ABL Kinase Domain Mutations Research Articles

Studying the impact of presence of point mutation, insertion mutation and additional chromosomal abnormalities in chronic myeloid leukemia patients treated with imatinib mesylate in the State of Qatar

Background: Imatinib is failing as a first line treatment in more than 40% of chronic myeloid leukemia (CML) patients in Qatar. We thus investigated ABL1 kinase domain mutations and additional chromosomal abnormalities (ACAs) as underlying mechanisms to explain this high rate of treatment failure. Methods: Between November 2006 and December 2011, all CML patients in Qatar were studied for BCR-ABL1 kinase domain mutations and ACAs. Total RNA was extracted and cDNA was produced via reverse transcriptase polymerase chain reaction (RT-PCR). PCR was used with special precautions to avoid amplification of wild type ABL1; the ABL1 kinase domain was then screened for mutations by direct DNA sequencing technology to detect the emergence of mutant clone. Cytogenetic analysis of bone marrow (BM) metaphases and fluorescence in situ hybridization (FISH) of peripheral blood (PB) and BM interphases were performed according to standard protocols. European Leukemia Net (ELN) response criteria were employed to identify the failing cases. Results: 26 out of 33 CML patients were eligible for the study, 22 CP and 4 AP. 14 failed Imatinib treatment, 2 had BCR-ABL1 kinase domain mutations; one patient had the G1739A mutation which leads to the exchange of glutamic acid at position 459 to lysine (E459K) (rs1064156) in the c-terminal loop while the other patient had a unique insertion of three nucleotides (AAG) at position 1432 which adds an amino acid Lysine to position 356 of the catalytic domain and a complex karyotyping at diagnosis, 6 had additional chromosomal abnormalities as an underlying mechanism of resistance, 4 patients had no identifiable cause of resistance and 2 patients were intolerant to treatment. There was a significant difference in median overall survival between patients with Ph chromosome only and patients with ACAs. Conclusions: In this study as we continued observing CML patients for nearly 5 years, the Imatinib failure reached as high as 54%. The resistance rate observed in Qatar is still higher than that reported by the IRIS study which is as high as 35%. In our cohort of CML patients, point mutation and unique tri-nucleotide insertions were identified. However, these mutations could explain only 14% of treatment failure. Additional chromosomal abnormalities were the most common cause of Imatinib failure in our patients' cohort and were documented in 50% of cases. 14% of patients stopped IM due to intolerance; and the mechanisms of resistance remained unknown in 28% of patients. Other causes such as patients' adherence to Imatitinb is being prospectively investigated.

Clinical significance of analysis of BCR- ABL kinase domain mutation in chronic myeloid leukemia patients

Clinical significance of analysis of BCR- ABL kinase domain mutation in chronic myeloid leukemia patients

Bosutinib Treatment for Philadelphia Chromosome-Positive Leukemias

The SRC-ABL inhibitor bosutinib is one of the five tyrosine kinase inhibitors currently approved for the treatment of Philadelphia chromosome-positive leukemias. Bosutinib has shown activity against all phases of resistant chronic myeloid leukemia that do not harbor the T315I or V299L ABL kinase domain mutations. Bosutinib is overall well tolerated; transient diarrhea is the most common side effect. This article summarizes the pharmacokinetics, pharmacodynamics, safety and efficacy of bosutinib for the treatment of Philadelphia chromosome-positive leukemias.

Bosutinib in the treatment of patients with Philadelphia chromosome-positive (Ph+) chronic myelogenous leukemia: an overview

Bosutinib is an orally bioavailable SRC/ABL tyrosine kinase inhibitor with activity against all phases of resistant chronic myeloid leukemia that do not express the T315I or V299L ABL kinase domain mutations. Bosutinib has a unique toxicity profile that is manageable. This paper provides an overview of bosutinib, covering pharmacodynamics and pharmacokinetic properties, results of treatment in newly diagnosed and previously treated chronic myeloid leukemia patients, as well as common side effects.

Modeling Resistance To Tyrosine Kinase Inhibitors In TEL/ABL-Positive Acute Lymphoblastic Leukemia

TEL/ABL-positive (TEL/ABL+) acute lymphoblastic leukemia (ALL) is a very rare prognostically unfavorable disease. Its pathogenesis is similar to the BCR/ABL-positive (BCR/ABL+) ALL. TEL/ABL kinase activity can be inhibited by tyrosine kinase inhibitors (TKI), which may be used in the therapy of TEL/ABL+ ALL. Thanks to the rareness of TEL/ABL+ ALL there is a lack of knowledge on potential development of TKI resistance and its mechanisms.We have used previously established TEL/ABL+ ALL cell line (Baeumler et al., Cancer Genetics and Cytogenetics, 2008) to create a TKI resistant daughter cell line and study the kinetic and mechanism of its resistance. Resistance to IM was acquired after a very long term (16 months) exposure to slowly increasing doses of imatinib mesylate (IM). We have tested the sensitivity of the newly established resistant cell line against a panel of 7 kinase inhibitors and we found that the cell line acquired resistance not only to IM but also to dasatinib, nilotinib and ponatinib, while it remained sensitive to bosutinib, sunitinib and sorafenib. The resistance to IM remained stable even after the withdrawal of IM from the growth medium and was not abrogated by the co-treatment with valproic acid, which has potential to reverse imatinib resistance in BCR/ABL-positive leukemia (Morotti et al., Cancer, 2006). These findings imply that the cells acquired de novo resistance rather than that primarily resistant subclone was selected, and that the acquired resistance has a genetic background.We have studied several potential mechanism of IM-resistance described in BCR/ABL+ leukemia - mutations in ABL kinase domain, genomic amplification and enhanced expression/autophosphorylation of chimeric protein - but we did not find any of them to be acquired by our resistant cell line. To screen for other potential causes of IM resistance we have performed whole genome genomic profiling by high density SNP arrays, whole genome gene expression profiling and whole exome sequencing. Genomic profiling and subsequent FISH analysis of parental versus resistant cell line revealed 3 discordant aberrations: amplification of 8q24.13-24.3, deletion of 10q26.13-26.3 and deletion of 13q12.12-12.13. Gene expression profiling showed broad changes in expression pattern with at least 2 fold change of expression between parental and resistant cell line in 566 probes representing 469 genes. Interestingly, this 469-gene set contained 5/27 genes from a published gene set which expression correlates with survival in BCR/ABL-positive ALL (Juric et al, Journal of Clinical Oncology, 2007) - the changes in their expression in the resistant versus parental cell line correspond to the changes between the patients with the worse versus better survival. Functional annotation of these 469 differentially expressed genes covers spectrum of diverse biological processes and pathways. Of interest, 2/5 top down-regulated genes in resistant cell line are pro-apoptotic protein PAWR and negative regulator of cytokine signaling SOCS2, both involved in pathways with potential biological relevance for the studied resistance. The data from exome sequencing are currently being analyzed.To summarize, we have successfully established multi-resistant TEL/ABL+ ALL cell line model to study the kinetics and mechanism of TKI resistance. We have demonstrated a very slow kinetics of the IM-resistance induction and excluded several resistance mechanisms typically found in BCR/ABL+ leukemia such as ABL kinase domain mutations, genomic amplification or enhanced expression/phosphorylation of the chimeric protein. We found 3 genomic and multiple gene expression changes to be associated with the resistant phenotype, their contribution to the mechanism of the resistance will be subjected to future studies.Grant support: IGA MZ NT/2121167; UNCE 204012; RVO-FNM64203; RVO-VFN64165 Disclosures:No relevant conflicts of interest to declare.

Patients With Philadelphia-Positive Leukemia With BCR-ABL Kinase Mutations Prior To Allogeneic Transplantation Predominantly Relapse With The Same Mutation

IntroductionTyrosine kinase inhibitors (TKIs) remain the front-line therapy for chronic myeloid leukemia (CML). TKIs also improve remission rates when incorporated into induction and maintenance regimens for Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL). Unfortunately, resistance to TKIs can occur, and is commonly associated with a point mutation in the ABL kinase domain of BCR-ABL. Allogeneic hematopoietic stem cell transplant (HSCT) is indicated for patients with advanced phase CML or for those who fail TKI therapy, and is also often used in Ph+ ALL patients with suitably matched donors. Sadly, relapse after HSCT is fairly common in both advanced phase CML and Ph+ ALL. Studies have explored the benefit of TKIs given post-HSCT to prevent relapse, but there is limited data as to guide their selection and administration. An important consideration is that the “second generation” TKIs may be associated with more toxicity, particularly cytopenias, in the post-HSCT setting. The question arises whether pre-HSCT mutation status should guide prophylactic TKI selection. Given that resistance can occur with a mix of wild type and mutant clones, which clones “win” in the post-transplant setting? Thus, the purpose of this study is to investigate if specific ABL kinase domain mutations persist or recur in CML and Ph+ ALL patients after HSCT. MethodsIn this retrospective analysis, subjects were at least 18 years of age, had undergone allogeneic HSCT at our center between 2000 and 2010 for CML or Ph+ ALL, and had a positive p210 or p190 BCR-ABL transcript by polymerase chain reaction (PCR) in both the pre- and post-HSCT settings. Patients without available records for chart review and those without available pre- and post-HSCT RNA were excluded. Bone marrow and peripheral blood samples obtained prior to HSCT were analyzed for mutations in BCR-ABL. Total RNA was extracted using a TRIzol reagent (Invitrogen, CA, USA) method. A nested PCR, including an initial RT-PCR step for p210 and/or p190 transcripts, was used to amplify a 928-bp product spanning exons 4-9 of the ABL kinase domain. Sanger sequencing of the PCR product was performed using an Applied Biosystems (ABI, CA, USA) 3730xl Analyzer. In patients with a mutation identified, post-HSCT samples were then sequenced. ResultsA total of 95 CML patients and 20 Ph+ ALL patients who underwent HSCT were included in the study. History was notable for pre-HSCT TKI therapy in 64.2% of CML and 90.0% of Ph+ ALL patients, correlating with a mean duration of pre-HSCT TKI exposure of 12.3 and 10.6 months, respectively. At a mean of 2.1 years of follow-up, 30.5% of CML and 70.0% of Ph+ ALL patients had received a TKI post-HSCT either for prophylaxis or relapsed/refractory disease.Sequencing revealed pre-HSCT ABL kinase mutations in 10 (10.5%) of CML and in 4 (20.0%) of Ph+ ALL patients. All 14 harbored at least one mutation known to be associated with resistance to one or more TKIs. Mutations occurred across the kinase domain, including the TKI-binding site, P-loop and A-loop: L248V, G250E, Q252H, Y253H, T315I, F317L, M351T, F359V, R362G, E450K, E459K and F486S. In 9 of the 14 (64.2%), the mutation conferring TKI resistance was also detectable post-HSCT at an average of day +191 (range +25 to +559). Seven of the 14 patients had refractory or relapsed disease by last follow-up and, of these, 5 (71.4%) received a TKI known to be resistant to the pre-HSCT mutation and failed to respond. DiscussionWe have shown that resistance mutations often continue to be detectable in patients with molecular relapse or persistently positive BCR-ABL transcripts after HSCT, attributable to persistence or relapse of a TKI-resistant leukemic clone. This finding suggests that in these patients at greatest risk for disease relapse, resistance patterns may be greatly influenced by any drug-resistant clone present before transplant. Thus, in choosing a TKI for use in the prophylactic setting, if no other clinical features (such as co-morbidities) force the exclusion of a particular TKI, the selection of the TKI with predicted activity against the mutant clone might “hedge the bet.” Notably, our overall rate of ABL mutations in CML is somewhat lower than expected, though this may be attributable to the number of patients without TKI exposure. To our knowledge, this is the largest analysis of ABL kinase domain mutations in a transplant population. Further analysis of additional samples is also underway. Disclosures:No relevant conflicts of interest to declare.

Bone Marrow Beta-Catenin Expression In Chronic Myelogenous Leukemia Patients Treated With Imatinib

Background Imatinib is the standard of care for treating chronic myelogeneous leukemia (CML). However, it is rarely curative and patients often show resistance to the drug. ABL-kinase domain mutations are one mechanism of resistance in CML. Regulation of hematopoietic stem cell survival, as well as disease progression in CML has been linked to the Wnt/beta-catenin pathway. Our study investigated the expression of beta-catenin in the bone marrow (BM) of CML patients treated with imatinib as well as its possible application as a prognostic marker for response to therapy.

Phasing Analysis Of TKI Resistance Mutations In The BCR-ABL1 Kinase Domain and Neighboring Domains Using Next-Generation Sequencing

BackgroundMany CML patients treated with tyrosine kinase inhibitors (TKIs) eventually develop resistance as a result of ABL1 kinase domain (KD) mutations, and sequential treatment with different TKIs may select for multiple BCR-ABL1 mutations. Whether multiple mutations arise in distinct clones (in trans, or polyclonal mutations) or instead are present within the same BCR-ABL1 molecule (in cis, or compound mutations), has been shown to have important implications with respect to TKI sensitivities (Eide, C.A. et al., Blood 2011). Distinguishing between polyclonal and compound mutations, or mutation phasing, for the ABL1 KD has not been clinically practical with standard mutation detection methods. Here we have developed a highly sensitive next-generation sequencing (NGS) assay on the Ion Torrent PGM along with a proprietary data analysis pipeline that together enable deep sequencing of the BCR-ABL1 KD and neighboring domains with a 1% limit of detection and quantitative reporting of mutation phasing. MethodsRT and long range PCR was performed to amplify BCR-ABL1 e1a2/3, e13a2/3, and e14a2/3 fusion transcripts and the PCR products were enzymatically randomly fragmented and ligated with Ion Torrent sequencing adaptors. Size-selected libraries were quantified, pooled, amplified with the OneTouch system and sequenced with the Ion Torrent PGM using 400 bp sequencing chemistry. Sequencing data were analyzed with Torrent Suite 3.4.2 with variant frequency cutoff adjusted to 1%. Variants were further annotated with a proprietary analysis pipeline and variant report was produced after manually reviewing variants by Integrative Genomics Viewer. If more than one non-synonymous variant was reported in a sample, a proprietary phasing analysis pipeline was applied to report the mutation spectrum of all of the combinations of multiple mutations in the sample. ResultsTo validate the accuracy of the sequencing method which employs 400 bp sequencing chemistry, we compared this assay with our previously validated BCR-ABL1 NGS assay based on Ion Torrent 200 bp sequencing chemistry for a set of clinical specimens from CML patients previously treated with TKI. Results were highly concordant and similarly sensitive, with 11/11 variants (frequencies ranging from 2% to 100%) identified with comparable frequencies by both methods.To evaluate the specificity of the phasing analysis, an artificial sample was created by mixing two samples (b2_10 and b2_6) with 6 distinct variants present at ratio of 1:19. Because variants are unique in each sample, any compound mutation composed of b2_10 variant and b2_6 variant identified would be false positive. The false positive error rate (percentage of b2_6 variant as compound mutation with b2_10 variant and vice versa) ranged from 0 – 0.6%, which was consistent with sequencing error rate. We conservatively define a compound mutation as true if it is present in at least 5% of any one of the component variants in the compound mutation. Mutation detection and phasing analysis were reproducible on different chips (314 v2 and 318 v2) and different library preps from the same long range PCR product of BCR-ABL1.Table 1 shows the mutation spectrum from sample b2_6. Of the four variants detected, L248V and G250E were mutually exclusive (in trans), while T315I and M351T were present as compound mutations with each other and, separately, with either L248V or G250E. Notably, >86% of the molecules harbored single mutations, and no compound mutations containing more than 2 variants were observed.Table 1Phasing analysis of sample b2_6L248G250T315M351% of moleculesL248VWTT315IWT2.38%L248VWTWTM351T0.84%L248VWTWTWT32.78%WTG250ET315IWT2.80%WTG250EWTM351T1.20%WTG250EWTWT43.00%WTWTT315IM351T0.16%WTWTT315IWT8.7%WTWTWTM351T1.80%WTWTWTWT6.34% ConclusionsWe have developed and validated a sensitive NGS assay that enables deep sequencing of the BCR-ABL1 KD and neighboring domains along with quantitative mutational phasing. This method has been applied in evaluating >250 clinical specimens for a clinical trial of a third-generation TKI(results reported separately). The ability to easily determine the mutation phasing of a CML patients' mutation profile using this assay will allow for investigations into compound mutation-based resistance mechanisms and may be used to better guide treatment decisions. Disclosures:Li:MolecularMD: Employment. Yan:MolecularMD: Employment. Darwanto:MolecularMD: Employment. Fang:MolecularMD: Employment. Liu:MolecularMD: Employment. Drafahl:MolecularMD: Employment. Toplin:MolecularMD: Employment. Spittle:MolecularMD: Employment. Galderisi:MolecularMD: Employment.

Evaluation Of Multidrug Resistance (MDR) Functional Activity and Expression Levels Of P-Glycoprotein and MDR Related Protein-1 For The Prediction Of Treatment Failure In Chronic Myeloid Leukemia Patients

▪ BackgroundOne of the resistance mechanisms of chronic myeloid leukemia (CML) is multidrug resistance (MDR) mediated by overexpression of P-glycoprotein (P-gp) and MDR related protein-1 (MRP1). The flow cytometric functional assay using efflux inhibitor cyclosporine and Rhodamine-123 (Rho-123 efflux assay) detects MDR functional activity. In this study, we performed Rho-123 efflux assay as well as MRP1 and P-gp expression assays by flow cytometry, and evaluated the performance of each test for the prediction of treatment failure in CML patients. MethodsA total of 229 peripheral blood samples (19 diagnosis and 210 follow-up) from 94 CML patients treated with TKIs were enrolled. These samples were categorized into three subgroups [optimal response (N=120), suboptimal response (N=54), treatment failure (N=36)] defined by the response criteria to treatment with TKIs in CML patients (Best Pract Res Clin Haematol 2009;22:331-41), and ABL1 mutation analysis was performed in some patients (N=18) with treatment failure. In addition, cutoff values (mean + 1.65SD) for each test were determined from 35 normal controls. The Rho-123 efflux assay, MRP1 and P-gp expression assays were performed separately with all samples. In the Rho-123 efflux assay, the MDR functional activity was measured as the ratio of mean fluorescence intensity (mean channel fluorescence, mcf) with and without cyclosporine. In the P-gp and MRP1 expression assays, MRP1 and P-gp expression levels were expressed as the positivity (%) with each representative monoclonal antibody. The results were compared between patients with treatment response (optimal and suboptimal) and failure. The sensitivity/specificity/negative predictive values (NPV)/positive predictive values (PPV) of 3 test results were calculated using estimated cutoff values and compared. ResultsThe cutoff values of Rhodamine-123 efflux assay, MRP1, and P-gp expression assays were calculated to be > 1.68, > 4.18%, and > 5.07%, respectively. The patients with treatment failure showed significantly higher MRP1 expression [mean 14.08% vs. 4.48%, P = 0.038 (diagnosis); 5.24% vs. 3.54%, P = 0.006 (follow-up)] and P-gp expression [12.27% vs. 3.41%, P = 0.027 (diagnosis); 5.25% vs. 3.48%, P = 0.005(follow-up)] than those with treatment response. However, there were no significant differences in the Rhodamine-123 efflux assay (P = 0.769 and 0.199 at diagnosis and follow-up) between 2 groups. Subsequent analysis revealed that 58.7% of follow-up patients with treatment failure show positive results in at least one test, and 53.3% of these patients with negative for the ABL1 kinase domain mutation also show positive results in at least one test. The sensitivity/specificity/NPV/PPV of 3 test results for the prediction of treatment failure were estimated as 22.9%/85.1%/84.6%/23.5% (Rhodamine-123 efflux assay), 37.1%/69.7%/83.9%/20.6% (MRP1 expression assay), and 31.4%/78.8%/84.4%/23.9% (P-gp expression assay). ConclusionMRP1 and P-gp expression were significantly higher in follow-up CML patients with treatment failure than those with treatment response. The patients who experienced treatment failure, had shown significantly higher MRP1 and P-gp expression at diagnosis than those who did not. Low sensitivity and PPV are inevitable for assays measuring MDR, because MDR is only one of mechanism that causes treatment failure. Our study revealed relatively high specificity and NPV in both MRP-1 and P-gp expression assay, and at least half of ABL1 mutation negative patients with treatment failure possessed positivity in MDR tests. Therefore, it can be speculated that the estimation of MRP-1 and P-gp expression levels can provide useful informations for the prediction of treatment failure in CML patients. Disclosures:No relevant conflicts of interest to declare.

Acquisition Of Compound BCR-ABL1 Alleles As A Mechanism Of Resistance To Ponatinib In Chronic Myeloid Leukemia

BACKGROUNDPonatinib targets the inactive conformation of the ABL1 kinase and avoids interacting with the side chain of the mutated 315 residue. In vitro, ponatinib inhibits all single-point BCR-ABL1 mutations. Yet, a significant proportion of patients with chronic myeloid leukemia in chronic phase (CML–CP) do not respond to ponatinib and a subset loses their response during the course of treatment. The mechanisms of resistance to ponatinib are currently not well characterized. OBJECTIVETo determine the impact of compound BCR-ABL1 mutations (polymutants) on the activity of ponatinib. METHODSBCR-ABL1 mutational status was determined in 70 pts with CML-CP post imatinib failure and during dasatinib therapy by DNA expansion of specific clones followed by DNA sequencing of ≥10 clones. Free energy of binding (DGbind) for the unmutated and all mutant BCR-ABL1 kinase/inhibitor complexes were obtained using Molecular Mechanics/Poisson-Boltzmann Surface Area (MM-PBSA) methodology. Single and polymutant BCR-ABL1 alleles obtained by direct mutagenesis and their expression was forced into Ba/F3 cells by electroporation by the pMX/eGFP-BCR-ABL1 expression vector using the Amaxa System. RESULTSAfter imatinib failure, 125 ABL1 kinase domain mutations at 113 amino acid positions were detected in 61/70 (87%) pts, including 38 (54%) with mutations in ≥20% of sequenced clones. Mutations conferring resistance to >1µM imatinib were detected in 30 (43%) pts. Polymutant BCR-ABL1 alleles were detected in 29/70 (41%) pts. These patients received dasatinib for a median of 19 mos (range, 2-52), during which dasatinib-resistant mutations were detected in 10/32 (31%) assessable cases (5 with T315I). Polymutants were present in 16/32 (50%) pts (all of them dead in blast phase). The proportion of clones carrying unmutated BCR-ABL1 was markedly lower in patients who only achieved a minor or no cytogenetic response compared to those achieving a major cytogenetic response (p=0.0001), suggesting exhaustion of unmutated clones and expansion of mutant (and polymutant) clones linked to clinical dasatinib resistance.Then, we performed 3D structural analyses to determine the thermodynamic impact of 21 BCR-ABL1 mutants (11 single and 10 double mutants) in the ability of ponatinib to bind the kinase domain (Table). Most single mutants did not result in high ponatinib resistance (except for E255K, IC50=8.8nM; DGbind=-10.99±0.01). However, the association of any 2 of 3 point mutants (T315I, F317L, V299L) in a dual polymutant produced highly resistant BCR-ABL1 proteins that exhibited fold change values from 19 to 40, compared to the unmutated protein, with T315I/F359V displaying the highest resistance (IC50=61nM; DGbind=-10.23±0.03 kcal/mol), unveiling a mechanism of escape to ponatinib.In Ba/F3-based assays, ponatinib (but not imatinib or dasatinib) was active against Ba/F3-BCR-ABL1T315I cells. Polymutants exhibited very high ponatinib resistance (10-fold higher than that of cells carrying BCR-ABL1T315I). As predicted in silico, BCR-ABL1T315I/F359V was the most resistant polymutant tested. Cell growth inhibition was coupled with CrkL and STAT5 phosphorylation inhibition. Ponatinib, while suppressing STAT5 phosphorylation, could not suppress CrkL phosphorylation in cells expressing the BCR-ABL1T315I/F359V polymutant kinase, even at 100 nM (50-fold the IC50 required to inhibit BCR-ABL1T315I). CONCLUSIONSPolymutants are very frequent in pt samples after TKI failure (particularly after sequential TKI therapy) and tend to induce high ponatinib resistance. Our in silico platform predicted very accurately TKI sensitivity in cells carrying different BCR-ABL1 mutations, which makes it clinically applicable for matching specific mutations to the most effective TKI. Some polymutants require ponatinib concentrations not clinically reachable, thus representing a mechanism of escape to ponatinib therapy through selection and expansion of refractory clones. [Display omitted] [Display omitted] [Display omitted] Disclosures:Talpaz:ariad: Research Funding. Cortes:Ariad: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Teva: Consultancy, Honoraria, Research Funding. Quintas-Cardama:ariad: Consultancy.

A T315I Gate Keeper Mutation Responding To Pegylated Interferon Alfa-2a (Peg-IFN) Monotherapy With Major Molecular Response (MMR4) At 12 Months In An Imatinib-Resistant Chronic Myeloid Leukemia (CML) Patient

Since the IRIS Study in 2000, tyrosine kinase inhibitors (TKI) have become a standard therapy for CML patients in lieu of interferon based therapy. Nevertheless, resistance occurs in roughly 30% of imatinib-treated patients. This resistance is attributable to ABL kinase domain mutations in 50% of cases, in which T315I mutation accounts for 10 to 15%. This latter is resistant to all TKIs available except for ponatinib which enables 75% of complete cytogenetic response (CCyR) in this setting (NEJM November 29, 2012). Before Ponatinib, various strategies were proposed to overcome the T315I mutation including more importantly ‘non-targeted' therapy such as Omacetaxine. IFN is also a ‘non-targeted therapy' known to have a well-established immunological effect. There are also clues about its cellular effects: microarray analyses have shown that IFN alfa can upregulate genes that encode for apoptotic proteins (i.e TRAIL, Fas, caspases 4 and 8 and XAF-1…) conferring the anti-growth effect different from ATP competition specific to TKIs.We report here an 80 years-old imatinib-resistant CML patient harboring the T315I mutation who has been successfully treated with Peg-IFN (Pegasys).His past medical history involved hypertension, asthma and chronic obstructive pulmonary disease. He was diagnosed in July 2008 with a chronic phase CML when he presented with weight loss, splenomegaly, leukocytosis (WBC 80G/l), slight anemia (Hemoglobin 112G/l) and normal Platelet count 240G/L. Karyotyping of 26 metaphase cells in the bone marrow showed Philadelphia (Ph1) chromosome without any additional abnormality. The molecular analysis by real time PCR (Light Cycler 480) detected a BCR-ABL p210 transcript. The Sokal score was high (at 1.24) while the Eutos score was low (31).He started Imatinib 400mg daily in July 2008 and achieved 5% IS BCR-ABL ratio at 3 months and complete cytogenetic response (CCyR) at 12 months. But the patient had never reached the major molecular response (MMR) and ended up losing the cytogenetic response in February 2012 (at 40 months) with an increase of 1.5log in the BCR-ABL ratio. Sequencing analysis for ABL mutation was carried out using the applied biosystems 3500 gentic analyzer. It revealed the T315I mutation at 20%. After a request for Ponatinib was dismissed by our health authorities, we decided to treat the patient with Peg-IFN in June 2012 at the dose of 90 µg per week. At this point, the imatinib had been stopped for 3 months, 8 metaphase cells out of 20 showed Ph1 chromosome without additional abnormality, BCR-ABL ratio was 3% IS, the mutational analysis showed 50% of T315I and 20% of another acquired E255K mutation. But the patient was still in complete haematologic response. figure 1 highlights the decline in the BCR-ABL ratio with MMR reached for the first time at 9 months (from Peg-IFN treatment). Of note, the CCyR was obtained again at this landmark. At 12 months from the Peg-IFN the CCyR was confirmed, the BCR-ABL ratio came very close to MMR4 (0.02% IS) while both T315I and E255K mutations disappeared on mutational analysis. [Display omitted] No side effect was reported except for weight loss when the dose of Peg-IFN was increased to 180 µg per week which was the dose suggested by J.H. Lipton (Leukemia and Lymphoma, March 2007). The average dose in the past 12 months was 90 µg per week. The latter dose is well tolerated and is still ongoing.This observation shows that, though we are in the ponatinib era, there still is a room for interferon, in the management of CP CML patients even in BCR-ABL domain mutated patients because of its different mechanism of action. In this case the Pegylated Interferon a-2-a at the dose of 90µg per week has allowed clearance of both T315I and E255K mutations with a response close to MMR4. Taking into account the slope of the BCR-ABL decline, deeper molecular response is expected before the ASH meeting in December 2013. Disclosures:No relevant conflicts of interest to declare.

Prospective Analysis Of Plasma Cholesterol and Triglycerides In Patients (pts) With Chronic Phase (CP)-Chronic Myeloid Leukemia (CML) During Treatment With The 2nd Generation Tyrosine Kinase Inhibitor (TKI) Nilotinib

BackgroundNilotinib is approved for use in pts with CML after failure of imatinib and in newly diagnosed CP-CML. In the ENESTnd trial, 1st line nilotinib was associated with a higher frequency of hypercholesterolemia than imatinib. Moreover, several studies reported a nilotinib-associated risk of artery occlusive disease (AOD). In ENESTnd, pre-existing treatment for dyslipidemia did not preclude pts from enrolment. To avoid this pitfall and to determine which cholesterol fractions are affected and whether triglycerides (TG) are modified, we prospectively studied the plasma lipid profile of nilotinib-treated pts without drug treatment for dyslipidemia. MethodsPts (n=25) in our center with CP-CML either newly diagnosed or after failure of imatinib were proposed 1st or 2nd line nilotinib provided that neither very high risk for fatal cardiovascular disease (CVD) nor nilotinib-resistant ABL kinase domain mutation were present at baseline. Pts with drug treatment for dyslipidemia at baseline were excluded from this study. Total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C) and TG were measured prior to the start of nilotinib and at 3, 6, 9 and 12 months. Fasting glucose (FG) and glycosylated hemoglobin (HbA1C) were also measured. Results are shown as medians (min-max). ESC/EAS 2012 guidelines were used to determine CVD risk categories and for the management of dyslipidemias. ResultsMedian age at nilotinib initiation was 52 years (19-69), 13 were males (52%). Sokal risk group was low in 11 pts (44%), intermediate in 11 (44%) and high in 3 (12%). Nilotinib was given upfront in 20 pts (80%) and after 1st line imatinib in 5 pts (20%). Median time from diagnosis was 0.8 month (0.4-4.3) in the former and 15.7 months (2.5-47.9) in the latter. Initial nilotinib dosing regimen was 300mg BID in 22 pts (88%) and 400mg BID in 3 pts (12%). Baseline CVD risk factors included age ≥ 45 years in men and ≥ 55 years in women in 33 pts (52%), active smoking or ceased during the previous year in 6 pts (24%), body mass index (BMI) ≥ 25 kg/m2in 14 pts (56%), hypertension in 1 pt (4%), type 2 diabetes mellitus (DM) in 1 pt (4%). Two pts were discovered pre DM. The CVD risk was low in 12 pts (48%), moderate in 11 pts (44%) and high in 2 pts (8%).At baseline, no pt required drug intervention by lipid lowering agents based on LDL-C, 11 pts (44%) had low HDL-C (≤0.4g/L) and 7 (28%) had high TG (≥1.5g/L). Median FG and HBA1C were 0.88g/L (0.63-1.26) and 5.5% (4.7-6.5), respectively. All pts were given lifestyle medical advice. At M3, TC, LDL-C and HDL-C had increased by a mean of 26.9%, 31.8% and 36.3%, respectively. Median TC at baseline and M3 was 1.84g/L (1.17-2.74) and 2.27g/L (1.08-3.62) (p<0.0001). Median LDL-C at baseline and M3 was 1.13g/L (0.69-2.02) and 1.45g/L (0.68-2.7) (p=0.00012). Median HDL-C at baseline and M3 was 0.42g/L (0.16-0.77) and 0.55g/L (0.27-0.99) (p<0.0001). Only 3 pts (12%) had low HDL-C at M3. In contrast, TG decreased by a mean of -22.3%. Median TG at baseline and M3 was 1.02g/L (0.38-4.18) and 0.8g/L (0.41-3.83) (p=0.0003). Only 2 pts (8%) had high TG at M3. No further significant change was observed beyond M3 with median TC, LDL-C, HDL-C and TG at M12 of 2.2g/L (1.15-2.9), 1.3g/L (0.69-1.84), 0.52g/L (0.38-1.27) and 0.83g/L (0.37-1.61), respectively. Finally, 1 pt developed type 2 DM at M12.Three pts (12%) required statins during the first year of nilotinib therapy: 2 pts with a moderate CVD risk at baseline and in whom LDL-C rose above 1.9g/L and 1 pt with a moderate CVD risk at baseline who developed peripheral artery disease (PAD) at M12, leading to nilotinib discontinuation. By M12, the CVD risk changed from low to high in 1 pt due do to the onset of DM and from moderate to very high in the pt with PAD. ConclusionsNilotinib is associated with a significant and early increase in TC, LDL-C and HDL-C. In contrast, a decrease in TG occurs but this may be due to lifestyle medical advice given to our patients. All patients considered for nilotinib therapy should have an evaluation of blood lipids at baseline and early after the start of therapy. Requirement for lifestyle modifications and lipid lowering treatment by statins should be determined at baseline and throughout therapy according to TC, LDL-C and CVD risk category. The role of nilotinib-induced high LDL-C in the onset of AOD needs to be investigated. Disclosures:Rea:Novartis: Honoraria; BMS: Honoraria; Teva: Honoraria; Ariad: Honoraria; Pfizer: Honoraria. Gautier:Novartis: Honoraria. Messas:Novartis: Honoraria. Dombret:Novartis: Research Funding.

Efficacy of the dual PI3K and mTOR inhibitor NVP-BEZ235 in combination with nilotinib against BCR-ABL-positive leukemia cells involves the ABL kinase domain mutation

Imatinib, an ABL tyrosine kinase inhibitor (TKI), has shown clinical efficacy against chronic myeloid leukemia (CML). However, a substantial number of patients develop resistance to imatinib treatment due to the emergence of clones carrying mutations in the protein BCR-ABL. The phosphoinositide 3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway regulates various processes, including cell proliferation, cell survival, and antiapoptosis activity. In this study, we investigated the efficacy of NVP-BEZ235, a dual PI3K and mTOR inhibitor, using BCR-ABL-positive cell lines. Treatment with NVP-BEZ235 for 48 h inhibited cell growth and induced apoptosis. The phosphorylation of the AKT kinase, eukaryotic initiation factor 4-binding protein 1 (4E-BP1), and p70 S6 kinase were decreased after NVP-BEZ235 treatment. The combination of NVP-BEZ235 with a BCR-ABL kinase inhibitor, imatinib, or nilotinib, induced a more pronounced colony growth inhibition, whereas the combination of NVP-BEZ235 and nilotinib was more effective in inducing apoptosis and reducing the phosphorylation of AKT, 4E-BP1, and S6 kinase. NVP-BEZ235 in combination with nilotinib also inhibited tumor growth in a xenograft model and inhibited the growth of primary T315I mutant cells and ponatinib-resistant cells. Taken together, these results suggest that administration of the dual PI3K and mTOR inhibitor NVP-BEZ235 may be an effective strategy against BCR-ABL mutant cells and may enhance the cytotoxic effects of nilotinib in ABL TKI-resistant BCR-ABL mutant cells.

FTY720 induces apoptosis of chronic myelogenous leukemia cells via dual activation of BIM and BID and overcomes various types of resistance to tyrosine kinase inhibitors

PP2A activator FTY720 has been shown to possess the anti-leukemic activity for chronic myelogenous leukemia (CML), however, the cell killing mechanism underlying its anti-leukemic activity has remained to be verified. We investigated the precise mechanisms underlying the apoptosis induction by FTY720, especially focusing on the roles of BH3-only proteins, and the therapeutic potency of FTY720 for CML. Enforced expression of either BCL2 or the dominant-negative protein of FADD (FADD.DN) partly protected CML cells from apoptosis by FTY720, indicating the involvement of both cell extrinsic and intrinsic apoptosis pathways. FTY720 activates pro-apoptotic BH3-only proteins: BIM, which is essential for apoptosis by BCR-ABL1 tyrosine kinase inhibitors (TKIs), and BID, which accelerates the extrinsic apoptosis pathway. Gene knockdown of either BIM or BID partly protected K562 cells from apoptosis by FTY720, but the extent of cell protection was not as much as that by overexpression of either BCL2 or FADD.DN. Moreover, knockdown of both BIM and BID did not provide additional protection compared with knockdown of only BIM or BID, indicating that BIM and BID complement each other in apoptosis by FTY720, especially when either is functionally impaired. FTY720 can overcome TKI resistance caused by ABL kinase domain mutations, dysfunction of BIM resulting from gene deletion polymorphism, and galectin-3 overexpression. In addition, ABT-263, a BH3-mimetic, significantly augmented cell death induction by FTY720 both in TKI-sensitive and -resistant leukemic cells. These results provide the rationale that FTY720, with its unique effects on BIM and BID, could lead to new therapeutic strategies for CML.

Kinase domain mutations and responses to dose escalation in chronic myeloid leukemia resistant to standard dose imatinib mesylate

Imatinib has shown unprecendeted success in the treatment of chronic myeloid leukemia (CML). However, over few years there have been reports regarding the primary and secondary resistance to Imatinib dampening the overall outcome in CML patients. In this study we have tried to assess the effect of dose escalation in patients resistant to standard dose of Imatinib and correlate it with presence of ABL kinase domain (KD) mutations. There were 90 patients resistant to imatinib, out which 29 patients were identified with KD mutations. The most common mutation was T315I , 9 out of 29 patients had it. 35 (38%) responded to dose escalation and had 67% event free survival (EFS) at estimated 2 years. Our results showed that dose escalation can over come resistance in some patients especially those in cytogenetic failure.

Overcoming Resistance in Chronic Myelogenous Leukemia

Resistance in chronic myelogenous leukemia is an issue that has developed in parallel to the availability of rationally designed small molecule tyrosine kinase inhibitors to treat the disease. A significant fraction of patients with clinical resistance are recognized to harbor point mutations/substitutions in the Abl kinase domain, which limit or preclude drug binding and activity. Recent data suggest that compound mutations may develop as well. Proper identification of clinical resistance and prudent screening for all causes of resistance, ranging from adherence to therapy to Abl kinase mutations, is crucial to success with kinase inhibitor therapy. There is currently an array of Abl kinase inhibitors with unique toxicity and activity profiles available, allowing for individualizing therapy beginning with initial choice at diagnosis and as well informed choice of subsequent therapy in the face of toxicity or resistance, with or without Abl kinase domain mutations. Recent studies continue to highlight the merits of increasingly aggressive initial therapy to subvert resistance and importance of early response to identify need for change in therapy. Proper knowledge and navigation amongst novel therapy options and consideration of drug toxicities, individual patient characteristics, disease response, and vigilance for development of resistance are necessary elements of optimized care for the patient with chronic myelogenous leukemia.

Frequency of ABL gene mutations in chronic myeloid leukemia patients resistant to imatinib and results of treatment switch to second-generation tyrosine kinase inhibitors

Background and objectivesTyrosine kinase inhibitors (TKI) have improved the management of patients with chronic myeloid leukemia (CML). However, a significant proportion of patients do not achieve the optimal response or are resistant to TKI. ABL kinase domain mutations have been extensively implicated in the pathogenesis of TKI resistance. Treatment with second-generation TKI has produced high rates of hematologic and cytogenetic responses in mutated ABL patients. The aim of this study was to determine the type and frequency of ABL mutations in patients who were resistant to imatinib or had lost the response, and to analyze the effect of second-generation TKI on their outcome. Patients and methodsThe presence of ABL mutations in 45 CML patients resistant to imatinib was evaluated by direct sequencing and was correlated with the results of the cytogenetic study (performed in 39 cases). The outcome of these patients after therapy with nilotinib or dasatinib was analyzed. ResultsABL mutations were detected in 14 out of 45 resistant patients. Patients with clonal cytogenetic evolution tended to develop mutations more frequently than those without clonal evolution. Nine out of the 15 patients with ABL mutation responded to a treatment switch to nilotinib (n=4), dasatinib (n=2), interferon (n=1) or hematopoietic stem cell transplantation (n=2). ConclusionThe frequency of ABL mutations in CML patients resistant to imatinib is high and is more frequent among those with clonal cytogenetic evolution. The change to second-generation TKI can overcome imatinib resistance in most of the mutated patients.

Abl Kinase Domain Mutations in Imatinib-treated Egyptian Patients with Chronic Myeloid Leukemia

Background: Point mutations within ABL kinase domain (AKD) of the BCR-ABL gene are the most common cause of resistance to Imatinib Mesylate (IM) treated Chronic Myeloid Leukemia (CML) patients. Objectives: To assess the frequency, type and impact of AKD mutations on prognosis in a cohort of Egyptian CML patients. Patients and methods: Serial measurements of BCR-ABL transcripts level in 175 IM treated CML patients were performed using real time quantitative polymerase chain reaction (RQ-PCR). Mutation screening was performed by allele specific oligonucleotide polymerase chain reaction (ASO-PCR) in 72 patients including all 42 non-optimal responders; 28 resistant patients, 18 suboptimal responders in addition to 30 patients randomly selected with stable/ decreasing transcript level representing an optimal responder category. Results: AKD mutations were detected in 16/28 resistant patients (57%) at time of >2-fold rise in BCR-ABL transcript and in none of the 44 optimal or suboptimal responders (0%) with decreasing or stable transcript levels. From 16 positive patients, P-loop mutations were detected in 9 patients; Q252H in 3 patients (19%), Y253H in 2 patients (12%), Y253F in 2 patients (12%) and E255K in 2 patients (12%). T315I was detected in 1/16 (6%) patient. Regarding non P-loop mutation; V299L was detected in one patient (6%), M351T in 4 patients (25%), F359V in 2 patients (12%). One patient had both Y253H and E255K mutations. Ten/16 (62%) patients carrying mutations experienced disease progression versus 1/56 (2%) in non mutation group (p=0.001). Median progression free survival (PFS) and overall survival (OS) of the mutation group was 13.5 months and 37.5 months, respectively versus 42.6 months in non mutation group (p=0.001). The estimated PFS and OS at 49 months in patients with mutations were 37.5% and 56.3% respectively versus 98.2% in non mutation carriers (p 2-folds in IM resistant patients may signal progression that implies testing for AKD mutations and early planning for second generation tyrosine kinase inhibitors (TKIs). P-loop mutations are significantly associated with advanced CML phases and poorer OS than non-P loop mutations. ASOPCR is a valuable tool for detection of mutations in countries where sequencing facilities are not available.

Ponatinib for the treatment of chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia

Ponatinib is a novel, next-generation, small-molecule tyrosine kinase inhibitor with potent activity against the BCR-ABL fusion oncogene as well as all other ABL kinase domain mutations that confer resistance to earlier generation tyrosine kinase inhibitors. Due to its unique structure, it is the only tyrosine kinase inhibitor with the capability to counter the highly resistant T315I or gate-keeper mutation in leukemic cells that express the Philadelphia chromosome. This review will focus on the preclinical pharmacology, pharmacokinetics and clinical utility of ponatinib in the treatment of chronic myeloid leukemia and Philadelphia chromosome-positive adult acute lymphoblastic leukemia.

Overcoming Resistance in Chronic Myelogenous Leukemia

Resistance in chronic myelogenous leukemia is an issue that has developed in parallel to the availability of rationally designed small molecule tyrosine kinase inhibitors to treat the disease. A significant fraction of patients with clinical resistance are recognized to harbor point mutations/substitutions in the Abl kinase domain, which limit or preclude drug binding and activity. Recent data suggest that compound mutations may develop as well. Proper identification of clinical resistance and prudent screening for all causes of resistance, ranging from adherence to therapy to Abl kinase mutations, is crucial to success with kinase inhibitor therapy. There is currently an array of Abl kinase inhibitors with unique toxicity and activity profiles available, allowing for individualizing therapy beginning with initial choice at diagnosis and as well informed choice of subsequent therapy in the face of toxicity or resistance, with or without Abl kinase domain mutations. Recent studies continue to highlight the merits of increasingly aggressive initial therapy to subvert resistance and importance of early response to identify need for change in therapy. Proper knowledge and navigation amongst novel therapy options and consideration of drug toxicities, individual patient characteristics, disease response, and vigilance for development of resistance are necessary elements of optimized care for the patient with chronic myelogenous leukemia.