ABL Kinase Domain Mutations Research Articles

Tyrosine kinase inhibitor treatment outcome in a single center cohort of chronic myeloid leukemia patients. The role of the T315I ABL kinase domain mutation

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Principles and Current Topics Concerning Management of Tyrosine Kinase Inhibitor Therapy for Chronic Myelogenous Leukemia

Chronic myeloid leukemia (CML) is a chronic myeloproliferative disorder with leukemic cells featuring the Philadelphia (Ph1) chromosome comprising the reciprocal chromosomal translocation t(9;22)(q34;q22) and the resultant constitutive active Bcr-Abl tyrosine kinase (TK). The introduction of disease-specific molecular targeted agents, that is, TK inhibitors (TKIs) for Bcr-Abl TK, such as the first-in-class TKI imatinib mesylate (IM) or the secondgeneration TKIs (SGIs) nilotinib and dasatinib, has dramatically improved the long-term treatment outcome for CML in this century. In addition, several new SGIs, such as bosutinib or bafetinib, are under development, while ponatinib, which is active against CML refractory for all preceding TKIs, for example, CML with T315I Abl kinase domain mutation, is currently being evaluated in clinical trials. Because those TKIs have different sensitivity for Bcr- Abl mutation and profiles for adverse effects, a thorough understanding of the pharmacologic characteristics of TKIs is mandatory for their safe and effective clinical use. Recent studies have clearly shown the faster and deeper responses to SGIs, both nilotinib and dasatinib, compared with those to IM, indicating the need for a paradigm shift in approaches to TKI therapy for treatment-naive CML. In addition, evidence accumulated during the past decade has indicated optimal methodologies for monitoring the treatment effect of TKIs, selecting the appropriate therapeutic strategies, and predicting the outcome for treatment with TKIs for individual patients with CML. In this article, we review the principles and current knowledge and topics of the various uses of TKIs for CML. We also touch upon the reason why the faster and the deeper responses to TKIs is the prerequisite for their safer use and longer-lasting positive treatment outcome for CML.

Fingolimod (FTY720) Overcomes the Resistance to Tyrosine Kinase Inhibitors Via Dual Activation of BIM and BID in Chronic Myelogenous Leukemia

Abstract 3744 [Introduction]Tyrosine kinase inhibitors (TKIs) for BCR-ABL have dramatically improved the treatment outcome of chronic myelogenous leukemia (CML) during this decade, however, the resistance to TKIs due to various molecular mechanisms, such as BCR-ABL mutations, dysfunction of cell killing mechanisms like BIM deregulation, or the supports by leukemia microenvironment factors, has remained to be overcome. We have identified that BIM, a member of BH3-only proteins, is the essential pro-apoptotic mediator under the blockade of BCR-ABL signaling (Kuroda J, PNAS, 2006). One of the attractive therapeutic strategies for complete eradication of CML cells by simultaneously targeting both TKI-sensitive and TKI-resistant CML cells may be the use of agents that can activate BIM as well as other pro-apoptotic BH3-only protein which is not utilized in apoptosis by TKIs. In this study, we assessed the therapeutic potential of the protein phosphatase 2A (PP2A) activator Fingolimod (FTY720), an immunosuppressive agent currently used for multiple sclerosis, in CML. [Materials and methods]Human CML-derived cell lines, K562, MYL, KBM5, KCL22, and their various subclones, were utilized in this study. Murine Ba/F3 lymphoid cells were transfected with either wild type or mutated bcr-abl gene. FTY720 was purchased from Cayman Chemical (Ann Arbor, MI). Cell death analyses were performed using flow cytometry. Protein levels were determined by conventional Western blotting. [Result and discussion]Treatment with FTY720 impairs phosphorylation of BCR-ABL and its downstream targets, AKT, ERK and STAT5, and inhibited the growth of CML cell lines through the induction of apoptosis. The apoptosis induced by FTY720 was accompanied by the activation of both caspase-8 and caspase-9, while enforced expression of either BCL2 or dominant-negative protein of FADD (FADD.DN) by gene introduction partly protected K562 cells from apoptosis by FTY720, indicating the involvement of both cell extrinsic and intrinsic apoptosis pathways in FTY720-induced apoptosis. FTY720 treatment resulted in the activation of Bim as well as the processing of Bid into tBid, a BH3-only protein which plays crucial role in accelerating death-receptor-mediated extrinsic apoptosis pathway and is not utilized in apoptosis by TKIs. Importantly, gene knockdown of either BIM or BID partly protected K562 cells from apoptosis by FTY720, but the degrees of cell protection by those were not as much as the degree of cell protection by overexpression of either BCL2 or FADD.DN. Moreover, double knockdown of BIM and BID did not further protect K562 cells from apoptosis by FTY720, when compared with single knockdown of BIM. These results indicate that BIM and BID compensate each other for promoting apoptosis by FTY720 in CML cells, especially when either of them is functionally disturbed. Through these mechanisms, FTY720 overcomes TKI resistance by various mechanisms, such as ABL kinase domain (KD) mutations in BCR-ABL-transfected Ba/F3 cells (T315I, G250E, E255K or H396P) and in KBM5 cells with T315I. FTY720 also overcomes TKI resistance due to dysfunction of BIM by gene deletion polymorphism in KCL22 cells. Also, although galectin-3 overexpression has been shown to mediate bone marrow environment-mediated drug resistance in CML cells (Yamamoto-Sugitani M, PNAS, 2011), the addition of FTY720 to imatinib overcomes resistance to imatinib in galectin-3 overexpressing K562 cells and MYL cells. In conclusion, FTY720 induces apoptotic cell death in CML via dual activation of both BIM and BID, and is potent in overcoming various types of cell intrinsic and tumor microenvironment-mediated resistance to TKI. Disclosures:No relevant conflicts of interest to declare.

Is Major Molecular Response a Safe Haven Against Blast Crisis in Cml Patients Treated with Imatinib?

Abstract 3760The introduction of tyrosine kinase inhibitors (TKIs) has proved to be a major advance in the management of patients with chronic myeloid leukemia in chronic phase (CML-CP) although the clinical benefit seems to be limited to those patients who achieve complete cytogenetic remission (CCyR). Patients who achieve CCyR are believed to have an excellent prognosis, but a very small proportion of those patients may lose the response and progress to blastic phase. In this work we studied a cohort of 210 patients who achieved CCyR (142 of them were in MMR) on imatinib as first line therapy in order to identify the incidence of blastic transformation in this population.Between June 2000 and December 2010, 282 consecutive adult patients with CML-CP were seen at our institution, of whom, 210 achieved CCyR on imatinib 400 mg daily as first-line therapy. The median follow-up was 69 months. Of the 210 patients in CCyR, 5 (2.3%) progressed to blastic transformation (BT) (3 myeloid and 2 lymphoid). The Sokal risk was low in 3 patients and intermediate in two. The median time to achievement of CCyR in these 5 patients was 18 months (range 9–20 months). Two of these patients also achieved a major molecular response (MR3), one achieved a 4 log reduction on the international scale and two patients had a CCyR with no MR3. The median time to achievement of MR3 (or better) in the three patients was 24 months (range 18–28 months). After achieving CCyR all 5 patients had progressive increases in their BCR-ABL1 transcript levels over a period of a few months leading to BT without an intervening accelerated phase. None of the patients had a ABL1 kinase domain mutation prior to transformation to advanced phase, but two patients subsequently developed a new mutation at the time of BT (M244V and T315I, respectively). The 8-year cumulative incidence (CI) of BT after reaching CCyR was 2.3% and was 2.8% when calculated from diagnosis (Figure 1). The median time from achieving CCyR to BT, was 18 months (range 12–40 months). The median time from the onset of an increase in BCR-ABL1 transcripts to BT was 6 months (range 4–7 months). All 5 patients had good compliance with TKIs.All 5 patients were treated by allogeneic stem cell transplantation after BT in their second chronic phase. One patient proceeded to a transplant after combination chemotherapy and TKI, but 4 others only received a TKI prior to transplant (3 dasatinib and 1 nilotinib). All 5 patients subsequently died due to transplant related causes. The 8-year CI of BT after attaining CCyR was 2.3%. MR3 does not appear to completely protect against BT as 3 of the 5 patients with MR3 lost the response and progressed to BT. In our series we could not identify a time point beyond which patients may be safe from blastic transformation, which continues to be a rare but catastrophic event in responding patients. [Display omitted] Disclosures:Goldman:Novartis, Bristol Myers-Squibb, and Amgen: Honoraria. Marin:Novartis: Research Funding; BMS: Research Funding.

ABL Kinase Domain Mutations in Patients with Chronic Myeloid Leukemia in Jordan

Mutations of the BCR-ABL tyrosine kinase domain constitute a major cause of resistance to tyrosine kinase inhibitors in patients with chronic myelogenous leukemia (CML). In this study, we analyzed peripheral blood samples from 185 Jordanian CML patients for ABL mutations, who were on imatinib for a minimum of 6 months regardless of their disease status and over a period of 5 years. Mutations were detected by nested RT-polymerase chain reaction, followed by direct sequencing of the ABL kinase domain. Twelve different point mutations were detected 25 times in 21 patients. The resultant mutations were as follows: four patients have T315I, three of each of the following: L248V, F317L, and G250E, two of each of the following: H396R, M244V, and T277A, and one of each of the following: F311I, M318T, Q252H, F359A, F359I, and Y326H. After patient follow-up, the mutation had disappeared in 12 patients; 3 patients died; 3 patients were not retested; and 3 patients had persistent mutation. The finding of our study is in line with what has been described in the literature. Detecting ABL mutations in chronic phase may lead to positive outcome by modifying treatment.

Impact of ABL kinase domain mutations on the outcome of patients with chronic myeloid leukemia (CML).

6588 Background: Patients with CML who develop resistance to imatinib commonly have mutations in the BCR-ABL kinase domain (KDM). Studies looking at outcomes in patients with P-loop versus non-P-loop mutations within the ABL-Kinase Domain have produced conflicting results. Methods: The Total Cancer Care (TCC) database was used to identify patients with CML treated at Moffitt Cancer Center (MCC). Descriptive data were reported, chi square test was used for categorical variables, and Kaplan Meier curves were used for OS and PFS. Log rank test was used to compare survival times between groups. Results: Between 1992 and 2011, 540 CML patients were treated at MCC. Of those, 51% were male and 71% were under the age of 60. Sixty percent (n=322) were diagnosed after 2001. Of the 540 patients, 6.5% (n=35) were found to have mutations of which 26 were detected in patients diagnosed after 2001. Of the 35 patients, 74% (n=26) had single mutations and 26% (n=9) had compound mutations. P-loop mutations were seen in 17% (n=6) and 43% (n=15) had T315I mutations. Patients with KDM progressed to accelerated or blast phase in 46% (n=16) of cases compared to 27% (n=136) without mutations (p=0.03). Median OS was 126 months, 109 months, and not reached in patients with P-loop, T315I, and non-P-loop mutations respectively (p=0.17). The corresponding median PFS was 85 months, 89 months, and not reached (p=0.20). In patients with one mutation median OS was not reached compared to 105 months in patients with compound mutations (p=0.27). After 2001, patients with KDM had a median PFS of 75 months and OS of 126 months while neither was reached in the non-mutation cohort (p=0.007, p=0.26 respectively). Median PFS in patients with single mutations was 85 months versus 10 months in those with compound mutations (p=0.037). Patients with KDM had additional Ph+ clones on cytogenetics in 49% of cases compared with 19% of cases in the non-mutation group (P &lt; 0.005). Conclusions: T315I and P-loop KDM predict PFS and OS in CML patients, and convey a trend for worse prognosis. The presence of additional Ph+ clones in patients with BCR-ABL KDM indicates a higher level of genetic instability and clonal evolution, which may be the contributing factor to poor outcomes.

Genetic mutations in chronic myelogenous leukemia

The review will appraise the literature concerning ABL kinase domain mutations that has appeared over the last year and identify new questions, answers to old questions, and discuss new trends in clinical and laboratory based research. A concise summary of European LeukemiaNet guidelines for kinase domain mutation studies was published this year. A new controversial topic emerged: the relevance of IC50 data to guide second-line tyrosine kinase inhibitor (TKI) therapy. Although flaws in the methodology have been acknowledged, one group summarily rejected IC50 data and recommended that clinicians use individual patient comorbidities and drug safety profiles. The influence of kinase domain mutations on response to second-line and third-line TKI therapy was also published this year; unexpectedly, kinase domain mutations were found to have no effect on response or survival. However, the presence of a kinase domain mutation did influence survival following hematopoietic stem cell transplantation. Lastly, new findings from laboratories identified transcription factors BCL6 and STAT5 as potential new treatment targets. The last 12 months has brought much attention to clinical management of patients with kinase domain mutations and identified a new controversy concerning IC50 data use in the clinic. Kinase domain mutations do not appear to influence response to second-line and third-line response to TKI therapy. New targets that do not directly involve BCR-ABL added potential new therapeutic approaches.

Bortezomib has little ex vivo activity in chronic myeloid leukemia: individual tumor response testing comparative study in acute and chronic myeloid leukemia

Aim of the studyResistance to imatinib is one of the most important issues in treatment of chronic myeloid leukemia (CML) patients. The objective of the study was to analyze the ex vivo drug resistance profile to bortezomib and 22 other antileukemic drugs, including three tyrosine kinase inhibitors (TKIs), in CML in comparison to acute myeloid leukemia (AML).Material and methodsA total of 82 patients entered the study, including 36 CML and 46 AML adults. Among CML patients, 19 had advanced disease, 16 were resistant to imatinib, and 6 had ABL-kinase domain mutations. The ex vivo drug resistance profile was studied by the MTT assay.ResultsCML cells were more resistant than AML blasts to the following drugs: prednisolone, vincristine, doxorubicin, etoposide, melphalan, cytarabine, fludarabine, thiotepa, 4-HOO-cyclophosphamide, thioguanine, bortezomib, topotecan, and clofarabine. CML cells were 2-fold more sensitive to busulfan than AML cells. CML patients with clinical imatinib resistance had higher ex vivo resistance to vincristine, daunorubicin, etoposide, and busulfan. No significant differences to all tested drugs, including TKIs, were observed between CML patients with non-advanced and advanced disease. CML patients with mutation had higher ex vivo resistance to vincristine, idarubicin, thiotepa, and busulfan.ConclusionsCML cells are ex vivo more resistant to most drugs than acute myeloid leukemia blasts. Busulfan is more active in CML than AML cells. In comparison to AML cells, bortezomib has little ex vivo activity in CML cells. No differences between CML subgroups in sensitivity to 3 tested TKIs were detected.

Abl Kinase Domain Mutations Leading to Relapse of Ph+ Acute Lymphoblastic Leukemia (ALL) Occur Commonly and Can Be Detected At Initial Diagnosis: Molecular Results From CALGB 10001

Abstract 2541CALGB 10001 tested the feasibility of performing allogeneic or autologous stem cell transplantation (SCT) following combination chemotherapy and tyrosine kinase inhibition with imatinib in patients <60 years old with newly diagnosed Ph+ ALL. Patients received imatinib with each course of chemotherapy prior to SCT. A minimum of 12 months of maintenance therapy with imatinib was recommended following SCT or until minimal residual disease was no longer detected using quantitative real-time PCR (q-PCR). One goal of this study was to determine the incidence of ABL kinase domain mutations in patients who relapsed during or following treatment on CALGB 10001, and to evaluate the kinetics of the resistant clone. 58 patients (median age, 44 yrs) enrolled on CALGB 10001 between 11/2003 and 2/2010. After 3–4 courses of imatinib plus chemotherapy, 15 patients received TBI/etoposide and an alloSCT from a matched sibling donor; 9 (60%) remain in CR (median, 665+ days; range, 152+ to 1208+ days), 2 subsequently relapsed (days +236 and +354), and 4 had transplant-related mortality (TRM). 17 patients received TBI/etoposide/cyclophosphamide and autologous SCT; 10 (59%) remain in CR (median, 742+ days; range, 196+ to 1656+), 9 relapsed (range, 257–522 days), and none had TRM (updated from Wetzler et al, ASH 2007; abstract #2869). Thus, a total of 19 patients have relapsed (8 prior to SCT; 2 after alloSCT; 9 after autoSCT,) at a median of 5.9 months following SCT. All patients were monitored for BCR/ABL1 by q-PCR at sequential time points during and following treatment. Using direct sequencing methods (sensitivity 15–25%), an ABL kinase domain mutation was found in 12 of the 19 (63%) at relapse. The mutations included: 4 with Y253H, 3 T315I, 3 E255K, and 1 E255V. One patient had both a T315I and E255V mutation. All of these mutations are associated with imatinib resistance. To evaluate the kinetics of the resistant clone, we used a quantitative mutation-specific PCR assay (Gruber et al, Leukemia 19:2159, 2005). The sensitivity of the assay was shown to be 1% for T315I and E255V and 0.1% for Y253H and E255K. Using the q-PCR method, we found the resistant clone was detectable at diagnosis in 6 of the 12 patients with kinase domain mutations. In 4 patients, the resistant clone was not detectable until the time of relapse; however, for 3 of these cases the relapse occurred very early, within 2 months of starting treatment. In the remaining 2 cases, the resistant clone was first detected during treatment at 1 and 9 months prior to relapse (Figure 1). In most cases, the kinetics of the resistant clone mirrored the overall BCR/ABL1 population (Figure 2). Nevertheless, the resistant clone proportionally increased in size over time, and at relapse, the resistant clone became the dominant BCR-ABL1 clone. In summary, our series demonstrates that ABL kinase domain mutations occur commonly at relapse in Ph+ ALL despite aggressive therapy with imatinib plus combination chemotherapy followed by autoSCT and in some cases, alloSCT. The relapses appear to be due to the emergence of an imatinib-resistant subclone that is frequently present but subclinical at the time of diagnosis. Improved TKI-based therapies with activity against imatinib-resistant subclones instituted early during treatment of Ph+ ALL are needed to prevent emergence of clonal resistance and eventual relapse. Use of sensitive q-PCR assays to screen for these commonly pre-existing mutations at diagnosis, perhaps using a multiplex method, would provide useful clinical information to allow for optimal therapeutic management of Ph+ ALL. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Outcome of Patients with Philadelphia Chromosome-Positive (Ph+) Acute Lymphoblastic Leukemia (ALL) with Relapse After Tyrosine Kinase Inhibitor (TKI) Therapy

Abstract 1518 Background:Treatment with TKIs has greatly improved the outcome of patients with Ph+ ALL. However, many patients treated with TKI-based therapy eventually have a relapse. The response to salvage therapy and long-term outcomes of these patients are unknown. Aims:Describe the outcomes of patients with Ph+ ALL with resistance to or relapse after frontline TKI-based chemotherapy. Methods:We analyzed the outcome of patients who were treated in clinical trials at our institution between February 2001 and July 2008 with TKI-based chemotherapy for newly diagnosed Ph+ ALL who had refractory or relapsed disease. Results:One hundred thirteen patients were treated with frontline hyperfractionated cyclophosphamide, doxorubicin, vincristine, and dexamethasone (HCVAD) plus imatinib (HCVAD+I; n=54) or HCVAD plus dasatinib (HCVAD+D; n=59). Of these, 35 (31%) experienced primary resistance (n=1) or relapse (n=34). The median age was 51 years [range (r): 20–85]; 12 patients (34%) were older than 60 years. Median follow-up was 21.1 mo (r: 4.2–56.7). Median white blood cell and platelet counts at diagnosis were 14.4 × 109/L (r: 1.2–292.9) and 48 × 109/L (r: 4–425), respectively. White blood cell count was >30 × 109/L in 13 patients (37%). Median peripheral and bone marrow blast percentages were 53% (r: 0–97%) and 80% (r: 1–98%), respectively. Twenty-two patients (63%) had received HCVAD+I and 13 (37%) HCVAD+D. Twenty-three patients (66%) had experienced first complete remission (CR1) with 1 cycle of induction. Median CR1 duration was 12 mo (r: 1.9–42). Four patients underwent allogeneic stem cell transplantation (ASCT) in CR1. ABL kinase domain mutations were investigated in 28 patients (80%) at relapse; 16 (57%) had mutations, including 5 (14%) with T315I (all had received HCVAD+D).Upon relapse, 31 patients received first salvage therapy (S1), 24 with chemotherapy [HCVAD+D, n=8; HCVAD+I, n=3; HCVAD+nilotinib (N), n=1; HCVAD+asparaginase (Asp), n=1; methotrexate, vincristine, Asp, and dexamethasone (MOAD), n=2; others, n=9]; 6 with a TKI only (I, n=2; D, n=1; N, n=1; others, n=2); and 1 with ASCT. Three patients were unfit for treatment. Median cycles of S1 were 2 (r: 1–8). Thirteen patients (42%) had second complete remission (CR2) (HCVAD+D, n=6; HCVAD+I, n=2; HCVAD+N, n=1; HCVAD+Asp, n=1; others, n=3). Median time to CR2 was 1.5 mo (r: 0.7–8.8). Five patients underwent ASCT in CR2. Median CR2 duration was 7.3 mo (r: 1.4–36.2). Complete cytogenetic response was seen in 11 patients (35%); major molecular response (BCR-ABL/ABL ratio <0.05%) in 9 (29%); and complete molecular response in 7 (22%); and complete hematologic response in 15 (48%). Times to complete cytogenetic response and complete molecular response were 1.3 mo (r: 0.7–10.6) and 3 mo (r: 1.5–8.7), respectively. Seven patients had second relapse. Fifteen patients (7 relapse, 8 refractory) received second salvage therapy (S2) with systemic chemotherapy (MOAD, n=2; phase I/single-agent TKI, n=8; others, n=5); 1 patient had solitary central nervous system (CNS) relapse treated with intrathecal cytarabine and methotrexate. CR3 was obtained in 1 patient, the patient with sole CNS relapse.Median disease-free survival (DFS) and overall survival (OS) after S1 were 6.5 mo (r: 0.5–45) and 7.3 mo (r: 1.4–36.2), respectively. At last follow-up, 2 patients (6%) were alive and 33 had died, 11 (33%) of infectious complications, 5 (15%) of organ failure, 3 (9%) of bleeding complications, 2 (6%) of graft-versus-host disease complications, 2 (6%) of CNS relapse, and 10 (30%) of other or unknown causes. Median OS after S2 was 2.1 mo (r: 1.4–2.6). In univariate analysis, age >60 years was associated with worse OS after S1 [4.2 vs. 12.7 mo; 95% confidence interval (CI) 1.8 to 6.7 vs. 7.5 to 17.9 (P=0.006)]. Complete hematologic response was associated with improved OS after S1 [15.4 vs. 4.3 mo; 95% CI 9.1 to 21.8 vs. 2.5 to 6.0 (P<0.001)]. Major molecular response was associated with improved OS after S1 [18.1 vs. 5.7 mo; 95% CI 9.3 to 26.8 vs. 3.6 to 7.8 (P=0.003)]. Choice of prior TKI (HCVAD+I vs. HCVAD+D) did not significantly influence CR and OS after relapse. Conclusion:Patients with refractory or relapsed Ph+ ALL after TKI-based therapy have poor outcome, particularly those who are older or have persistent BCR/ABL transcripts. New agents are needed to improve the outcome in this population. [Display omitted] Disclosures:Kantarjian:BMS: Research Funding. Ravandi:Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria. Cortes:Chemgenex: Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Role of ABL Gene Mutations in Chronic Myeloid Leukemia Patients Resistant to Imatinib and Results of Treatment Shift to Second-Generation Tyrosine Kinase Inhibitors

Abstract 4442 Introduction:Chronic myeloid leukemia (CML) is a model of disease in the development of targeted therapies. Tyrosine kinase inhibitors (TKIs) have transformed the approach to management of CML and have dramatically improved patients' outcome. Clinical response is obtained in the majority of patients. However, a significant proportion of patients do not achieve the optimal desirable outcome or are completely resistant to this treatment. ABL kinase domain mutations have been extensively implicated in the pathogenesis of TKI resistance. Treatment with second-generation TKIs has produced high rates of hematologic and cytogenetic response in mutated ABL patients.The aim of this study was analyzed the presence of ABL mutations in imatinib resistant patients and determine the importance of changing to second-generation TKIs treatment as soon as failure or suboptimal response is recognized. Patients and methods:From 420 CML patients diagnosed in 6 centers between 2004 and 2010, we have amplified and sequenced the ABL1 domain from BCR-ABL1 amplicon of 45 imatinib resistant patients (23 patients with suboptimal response, 14 with treatment failure, 4 who lost the molecular response and 4 patients who progressed to blast phase). The obtained sequences were compared with the published ABL1 sequence, GenBank U07563, using BLAST 2 software. Results:We have detected mutations in 15 of 45 patients (33%), some of them with more than one mutation (Table 1). Seven of these patients were treated with second-generation TKIs as a single treatment. Three of them achieve a major molecular response (MMR), one patient is in complete cytogenetic response (CCyR) and the other two patients are in major (MCyR) and partial (PCyR) cytogenetic response. Another patient received nilotinib followed by hematological stem cell transplantation (HSCT) and is in MMR. Two patients were submitted to a HSCT and achieve MMR. Only one patient treated with nilotinib as second option has not reach a cytogenetic response one year after detection of the mutation.Two of the patients with the T315I mutation were treated with IFN and nilotinib achieving PCyR and MCyR, respectively, and are still alive. The other T315I patient, and two patients in blast-crisis (BC) disease with the F317L mutation who received dasatinib prior to the study of ABL mutations, died before a change of treatment could have been performed. Conclusions:1.In our series, 10 out of the 15 patients who carried an ABL mutation benefit of treatment shift, suggesting that change of therapy as soon as either failure or suboptimal response is recognized seems to be better than waiting until hematologic response is also lost.2.F317L mutation in the two patients in BC appeared, probably, as a consequence of dasatinib treatment.3.We could identify the T315I mutation by early study of the ABL mutational status in patients with suboptimal response or failure to treatment in chronic-phase CML before their transformation to an advanced-phase disease.Table 1Characteristics of the 15 analyzed patients harboring ABL mutations.UPNFASEINITIAL TREATMENTRESPONSEMUTATIONSECOND LINE TREATMENTRESPONSE1APIM 400mg/24h - DA 70mg/12hFailureM351T and Y253HNI 400mg/48hCCyR2CPIM 400mg/24h – DA 70mg/12hSuboptimal ResponseM244VNI 800mg/12hHR3CPIM 400mg/24hSuboptimal ResponseS417YIM 400mg/24hMMR4CPIM (400-600-800mg/24h)Suboptimal ResponseL340LDA 70mg/12hMMR5BCIM 400mg/24hOtherdel exon 7DA 70mg/12hMMR6BCIM (400-600-800mg/24h)FailureT277AHSCTMMR7CPIM 400mg/24h - NI 200mg/12hFailureV379IHSCTMMR8CPIM 400mg/24hSuboptimal ResponseT315IIFNPCyR9BCIM 400mg/24h -DA 70mg/12hFailureF317LNoneExitus10BCIM 400mg/24h - DA 70mg/12hFailureT315INoneExitus11CPIFN – IM 400mg/24hSuboptimal ResponseD276ADA 50mg/48hPCyR12CPIM 400mg/24hSuboptimal ResponseT315I and F359VNI 200mg/12hMCyR13CPIM – NI - DAProgression to BCF317LNoneExitus14CPIM 400mg/24hTransformation to LAL-BCG250E and F311LNI + HSCTMMR15CPIM 400mg/24hSuboptimal ResponseLoss of 1 nucleotideNI 400mg/12hMMRAbbreviations: AP, Accelerated phase; CP, Chronic phase; BC, Blast crisis, IM, imatinib; DA, dasatinib; NI, nilotinib; del, deletion; HSCT, hematopoietic stem cell transplantation; IFN, interferon; MMR, major molecular response; CCyR, complete cytogenetic response; MCyR, major cytogenetic response; PCyR, partial cytogenetic response; HR, hematologic response. Disclosures:No relevant conflicts of interest to declare.

Long-Term Follow-up of Combined Hypercvad (hCVAD) Regimen with Dasatinib (Db) in the Front Line Therapy of Patients (pts) with Philadelphia Chromosome Positive (Ph+) Acute Lymphoblastic Leukemia (ALL)

Abstract 1512 Background:The introduction of tyrosine Kinase Inhibitors (TKI) has significantly improved the outcome of patients (pts) with Ph+ ALL. Dasatinib (Db) is a second generation dual SRC/ABL TKI with greater potency compared to Imatinib in inhibiting BCR/ABL. Aim:To determine the outcome of pts with Ph+ ALL treated with hCVAD + Db. Method:Between 9/06 and 7/09, pts with newly diagnosed Ph+ ALL received Db 50mg oral (PO) twice daily (BID) or 100mg PO daily for the first 14 days of each of 8 cycles of alternating hCVAD, and high dose cytarabine and methotrexate. Pts in complete remission (CR) continued to receive maintenance Db 50mg PO BID or 100mg PO daily, as well as monthly prednisone and vincristine for 2 years, followed by Db indefinitely. From 8/09 protocol was amended and pts received 100mg Db for the first 14 days of cycle #1 and then 70mg daily continuously for the next 7 cycles, as well as 2 doses of rituximab 375 mg/m2 during each of the first 4 cycles. Maintenance was with Db, vincristine and prednisone. Results:Sixty-one pts with newly diagnosed Ph+ ALL have been treated to date. Median age was 56 years (yrs) (range (r), 22–80) and 41 (67%) pts were >50 yrs. The median follow up is 26.1 months (mo) (r, 4–58). Central nervous system (CNS) involvement was noted in 9 (14%) pts at diagnosis. Sixteen (26%) pts had Ph+ alone, 38 (62%) pts had Ph+ with additional abnormalities, and 7 (12%) pts were Ph negative, and BCR/ABL positive. Median white blood cell count (WBC) at diagnosis was 13.4 × 109/L (r: 0.4–658), and 22 (36%) pts had WBC >30 × 109/L at diagnosis. BCR/ABL transcript was identified in 60 (98.3%) pts at diagnosis, including e1a2 in 46 (76%) pts, b2a2 in 10 (17%) pts, b2a2+b3a2 in 2 (3%) pts, b3a2 and e1a3 in 1 (1.6%) pt each. One pt had a variant transcript that was not detectable with the standard primers. The median number of induction and maintenance cycles received were 6 cycles (r: 1–8) and 13.5 cycles (r: 1–24), respectively. Fifty seven (94%) pts achieved CR1 and 1 (1.5%) pts achieved CR with incomplete platelet recovery with first induction cycle of chemotherapy. Three (4.5%) pts died before response assessment could be performed due to infections. Thirty-nine (64%) pts received maintenance, 3 (5%) pts are currently receiving induction and 19 (31%) pts had no maintenance [9 pts received allogeneic stem cell transplant (ASCT) prior to maintenance, 10 pts had progression of disease]. To date, twelve (19%) pts have relapsed and Abl kinase domain mutations were analyzed in 7 pts; mutations were noted in 4 pts. These included T315I in 2 pts, and F359V and V299L in 1 pt each. CNS relapse occurred in 5 pts. Salvage (S1) regimens included [hCVAD + another TKI in 7 pts, single agent TKI in 2 pts, single agent monoclonal antibody in 1 pt, methotrexate, vincristine, asparginase, dexamethasone (MOAD) in 1pt, intrathecal cytarabine/methotrexate plus CNS radiation and Db in 1 pt]. Eight pts achieved CR2, 3 pts were refractory (2 pts with T315I and 1 pt with F359V) and one is still undergoing salvage treatment. Median DFS and OS after S1 were 5.3 mo (r: 0.7–17.3) and 6.7 mo (r: 0.6–24.4), respectively. ASCT was performed in 15 (24%) pts, including 10 pts in CR1 and 5 pts in CR2. Donors were related in 8 (53%) and unrelated in 7 (47%) transplants. Sixteen pts have died 11 (68%) pts from infectious complications, 2 (13%) pts from multi-organ failure, 1 (6%) pt with graft versus host disease, and 2 (13%) pts from unknown causes. Three-year disease free survival (DFS) and overall survival (OS) (n=61) were 49% and 62%, respectively. Conclusion:Db plus hCVAD is an effective regimen with durable responses in pts with newly diagnosed Ph+ ALL. [Display omitted] [Display omitted] Disclosures:Kantarjian:BMS: Research Funding. Jabbour:Pfizer: Honoraria; BMS: Honoraria; Novartis: Honoraria. Cortes:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Membership on an entity's Board of Directors or advisory committees, Research Funding; Chemgenex: Membership on an entity's Board of Directors or advisory committees, Research Funding. Ravandi:Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria.

Activity of Bosutinib by Baseline and Emergent Mutation Status in Philadelphia Chromosome–Positive Leukemia Patients with Resistance or Intolerance to Other Tyrosine Kinase Inhibitors

Abstract 110Bosutinib is an orally active, Src/Abl tyrosine kinase inhibitor (TKI) that has demonstrated clinical activity and an acceptable safety profile in an open-label, phase 1/2 study of patients (pts) with Philadelphia chromosome–positive (Ph+) leukemia following resistance or intolerance to imatinib. Abl kinase domain mutations may affect drug binding and subsequent TKI efficacy. Responses to bosutinib by Bcr-Abl baseline mutation status and the emergence of mutations during bosutinib treatment in this phase 1/2 study are reported here.A total of 570 pts in 3 cohorts received bosutinib: chronic phase chronic myeloid leukemia (CP CML) following prior imatinib only (CP2L cohort; n = 288); CP CML following prior imatinib plus dasatinib and/or nilotinib (CP3L cohort; n = 118); and advanced leukemia (ADV cohort: accelerated and blast phase CML and acute lymphoblastic leukemia; n = 164) following prior treatment with imatinib only or with other TKIs. For the CP2L, CP3L, and ADV cohorts, the respective median times since diagnosis were 3.6 y (range, 0.1–15.1 y), 6.5 y (range, 0.6–18.3 y), and 3.7 y (range, 0.1–22.1y). Per protocol, Bcr-Abl mutational status was evaluated at baseline (pre-dose) and at the end of treatment (EOT).Mutations were assessed at baseline in 412 pts; of these, 79/212 (37%) CP2L pts, 39/83 (47%) CP3L pts, and 65/117 (56%) ADV pts had ≥1 baseline mutation, and 11 (5%), 9 (11%), and 7 (6%) pts, respectively, had ≥2 baseline mutations. The most common mutations were T315I (n = 31; prior to eligibility exclusion), F359C/I/S/V (n = 23), F317L (n = 21), G250E (n = 19), Y253F/H (n = 16), and M351T (n = 15). Rates of confirmed complete hematologic response (CHR) and major cytogenetic response (MCyR) were generally similar among chronic phase pts (CP2L, CP3L) with and without baseline mutations, although the presence of a mutation negatively impacted response rates in ADV pts (CHR, 17% vs 39%; MCyR, 24% vs 37%; see Table). CHR and/or MCyR were observed broadly across mutations, including those associated with dasatinib (F317L) or nilotinib (Y253F/H, E255K/V, and F359C/I/S/V) resistance, with the exception of the T315I mutation in all cohorts and the F317L and Y253H mutations in the ADV cohort (see Table).Of the 140 pts who were assessed for emergent mutations at treatment completion, a new mutation was identified in 39 (28%) pts. Pts with a new mutation were more likely to have discontinued treatment due to PD/unsatisfactory response (UR; n = 34/39 [87%]) than those without a new mutation (n = 47/101 [47%]). The most common emergent mutations were T315I (n = 14) and V299L (n = 12), and nearly all pts who developed these mutations (93% and 100% of pts, respectively) discontinued treatment due to PD/UR. New mutations were more common in pts with ≥1 baseline mutation (n = 26) versus those without a baseline mutation (n = 13); of those pts, 21/26 (81%) and 13/13 (100%), respectively, discontinued treatment due to PD/UR. Eight pts discontinued treatment due to PD/UR within 6 months of starting bosutinib; all but 1 patient had a T315I or V299L mutation present at baseline or identified at treatment completion.In conclusion, clinical activity to bosutinib was observed in CML pts across baseline Bcr-Abl kinase domain mutations, including those associated with resistance to other second-generation TKIs, except typically for T315I. These results support the observed benefit of bosutinib in pts with Ph+ leukemia following prior treatment with TKIs. Pts in whom new mutations were identified at treatment completion were more likely to have pre-existing mutations and to discontinue treatment due to PD/UR. The most common mutations to emerge during bosutinib therapy were T315I and V299L, some of which may have been present but not detected at baseline.Baseline mutation status, n/n evaluablea (%)IC50 fold increaseCP2L cohortCP3L cohortADV cohortCHRMCyRCHRMCyRCHRMCyRNo mutation120/133 (90)65/121 (54)34/44 (77)15/43 (35)19/49 (39)16/43 (37)≥1 mutation65/78 (83)44/78 (56)26/39 (67)11/35 (31)10/59 (17)13/55 (24)Y253H0.52/22/25/64/61/72/7F359V0.98/94/90/21/20/20/1Y253F1.01/10/10000F317L2.44/43/44/81/70/90/6F359I2.41/11/12/22/20/21/2G250E4.35/63/53/60/54/62/7E255V5.52/21/21/10/10/41/3E255K9.50/22/30/10/10/41/3V299L26.1001/20/200T315I45.42/92/92/70/60/131/13F359CNA002/21/20/10/1F359SNA1/11/1001/10/1NA, not available.aTreated pts with adequate baseline response assessment. Disclosures:Cortes:Pfizer Inc: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Gambacorti-Passerini:Pfizer Inc: Honoraria, Research Funding; BMS: Research Funding; Novartis: Honoraria; Biodiversity: Honoraria. Kim:Pfizer Inc: Consultancy, Research Funding; BMS: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Ariad: Research Funding. Hochhaus:Pfizer Inc: Research Funding; BMS: Research Funding; Novartis: Research Funding; Ariad: Research Funding. Leip:Pfizer Inc: Employment. Besson:Pfizer Inc: Employment. Kelly:Pfizer Inc: Employment. Brümmendorf:Pfizer Inc: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Patents & Royalties, Research Funding; BMS: Consultancy, Honoraria.

Statins Increase Antileukemic Potency of Imatinib Through the Inhibition of MDR/ABCB1 and BCRP/ABCG2 Drug Transporters Activity

Abstract 2742The treatment of chronic myeloid leukemia (CML) was revolutionized by the introduction of tyrosine kinase inhibitors (TKIs) to the clinical practice. However, there is still a significant group of patients who due to drug resistance do not fully benefit from the therapy. Among various reasons of drug resistance, the changes of intracellular concentration of TKIs caused by the activity of drug transporters may have profound effect on clinical activity of the drug. Intracellular concentration of imatinib is a result of: drug influx into the cell and drug efflux out of the cell. The latter is mediated by ATP-binding casette (ABC) transporters - MDR/ABCB1 and BCRP/ABCG2. According to the recent knowledge modulation of membrane cholesterol level may change the activity of ABC transporters by changing their conformation. Hence we decided to evaluate potential effect of statins, widely used hypocholesterolemic drugs, on antileukemic activity of imatinib in cell lines transformed with BCR/ABL oncoprotein and primary human CML CD34+ cells.In a set of experiments involving cytotoxic assays (i.e. clonogenic assay, XTT, trypan blue exclusion assay) we revealed that lovastatin, a member of statin family, is able to enhance imatinib cytotoxicity both in established cell lines (32Dcl3 BCR/ABL+, K562) as well as in primary CML CD34+ cells obtained from patients in various stages of the disease, including those clinically resistant to imatinib with no detectable ABL kinase domain mutations. In the same time, lovastatin alone and in combination with imatinib did not influence BCR/ABL negative cells (32Dcl3 wild type, HL-60, CD34+ cells obtained from healthy blood donors). As measured using specific, fluorescent-labeled substrates for ABC transporters (rhodamine-123 and Bodipy-FL-prazosin respectively), lovastatin significantly inhibits MDR/ABCB1- and BCRP/ABCG2-mediated efflux capacity. Similar effects were observed for other statins. The addition of cholesterol to the samples previously incubated with lovastatin completely reversed inhibitory activity of statins. To confirm these results we examined direct changes of intracellular concentration of imatinib itself using radiolabeled 14C-imatinib. In these settings, statins caused 2–3-fold increase in intracellular imatinib concentration in murine and human cell lines (32Dcl3 BCR/ABL+, K562) and also in primary CML CD34+ cells including clones resistant to imatinib (Figure 1). Statins did not influence initial concentration of imatinib what may suggest that their effects on imatinib concentration are not mediated by changes in OCT-1 activity. In addition, statins did not influence expression of MDR/ABCB1 and BCRP/ABCG2 drug transporters measured using qPCR nor ABC proteins levels measured using Wester blotting. Combination of imatinib and lovastatin induced cell cycle arrest and increased percentage of apoptotic leukemic cells measured using flow cytometric analysis after propidium iodide staining. Lovastatin also reduced imatinib-induced phosphorylation of the adaptor protein CrkL.In summary, we show that statins are able to increase therapeutic efficacy of imatinib through the inhibition of drug efflux mediated by MDR/ABCB1 and BCRP/ABCG2 transporters. Our results suggest that statin-mediated depletion of cholesterol in the cell may change conformational status of the efflux pumps, including MDR/ABCB1 and BCRP/ABCG2 pumps involved in the transport of imatinib, and in that way modulate intracellular concentration of the drug. The addition of statins may become attractive treatment modality for the selected group of patients in chronic phase and blast crisis. [Display omitted] CML-CP, CML-BP–CD34+ cells obtained from CML patient in chronic phase or blast crisis respectively; *p<0,05 Disclosures:No relevant conflicts of interest to declare.

Rapid automated detection of ABL kinase domain mutations in imatinib-resistant patients

ABL tyrosine kinase inhibitor (TKI), imatinib is used for BCR–ABL + leukemias. We developed an automatic method utilizing guanine-quenching probes (QP) to detect 17 kinds of mutations frequently observed in imatinib-resistance. Results were obtained from 100 μL of whole blood within 90 min by this method. Detected mutations were almost identical between QP method and direct sequencing. Furthermore, the mutation-biased PCR (MBP) was added to the QP method to increase sensitivity, resulting earlier detection of T315I mutation which was insensitive to any ABL TKIs. Thus, the QP and MBP-QP may become useful methods for the management of ABL TKI-treated patients.

Low-level Bcr–Abl mutations are very rare in chronic myeloid leukemia patients who are in major molecular response on first-line nilotinib

We investigated whether low-level Bcr–Abl kinase domain mutations can be detected in patients who have stable responses to first-line nilotinib–like it is known to happen in patients receiving imatinib. We screened for mutations twelve such patients by cloning and sequencing. Only one case was found to harbor mutations at low levels (including a T315I). However, major molecular response (MMR) was maintained and it even improved to complete molecular response. Our results suggest that a) Bcr–Abl mutations, even at low level, seem to be very rare in patients in MMR on first-line nilotinib; b) low-level mutations do not always predict for subsequent relapse.

Tyrosine kinase inhibitor resistance in chronic myeloid leukemia cell lines: investigating resistance pathways

There are three currently identified secondary resistance mechanisms observed in patients with chronic myeloid leukemia (CML) receiving tyrosine kinase inhibitors (TKIs). These are BCR–ABL kinase domain (KD) mutations, increased BCR–ABL expression, and overexpression of drug-efflux proteins (ABCB1 and ABCG2). To investigate the interplay between these three modes of resistance, three CML blast crisis cell lines (K562, its ABCB1-overexpressing variant K562 Dox, and KU812) were cultured in gradually increasing concentrations of imatinib to 2 μM, or dasatinib to 200 nM. Eight imatinib- and two dasatinib-resistant cell lines were established. Two imatinib-resistant K562 lines both had increased BCR–ABL expression as the apparent mode of resistance. However, when a dasatinib-resistant K562 culture was generated we observed gradually increasing BCR–ABL expression which peaked prior to identification of the T315I mutation. BCR–ABL overexpression followed by mutation development was observed in a further 4/10 cell lines, each with different KD mutations. In contrast, three imatinib-resistant K562 Dox lines exhibited only a further increase in ABCB1 expression. All TKI-resistant cell lines generated had increased IC50 (dose of drug required to reduce phosphorylation of the adaptor protein p-Crkl by 50%) to imatinib, dasatinib, and nilotinib, regardless of which TKI was used to induce resistance. This suggests that currently available TKIs share the same susceptibilities to drug resistance.

Clinical outcome of chronic myeloid leukemia imatinib-resistant patients: do BCR–ABL kinase domain mutations affect patient survival? First multicenter Argentinean study

In imatinib-treated patients with chronic myeloid leukemia (CML), BCR–ABL mutations are the most common mechanism of resistance. Here we report the first multicenter Argentinean study investigating mutations in those patients with CML who fail or lose response to imatinib, with or without previous interferon treatment. Point mutations were detected in 36 of 154 patients by direct sequencing. In our series, the single most common mutations were G250E, E255K/V, and M351T. The presence of mutations correlated significantly with accelerated phase, lack of molecular response, and lower cytogenetic and hematological responses. While overall survival did not differ between patients with or without mutations, the probability of progression was higher in patients with mutations. Cases with non-P-loop mutations showed a significantly better overall survival from diagnosis. Multivariate analysis showed that the most significant variables related to the development of mutations were accelerated phase, duration of imatinib treatment, and time delay to starting imatinib. Our results demonstrated that mutation frequency increased with the progression of disease, and suggest that imatinib treatment should be started early.

Conformational Control Inhibition of the BCR-ABL1 Tyrosine Kinase, Including the Gatekeeper T315I Mutant, by the Switch-Control Inhibitor DCC-2036

Acquired resistance to ABL1 tyrosine kinase inhibitors (TKIs) through ABL1 kinase domain mutations, particularly the gatekeeper mutant T315I, is a significant problem for patients with chronic myeloid leukemia (CML). Using structure-based drug design, we developed compounds that bind to residues (Arg386/Glu282) ABL1 uses to switch between inactive and active conformations. The lead "switch-control" inhibitor, DCC-2036, potently inhibits both unphosphorylated and phosphorylated ABL1 by inducing a type II inactive conformation, and retains efficacy against the majority of clinically relevant CML-resistance mutants, including T315I. DCC-2036 inhibits BCR-ABL1(T315I)-expressing cell lines, prolongs survival in mouse models of T315I mutant CML and B-lymphoblastic leukemia, and inhibits primary patient leukemia cells expressing T315I in vitro and in vivo, supporting its clinical development in TKI-resistant Ph(+) leukemia.

Outcome of patients with chronic myeloid leukemia with multiple ABL1 kinase domain mutations receiving tyrosine kinase inhibitor therapy.

We investigated the impact of carrying more than one BCR-ABL1 mutation in 207 patients with chronic myeloid leukemia (102 chronic, 61 accelerated, and 44 blast phase) post-imatinib failure. Seven (8%) of 92 patients carrying mutations had more than one mutation: 4 (4%) in chronic phase, 2 (2%) in accelerated phase, and one (1 %) in blast phase. The cytogenetic response rate to second generation TKIs for patients with no, one, or more than one mutation was 88%, 69%, 50% (P=0.03) in chronic phase, 54%, 42%, 50% in accelerated phase (P=0.67), and 35%, 25%, 0% (P=0.63) in blast phase, respectively. No differences were observed in event free survival or overall survival in accelerated or blast phase according to their mutational status. However, the 4-year event free survival rates among patients in chronic phase with no, one, or more than one BCR-ABL1 mutation were 56%, 49%, and 0%, respectively (P=0.02), with overall survival rates of 91%, 69%, and 75%, respectively (P=0.13). In conclusion, patients with more than one BCR-ABL1 mutation fare worse than those with no or one mutation.