BCR-ABL Tyrosine Kinase Domain Mutations Research Articles

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

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.

Study of different mutations in chronic myeloid leukemia in India and their co-relation with drug resistance.

7083 Background: Emergence of ABL point mutations is the most frequent cause for imatinib resistance in CML. Aim of our study is to investigate two potential resistance mechanisms i.e.,mutations of BCR-ABL tyrosine kinase domain (TKD) and Additional Chromosomal Abnormalities during TKI treatment in CML. Methods: Karyotyping and BCR-ABL TKD mutation screening are performed in 100 imatinib resistant CML patients who were on imatinib at the time of loss of hematologic response, cytogenetic or molecular response. Imatinib–Resistance Mutation Analysis (Qualitative) were detected by Nested RTPCR and Sanger’s Sequencing. In 100 cases, 34 received escalated imatinib, 34 nilotinib and another 32 dasatinib. Results: In 100 BCR-ABL positive imatinib, nilotinib and dasatinib resistant cases, 11 different BCR-ABL TKD mutations were detected. Analysis revealed no mutations-43 cases, M351T-12 cases, G250E-10 cases, F317L-8 cases, M244V-5 cases, E255K-4 cases, V379I-4 cases, F359V-3 cases, H396R-3 cases, Y253F-3 cases, E355G-3 cases, T315I-2 cases. 11 novel mutations (F317L, G250E, M244V, Y253F, E255K, M351T, F359V, H396R, V379I, E355G, T315I) conferring imatinib resistance, 10 nilotinib–resistant mutations (M244V, F359V, T315I, E355G, G250E) and 8 dasatinib-resistant mutations (H396R, F317L, H396R, T315I, M351T) were seen in our patient population. T315I was found more frequently in cases on dasatinib than on imatinib therapy. Conclusions: T315I which confers resistance to all TKIs was detected only in 2/100 patients who demonstrated loss of response in our population. As compared with other western studies, incidence of T315I mutation was very low in our study. In addition analysis of mutation patterns at baseline may help in stratifying patients for treatment. For cases with TKI resistance, mutation and ACA screening may play role in identifying patients with poorer prognosis. In our practice if nilotinib–resistant mutation was detected, dasatinib was preferred and for dasatinib-resistant mutation, nilotinib was preferred. We are planning for using bosutinib, panotinib and omacetaxine (SC route) in third line therapy in imatinib resistant different mutation positive chronic myeloid leukemia.

Characterization of ABL exon 7 deletion by molecular genetic and bioinformatic methods reveals no association with imatinib resistance in chronic myeloid leukemia

In chronic myeloid leukemia (CML), the best characterized imatinib resistance mechanisms are BCR-ABL tyrosine kinase domain mutations and clonal evolution, but recently alternative splicing of BCR-ABL was also proposed as a mechanism for imatinib resistance. Among recently reported BCR-ABL splice variants, exon 7 deletion (Δexon7) was characterized in this study. The frequency of Δexon7 was investigated in 30 healthy controls and in 76 CML patients at different time points of the disease course by four different molecular genetic methods (direct sequencing, fragment analysis, allele-specific and quantitative PCR). The functionality and viability of the variant protein was tested by bioinformatic prediction. The Δexon7 was abundantly detected with similar frequency in healthy controls, in imatinib naive and resistant CML patients on BCR-ABL and also on the nontranslocated ABL. The detection rate of Δexon7 (varying between 17 and 100%) was highly dependent on the expression levels of BCR-ABL or ABL and the sensitivity of detection method. According to secondary structure prediction by bioinformatic methods, the exon 7 deleted mRNA is a target for nonsense-mediated decay, and the translated protein is likely to be nonfunctional and unstable. Taken together all the above observations, we concluded that Δexon7 is a common splice variant not associating with imatinib resistance.

Additional Chromosome Abnormalities, BCR-ABL Tyrosine Kinase Domain Mutations and Clinical Outcome in Hungarian Tyrosine Kinase Inhibitor-Resistant Chronic Myelogenous Leukemia Patients

Background: Additional chromosome abnormalities (ACAs), mutations of the BCR-ABL tyrosine kinase domain (TKD) and BCR-ABL splice variants may cause resistance to first- and second-generation tyrosine kinase inhibitors (TKIs) in chronic myelogenous leukemia (CML) and Philadelphia-positive (Ph+) acute lymphoid leukemia (ALL). Methods: Karyotyping and BCR-ABL TKD mutation screening were performed in 71 imatinib-resistant CML patients and 6 Ph+ ALL patients. A total of 56 out of these 77 patients received second-generation TKI. Results: ACAs were present in 30 of 65 imatinib-resistant patients (46%). In 27 of 74 imatinib-resistant patients (36%), 15 different BCR-ABL TKD mutations were detected. Mutations were found in 25% of chronic-phase patients (12/47), 33% of accelerated-phase patients (5/15), 71% of blast crisis CML patients (5/7) and 100% of ALL patients. In nilotinib-resistant patients, Y253H, T315I and F359I/V mutations were detected; in dasatinib-resistant patients, L248M, E279K and T315I mutations were detected. T315I was found more frequently in patients on dasatinib than on imatinib therapy. The presence of ACAs predicted shorter survival during first- and second-generation TKI therapy, while TKD mutations only influenced survival during second-generation TKI therapy. Conclusion: For patients with TKI resistance, mutation and ACA screening may play a role in identifying patients with poorer prognosis.

Adult Philadelphia-Positive Acute Lymphoblastic Leukemia (Ph + ALL) Treated at Diagnosis with Imatinib Followed by 2nd Line TKI (Imatinib or Dasatinib) and Stem Cell Transplantation.

Abstract 4109Imatinib has become an integral part of front-line therapy for Ph+ ALL, with remission rate exceeding 90% irrespective of whether Imatinib is given alone or combined with chemotherapy. Treatment outcome with Imatinib-based regimens has improved survival compared with historic controls, but most patients who do not undergo allogeneic stem cell transplantation (SCT) eventually relapse. Second generation TKI, e.g. Nilotinib and Dasatinib, show activity against most of the BCR-ABL tyrosine kinase domain mutations involved in acquired Imatinib resistance, but clinical benefit is generally short lived. In the attempt to improve the prognosis of these patients, we started a new intensive protocol consisting of induction phase with Imatinib (600 mg po, daily) in combination with conventional chemotherapy; moreover, two intrathecal methotrexate will be administered at the day +8 and +22. As known, this approach will determine an high number of complete hematologycal (CHR) and cytogenetic remissions (CcyR). Soon after, Imatinib will be discontinued and a 2nd TKI (Nilotinib or Dasatinib) will be offered to the patients. The idea is to reduce/eliminate as soon as possible the mutations generating under Imatinib. The primary objective of the study will be to assess the activity of sequential TKI to induce molecular remission; secondary objectives will be DFS, relapse rate, OS. This protocol will be designed for patients >18 years. Recruitment started in June 2008. Minimal residual disease will be investigate by RQ-PCR at day +30, +60, +90, +120, +150 and so on. The protocol has been designed for 20 patients. Three evaluable patients entered until now this trial; the median age was 47 years (range, 39-64 years). All 3 patients achieved CHR and CCyR at a median of 26 days (range, 18-31 days) and 46 days (range, 39-59 days), respectively. Monitoring RQ-PCR, a marked clearance of leukemic cells was found at d+60; when CCyR was achieve, Imatinib was discontinued and 2nd TKI was started: 2 patients received Dasatinib 100 mg po daily and 1 patient Nilotinib 800 mg po twice daily. Between d+100 and d+120, RQ-PCR became negative in the peripheral blood of all patients. One patient with an HLA+ sibling donor is waiting to receive an allograft; the other 2 patients, who did not find an HLA+ donor, received an autograft with molecular negative PBPC. The conditioning regimen consisted of the BU-Cy regimen. Both patients are alive and in CMR at 3 and 10 months after autografting. More patients and more time will confirm the efficacy of this new approach. Disclosures:No relevant conflicts of interest to declare.

First-line treatment of Philadelphia chromosome-positive acute lymphoblastic leukaemia in adults

The tyrosine kinase inhibitor (TKI) imatinib has become an integral part of front-line therapy for Philadelphia chromosome-positive acute lymphoblastic leukaemia, with remission rates exceeding 90% irrespective of whether imatinib is given alone or combined with chemotherapy. Treatment outcome with imatinib-based regimens has improved compared with historic controls, but most patients who do not undergo allogeneic stem cell transplantation (SCT) eventually relapse. Second-generation TKI, e.g. dasatinib and nilotinib, show activity against most of the bcr-abl tyrosine kinase domain mutations involved in acquired imatinib resistance, but clinical benefit is generally short lived. Accordingly, SCT in first complete response is considered to be the best curative option. Strategies to improve outcome in patients ineligible for transplantation as well as after SCT include front-line treatment with more effective TKI to increase molecular response rates. Following SCT, the pre-emptive use of imatinib appears to reduce the relapse rate. Novel immunotherapeutic interventions and combinations of TKI are also being explored.

Bcr-Abl Tyrosine Kinase Domain Mutations Both Pre-Exist and Develop during Imatinib Treatment and Are Responsible for Resistance in Patients with Philadelphia-Chromosome Positive Acute Lymphoblastic Leukemia (Ph+ALL).

Background: Point mutations in the tyrosine kinase domain (TKD) of BCR-ABL are an important cause of resistance to imatinib (IM) in pts. with CML and Ph+ ALL. The significantly inferior response to IM in Ph+ALL pts. who failed prior chemotherapy compared to those with de novo Ph+ALL suggests that treatment with cytotoxic drugs may promote the development of TKD mutations. However, it is not known whether the frequency and pattern of TKD mutations at the time of treatment initiation with TK inhibitors are related to disease stage or prior anti-leukemic therapy. Moreover, the potential of combined treatment with IM and multi-agent chemotherapy to influence the development of mutational resistance, as compared to IM alone, has not been determined.Patients and methods: 51 pts. with newly diagnosed Ph+ALL (>55 yrs.) enrolled in a GMALL study of combined IM and chemotherapy, and 68 Ph+ALL pts. who had failed prior chemotherapy and received single-agent IM as salvage therapy were analysed for the occurrence of point mutations within the TKD. Bone marrow samples collected pre-treatment, during therapy and at relapse were examined by denaturing high-performance liquid chromatography (D-HPLC) and cDNA sequencing.Results: The frequency of TKD mutations pre-IM was 44% in newly diagnosed Ph+ALL and 53% (34/64) in pts. with advanced Ph+ALL. At relapse after combination therapy (n=19), the frequency of de novo ALL pts. harbouring a TKD mutation had increased to 89% (P-loop 47%, T315I 29%, A-loop 24%), 2 pts. (11%) showed wild-type BCR-ABL. The frequency of TKD mutations in pts. with advanced disease who relapsed after IM was 55% (P-loop 73%, T315I 23%, A-loop 4 %).In both patient groups, the D-HPLC pattern showed concordance between the mutation detected in pre-therapeutic specimens and the dominant mutation detected at relapse.The CR rate in de novo pts. receiving IM induction was 90 % irrespective of detectable mutations pre-study. Bcr-abl transcripts became undetectable during the course of therapy in 40% of pts. with and 37% of pts. without a mutation. Median remission duration in pts. with a T315I mutation (n=4) was 130 d (range: 53–319d), in contrast to 526 d (range: 504–549d) with activation loop and 411 d (range: 106–745d) with P-loop mutations. To date, 7 pts. with an initially detected mutation remain in CR after median FU of 12.8 mo (range 2.4–24.5 mo.).Conclusions: Bcr-abl TKD mutations are detectable prior to first imatinib exposure in approximately 50% of Ph+ALL patients. Clinical imatinib resistance is in most cases associated with the identical mutation detected pre-IM, which is not eradicated by the combination of chemotherapy and IM. Identification and elimination of TKD mutations during early stages of treatment is essential to improve treatment.

Chronic Myeloid Leukemia Stem Cells Possess Unique Properties That Predict Intrinsic and Acquired Resistance to Imatinib Mesylate.

Chronic myeloid leukemia (CML) is sustained by a clonally amplified, but still rare population of BCR-ABL+ hematopoietic stem cells (HSCs). Understanding their properties is assuming increasing importance in light of recent evidence that they are innately resistant to BCR-ABL−targeted therapies and that, over time, the CML clone acquires BCR-ABL tyrosine kinase domain mutations. The relative resistance of CML HSCs (lin−CD34+CD38− cells) to imatinib mesylate (IM) may be explained at least in part by their elevated expression of BCR-ABL and higher tyrosine kinase activity than is seen in the more prevalent lin−CD34+CD38+ leukemic cells. We have now obtained formal support for this concept from experiments with a BCR-ABL−transduced BaF3 cell line in which p210BCR-ABL expression can be regulated by doxycycline (dox)-mediated activation of an upstream tet-regulated repressor. Treatment of these cells for 48 hr with 0.1–5 μM IM in the absence of IL-3 and in the presence of 0.1–1 μg dox/ml confirmed the expected dox-control of intracellular phospho-CrkL levels and demonstrated a corresponding p210BCR-ABL kinase activity-dependent effect on IM sensitivity. To investigate the possibility that CML HSCs possess other unique properties that contribute to their relative resistance to many therapies, we compared the expression of 3 transporter genes (OCT1, ABCB1 and ABCG2) in CML cells at different stages of differentiation. Interestingly, we found that transcript levels for OCT1 (which regulates IM uptake) were very low in the most primitive (lin−CD34+CD38−) normal bone marrow cells and progressively increased (>100-fold) as these cells differentiate into mature (lin+CD34−) cells (n=4). Importantly, this difference in OCT1 expression was exaggerated in CML samples where OCT1 transcripts were even lower in the most primitive populations (below the level of detection in 2 of the 4 CML HSC isolates analyzed). Conversely, transcript levels for ABCB1 and ABCG2 (which regulate the efflux of many drugs) were highest in the normal lin−CD34+CD38− (HSC) population and lowest (>6-fold) in the most mature lin+CD34− normal cells, and again this difference was enhanced in the CML samples. The combination of a very low expression of OCT1 (low IM uptake) and highly elevated expression of ABCB1 and ABCB2 (high drug efflux) and BCR-ABL (elevated kinase activity) in CML HSCs identifies multiple mechanisms that would predict their broad insensitivity to IM and other therapeutics. In addition, we have found both by direct sequencing of cloned transcripts and allele-specific RT-PCR analyses, that a significant fraction of BCR-ABL transcripts in CML HSCs from chronic phase patients who have never received BCR-ABL-targeted therapy contain readily detectable kinase domain mutations (20–33%, n=3). A high incidence of mutations was also found at 2 additional sites in the BCR-ABL gene 5′ to the kinase domain and extending into the region encoded by BCR. After primitive CML cells had been cultured for 3 weeks (± 5 μM IM), we frequently found new BCR-ABL mutants in their clonal progeny. These in vivo and in vitro findings suggest that primary CML HSCs are also characterized by a high degree of genetic instability. We have thus identified multiple unique features of chronic phase CML HSCs that underscore the importance of evaluating these critical cells when considering new therapeutic approaches.

Leukemic Stem Cells of Chronic Phase CML Patients Possess Uniquely Elevated BCR-ABL Kinase Activity and Acquire Spontaneous BCR-ABL Kinase Domain Mutations at a High Frequency.

Growing evidence indicates that the therapeutic potential of imatinib mesylate (IM) for the treatment of CML may be limited initially by a relative innate resistance of the leukemic stem cells and eventually by an accumulation of cells with BCR-ABL tyrosine kinase domain mutations. We now show that the amount and tyrosine kinase activity of p210-BCR-ABL in the most primitive and relatively IM-unresponsive lin−CD34+CD38− CML cells is 3 to 10-fold higher than in the majority of the lin−CD34+CD38+ CML progenitors (n=3). These results confirm previous BCR-ABL transcript data and identify elevated p210-BCR-ABL expression to be a likely important factor in the characteristic IM-insensitivity of very primitive CML cells. To determine whether in vivo, CML stem cells also accumulate gene mutations affecting the BCR-ABL kinase domain, cDNAs were prepared from RNA extracts of purified lin−CD34+CD38− cells isolated from 3 chronic phase patients that had not received IM therapy. Bidirectional sequencing of individually cloned cDNAs from these samples revealed BCR-ABL kinase domain mutations in 2 of the 3 patients at frequencies of 10% (1/10), 20% (2*/10,*identical mutations). Incubation of these lin−CD34+CD38− cells in vitro for 2–3 wk ± a high concentration of IM (up to 10 μM, which was sufficient to reduce the tyrosine kinase activity in the input cells by 70±12% and in their 2 wk progeny by 10±5%) selected a subpopulation of more differentiated and completely IM-resistant cells. This was shown in Western blots by the inability of 10 μM IM to reduce either their p210-BCR-ABL tyrosine kinase activity or CrkL phosphorylation and in methylcellulose assays ±5 μM IM. As predicted, IM-selected cells showed a higher frequency of kinase domain mutations (13–20% vs 0–20% of cDNA clones analyzed from 3 wk cells cultured ±IM). Analysis of individual colonies produced from CFCs in the cultured cells showed all (21/21) colonies from IM-selected cells had mutations vs 50% (5/10) in those cultured without IM. The total frequency of mutant cDNAs detected was also increased in the IM-resistant cells (35–55% vs 10–25% mutant cDNAs in selected vs control cells). Interestingly, in most cases, both wild-type and mutant cDNAs were identified in the same colony, indicating de novo generation of mutations in vitro. Overall, >50 different mutations were identified. These included 10 point mutations previously associated with clinical IM resistance (including G250 and T315), another 13 point mutations previously identified in a comprehensive mutational screen, and >20 previously undescribed mutations. Several of the latter affect the critical region of the P loop, the c-helix and the activation loop and would be predicted to confer significant IM resistance. To investigate the possibility that the observed genomic instability of very primitive CML cells might be related to their elevated innate p210-BCR-ABL activity, BCR-ABL transcript levels in individual IM-selected, fully resistant and control (similarly treated but no IM exposure) colonies were compared. This showed that BCR-ABL transcripts were ~20-fold higher (P<0.05) in the resistant colonies (30 assessed from 3 patients). These findings suggest that the increased BCR-ABL expression and activity that uniquely characterizes the most primitive CML cells may contribute not only to their innate insensitivity to IM but also to a deregulation of genomic stability leading to the emergence of IM-resistant mutants and other subclones associated with disease progression.

A Randomized Phase II Study of Single-Agent Imatinib Versus Chemotherapy Therapy in Elderly Patients with Philadelphia-Positive Acute Lymphoblastic Leukemia (Ph+ALL) Including Analysis of Resistance Patterns.

Background: Elderly pts. with Ph+ALL have a very poor prognosis with a low CR rate, high induction mortality and short remission duration. Single-agent Imatinib (IM) has shown rapid anti-leukemic activity and a favorable toxicity in advanced Ph+ALL.Study design: To determine the value of single-agent IM as induction (ind.) therapy, we designed a prospective, randomized multicenter trial comparing a 4 week course of IM at 600 mg/d (IndIM) with age-adapted multi-agent chemotherapy (Indchemo) as front-line treatment of elderly Ph+ALL (>55 yrs.) patients. In addition, we investigated the tolerability and efficacy of an IM-based consolidation therapy in which responding pts. received successive chemotherapy cycles parallel to IM, given continuously for one year.Methods: As mutations of the BCR-ABL tyrosine kinase domain (KD) constitute the leading cause of resistance to IM, we evaluated 17/21 responding pts. at the time of hematologic relapse for the presence of these mutations using direct sequencing and denaturing HPLC.Results: To date, 52 pts. with newly diagnosed Ph+ALL (49) or lyBC (n=3) and a median age of 68 (58–79) yrs. were randomly assigned to receive IndIM (n=25) or Indchemo (n=27). Three pts. not entered in the study but treated according to the Indchemo regimen are included in the analysis. Twenty-four of 25 pts. assigned to IndIM received a CR (96%) and 1 pt. a PR (4%). In comparison, 14 of 23 evaluable pts. allocated to Indchemo achieved a CR (61%) and 2 pts. a PR (9%), while 7 pts. failed treatment (30%) (p=0.004). Two deaths occurred during Indchemo and none during IndIM. Severe infections during ind. developed in 13 of 23 evaluable pts. (57%) assigned to Indchemo and in 3 of 25 pts.(12%) receiving IndIM (p=0.002). After a median follow-up of 8.2 (0.6–35.4+) mo. 17 pts. are in ongoing CR and 21 pts. have relapsed (10 pts. during study, 7 pts. after end of study and 4 pts. after discontinuation). Ten pts. died in CR a med. of 4.1 (0.4–11.7) mo. after starting treatment and 1 pt. died after allogeneic SCT. Estimated DFS and OS at 18 mo. is 27±8% and 43±8%, respectively. Seventeen of 45 pts.(38%) with serially analysed MRD-levels achieved a molecular remission during treatment. Med. DFS was 15,7 mo. in molecular responders compared to 8.3 mo. in pts. remaining MRD positive (p=0.03). At the time of relapse 10 different BCR-ABL tyrosine KD mutations were detected in 13 of 17 evaluated pts. (76.5%). P-loop mutations were detected in 77% (10 of 13 pts.), activation loop and the T315I mutation each in 15% (2 of 13) of pts. In 3 pts. (23%) two different mutations were detected. In both pts. harboring the T315I mutation, it was already present at the time of diagnosis conferring an inferior remission duration of 1.8 and 5.2 mo.Conclusion: Single-agent IM is highly effective as ind. therapy in elderly pts. with Ph+ALL and is superior to standard chemotherapy induction with respect to tolerability, induction mortality and CR-rate. Monitoring of resistance patterns will help to identify candidates at risk of relapse and can guide treatment with 2nd generation tyrosine kinase inhibitors.

Imatinib (STI571) resistance in chronic myelogenous leukemia: molecular basis of the underlying mechanisms and potential strategies for treatment.

Following the paradigm set by STI571, protein tyrosine kinase inhibitors are emerging as a promising class of drugs, capable of modulating intracellular signaling and demonstrating therapeutic potential for the treatment of proliferative diseases. Although the majority of chronic phase CML patients treated with STI571 respond, some patients, especially those with more advanced disease, relapse. This article reviews the reasons for relapse and, in particular, analyses resistance resulting from Bcr-Abl tyrosine kinase domain mutations at the molecular level. Arguments are based upon the structure of the STI571-Abl complex, which is compared to the crystal structures of PD173955-Abl and PD180970-Abl, which bind to the kinase differently. Strategies to potentially circumvent or overcome resistance are discussed.