Clinical & Experimental Pathology

Singh et al., J Clin Exp Pathol 2012, 2:4

http://dx.doi.org/10.4172/2161-0681.1000113

Review Article

Open Access

Molecular Monitoring and Treatment of Chronic Myeloid Leukemia (CML)

Zeba Singh1*, Stephen Medlin2 and Saad Z Usmani2
1
2

Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, AR, USA
Myeloma Institute for Research & Therapy, University of Arkansas for Medical Sciences, Little Rock, AR, USA

Abstract
The advances in the treatment strategies in CML epitomize the concept of personalized medicine. These rapid
developments towards cure of CML are acutely dependent on our ability to monitor therapeutic response to the drugs
and fine-tune the therapy to the challenges posed by drug resistance from development of target site mutations.
The fundamental requirement for success of targeted therapy in CML is standardization of methods for molecular
monitoring such that there is a uniform understanding of response therapies and results of clinical trials. This review
discusses the aspects of molecular monitoring of CML in the context of recent advances in its treatment strategies. It
provides guidelines to both clinicians and laboratory physicians in choosing the appropriate methodology for laboratory
monitoring and in interpretation of the results for effective therapeutic decision-making.

Keywords: CML; Targeted therapy; Tyrosine kinase inhibitors;

Drug resistance; Molecular monitoring

Introduction
In 1960, Peter Nowell and David Hungerford made the seminal
observation of an abnormally small chromosome in patients with
Chronic Myeloid Leukemia (CML), which they named the Philadelphia
or Ph1 chromosome [1] for the city where the discovery was made. In
the early 1970s, Janet Rowley identified the underlying non-random
balanced chromosomal translocation between chromosomes 9q34 and
22q21 [2]. Molecularly, this translocation fuses the 5 end of the BCR
gene on chromosome 22 with 3 end of the ABL1 gene on chromosome
9 and gives rise to the chimeric BCR-ABL gene [3]. Ph1 was the first
disease-specific chromosomal abnormality described in cancer and
is the disease defining abnormality for Chronic Myeloid Leukemia
(CML). The BCR-ABL1 chimeric gene on derivative chromosome 22
has since been established to drive the oncogenesis.
In the fifty years since the first identification of the Ph chromosome,
the sequence of discoveries and inventions made in understanding
the biology, detection strategies, and treatment of CML exemplify
the concept of molecular medicine. The increase in understanding
of the disease has resulted in a paradigm shift in treatment from
the traditional combination of Hydroxyurea, interferon-alpha, and
Ara-C to a targeted approach by imatinib mesylate, (Glivec/Gleevec;
formerly STI571; Novartis Pharmaceuticals, East Hanover, NJ), a
tyrosine kinase inhibitor (TKI) that targets the breakpoint cluster
region-v-abl, the Abelson murine leukemia viral oncogene homolog
1, also known as ABL1, and by competitive inhibition, disrupts the
downstream oncogenic signals. In the IRIS (International Randomized
Study of Interferon and STI571) trial, imatinib proved superior to a
combination of interferon-alpha and cytarabine in having statistically
significant higher rates of hematological and complete cytogeneic
responses, lower risk of progression to advanced disease states, and
increased progression free survival [4]. Imatinib has thus revolutionized
the treatment and outcome of CML, making it the paradigm of
molecularly targeted cancer therapies. The success of imatinib in the
IRIS trial necessitated the development of sensitive laboratory methods
for molecular monitoring of the disease. This review summarizes the
recent advances in the treatment of CML and the current appropriate
methods for establishing diagnosis and molecular monitoring of the
disease.
1

Natural History of the Disease

CML is characterized by uncontrolled proliferation of primarily the
J Clin Exp Pathol
ISSN: 2161-0681 JCEP, an open access journal

granulocytic and megakaryocytic lineages resulting in accumulation

of immature granulocytic precursors in the one marrow, blood and
other organs. If left untreated, the natural history of CML follows
a course progressing through a chronic phase to an accelerated
phase (in most cases) and terminating in blast crisis over 3-5 years.
Disease progression is heralded by increasing genomic instability
and acquisition of additional chromosomal abnormalities, the most
common of which are +8, +19, del(9)(q34), isochromosome 17,
and additional Ph1 chromosome. Some recent studies have shown
emergence of the RUNX1 mutations during progression of CP-CML
to blast crisis [5]. The blast transformation is myeloblastic in twothirds, and lymphoblastic (precursor B-) in one-third of the patients.
Blasts with mixed phenotypes representing different lineages are not
uncommon, consistent with the stem cell origin of the disease [6-8].

Molecular Pathogenesis
Studies in mice have shown that translocation between BCR and
v-ABL is sufficient to drive oncogenesis [9]. The chimeric BCR-ABL
gene encodes for an abnormal, constitutively active tyrosine kinase,
which phosphorylates and activates downstream signaling pathways
including PI3K, AKT, SRC-kinase, and STAT that in turn modulate
transcription factors related to cell proliferation, survival, protection
from apoptosis, and differentiation. In addition, the BCR-ABL
protein causes oxidative damage to the DNA by induction of reactive
oxygen species and also interferes with the DNA-repair pathway [10],
thus making the cells genetically unstable and prone to accumulate
deleterious mutations. Nearly 80% of patients with CML have
additional chromosomal abnormalities, many of which are associated
with disease progression.

Laboratory Diagnosis of CML

The WHO classification has laid down definite criteria for the

*Corresponding author: Zeba Singh, University of Arkansas for Medical

Sciences, 4301 W. Markham Street, Mail Slot 502 Little Rock, AR 72205, USA,
E-mail: zsingh@uams.edu
Received May 09, 2012; Accepted June 14, 2012; Published June 17, 2012
Citation: Singh Z, Medlin S, Usmani SZ (2012) Molecular Monitoring and Treatment
of Chronic Myeloid Leukemia (CML). J Clin Exp Pathol 2:113. doi:10.4172/21610681.1000113
Copyright: 2012 Singh Z, et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and
source are credited.

Volume 2 Issue 4 1000113

Citation: Singh Z, Medlin S, Usmani SZ (2012) Molecular Monitoring and Treatment of Chronic Myeloid Leukemia (CML). J Clin Exp Pathol 2:113.
doi:10.4172/2161-0681.1000113

Page 2 of 9

chronic phase, accelerated phase, and blast crisis of CML. Whereas

demonstration of the defining BCR-ABL1 translocation is a must for
establishing the diagnosis of CML, the diagnosis can easily be made
from the peripheral smear in most cases. Leukocytosis with circulating
granulocytic precursors, absolute basophilia, and thrombocytosis form
the backbone of morphological diagnosis of chronic phase CML (CPCML). In CP-CML, the peripheral blood and bone marrow have less than
10% blasts. Accelerated disease is associated with increasing leukocyte
count, increase in circulating or bone marrow blasts, basophilia of
>20% in the peripheral blood, thrombocytosis or thrombocytopenia,
and bone marrow fibrosis with proliferation and clustering of abnormal
megakaryocytes. The last mentioned morphological feature, while not
a definitive criterion, is often observed with the other defined WHO
criteria for accelerated disease. For details on the clinical features and
diagnostic criteria of the different stages of disease evolution, the reader
is referred to the WHO Blue book on the Classification of tumors of the
hematopoietic and lymphoid system [6].
Demonstration of the translocation t(9;22)(q34;q22) or the BCRABL1 chimeric gene product is quintessential to establish the diagnosis.
The Ph1 chromosome can be demonstrated by G-banded metaphase
cytogenetics or fluorescent in-situ hybridization (FISH), and the
abnormal BCR-ABL transcript by RT-PCR (reverse transcriptasePCR). Each of these methods has its pros and cons as a diagnostic tool
or for subsequent monitoring of the disease.

Conventional metaphase cytogenetics and fluorescent in-situ

hybridization
In 95% of patients, the Ph1 chromosome can be detected by
metaphase cytogenetics or G-banding [11]. Analysis of 20-25
metaphases for presence of Ph1 chromosome is standard practice.
A bone marrow sample is required for obtaining metaphases from
dividing cells, entailing the risk associated with an invasive procedure.
In about 5% cases the result may be negative due to the overgrowth
of normal bone marrow cells or inadequate metaphases for analysis.
The specific translocation may be masked in the presence of a complex
translocation involving three or more chromosomes, or when there is
no alteration in the size of chromosome 22 due to cryptic interstitial
insertions of the ABL1 within the BCR gene [12]. The main advantage
of G-banding is that no a priori information about the chromosomal
abnormalities is required and it can detect abnormalities that may be
present in addition to the Ph1 chromosome. The commonest secondary
abnormalities in CML are an additional Ph1 chromosome, +8, and
isochromosome 17q. Several previous studies had reported rapid
disease progression and worse outcome in the 10-15% of patients
who develop deletions of the derivative chromosome 9 [13-15]; in
recent studies however, authors show that the adverse effect of this
chromosomal abnormality on the EFS in chronic phase patients can
be mitigated by second generation TK inhibitors [4-16]. Fluorescent
in-situ hybridization (FISH) has a higher sensitivity for detection of
Ph1 than metaphase cytogenetics. The dual-color, dual-fusion FISH
probe set is very sensitive and has high specificity of 1.0 to 0.1%. The
major advantage of FISH is that it can be performed on interphase,
non-dividing cells from peripheral blood, precluding requirement for
the invasive, costly, and labor intensive bone marrow procedure. The
results obtained from the peripheral blood sample are comparable in
sensitivity to those obtained from the bone marrow cells [17]. FISH
should however not be considered an alternative for metaphase
cytogenetics. Current guidelines recommend that cytogenetic analysis
be performed at baseline, and then at 3 to 6 month intervals after
initiation of TK inhibitor therapy [18,19]. Attaining the defined levels
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of cytogenetic responses at specific time points correlates with response

to therapy and likelihood of subsequent relapse or disease progression.
The definitions for hematological, cytogenetic and molecular remission
are depicted in 9 (Table 1). (Table 2) shows the NCCN and ELN
recommendations for time points for metaphase cytogenetics, FISH,
and quantitative PCR. Patients with a major cytogenetic response
(MCyR) including those with a partial response (1-34% Ph1 positive
metaphases), or a complete cytogenetic response (CCyR: no Ph1
positive metaphases) are less likely to progress to advanced phase CML.
Achieving CCyR at 12 months is a major landmark in therapeutic
response to imatinib [20-21].

Qualitative and quantitative PCR

Qualitative testing by PCR analysis of the BCR-ABL transcript
is required to establish diagnosis only in the rare situations when
interphase FISH is inconclusive due to failure of probe hybridization
or sample inadequacy. At the molecular level, the translocation
t(9;22)(q34;q22) consistently involves exon 2 of the ABL gene, but
occurs in different exons of the BCR gene. The fusion involving the
major breakpoint region (MBR) between exons 12 and 13 (or e13a2;
formerly called b2a2) or 13 and 14 (or e14a2; formerly called b3a2)
leads to expression of a transcript that codes for a 210kD fusion protein
(p210BCR-ABL1). The breakpoint in the minor breakpoint region (mBR) between alternate exon1 and exon 2 (e1a2) results in a smaller
transcript coding for a 190 kD protein (p190BCR-ABL1) [22,23]. Most
adult CML have the b2a2 or b3a2 transcript (p210BCR-ABL1), whereas the
e1a2 (p190BCR-ABL1) is usually present in acute lymphoblastic leukemia
and the lymphoid blast crisis of CML. Chronic phase CML with p190
BCR-ABL1
have a myelomonocytic morphology. Less commonly, the break
point involves the -BCR (micro-BCR) or exon 19(e19a2) resulting in
a p230BCR-ABL1 kD product [23]. Co-expression of the p210BCR-ABL1 and
p190BCR-ABL1 encoding transcripts can occur as a result of alternative
splicing in the M-BR of BCR [24-27]. Quantitative monitoring is
the basis of molecular monitoring of CML and is discussed in a later
section.

Treatment Strategies
Prior to the advent current therapeutic modalities, the treatment for
CML was restricted to lowering the high cell counts by chemotherapy,
or managing the symptoms arising from an enlarging spleen with local
radiotherapy [28]. Interferon-alpha (IFNa) based regimens were the
HEMATOLOGICAL
Complete (CHR)

WBC< 1x109/L; basophils <5%; no circulating granulocyte

precursors; platelet count <450x109/L; no splenomegaly

Partial (PHR)

Levels less than the above

CYTOGENETIC
Partial (PCyR)

1%-35% Ph1 positive metaphases

Major

Combination of PCyR and CCyR (35% Ph1 positive

metaphases)

(MCyR)

Complete (CCyR)

No Ph1 positive metaphases detected in 2 successive bone

marrow samples after counting 30 metaphases

MOLECULAR
Major (MMR)

3-log reduction in transcript level based on 2-consequtive

molecular analyses

Complete (CMR)

2 consecutive samples with no detectable transcripts

provided that control gene copy numbers are adequate

Table 1: Definitions for hematological, Cytogenetic and Molecular remission.

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Citation: Singh Z, Medlin S, Usmani SZ (2012) Molecular Monitoring and Treatment of Chronic Myeloid Leukemia (CML). J Clin Exp Pathol 2:113.
doi:10.4172/2161-0681.1000113

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Response
Suboptimal response

3 months

6 months

12 months

18 months

CHR

PCyR

CCyR

MMR

<CHR

<PCyR
(35% Ph1 positive metaphases)

<CCyR

<MMR

Failure

No hematological response, stable <CHR or no CyR (95% Ph1

disease, or disease progression
positive metaphases)

Failure

At any time: loss of CHR, or loss of CCyR, or Abl kinase Mutation

<PCyR
(35% Ph1 positive
metaphases)

<CCyR

Table 2: NCCN and European LeukemiaNet criteria for remission, suboptimal response and failure for previously untreated chronic-phase CML patients on standard dose
of imatinib.

first to offer the possibility of eliminating the malignant clone, with

20-30% of patients achieving CCyR [29] and were distinctly superior
to the primarily leukostatic hydroxyurea [30,31]. However, the benefit
with IFNa was mostly in patients with a low Sokal risk score, and that
too at the expense of profound toxicity.
Allogeneic hematopoietic stem cell transplant is curative, especially
when performed early during the disease course and was very popular
in the 1990s. The advantage from HSCT was however restricted to
the younger patients with a suitable donor. The outcome in HSCT
depends on several factors including the age of the patient, the stage
of the disease, time since diagnosis, type of donor, source of stem
cells, post-transplant management of infections and graft versus host
disease [32]. Younger patients in chronic phase disease with matched
related donors have the best outcome [33,34]. Better supportive care
has largely mitigated the negative impact of age in the matched related
transplant group, but not for those with matched unrelated transplants
[35]. With advances in HLA typing and GVHD management, the
results for fully matched unrelated transplants are now nearly similar
to that for matched related donor transplants [36]. Currently, the
survival following HSCT is nearly 80% in chronic phase patients, about
40%-50% in accelerated phase, and 20% in patients who receive it
after blast crisis sets in [37]. The therapeutic landscape for CML has
undergone a paradigm change with the advent of TK inhibitors. HSCT
is no longer the choice for first-line treatment even in younger patients.
At this time, HSCT is recommended for patients who develop resistant
mutations that fail to respond to second line TK inhibitors as well, or
for patients in the advanced stage of disease [37,38].

Imatinib as first-Line therapy

Imatinib was initially approved over a decade ago for treatment
of CML patients who had failed prior therapy with IFNa. The results
of this Phase I study demonstrated a remarkable response with 53/54
patients achieving complete hematological remission (CHR), and
31% achieving MCyR (major molecular remission) (13% with CCyR)
[39]. In the subsequent Phase II study, the efficacy of imatinib was
established in patients in all stages of the disease. The response in
CP-CML was more impressive (CHR 95%, MCyR 60%, CCyR 41%)
than in AP-CML and BP-CML in whom CHR was obtained in 34%
and 8% patients respectively [40]. Shortly thereafter, the International
Randomized Study of Interferon and STI571 (IRIS) study (Phase
III, randomized, double blind crossover study) demonstrated the
superiority of imatinib over the IFNa/Ara-C combination in newly
diagnosed chronic phase patients [20]. Results of the IRIS study
showed 87% MCyR rate in patients on imatinib versus 35% on IFNa/
Ara-C (P < 0.001), and a CCyR of 76% in the imatinib group compared
with 15% in the IFNa/Ara-C group (P < 0.001). The progression free
survival (PFS) at 18 months was significantly higher in the imatinib
group versus IFNa/Ara-C (97% vs. 91.5%; P < 0.001) [20]. Based
on the impressive results of the IRIS trial, imatinib received a fast
approval by FDA for use as first line therapy of CML patients in May
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2001. FDA also approved imatinib for treatment of AP and BP-CML,

relapsed CML following HSCT, and Ph1 positive acute lymphoblastic
leukemia. Long term follow up of the IRIS trial confirmed that the
responses obtained with imatinib were durable over time and clearly
improved the overall survival (OS) and PFS in patients with CML [41].

Management of imatinib resistance

Whereas the majority of patients achieve sustained remission with
imatinib, an 8-year follow up study showed that it had to be discontinued
in 30%-40% of patients because of intolerance or refractoriness to the
drug [42]. Resistance to imatinib is more commonly acquired and
mostly due to additional genetic abnormalities, or resistant mutations
in the ABL kinase domain. Primary resistance is less common and
may be due to polymorphisms in drug metabolizing enzymes [43].
Secondary resistance or refractoriness to imatinib is most often due
to ABL kinase mutations, from alterations in the intracellular drug
availability due to overexpression of the multidrug resistance (MDR1)
gene product P-glycoprotein, or decreased expression of the octamer
binding protein (OCT1) gene [44].
Increasing the dose of imatinib is a rational first step in countering
resistance. The effect of many of the earlier mentioned factors for
resistance, including some of the ABL kinase mutations (e.g., M351T,
V299L, L384M, G398R, M244V, M244I, Q252H, E355G, L387M), can
be overcome by escalation of the dose from the standard 400 mg daily to
600 mg or even 800 mg per day. However, in these dose-escalation trials
only a small number of patients achieved CCyR, which is predictive of
a sustained favorable outcome [45-47]. The T315I is a highly resistant
mutation that fails to respond to imatinib dose escalation as well as to
second generation TKIs, as discussed further.

Second-generation TK inhibitors
Second line TK inhibitors- dasatinib and nilotinib are effective
in patients who fail to respond to dose escalation or develop adverse
effects to it. Dasatinib and nilotinib are structurally different from
imatinib, hence are able to overcome the resistance induced as a result
of conformational change in the molecule by ABL1 kinase mutations.
In addition, dasatinib and nilotinib do not require the OCT1 drug
transport protein and are not affected by a decrease in its level [48,49].
Dasatinib is several hundred times more potent than imatinib and
is also effective against the Src Family Kinases. Nilotinib is 30 times
more effective than imatinib against BCR-ABL1 kinase. Both dasatinib
and nilotinb can counter the negative effect of many of the ABL
kinase mutations including several that do not respond to escalation
of imatinib dose (e.g., G250E, E255K, E255V, F486S, Y253H, Y253F,
F317L, F311L) [50]. Clinical trials using dasatinib and nilotinib have
demonstrated durable overall response rates, improved PFS rates,
and tolerability of these second line TK inhibitors in patients with
imatinib resistant or intolerant CML in CP, AP, and BP-CML resulting
in FDA-approval for use of dasatinib and nilotinib in treatment of

Volume 2 Issue 4 1000113

Citation: Singh Z, Medlin S, Usmani SZ (2012) Molecular Monitoring and Treatment of Chronic Myeloid Leukemia (CML). J Clin Exp Pathol 2:113.
doi:10.4172/2161-0681.1000113

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patients resistant or intolerant CML in all stages of the disease [51-54].

The choice between dasatinib and nilotinib is generally based on the
adverse effect profile of these drugs in the context of a specific patient,
and the nature of the specific mutations [55]. Secondary resistance
from additional mutations may develop under drug pressure from
second line TK inhibitors [56,57]. Mutations such as Q252H, E255K/V,
V299L, F317L, and T315A are more likely to develop resistance to
dasatinib, whereas others including Y253H, F359C/V, and E255K/V
to nilotinib. The little overlap between mutations insensitive to both
drugs allows, replacing one by the other to restore effectiveness against
the mutation. The highly resistant T315I mutation however fails to
respond to both dasatinib and nilotinib, requiring recourse to allogenic
BMT or other alternative therapies for subsequent management [58].

Third -generation TKI

A future option may be a third-generation TK inhibitor ponatinib.
Pontinib is an oral pan-BCR-ABL inhibitor developed to specifically
counter the steric hindrance induced by the T315I mutation. Ponatinib
overlaps the gatekeeper region of the ATP-binding site, and has
an extensive network of high-potency hydrogen and triple carbon
bonds that form multiple high-affinity contact points preventing
destabilization of the ABL-kinase domain by the mutation [59]. It is
effective against several common BCR-ABL1 mutations as well. In a
cell-based mutagenesis screen, Ponatinib in therapeutic doses was
able to inhibit growth of resistant BCR-ABL1 mutations, offering the
possibility that it may prevent emergence of resistant mutations [60].
Initial results of a 3-month evaluation in the pivotal phase II trial of
ponatinib (AP24534) in patients with refractory CML show MCyR in
57% of the 23 patients with the T315I mutation, of whom 11 achieved
CCyR [59].

Second-generation TK Inhibitors as frontline therapy for

CML
Results of recent clinical trials comparing the efficacy of dasatinib
and nilotinib with standard dose imatinib in the first-line setting in
CP-CML clearly demonstrate significantly higher rates and depth
for achieving CCyR and MMR with both nilotinib and dasatinib
compared with standard dose imatinib, with lesser rates of progression
to accelerated phase or blast crisis, and with similar safety profiles
[61,62]. Compared with imatinib, the time to attain CCyR and MMR
was significantly shorter with dasatinib (p < 0.0001) [62]. FDA has
approved both dasatinib and nilotinib for front line treatment of CPCML.
Overall, for most patients, the treatment options for CML are
rendering it more of a chronic disease with the median survival estimated
at greater than 20 years [63]. With improved overall survival for most
patients, quality of life issues require more attention. In addition to
common class-related adverse effects, each TK kinase inhibitor is
characterized by a unique side effect profile [64]. The importance of
being cognizant of these adverse effects and taking them along with
the patient-specific comorbidities into account while tailoring the
TK inhibitor therapy cannot be overemphasized. Early management
of adverse effects is important in preventing non-compliance and to
ensure maintenance of remission status in patients [65].

Molecular monitoring of CML

With the current molecularly targeted therapy for CML the
majority of patients in chronic phase achieve CCyR at the end of one
year [20,66]. At the time of CCyR the tumor burden in the bone marrow
may be as high as 109 leukemic cells. In the IRIS study, quantitative
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real time PCR testing for the BCR-ABL transcript was performed in all
patients who achieved CCyR [66]. Results of the study showed that the
likelihood of progression free state at 24 months was 100% in patients
who additionally achieved a 3-log reduction in the BCR-ABL transcript
at 12 months, compared with 95% in those who did not achieve
3-log reduction (P < 0.001) [66]. These results made it incumbent on
laboratories to develop more sensitive, reproducible, and standardized
methods for subsequent monitoring of the disease. Quantitative
reverse transcriptase PCR (RQ-PCR) performed on real time platforms
can detect presence of abnormal transcripts with a sensitivity of 1
in 104 or 1 in 105 cells, and is currently the most sensitive laboratory
technique for detection of the BCR-ABL1 transcript [67-70]. RQ-PCR
is particularly useful for subsequent monitoring of patients who have
achieved CCyR. The assay is equally sensitive on peripheral blood and
bone marrow samples and either specimen can be used for the assay, as
long as the follow up is done using the same sample type for comparable
results [71]. The levels of BCR-ABL transcript in the peripheral
blood by RQ-PCR show excellent congruity with those of metaphase
cytogenetics [71]. Quantitative reverse-transcriptase PCR (RQ-PCR)
involves extraction of total RNA from the peripheral blood or bone
marrow specimen, reverse-transcription of the mRNA so obtained
into cDNA, and quantitative (real time) co-amplification of the target
BCR-ABL cDNA and cDNA of an internal control gene (to control for
RNA integrity, sample preparation, extraction and loading). Standard
curves are constructed by serial dilutions of known amount of cloned
plasmid containing the fusion DNA, or from serial dilutions of K562
cells in normal DNA. The value of BCR-ABL transcript extrapolated
from the standard curve is expressed as a normalized ratio of the BCRABL transcript to the control gene transcript [72,73]. Despite the fact
that RQ-PCR is currently performed by most laboratories nation-wide,
there is a lack of uniformity in the way the results are obtained and
the data expressed. This inherent variability is due to several factors
that affect the performance of RQ-PCR including methods of specimen
transport, storage, RNA extraction procedures, reverse transcription
and PCR efficiency, and the type of real-time platform used. The use
of different control genes by the laboratories can also significantly
alter the BCR-ABL/control gene ratios. To develop international
treatment guidelines it is essential that the molecular end-points for
therapy protocols be comparable. For harmonizing the molecular
monitoring of CML by RQ-PCR, recommendations were made at
the CML meeting at the National Institutes of Health in Bethesda in
2005 to establish guidelines for specimen collection and transport,
appropriate RNA quality, methodology for reverse transcription, PCR
amplification efficiency, use of suitable control genes, test sensitivity
for reporting negative results, quality assurance of the assay, generation
of international reference, and a standardized method for expressing
the result on an international scale [71]. To achieve this objective, a
proposal was made to develop laboratory-specific conversion factors to
convert the results obtained locally to a standardized international scale
(expressed as % or IS units) [66]. The international scale was derived
from the IRIS study in which the baseline median transcript level in 30
pre-treatment patient samples by RQ-PCR performed independently
in the three principal laboratories was defined as 100% BCR-ABL level.
Using this international scale, 1% BCR-ABL level correlated with the
state of complete cytogenetic remission, and 1 log below this level
(0.1% BCR-ABL) was defined as major molecular remission (MMR).
In the IRIS study, no patient in MMR had positive Ph1 on conventional
karyotyping [71]. According to the current guidelines laid by the
NCCN and the European Leukemia Net [18,19], optimum response to
imatinib therapy is indicated by attainment of MMR by 18 months of
start of therapy. The NCCN and ELN criteria for remission, suboptimal

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Citation: Singh Z, Medlin S, Usmani SZ (2012) Molecular Monitoring and Treatment of Chronic Myeloid Leukemia (CML). J Clin Exp Pathol 2:113.
doi:10.4172/2161-0681.1000113

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response and failure for previously untreated patients on standard dose

of imatinib are shown in (Table 3). Data from several recent studies
have shown that time to response is important and the probability of
a favorable long-term outcome is best for patients who have the best
responses at 3 months after the start of therapy [74-76]. Using receiveroperated characteristic curves, [76] authors have confirmed that the
BCR-ABL1 transcript ratio of less than 9.84% at 3 months correlates
with best outcome, and compared with current NCCN and ELN
cytogenetic milestones (Table 2), molecular determination of transcript
levels at 6 and 12 months levels was the only independent of overall
survival (p < 0.001 for both time points). These data have not yet been
translated into guidelines for testing. It is clear that the success of these
predictive time points depends on standardization of methodology
for determining the BCR-ABL1 transcript and subsequent reporting
of the results. The first World Health Organization International
Genetic Reference Panel for quantitation of BCR-ABL mRNA was
approved by the Expert Committee on Biological Standardization
of the World Health Organization in November 2009 and is now
available commercially [77,78]. It consists of 4 dilutions of lyophillized
preparations of K562 cells with assigned, fixed BCR-ABL/control gene
values according to the IS. This development is a major milestone
towards standardized molecular monitoring of therapeutic response to
tyrosine kinase inhibitors in CML.

Mechanism of resistance to imatinib and molecular testing

for ABL kinase inhibitors
The majority of CML patients in chronic phase achieve an
optimum response with the standard recommended dose of imatinib.
A minority (20%), patients develop primary resistance to imatinib,
defined by failure to achieve the different levels of response i.e.
hematological, cytogenetic, or molecular, at the expected time points
[19]. The resistance is acquired in the majority of patients, with loss
of a previously obtained hematological or cytogenetic remission, or
loss of MMR fifty- ninety percent of the acquired resistance in CML
is due to BCR-ABL-kinase domain mutations. According to a recent
publication from 2011, nearly 100 different ABL kinase mutations
have been identified [79]. The mutations involve the catalytic domain,
the highly conserved p-loop, or the activation loop, with two different
mechanisms of causing resistance [58,80,81]: direct interference in
binding of the drug (eg, T315I, F317L, and F359C/V), or by a causing
a conformational change in the ABL kinase ATP-binding site that
prevents access to the drug (eg, G250E, Q252H, Y253H, E255K/V)
[80]. The mutations have different level of sensitivity or resistance to
the different TK inhibitors based on the in vitro IC50 determinations.
Mutations resistant to imatinib may be sensitive to second-generation
Test

TK inhibitors, underscoring the importance of mutation testing.

The T315I mutation present in 4-15% of imatinib-resistant patients
however, is resistant to imatinib as well as the second-generation TK
inhibitors dasatinib and nilotinib [58]. Other mechanisms of acquired
resistance include increased dosage of the BCR-ABL1 transcript
by amplification or additional copies of the Ph1 chromosome, less
commonly by involvement of different pathways such as the SRK kinase
pathway that are not inhibited by imatinib, or drug efflux mechanisms
that decrease the effective concentration of the drug within the cells
[82,83].

The Pertinent Questions Regarding Mutation Testing

for BCR-ABL1 Are Discussed Below
When should the testing be done and who should be tested?
Treatment naive patients: There are a few reports of ABL1 kinase
mutations present in newly diagnosed treatment nave patients. Most
of them are in patients who present in accelerated phase or blast crisis
at diagnosis [84-87]; they are rare in chronic phase treatment nave
patients [88,89]. Given the rare occurrence, and lack of any clinical
evidence of an adverse outcome from mutations in chronic phase
patients, routine testing of all chronic-phase patients for mutations
is not recommended at this time. The present recommendation to
perform BCR-ABL (kinase domain) KD mutations in treatment nave
patients is restricted to those who present with advanced disease in
accelerated phase or blast crisis [87].
In patients on imatinib ss first line therapy: Per the NCCN practice
guidelines [18] and ELN [19], testing for BCR-ABL KD mutations
in patients on imatinib as first line therapy is to be performed in the
following circumstances: 1) Failure to respond or suboptimal response
at the defined time points i.e. complete hematological remission at 3
months, minor cytogenetic remission at 6 months, and CCyR at 12
months of initiation of imatinib therapy 1 [8,19]. Twenty-nine percent
of chronic phase non-responders and 16% of patients with suboptimal
response harbor BCR-ABL KD mutations [90-92]. 2) Confirmed
loss of previously attained optimum response demonstrated by loss
of MMR or disease progression [86]. Fluctuations in levels of BCRABL transcript may occur when the levels are very low and are not
considered clinically significant. A trend of increasing BCR-ABL
transcript level by analysis of sequential samples, or a two-fold (or
one log) increase in BCR-ABL transcript level is more consistent with
definitive loss of MMR and predictive of relapse [93].
Mutation analysis in patients on second-line TKIs: KD mutations
arise under selective drug pressure. Thus, it is not unexpected to see

Time Point

A. Metaphase cytogenetics

1. At diagnosis and 6, 12, and 18 months until CCyR achieved. If CCyR achieved at an earlier time point,
then there is no need to perform metaphase cytogenetics in a stable patients.
B. FISH (peripheral blood sample is acceptable if bone
marrow specimen not available) using dual color, dual fusion
2. Rising level of BCR-ABL1 transcript (1 log increase) without achieving MMR
probes and standard technique
1. At diagnosis to establish baseline transcript level.
QRT-PCR for BCR-ABL transcript

2. Every 3 months thereafter until patient is responding to TK inhibitors, and every 3-6 months after
achieving CCyR
3. If level of BCR-ABL1 transcript is rising (1 log increase) after achieving MMR, then QRT-PCR should be
performed every 1-3 months

Kinase domain mutation testing

1. At time of suboptimal response or failure

2. Before switching to another TK inhibitor

Table 3: Recommendations of NCCN and European LeukemiaNet for monitoring of CML patients on Tyrosine kinase inhibitors.

J Clin Exp Pathol

ISSN: 2161-0681 JCEP, an open access journal

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Citation: Singh Z, Medlin S, Usmani SZ (2012) Molecular Monitoring and Treatment of Chronic Myeloid Leukemia (CML). J Clin Exp Pathol 2:113.
doi:10.4172/2161-0681.1000113

Page 6 of 9

emergence of additional resistant mutations in patients who are put

on second-generation TK inhibitors following resistance to imatinib
[94]. Mutations resistant to dasatinib (V299L, T315A, F317L/V/I/C)
or to nilotinib (E255K/V, Y253H, F359V/C/I) have been described in
patients developing secondary response failure with second-generation
TK inhibitors [95,96]. The data on resistant mutations to the second
generation TKIs is limited; currently KD mutation testing is done in
event of failure to respond, or suboptimal response, similar to that for
imatinib. Preliminary data show a better projected one-year survival
in patients receiving dasatinib as second or subsequent-line therapy if
they attain MCyR at 12 months, and suggest alternative therapies if
there is no cytogenetic response in 3 to 6 months [97,98].

Methods for Mutation Analysis

Many different approaches can be applied for mutation detection
with sensitivities varying from 20% (one mutant allele in a background
of 5 normal alleles) by Sanger sequencing to 0.01%-0.1% by allelespecific PCR(AS-PCR) [71-79,99-101]. Despite the low sensitivity of
Sanger sequencing, it is the most commonly used method because of
its availability in most molecular laboratories, and its ability to detect
a large number of mutations in the activation-loop, the P-loop, as
well as the catalytic loop [71-79,99,100]. Bidirectional sequencing is
recommended to avoid false positive calls [102]. A two-step process
of initial screening by DHPLC (Denaturing High Performance Lliquid
Chromatography) increases the sensitivity of subsequent sequencing
[99-101]. The threshold of detection at 20% is acceptable since the
clinical significance of detecting very low-level mutations is unclear
[101]. According to a recent study, sensitive detection methods such
as mass-spectrometry can predict a sub group of poor-responders to
dasatinib/nilotinib at the time of switchover, by detecting multiple
low-level mutations that are not detected by routine direct sequencing
method [103]. The clinical validity of these results is yet to be seen in
large multicenter studies. The sensitivity of detection will be greatly
increased when the high-throughput deep sequencing technology is
established in the clinical laboratory.

Reporting of Results and Interpretation

for 2 years after discontinuing imatinib [103,104]. Longer-term data

are not yet available. Early molecular relapse in 60% of these patients
with stable CMR, relapses occurring after nearly a decade in patients
who underwent allogenic stem cell transplant [105] clearly indicate
persistence of leukemic cells at a very low level. Indeed, the unique
genomic BCR-ABL1 fusion of the original leukemic clone could be
detected by patient-specific, highly sensitive, DNA quantitative PCR
in patients who were transcript negative and in functional remission
[106,107]. The factors determining sustained remission, or relapse in
a patient after stopping TK inhibitor therapy are presently unclear.
It is believed that low level persistence of leukemic stem cells that are
independent of BCR-ABL1 for their survival, and hence immune to the
TK inhibitors, are responsible for subsequent relapses [108], implying
that cure in CML is dependent on eradication of this resistant leukemic
stem cell clone. Three hypothetical models of operational cure have been
proposed that include depletion of the stem cell clone by prolonged
imatinib therapy in combination with therapies that target pathways
other than those mediated by BCR-ABL1, exhaustion of the stem cell
from asymmetric division by depleting the mutable mature leukemic
clone that replenishes the self renewing cells, and third by boosting the
host T- cell dependent cancer surveillance by immunomodulation to
maintain the initially established remission [103].
In summary, the current management of CML patients is able to
provide sustained CCyR and MMR in a significant number of patients.
The goals are to maintain the remission status in these patients,
recognize causes of relapse in the remaining subset of patients, and
eventually gear towards a cure for this disease. An approach to achieve
PCyR or reducing the BCR-ABL transcript to 10% within 3 months
by newer second-generation TK inhibitors, assiduous monitoring for
early detection of resistance by standardized sensitive methodology,
judicious use of alternate TK inhibitors and HSCT to counter resistant
mutations, and ensuring compliance by management of adverse effects
is required to meet these goals.
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Citation: Singh Z, Medlin S, Usmani SZ (2012) Molecular Monitoring and Treatment of Chronic Myeloid Leukemia (CML). J Clin Exp Pathol 2:113.
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