kinase domain testing (age range 7–73 years, male
predominant M:F ¼ 3:1). Patients were tested for a var-
iety of reasons, such as presentation in accelerated
phase(AP)/blast crisis(BC) (14%), loss of cytogenetic
response (10.4%), loss of major molecular response
(MMR; 5.7%), loss of complete hematological response
(CHR; 7.5%). Suboptimal responses to IM were seen in
30.6% of patients. In six patients, limited clinical details
were available (referral patients), whereas 31.7% of
patients never went into cytogenetic remission.
Best response to imatinib
This was noted as follows: did not achieve CHR
(n ¼ 4.1%), CHR (n ¼ 26, 6.8%), complete cytogenetic
response: CCgR (n ¼ 178, 46.2%), partial
cytogenetic response: pCgR (n ¼ 80, 20.8%), minor cyto-
genetic response: mCgR (n ¼ 30, 7.8%), minimal cytogen-
etic response: minCgR (n ¼ 33, 8.6%), major molecular
response: MMR (n ¼ 9, 2.3%)
Characteristics of BCR-ABL kinase domain
mutations
Two hundred (51.9%) patients had a detectable abnor-
mality in the ABL KD. Predominantly, three classes of
sequence variations were seen in varying combina-
tions, namely singly occurring missense mutations (98

patients, 25.5%), polyclonal/compound point mutations
(19 patients, 4.9%), insertions and deletions (with/with-
out point mutations) (114 patients, 29.6%). These
classes are described in detail below. The characteris-
tics of BCR-ABL point mutations can be seen in
Figure 1
 Singly occurring missense mutations: Mutations
commonly involved the imatinib-binding region fol-
lowed by the P-loop domain. The commonest muta-
tion was the p.T315I (20.7% of all point mutations)
followed by p.Y253H (10.1%) and p.M244V and
p.F317L (7.2% each).
 Polyclonal/compound point mutations: Descriptions
of polyclonal/compound point mutations as well as
patient outcome can be seen in Table 1. Majority of
the patients had two missense mutations, whereas
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three patients harbored three missense mutations.
 Insertions and deletions: A total of 114 patients har-
bored insertions/deletions with/without point muta-
tions (single as well as polyclonal/compound). The
commonest was a 185bp deletion (p.R362fs*21/
c.1086_1270del185) involving exon 7 of the ABL
kinase domain, seen in 98 patients (25.5%).[18,19]
Intron-derived insertion/truncation mutation
(p.C475fs*11; c.1423_1424ins35) in which a 35bp
intronic sequence gets inserted into the exon 8/9
Figure 1. A total of 139 point mutations were detected. Frequencies of BCR-ABL point mutations and their distribution in the kinase
domain are appreciated in the figure above.
BCR-ABL KINASE DOMAIN MUTATIONS FROM INDIA 3
junction was seen in 12 patients (3.1%).[20] We also
detected DL248-K274 (p.L248_K274del) that has
been described previously by Sherbenou et al., as
well as by Vaidya et al., from India.[21,22] Another
patient had a recently described K294RGG insertion
with p.G250E.[23] The rest are described in Table 2.
 Novel mutations: We detected 11 novel mutations
that included missense mutations as well as inser-
tions and deletions as can be seen in Table 2.
These mutations have not been described in the
Catalog of Somatic Mutations in Cancer (COSMIC)
database as well as a literature search on PUBMED.
They have also not been described as ABL single-
nucleotide polymorphisms (SNPs).[24]
 Pediatric CML KDM mutations: Out of 17 patients,
10 cases (58.8%) showed a kinase domain abnor-
mality. Six patients (35.3%) showed a missense
mutation, whereas four showed the presence of
insertions/deletions (p.C475fs*11 c.1423_1424ins35
in two patients, p.R362fs*21, one patient,
p.L248_K274del in one patient). Four patients
(23.5%) showed compound/polyclonal mutations.
Patient characteristics and response to therapy
Majority of patients presented in the chronic phase,
whereas 6.5% and 7.5% of patients presented with de
 4 N. PATKAR ET AL.

Table 1. Characteristics of polyclonal/compound point mutations seen in 19 patients.

Nature of
polyclonal/compound No. of
mutation cases Description of mutations Outcome of patient
Three mutations 3 E355G F359C E453K Alive, loss of CHR, on dasatinib
M244V K247R E459K Alive, in CHR on nilotinib, cannot afford dasatinib
L387M E453V G442E Alive, in CHR, cannot afford 2nd line TKI, on hydroxyurea
Two mutations 16 T315I E459K Alive, cannot afford other treatment, not in CHR
F317L E459K Died, treated with nilotinib, dasatinib, and hydroxyurea
G250E F317L Alive at last follow-up, on dasatinib, not in CHR
E255K T315I Alive, cannot afford other treatment, in CHR
Y253H D325N Alive on dasatinib, in deep MR (NCN:0.008%)
E238D V448A Alive, treated with 7 þ 3 (AML transformation) chemotherapy,
subsequent immunophenotypic MRD negative
G250E F311L Alive, on palliation, transformation to blast crisis on dasatinib
M244V H246R Alive on increased dose imatinib, cannot afford other TKI/BMT, loss of CHR
M244V V299L Lost to follow up
R239G L384P Died, treated with dasatinib
T315I E355G Alive, treated as lymphoid blast crisis, in MMR
T315I M351T Alive, cannot afford other treatment, not in CHR
E255K T315I Alive, cannot afford other treatment, not in CHR
V299A Q477X Increased dose of IM, almost in MMR (NCN:0.2%)
Y253H Y353H Alive, cannot afford other treatment, in CHR (NCN:55%)
H396L V506E Alive, cannot afford second-line
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TKI, blood counts controlled with hydroxyurea

Table 2. Characteristics of novel mutations seen this study.

Sr No Description Domain PolyPhen-2 score
Missense mutations
1 c.897 G > C, p.V299A SH3 contact domain 0.99 (probably damaging)
2 c.1014 T > G, p.V338G SH2 contact domain 0.97 (probably damaging)
3 c.1187 A > T, p.H396L A-loop 0.02 (benign)
4 c. 1325 G > A, p.G442E C-terminal 0.99 (probably damaging)
5 c.1429 C > A, p.Q477X C-terminal NA
6 c.1518 G > A, p.V506E C-terminal 0.63 (probably damaging)
7 c.1001 A > G, p.E334G SH2 contact domain 0.63 (probably damaging)
Insertions/Deletions
Sr No Description Comments
1 Complex indel, c.1103_1116delins216 Mutation deletes 13bp from catalytic domain and insertion of
216 bp from BCR exons 1 and 2. Disrupts part of catalytic domain,
A-loop, and C-terminal
2 p.Ala424Glyfs*3, c.1271_1342del71 71bp deletion in C-terminal, abrupt termination of AA synthesis
3 p.Val371fs*, c.1114_1287del173 Mutation deletes 173bp between catalytic domain and A-loop
4 p.K356L, c.1065_1066 ins CTC In-frame insertion in the catalytic domain

novo blast crisis or accelerated phase, respectively.
Sokal risk was not consistently noted in these patients
due to nondocumentation of spleen size. However,
data from our center on CML-CP (observations from
physicians) shows that high-risk Sokal constitutes
56.3% of patients, intermediate and low-risk constitutes
23.7% and 32.6%, respectively. In our series, 98
patients (25.5%) were counseled second-line TKIs but
could not afford them. As seen in Table 3, a total of
113 patients (29.4%) switched to second-line TKIs,
whereas in 86 patients (22.3%), dose escalation of IM
was done. Seventeen (4.4%) patients were treated with
stem cell transplantation, whereas for 36 (9.4%)
patients salvage therapy was the only feasible option.
Most of these salvage therapy patients were treated
with hydroxyurea except two patients who were
treated with interferon. Majority of deaths occurred in
the CML blast crisis group (40%). At the last follow–up,
three patients has transformed into blast crisis, one of
which was acute promyelocytic leukemia. Correlation
of different phases of disease, types of mutations
and treatment responses can be seen in Table 3. As
Table 3 indicates, only a small fraction of patients with
isolated insertions/deletions responded to increased IM
dose or second-line TKIs. Patients with compound/
polyclonal mutations had a distinctly worse outcome
as compared with other patients (Table 1). In this
group, seven patients had loss of CHR (including one
who had transformed to blast crisis) and two patients
had died.
Discussion
CML is a relatively common myeloproliferative neo-
plasm in India.[25] India has an ‘out of pocket’ health
care system where most patients cannot afford
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Table 3. Patients in various phases of disease and nature of kinase domain mutations, type of treatment and responses can be seen in the first part; association of mutations in
patients who were in chronic phase with responses is seen in second part; the third part shows different types of kinase domain mutations and treatment responses; the last part of
the table looks at duration of treatment with IM in patients of chronic phase and types of mutations.
Missense Isolated insertions/ Compound/poly- Increased dose of
Phase of disease mutations deletions Negative for KDM clonal mutations IM Second-line TKI Salvage treatment BM transplantation In MMR Outcome
Blast crisis (n ¼ 25) 9 (36%) 6 (24%) 10 (40%) 3 (12%) 3 (12%) 9 (36%) 2 (8%) 1 (4%) 3 (12%) 40% deaths
Accelerated phase 10 (34.5%) 11 (37.9%) 8 (27.6%) 1 (3.4%) 8 (27.6%) 9 (31%) 2 (6.7%) 3 (10.3%) 2 (6.9%) 6.9% deaths
(n ¼ 29)
Chronic phase 98 (29.6%) 66 (19.9%) 167 (50.5%) 16 (4.8%) 75 (22.7%) 95 (28.7%) 30 (9.1%) 14 (4.2%) 17 (5.1%) 4.9% deaths
(n ¼ 331)
Missense Isolated insertions/ Negative Compound/poly- Common mutations
mutations deletions clonal mutations
Never went into 30 (24.6%) 31 (25.4%) 61 (50%) 7 (5.7%) T315I, Y253H, G250E
cytogenetic remis-
sion (n ¼ 122)
Loss of cytogenetic 11 (27.5%) 7 (31.8) 22 (5.7%) 1 (4.5%) T315I
response (n ¼ 40)
Loss of MMR 11 (50%) 1 (4.5%) 10 (45.5%) – E255K, M244V, Y253H
(n ¼ 22)
Loss of CHR 18 (62%) 3 (13.6%) 8 (27.6%) 3 (13.6%) T315I, E459K, F317L
(n ¼ 29)
Suboptimal 28 (23.7%) 24 (10.3%) 66 (55.9%) 5 (4.2%) T315I, Y253H, M244V
response to IM
(n ¼ 118)
Dead Alive Increased dose of MMR post Second-line TKI MMR post second- BMT MMR post-BMT Salvage MMR postsalvage
IM increased dose of line TKI
TKI
Single missense 9 (9.2%) 89 (90.8%) 20 (20.4%) 0 39 (39.8%) 10 (25.6%) 9 (9.2%) 4 (44.4%) 14 (14.3%) 1 (7.1%)
mutations (n ¼ 98)
Compound/poly- 3 (15.8%) 16 (84.2%) 2 (10.5%) 0 7 (36.8%) 2 (28.6%) 1 (5.2%) 1 (100%) 3 (15.8%) 0
clonal mutations
(n ¼ 19)
Isolated insertions/ 6 (7.2%) 77 (92.7%) 18 (21.7%) 1 (5.5%) 23 (27.7%) 0 3 (3.6%) 1 (33.3%) 4 (4.8%) 0
deletions (n ¼ 83)
Duration of imati- Number Negative for Isolated insertions/ Compound/poly- Point mutations Commonest Second most Third most
nib treatment for mutation deletions clonal point mutation common common
CML chronic phase
(n ¼ 325)
Less than one year 26 15 (56.7%) 8 (30.8%) 0 3 (11.5%) T315I F317L M244V
One to 5 years 151 74 (49%) 35 (23.2%) 8 42 (27.8%) T315I (n ¼ 10) Y253H M244V
More than 5 years 148 74 (50%) 21(14.2%) 8 53 (35.8%) T315I (n ¼ 13) Y253H F317L
BCR-ABL KINASE DOMAIN MUTATIONS FROM INDIA
5
 6 N. PATKAR ET AL.
Table 4. Comparison of our data with other studies.
Number of patients
Tata Memorial Center, India 385
GIMEMA CML WP[36] 297
MD Anderson Cancer Center[37] 112
Argentinean study[38] 154
Chinese study[39] 127
Australian study[12] 144
Malaysian study[40] 125
Thai Study[41] 171
Korean study[42] 137
Hungarian study[43] 71
French study[44] 89
CMC, Vellore, India[45] 76
modern investigations and advanced treatments. Most
Indians cannot afford to buy the original IM molecule,
Gleevec (Novartis). However, because of support
through the GIPAP, many nonaffording Indian patients
have been receiving IM. After the availability of generic
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IM, majority of patients are on IM. However, emer-
gence of resistance poses a unique dilemma in a
resource-constrained setting as very few patients can
afford second-generations TKIs – nilotinib or dasatinib
– especially since the generic molecules of these drugs
are not available presently. The third-generation TKIs,
such as ponatinib and bosutinib, are not even available
on sale presently in India. Stem cell transplantation
(SCT), especially unrelated/haploidentical transplants,
can be afforded only a fraction of patients and
because of very few centers offering SCT, the waiting
period is long. Because of these socio-economic rea-
sons, leading to lack of therapeutic options, the detec-
tion of KDM is a potential cause of concern. Thus, at
many centers, the presence of KDM is not even tested,
leading to paucity of Indian data in this respect.
There have been studies published from across the
world that have documented the frequencies of BCR-
ABL kinase domain mutations. Results from our study
can be compared to some of these studies in Table 4.
Many studies have mutations in common. The com-
monest mutation in most is p.T315I. This may change
in the future if ponatinib is approved as the first line
of management of CML-CP. A majority of published
studies have employed Sanger sequencing as a tech-
nique to detect mutations. This technique has been
recommended by ELN.[26] Whereas the sensitivity of
mutation detection can be increased by use of sensi-
tive techniques, such as allele specific PCR, digital PCR
or next-generation sequencing (NGS), the clinical utility
of a highly sensitive assay may be questioned.[27,28]
Among the emerging technologies NGS may offer an
ideal trade-off between sensitivity and specificity keep-
ing in mind, its obvious advantage of detecting
unknown mutations. Recent work in this field by
Soverini et al., showed that NGS identified KDM in
Patients with missense mutations Common mutations
114(29.6%) T315I, Y253H, M244V, F317L
127 (42.7%) E255K/V, Y253F/H, T315I, M351T
61 (54%) G250E, T315I, F317L, E355G
36 (23%) G250E, M351T, T315I, Y253H
74 (58%) M244V, Y253H, G250E, T315I
27 (19%) M351T, E255L, Q252H, E355G
28 (22.4%) T315I, E255K, G250E, M351T, F359C
37 (21.6%) T315I, M351T, Y253H, G250E
70 (51%) T315I, E255K, G250E, Y253H
27 (36%) M244V, T315I, M351T, E255V
NA T315I, M244V, M351T, E255V/K
25 (33%) T315I, M244V, Y253F/H, E255K
TKI-resistant patients with CML which would have
been misclassified or missed using Sanger sequenc-
ing.[29] They also stated that such a laboratory
approach may also be used to guide therapy.[28]
Compound KD mutations are multiple mutations
present within the same mRNA molecule (and presum-
ably the same clone), whereas polyclonal mutations
arise across different mRNA molecules and therefore
different clones.[26] The distinction between the two
can be made by single-molecule-sequencing techni-
ques (e.g. NGS) or by cloning PCR products followed
by sequencing of cultured colonies. The latter is tech-
nically cumbersome in a diagnostic setting and there-
fore was not done in our center, one of the
shortcomings of the study. In a recent study by
Khorashad et al.,[30] multiple mutations were often
compound in nature. In our study, the commonest
mutation in the compound/polyclonal group were
p.T315I (4 cases, 20%), p.G250E, and p.M244V (3 cases
each, 15%). In all these, mutations accounted for 50%
of the total. This paper is the first from India to docu-
ment compound/polyclonal point mutations and show
their relatively poor outcome. A recent study by Kagita
et al.,[31] demonstrated that T315I and P-loop muta-
tions were common in advanced phase and were asso-
ciated with a poor outcome.
Additionally, we also document a high frequency
of the 185bp deletion (exon7 deletion). This mutation
has probably been underreported by some studies
due to the position of reverse primer in KDM-
sequencing assays.[19] The contribution of this muta-
tion to IM resistance is controversial as some studies
have suggested that it is merely a result of alternative
splicing.[32] Others have even reported it in the nor-
mal ABL KD.[33] However, awareness about this muta-
tion is necessary, especially amongst laboratory
personnel.[34] Unlike other studies that have reported
a low percentage on Sanger sequencing, we report a
high frequency of this mutation, pointing toward a
geographical bias in Indian patients, which has not
been reported.

Pediatric CML is a relatively rare subset of hematol-
gical malignancies and there is a paucity of literature
with respect to treatment responses and resistance to
TKI therapy.[35] With that analogy, description of KDM
in the pediatric subgroup is even scarcer. In our study,
37% of pediatric patients showed missense mutations,
including polyclonal/compound mutations. A small
subset even showed the intron-derived insertion/trun-
cation mutation. To the best of our knowledge, this
has not been described in the pediatric population.
In this manuscript (Table 2), we describe 11 novel
mutations; however, we could not functionally validate
them. However, molecular diagnosticians need to be
aware of such undescribed mutations.
To conclude, when a large series of patients were
studied we have found a similar repertoire of missense
mutations in our patient population as compared to
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global literature. For the first time from India, we
report that not just missense mutations but also inser-
tions/deletions are prevalent and may be associated
with resistance to TKI therapy. We also describe the
profile of KDM in pediatric CML patients and describe
a relatively large number of polyclonal/compound
mutations.
Potential conflict of interest: Disclosure forms pro-
vided by the authors are available with the full text of
this article at http://dx.doi.org/10.3109/10428194.2016.
1157868.
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