Nilotinib-resistant Mutations Research Articles

Ultra-deep sequencing mutation analysis of the BCR/ABL1 kinase domain in newly diagnosed chronic myeloid leukemia patients

Ultra-deep sequencing detects low-frequency genetic mutations with high sensitivity. We used this approach to prospectively examine mutations in the BCR/ABL1 tyrosine kinase from patients with newly diagnosed, chronic-phase chronic myeloid leukemia (CML) treated with the tyrosine kinase inhibitor nilotinib. Between May 2013 and November 2014, 50 patients from 18 institutions were enrolled in the study. We screened 103 somatic mutations and found that mutations in the P-loop domain were the most frequent (173/454 mutations in the P-loop) and noted the presence of the V299 L mutation (dasatinib-resistant/nilotinib-sensitive) in 98 % of patients (49/50). No patients had Y253H, E255 V, or F359 V/C/I mutations, which would recommend dasatinib rather than nilotinib treatment. The S417Y mutation was associated with lower achievement of a major molecular response (MMR) at 6 months, and the V371A mutation was associated with reduced MMR and MR4.5 durations (MMR for 2 years: 100 % for no mutation vs. 75 % for mutation, P=0.039; MR4.5 for 15 months: 94.1 % vs. 25 %, P=0.002). Patients with known nilotinib-resistant mutations had lower rates of MR4.5 achievement. In conclusion, ultra-deep sequencing is a sensitive method for genetic-based treatment decisions. Based on the results of these mutational analyses, nilotinib treatment is a promising option for Korean patients with CML.

In chronic myeloid leukemia patients on second-line tyrosine kinase inhibitor therapy, deep sequencing of BCR-ABL1 at the time of warning may allow sensitive detection of emerging drug-resistant mutants.

BackgroundImatinib-resistant chronic myeloid leukemia (CML) patients receiving second-line tyrosine kinase inhibitor (TKI) therapy with dasatinib or nilotinib have a higher risk of disease relapse and progression and not infrequently BCR-ABL1 kinase domain (KD) mutations are implicated in therapeutic failure. In this setting, earlier detection of emerging BCR-ABL1 KD mutations would offer greater chances of efficacy for subsequent salvage therapy and limit the biological consequences of full BCR-ABL1 kinase reactivation. Taking advantage of an already set up and validated next-generation deep amplicon sequencing (DS) assay, we aimed to assess whether DS may allow a larger window of detection of emerging BCR-ABL1 KD mutants predicting for an impending relapse.Methodsa total of 125 longitudinal samples from 51 CML patients who had acquired dasatinib- or nilotinib-resistant mutations during second-line therapy were analyzed by DS from the time of failure and mutation detection by conventional sequencing backwards. BCR-ABL1/ABL1%IS transcript levels were used to define whether the patient had ‘optimal response’, ‘warning’ or ‘failure’ at the time of first mutation detection by DS.ResultsDS was able to backtrack dasatinib- or nilotinib-resistant mutations to the previous sample(s) in 23/51 (45 %) pts. Median mutation burden at the time of first detection by DS was 5.5 % (range, 1.5–17.5 %); median interval between detection by DS and detection by conventional sequencing was 3 months (range, 1–9 months). In 5 cases, the mutations were detectable at baseline. In the remaining cases, response level at the time mutations were first detected by DS could be defined as ‘Warning’ (according to the 2013 ELN definitions of response to 2nd-line therapy) in 13 cases, as ‘Optimal response’ in one case, as ‘Failure’ in 4 cases. No dasatinib- or nilotinib-resistant mutations were detected by DS in 15 randomly selected patients with ‘warning’ at various timepoints, that later turned into optimal responders with no treatment changes.ConclusionsDS enables a larger window of detection of emerging BCR-ABL1 KD mutations predicting for an impending relapse. A ‘Warning’ response may represent a rational trigger, besides ‘Failure’, for DS-based mutation screening in CML patients undergoing second-line TKI therapy.

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.

Bcr-Abl Kinase Domain Mutations and Other Mutations on the Response to Nilotinib in Bcr Abl –Positive Chronic Myeloid Leukemia - a Study from Eastern India

ABSTRACT Aim: The emergence of ABL point mutations is the most frequent cause for imatinib resistance in CML patients and can occur during any phase of the disease. The treatment of CML has changed with the introduction of tyrosine kinase inhibitors (TKI).The aim of the study is to investigate two potential resistance mechanisms i.e., mutations of the BCR-ABL tyrosine kinase domain (TKI) and additional chromosomal abnormalities (ACA), during 1st and 2nd generation TKI treatment in CML. Methods: Between September 2002 and February 2012, 66 patients with Ph-positive CML and imatinib resistance or intolerance were treated with nilotinib and were analyzed. Karotyping and BCR-ABL TKD mutation screening are performed in 66 imatinib-resistant CML patients who were on imatinib at the time of loss of hematologic response (HR), cytogenetic (CyR)or molecular response (MR).Imatinib-resistance mutation analysis (Qualitative) were detected by nested RT-PCR and Sanger sequencing. Out of 66 patients, 34 received escalated imatinib, 32 received nilotinib. Results: In 66 BCR-ABL-positive imatinib-resistant patients,11 different BCR-ABL TKD mutations were detected. Lack of HR was noted in 20 patients where CyR was not evaluated. Imatinib resistance mutation analysis was promoted by no HR (n = 12); loss of HR after achieving CyR (n = 27); failure to achieve CyR milestones (n = 19); loss of CyR (n = 6); development of extra medullary BC (n = 2),The analysis revealed no mutations in 33 patients, M351T in10 patients, G250E in 7 patients, F317L in 7 patients, M244V in 4 patients, E355G in 3 patient, and T315I in 2 patients. The seven novel mutations (F317L, G250E, M244V, M351T, F359V, T315I, E355G) were seen in our patient population. T315I was found in 2/66 patients who demonstrate loss of response. Conclusions: Compared with other western studies the incidence of the T315I mutation is very low in our study population. For patients with TKI resistance, mutation and ACA screening may play a role in identifying patients with poorer prognosis.If nilotinib-resistant mutation was detected, we preferred dasatinib .We plan for bosutinib, panotinib omacetaxine (SC route) in third line therapy in nilotinib-resistant, different mutation-positive CML. Disclosure: All authors have declared no conflicts of interest.

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.

Efficacy and Safety Of Ponatinib Following Failure Of Nilotinib In Patients With Chronic Phase Chronic Myeloid Leukemia (CP-CML) In The PACE Trial

Efficacy and Safety Of Ponatinib Following Failure Of Nilotinib In Patients With Chronic Phase Chronic Myeloid Leukemia (CP-CML) In The PACE Trial

4-Year Outcome Of 215 Patients With Newly Diagnosed Chronic Myeloid Leukemia (CML) Treated Frontline With Nilotinib In Investigator-Sponsored Studies. A Report From The Gimema CML Working Party

BackgroundNilotinib is a potent and selective BCR-ABL inhibitor approved for the frontline treatment of CML. The latest update (4-yr follow-up) of the ENESTnd study demonstrated sustained superiority of nilotinib vs. imatinib (Hochhaus et al, EHA 2013, abstract 712). The CML Italian Registry of Nilotinib is the largest series of patients treated frontline with nilotinib-based regimens, outside of Company-sponsored trials. Therefore, it represents an important resource for an independent evaluation of the outcome of such patients. Aimsto analyze the response rates and outcome in an independent cohort of patients treated frontline with nilotinib-based regimens in Italy. MethodsThe CML Italian Registry of Nilotinib includes 215 patients, enrolled in 2 multicenter phase II studies conducted by the GIMEMA CML WP (ClinicalTrials.gov. NCT00481052 and NCT00769327) with nilotinib 300 mg or 400 mg BID as initial treatment; 123 patients received a sequential treatment with nilotinib and imatinib, with a 3-mo rotation period. The median age was 53 years (range 18–86). Ten out of 215 patients (5%) had a high EUTOS score. The median follow-up was 43 months (range 18–69 months). We analyzed: the rates of Complete Cytogenetic Response (CCyR) and Major Molecular Response (MMR); the overall survival (OS; any death included), progression-free survival (PFS; progression to accelerated/blast phase [AP/BP] and deaths for any cause), failure-free survival (FFS; failures according to ELN 2013 recommendations and deaths for any cause), and event-free survival (EFS; events: failures, permanent discontinuation of nilotinib for any cause, including deaths). ResultsRates of CCyR were 72% and 92% by 3 months and 12 months, respectively. Rates of MMR were 56% and 83% by 3 months and 12 months, respectively. The cumulative rates of CCyR and MMR were 93% and 92%, respectively. Overall, events were recorded in 64 (30%) patients: 31 (14%) patients permanently discontinued nilotinib for adverse events or intolerance; 22 (10%) patients failed therapy according to ELN 2013 recommendations, including 8 (3.7%) patients that progressed to AP/BP; 11 (5%) patients permanently discontinued nilotinib for other reasons. All progressions to AP/BP occurred within the first year of therapy, and all patients subsequently died. Nilotinib-resistant mutations were identified in 5 of these patients (4 T315I; 1 Y253H). No difference in the rate of progression to AP/BP was observed between patients receiving nilotinib alone or nilotinib and imatinib in sequential schedule. Overall, 15 (7%) patients died, in 7 cases for reasons unrelated to CML progression. The 4-year OS, PFS, FFS, and EFS were 93%, 93%, 86%, and 69%, respectively. ConclusionThese Italian nilotinib registry data provide an independent and unbiased overview of the therapeutic effects of nilotinib, with high and early rates of complete cytogenetic and major molecular response. All progressions (3.7%) to AP or BP occurred within the 1st year of therapy; however, all cases were fatal, emphasizing how crucial is the prevention of AP/BP. With a median follow-up of 43 months, 69% of patients were still on nilotinib, and 93% were alive and progression-free. AcknowledgmentsEuropean LeukemiaNet, COFIN, Bologna University, BolognAIL [Display omitted] Disclosures:Gugliotta:Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria. Castagnetti:Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria. Bocchia:Novartis and Bristol Mayer Squibb: Honoraria. Soverini:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Saglio:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Cavo:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Onyx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Baccarani:Novartis: Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Pfizer: Honoraria; Ariad: Honoraria. Rosti:Novartis: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Speakers Bureau; Ariad: Consultancy; Roche: Speakers Bureau.

BCR-ABL1 kinase domain mutations may persist at very low levels for many years and lead to subsequent TKI resistance

Background:BCR-ABL1 mutation analysis is recommended for chronic myeloid leukaemia patients. However, mutations may become undetectable after changing therapy, and it is unknown whether they have been eradicated.Methods:We examined longitudinal data of patients with imatinib-resistant mutations, which became undetectable by Sanger sequencing to determine whether mutations could reappear, and the related circumstances.Results:Identical imatinib- and nilotinib-resistant mutations reappeared following further therapy changes in five patients, and was associated with subsequent nilotinib resistance in four.Conclusion:The data suggest that some BCR-ABL1 mutations may persist at undetectable levels for many years after changing therapy, and can be reselected and confer resistance to subsequent inhibitors.

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

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

Chronic Myelogenous Leukemia Patients with Highly Nilotinib-Resistant BCR-ABL Mutations Demonstrate Similar Efficacy to Dasatinib, but Have a Higher Likelihood of Developing New Mutations in the Event of Clinical Resistance

AbstractAbstract 3755 Objective:Assess the outcome of imatinib- and/or nilotinib-failure chronic myelogenous leukemia (CML) patients with pre-existing, highly nilotinib-resistant mutations (Y253H, E255K/V and F359V/C), during dasatinib therapy. Methods:Sixty-one CML patients who failed imatinib (n=44) or imatinib and nilotinib (n=17), including 20 in chronic phase (CP), 16 in accelerated phase (AP) and 25 in blast phase (BP), were switched to dasatinib, except for patients with highly dasatinib-resistant mutations (T315I/A, F317L/V/I/C and V299L). Patients were grouped into 3 cohorts: no mutation (n=22), no-specific mutation (any mutation except highly nilotinib-resistant mutations; n=15), or specific mutation (Y253H, E255K/V or F359V/C; n=24), according to baseline BCR-ABL mutation status. Cumulative incidence frequencies of hematological response (HR), complete hematological response (CHR), major cytogenetic response (MCR), complete cytogenetic response (CCR) and major molecular response (MMR) were compared. Probabilities of clinical resistance to dasatinib (according to European LeukemiaNet recommendations), progression-free survival (PFS) and overall survival (OS), and dynamics of BCR-ABL mutations during dasatinib therapy were also assessed. Results:Prior nilotinib was associated with a higher proportion of specific mutation versus no mutation (50.0% vs 13.6%, P=0.001) and non-specific mutation (50.0% vs 13.3%, P=0.02). Patients harboring specific mutation also had a higher frequency of advanced phase CML before dasatinib treatment than patients with no mutation (83.3% vs 50.0%, P=0.008), and a similar frequency to those with non-specific mutation (83.3% vs 66.7%, P=0.266). Response, PFS and OS rates, and follow-up duration were similar among the 3 cohorts by baseline mutation status, as shown in Table. Probability of clinical resistance to dasatinib in patients with specific mutation was not significantly different to those with no mutation (54.2% vs 77.3%, P=0.100) or non-specific mutation (54.2% vs 33.3%, P=0.204) despite that there was a significant difference between the cohort with no mutation and non-specific mutation (77.3% vs 33.3%, P=0.008). Among 32 patients who developed clinical resistance to dasatinib and were assessed for BCR-ABL mutation status, newly detectable mutations were identified in 14 patients and most frequently occurred in those already harboring specific mutation versus those with no mutation (76.9% vs 26.7%, P=0.008) and non-specific mutation (76.9% vs 0%, P=0.015) at baseline. T315I (9/14, 64.3%) was the most common newly acquired BCR-ABL mutation. Harboring specific mutation compared with other mutation states at baseline (76.9% vs 16.7%, P=0.001), and developing hematological resistance rather than cytogenetic resistance during dasatinib therapy (65.0% vs 8.1%, P=0.002), were identified as risk factors associated with the development of new mutations. Conclusions:Imatinib- and/or nilotinib-failure patients with highly nilotinib-resistant BCR-ABL mutations demonstrate similar efficacy to dasatinib as those with no or any other mutation. However, patients with highly nilotinib-resistant mutations had a higher likelihood of developing new mutations at the time of clinical resistance. More extensive studies are needed to assess the significance of various BCR-ABL mutations prior to and during tyrosine kinase inhibitor therapy for CML.Table:Response, survival and clinical resistance to dasatinib by baseline BCR-ABL mutation statusMutation statusP valueNo mutation n=22Non-specific mutation n=15Specific mutation n=24HR, n (%)16 (72.7)14 (93.3)21 (87.5)0.202CHR, n (%)16 (72.7)12 (80.0)19 (79.2)0.832MCR, n (%)a9 (69.2)8 (80.0)12 (70.6)0.826CCR, n (%)a8 (61.5)8 (80.0)10 (58.8)0.511MMR, n (%)a3 (23.1)5 (50.0)4 (23.5)0.2813-year PFS, %84.673.765.20.4693-year OS, %75.680.854.00.798Clinical resistance to dasatinib, n (%)17 (77.3)5 (33.3)13 (54.2)0.027Follow-up (months), median (range)31 (2..C48)24 (2..C48)7 (1..C48)0.277a40 patients were evaluated including 13 with no mutation,10 with non-specific mutation and 17 with specific mutation Disclosures:No relevant conflicts of interest to declare.

Low-Level Bcr-Abl Kinase Domain Mutations Are Very Rare In Chronic Myeloid Leukemia Patients Who Are In Major Molecular Response After 12 Months of First-Line Nilotinib Therapy.

AbstractAbstract 1666▪Bcr-Abl kinase domain (KD) mutations have been documented in a variable proportion of residual Ph+ cells in some patients (pts) with stable response to imatinib. In general, the higher the sensitivity of the screening method, the greater the number of pts found to harbour mutations – although the clinical significance of low-level mutations is still controversial. Nilotinib is an imatinib derivative more selective and more potent in Bcr-Abl inhibition – with reported major molecular response (MMR) rates at 12 months ranging between 44 and 85%. We wondered whether low-level Bcr-Abl KD mutations are detectable in pts in MMR on nilotinib. To address this issue, we retrospectively analyzed the samples collected after 12 months of therapy from 12 pts enrolled on the GIMEMA CML working party study of nilotinib 400 mg BID as frontline treatment of CML. These pts had all achieved MMR (>3-log reduction in Bcr-Abl transcript according to the IS) between 3 and 6 months from nilotinib start and had a Bcr-Abl/Abl ratio ranging from 0.009%IS and 0.02%IS at the time of analysis. In all 12 pts, MMR was maintained at last follow up (24 months after nilotinib start), with four pts achieving complete molecular response (CMR; >4.5-log reduction) at 18 months and one at 24 months. Pts were equally distributed across Sokal risk categories (low Sokal risk, n=4; intermediate Sokal risk, n=4; high Sokal risk, n=4). Screening for low level mutations was performed by cloning the Bcr-Abl KD (a.a.240-502) in a bacterial vector and sequencing 150 independent clones for each patient. To rule out false positive results, a mutation was considered to be present in a sample if it was detected on both strands of two or more independent clones. The KD of Abl in 3 healthy individuals was analyzed in parallel. Our approach showed evidence of Bcr-Abl KD mutations in only 1 out of 12 pts analyzed. In this high Sokal risk patient, a Q346L mutation was detected in 3/150 independent clones, and an additional T315I mutation was present in 2 out of these 3 clones. The Q346L has never been reported in imatinib-resistant pts, neither is it among the mutants emerged in the in vitro random mutagenesis screenings for nilotinib-resistant mutations – hence it should be devoid of any clinical relevance. The T315I, in contrast, is known to be highly insensitive to nilotinib both in vitro and in vivo. Nevertheless, in this patient the Bcr-Abl transcript level continued to decline down to CMR (achieved after 24 months from nilotinib start). It may also be hypothesized that the Q346L-T315I double mutant may have decreased degree of resistance with respect to the T315I mutant. The remaining 11 pts scored negative for mutations – showing only evidence of some single, mutated clones as also the three healthy individuals did. To increase the number of pts analyzed and assess whether high Sokal risk pts are more prone to develop low level mutations, we have now set up a massively parallel amplicon sequencing approach of the Abl KD on the Roche 454 GS FLX instrument, allowing to increase both throughput and sensitivity (a 100.000x coverage will allow to detect mutations with a lower detection limit of 0.01%). Our current results suggest that a) low level Bcr-Abl KD mutations seem to be very rare in pts in MMR after 12 months of nilotinib therapy, a milestone achieved by the majority of pts. This may be due to the higher efficacy of nilotinib resulting in a more rapid clearance of the reservoir of Ph+ cells where mutations arise; b) as hypothesized by some authors, tyrosine kinase inhibitor-resistant mutations at low levels do not always predict for subsequent relapse and should not trigger changes in therapy.Supported by: European LeukemiaNet, AIL, AIRC, PRIN 2008, Fondazione del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integrated program (PIO), Programma di Ricerca Regione – Università 2007 – 2009. Disclosures:Rosti:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Baccarani:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Martinelli:Novartis: Consultancy, Honoraria; BMS: Honoraria; Pfizer: Consultancy.

Prevalence of T315I, Dasatinib-Specific Resistant Mutations (F317L, V299L, and T315A), and Nilotinib-Specific Resistant Mutations (P-loop and F359) at the Time of Imatinib Resistance in Chronic-Phase Chronic Myeloid Leukemia (CP-CML).

Mutations in the catalytic domain of ABL kinase (AKD) are a major mechanism of resistance to imatinib. Over 70 mutations in more than 50 amino acid residues have been reported to date. The ‘gatekeeper’ mutation, T315I, which causes complete resistance to all three FDA-approved tyrosine kinase inhibitors (TKIs), was reported to be not uncommon among a heterogeneous set of patients who had failed first-line imatinib therapy. In addition, the F317L, V299L, and T315A mutations were reported to convey a high degree of resistance to dasatinib, and higher frequency mutations within the P-loop (Y253H/F, E255V/K) and F359 mutations were associated with a high degree of resistance to nilotinib. We studied the prevalence of AKD mutations in the START-C phase II trial of dasatinib in patients who have failed imatinib (resistance or intolerance). Baseline mutation data were available for 95 of 99 patients with imatinib intolerance, and 274 of 288 patients with imatinib resistance. Of these patients, 13 (14%) with imatinib intolerance and 136 (50%) with imatinib resistance had AKD mutations. Of the 149 patients with mutations, only 3 (2%) had the T315I mutation. A total of 57 (38%) subjects had mutations in the P-loop (between 248–256): 13 patients with Y253H/F (9%), and 6 patients with E255V/K (4%). Four subjects (3%) had the F317L mutation, and 8 (5%) had F359 mutations. No subjects with V299L or T315A mutations were detected at baseline. The rates of complete cytogenetic response (CCyR) were 52% in patients with any mutation, 69% in patients with Y253H/F, 40% among those with E255V/K, 0% for T315I mutations, 0% for F317L mutations, and 50% for F359 mutations. Patients without mutations achieved a 55% rate of CCyR. These results confirm that select P-loop and F359 mutations are sensitive to dasatinib, while F317L and T315I mutations are resistant to dasatinib treatment. However, the overall incidence of these dasatinib-resistant mutants is low. In contrast, nilotinibresistant mutations in the P-loop (Y253H/F, E255V/K) or at F359 are more common, representing 15% and 5% of all patients with mutations, respectively. Therefore, the likelihood of harboring a nilotinib-resistant mutation at the time of imatinib resistance appears higher than the likelihood of harboring a dasatinib-resistant mutation, and suggests mutation testing may become instrumental for choosing between the various second-line TKI inhibitors to optimize outcomes.

Correlation of Clinical Response to Nilotinib with BCR-ABL Mutation Status in Advanced Phase Chronic Myelogenous Leukemia (CML-AP) Patients with Imatinib-Resistance or Intolerance.

The 2nd-generation bcr-abl inhibitor nilotinib is more potent than imatinib (IC50 <30 nM) against unmutated bcr-abl and active against 32/33 imatinib-resistant BCR-ABL mutants in vitro. We investigated the in vivo activity of nilotinib stratified by the baseline BCR-ABL mutation status in 127 imatinib-resistant or -intolerant CML-AP patients (pts) enrolled in an open-label phase II trial of nilotinib. Eighty-five pts (85/127, 67%) were screened prior to nilotinib therapy for BCR-ABL kinase domain mutations by direct sequencing. Of the 85 pts, 75 (88%) were resistant to imatinib and 10 (12%) were intolerant using standard published criteria. Twenty-two different baseline mutations involving 19 amino acids were identified in 50 (59%) pts analyzed. Other 35 (41%) pts did not have a baseline mutation. The most frequent mutation types identified included M351T (8 pts), G250E (7 pts), Y253H (6 pts), M244V (5 pts), F359V (5 pts) and T315I (5 pts). Twenty-two percent of pts with baseline mutations (11/50) showed more than one mutation (9 with two, 1 with three, and 1 with four mutations). All baseline mutations occurred in imatinib-resistant pts but none in intolerant pts. After 12 months of therapy, confirmed (confirmed in two consecutive analyses 4 week apart) hematologic response (HR) was achieved in 48% (21/50), major cytogenetic response (MCR) in 20% (10/50), and complete cytogenetic response (CCR) in 16% (8/50) of imatinib-resistant pts with baseline mutation versus 44% (12/25), 40% (10/25), and 20% (2/25) of imatinib-resistant pts without baseline mutation, respectively. Responses appeared to be affected by the in vitro sensitivity of the mutant clone against nilotinib. Pts with less sensitive mutation (cellular IC50 of >200nM: Y253H, E255K, E255V, F359C) representing 13% (11/85) of all patients assessed for baseline mutation, showed 13% (1/11) HR and 13% (1/11) MCyR compared to 74% (17/28) and 18% (5/28) respectively in the mutant group with IC50 of ≤200 nM. The nilotinib resistant T315I mutation occurred in 5 pts. Only one of these 5 pts who had T315I and G250E dual mutation achieved HR conceivably reflecting the sensitivity of G250E or non-mutant clone to nilotinib. At the time of data analyses, 50% of pts with baseline mutation were free of disease progression versus 62% of pts without baseline mutation. Rate of progression was 64% (7/11) in the group with less sensitive mutations and 60% (3/5) in pts. with T315I. However, the mutants most frequently associated with progression were F359V and M244V both having 4/5 pts (80%) progressed. In summary, BCR-ABL kinase domain mutations were identified at baseline in 59% of all pts in this cohort and in 67% of pts with imatinib resistance. Responses were observed across a broad spectrum of mutant genotypes. The rate of responses and disease progression may be affected by the baseline mutation types, although a larger data set with longer follow up is needed to further establish the correlation.