Detection of BCR-ABL kinase domain mutations in patients with chronic myeloid leukemia on imatinib

Abstract

BCR-ABL tyrosine kinase domain mutations are the most important factor contributing to imatinib-resistance in patients with chronic myeloid leukemia. We used a semi-nested reverse transcriptase-polymerase chain reaction followed by bidirectional sequencing to detect mutations in a cohort of 110 chronic myeloid leukemia patients. In total, 34 mutations in 19 distinct codons were identified in 32 patients, of which D276N and E279A were novel. The most commonly mutated region was drug-binding site (29%) followed by P-loop region (26%) and most patients bearing them were in accelerated phase and blastic phase. This report expands the spectrum of BCR-ABL mutations and stresses the use of mutation testing in imatinib-resistant patients for continuation of treatment procedure.

Keywords:

• Chronic myeloid leukemia
• BCR-ABL
• TKD mutation
• Imatinib resistance

Introduction

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder that originates from the transformation of a multipotent hematopoietic progenitor. CML is characterized cytogenetically by t(9; 22) (q34;q11) that yields the Bcr-Abl fusion oncoprotein with constitutive tyrosine kinase activity.¹ Eventually, the CML cells undergo more changes, and the disease progresses to an accelerated phase (AP) and then to blast crisis.

Given the central role of the Bcr-Abl oncoprotein in CML pathogenesis, tyrosine kinase inhibitors (TKIs) have been introduced in the last decade. Imatinib mesylate, a TKI, has become as the standard in the treatment of CML patients.² Imatinib binds to the inactive conformation of ABL protein tyrosine kinase, competitively blocks ATP-binding site and prevents its conformational switch to the active form, resulting in limitation of downstream signals and selectively induces cell growth inhibition and apoptosis of malignant cells harboring the oncoprotein.³ Imatinib causes more effective cytogenetic remission compared to previous treatments.⁴ At the end of a 7-year follow-up period, the overall survival rate and progression-free survival in patients with complete cytogenetic response (CCyR) were over 85 and 90%, respectively.⁵

Despite the success of imatinib therapy in improving survival in CML, treatment failure remains a significant challenge and some patients ultimately develop resistance to imatinib treatment and undergo clinical relapse.⁶ Point mutations within the BCR-ABL kinase domain and protein overexpression by gene amplification are the most common mechanism of imatinib resistance.⁷ To date, more than 90 different amino acid substitutions in ABL tyrosine kinase domain have been identified, which result in variable degrees of resistance to imatinib.⁸ Many of these mutations are associated with an increased risk of relapse and in particular, mutations in ATP-binding loop are associated with poor prognosis of CML.⁹

European Leukemia Net (ELN) Consortium has provided guidelines for the diagnosis and management of CML patients. They have presented definitions of optimal response, suboptimal response, and treatment failure linked to TKI-based treatments according to time-dependent hematological and cytogenetic responses. Patients failing to achieve the specified points within the designated period are defined as primary resistance and those losing the former responses as secondary resistance.^2,10 Hence, early identification of BCR-ABL mutations can help identify patients who are at an increased risk for developing resistance to imatinib. As a result, therapeutic interventions including dose escalation, combination treatments, or prescription of second-generation TKIs may be needed.¹¹

In this study, we used bidirectional DNA sequencing methods to identify the tyrosine kinase domain mutations in a large series of Iranian patients with CML. In addition, we evaluated the clinical findings in the patients with mutation during the imatinib treatment.

Materials and methods

Patients

Over 400 CML patients are currently under treatment with imatinib in the Hematology, Oncology, and Stem Cell Transplantation Research Center in Shariati Hospital. The criteria for monitoring patients response to therapy was based on ELN guideline.^2,10 As of 2009, 110 patients have been enrolled into the study who were resistant to treatment or had suboptimal responses. Of whom, 25 patients (23%) were pretreated with hydroxyurea and/or interferon-alpha before imatinib, and the remaining 85 patients (77%) received imatinib as first-line therapy. These patients were in different phases of the disease and received treatment with high-dose imatinib (600–800 mg/day). Patients received imatinib for at least 6 months. Based on patient's conditions, one to four samples were taken at intervals of at least 3 months. We performed mutation analysis of BCR-ABL gene on 195 samples taken from these patients.

Mutation analysis

Total RNA was extracted from peripheral blood using QIAzol Lysis Reagent (Qiagen, Hilden, Germany). First strand cDNA was synthesized from 2 µg RNA using random hexamers and MMLV reverse transcription reagents (Fermentas, Life Sciences, Vilnius, Lithuania) in total volume of 20 µl. The first step of PCR amplified BCR-ABL using the forward primer F1 and the reverse primer R1. Then, the semi-nested PCR has been applied to the amplification of the ABL kinase domain sequence using the forward primer F2 and the reverse primer R1. All primers were used based on a report by Branford et al.^12,13 An 863-bp PCR product, which codes for amino acids 220–506 of the ABL protein, was purified using QIAquick PCR purification kit (Qiagen) and was then sequenced using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Sequencing products were purified by the DyeEx 2.0 Spin kit (Qiagen). The samples were then dried, dissolved in 15 ml formamide (Applied Biosystems) and denatured. Bidirectional sequencing was carried out in an ABI Prism 3130 Genetic Analyzer (Applied Biosystems). The sequences were compared to the wild-type ABL sequence (GenBank accession number X16416). All mutations were confirmed by independent sequencing reactions in both the forward and reverse strands, with a sensitivity of 10–20% mutation-bearing cells in the analysed population.

Quantitative reverse transcriptase-polymerase chain reaction for BCR-ABL

Quantification of BCR-ABL fusion transcript and ABL control gene were performed using BCR-ABL FusionQuant® Kit (Ipsogen, Marseille, France) according to a standardized protocol.¹⁴ All standard and patient samples were run in duplicate. The copy number of BCR-ABL and ABL were calculated based on the standard curve. Finally the BCR-ABL copy numbers were expressed as percentage ratio of BCR-ABL/ABL.

The BCR-ABL values converted to the International scale (IS) equate approximately to cytogenetic response levels based on a report by Ross DM et al.¹⁵ The BCR-ABL values of more than 1–10% IS, the corresponding Philadelphia chromosome percentage was within the range of an MCR or partial cytogenetic response (PCyR) and BCR-ABL values of 1.0% or less IS, indicated a CCyR (Table 1).

Table 1. Characteristics of 110 CML patients with resistance or suboptimal response to imatinib

Parameter	Median (range) or no. (%)
Patients	n = 110
Age (years)	42(10–83)
Sex	M:67(61)
	F: 43(39)
Therapy before imatinib	25(23)
Disease phase	CP: 93(84)
	AP: 4(4)
	BP: 13(12)
Response to imatinib at time of mutation analysis according to BCR-ABL/ABL in percent	0.1–1 (CCyR): 22(20)
	1–10 (PCyR): 29(26)
	>10 (<PCyR): 59(54)
Duration of disease (months)	54(7–192)
qRT-PCR (percentage ratio of BCR-ABL/ABL)	27(0.1–258)

CP, chronic phase; AP, accelerated phase; BP, blastic phase; PCyR, partial cytogenetic response; CCyR, complete cytogenetic response.

Results

Frequency and distribution of mutations in relation to disease phase

The clinical characteristics of the 110 patients are summarized in Table 1.

ABL mutations were found in 32 (29%) out of 110 patients (Table 2). The frequencies of mutations in chronic phase (CP), AP, and blastic phase (BP) were 23 (21/93), 75 (3/4), and 62% (8/13), respectively. The frequencies of mutations based on the type of response during mutation analysis in patients with CCyR (0.1–1% of BCR-ABL copy number), PCyR (1–10% of BCR-ABL copy number), and less than PCyR (>10% of BCR-ABL copy number) were 9 (2/22), 10 (3/29), and 46% (27/46), respectively.

Table 2. Patient's details with detection of kinase domain mutations

Patient number	Sex	Age (years)	Phases	Response to imatinib at time of mutation analysis	Duration of disease (months)	Nucleotide exchange in BCR-ABL	Codon exchange in BCR-ABL	Mutation	Amino acid change	BCR-ABL/ABL ratio (%)
1	M	42	CP	<PCyR	36	A1094G	ATG to GTG	M244V	Met to Val	11.7
						A1722C	GAG to GCG	E453A	Glu to Ala
2	M	45	CP	<PCyR	72	A1094G	ATG to GTG	M244V	Met to Val	14.8
3	M	29	CP	PCyR	21	A1094G	ATG to GTG	M244V	Met to Val	6.16
4	F	47	AP	<PCyR	30	G1113A	GGG to GAG	G250E	Gly to Glu	23.5
5	M	35	CP	<PCyR	12	G1113A	GGG to GAG	G250E	Gly to Glu	19.3
6	M	74	CP	<PCyR	71	G1113A	GGG to GAG	G250E	Gly to Glu	60
7	M	55	CP	<PCyR	48	G1113A	GGG to GAG	G250E	Gly to Glu	21
8	M	51	BP	<PCyR	118	G1113A	GGG to GAG	G250E	Gly to Glu	19.1
9	M	65	AP	<PCyR	60	T1121C	TAC to CAC	Y253H	Tyr to His	60
10	M	50	CP	<PCyR	70	A1128T	GAG to GTG	E255V	Glu to Val	70.2
11	M	35	CP	PCyR	32	G1127A	GAG to AAG	E255K	Glu to Lys	1.1
12	M	64	BP	<PCyR	54	G1127A	GAG to AAG	E255K	Glu to Lys	46.9
13	F	48	CP	<PCyR	118	G1190A	GAC to AAC	D276N	Asp to Asn	18.90
14	M	43	BP	<PCyR	45	A1200C	GAG to GCG	E279A	Glu to Ala	258
15	F	50	BP	<PCyR	121	C1308T	ACT to ATT	T315I	Thr to Ile	86.6
16	M	54	AP	<PCyR	29	C1308T	ACT to ATT	T315I	Thr to Ile	21.8
17	F	62	AP	<PCyR	15	C1308T	ACT to ATT	T315I	Thr to Ile	67.60
18	M	12	BP	<PCyR	36	C1308T	ACT to ATT	T315I	Thr to Ile	45
19	M	35	CP	<PCyR	50	C1315G	TTC to TTG	F317L	Phe to Leu	42
20	F	27	CP	<PCyR	118	T1416C	ATG to ACG	M351T	Met to Thr	75.5
						G1525C	TTG to TTC	L387F	Leu to Phe
21	M	41	CP	<PCyR	44	T1416C	ATG to ACG	M351T	Met to Thr	50
22	M	42	CP	<PCyR	18	A1428G	GAG to GGG	E355G	Glu to Gly	52
23	F	30	BP	<PCyR	34	T1439G	TTC to GGC	F359V	Phe to Val	55
24	M	47	CP	CCyR	45	T1439G	TTC to GTC	F359V	Phe to Val	0.1
25	F	60	CP	<PCyR	7	T1440G	TTC to TGC	F359C	Phe to Cys	69
26	F	42	CP	<PCyR	60	T1440G	TTC to TGC	F359C	Phe to Cys	45.8
27	M	60	CP	<PCyR	24	T1440G	TTC to TGC	F359C	Phe to Cys	37.40
28	M	25	CP	<PCyR	32	T1523A	TTG to ATG	L387M	Leu to Met	52.8
29	F	32	CP	CCyR	36	A1551G	CAT to CGT	H396R	His to Arg	0.28
30	M	45	CP	<PCyR	84	C1677G	TCC to TGC	S438C	Ser to Cys	27.3
31	F	49	AP	<PCyR	100	A1740G	GAG to GGG	E459G	Glu to Gly	34.2
32	F	44	CP	CCyR	36	A1740G	GAG to GGG	E459G	Glu to Gly	0.2

Nucleotide positions in coding DNA, according to GenBank accession number X16416.

CP, chronic phase; AP, accelerated phase; BP, blastic phase; PCyR, less than partial cytogenetic response; CCyR, complete cytogenetic response.

The type and frequency of mutations

A total of 19 types of mutations were found including 17 previously reported and two newly identified ones. A total of 34 mutations of 19 types were detected in 16 amino acid positions. The most frequent mutations in patients were G250E (n = 5) followed by T315I mutation (n = 4), M244V and F359C mutations (n = 3 each), and E255K, M351T, F359V, and E459G mutations (n = 2 each). The remaining mutations included Y253H, E255V, D276N, E279A, F317L, E355G, L387M, L387F, H396R, S438C, and E453A, each occurred in only one patient. D276N and E279A mutations (GenBank accession numbers JX565024 and JX565025, respectively) were detected for the first time and were not reported in previous studies. In Table 3, distribution of kinase domain mutations are shown.

Table 3. Distribution of kinase domain mutations

Site of mutation	Mutation	Frequency
N-terminal	M244V	3
P-loop	G250E	5
	Y253H	1
	E255K	2
	E255V	1
C-helix	D276N	1
	E279A	1
ATP-binding region (drug contact sites)	T315I	4
	F317L	1
SH2-contact	M351T	2
	E355G	1
Substrate-binding region (drug contact site)	F359V	2
	F359C	3
A-loop	L387M	1
	L387F	1
	H396R	1
C-terminal	S438C	1
	E453A	1
	E459G	2

Among these 32 patients, two patients had two mutations simultaneously. One of them had M244V and E453A mutations and the other one had M351T and L387F mutations. However, M244V mutation from the first patient and L387F from the second one disappeared in their subsequent samples and the dominant mutations became E453A and M351T, respectively. The mutations can be divided into seven groups based on occurrence in BCR-ABL sites (Table 3). Twenty-nine percent of mutations (10/34) were in the drug-binding site, 26% (9/34) in the P-loop region, and 12% (4/34) in the C-terminal region. Nine percent of mutations occurred in each of A-loop, SH2-contact, and N-terminal, and 6% in the C-helix region.

Additionally, two polymorphisms at codons 284 (TTG → CTG, Leu → Leu) and 438 (TCC → TCT, Ser → Ser) were detected.

Kinetic studies

Kinetic studies in individual patients at intervals of 3 months during treatment showed the appearance and the expansion of G250E and M244V mutated cells in parallel to the increase in BCR-ABL copy number during the follow-up period of two patients. In addition, in five patients who had a mix of mutant and wild-type BCR-ABL, an increase in mutated clones (T315I, H396R, G250E, M351T and L387M) from 10–70% to 50–100% was observed during the follow-up.

Mutational status and clinical response to imatinib

Of two patients who had novel mutations, one with E279A mutation was in BP and the second one with D276N mutation who had lost former responses had less than PCyR (BCR-ABL copy number were 18.90). All nine patients with mutations in P-loop region had lost their responses, of whom eight had less than PCyR and no hematologic response (four out of eight were in AP and BP) and one showed PCyR. Two out of four patients with T315I mutations in drug-binding site were in BP and two were in AP. The remaining patients (n = 6) with mutations in the same site had lost their responses and had less than PCyR and no hematologic response, except one with PCyR. Two patients had a hematologic response and were in CP, whereas three had no hematologic response (one was in BP).

All the three patients with M244V mutations showed PCyR and were resistant to treatment. They had lost or failed to achieve CCyR. Three patients with a mutation at SH2-contact site were in CP and also lost their responses and had less than PCyR. Among patients with mutation in C-terminal position, one with S438C mutation had previously lost CCyR and PCyR. The patient with the E453A mutation was in PCyR, and of two patients with the E459G mutation, one was in AP and the other patient had a CCyR. Both patients with a mutation in A-loop region were in CP, of whom the patient with L387M mutation who had lost his response had less than PCyR, another with H396R mutation had a CCyR.

Discussion

The most common mechanism of resistance to imatinib is the acquisition of mutations within the kinase domain. Mutations can affect the TKI and Bcr-Abl tyrosine kinase interaction directly as well as indirectly if their presence shifts the thermodynamic equilibrium from the inactive toward the active conformation of the enzyme.¹⁶ Availability of second-generation TKI has provided new therapeutic agents for imatinib-resistant patients, as these agents are effective against all imatinib-resistant kinase domain mutants except T315I.^17,18 Therefore, mutational analysis of ABL kinase domain in imatinib-resistant patients is of tremendous importance. This is the first comprehensive BCR-ABL gene mutation analysis in a large group of Iranian CML patients treated with imatinib.

Among 110 patients with imatinib resistance or suboptimal responses, 29% had mutations. Frequency of BCR-ABL gene mutations in imatinib-resistant patients has been reported up to 90%.¹⁹ In recent studies in several Asian countries including Korea,²⁰ China,²¹ and India,²² kinase domain mutations in CML patients have been reported to be 63, 58, and 33%, respectively. We found a lower incidence of mutations compared to those described by the authors set out above. These discrepancies may be due to differences in sensitivity of tests applied in different studies, including the use of more sensitive methods like allele-specific oligonucleotide-polymerase chain reaction or denaturing high-performance liquid chromatography, as well as the higher percentage of patients with more advanced disease (AP and BP). This study included a high proportion of patients in CP (85%) and nonadherence of patients to imatinib was common in patients with a suboptimal response. Ethnic origin of patients and differences in pharmacokinetic profiles should also be considered.²²

Seventy-eight percent (25/32) of patients with mutations losing former responses, indicating clinical compatibility with the presence of mutation. The most commonly mutated region was drug-binding site (29% of cases), followed by P-loop region (26%). Fifty-five percent of mutations were in these two positions, and most patients bearing them were in AP and BP, confirming these mutations are associated with disease progression and poor prognosis in CML patients.^{9, 23–25} Twelve different amino acid substitutions (85% of all the mutations) in this study have also been reported by others.^26,27

Despite the high frequency of the T315I mutation reported in other studies,^20,28 in our study the mutation was identified in only 12% (4/32). It might be due to the small number of patients in advanced stages of disease. Furthermore, two novel mutations (D276N and E279A) were identified for the first time among Iranian CML population. Both mutations were located in the C-helix region of kinase domain and patients with these mutations were resistant to imatinib, of whom one with E279A mutation was in BP, and the other with D276N lost his molecular and cytogenetic response. Like previous studies, the prevalence of mutation increased in AP and BP.^20,27

Two patients with F359V and H396R mutations were in CCyR at the time of mutation analysis, and the BCR-ABL copy numbers were 0.1 and 0.28, respectively. Further follow-up studies are needed for these patients and disease progression may be indicated by rising amounts of BCR-ABL transcripts which would point out the need for further therapy. Continued molecular monitoring during imatinib therapy is an efficient method to follow the treatment response and can predict early stages in the development of imatinib resistance in patients with CML. Patients with treatment failure or suboptimal response to imatinib often demonstrate a steady trend of BCR-ABL level.²⁹ According to the report of Marin et al.,³⁰ the patients who are in CCyR stage and cannot achieve MMolR stage within 12–18 months are more likely to lose CCyR compared with patients who have obtained MMolR. In another study, it was found that patients with constant levels of BCR-ABL above 0.1% up to 18 months are more likely to have mutation followed by disease progression.³¹ Hence, monitoring BCR-ABL transcript levels by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) can specify the patients with indication to evaluate mutation in case of significant increase in the number of copies in successive assays, or patients who have not achieved the desirable responses, or who have lost their responses. Based on expert advice, mutation identification should be performed as far as possible in the early stages to consider other therapeutic strategies.¹⁴ Kinetic studies of mutation revealed that the mutated clone which was present at low levels at the time of initial evaluation would be replaced by mutant clone over time. Therefore, timely therapeutic interventions are helpful for these patients.

In conclusion, the results of this study showed that mutations in imatinib-resistant patients occurred with a higher frequency in patients with advanced phases. The most frequent and clinically important mutations are in drug-binding site and P-loop region, and patients with such mutations have been shown to have an inferior outcome. Therefore, it seems that the patient's response to treatment should be regularly assessed using qRT-PCR and mutation test should be performed in all individuals who have significant increases in the BCR-ABL level. This may support the decision for therapeutic interventions before clinical symptoms arise.

Acknowledgments

This study was supported and performed in collaboration with the Department of Hematology, School of Allied Medical Sciences, Tehran University of Medical Sciences and the Hematology, Oncology, and Stem Cell Transplantation Research Center in Shariati Hospital.