Imatinib-resistant Pts Research Articles

Treatment-Free Remission in Ponatinib-Treated CML Patients: The Italy-Tfr Experience

Background: In recent years, treatment-free remission (TFR) has become a new therapeutic goal and is considered feasible even by international expert guidelines, at least in patients (pts) with a stable deep molecular response (DMR). From the EPIC study, we learned that many pts achieve a DMR very early, regardless of Sokal risk (Lipton, Lancet Oncol 2016). The Optic study showed that early dose reduction is associated with a containment of cardiovascular (CV) risk while maintaining therapeutic efficacy. In the cohort of pts initially treated with 45 mg QD, the number of events remained around 10% (Cortes, Blood 2021). Recommendations for treatment discontinuation suggest that it should be considered only after one line of treatment or after a second line if first-line TKI was switched for intolerance. Since 2016, there are case reports in the literature of imatinib-resistant pts, treated with ponatinib in second or following lines due to intolerance or resistance to previous TKIs, with or without ABL1 mutations, who reduced the daily dosage from 45 to 15 mg, still achieving a DMR and then discontinued ponatinib due to toxicity, obtaining a persistent TFR (Wisniowski, Blood 2016; Engel, J Oncol Pract 2016; Cohen, Leuk Lymphoma 2020). Methods: Within the context of the Italy-TFR retrospective and prospective study (NCT04769947) which is ongoing and actually includes 467 pts, we collected the data of 19 pts who attempted TFR after ponatinib treatment. Results: Median age was 44 years at diagnosis, 60 years at ponatinib discontinuation. 8 pts were male (42%); Sokal score was available for 14 pts: 5, 3 and 6 pts were low, intermediate and high risk, respectively. ELTS score was available for 11 pts: 6 pts, 4 and 1 were low, intermediate and high risk, respectively. 6 pts were treated with ponatinib in second line, 9 in third line, 3 in forth line. First-line therapy was available for 16 pts and was imatinib for 11 pts, nilotinib for 2 pts, dasatinib for 3 pts. Median duration of front-line treatment was 16 months (mos, IQR 11-34). Data on second line therapy was available for 11/12 pts: 5 pts were treated with nilotinib, 6 with dasatinib. Median duration of second-line treatment was 47 mos (IQR 19-58). Third line therapy was dasatinib for all 3 pts, with a duration of 1, 18 and 139 mos. Reason for starting ponatinib was intolerance for 8 pts, resistance for the others. For 7/11 resistant pts response at ponatinib start was CHR for one pt, MR1 for 3 pts, MR2 for 3 pts. 3 pts carried 1 ABL1 mutation at ponatinib start: 1 F359V, 1 M244V and 1 T315I; 1 pt carried 2 mutations: 1 Y253H and 1 E499E. Patients received ponatinib for a median of 47 mos (IQR 34-54). Starting dose was 45 mg for 6 pts, 30 mg for 8 pts and 15 mg for 5 pts. Response at 3 mos was available for 17 pts and was MR2 for 2 pts, MR3 for 9 pts and DMR for 6 pts. Median time to DMR was 18 mos (IQR 6-36). Ponatinib dose before treatment discontinuation was 15 mg for all but 4 pts: 1 was dose reduced from 45 mg to 30 mg, 1 stayed on 45 mg; 2 pts who had started with 15 mg dose reduced to 15 mg every other day. 11 pts discontinued in MR4, 7 pts in MR4.5 and 1 pt in MR5. Median duration of DMR before discontinuation was 25 mos (IQR 14-37). Reasons for discontinuation were: physician-patient shared choice for 9 pts, intolerance for 6 pts (among whom 1 had pancreatitis, 1 worsening of cardiac heart failure, 1 tachycardia, and 3 low-grade GI symptoms), need to start chemotherapy for a second neoplasia for 2 pts, pregnancy for 1 pt and auto-suspension for 1 pt. Only one pt had a CV adverse event (stroke) during treatment with ponatinib. 16/19 pts maintained TFR for a median time of 19 mos (IQR 8-32) and were still off treatment at the last follow-up. 3 pts had to resume treatment after 3, 4 and 9 mos. All 3 restarted treatment with ponatinib and reobtained DMR. All pts were alive at last follow-up, except for 2 pts who died for a gastric and a lung cancer. Conclusions: Our data confirmed the efficacy of ponatinib in second or later lines of treatment. All pts, including those treated for resistance, achieved optimal responses at 3 mos. We also showed that TFR may be feasible for some pts treated in second or subsequent lines, not only for intolerance. Follow-up time extension and collection of data from additional pts are desired to confirm our observations, and seem to be of utmost importance to better define the characteristics of pts who might benefit from ponatinib with the purpose of TFR attempt.

Detection of BCR-ABL1 Compound and Polyclonal Mutants in Chronic Myeloid Leukemia Patients Using a Novel Next Generation Sequencing Approach That Minimises PCR and Sequencing Errors

BackgroundBCR-ABL1 kinase domain (KD) mutations are the most common known cause of resistance to tyrosine kinase inhibitors (TKIs) in CML. Mutation analysis is critical for selection of subsequent TKI therapy after treatment failure. Low level and compound mutants (>1 KD mutation in the same molecule) may also lead to therapy failure. However, compound and multiple polyclonal mutants cannot be distinguished by conventional methods as they determine the average genotype of all molecules. Next generation sequencing (NGS) has the potential to sensitively detect these mutants, however sequencing and PCR errors confound the detection of true, low level mutants using current approaches. Indeed, we demonstrated that the reported frequency of BCR-ABL1 compound mutants may be over estimated due to PCR recombination artifacts that mimic compound mutations (Parker Blood 2014). More reliable methods are needed to appropriately assess the impact of various mutations on patient (pt) outcome.AimTo develop a clinically applicable NGS assay that can robustly distinguish BCR-ABL1 compound and polyclonal mutants.MethodWe have developed a novel NGS assay termed Single Molecule Consensus Sequencing (SMCS) that involves tagging individual BCR-ABL1 cDNA molecules before library amplification, enabling identification and elimination of most PCR and sequencing errors. NGS was performed on the Illumina MiSeq, 2 x 300 bp; aa 244 - 407 of the KD was examined. Reads derived from an initial BCR-ABL1 molecule are identified bioinformatically by virtue of sharing the same tag sequence. The consensus sequence of reads with the same tag is determined using automated variant calling and filtering algorithms. The consensus sequence represents the sequence of the initial BCR-ABL1 cDNA molecule (Fig A).ResultsTo test the validity of SMCS, we examined 10 samples lacking KD mutations and 5 mock samples created by mixing compound mutant plasmids or pt samples. Examination of raw sequencing reads revealed a complex spectrum of mutants, similar to previous clinical reports. SMCS enabled bioinformatic filtering of these artifacts, largely eliminating PCR and sequencing error, and exclusively reported the compound and polyclonal mutants known to be present in the mock samples. We estimated the background error rate to be ~2x10-5 per base. The error spectrum was consistent with DNA damage causing first round PCR errors.SMCS was used to retrospectively examine samples of 46 pts (36 CP, 2 AP, 8 BP) who were resistant to ≤4 TKIs (1st and 2nd generation). 71 mutations were previously detected by Sanger sequencing in these samples, collected before starting next line TKI. Within the region examined using SMCS, there was 100% detection concordance with Sanger sequencing.We compared the results of SMCS with an amplicon NGS method performed at another centre for 24/46 pts (Ion Torrent, depth ~10000). Ion Torrent detected 34 compound mutants in 24 pts. Of the 30/34 that were within the region examined by SMCS we only detected 8. Based on observations in Parker Blood 2014, 14 of the 22 compound mutants not detected by SMCS were likely to be PCR recombination artifacts. The other 8/22 were low level (1 - 4%) and most (6/8) involved mutations rarely/never reported in TKI resistant pts so may also be artifacts (Fig B). We detected 3 additional compound mutants in these 24 pts, plus 5 in the remaining 22/46 pts. The compound mutants detected by SMCS were consistent with the pts’ TKI treatment history.ConclusionWe demonstrated detection of BCR-ABL1 compound and polyclonal mutants in pt samples using a novel NGS assay that has the potential to overcome technical artifacts generated with other published methods. Whilst there is no gold standard method that can accurately detect low level compound mutations, SMCS has correctly identified sequencing and PCR recombination artifacts using mock samples. The accuracy and clinical utility of SMCS for sensitive compound and polyclonal mutant detection is currently being validated in another group of 200 imatinib resistant pts. The frequency of compound mutants detected in pts with >1 mutation by SMCS in the current analysis (35%) is approximately half of that reported previously, which suggests the published frequency may have been overestimated. Our novel assay takes an important step towards enabling a more concrete understanding of the mutation spectra in pts and their association with resistance. [Display omitted] DisclosuresYeung:Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Lustgarten:ARIAD Pharmaceuticals Inc: Employment, Equity Ownership. Hodgson:ARIAD Pharmaceuticals, Inc.: Employment, Equity Ownership. Rivera:ARIAD Pharmaceuticals Inc: Employment, Equity Ownership. Hughes:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Honoraria, Research Funding. Branford:Novartis: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Otsuka: Honoraria, Research Funding.

Ultra Deep Sequencing (UDS) Allows More Sensitive Detection Of Tyrosine Kinase Inhibitor (TKI)-Resistant BCR-ABL Mutations That Would Influence Therapeutic Decision At The Time Of Switchover To Second- Or Third-Line Therapy

Ultra Deep Sequencing (UDS) Allows More Sensitive Detection Of Tyrosine Kinase Inhibitor (TKI)-Resistant BCR-ABL Mutations That Would Influence Therapeutic Decision At The Time Of Switchover To Second- Or Third-Line Therapy

Multiple Low Level Mutations Identifies Imatinib Resistant CML Patients At Risk of Poor Response to Second-Line Inhibitor Therapy, Irrespective of the Resistance Profile of the Mutations

Abstract 111▪▪This icon denotes a clinically relevant abstractSpecific imatinib resistant BCR-ABL1 mutations confer clinical resistance to nilotinib (NIL; Y253H, E255K/V, T315I, F359V/C) and/or dasatinib (DAS; V299L, T315I/A, F317L/I/V/C). Therefore, mutation analysis is recommended for CML patients (pts) after imatinib failure to facilitate selection of appropriate therapy. However, around 40% of chronic phase (CP) pts without these NIL/DAS resistant mutations also fail second line inhibitor therapy. For imatinib resistant pts without these mutations at the time of commencing NIL/DAS therapy (switchover) we investigated whether sensitive mutation analysis could identity pts at risk of poor response to subsequent therapy.Switchover samples of 220 imatinib resistant pts (DAS n=131, NIL n=89) were analysed by direct sequencing (detection limit 10–20%) and sensitive, high throughput mass spectrometry (mass spec; Sequenom MassARRAY, detection limit 0.05–0.5%), which detects 31 common BCR-ABL1 mutations (approximately 89% of mutations detected in pts receiving imatinib). We previously demonstrated that mass spec could detect NIL/DAS resistant mutations at switchover in an extra 9% of pts compared to sequencing and that these low level resistant mutations were associated with subsequent failure of these inhibitors (Parker et al, JCO. 2011 In Press). Therefore, for the current analysis, pts with NIL/DAS resistant mutations detected by either method (n=45) were excluded since response is already known to be poor in these cases.In the switchover samples of the remaining 175 pts, 159 mutations were detected in 86 pts by mass spec, but just 108 mutations were detected in 89 pts by sequencing. Thirteen rare mutations detected by sequencing were not included in the mass spec assay design. Mass spec detected all other mutations detected by sequencing, plus an additional 64 low level mutations. Multiple NIL/DAS sensitive mutations (≥2 mutations) were detected at switchover in more of the 175 pts by mass spec (34/175, 19%; 2–9 mutations per pt) than sequencing (16/175, 9%; 2–3 mutations per pt), P=.009. We divided pts into 2 groups; those with multiple mutations detected by mass spec at switchover (n=34) and those with 0/1 mutation (n=141), and investigated the impact of multiple mutations on response to subsequent NIL/DAS therapy. Pts with 0 or 1 mutation, and similarly pts with 2 or >2 mutations, were grouped together, as no difference in response was observed. The median follow up for CP, accelerated phase and blast crisis pts was 17 (2–33), 18 (1–33) and 3 (1–27) mo, and the frequency of multiple mutations was 18%, 24% and 18%, respectively. During follow up, multiple mutations at switchover was associated with lower rates of complete cytogenetic response (CCyR; 21% vs 50%, P=.003, Fig 1A) and major molecular response (MMR; 6% vs 31%, P=.005, Fig 1B), and a higher incidence of acquiring new NIL/DAS resistant mutations detectable by sequencing (56% vs 25%, P=.0009, Fig 1C). At 18 mo, the failure-free survival rate (European LeukemiaNet recommendations) for CP pts with multiple mutations at switchover was 33% compared to 51% for CP pts with 0 or 1 mutation (P=.26, Fig 1D).The number of mutations detected per pt by mass spec at switchover (max of 9, 8 of 86 pts with mutations had ≥4, 9%) far exceeded the number concurrently detected by sequencing (max of 3). This suggests that mass spec detected a pool of subclonal mutants, each with a small survival advantage after imatinib therapy that was insufficient for their clonal predominance. Multiple low level mutations may be a marker of an increased propensity for subsequent selection of resistant mutations, possibly driven by genetic instability, demonstrating the advantage of a sensitive multiplex mutation assay.In conclusion, sensitive mutation analysis identified a poor-risk subgroup with multiple mutations that were not identified by sequencing. This subgroup represented 15.5% of the total cohort (34/220), who would not otherwise be classified as being at risk of poor response on the basis of their mutation status. These pts did not have NIL/DAS resistant mutations at switchover; however, they had a lower incidence of CCyR and MMR, and higher incidence of acquiring new NIL/DAS resistant mutations during NIL/DAS therapy compared to pts with 0 or 1 mutation. This poor-risk subgroup may warrant closer monitoring or experimental approaches to reduce the high risk of kinase inhibitor failure after imatinib resistance. [Display omitted] Disclosures:Hughes:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees. Branford:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Research Funding; Ariad: Research Funding.

Incidence of the BCR-ABL Kinase Domain Mutations in Imatinib Treated CML Patients: Russian Experience

Abstract 4420In the period from 18.01.06 to 25.05.11 we have performed mutation analysis in CML pts with the obvious manifestation of resistance against imatinib. Chronic phase CML patients have been treated by imatinib and monitored by means of RQ-PCR using IS units. At first signs of the evident BCR-ABL level increase (more than 5–10% IS), the mutation analysis has been performed by means of direct PCR fragment sequencing. We have used original primers that enable to search for mutations in the area spanning from a3 to a11 ABL exons of BCR-ABL. Sequencing has been performed for 274 CML pts that were suspected to acquire imatinib resistance. Among them there were 47,45% (130/274) of females (median age 51 years, from 15 to 73), 52,55% (144/274) of males (median age 50 years, from 15 to 74). In the group of mutation negative pts (80/274 – 29,2%) the BCR-ABL level was more than 9% (IS). Interestingly, in this group there were a little bit more females than in the total cohort of pts (58,7% against 47,45%, p=0,034). The median age of the mutation negative females (51 years) was quite the same as in the total cohort, whereas the median age of mutation negative males significantly differed from the one in the total cohort (58 vs. 51 year, p=0,041). 70,8% (194/274) pts tested occurred to be mutation positive (42,8% females (83/194), 57,2% males (111/194)). 214 point mutations of 38 different types have been detected, among them T315I (26/194 – 12%), G250E (26/194 – 12%), T317L (16/194 – 7,4%), F359V (15/194 – 7,0%), H396R (15/194 – 7,0%), M244V (15/194 – 7,0%), E255K (12/194 – 5,6%), Y253H (12/194 – 5,6%), E255V (9/194 – 4,2%), L248V (8/194 – 3,7%), E355G (6/194 – 2,8%), M315T (6/194 – 2,8%), Q252H (5/194 – 2,3%), L387F (4/194 – 1,8%), S348L (4/194 – 1,8%), F311I (3/194 – 1,4%), F359C (3/194 – 1,4%), E255D (2/194 – 0,9%), E275K (2/194 – 0,9%), E279A (2/194 – 0,9%), K247R (2/194 – 0,9%), E292V (1/194 – 0,46%), E334G (1/194 – 0,46%), E450K (1/194 – 0,46%), E459A (1/194 – 0,46%), E459K (1/194 – 0,46%), F359I (1/194 – 0,46%), F486S (1/194 – 0,46%), L383F (1/194 – 0,46%), P441L (1/194 – 0,46%), Q252M (1/194 – 0,46%), Q491L (1/194 – 0,46%), T305I (1/194 – 0,46%), T345I (1/194 – 0,46%), V299A (1/194 – 0,46%), Y312C (1/194 – 0,46%), T520S (1/194 – 0,46%). As rare events we have observed some other types of mutations. In one CML patient with primary imatinib resistance there was a G425Stop (1/194 – 0,46%) mutation which caused a significant truncation of the C-end of the BCR-ABL protein. There were also 2 highly imatinib resistant pts with deletions of exon a7 ABL (2/194 – 0,9%). One more unusual mutation was an insertion of 72 bp between the a8 and a9 exons of ABL. The origin of this insertion was a middle part of the 8th intron of the ABL gene. This sequence is arranged as a typical human exon because it is flanked by donor and acceptor splicing signals. We found this mutation in 3 imatinib resistant CML pts (3/194 – 1,4%). This mutation gives rise to the abnormal stop-codon within the exon a9 due to a CD's frameshift. In all pts with point L248V mutation (8/194 – 3,7%) we have also observed additional abnormal variant of BCR-ABL gene as a splicing isoform lacking 81 bp of exon a4. The point mutation L248V (742C>G) exchange the motif CAAGCT for CAAGGT and the latter is a strong splice acceptor site. It seems that this cryptic splice acceptor site in the middle of 4a exon carrying 742C>G point mutation may compete with natural splice acceptor site from 4th intron. It gives rise to two BCR-ABL transcripts: one carrying a point mutation and the other one with 81 bp's deletion of the 3'end of exon 4a, without frameshift. These findings suggest that in the case of mutational analysis the considerable attention should be paid not only to BCR-ABL point mutations but also to the possible emergence of the other changes of the BCR-ABL mRNA structure which may occur due to arbitrary activity of splicing machinery. Our findings also suggest that among imatinib resistant CML pts some of them already acquired mutations that prevented adequate response for the 2nd generation of TKI. In our study 12% of BCR-ABL mutation positive CML pts resistant to imatinib (n=26/194) had T315I which also render nilotinib and dasatinib resistance. 23,8% (n=51/194) were positive for mutations that decreases nilotinib response (E255K/V, F359C/V, Y253H) and 7,5% (n=16/194) were positive for T317L that hinders dasatinib response. Disclosures:No relevant conflicts of interest to declare.

Drug Resistance and Bcr-Abl Kinase Domain Mutations In Philadelphia-Positive Acute Lymphoblastic Leukemia From the Imatinib to the 2nd-Generation Tyrosine Kinase Inhibitor Era: The Main Changes Are In the Type of Mutations, but Not In the Frequency of Mutation Involvement

Abstract 575Incorporation of the tyrosine kinase inhibitor (TKI) imatinib in the frontline treatment of Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) patients (pts) has significantly improved the anti-leukemic efficacy of induction therapy. In contrast to chronic myeloid leukemia (CML), however, responses are short-lived and relapse is frequently associated with the selection of Bcr-Abl kinase domain (KD) mutations, fostered by the high genetic instability of Ph+ ALL cells. The advent of the 2nd-generation TKIs dasatinib and nilotinib has brought additional treatment options both for newly diagnosed and for imatinib-resistant pts. To analyze the changes they have brought in mutation frequency and type, we have reviewed the database recording the results of BCR-ABL KD mutation analyses done in our laboratory from January 2004 to June 2011. Overall, 781 tests on 258 Ph+ ALL pts (number of tests per pt, range: 1–15) were performed by denaturing high-performance liquid chromatography (D-HPLC) followed by direct sequencing of D-HPLC-positive cases.One hundred and fourty-three pts were analyzed because of imatinib resistance. One hundred and one out of 143 (71%) pts scored positive for one or more KD mutations. Similarly to what is know to occur in CML, hematologic and cytogenetic resistance were by far more frequently associated with mutations than molecular resistance (Bcr-Abl transcript increase as assessed by RT-Q-PCR). Overall, mutations at thirteen residues were detected. In contrast to what can be observed in CML, three mutations were by far the most frequent, accounting for almost 75% of the mutated cases: T315I (n=38 pts, 37%), E255K/V (n=19 pts, 18%) and Y253H (n=19 pts, 18%). The other mutations were, in order of frequency: F359V/I, M244V, M351T, F317L, G250E, Q252H, L387M, D276G, L248R, E279K. Nine out of 103 (9%) pts had two mutations, in the same (2 pts) or in different (7 pts) subclones.In 84 pts who were analyzed because they were reported to have developed resistance to dasatinib (n=72) or nilotinib (n=12) as 2nd- or 3rd-line TKIs, 65 (77%) had newly acquired mutations (57/72 dasatinib-resistant pts and 8/12 nilotinib-resistant pts). The most frequent newly acquired mutation in this setting was the T315I, detected in 35/57 (61%) cases acquiring mutations on dasatinib and in 2/8 cases acquiring mutations on nilotinib. Other recurrent newly acquired mutations were F317L, V299L, T315A in dasatinib-resistant pts and Y253H and E255K in nilotinib-resistant pts. Thirty out of 65 pts (46%) were positive for multiple mutations (2 to 4 mutations, in the same or in different subclones or both) that emerged under the same TKI in 11 cases (37%) and accumulated as a consequence of multiple lines of TKI therapy in the remaining 19 (63%) cases.Mutation analysis was also performed in 15 resistant pts enrolled in a clinical trial of dasatinib as first-line treatment for Ph+ ALL. Twelve pts were positive for mutations; 11/12 had a T315I.Sixty-one pts were analyzed at the time of diagnosis in order to assess whether TKI-resistant mutations could already be detectable. Only two pts (3%) were positive for mutations: one patient had an F311L that disappeared after one month of nilotinib treatment; an additional patient was positive only by D-HPLC, but not by the less sensitive direct sequencing – most likely for the T315I mutation that shortly after the start of dasatinib treatment outgrew and led to resistance. Taking advantage of the recent availability of a next-generation sequencing platform (Roche 454), allowing high sensitivity (0.01%) mutation scanning of the KD, samples collected at the time of diagnosis and during follow-up from selected Ph+ ALL cases who developed mutations and resistance to TKI therapy were retrospectively analyzed – but the mutations were not always already detectable at diagnosis.In conclusion: a) although 2nd generation TKIs may ensure a more rapid debulking of the neoplastic clone and have much fewer insensitive mutations, long-term disease control remains a problem and the T315I becomes an even tougher enemy; b) the clinical usefulness of mutation screening of Ph+ ALL pts at diagnosis before TKI start, even with highly sensitive approaches is low – not all mutations pre-exist since genetic instability remains high and fosters mutational events anytime during treatment.Supported by PRIN, FIRB, AIL and AIRC. Disclosures:Soverini:ARIAD: Consultancy; Novartis: Consultancy; Bristol-Myers Squibb: Consultancy. Luppi:CELGENE CORPORATION: Research Funding. Foà:Bristol-Myers Squibb: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Baccarani:Bristol-Meyers Squibb: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.

Health-related quality of life (HRQoL) of bosutinib (SKI-606) in imatinib-resistant (IM-R) or imatinib-intolerant (IM-I) chronic phase chronic myeloid leukemia (CP CML).

6620 Background: Bosutinib, a dual Src/Abl tyrosine kinase inhibitor (TKI), has demonstrated efficacy in a phase 1/2 single-arm study of patients (pts) with IM-R or IM-I CP CML. Given the potential for CP CML pts receiving 2nd-generation TKIs to be treated for significant durations, evaluation of concomitant HRQoL is important. The effect of bosutinib on HRQoL was assessed as an exploratory endpoint. Methods: In the phase 2 part of the trial, 253 pts with IM-R (n = 170) or IM-I (n = 83) CP CML were treated with bosutinib 500 mg/d. HRQoL was measured using the 44-item Functional Assessment of Cancer Therapy–Leukemia (FACT-Leu), which consists of 5 domains: Physical Well-being (PWB), Social/Family Well-being (SFWB), Emotional Well-being (EWB), Functional Well-being (FWB), and Leukemia Subscale (LeuS); and 3 summary scales: Treatment Outcome Index (TOI), FACT-General, and FACT-Leu Total. FACT-Leu items are rated on a scale of 0-4. Scores are obtained for each scale by summing item responses; higher scores reflect better HRQoL. The FACT-Leu was completed at baseline, wks 4, 8, and 12, every 12 wks thereafter, and at treatment completion. Within-cohort comparisons were assessed using paired t-tests. Results: In this interim analysis, median follow-up was 97 wks (range, 2-215 wks); 51% of pts were still receiving treatment. At 12 wks, IM-R pts reported significant improvements (P <0.05 to P <0.01) in the PWB, EWB, LeuS, Total, and TOI; IM-I pts reported improvement in the LeuS (P <0.05). At 24 wks, IM-R pts reported improvements from baseline in the EWB, LeuS, Total, and TOI; IM-I pts noted significant improvements (P <0.001 to P <0.05) in the PWB, EWB, LeuS, Total, and TOI. At 48 and 96 wks, the LeuS (P <0.01) and TOI (P <0.05) remained improved for IM-R pts, with additional improvements in PWB, Total (P <0.05), and EWB (P <0.01) observed at 96 wks. EWB, LeuS, Total, and TOI were improved for IM-I pts (P <0.001 to P <0.05) at 48 wks, although not at 96 wks. Conclusions: When examined in the context of previously reported efficacy, these early HRQoL data suggest that bosutinib is associated with improved HRQoL among pts with IM-R or IM-I CP CML who remained on therapy.

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.

Patients with Chronic Myeloid Leukemia In Chronic Phase Carrying More Than One BCR-ABL Kinase Domain Mutation Exhibit Poorer Response Rates and Outcomes to Second-Line Dasatinib Compared to Those with No or Only One BCR-ABL Mutation

AbstractAbstract 2297 Background:BCR-ABL kinase domain mutations occur in 30%-90% of patients (pts) who develop resistance to imatinib. A recent analysis reported that 48% of pts with imatinib resistance or suboptimal response had a baseline mutation at the start of dasatinib therapy (Müller 2009). After 2 years of follow-up, dasatinib treatment of imatinib-resistant pts resulted in notable response rates (complete cytogenetic response [CCyR]: 43% vs 47%) and promising progression-free survival (PFS, 70% vs 80%) in pts with or without baseline mutations, respectively. However, some pts present with two or more co-existing mutations in one or more clones. The outcome of these pts after dasatinib therapy has not been described in detail. Methods:This is a retrospective analysis of pts with CML in chronic phase (CML-CP) receiving dasatinib who were imatinib resistant or intolerant and who had >1 BCR-ABL mutation at baseline. Pts with CML-CP from the phase 3 dose optimization trial (-034) and phase 2 START-C (-013) and START-R (-017; dasatinib only population) studies were evaluated. Since the frequency of >1 mutation was expected to be lower than the rates reported for the presence of any mutation, the inclusion of all 3 studies provided a larger population to determine pt responses and outcomes. A 2-year database was used for all 3 studies. Mutations were detected by conventional Sanger sequencing (sensitivity 10%-20%) after nested RT-PCR amplification of the BCR-ABL transcript. BCR-ABL polymorphisms were excluded from analysis. 1150 pts were included in the analysis: -034 (n=662), -013 (n=387), and -017 (n=101). Similar baseline characteristics among those analyzed included: median age (51-55 years), male (47%-53%), median duration of CML (54-64 months [mos]), prior imatinib therapy lasting >3 years (41%-53%) and prior response rates to imatinib (complete hematologic response [82%-92%], major cytogenetic response [MCyR, 28%-42%], and CCyR [15%-21%]). Only the rate of imatinib intolerance differed among the 3 trials: 26% for -034 and -013, whereas imatinib-intolerant pts were not eligible for the -017 study. Results:Of 1150 pts analyzed, baseline mutation data were available for 1043 dasatinib-treated pts (Table); 641 (61%) had no baseline mutation (202 intolerant) and 402 (39%) had a baseline mutation (18 intolerant). Of those with mutations, 70 (17%) had >1 mutation (4 intolerant) and 16 (4%) had >2 mutations. The TKI-resistant T315I mutation occurred in 21/402 (5%) and 5/70 (7%) of those with 1 or >1 mutation, respectively–with G250E also occurring in 2 of those 5 pts. The 24-mo response rates for pts with >1 mutation at baseline were lower than the rates in those who had only 1 mutation, or no mutations at baseline for MCyR (52.2% vs 56.4% vs 65%) and CCyR (36% vs 45% vs 56%). The 24-mo PFS rates on dasatinib for those with >1, 1, and no mutation were 57%, 73%, and 83%, respectively. While pts who achieved CCyR at 12 mos had excellent 2-year PFS, regardless of whether they had no, 1, or >1 mutation, pts who achieved partial cytogenetic response (PCyR) or less than PCyR at 12 mos had lower 2-year PFS if they carried >1 mutation compared to those with no or 1 mutation. The 2-year overall survival (OS) rates were 93.5% for those without a mutation and similar for those with 1 or >1 mutation (89%). Conclusion:Dasatinib shows considerable efficacy in pts with or without baseline BCR-ABL mutations. However, pts with baseline mutations tended to have lower rates of response and PFS compared with those without mutations at baseline. In addition, the presence of >1 mutation compared with the presence of only 1 mutation yielded the lowest rates of response and PFS. Disclosures:Quintás-Cardama: Bristol-Myers Squibb: Consultancy; Novartis: Consultancy. Kantarjian: Bristol-Myers Squibb: Research Funding; Pfizer: Research Funding; Ariad: Research Funding; Novartis: Consultancy, Research Funding. Shah: Bristol-Myers Squibb: Consultancy; Novartis: Consultancy; Ariad: Consultancy. Schiffer: Bristol-Myers Squibb: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy; Cellgenix: Consultancy. le Coutre: Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria. Saglio: Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria. Guilhot: Bristol-Myers Squibb: Consultancy; Novartis: Consultancy. Hochhaus: Bristol-Myers Squibb: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Bahceci: Bristol-Myers Squibb: Employment. Lambert: Bristol-Myers Squibb: Employment. Cortes: Bristol-Myers Squibb: Research Funding; Novartis: Research Funding; Pfizer: Research Funding.

The Novel BCR-ABL1 V304D Mutation Induces Pan-Tyrosine Kinase Inhibitor Resistance by a Unique Kinase Lateral Escape Mechanism and Is Associated with Very Poor Prognosis In Patients (PTS) with Chronic Myeloid Leukemia (CML).

AbstractAbstract 3402We discovered a novel BCR-ABL1 mutation (V304D) in pts with CML failing imatinib by DNA expansion of specific clones followed by DNA sequencing of ≥10 clones. BCR-ABL1V304D was detected in a median of 37% (range, 20% to 80%) resistant clones from 13 (18%) of 70 imatinib-resistant pts with CML in chronic phase (CP). Pts received imatinib for a median of 35 months (range 2 to 66) at doses ≥600 mg/d. No pt achieved a cytogenetic response. Four received nilotinib: 3 had hematologic resistance and 1 progressed to blast phase (BP). All pts died with negligible response to second-line TKIs: 8/12 pts on dasatinib had disease progression and 4 responded (2 hematologic and 2 transient minor cytogenetic responses). BCR-ABL1V304D failed to induce cytokine-independence or activate Stat5 in Ba/F3 cells. Phosphorylation of CrkL and specific BCR-ABL1 substrates were detectable but diminished compared to unmutated BCR-ABL1-transduced cells. BCR-ABL1V304D failed to catalyze autophosphorylation and the catalytic domain of ABL1V304D demonstrated deficient kinase activity. Enforced expression of BCR-ABL1V304Din CML cells induced quiescence and protection from imatinib-induced apoptosis. In vitro analyses of cells from a pt in CP expressing BCR-ABL1V304D in 50% of clones failed to detect CrKL phosphorylation in the presence of normal BCR-ABL1 protein levels, suggesting that BCR-ABL1V304D encodes a kinase-deficient protein and is associated with remarkable TKI resistance and extremely poor prognosis.To determine the mechanism of resistance imposed by BCR-ABL1V304D, we modeled this mutation in water and counterions and compared it to unmutated and mutant BCR-ABL1 isoforms. We first correlated the free energy of binding (DGbind) to the corresponding IC50 (DGbind = -RT lnIC50) and calculated the difference in free energy of binding between wild-type and mutant kinases (DDGbind = DGbind(WT) – DGbind(MUT)). DGbind <0 indicates a tighter binding to a TKI of the unmutated kinase relative to the mutant kinase. A negative increase of 1.4 kcal/mol in DGbind corresponds to a decrease by a factor of 10 in the IC50 value. The DGbind (IC50) values of imatinib for wild-type, Y253H, and T315I kinases were -10.47kcal/mol (21nM), -7.45 kcal/mol (3.4mM), and -6.38 kcal/mol (21mM), similar to published experimental data (25nM, 1.8–3.9mM, and >10mM, respectively), thus validating our modeling. DGbind and IC50 values for imatinib and dasatinib against V304D are -9.86kcal/mol (59nM) and -12.27 kcal/mol (1.02nM), respectively. 3D images generated from an equilibrated frame of 10 ns molecular dynamics (MD) simulations demonstrated that the 304 position is not in direct contact with imatinib, nor does it directly alter imatinib binding. Rather, V304D disturbs the position of the regulatory αC helix (Figure1). Longer standard molecular dynamics simulations coupled with steered MD recipes indicate that V304D induces a rearrangement of the ATP/drug binding pocket and water-mediated disruption of some fundamental hydrogen bonds regulating the transition of the activation loop to a “semi-open” conformation and the apt overall conformation of the SH3-binding segment of the TK (residues K294-F311). Furthermore, a decrease in the number of total interactions causes unidirectional drug translation toward the binding site exit. Iterative simulations revealed significant ATP/inhibitor diversion with subsequent complete imatinib expulsion. Thus, the V304D-induced semi-opened conformation of the activation loop favors 1) the lateral escape of imatinib, thus increasing the rate of TKI dissociating from the kinase and 2) does not allow the passage of ATP to reach deep into the binding pocket, thus hampering tyrosine phosphorylation. A similar phenomenon is observed in the activation loop in the active conformation of the V304D kinase bound to dasatinib, which results in greater exposure to water solvent of a part of the binding site and almost complete loss of hydrophobic contacts in the opposite end of the binding site. [Display omitted] In summary, BCR-ABL1V304D results in kinase inactivation, pan-TKI resistance mediated by a novel mechanism of lateral escape at the kinase domain, less control of protein autoinhibition via perturbation of the SH3 binding domain and very poor prognosis. Complete modeling data against a panel of novel TKIs and potential modes of overcoming this novel mechanism of resistance will be presented. Disclosures:Kantarjian:Bristol Myers Squibb: Research Funding; ARIAD: Research Funding; Nerviano: Research Funding. Cortes:Bristol Myers Squibb: Research Funding; ARIAD: Research Funding; Nerviano: Research Funding.

Experience in Second Line TKI Treatment in 6 Million Region of Russia (St-Petersburg and Leningrad region).

Abstract 4288Resistance to imatinib could be overcome by new generations of TKIs. Data about the efficacy of second line TKI treatment may help to create a system for prognosing the duration of 2nd line TKI treatment and time of switching from second line TKI treatment to hematopoietic stem cell transplantation.The aim of the study was to evaluate the results of 2nd line TKI treatment in patients resistant or intolerant to imatinib. Patients and methods44 resistant and 3 intolerant to imatinib pts were included. Cytogenetics with G-banding and PCR with sequencing were performed for evaluation the response and mutations. There were 39, 7 and 1 pts in CP, AP and BP respectively, Patients were treated by three different ATP-pocket inhibitors. The follow-up on 2nd line TKIs was 0.9-48mons (Median - 15.9 mons), median follow-up from diagnosis was 73,9 mons. In prognosis analysis (CHR, cytogenetics, Sokal) intolerant patients were not included. ResultsProbability of overall survival from diagnosis by 10 years was 85%. Probability of survival from the start of TKI2 by 4 y -90%. There were no differences in survival in pts with primary and secondary resistance, in CP and AP. Patients with low and intermediate Sokal risk did better than patients with high risk (p=0.014, Fig. 1.).8 pts had T315I mutation when studied on second line TKI therapy.Achievement of CCyR was 50% (58% when T3151 patients were excluded), it was higher in low+intermediate in comparison with high risk pts (60% vs 30%, p=0.091), in CP than in AP (56% vs 20%,p=0.07), in patients with CHR at switching than in those without CHR (72% vs 18%, p=0.014). We failed to find any differences in CCyR achievement in pts with different best cytogenetic response on imatinib (complete, major, minor, minimal responses, absence of cytogenetic response).MCyR depended on the type of resistance - it was higher in secondary, than in primary resistant pts (80% vs 45%,p=0.06, Fig2). Probability of MCCyR loss was 20%, all of them appear during the first year of treatment. It was higher in AP than in CP (40% vs 12%,P=0.04).Probability of 4 years PFS was high - 75%, also without differences in primary vs secondary resistance and even in AP vs CP. ConclusionSecond line TKI treatment is very efficacious in imatinib resistant pts. The effect depended on the type of resistance and phase of the disease, CHR at the moment of switching to 2nd line treatment. 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.

Nilotinib Efficacy According to Baseline BCR-ABL Mutations in Patients with Imatinib-Resistant Chronic Myeloid Leukemia in Chronic Phase (CML-CP)

Resistance or intolerance to imatinib in CML-CP occurs in ~20–30% of cases. The most frequent cause of resistance is clonal selection of cells harboring BCR-ABL kinase domain mutations. Nilotinib is a rationally designed, selective and potent BCR-ABL inhibitor with activity against most BCR-ABL mutants (not T315I) indicated for the treatment of Ph+ CML patients (pts) in chronic (CP) or accelerated phase (AP) resistant or intolerant to prior therapy including imatinib. This subanalysis of a phase II study of nilotinib in imatinib-resistant CML-CP pts assessed the occurrence of BCR-ABL mutations at baseline and during nilotinib treatment and their impact on treatment outcome after 12 months of nilotinib therapy. Of 321 CML-CP pts, 281 (88%) had baseline mutation data available, 114/281 (41%) had detectable BCR-ABL mutations prior to nilotinib therapy. The frequency of mutations at baseline was 55% among imatinib-resistant pts (n=192) and 10% among imatinib-intolerant pts (n=89). 23% of imatinib-resistant pts had mutations that were sensitive to nilotinib in vitro (IC50 ≤150 nM). These 12 different mutations (n=44) spread across the entire BCR-ABL kinase domain including P-loop, A-loop, and other regions. 14% of imatinib-resistant pts had 3 mutations that were less sensitive to nilotinib in vitro (IC50 >150 nM; Y253H, E255K/V, and F359C/V) and another 15% had a total of 16 mutations with unknown sensitivity to nilotinib. In imatinib-resistant pts lacking baseline mutations, after 12 months of therapy, major cytogenetic response (MCyR) was achieved in 60%, complete cytogenetic response (CCyR) in 40%, and major molecular response (MMR) in 28% of pts. In pts with detectable mutations, 51% achieved MCyR, 32% CCyR, and 20% MMR. Cytogenetic response rates in pts harboring mutations sensitive to nilotinib (MCyR 59%; CCyR 41%) or mutations with unknown sensitivity to nilotinib (MCyR 63%; CCyR 50%;) were comparable to those for pts without baseline mutations (MCyR 60%; CCyR 40%). Pts with mutations less sensitive to nilotinib in vitro had less favorable response after 12 months of therapy (23% MCyR). Pts with baseline mutations had a higher rate of disease progression during nilotinib treatment compared to pts without baseline mutations (46% vs. 26%). Different rates of progression were also observed with different mutations: 34% (15/44) of pts with mutations sensitive to nilotinib vs. 69% (18/26) with mutations less sensitive to nilotinib progressed. Mutations most frequently associated with progression were E255K/V (6/7) and F359C/V (9/11). Progression was defined as any of the following: investigator's evaluation as progression, development of CML-AP or blast crisis, loss of CHR, loss of MCyR. During nilotinib therapy, 48/281 (17%) pts had newly detectable mutations, which were more frequent in pts with baseline mutations than in pts without baseline mutations (29% vs. 9%, respectively). The majority of pts without baseline mutations also did not have newly detectable mutation at the time of progression (n=14/18) suggesting that pts without baseline mutations are less likely to progress due to newly detectable mutations. In the 63 pts who progressed, 29% had no detectable mutation at progression, suggesting the involvement of alternative mechanisms of resistance in these pts. Overall, nilotinib treatment results in significant cytogenetic responses in pts with imatinib-resistant CML-CP with or without BCR-ABL mutations. The majority of imatinib-resistant pts with detectable BCR-ABL mutations at baseline also responded to nilotinib. Pts with BCR-ABL mutations sensitive and with unknown sensitivity to nilotinib in vitro achieved significant response rates with nilotinib therapy, comparable to those for pts without baseline mutations.

Efficacy and Safety of Bosutinib (SKI-606) in Patients with Chronic Phase (CP) Ph+ Chronic Myelogenous Leukemia (CML) with Resistance or Intolerance to Imatinib.

Bosutinib (SKI-606) is an orally bioavailable dual Src/Abl inhibitor demonstrating inhibitory activity against BCR-Abl phosphorylation, and is 200 times more potent than imatinib but with minimal inhibition of platelet-derived growth factor receptor (PDGFR) or c-kit. The phase I portion of this study identified a treatment dose of 500 mg daily and showed evidence of clinical efficacy. The phase II portion of the study to investigate the efficacy and safety of bosutinib in patients (pts) with CP Ph+ CML who have failed imatinib therapy is ongoing. Preliminary data for 283 treated pts, median age 54 yrs (range 18 – 91 yrs) and 52% male are reported. A subset of pts received treatment in addition to imatinib, including interferon (91 pts), dasatinib (71 pts), nilotinib (7 pts) and stem cell transplant (13 pts). Among pts who failed imatinib (and received no other tyrosine kinase inhibitor treatment), 137 were imatinib-resistant (all received imatinib ≥600mg) and 64 pts were imatinib-intolerant; median duration of bosutinib treatment to date is 7.7 mos (range 0.2 – 28.2 mos) and 4.5 mos (range 0.5 – 21.5 mos), respectively. Among 67 imatinibresistant pts evaluable for hematological response, 53 (79%) had complete hematological response (CHR). Of 84 imatinib-resistant pts evaluable for cytogenetic response, 34 (40%), achieved a major cytogenetic response (MCyR), including 24 (29%) with a complete cytogenetic response (CCyR). Of 34 pts with MCyR, 31 have maintained their response to date. Of 60 evaluable imatinib-resistant pts, 20 (33%) achieved major molecular response, 10 (17%) of which were complete. Among imatinib-intolerant pts, 22 of 29 evaluable (76%) achieved CHR, and 13 of 22 evaluable (59%) achieved MCyR, including 11 (50%) with CCyR. Of 25 evaluable imatinib-intolerant pts, 7 (28%) achieved major molecular response, 5 (20%) of which were complete. Of 105 pts with baseline samples tested for mutations, 17 different mutations were found in 45 pts (43%). CHR occurred in 5/6 pts (83%) with P-loop mutations and 13/17 (76%) with non-P-loop mutations; MCyR occurred in 3/6 pts (50%) and 11/24 pts (46%), with P-loop and non-P-loop mutations, respectively. Treatment was generally well tolerated. The most common adverse events among treated pts (n=283) were gastrointestinal (nausea, vomiting, diarrhea), these were usually grade 1 – 2, manageable and transient, diminishing in frequency and severity after the first 3 – 4 weeks of treatment. Grade 3 – 4 non-hematologic toxicity occurring in ≥5% of pts were diarrhea (8%), rash (8%) and increased ALT (5%). 27 pts (10%) reported grade 1/2 fluid retention adverse events, including 21 pts with edema, and 6 pts with effusions: 4 pleural, 1 pericardial, and 1 pleural and pericardial. A single patient experienced grade 3 pleural effusion possibly related to bosutinib with concomitant pneumonia and a pre-treatment history of recurrent pleural effusions. Grade 3 – 4 hematologic laboratory abnormalities included thrombocytopenia in 65 pts (23%), neutropenia in 37 pts (13%) and anemia in 17 pts (6%). 124 pts (44%) had at least 1 temporary treatment interruption and 85 pts (30%) had at least 1 dose reduction due to toxicity. 37 pts (13%) have permanently discontinued treatment due to adverse event. Bosutinib is effective in pts with CP CML with resistance or intolerance to imatinib across a range of mutations. Unlike other tyrosine kinase inhibitors, bosutinib does not significantly inhibit PDGFR or c-kit, and this may be responsible for the relatively favorable toxicity profile with few pts experiencing hematologic toxicity or fluid retention.

Bosutinib (SKI-606) Demonstrates Clinical Activity and Is Well Tolerated in Patients with AP and BP CML and Ph+ ALL.

Bosutinib (SKI-606) is an orally available, Src/Abl kinase inhibitor with minimal activity against platelet derived growth factor receptor (PDGFR) and c-kit. An open-label study of patients (pts) with Philadelphia chromosome positive (Ph+) accelerated phase (AP) and blast phase (BP) CML and Ph+ ALL who failed prior imatinib therapy is ongoing. Patients receive 500 mg/day of bosutinib. Preliminary data for 101 subjects, median age 51.5 yrs (range 18 – 84 yrs) and 56% male are reported. 44 pts were in AP, 35 in BP, 21 had Ph+ ALL, and 1 was unclassified. Prior therapy in addition to imatinib included interferon (35 pts), dasatinib (40 pts), nilotinib (15 pts) and stem cell transplant (11 pts). 49 pts failed imatinib (and received no other tyrosine kinase inhibitor [TKI]) and 52 pts failed both imatinib and other TKIs, with median duration of bosutinib treatment to date 4.4 mos (range 0.3 – 21.3 mos) and 2.0 mos (range 0.3 – 18.8), respectively. Among pts with no TKI exposure other than imatinib, complete hematological response (CHR) was obtained in 12/25 evaluable pts (48%), including 7/11 pts (64%) with AP-CML, 4/11 pts (36%) with BP-CML and 1 pt with Ph+ ALL. Major cytogenetic response (MCyR) was achieved in 16/22 evaluable pts (73%) with no TKI exposure other than imatinib, including 9/13 pts (69%) with AP-CML and 6/8 pts (75%) with BP-CML; 1 pt with Ph+ ALL achieved MCyR. Major molecular response (MMR) was achieved in 9/25 evaluable pts (36%) with no TKI exposure other than imatinib, including 1/7 pts (14%) with AP-CML, 4/10 pts (40%) with BP-CML and 4/8 pts (50%) with Ph+ ALL. Among pts with other TKI exposure in addition to imatinib, CHR was obtained in 3/15 evaluable pts (20%), all with AP-CML; MCyR was achieved in 6/20 evaluable pts (30%), including 3/12 pts (25%) with AP-CML and 2/7 pts (29%) with BP-CML; 1 pt with Ph+ ALL achieved MCyR. Of the 20 pts with other TKI exposure in addition to imatinib who were evaluable for MMR, 1 pt with Ph+ ALL (5%) achieved this response. Of 60 pts with baseline samples tested for mutations, 15 different mutations were found in 32 pts (53%), including 8 instances of T315I. CHR occurred in 2/8 evaluable pts (25%) with non-P-loop mutations; the 1 evaluable pt with a P-loop mutation did not achieve CHR. MCyR occurred in 4/11 evaluable pts (36%) with non-P-loop mutations and in 1/2 evaluable pts (50%) with P-loop mutations. Treatment was generally well tolerated. The most common adverse events among treated pts (n=101) were gastrointestinal (diarrhea [66%], nausea [46%] and vomiting [42%]) but these were usually grade 1 – 2, manageable and transient, reducing in frequency and severity after the first 3 – 4 weeks of therapy. Grade 3 – 4 non-hematologic toxicities occurring in ≥ 5% of pts were diarrhea (7%), vomiting (6%), pneumonia (6%) and increased ALT (5%). Fluid retention was reported as grade 1 – 2 in 18 pts and grade 3 – 4 in only 3 pts (including 2 pleural effusions, neither related to bosutinib). Grade 3 – 4 hematologic laboratory abnormalities reported include thrombocytopenia (68%), neutropenia (48%) and anemia (37%). 38 pts had at least 1 temporary treatment interruption and 22 pts had at least 1 dose reduction due to toxicity. 11 pts have permanently discontinued treatment due to adverse event. Bosutinib is effective in imatinib-resistant pts with advanced CML. Responses were observed across a wide range of Bcr/Abl mutations.

Long-Term Mutation Follow-up of Philadelphia-Chromosome Positive Leukemia Patients Treated with Second-Generation Tyrosine Kinase Inhibitors after Imatinib Failure Shows That Newly Acquired Bcr-Abl Kinase Domain Mutations Leading to Relapse Are Mainly Detected during the First Year.

Resistance to imatinib in Philadelphia-positive (Ph+) leukemia patients is often associated with selection of point mutations in the Bcr-Abl kinase domain (KD). Dasatinib and nilotinib are second-generation tyrosine kinase inhibitors (TKIs) with different binding modes with respect to imatinib, that have been shown to confer in vitro and in vivo activity against many Bcr-Abl mutated forms. However, both dasatinib and nilotinib have been shown to retain some ‘Achilles heels’, and they include both imatinib-resistant mutations (e.g., T315I) and some novel, inhibitor-specific ones. Selection of either type of KD mutations has frequently been observed in patients (pts) who relapse after an initial response to dasatinib or nilotinib and represents one of the major hurdles on the road to successful treatment of imatinib-resistant pts. We have monitored Abl KD mutation status in a total of 121 pts who received dasatinib (n= 78) or nilotinib (n=43) as 2nd TKI after imatinib failure since February 2005. Fifty-eight (48%) pts had chronic phase (CP) chronic myelogenous leukemia (CML), 63 pts (52%) had accelerated phase (AP) or blast crisis (BC) CML or Ph+ acute lymphoblastic leukemia (ALL). Median age was 55 years (range, 18–76); median time from diagnosis was 49 months (range, 4–181); median time on imatinib was 32 months (range, 4–66). Median follow-up of all pts who received a 2nd TKI is 7 months (range, 1–38). Median follow-up of pts who are still on 2nd TKI treatment is 32 months (range, 28–38). Relapses after an initial response have so far been observed in 46/121 pts. Thirty-eight out of these 46 pts had AP/BC CML or Ph+ ALL at the time 2nd TKI was started. Forty-one out of 121 (34%) pts have experienced relapse after an initial response during the first 12 months of 2nd TKI treatment (median time to relapse, 6,5 months; range 4–12 months), while only five of the 45 (11%) pts who were still on 2nd TKI treatment after >12 months have relapsed (at 13, 15, 18, 20 and 33 months, respectively). Interestingly, none of these 5 pts had never achieved more than a minor cytogenetic response (CgR), and 4/5 pts were receiving a reduced TKI dose because of toxicity. In 36/46 (78%) cases, relapse was associated with newly acquired Abl KD mutations. In particular 26/30 (87%) pts who relapsed on dasatinib and 10/16 (63%) pts who relapsed on nilotinib had evidence of a newly acquired KD mutation presumably responsible for treatment failure. Newly acquired mutations in pts who relapsed on dasatinib as 2nd TKI were T315I (n= 12 pts) F317L (n= 8 pts) T315A (n=3 pts); V299L (n=3 pts); F317I (n=2 pts); 2 pts had multiple mutations. Newly acquired mutations in pts who relapsed on nilotinib as 2nd TKI were E255K (n=3); E255V (n=2); Y253H (n=2); T315I (n=1); F359V (n=1); F359C (n=1). Sixteen pts (but none of those harboring the T315I) switched to dasatinib or nilotinib or high-dose imatinib as 3rd TKI and this rescued hematologic or even cytogenetic responses in a proportion of cases. Our observations suggest that:newly acquired mutations leading to relapse in Ph+ leukemia pts receiving dasatinib or nilotinib as 2nd TKI usually arise rapidly; the likelihood of mutation selection consistently decreases over time, and seems mainly confined to advanced phase pts and to pts with no or minor CgR;almost all (87%) cases who developed resistance to dasatinib had newly acquired KD mutations - suggesting that the higher potency with respect to imatinib can overcome Bcr-Abl gene amplification and that Src kinase inhibition may turn off Bcr- Abl-independent resistance mechanisms;a lower incidence (63%) of newly acquired KD mutations was observed in pts who developed resistance to nilotinib;with the exception of T315I, there is little if no overlap between dasatinib and nilotinib-resistant mutants, which may allow to regain responses by switching TKIs.

Mutational Analysis of Chronic Phase Chronic Myeloid Leukemia (CMLCP) Clones Reveals Heightened BCR-ABL1 Genetic Instability in Patients Failing Sequential Imatinib and Dasatinib Therapy.

ABL1 kinase domain (AKD) mutations are the most important mechanism of resistance to tyrosine kinase inhibitors (TKIs) in CML. Direct sequencing (DS) techniques detect AKD mutations in 20%–40% of imatinib-resistant pts. Therefore, most pts fail TKI therapy for unknown reasons. We evaluated the incidence and clinical consequences of AKD mutations among 70 pts in CML-CP after imatinib failure (13 intolerant) enrolled in a phase I study of dasatinib. Mutations were studied by DS of nested PCR-amplified BCR-ABL1 products and by DNA expansion of specific clones (DESC) followed by DNA sequencing of ≥10 clones. Patients had received imatinib at 400 mg/d (n=60), 600 mg/d (n=8), or 800 mg/d (n=2). Prior to dasatinib, AKD mutations were detected in 61/70 (87%) pts by DESC, including 38 (54%) with mutations in ≥20% of sequenced clones. Mutations were only detected in 18/38 (47%) by DS. Overall, 125 mutations at 113 amino acid positions were detected by DESC (78 previously unreported). Mutations conferring resistance to >1μM imatinib (M244V, G250E, Q252H, Y253H, E255K/V, F359V, H396R, and T315I) were detected in 30 (43%) pts by DESC, but only in 5 (7%) by DS. Two or more mutations within the same clone (polymutants) were detected in 29/70 (41%) pts by DESC, with clones expressing 2 (n=38), 3 (n=11), 4 (n=1), or even 5 (n=2) distinct mutations. By contrast, only 1 pt was found to carry 2 different mutations (M244V and M351T) by DS. Pts received dasatinib for a median of 19 months (range, 2–52), of whom 68 (97%) are evaluable for response. DESC available in 32 pts during dasatinib therapy revealed 20 additional mutations not present at dasatinib start (19 amino acid positions), including 5 previously not reported (all in polymutant clones). Dasatinib-resistant mutations (L248V/R, Q252H, E255K, V299L, T315I/A, and F317L/C/I/S/V) were detected in 10/32 (31%) cases (5 with T315I) by DESC (but only in 3/16 [19%] by DS). Of these 16 pts 13 died (all in BP) and 3 are alive in CP carrying E255K, T315I, and F317L respectively.The percentage of clones with unmutated BCR-ABL1 before dasatinib decreased significantly compared to those present after a median of 16 wks (range, 4–84) during dasatinib (p=0.001), particularly in pts carrying highly dasatinib-resistant mutants. No differences were seen in the proportion of unmutated BCR-ABL1–expressing clones between pts with no cytogenetic (CG) response and those who achieved a partial (PCyR) or a complete CG (CCyR) response prior to dasatinib therapy. Conversely, DESC during dasatinib therapy showed the proportion of unmutated clones was lower among pts who failed to achieve a CG response compared to those who had a PCyR or CCyR (p=0.0001).AFTER IMATINIBON DASATINIBNo. Evaluable for Response to dasatinibNo. clones sequencedNo. unmutated Clones (%)No. Evaluable PtsNo. clones sequencedNo. unmutated Clones (%)DASATINIB RESPONSE68680311 (46)3230594 (30)No CG Response30309149 (48)1312017 (14)CG ResponseMinor1191 32 (35)5474 (8)Partial76938 (55)32813 (46)Complete2021192 (43)1111060 (55)In conclusion, DESC demonstrates a high prevalence of AKD mutations among pts who fail imatinib, revealing heightened BCR-ABL1 genomic instability in this setting. This high incidence of mutations might partly explain TKI resistance in pts found to carry unmutated BCR-ABL1 by DS. The latter might be mediated by generation of resistant polymutant clones that perpetuate a “mutator phenotype” and by exhaustion of clones carrying unmutated BCR-ABL1 alleles.

Nilotinib in accelerated phase chronic myelogenous leukemia (CML-AP) patients with imatinib-resistance or -intolerance: Update of a phase II study

7050 Background: The potent and highly selective BCR-ABL inhibitor nilotinib has been approved in both the US and Europe for the treatment of patients (pts) with Philadelphia chromosome-positive (Ph+) chronic phase chronic myelogenous leukemia (CML- CP) and CML-AP who are resistant or intolerant to prior therapy, including imatinib. Methods: This phase 2, open-label study evaluated the efficacy and safety of nilotinib in pts with Ph+ CML-AP with imatinib resistance or intolerance. Primary and key secondary endpoints were rate of confirmed hematologic response (HR) and MCyR, respectively. Nilotinib was started at 400 mg twice daily (BID) and escalated to 600 mg BID for inadequate responses. Results: Included in the analysis were 136 pts who received at least 1 dose of nilotinib. Median age was 58 (22–82) yrs; median dose intensity was 781 mg/day; median treatment duration was 210 days. Confirmed HR occurred in 69/129 pts (54%); 34 (26%) had complete HR. Of imatinib-resistant pts, 55/104 pts (53%) had a confirmed HR, and 14/25 imatinib- intolerant pts (56%) had a confirmed HR. A majority of responding pts (82%) maintained HR for at least 12 months. MCyR occurred in 40/129 pts (31%); 24/129 pts (19%) had complete CyR. Of imatinib-resistant pts, 30/104 pts (29%) had a MCyR, and 10/25 imatinib-intolerant pts (40%) had a MCyR. Time to first HR and MCyR was 1 and 2.8 months, respectively. At 12 months, it is estimated that 57% of pts were without progression and the estimated overall survival rate was 81%. At data cutoff, treatment was ongoing for 57/136 pts (42%). The most common grade 3/4 laboratory abnormalities were thrombocytopenia (41%), neutropenia (39%), anemia (25%), and elevated serum lipase (16%). Grade 3/4 non-hematologic AEs were rare and included nausea, fatigue, headache, and diarrhea. Conclusions: The results of this study confirm that nilotinib induces significant and durable responses in CML-AP pts with imatinib resistance or intolerance. There was minimal nilotinib cross- intolerance in patients intolerant to prior imatinib treatment. Nilotinib is well tolerated, with minimal occurrence of grade 3/4 AEs, and is an effective treatment option for this pt population. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Novartis Bristol-Myers Squibb, Innovive, Novartis Bristol-Myers Squibb, Novartis Bristol-Myers Squibb, Novartis, Wyeth

Dasatinib 2-year efficacy in patients with chronic-phase chronic myelogenous leukemia (CML-CP) with resistance or intolerance to imatinib (START-C)

7009 Background: Dasatinib is 325-fold more potent than imatinib and 16-fold more potent than nilotinib against BCR-ABL in vitro. Extended 2-year follow-up data are available for the START-C study, an international, 75-center, phase II study in patients (pts) with CML-CP resistant or intolerant to imatinib. Methods: Pts were required to be either imatinib-resistant or -intolerant. The starting dose was dasatinib 70 mg BID, with dose escalations (90 mg BID) or reductions (50 or 40 mg BID) allowed for lack of response or toxicity. The primary endpoint was major cytogenetic response rate (MCyR). Results: Median time from CML diagnosis at baseline was 61 mos (range 3- 251). Best response to prior imatinib therapy was complete hematologic response (CHR) in 82%, complete cytogenetic response (CCyR) in 19%, and MCyR in 37% of pts. After a minimum follow up of 24 mos (LPFV to database closure; n=387, 288 imatinib-resistant, 99 imatinib-intolerant), CHR was noted in 91% (95% CI 88–94%), MCyR in 62% (95% CI 57–67%), CCyR in 53% and major molecular response (MMR) in 47% of pts. Rates of MCyR were 55% for imatinib-resistant pts, and 63% for pts with baseline BCR-ABL mutations; responses were seen across all mutations except T315I. In imatinib-intolerant pts, the CCyR (n=99) rate and MMR (n=94 evaluable) rate were both 78%. MCyRs were durable, with 88% of pts maintaining response at 24 mos. Progression-free survival (PFS) at 24 mos was 80% (75% in imatinib-resistant and 94% in imatinib-intolerant pts). Overall survival at 24 mos was 94% (92% in imatinib -resistant and 100% in imatinib-intolerant pts). Grade 3–4 toxicities included thrombocytopenia (49%) and neutropenia (50%), pleural effusion (9%), dyspnea (6%), bleeding (4%), diarrhea (3%), and fatigue (3%). 3% of imatinib-intolerant pts developed similar grade 3–4 toxicities; all were able to continue treatment after dose reduction, demonstrating lack of cross intolerance. The appearance of new higher grade toxicity between 12 and 24 months was uncommon. Conclusions: Dasatinib is well-tolerated in pts with CML-CP and induces cytogenetic responses that are highly durable and result in a PFS of 80% at 24-mos. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Bristol-Myers Squibb Bristol-Myers Squibb, Novartis Bristol-Myers Squibb Bristol-Myers Squibb, Novartis Bristol-Myers Squibb, Novartis Bristol-Myers Squibb

Nilotinib in patients with imatinib-resistant or -intolerant chronic myelogenous leukemia in chronic phase (CML-CP): Updated phase II results

7010 Background: Nilotinib, a potent and highly selective BCR-ABL inhibitor, has been approved in both the US and Europe for the treatment of patients (pts) with CML-CP and accelerated phase CML (CML-AP) who are resistant or intolerant to prior therapy, including imatinib. Methods: This phase 2, open-label study was designed to evaluate the efficacy and safety of nilotinib in pts with Philadelphia chromosome-positive (Ph+) imatinib-resistant or -intolerant CML-CP. Nilotinib was administered at 400 mg twice daily (BID), with the possibility to dose escalate to 600 mg BID for inadequate responses. The primary endpoint was the rate of major cytogenetic response (MCyR). Results: Included in this analysis were 321 pts (71% imatinib-resistant; 29% imatinib-intolerant). 72% of pts received prior imatinib doses > 600 mg. Of the 206 pts who did not have complete hematological remission (CHR) at baseline, 158 (77%) achieved CHR during nilotinib therapy. Overall, MCyR was observed in 184 pts (57%); 41% had complete CyR. MCyR was observed in 125 (55%) of the 227 imatinib-resistant pts and in 59 (63%) of the 94 imatinib-intolerant pts. The median time to CHR and MCyR was 1.0 and 2.8 months, respectively. The majority of pts (84%) maintained MCyR for at least 18 months. At 18 months, the estimated overall survival rate was 91%. Treatment with nilotinib is ongoing in more than half of pts enrolled. The most frequent grade 3/4 laboratory abnormalities were thrombocytopenia (28%), neutropenia (30%), and asymptomatic serum lipase elevation (15%). Grade 3/4 non-hematologic AEs were rare and included rash, headache, and diarrhea. A low incidence of QT prolongation (QTcF > 500 msec, < 1%) was observed overall. The safety profile of nilotinib has not changed with increased follow up. There was minimal cross-intolerance between nilotinib and imatinib in pts intolerant to prior imatinib treatment. Conclusions: The present study confirms that nilotinib induces significant and durable responses in CML- CP pts with imatinib-resistance or -intolerance. Nilotinib is well tolerated, with minimal occurrence of grade 3/4 AEs and minimal cross-intolerance to imatinib, and is a valuable new treatment option for this pt population. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Novartis Bristol-Myers Squibb, Novartis Bristol-Myers Squibb, Novartis Bristol-Myers Squibb, Innovive, Merck, Novartis, Wyeth