F359V Mutation Research Articles

Impact of Treatment Cessation on Overall Disease Outcomes in Patients with Chronic Myeloid Leukemia in Chronic Phase (CML-CP) Attempting Treatment-Free Remission (TFR): Findings from Enestfreedom and Enestop

Impact of Treatment Cessation on Overall Disease Outcomes in Patients with Chronic Myeloid Leukemia in Chronic Phase (CML-CP) Attempting Treatment-Free Remission (TFR): Findings from Enestfreedom and Enestop

High Frequency of Point Mutations of BCR/ABL Gene in CML Patients Resistant to Imatinib Therapy - Report of a Novel Mutation.

Imatinib is a drug that competitively targets the adenosine 5′-thriphosphate (ATP)-binding site of the kinase domain of ABL with high specificity and has been stablished as the new standard care in CML. However, resistance to imatinib develops in all phases of the disease, mainly in accelerated phase (AP) and blast crisis (BC). Mutations in the kinase domain of BCR/ABL are the most frequent mechanisms associated to resistance in vivo.Aims: detect mutations in the kinase domain of BCR/ABL in CML patients with primary or secondary resistance to Imatinib.Patients and Methods: we evaluated five patients in blast crisis followed in our institution with primary (1) or secondary resistance (4) to Imatinib. After RNA extraction from peripheral blood samples, amplification of the kinase domain of ABL from BCR/ABL was performed, using a semi-nested RT-PCR. For the first PCR, primers PCR1 were used and for the second PCR, PCR2 primers; PCR 1 - BCRF (sense - 5′ TGA CCA ACT CGT GTG TGA AAC TC) and ABLKinaseR (anti-sense - 5′ TCC ACT TCG TCT GAG ATA CTG GAT T); PCR 2 ABLKinaseF (sense - 5′ CGC AAC AAG CCC ACT GTC T) and ABLKinaseR (anti-sense - 5′ TCC ACT TCG TCT GAG ATA CTG GAT T). PCR product was submitted to direct automated sequencing and compared with normal sequences of BCR-ABL gene (M14752, GenBank).Results: We found mutations in four of five CML patients in blast crisis with secondary resistance to Imatinib (E255K, E279K, F359V). E279K mutation was found in a patient in myeloid blast crisis which become resistant to imatinib after 18 months of therapy. He died of progressive disease(PD). This mutation was not previously described and additional studies are necessary to categorize it. Recently H-bonds were reported between Arg 386, located in A-loop and Asp276/Glu279 located in β3-αC-loop which stabilizes the inactive conformation of the A-loop, necessary for imatinib binding. E255K mutation was found in two patients with lymphoid BC. This mutation is located in P-loop and is related to a poor prognosis. These two patients died with PD. F359V mutation is found outside P-loop and related to a longer survival. In fact, the patient with this mutation was in second CP when mutation was detected and had a good response to dose increase. Later, she was submitted to non-myeloablative BMT and is still alive. Mutations were not identified in one patient, a case with primary resistance, corroborating literature data. In conclusion, BCR/ABL kinase domain mutations are frequent in advanced phases of the disease and have prognostic implications. We described a novel mutation which may have clinical relevance. Early detection of BCR/ABL mutations may help in clinical decisions.

Asciminib, a First-in-Class STAMP Inhibitor, Provides Durable Molecular Response in Patients (pts) with Chronic Myeloid Leukemia (CML) Harboring the T315I Mutation: Primary Efficacy and Safety Results from a Phase 1 Trial

Asciminib, a First-in-Class STAMP Inhibitor, Provides Durable Molecular Response in Patients (pts) with Chronic Myeloid Leukemia (CML) Harboring the T315I Mutation: Primary Efficacy and Safety Results from a Phase 1 Trial

Tyrosine Kinase Inhibitor Resistance in Patients with Chronic Myeloid Leukemia: A 10-Year Study of BCR-ABL Gene Mutation Profile in Russia (2006-2016)

Background. Kinase domain mutations of BCR-ABL gene is the most common cause of tyrosine kinase inhibitor resistance. Aim. To present the data on prognostic value of BCR-ABL mutation burden in Russian patients over the last 10 years. Materials & Methods. The study included 1885 chronic myeloid leukemia (CML) patients with tyrosine kinase inhibitor resistance who were followed up from 2006 to 2016. BCR-ABL point mutations in mRNA samples were analyzed by means of polymerase chain reaction and subsequent Sanger sequencing. Results. In 1257 CML patients with signs of tyrosine kinase inhibitor resistance BCR-ABL expression level was > 1 %. BCR-ABL mutations were detected in 31.8 % of patients. Total mutation count was 467 (70 mutation types). Total count of patients with mutation-associated tyrosine kinase inhibitor resistance decreased from 36.6 % (2006-2008) to 24.95 % (2013-2016) and to marked decrease of 23.12 % in 2014. Detection rate of imatinib-resistant mutations and F359V mutation was shown to decrease within the period from 2010-2011 to 2014-2015. F317L level, which is responsible for dasatinib resistance, considerably increased in 2015. T315I frequency was the highest in 2014, afterwards it was gradually decreasing. Mutation-associated resistance rates varied by region of the Russian Federation. Conclusion. The analysis of trends of mutation incidence in patients with CML can be of extreme significance in longterm prognosis of resistance development and in improvement of treatment planning.

Chemotherapy a Last Resort for Cancer Treatment

Chemotherapy deals with various types of therapeutic techniques such as antimicrobial chemotherapies, gynecological cancers, squamous cell carcinoma, tumorigenesis, chemo resistance, radiotherapy, electro chemotherapy used to treat several dreadful diseases. Imatinib and Nilotinib resistant gene identified in the chronic myeloid leukemia patient. Authors H Cheng et al. found a new mutation N796S in the BCR (exon 10) domain with T315I and F359V mutations, it was evaluated by the next generation exon sequencing technique. The change imposed by mutations was evaluated by measurement of hematologic, cytogenetic and molecular responses. Many studies exerted to explore the function of this new mutation N796S, is unknown and hence study suggests further to be explored in TKIs resistance [1].

Bull's-Eyes and Blind Spots: Pre-Clinical Studies with the Allosteric Inhibitor ABL001 in BCR-ABL1 Compound Mutation-Based Resistance

▪ABL001 (Fig. 1) is a first-in-class allosteric inhibitor of BCR-ABL1 that occupies the vestigial myristoyl pocket, inducing an autoinhibited kinase conformation (Sellers, et al. Proceedings of the 106th Annual Meeting of the AACR; 2015 Apr 18-22; Philadelphia, PA). Phase 1 clinical trials with single-agent ABL001 in relapsed patients with chronic or accelerated phase chronic myeloid leukemia (CML) previously treated with at least two different tyrosine kinase inhibitors (TKIs) are underway. Patients who exhibit relapsed disease associated with the presence of the T315I mutation after at least one TKI are also eligible if no other effective therapy exists. The unexplored possibility of combining an allosteric inhibitor (e.g. ABL001) with a clinically proven BCR-ABL1 TKI such as nilotinib could have important implications for maximum disease control.Given the remote location of the myristoyl allosteric pocket relative to the catalytic site in the kinase domain of BCR-ABL1, a reasonable expectation is that ABL001 should be insulated from point mutations that impart resistance to TKIs such as nilotinib, dasatinib or ponatinib. However, pre-clinical studies demonstrate that the high level of potency exerted on native BCR-ABL1 is not uniformly maintained against a panel of clinically observed point mutations that impart resistance to non-allosteric BCR-ABL1 TKIs (Wylie, et al. Blood (2014) 124, 21, Abstract 398). For example, the T315I and F359V mutations confer substantial in vitro resistance to ABL001 in cell line experiments. The mechanistic reasons for these differences are under investigation.We will report on: (1) Ba/F3 cell line resistance assays predictive of clinical mutation-based escape mechanisms starting from cells expressing native BCR-ABL1 or BCR-ABL1T315I, for ABL001 as a single agent and in combination with a BCR-ABL1 TKI (nilotinib, dasatinib or ponatinib) (2) profiling of ABL001 against T315I-inclusive and non-T315I compound mutations, as a single agent and in combination with a BCR-ABL1 TKI (nilotinib, dasatinib or ponatinib) and (3) Molecular Dynamics studies probing the structural reasons for the selectivity profile of ABL001.In summary, our results to date on ABL001 suggest that single-agent ABL001 retains activity against a subset of non-T315I compound mutants. Preliminary findings indicate that the in vitro resistance profile of ABL001 is non-overlapping with that of TKIs that bind in the catalytic site. In contrast to ABL001, it is known that some myristoyl pocket binders activate rather than induce autoinhibition of BCR-ABL1. Current computational modeling efforts are focused on exploring a similar bifurcation from the perspective of native BCR-ABL1 as compared to ABL001-resistant BCR-ABL1 mutants such as T315I and F359V. Results of ongoing experiments with T315I-inclusive compound mutants will be reported. Our ultimate interest is to identify ABL001-sensitive compound mutants and make this information available to clinicians. [Display omitted] DisclosuresDruker:Millipore: Patents & Royalties; ARIAD: Research Funding; Fred Hutchinson Cancer Research Center: Research Funding; Aptose Therapeutics, Inc (formerly Lorus): Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; Henry Stewart Talks: Patents & Royalties; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; McGraw Hill: Patents & Royalties; Cylene Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; CTI Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Oncotide Pharmaceuticals: Research Funding; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals: Research Funding; Roche TCRC, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sage Bionetworks: Research Funding; AstraZeneca: Consultancy. Deininger:Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Patients with Philadelphia-positive leukemia with Y253H or F359V mutation have a high risk of developing new mutations in the setting of dasatinib resistance

The treatment outcome and development of new mutations in patients with imatinib- and/or nilotinib-failure Philadelphia chromosome positive (Ph+) leukemia with highly nilotinib-resistant mutations (Y253H, E255K/V and F359V/C) were assessed on dasatinib therapy. A total of 111 patients with Ph+ leukemia were grouped into three cohorts by baseline BCR–ABL kinase domain mutation status: no mutation (n = 44), non-nilotinib-resistant mutations (n = 26) or nilotinib-resistant mutations (n = 41). The frequencies of hematological, cytogenetic and molecular responses and clinical resistance to dasatinib were similar among the three cohorts during dasatinib therapy. In dasatinib-resistant patients, new mutations were most frequently observed in the cohort with nilotinib-resistant mutations (p = 0.001). Multivariate analysis revealed that advanced disease and harboring Y253H or F359V mutation before dasatinib were independent predictors of developing new mutations. We conclude that patients with Ph+ leukemia with Y253H or F359V mutation have a high likelihood of developing new mutations in the setting of dasatinib resistance.

Low Level BCR-ABL Mutations and Response to Treatment: A Substudy of the ENEST1st Trial

The presence of BCR-ABL kinase domain mutations below the detection limit of sanger sequencing (low level mutations, LLM) predicts the outcome of subsequent therapy in patients with imatinib resistance (Parker et al. JCO 2011, Blood 2012). We have evaluated LLM in the context of the ENEST1st trial, which addresses the frequency of deep molecular response (MR4) after 18 months on nilotinib 300mg BID (NI) in newly diagnosed patients with chronic myeloid leukemia (CML) in chronic phase (CP). We have investigated the incidence of detectable LLM in the CD34+ progenitor cell compartment and correlated the mutation status (LLM vs. no LLM) with the primary endpoint of the ENEST1st trial, MR4at 18 months. Additionally, we evaluated MMR at 18 months.Seventy eight ENEST1st study patients (pts) provided 10ml of peripheral blood or 2ml bone marrow after written informed consent. CD34+ selection was carried out by MACS® (Miltenyi Biotec) and the CD34+purity was subsequently determined by fluorescent activated cell sorting (FACS).Aliquots of 105 CD34+ were used for RNA extraction, cDNA synthesis and BCR-ABL amplification followed by Ligation PCR (L-PCR) for mutations T315I, Y253H, E255K/V, and F359V. This method has previously been shown to achieve a dynamic detection range of 100% to <0.1% mutant allele (3–3.5 log). No patient showed BCR-ABL kinase domain mutations detected by Sanger sequencing spanning ABL exons 4–9. Fifty one of 78 pts (65%) with 105 CD34+ cells and a documented CD34+ purity of >50% were available for BCR-ABL amplification. Amplification was successful from 41 (52%) of these CD34+samples.A total of 205 L-PCR assays of CD34+ cells identified 29 (14%) mutations (T315Ix12, Y253Hx7, E255Kx8/Vx1 and F359Vx1) from 21/41 pts (51%). The median quantitative level of mutant alleles in CD34+ cells was 0.13 (range 0.06–0.54) BCR-ABLmutant/ BCR-ABLunmutated.One pt was taken off the study because of adverse events before the 3 months evaluation, 2 pts between 3 and 6 months and 1 after the 6 months evaluation (9.7%). Two pts were screened for the trial and did not fulfil the inclusion criteria (4.9%). One pt lost complete hematological response after 6 months and was switched to a different TKI and one pt had a persistent suboptimal response and switched TKI after 12 months on NI (4.9%). These two pts were considered as non-optimal responders at all time points after the change of treatment. All patients except one had typical transcripts (b2/a2 or b3/a2). One pt had an atypical transcript and was not available for MR4 assessment.At 18 months, 31 of 41 patients were evaluable for BCR-ABL levels. Thirteen out of 31 (42%) pts had MR4 or higher at 18 months (6 pts in the LLM group). Twenty six (84%) pts had MMR (10 in the LLM group), while five pts did not reach MMR at 18 months (4 pts with LLM). No statistically significant differences in the proportion of patients achieving MR4 or MMR could be detected between the two groups.In conclusion, T315I, Y253H, E255K or F359V mutations in CD34 selected cells below the detection limit of sanger sequencing do not predict the achievement of MR4 or higher in patients on nilotinib in the ENEST1st study. Therefore, screening for low level BCR-ABL mutations at diagnosis of CML is currently not recommended. DisclosuresGiles:Novartis: Honoraria, Research Funding. Hochhaus:Novartis: Research Funding; BMS: Research Funding; MSD: Research Funding; Ariad: Research Funding; Pfizer: Research Funding. Frank:Novartis: Employment. Lange:Novartis: Consultancy, Honoraria, Research Funding.

Molecular Screening for E255K and F359V Mutations in Non-Responders Iraqi Chronic Myeloid Leukemia Patients to Imatinib Mesylate Therapy

Mutations in the kinase domain (KD) of BCR-ABL are the most prevalent mechanism of acquired resistance to first generation of tyrosine kinase inhibitor Imatinib Mesylate (IM) in chronic myeloid leukemia (CML) patients. Dasatinib and Nilotinib, the second generation of tyrosine kinase inhibitors (SGTKI), have been approved for second-line treatment of CML patients who demonstrate resistance to IM. The identification of E255K and F359Vmutations, which are considered highly resistant to SGTKI nilotinib, lead to a clear-cut decision in the choice of the appropriate SGTKI dasatinib. The aim of this study is to assess the frequency of mutations E255K and F359V in Iraqi CML patients who showed criteria of failure or suboptimal response to IM by using Allele Specific Oligonucleotid-Polymerase Chain Reaction (ASO-PCR). In this cross-section study, 70 patients were diagnosed clinically and hematologically as CML, who attended the Baghdad Medical City /Teaching Hospital/Hematology Unite, during the period between September 2011 to June 2012. They were on IM in different doses (400-800 mg/day) for at least one and a half year. Those patients were classified according to their responsiveness to IM into four groups according to European Leukemia Net. Also, 10 healthy age match subjects were included who served as technical negative control. Peripheral blood (PB) samples were taken from each subject. DNA was extracted using commercial available DNA extraction kit. Molecular screening for the presence of E255K and F359V mutations were done using ASO-PCR. The mean value of age of CML patients was 40.5 ± 2.56 years. Male to female ratio of CML patients was 1.12:1. The result of molecular screening for two mutations showed that all seventy CML patients were negative for both mutations. We conclude that the presence of other variants of studied mutations, another types of point mutations or other mechanisms for resistance to IM such as over expression of BCR-ABL, drug influx and efflux could be the causes of IM resistance.

Philadelphia-Positive Leukemia Patients With The Y253H Or F359V Mutations Have a High Risk Of Developing New Mutations In The Setting Of Dasatinib Resistance

ObjectiveThe clinical significance of individual mutations in Philadelphia-positive (Ph+) leukemia patients is not well-characterized on dasatinib therapy. The treatment outcomes and development of new mutations in imatinib- and/or nilotinib-failure Ph+ leukemia patients with highly nilotinib-resistant mutations (Y253H, E255K/V and F359V/C) were assessed during dasatinib therapy. MethodsOne hundred and eight Ph+ leukemia patients, including 36 with chronic myeloid leukemia in the chronic phase, 18 in the accelerated phase, 40 in the blastic phase and 14 with Ph+ acute lymphoblastic leukemia, who failed imatinib (n=79) or imatinib and nilotinib (n=29) were treated with dasatinib. Those harbored highly dasatinib-resistant mutations (T315I/A, V299L and F317L/V/I/C) were excluded. The patients were grouped into 3 cohorts by baseline BCR-ABL kinase domain (KD) mutation status: no mutation (n=42), non-nilotinib-resistant mutation (any mutation except highly nilotinib-resistant mutations; n=25), or nilotinib-resistant mutation (Y253H, E255K/V or F359V/C; n=41). BCR-ABL KD mutation analysis was detected by direct sequencing at the time of dasatinib resistance. ResultsWith a median follow-up of 6 months (range 2-60 months), the frequencies of hematological and cytogenetic responses and clinical resistance to dasatinib, failure-free survival, progression-free survival and alternative treatment-free survival were comparable by baseline BCR-ABL KD mutation status. Sixty of sixty-four patients (93.8%) underwent BCR-ABL KD mutation analysis at the time of developing clinical resistance to dasatinib. Among the 60 evaluated patients, 33 new mutations were first identified in 29 (48.3%) patients at a median of 3 months (range, 2-26 months) of dasatinib therapy, including 8 of 28 (28.6%) with no mutation, 4 of 9 (44.4%) with non-nilotinib-resistant mutations and 17 of 23 (73.9%) with nilotinib-resistant mutations at baseline. New mutations were most frequently observed in the cohort with nilotinib-resistant mutations (P=0.031). Specially, the subset with the Y253H (12/13, 92.3%) or F359V (5/5, 100%) in the cohort with nilotinib-resistant mutations was more likely to acquire new mutations. Multivariate analyses revealed that harboring the Y253H (HR=9.0, 95%CI: 2.7-30.5; P<0.001) or F359V (HR=7.6, 95%CI: 1.5-39.4; P=0.016) mutation at baseline and a lack of any hematologic response to dasatinib (HR=2.1, 95%CI: 1.4-3.1; P<0.001) were identified as independent predictors of developing new mutations during dasatinib therapy. ConclusionsOur study suggested that Ph+ leukemia patients with the Y253H or F359V mutation had a high likelihood of developing new mutations in the setting of dasatinib resistance. Our data highlight the importance of closer monitoring and mutation follow up for therapeutic choices, especially alternative therapies, which should be considered for patients with the Y253H or F359V mutation during dasatinib therapy. Larger studies are needed to assess the significance of various BCR-ABL KD mutations prior to and during sequential TKI therapy for Ph+ leukemia patients. Disclosures:No relevant conflicts of interest to declare.

ABL Alternative Splicing Is Quite Frequent in Normal Population - Letter

In the article by Lee et al. (1), an alternative splicing of BCR-ABL is proposed as a mechanism for imatinib resistance in chronic myeloid leukemia (CML) patients. This splicing (2), which inserts 35 bp between exons 8 and 9 of ABL (35INS), results in a truncated BCR-ABL protein that the authors compare dynamically with the native protein. We found their work very relevant, especially the finding on the conformational change and its similarity with tertiary structures due to resistance mutations. We were nonetheless surprised by the high proportion of patients who became resistant to imatinib with the alternative splicing; however, the authors did not present data on the presence of the 35-bp insertion in other populations or native ABL mRNA.Using a similar experimental approach to the one described in their article, we analyzed 18 samples from CML patients treated with kinase inhibitors and 24 samples from a normal population. The analysis of the normal population may serve as an estimation of the alternative splicing frequency of the ABL gene. The results showed that 27% of the patients with CML and 41.6% of the normal population expressed 35INS in different degrees (only those with &gt;5% alternatively spliced were considered positive, as described by Lee et al.). In the samples from CML patients, we were unable to correlate either the presence or relative levels of expression of INS35 to tyrosine kinase inhibitor resistance. Several patients who had suboptimal responses to imatinib and an absence of ABL mutations were negative for INS35. One patient, who was marginally positive (5.1%) for INS35 and have a F359V mutation, presented a BCR-ABL/GUS ratio of 22.3%; after being switched to dasatinib treatment, his BCR-ABL ratio decreased to 0.08% in 4 months, but showed an INS35 presence of 24%, which indicates that the splicing was occurring in nonmutated Ph+ cells. The BCR-ABL ratio of the patient, however, further decreased to undetectable levels in the following 4 months. Another patient, who failed to achieve a complete molecular response, was negative for INS35 or ABL mutation at that point; previous samples showed the presence of the alternative splicing in 48.4% of the BCR-ABL mRNA, which indicates that the splicing has decreased over time while the resistance has endured.All these data seem to confirm that INS35 may just be a pseudo-exon not uncommon in the ABL gene processing (3) and therefore unrelated to resistance or sensitivity (4) to inhibitors in CML treatment.No potential conflicts of interest were disclosed.

Combined Effects of novel Heat Shock Protein 90 Inhibitor NVP-AUY922 and Nilotinib in a Random Mutagenesis Screen.

Abstract 3765Poster Board III-701To overcome imatinib resistance, more potent ABL TKIs such as nilotinib and dasatinib have been developed, with demonstrable preclinical activity against most imatinib-resistant BCR-ABL kinase domain mutations, with the exception of T315I. However, imatinib-resistant patients already harbouring mutations have a higher likelihood of developing further mutations under the selective pressure of potent ABL TKIs. The challenge for development of an effective Ph-positive leukemia therapy is therefore to develop an alternative treatment strategy that does not rely solely on kinase domain inhibition but rather results in degradation of the offending BCR-ABL protein regardless of its mutation status. NVP-AUY922 (Novartis) is a novel 4,5-diaryloxazole ATP-binding site heat shock protein 90 (HSP90) inhibitor, which has been shown to inhibit the chaperone function of HSP90 and deplete the levels of HSP90 client protein. In the present study, we investigated the combined effects of AUY922 and Nilotinib on random mutagenesis for BCR-ABL mutation (Blood, 109; 5011, 2007). We performed a comprehensive drug combination experiment using a broader range of concentrations for AUY922 and Nilotinib. Compared with single agents, combination with AUY922 and Nilotinib was more effective at reducing the outgrowth of resistant cell clones. At the highest concentration of Nilotinib, the mutation spectrum narrowed to T315I and E344V by direct sequencing, whereas, at intermediate concentration of AUY922, the resistant clone was recovered by wild-type BCR-ABL only. No outgrowth was observed in the presence of 2 μM Nilotinib and 20 nM AUY922. To assess the in vivo efficacy of AUY922 and Nilotinib, athymic nude mice were injected s.c. with BaF3 cells expressing random mutagenesis for BCR-ABL mutation. 7 days after injection (average tumor volume, 100 mm3), the mice were divided four groups (5 mice per group), with each group receiving either vehicle, AUY922 (50mg/kg; i.p. weekly), Nilotinib (30mg/kg; p.o. once every day), AUY922 (50mg/kg; i.p. weekly) + Nilotinib (30mg/kg; p.o. once every day). Combination with AUY922 and Nilotinib effectively inhibited tumor growth in mice compared with vehicle- or Nilotinib- or AUY922-treated mice. Histopathologic analysis of tumor tissue from AUY922 plus Nilotinib-treated mice demonstrated an increased number of apoptotic cells detected by TUNEL stain. To investigate combined effects of AUY922 and Nilotinib on primary CML cells, NOD/SCID mice were injected i.v. with bone marrow mononuclear cells from a CML patients with F359V mutation. Treatment with AUY922 and Nilotinib demonstrated a marked segregation of apoptotic cells in both the central bone-marrow cavity and the endosteal surface. Furthermore, treatment with AUY922 and Nilotinib inhibited the phosphorylation of GSK-3β and Stat-3 in the endosteal surface. These results suggest that the combination with a HSP90 inhibitor and ABL TKIs may eliminate the CML initiating cells. Taken together, the present study shows that the combination of AUY922 and Nilotinib exhibits a desirable therapeutic index that can reduce the in vivo growth of mutant forms of BCR-ABL-expressing cells. Disclosures:No relevant conflicts of interest to declare.

Comparative in Vitro Cellular Data Alone Is Insufficient to Predict Clinical Responses and Guide Choice of BCR-ABL Inhibitor to Treat Imatinib-Resistant Chronic Myeloid Leukemia.

Abstract 510 Background:Recently, Redaelli et al (J Clin Oncol. 2009;27:469) compared the in vitro inhibitory activity of imatinib, dasatinib, nilotinib, and bosutinib against 18 mutant forms of BCR-ABL (expressed in transfected Ba/F3 cells) associated with imatinib resistance and proposed a chart to assist in the selection of second-generation tyrosine kinase inhibitors (2TKIs) for the treatment of imatinib-resistant CML associated with mutations. However, the predictability of this chart has neither been clinically evaluated nor does it take into account other important clinical factors (e.g. pharmacokinetics (PK)/pharmacodynamics) that may impact response rates to 2TKIs in the presence of mutations. The purpose was to assess the impact of 2TKIs' in vivo plasma levels on the in vitro GI50 data, and to determine if in vitro GI50 data with or without plasma levels correlates with observed clinical responses in imatinib-resistant patients (pts) with mutations. Methods:To enable appropriate comparison of the activity of 2TKIs against specific mutations we modified the original in vitro GI50 data by adjusting it to include an estimate of in vivo Cmax exposure data for each 2TKI. Further refinement was achieved by calculating the Cmax/GI50 values for each agent and normalizing these against imatinib vs wild-type BCR-ABL. To assess the correlation between patient response and in vitro GI50 data, the previously published CCyR rates for pts with specific mutations were plotted according to in vitro GI50 values alone and against the adjusted Cmax/GI50 values. Results:The adjusted Cmax/GI50 data suggest that nilotinib delivers the most potent inhibition of most BCR-ABL mutations in vivo, with dasatinib being the next most potent. However, when either in vitro GI50 data alone or the modified Cmax/GI50 data are considered, there is poor correlation of clinical responses to both nilotinib and dasatinib against several of the mutations in vivo (Figure). Overall, activity of 2TKIs against all mutations was less than expected based on original in vitro GI50 or Cmax/GI50 calculations of systemic exposure. For example, the G250E mutation has similar systemic exposure to nilotinib as the F359V mutation as indicated by Cmax/GI50, but substantial differences are observed in the CCyR rate (60% vs 0%). For dasatinib, the same was observed for the F317L and L248V mutations which have similar exposures to dasatinib but have different CCyR rates (7% vs 41%). Similarly, several mutations with comparable exposure to nilotinib and dasatinib had substantial differences in CCyR rates, suggesting that other factors were influencing responses. For example, the G250E mutation was considered moderately sensitive to both nilotinib and dasatinib based on the adjusted Cmax/GI50; however, CCyR rates on nilotinib were much higher (60%) compared with dasatinib (34%). Similarly, the E255K mutation was considered moderately sensitive to both agents based on the adjusted Cmax/GI50; however, CCyR rates on dasatinib were much higher (38%) compared with nilotinib (13%). [Display omitted] Conclusions:This analysis illustrates the limitations of in vitro inhibition data alone or in combination with PK exposure data in the selection of 2TKI therapy for imatinib-resistant pts with mutations. The current analysis still does not consider parameters such as protein binding and intracellular influx/efflux, among a variety of other clinical factors that could further influence response rates. This tool is also not useful for pts with mutations of unknown in vitro sensitivity, which may represent 15% of all pts with mutations. Currently, clinical responses remain the best approach for selection of 2TKIs in pts with mutations, with only a small subset of mutations having low sensitivity mutations existing for each 2TKI. Other factors, such as patient medical history, comorbidities, and the agents' safety profiles, are also important in selection of 2TKIs. Disclosures:Laneuville:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Wyeth: Research Funding; ChemGenex: Research Funding. DiLea:Novartis: Employment. Mestan:Novartis: Employment. Yin:Novartis: Employment, Equity Ownership. Woodman:Novartis: Employment. Manley:Novartis: Employment.

Screening of Mutations in BCR-ABL Kinase Domain in Chronic Myeloid Leukemia (CML) Patients Treated with Kinase Inhibitors by Denaturing High-Performance Liquid Chromatography (D-HPLC).

Point mutations within the ABL kinase domain are the most frequent mechanism for reactivation of kinase activity of the BCR-ABL gene and have been associated with clinical resistance to tyrosine kinases (TK) inhibitors in CML patients conferring in some of them a poor prognosis. The T315I (Treonine → Isoleucine) is a mutation described in exon 6 of BCR-ABL gene that makes the protein resistant to all kinase inhibitors most currently used for treating CML (imatinib, nilotinib and dasatinib). D-HPLC allows for high throughput mutation screening. This technique is based on heteroduplex formation by PCR products amplified from wild type and mutant alleles. Under optimized denaturing conditions, these heteroduplexes can be distinguished from homoduplex. In this study we screened mutations in exon 6 of BCR-ABL gene in patients treated with kinase inhibitors, in different phases of the disease. We evaluated 85 patients: 9 at diagnosis, 81 in chronic phase, 3 in accelerated phase, one in blast crisis. Thirty four were resistant to imatinib, 10 of them to dasatinib and three had suboptimal response to imatinib. In 9 of 85 (10,5%) samples, D-HPLC showed an abnormal elution profile suggesting the presence of nucleotide changes. Automated sequencing confirmed the presence of two point mutations: T315I (two patients) and F359V (two patients). Five patients requires sequencing confirmation. Patients with T315I mutation failed to imatinib and dasatinib. One of them relapsed after bone marrow transplantation in blast crisis. Patients with F359V mutation were resistant to imatinib. One of them has partial hematological response with dasatinib and the other is in complete molecular response after bone marrow transplantation. D-HPLC seems to be a ship and practical method for routine clinical monitoring for emergence of kinase domain mutations and may be useful for optimizing therapy in CML. Early detection of emerging mutant clones may help in decision-making of alternative treatment.

BCR-ABL Kinase Domain Mutations in the P-Loop and at Imatinib Contact Sites in CML Patients with Imatinib Resistance Are Associated with Higher Maximum Imatinib Doses.

Objectives: Acquired resistance to imatinib (IM) in patients with chronic myeloid leukaemia (CML) is frequently due to mutations of the BCR-ABL kinase domain (KD). Current thinking holds that this results from the selection of pre-existing mutant clones on IM. As new BCR-ABL inhibitors with differential activity against KD mutant BCR-ABL have become available, precise quantification of key mutations even at a low level is required to adequately monitor responses. Here we report that the occurrence of G250E, Q252H, Y253F/H, E255K/V, T315I, F317L, F359V mutations at the time of IM resistance is independently associated with a high maximum IM dose.Patients and Methods: We have developed highly specific allele-specific ligation-PCR assays (L-PCR) to accurately quantify a panel of frequent KD mutations, including G250E, Q252H, Y253F/H, E255K/V, T315I, F317L, F359V. In limiting dilution experiments the L-PCR assays routinely detect between 0.05 and 0.1% of mutant allele in total BCR-ABL, and their average dynamic range is in the range of 4.5 log. Forty-three patients with imatinib failure were analyzed. The median age was 60 (range 20 – 75) years and the median disease duration 64 (range 3–213) months. Eleven patients were in blast crisis, 20 in accelerated phase and 12 in chronic phase.Results: Patients were treated with chemotherapy (48%) and/or interferon alpha (76%) prior to IM. The median maximal IM dose was 600 (range 500–800) mg and the median duration of total therapy until resistance was 15.5 (range 1 to 75) months. Eighteen patients (42%) had dose reductions due to toxicity. At the time of IM resistance, clonal evolution was present in 21/41 (47%) of the patients. L-PCR identified 50 mutations in 29/43 patients (67%) (table1). One, 2, 3 or 4 different mutations were identified in 14, 11, 2 and 2 patients, respectively. The T315I and E255K/V mutations accounted for 32/50 (64%) of all mutations, while Q252H and Y253H were not detected. Twelve mutations (24%) were confirmed by direct sequencing (DS) and an additional M315T mutation and a K247R polymorphism were detected. Thirty eight (76%) mutations were negative by DS for the corresponding mutation from the L-PCR panel but additional mutations (L248V, M351T, H396Rx2, L298Vx2) were identified. All 38 L-PCR positive mutations were confirmed in a second independent experiment. The mutated clone was significantly smaller in mutations with no confirmation by DS (median 0.2, range 0.05–12.63%) compared to positive by DS (median 40.76, range 13.58–100%, p=0.003, Wilcoxon test). The detection of one or more mutations was significantly more frequent in patients with a maximum IM dose of 800mg (n=15/17, 88%) compared to less than 800mg (14/26, 53%, p= 0.02 Fisher's exact test). Multivariate analysis (Wald forward) confirmed that a maximum IM peak dose of 800mg is an independent prognostic parameter to detect a mutation from the L-PCR panel at the time of IM resistance.Conclusions:1.High sensitive testing with L-PCR detects mutations with 4-fold increased frequency compared to direct sequencing.2.Mutations, including p-loop and T315I, may be selected by exposure to higher drug levels.3.The predominance of T315I and E255K/V mutations at a low level is consistent with the findings in newly diagnosed patients with Ph+ALL (Pfeifer et al. Blood 2007).

CMV Colitis in a Patient Receiving Dasatinib.

Introduction: CMV generally produces an asymptomatic or minimally symptomatic acute illness in immunocompetent patients. CMV reactivation commonly occurs in patients with CML after allogeneic transplant but has not been described in those who are treated with tyrosine kinase inhibitors. We report a case of a patient with CML in lymphoid blast phase treated with dasatinib to cytogenetic remission that presented with CMV Colitis.Method: 73-year-old male diagnosed with CML in lymphoid blast crisis 2/2006 treated with chemotherapy and imatinib with good response. He relapsed in 2/2007 with leukostasis and underwent leukopharesis. His BCR/ABL mutation analysis was positive for F359V mutation conferring resistance to imatinib. He was started on dasatinib 70 mg twice daily and had a cytogenetic remission within four months. He presented five months after starting therapy with four weeks of recurrent fevers, chills, malnutrition and diarrhea.Results: CBC and differential revealed only mild thrombocytopenia, Absolute CD4 count of 209; absolute CD8 count of 1495. CMV DNA PCR Quantification of 1500 copies/ml. CT chest abdomen and pelvis showed right sided pleural effusion but no pneumonitis. Flex sigmoidoscopy showed inflamed edematous mucosa with submucosal hemorrhages scattered throughout colon and rectum. Pathology showed chronic colitis, focal viral cytopathic changes consistent with CMV presence. Presence of CMV was confirmed by immunoperoxidase stain for CMV. Treatment was started with IV ganciclovir then switched to oral valganciclovir with good response and resolution of CMV infection.Conclusion: CMV colitis is a well-known complication in immunocompromised hosts after HCT. We are reporting a case of CMV colitis in a patient with CML and had hematologic and cytogenetic remission on a novel tyrosine kinase inhibitor - dasatinib. CMV reactivation may occur in patients who are receiving dasatinib. Furthur evaluation is necessary to determine the incidence of this complication.

Detection of BCR-ABL Point Mutations in Patients with Chronic Myeloid Leukemia (CML) Resistant to Imatinib.

Background: Imatinib is now the standard therapy for newly diagnosed CML patients. However, some patients develop resistance, mainly those treated in advanced stages. Mutations in the kinase domain of BCR/ABL are the most frequent mechanisms associated to resistance in vivo and some of them do not respond even to other kinase inhibitors (nilotinib and dasatinib).Aims: detect mutations in the kinase domain of BCR/ABL in CML patients with primary or secondary resistance to Imatinib and describe their clinical outcome after mutation detection.Patients and Methods: we evaluated 17 CML patients with primary or secondary resistance to Imatinib. After RNA extraction from peripheral blood samples, amplification of the kinase domain of ABL from BCR/ABL was performed, using a semi-nested RT-PCR, to cover amino acids 244–486. PCR product was submitted to direct automated sequencing and compared with normal sequences of BCR-ABL gene (M14752, GenBank).Results: We found seven mutations in ten patients resistant to imatinib: 5 in blast crisis (BC), 3 in accelerated phase (AP) and 2 in chronic phase (CP).Mutations identified: T315I (3), L248V(1), G250E(1), F359V(1), M244V(1), E255K (2) and E279K (1). Five patients were treated with dasatinib (trials CA-180–005, 006, 034 and 035). Patients with T315I mutation (3) did not respond to treatment. One is dead due disease progression and two are currently been treated with hidroxiurea in CP and AP. Patient with mutation M244V is still using dasatinib, presenting major cytogenetic response. Patient with F359V mutation was submitted to a non-myeloablative stem cell transplantation from an identical sibling donor, in AP. The transplantation was uneventful, and no acute or chronic GVHD was observed. On day +57 the chimerism analysis showed a mixed chimerism followed by a hematological relapse. Two lymphocytes infusions were done, on days +77 and +114, with no response. The F359V mutation was still present after the SCT and the patient was included in a phase II trial of dasatinib (CA180-005), which is on going and is in partial hematological response. Patients with other mutations died in blast crisis. In seven patients mutations were not found, but two patients had clonal evolution.Summary/Conclusions: BCR-ABL mutations are frequent in patients resistant to imatinib. Patients with P-loop mutations presented a worse prognosis with rapid evolution to blast crisis and patients with T315I mutation had no response to dasatinib, as recently described. We identified two patients with non-P-loop mutations with good response to dasatinib. P-loop mutations have been associated with poor prognosis, but its prognostic impact depends on patient selection and treatment received after detection of mutation. Mutations detection is helpful in deciding strategies to overcome resistance to imatinib.