The Role of Mutation Testing in Patients with Chronic Myeloid Leukemia in Chronic Phase after Imatinib Failure and Their Outcomes after Treatment Modification: Single-institutional Experience Over 13 Years.

INTRODUCTION: BCR-ABL1 kinase domain mutations represent the most frequent mechanism of resistance to tyrosine kinase inhibitor (TKI) therapy, being detected in 40%-50% of imatinib-resistant patients with chronic myeloid leukemia in chronic phase (CML-CP). Over 100 BCR-ABL1 single-point mutations have been reported in patients with imatinib-resistant CML. There were few studies reported from India on BCR-ABL kinase mutations in imatinib failure patients. We present our data on imatinib resistance mutation analysis (IRMA) and use of imatinib dose hike and 2nd-generation TKI at our institute.
MATERIALS AND METHODS: All patients with a diagnosis of CML in a university hospital from June 2003 to July 2016 and who were tested for IRMA in view of imatinib failure, those in CP, and age <18 years were included in the study.
RESULTS: A total of 2110 cases of CML reviewed and 269 cases of CML with imatinib failure were analyzed. The male to female ratio was 1.7:1. The median age at presentation was 36 years (range: 18-66 years). Among these, 26% were primary failures and 74% were secondary failures. The treatment was modified either as imatinib dose hike or nilotinib/dasatinib. Molecular response at 12 months was achieved in 25.7% in imatinib dose hike, 46.6% in nilotinib, and 53.8% in dasatinib arms. The 4-year overall survival in mutation detected group was 37.5% and in nonmutated group was 87.7%.
CONCLUSION: Imatinib-resistant mutations were more common in the cases with secondary failure though not statistically significant. T315I mutation was the common mutation found in the study. Imatinib dose hike to the failure cases resulted in optimal hematological response rates.

Introduction

BCR-ABL protein, which results from a reciprocal translocation involving chromosomes 9 and 22, has a central role in the pathogenesis of chronic myeloid leukemia (CML). The BCR-ABL protein functions as a constitutively activated tyrosine kinase and the drugs are developed to specifically inhibit the BCR-ABL tyrosine kinase.[1]

BCR-ABL1 kinase domain mutations are the most frequent mechanism of resistance to tyrosine kinase inhibitor (TKI) therapy, which is detected in 40%–50% of imatinib-resistant patients with CML in chronic phase (CML-CP[23]). More than 100 BCR-ABL1 single-point mutations have been detected in patients with imatinib-resistant CML, which led to various levels of TKI resistance.[4] A threonine to isoleucine substitution at the 315 residue (T315I), which forms a key H-bond interaction with TKIs, results in clinical insensitivity to imatinib, nilotinib, and dasatinib.[25678910] Other mutations can be inhibited to some extent by 2nd-generation TKIs (SG-TKIs).

The different mutations may therefore require different strategies to overcome resistance such as dose escalation for those that confer moderate resistance or shifting to SG-TKIs or even transplantation for more resistant mutations.[11]

Aims and objectives

The aim of this study was to analyze BCR-ABL tyrosine kinase domain mutations in CML patients with imatinib failure and study the outcomes with imatinib dose escalation or SG-TKIs following imatinib resistance mutation analysis (IRMA).

Materials and Methods

All patients with a diagnosis of CML in tertiary care university hospital in South India between 2003 and July 2016 and a minimum follow-up of 12 months were included in the study. European LeukemiaNet (ELN) recommendations were used for the diagnosis and monitoring the response to treatment.[12] Sokal, Euro, and EUTOS scoring systems were used for risk stratification.[131415] Primary failure was defined as failure to achieve a given response at a given time and secondary failure as loss of initial response to TKI at anytime.

All patients who were tested for IRMA in view of imatinib failure, those in CP, and age <18 years were included in the study and those in CML-AP and BP, age <18 years, and pregnant were excluded from the study. Direct sequencing of the BCR-ABL transcript by the Sanger method was used for IRMA testing. Overall survival (OS) was defined from the date of imatinib resistance to the date of death or last follow-up.

Fisher's exact test using Prism version 7.02, Graphpad software, LaJolla, California, USA was used to analyze the proportion of mutations depending on the type of failure. To evaluate the association between mutation status and survival, log-rank test was used. Survival probabilities were estimated by the Kaplan–Meier method and compared by the log-rank test.[16]

Results

A total of 2110 patients of CML reviewed and 269 cases of CML with imatinib failure were analyzed. Baseline characteristics of these patients (n = 269) were tabulated in Table 1.

Table 1

Baseline characteristics (n=269)

The baseline hematological parameters assessed for these patients were represented in Table 2.

Table 2

Baseline hematological parameters

Molecular studies for BCR-ABL revealed the major transcript (p210 = e13a2, e14a2) in 138 cases, and in 131 cases, the transcript was not specified. Transcript type found in the cases is shown in Table 3.

Table 3

Molecular studies for BCR-ABL

The patients were risk stratified using three prognostic score systems – Sokal, Hasford, and EUTOS. There were some patients for whom risk stratification could not be done because of paucity of data which was depicted in Table 4.

Table 4

Risk stratification

The median duration of imatinib treatment before developing treatment resistance was 24 months (range: 3–60 months).

Based on the therapeutic response to imatinib, patients were categorized into primary and secondary failure taking into consideration the ELN guidelines. Both primary and secondary failures are subcategorized into hematological, cytogenetic, and molecular resistance. Categorization of imatinib failure is shown in Table 5. Of the 269 cases tested for imatinib resistance, mutations were detected in 88 (32.7%) cases and 181 (67.3%) cases were negative for mutations. The distribution of mutations in primary and secondary failure was depicted in Table 6. Fisher's exact test did not result in statistical significance with P = 0.0536.

Table 5

Categorization of imatinib failure (n=269)

Table 6

Mutation analysis and its distribution in imatinib failure

Mutations in kinase domain were detected in 88 (32.7%) cases. Mutations in ATP binding region (P-loop), other P-loop, substrate binding region, SH2 contact, A-loop, C-helix, and others were found in 28 (31.8%), 25 (28.4%), 14 (15.9%), 6 (6.8%), 3 (3.4%), 1 (1.1%), and 11 (12.5%) cases, respectively. In the ATP binding region, T315I mutation was seen in 28 cases (31.8%). Mutations in kinase domain based on the site and amino acid distribution were shown in Figures 1 and 2.

Figure 1

Mutations in kinase domain based on the site

Figure 2

Mutations in kinase domain based on amino acid substitution

Of the 269 cases evaluated for imatinib resistance, the treatment was modified either as imatinib dose escalation or second-line TKI for 205 cases. The remaining 64 cases were given supportive care. Imatinib dose hike (600/800 mg), nilotinib, and dasatinib were given to 162, 30, and 13 cases, respectively. Response evaluation was done for these cases at 3 months and 12 months. Of 205 cases, 105 were in hematological failure at the time of treatment modification and the remaining 100 cases were in complete hematological response (CHR). These 105 cases were assessed for CHR at 3 months and were achieved in 53 of the 80 cases (66.2%) in imatinib dose hike, 13 of the 17 cases (76.4%) in nilotinib, and 6 of the 8 cases (75%) in dasatinib arms.

Molecular response was assessed in 187 of 205 cases. For 18 cases, molecular response could not be assessed at the end of 1 year of treatment modification. Molecular response (<1% of BCR-ABL) at 12 months was achieved in 37 of the 144 cases (25.7%) in imatinib dose hike, 14 of the 30 cases (46.6%) in nilotinib, and 7 of the 13 cases (53.8%) in dasatinib arms. Response to treatment modification is shown in Table 7.

Table 7

Response to treatment modification

Median OS (since imatinib resistance) for T315I mutation group was 20.5 months (range: 1.4–42.8 months), for P-loop mutation group was 31 months (range: 1.4–57 months), and it was not reached for the group with non-P-loop mutation (3.9 months-NR) and nonmutated groups (5.3 months-NR) with median follow-up of 48 months from the date of imatinib resistance (range: 1.4–96 months).

Four-year OS in mutation detected group was 37.5% and in nonmutated group was 87.7%. OS from the date of imatinib resistance was shown in Figures 3 and 4.

Figure 3

Kaplan–Meier estimates of overall survival since imatinib resistance between various mutant groups

Figure 4

Kaplan–Meier estimates of overall survival since imatinib resistance between mutated and nonmutated group

The hematological and nonhematological adverse effects were shown in Tables 8 and 9.

Table 8

Hematological adverse effects

Table 9

Nonhematological adverse effects

Discussion

Our study comprised 269 CML patients with imatinib treatment failure. The median age was 36 years (18-66 years) and the sex ratio was 1.7:1, whereas in other studies by Quintas Cardama Alfonso et al.,[17] Jabbour et al.,[18] Cortes et al.,[19] Rajappa et al.,[20] and Mishra et al.,[21] the median age was 52 years (18–79), 57 years (25–82), 51 years (17–96), 36 years (18–65), and 35 years, respectively. The sex ratio was 1.08:1, 1.6:1, 2.7:1, and 1.6:1 in the studies done by Quintas Cardama Alfonso et al.,[17] Jabbour et al.,[18] Rajappa et al.,[20] and Mishra et al.,[21] respectively.

We stratified patients into different risk groups using three scoring systems; nevertheless, Sokal score was used in most of the studies[18212223] and in comparison to Western studies, this study had more number of intermediate and high-risk Sokal score.

In this study, the ratio of primary and secondary failure was 1:2.8 and the ratio of cytogenetic/molecular and hematologic failure was 1.57:1. The results of our study were similar to Jabbour et al.[18] which included all three phases of disease. Compared to our study, Jabbour et al.[23] showed higher cytogenetic failures. Rajappa et al.,[20] Cortes et al.,[19] and Kantarjian et al.[25] showed more hematological failures compared to our study.

However, Soverini et al.[24] showed similar ratio of hematological and cytogenetic failure when compared to our study, yet there is difference in the ratio of primary and secondary failure.

Although the frequency of mutations was higher in secondary failure cases, it was not statistically significant (P = 0.38) in this study. Al-Ali et al.[26] and Soverini et al.[24] demonstrated significantly higher frequency of mutations in secondary failure cases. In the study by Jabbour et al.,[18] it was observed that mutations associated with secondary imatinib resistance occur more frequently in later stages of the disease and are associated with older age, prior interferon therapy, initiation of imatinib therapy in AP or BC, development of clonal evolution, high-risk Sokal score at diagnosis, and failure to achieve complete cytogenetic response (CCyR) by 12 months.

T315I was the most commonly seen mutation in our study (31.8%), Rajappa et al.[20] (31%) which was our previous institutional study and Suresh-Babu et al.[27] (16.6%) studies. IRMA was directed to T315I presence or absence for many years at our institute that could have been the reason for high proportion of this mutation in our study.

E255 V/K mutation was the most common in Soverini et al.[24] study (16%) and it was seen in 5.7% of cases in our study. Quintas Cardama Alfonso et al.,[17] Jabbour et al.,[18] and Nicolini et al.[22] showed the most frequent mutation at G250 residue and this was the 3rd most common in our study and Rajappa et al.[20] and Srivastava and Dutt[31] studies.

Treatment modification after imatinib resistance mutation analysis result

In our study, CHR at 3 months to imatinib dose escalation was 66.2%. In various studies on imatinib dose escalation, the CHR at 3 months was between 45% and 86%.[2325] In our study, CCyR/< 1% of BCR-ABL at 12 months after imatinib dose escalation was 25.7% of cases. In other studies, it is between 17.5% and 59.5%.[202325]

For 2nd-line TKI, CHR at 3 months was attained in 75% of cases in our study. In other studies,[1719282930] CHR at 3 months ranged from 82% to 92%. In this study, CCyR/< 1% of BCR-ABL at 12 months to 2nd-line TKI was 46.6% to nilotinib and 53.8% to dasatinib. In other studies, it ranged from 28.4% to 69%.

Survival analysis

In this study, the median OS and range for T315I mutation and P-loop mutation were 20.5 months (range: 1.4–42.8) and 31 months (range: 1.4–57), respectively, and it was not reached for the group with non-P-loop mutation (3.9 months-NR) and nonmutated groups (5.3 months-NR). The 4-year OS in mutation-detected group was 37.5% and in nonmutated group was 87.7%.

Our study showed inferior survival for T315I and P-loop mutation patients compared to those with non-P-loop mutations and with no mutations. Similar results were seen in the study done by Nicolini et al.[22] There was no difference in survival between patients with T315I, other mutations, or no mutations in the study done by Jabbour et al.[30]

In this study, the 4-year OS was inferior in the group with mutations compared to the group with no mutations. Results were similar in the studies done by Quintas Cardama Alfonso et al.[17] and Nicolini et al.[22]

Limitations of the study

The drawbacks of this study were that it is retrospective study. Laboratory data were not available for all patients to stratify the risk. There was underreporting of both hematological and nonhematological adverse events because of inadequate documentation in case records. Second-generation TKI and hematopoietic stem-cell transplantation could not be provided for the eligible cases due to financial constraints.

Conclusion

Imatinib-resistant mutations were more common in the cases with secondary failure though it was not statistically significant. T315I mutation which is resistant to all second-generation TKIs was the common mutation found in the study.

Imatinib dose hike to the failure cases resulted in optimal hematological response rates which is on par with second-generation TKIs.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.