Point Mutations In Kinase Domain Research Articles

Molecular monitoring and mutations in chronic myeloid leukemia: how to get the most out of your tyrosine kinase inhibitor.

The course of chronic myeloid leukemia (CML) and the response to treatment with tyrosine kinase inhibitors (TKIs) are best monitored and assessed using two molecular tests: the first is real-time quantitative reverse transcription-polymerase chain reaction (RQ-PCR), which measures the size of residual disease that is expressed as BCR-ABL1% (the ratio between BCR-ABL1 and a control gene) and the other is mutational analysis by Sanger sequencing, which checks for the presence of BCR-ABL1 kinase domain point mutations. Both tests are technically demanding and require a high level of specialization and standardization. RQ-PCR, when performed on a regular basis, allows for the defining of molecular response (MR) levels as log reduction from a standardized baseline: major molecular response (MMR or MR(3)) that is the best predictor of survival; and the deeper molecular response (MR(4), MR(4.5), and MR(5)) that is necessary to enroll a patient in a trial aiming at treatment-free remission (TFR). Mutational analysis, to be performed in case of failure or warning by Sanger sequencing, allows for screening of the BCR-ABL1 kinase domain for mutations conferring resistance to TKIs. Since different mutations have different degrees of sensitivity to each of the currently available TKI, the knowledge of BCR-ABL1 kinase domain-mutation status is necessary for subsequent treatment choice. Optimal patient management requires that MR and mutational information be rationally interpreted at both the technical and at the biologic level, and put into context-therapeutic decisions also take into account other factors, such as age, comorbidities, side effects, compliance, and treatment-related complications.

Reversal of Acquired Drug Resistance in FLT3-Mutated Acute Myeloid Leukemia Cells via Distinct Drug Combination Strategies

FMS-like tyrosine kinase-3 (FLT3) internal tandem duplication (FLT3-ITD) mutations are common in patients with acute myeloid leukemia (AML). These patients regularly develop resistance to FLT3 inhibitors suggesting that targeted combination drug strategies are needed to enhance AML therapy efficacy. Acquired point mutations of FLT3-ITD gene were screened using cDNA-based sequencing approach in vitro sorafenib-resistant cells, which were developed by long-term exposure of Ba/F3-ITD to increasing doses of sorafenib, and in FLT3-ITD mutated AML patients, who developed relapse following sorafenib therapy. Drug effects (e.g., proliferation inhibition, apoptosis induction, and changes in signal transduction protein expression) were assessed in AML cells harboring the point mutations in vitro and in FLT3-ITD-mutated AML patient samples. We identified several acquired point mutations in the tyrosine kinase domains (TKD) of the FLT3 gene in sorafenib-resistant murine leukemia cell line carrying human FLT3-ITD mutations, which were also detected in two of four sorafenib-resistant patient samples. Engineering these point mutations into Ba/F3-ITD cells generated sublines that demonstrated varying degrees of sorafenib [a type II tyrosine kinase inhibitor (TKI)] resistance. A similar pattern of resistance could be observed by exposing these sublines to the other type II TKIs AC220 and MLN518. However, these sublines retained sensitivity to the type I TKIs PKC412 or crenolanib. The combination of crenolanib with sorafenib demonstrated marked cytotoxic effects in all of the sorafenib-resistant sublines. These combination strategies could be clinically important in reversing acquired resistance to FLT3 inhibition in AML.

FLT3 Inhibitors in AML: Are We There Yet?

FMS-like tyrosine kinase 3 (FLT3) is the most frequently mutated gene in AML. Thirty percent of patients with acute myeloid leukemia (AML) harbor activating mutations in FLT3, either internal tandem duplication mutations in the juxtamembrane domain (FLT3-ITD) or point mutations in the tyrosine kinase domain (FLT3 TKD). Small molecule FLT3 inhibitors have emerged as an attractive therapeutic option in patients with FLT3 mutations; however, the clinical activity of early inhibitors was limited by a lack of selectivity, potency and unfavorable pharmacokinetic properties. Newer agents such as quizartinib have improved potency and selectivity associated with much higher bone marrow response rates; however, response duration is limited by the development of secondary resistance. We will review here a number of FLT3 inhibitors that have been evaluated in clinical trials and discuss challenges facing the use of these agents in AML.

CAMKIIγ, HSP70 and HSP90 transcripts are differentially expressed in chronic myeloid leukemia cells from patients with resistant mutated disease

Patients with chronic myeloid leukemia (CML) can develop disease resistance to tyrosine kinase inhibitor (TKI) therapy, which is mainly attributable to the presence of point mutations in the tyrosine kinase domain of BCR–ABL1. In order to examine suitable markers to monitor treatment efficacy, we investigated transcript expression profiles of genes known to be involved in myeloid cell proliferation, such as CAMKIIγ and KI67, and in protein stability and ultimately cell survival under physiological and stress conditions, such as heat shock proteins HSP70 and HSP90. We studied 101 patients with CML in different stages of disease and with different responses to TKI treatment. The results of quantitative real-time polymerase chain reaction (qPCR) analyses showed that the expression levels of CAMKIIγ, KI67, HSP70 and HSP90 genes were up-regulated at diagnosis, and in cases with signs of treatment resistance both in chronic and advanced phases (accelerated and blastic phases) with respect to chronic phase in remission and healthy donors. When only 56 resistant cases, 31 with mutations (MT) and 25 without mutations (WT), in the BCR–ABL1 tyrosine kinase domain were considered, the transcript expression profile showed an unexpected significant increase in CAMKIIγ and HSP70, and a significant decrease in HSP90 in MT versus WT cases. This differential transcript expression prompted us to design an expression score, log(CAMKIIγ × HSP70/HSP90), which can be used to provide rapid screening to discriminate the presence or absence of mutations in resistant cells and to monitor TKI treatment efficacy in patients with CML.

Overexpression of Bcl2 protein predicts chemoresistance in acute myeloid leukemia: Its correlation with FLT3

Potential prognostic biomarkers in acute myeloid leukemia (AML) can be identified by understanding the cellular pathway and molecular changes underlying leukemogenesis. Deregulation of apoptosis is one of the important features of AML and to understand the molecular mechanism underlying apoptosis and its contribution to tumor progression, this study aimed to evaluate anti-apoptotic Bcl2 protein expression in AML and correlate with FLT3 parameters for their role in prognosis of disease.Bcl2 and FLT3 protein expression was quantified by flow cytometry on leukemic blasts in total 174 de novo AML, myelodysplastic syndrome (MDS) and aplastic anemia patients. FLT3 internal tandem duplication (ITD), Tyrosine kinase domain (TKD) point mutations and quantification of mRNA level was carried out using PCR and RT-PCR methods. The incidence of Bcl2 positivity was 71% in AML patients. Bcl2 positivity was significantly associated with CD34+ and CD117+ AML. Bcl2 positivity tended to be associated with reduced DFS while Bcl2 positivity with FLT3 protein positivity was significantly associated with reduced DFS. In multivariate analysis, Bcl2+ and combined Bcl2+/FLT3 protein+ along with high WBC count emerged as poor prognostic factors for reduced DFS and high blast count for predicting reduced OS. In MDS patients, the incidence of Bcl2 expression was high while in aplastic anemia patients, incidence of Bcl2 expression was low.Patients with Bcl2 and FLT3 protein positivity showed significantly reduced DFS suggesting parallel role of these proteins in imparting chemoresistance to the leukemic cells.

The Impact Of Novel Splicing Abnormalities Of BCR-ABL On The Pathogenesis Of CML

BackgroundChronic myeloid leukemia (CML) is effectively treated with tyrosine kinase inhibitors (TKIs), but reactivation of BCR-ABL frequently occurs through acquisition of kinase domain point mutations. The mechanism of resistance in patients without BCR-ABL kinase domain point mutation is still elusive. Previous studies have revealed the abnormal splicing of BCR-ABL kinase domain, including Exon8/9 junction 35bp insertion and exon-skipping of Exon 7 (T O'Hare et al, Blood 2011. Gaillard JB, et al. Mol Cancer Ther 2010). The insertion of 35 intronic nucleotides at the exon 8/9 splice junction introduces a stop codon after 10 intron-encoded residues and inactive tyrosine kinase activity. The effect of these splicing abnormalities on susceptibility of cells against TKIs is still controversial. Furthermore, the conventional direct sequence techniques could not evaluate splicing abnormalities in major molecular response (MMR)- complete molecular response (CMR) CML patients, who achieved clinically leukemia-free state with a small number residual CML stem cells. AimsThe aim of this study is to evaluate the frequency and the patterns of splicing abnormalities of BCR-ABL in CML patients, especially who achieved MMR-CMR by TKI treatment. MethodsWe analyzed peripheral blood samples from healthy individuals and CML chronic phase patients. We extracted total RNA from these samples and synthesized cDNA, and then performed PCR-amplification of BCR-ABL kinase domain in CML patients and ABL kinase domain in healthy individuals, respectively. PCR products were subjected to the amplicon sequence: We deeply sequenced BCR-ABL fusion gene transcripts, and evaluated splicing forms of BCR-ABL by using HiSeq 2000 (illumina). ResultsWe successfully established a novel analysis method, which can detect the pattern of splicing abnormalities even in MMR-CMR patients. Using the amplicon sequence technique, we detected abnormal splicing patterns of BCR-ABL in 5 out of 15 CML patients. We also found that the splicing abnormalities were not restricted to 35bp insertion at the exon8/9 junction, thus intronic retention of intron 8 and intron 9 could be frequently detected with or without the 35bp insertion in CML patients (Table 1). Of note, these abnormal splicing patterns always co-existed with wild type BCR-ABL transcripts in all 5 cases analyzed. In addition to the novel splicing abnormalities in CML, we unexpectedly found in healthy individuals that splicing abnormalities such as 35bp insertion at the exon8/9 junction and intronic retention could be detected in ABL1 transcripts (Table 1). This result suggests that this sort of splicing abnormalities could occur at a certain frequency in steady state human hematpoiesis, and is not specific to BCR-ABL. Summary / ConclusionWe have newly established an analysis system to efficiently detect splicing abnormalities of BCR-ABL even in MMR-CMR CML patients. Using this highly efficient amplicon sequence technique, we identified novel splicing abnormalities both in healthy individuals and CML patients, and found that the wild type BCR-ABL transcripts always co-exist with abnormally spliced BCR-ABL transcripts. These results collectively suggest that splicing abnormalities within the ABL1 kinase domain are not specific to CML patients treated with TKIs, and that the detection of such kinase domain splicing abnormalities do not reflect insusceptibility of the remaining cells during TKI treatment. Disclosures:No relevant conflicts of interest to declare.

Rapid Identification Of Compound Mutations In Patients With Ph-Positive Leukemias By Long-Range Next Generation Sequencing

Mutations in the BCR-ABL1 tyrosine kinase domain (TKD) are regarded as the most important mechanism of resistance to tyrosine kinase inhibitors (TKIs) in patients with Ph-positive leukemias. The occurrence of two or more mutations on the same DNA molecule, the so-called compound mutations, can be associated with particularly high resistance to multiple TKIs. Recent reports indicate that the frequency of compound mutations is rather high, thus rendering their reliable detection an important diagnostic challenge 1,2. Analysis of PCR amplicons of the BCR-ABL1 TKD by next generation sequencing (NGS) has become the method of choice for sensitive detection of compound mutations. This approach is, however, hampered by the requirement of 3-4 overlapping amplicons to cover the entire TKD due to the limited read length offered by most current NGS technologies. This prevents the assignment of nucleotide substitutions located on different amplicons to the same TKD/DNA molecule, and therefore requires additional laborious steps to facilitate unequivocal identification of such constellations. To overcome this limitation, we have established a long-range NGS approach on the FLX instrument (Roche) permitting the coverage of the entire TKD length of ∼0.9 kb in a single read. By testing a series of individual and consecutive specimens derived from five patients with chronic myeloid leukemia, we demonstrate that long-range NGS analysis readily permits the identification of mutations and their assignment to the same or to separate subclones at a limit of sensitivity comparable to NGS-based sequencing of shorter amplicons. In addition to the detection of individual and compound mutations, this approach also facilitates an interpretable documentation of insertions and deletions in the TKD. To address the possibility of artifacts inherent in the technique that could lead to incorrect identification of single and compound mutations, the NGS findings were reevaluated by independent technical approaches. Point mutations were confirmed by Sanger sequencing, LD-PCR 3 and pyrosequencing 4. In select cases, PCR amplicons of the BCR-ABL1 TKD derived from individual specimens were subcloned into pGEM®T easy plasmids, and >100 clones were subjected to analysis by Sanger sequencing. The observations made by NGS analysis including various single mutations (e.g. G250E, Y253H, T315A, F317I, Q252H, T315I), compound mutations (e.g. G250E/Y253H, G250E/T315A, G250E/F317I), and combinations of point mutations with small insertions or deletions (e.g. E459K/C475fs, Q252H/R362fs, T315I/R362fs) as well as large deletions involving multiple exons, could be confirmed in individual clones by Sanger sequencing, thereby documenting the reliability of the long-range NGS technology. The technical advancement presented therefore provides an economic approach to the identification of compound mutations and other genetic alterations in the entire BCR-ABL1 TKD, thus extending the diagnostic armamentarium for rapid assessment of impending resistant disease.1. Khorashad JS, Kelley TW, Szankasi P, et al. BCR-ABL1 compound mutations in tyrosine kinase inhibitor-resistant CML: frequency and clonal relationships. Blood. 2013;121(3):489-498.2. Soverini S, De Benedittis C, Machova Polakova K, et al. Unraveling the complexity of tyrosine kinase inhibitor-resistant populations by ultra-deep sequencing of the BCR-ABL kinase domain. Blood. 2013.3. Preuner S, Denk D, Frommlet F, Nesslboeck M, Lion T. Quantitative monitoring of cell clones carrying point mutations in the BCR-ABL tyrosine kinase domain by ligation-dependent polymerase chain reaction (LD-PCR). Leukemia. 2008;22(10):1956-1961.4. Alikian M, Gerrard G, Subramanian PG, et al. BCR-ABL1 kinase domain mutations: methodology and clinical evaluation. Am J Hematol. 2012;87(3):298-304. [Display omitted] Disclosures:Valent:Novartis: Honoraria, Research Funding. Lion:Novartis, Bristol-Myers- Squibb, Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Association Of FLT3 Mutations With Unfavorable Factors In AML and APL Patients

BackgroundMutations in the fms-like tyrosine kinase 3 (FLT3) gene have been considered to predict a poor prognosis in acute myeloid leukemia (AML). Internal tandem duplication (ITD) of the FLT3 is one of the most frequent mutations activating aberrant signal-transduction in AML. The impact of D835 point mutations in tyrosine kinase domain (TKD) on prognosis is less clear. AimsCharacterization of AML and APL (acute promyelocytic leukemia) patients with FLT3 mutations. MethodsFLT3 status in bone marrow samples of adult patients (pts) with AML (63 pts) (m\\28; f\\35; median 53 yrs, 19-73 yrs) and APL (14 pts) (m\\3; f\\11; median 43 yrs, 25-62 yrs) at diagnosis was examined. Genomic PCR amplification of ITD and TKD containing regions of the FLT3 gene was performed. The TKD amplicon product was restricted with the EcoRV enzyme. Results were detected by gel electrophoresis. ResultsFLT3\\ITD was found in 8 AML pts (12.7%) and in 5 APL pts (35.7%), p=0.053 (Fisher Exact test). FLT3\\TKD was found in 4 AML pts (6.4%) and in 1 APL pt (7.1%).In AML group there were 1 pt with t(6;9) in FLT3\\ITD+ subgroup and 1 pt with inv16 in FLT3\\TKD+. There were 1 pt with t(6;11), 1 with dupMLL, 3 with t(8;21), 7 with inv16 in AML pts without FLT3 mutations (FLT3-). There were no significant differences in sex, age, WT1 expression at diagnosis between AML pts with FLT3\\ITD, FLT3\\TKD and without FLT3 mutations. But the presence of FLT3 mutations was related to high peripheral white blood cell (WBC) count in AML pts at diagnosis. The median value (m) of WBC was 49.2 x109\\L (range 2.75-191) in FLT3\\ITD+; m= 81 x109\\L (range 7.2-153) in FLT3\\TKD+; m= 5.4 x109\\L (range 0.2-230) in FLT3-. The differences are statistically significant between subgroups with FLT3\\ITD+ and FLT3- (p<0.05, Mann–Whitney U test); FLT3\\ITD+TKD and FLT3- (p<0.01, Mann–Whitney U test). In FLT3\\ITD+TKD subgroup 66.7% of pts had WBC over 30 x109\\L, whereas in subgroup without FLT3 mutations only 23.4% of pts had the same unfavorable factor, p=0.012 (Fisher Exact test).In APL group the t(15;17) translocation was detected in 11 pts. 3 pts were without PML\\RARa, NPM1\\RARa, PLZF\\RARa fusions. One FLT3\\TKD+ pt had bcr1 PML breakpoint. In FLT3\\ITD+ subgroup 4 pts (80%) had bcr3 PML breakpoint, whereas in FLT3- subgroup only 1 pt (12.5%) had bcr3, p=0.032 (Fisher Exact test). In FLT3\\ITD+ subgroup all pts had WBC >10 x109\\L; moreover, 3 pts (60%) had two unfavorable factors in combination (WBC >10 x109\\L plus platelet count < 40 x109\\L) at diagnosis, and in FLT3- subgroup nobody had the same combination, p=0.035, (Fisher Exact test). Out of 5 pts with FLT3\\ITD 2 ones had death in induction, 1 pt had late molecular remission, 1 had molecular persistence and 1 pt without RARa fusion had failure in WT1 reduction after induction (under 2 log). One FLT3\\TKD+ pt had the early relapse. SummaryThe results confirm previous reports. FLT3\\ITD mutations are seen more frequently in APL than in other subtypes of AML. FLT3 mutations in AML have association with such unfavorable prognostic factor as high WBC count at diagnosis. FLT3\\ITD in APL associates with bcr3 and with combination of high WBC count plus low platelet count. The presence of FLT3 mutations is related to unfavorable events in APL pts. Disclosures:No relevant conflicts of interest to declare.

Philadelphia Chromosome (Ph)-Positive Leukemia Patients Who Fail ABL1 Tyrosine Kinase Inhibitors Without BCR-ABL1 Kinase Domain Point Mutations Demonstrate Sensitivity To PI3-K/AKT Inhibitors

Despite the impressive success of imatinib in chronic myeloid leukemia (CML), resistance to therapy remains an issue for approximately 15-20% of newly diagnosed chronic phase patients by 5 years and transient responses are the rule for patients with blast crisis CML or Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). The most frequent and well-characterized mechanism of resistance to imatinib is acquisition of point mutations in the BCR-ABL1 kinase domain which compromise drug binding. This form of resistance is largely well-handled by the newer, more potent ABL1 inhibitors nilotinib, dasatinib, and ponatinib. However, in the remainder of patients with resistance sustained inhibition of BCR-ABL1 kinase activity is necessary but no longer sufficient to inhibit cell growth, implicating activation of additional, thus far, poorly understood BCR-ABL1-independent mechanisms of growth and survival. In an effort to identify and validate novel pathways important in BCR-ABL-independent resistance to ABL1 kinase inhibitors, we screened a cohort of 30 patients with CML or Ph+ ALL exhibiting clinical resistance to at least one ABL1 kinase inhibitor without evidence of a drug-resistant BCR-ABL1 kinase domain mutation. Following informed consent, fresh primary mononuclear cells were isolated from each sample by Ficoll gradient centrifugation, plated ex vivo in the presence of a panel of clinical and pre-clinical small-molecule kinase inhibitors, and assessed for effects on viability after 3 days. Ex vivo resistance to ABL1 kinase inhibitors largely tracked with clinical resistance profiles, and considerable variation in sensitivity profiles for other inhibitors was observed. Among these data, we found a particularly interesting subset of patients whose cells demonstrated ex vivo sensitivity to one or more PI3-K/AKT inhibitors (n=6/30; 20%) including PI-103, BEZ235, CAL-101, and MK-2206. These findings are consistent with previous studies demonstrating PI3-K activation in select ABL1 kinase inhibitor-resistant CML cell lines (Quentmeier et al., J Hem Onc 2011) and in response to imatinib in pre-overt resistance (Burchert et al., Leukemia 2005). Among the effective PI3-K/AKT inhibitors, the most prominent were the dual class IA PI3-K/mTOR inhibitor PI-103 (IC50 range:38-110 nM) and BEZ235 (IC50 range: 20-210 nM). To further interrogate the mechanism(s) behind dependence on this pathway, samples of interest were analyzed by deep sequencing using a custom capture library encompassing ∼2000 kinases, phosphatases, and adaptor proteins. However, in contrast to previous reports of activating PIK3CA mutations in imatinib-resistant KCL-22 cells, we found PIK3CA to be wild-type in all samples. Despite a common sensitivity to PI3-K/AKT pathway inhibition, we found sequence variants were somewhat heterogeneous and direct kinase targets in this pathway were wild-type, suggesting other causative lesions which activate this pathway. Variants were prioritized based on known associations to this pathway, generated and evaluated in vitro for Ba/F3 cell transformation capacity, and validated for pathway activation and kinase inhibitor sensitivity. These results will be presented. Taken together, our findings suggest that a subset of patients with Ph+ leukemia who become refractory to ABL1 kinase inhibitors without a BCR-ABL1 kinase domain mutation demonstrate acquired dependence on the PI3-K/AKT axis, warranting further investigation of inhibitors of this pathway alone and in combination with ABL1 inhibitors as a molecularly targeted therapeutic strategy in patients. Disclosures:Off Label Use: Ruxolitinib - a JAK1/2 inhibitor that we propose can be used off-label for disease management of CSF3R-mutant neutrophilic leukemia. Deininger:BMS: Consultancy, Research Funding; ARIAD: advisory board, advisory board Other, Consultancy; Novartis: advisory board, advisory board Other, Consultancy, Research Funding; Celgene: Research Funding; Gilead: Research Funding. Tyner:Incyte Corporation: Research Funding. Druker:Novartis, Bristol-Myers Squibb, ARIAD & Incyte: Clin trial funding. OHSU holds contracts; Druker receives no salary/lab research funds. OHSU & Druker have financial interest in MolecularMD; technology used in some studies licensed to MolecularMD. This conflict has been reviewed and managed by OHSU. Other.

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

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

Targeting Tyrosine Kinase Receptors

Protein tyrosine kinases (PTKs) regulate cell growth and other key functions. Constitutive PTK activation by somatic mutations, overexpression, or abnormal upstream signaling is characteristic of many cancers, including hematologic malignancies, providing a rationale for therapeutically targeting PTKs with small molecules. Imatinib, an ATP-competitive inhibitor of BCR-ABL1, the PTK causal to chronic myeloid leukemia (CML), established a paradigm for tyrosine kinase inhibitors (TKIs) as cancer therapeutics. Although a relatively weak inhibitor, imatinib is effective in most patients with chronic phase CML (CML-CP), while responses are transient in blastic phase (CML-BP). Point mutations in the BCR-ABL1 kinase domain have emerged as a major mechanism of drug resistance. The more potent second-generation TKIs – dasatinib, nilotinib, and bosutinib – induce deeper and faster responses and are active against many imatinib-resistant mutants, with the exception of T315I in the gatekeeper position of the catalytic site. This problem was addressed with ponatinib, a third-generation TKI covering all single BCR-ABL1 mutants, including T315I. Ponatinib has excellent clinical activity in CML-CP patients who failed other TKIs, while responses in CML-BP are short-lived. Some patients fail ponatinib due to BCR-ABL1 compound mutations, suggesting even third-generation TKIs cannot completely prevent mutational escape by the disease-initiating kinase. Another unsolved problem is that TKIs fail to efficiently target CML stem cells, leading to recurrence of active leukemia upon discontinuation. Despite these shortcomings, TKIs have completely changed the face of CML. Unfortunately, repeating this success in other hematologic malignancies has been challenging, likely reflecting differences in disease biology as much as suboptimal design of early compounds. CML-CP represents one extreme of the spectrum, where a single genetic lesion is sufficient to produce the phenotype and the hierarchy of hematopoietic differentiation is maintained. The situation is different in acute myeloid leukemia (AML) with activating FLT3 mutations. Not only these AML cases have mutations in other genes, they typically acquire FLT3 mutations late during disease evolution, implying that the disease-initiating clone will be impervious to FLT3 inhibition. Progress has been made through successive development of more potent TKIs with improved pharmacology, leading to quizartinib. It is clear, however, that FLT3 inhibitors cannot be used as single agents if there is a curative intent and the same may be true for JAK2 inhibitors in myelofibrosis. The first approved JAK2 inhibitor, ruxolitinib, dramatically improves symptoms, but has yet to demonstrate a significant impact on the malignant clone and is certainly not curative. It remains to be seen whether this reflects the fact that JAK2 activation is not the disease–initiating event, lack of inhibitor specificity towards the mutant JAK2 kinase, or other undesirable off-target effects that may be overcome with improved drugs. A completely new chapter was opened with ibrutinib, an irreversible inhibitor of Bruton's tyrosine kinase (BTK), for the treatment of chronic lymphocytic leukemia (CLL). BTK is essential for signal transduction from the B-cell receptor (BCR). No activating mutations in BTK have been identified in lymphoma or CLL, but constitutive BCR signaling is critical to CLL cell survival in the microenvironment. Early studies show excellent clinical activity in patients with advanced CLL, although many responses are incomplete; much like the imatinib responses in late CML-CP. Ibrutinib may have a similarly profound effect upon CLL as imatinib on CML, but perhaps also similar limitations, such as the inability to eradicate residual leukemia; this of course needs to be tested in frontline studies. TKIs have had a significant albeit uneven impact upon treatment paradigms in hematologic malignancies. Future progress will involve optimizing compounds in terms of potency, selectivity, and pharmacokinetics. Allosteric inhibitors may add to the armamentarium. From the target perspective, it is likely that most activated kinase alleles have been discovered and the focus should shift to identification of disease-critical unmutated kinases. Lastly, identifying synthetically lethal inhibitor combinations will be critical to fully exploit the potential of TKI therapy. Disclosures:Deininger:BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; ARIAD: Consultancy, Membership on an entity's Board of Directors or advisory committees; NOVARTIS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; CELGENE: Research Funding; GENZYME: Research Funding; INCYTE: Consultancy, Membership on an entity's Board of Directors or advisory committees; GILEAD: Research Funding.

Kinetics Of Low-Level Mutant Clones Detected By Subcloning and Sequencing In Tyrosine Kinase Inhibitor Resistant CML

BackgroundBCR-ABL1 kinase domain (KD) point mutation causes resistance to tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML) patients through impaired binding of TKI to the target site. Recent studies have reported that multiple mutations detected in 2-9% of patients with imatinib (IM)-resistant CML were associated with poor response rate and survival outcomes. However, biological characteristics and dynamics of multiple low-level mutations are still not assessed with a quantitative serial follow-up data in the same populations. AimsThe aim of this study was to investigate biological characteristics and dynamics of low-level mutations in the serial samples from the patients carrying multiple mutations using subcloning and sequencing. MethodsSince 2002, 414 CML patients were screened for mutation analysis due to sign of resistance to TKIs including imatinib (IM), nilotinib (NIL), dasatinib (DAS), bosutinib (BOS), radotinib (RAD) or ponatinib (PON) at Seoul St Mary's Hospital using direct sequencing and allele specific oligonucleotide-polymerase chain reaction (ASO-PCR). Among them, 31 patients carried ≥ 2 BCR-ABL1 kinase domain mutations. We analyzed 137 samples from these 31 patients using subcloning and sequencing (in total, 2737 colonies were sequenced). By cloning and sequencing, two or more missense mutations present in the same clone were defined as compound mutation and co-existence of single missense mutations in the separated clones was defined as polyclonal mutation. Co-existence of single missense mutation and compound mutation harboring two or more missense mutations in the same clone was defined as mixed mutation. Missense mutations detected by direct sequencing are defined as predominant mutations. Missense mutations detected by cloning and sequencing but not by direct sequencing are defined as low-level mutations. ResultsIn a total of 2737 colonies from 137 samples, 1596 (58%) colonies harbored ≥ 2 missense mutations with a median 2 (range, 2 – 7) mutations, and 905 (33%) colonies with a single mutation and 236 (9%) colonies with wild type were observed.In 2737 colonies, 692 different low-level mutations were detected by cloning and sequencing but not by direct sequencing. Among them, M244V, G250E, Y253H, E279K, T315I, F317L, M351T, E355A, F359I, and F359V were detected by direct sequencing in the followed-up samples, and the others remain undetectable by direct sequencing. To address whether these low-level mutations were distinct in the patients with TKI resistance, we applied the cloning and sequencing to samples from 3 healthy controls and 3 patients with optimal response to IM. 38 different mutations were detected healthy controls. 52 different mutations were detected in optimal responders. S349P, N374S, E450G, and S485P were detected in both healthy controls and optimal responders. All mutations detected in optimal responders except 3 missense mutations (V335A, F382V, and A395S) were detected as low-level mutations in the 31 patients' cohort with TKI resistance. Of 38 different mutations detected in healthy control, 31 mutations except 7 missense mutations (Y264H, M343R, A350V, E373G, G298R, E462K, and T495K) were also detected as low-level mutations in the 31 patients' cohort. ConclusionsWe showed basic characteristics and dynamics of low-level mutations by subcloning and sequencing. Some of the low-level mutant clones harboring previously known (clinically common) TKI resistant mutations changes to the predominant in the followed-up samples. Except them, most low-level mutant clones were not detected repeatly and did not increase gradually in the serial samples, implying that they may not have clinical significance. Disclosures:No relevant conflicts of interest to declare.

New Strategies in Neuroblastoma: Therapeutic Targeting of MYCN and ALK

Clinical outcome remains poor in patients with high-risk neuroblastoma, in which chemoresistant relapse is common following high-intensity conventional multimodal therapy. Novel treatment approaches are required. Although recent genomic profiling initiatives have not revealed a high frequency of mutations in any significant number of therapeutically targeted genes, two exceptions, amplification of the MYCN oncogene and somatically acquired tyrosine kinase domain point mutations in anaplastic lymphoma kinase (ALK), present exciting possibilities for targeted therapy. In contrast with the situation with ALK, in which a robust pipeline of pharmacologic agents is available from early clinical use in adult malignancy, therapeutic targeting of MYCN (and MYC oncoproteins in general) represents a significant medicinal chemistry challenge that has remained unsolved for two decades. We review the latest approaches envisioned for blockade of ALK activity in neuroblastoma, present a classification of potential approaches for therapeutic targeting of MYCN, and discuss how recent developments in targeting of MYC proteins seem to make therapeutic inhibition of MYCN a reality in the clinic.

Activation of the Janus kinase/signal transducer and activator of transcription pathway in multiple myeloma is not related to point mutations in kinase and pseudokinase domains of JAK1

Considering the recent impact of tyrosine kinase inhibitors in the treatment of myeloproliferative disorders carrying a recurrent JAK2 mutation not identified in multiple myeloma (MM), this study aimed to search for mutations in kinase and pseudokinase domains of the JAK1 gene in an attempt to define any critical and recurring change that can be used as a therapeutic target. We obtained CD138 + purified cells from 27 bone marrow aspirates of untreated MM, four normal controls and four MM cell lines. After amplification of kinase and pseudokinase domains of JAK1 in cDNA samples, the fragments were automatically sequenced. Seventy-eight percent of MM cases showed at least one polymorphism, all being synonymous single nucleotide polymorphisms (SNPs), with allele frequencies consistent with previous studies in normal European, African American and Asian populations. The four cell lines also showed only synonymous SNPs. Mutations in the kinase and pseudokinase domains of the JAK1 gene do not seem to be important for activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway because we were not able to find any recurrent mutation in a case series of 27 patients and four MM cell lines.

Suppression of Survivin Induced by a BCR-ABL/JAK2/STAT3 Pathway Sensitizes Imatinib-Resistant CML Cells to Different Cytotoxic Drugs

The BCR-ABL oncoprotein of chronic myelogenous leukemia (CML) displays exclusive cytoplasmic localization and constitutive tyrosine kinase activity leading to the activation of different pathways that favor cell proliferation and survival. BCR-ABL induces survivin expression at both the mRNA and protein level, thus inhibiting the apoptotic machinery of CML cells and contributing to the expansion of the leukemic clone. We report that, in human CML cell lines, BCR-ABL-mediated upregulation of survivin involves the JAK2/STAT3 pathway since silencing of either protein caused a consistent reduction in survivin expression. Cell lines unresponsive to imatinib mesylate (IM) because of BCR-ABL gene amplification were not resensitized to the drug after survivin downregulation. However, cells insensitive to IM because of point mutations in the BCR-ABL kinase domain were highly responsive to hydroxyurea (HU) after survivin silencing. To address the possible clinical applications of our results, we used shepherdin, a cell-permeable peptidomimetic compound that downregulates survivin expression by preventing its interaction with Hsp90. Incubation with shepherdin of immortalized cell lines both sensitive and resistant to IM enhanced cell death induced by HU and doxorubicin. Similarly, the combination of shepherdin with first- and second-generation tyrosine kinase inhibitors reduced the colony-forming potential of human progenitors derived from both patients with IM-sensitive and IM-resistant CML. These results suggest that strategies aimed at reducing survivin levels may represent a potential therapeutic option for patients with CML unresponsive to IM.

Modeling the Regulation of the Kinase Domain of EGFR

Cell division is a tightly regulated process, and if not controlled, may lead to the formation of tumors. Epidermal growth factor receptor (EGFR) is a transmembrane receptor that causes cell division in a wide range of cell types in response to several different growth factors. Mutations that activate EGFR in the absence of growth factors can contribute to cancer. The Dalton School SMART Team (Students Modeling A Research Topic) modeled EGFR using 3D printing technology. EGFR is a kinase that phosphorylates other proteins to turn them on or off. EGFR consists of an extracellular ligand‐binding domain, an anchoring intramembrane segment, a juxtamembrane connector segment, and a phosphorylating intracellular kinase domain. Generally, EGFR remains in an inactive form and becomes active when a ligand growth factor binds to the extracellular domain. Ligand binding changes the shape of the intracellular kinase domain, including the active site, turning it on. Active EGFR initiates a signaling pathway that causes cell growth and division. However, mutations in EGFR, including deletion of the extracellular domain or kinase domain point mutations, can activate EGFR in the absence of a growth factor, which may cause the formation of tumors because cells grow and divide uncontrollably. Therefore, EGFR is a potential target for cancer therapy. Supported by a grant from the Camille and Henry Dreyfus Foundation.

Strategies to circumvent the T315I gatekeeper mutation in the Bcr-Abl tyrosine kinase

Despite the remarkable success of imatinib against Bcr-Abl, development of secondary resistance, most often due to point mutations in the Bcr-Abl tyrosine kinase (TK) domain, is quite common. Of these, the T315I “gatekeeper” mutation is resistant to all currently registered Bcr-Abl TK inhibitors (TKIs) with the notable exception of ponatinib (Iclusig™), which was very recently approved by the United States Food and Drug Administration (FDA). Besides ponatinib, numerous strategies have been developed to circumvent this problem. These include the protein synthesis inhibitor omacetaxine (Synribo®), and “switch-control” inhibitors. Dual Bcr-Abl and aurora kinase inhibitors represent another promising strategy. Finally, several promising synergistic combinations, such as TKIs with histone deacetylase inhibitors (HDACIs), warrant attention.

Contribution of BCR-ABL Kinase Domain Mutations to Imatinib Mesylate Resistance in Philadelphia Chromosome Positive Malaysian Chronic Myeloid Leukemia Patients

Development of resistance to imatinib mesylate (IM) in chronic myeloid leukemia (CML) patients is mediated by different mechanisms that can be classified as BCR-ABL dependent or BCR-ABL independent pathways. BCR-ABL dependent mechanisms are most frequently associated with point mutations in tyrosine kinase domain (TKD) of BCR-ABL1 and also with BCR-ABL gene amplification. Many different types and frequencies of mutations have been reported in different studies, probably due to the different composition of study cohorts. Since no reports are available from Malaysia, this study was undertaken to investigate the frequency and pattern of BCR-ABL kinase domain mutations using dHPLC followed by sequencing, and also status of BCR-ABL gene amplification using fluorescence in situ hybridization (FISH) on 40 IM resistant Malaysian CML patients. Mutations were detected in 13 patients (32.5%). Five different types of mutations (T315I, E255K, Y253H, M351T, V289F) were identified in these patients. In the remaining 27 IM resistant CML patients, we investigated the contribution made by BCR-ABL gene amplification, but none of these patients showed amplification. It is presumed that the mechanisms of resistance in these 27 patients might be due to BCR-ABL independent pathways. Different mutations confer different levels of resistance and, therefore, detection and characterization of TKD mutations is highly important in order to guide therapy in CML patients.

Dynamics and Characteristics of BCR-ABL1 Multiple Mutations in Tyrosine Kinase Inhibitor Resistant CML

AbstractAbstract 1677 Background:BCR-ABL1 kinase domain (KD) point mutation causes resistance to tyrosine kinase inhibitors (TKI) in CML patients through impaired binding of TKI to the target site. One of the characteristics of patients with BCR-ABL1 kinase domain point mutations is the fact that some patients have multiple mutations. However there have not been many studies showing that data about clinical relevance or dynamics of multiple mutation during CML treatment. Methods:Since 2002, 414 CML patients were screened for mutation analysis due to sign of resistance to TKI including imatinib, nilotinib, dasatinib, bosutinib, radotinib or ponatinib at Seoul St Mary's Hospital using direct sequencing and ASO-PCR. Among them, 31 patients showed multiple mutations. We analyzed serial samples from the 31 patients using subcloning and sequencing to investigate whether the multiple mutations are on same clone (defined as compound clone), separated clones (defined as multiple clone) or co-existent clones (defined as mixed clone) and characterize its clinical relevance and dynamics. Results:Status of the patients with multiple mutations is shown in Table 1. In order to investigate whether the multiple mutations are on same clone or on separated clone, we cloned serial samples from the 31 patients. Cloning of cDNA region corresponding to BCR-ABL1 KD into plasmid was performed and followed by transformation into competent cells, colony formation, plasmid preparation of 20 colonies from each sample, and then direct sequencing.Multiple mutations of 65% patients (20 out of 31) existed compound mutation which means the individual mutant types are located on the same BCR-ABL1 molecule. In addition of major mutation types which were detectable in direct sequencing analysis, all the patients showed to have minor types of mutations which were found only through BCR-ABL1 KD cloning and subsequent colony sequencing. To make sure that this minor mutation types were not caused by sequencing error, we also analyzed of 3 patients who showed TKI resistance, but had no BCR-ABL1 mutation. In addition, samples from 3 normal persons were analyzed with the same method. The frequency of appearance of the minor types of point mutation was reduced in the patient group who showed TKI resistance, but had no BCR-ABL1 mutation, and then dramatically decreased in the normal person group, indicating that BCR-ABL1 gene in patients with point mutation are relatively unstable.Among 20 patients with compound mutation, 9 patients were available for serial timepoint samples under same TKI therapy. In all nine patients (100%), portion of compound clone was increased as treatment went on. With a median follow-up 53.3 months (range, 0–113.2 months), of 31 patients with multiple mutation, 7 patients remained alive; 4 of 11 (36%) in the multiple clone group vs 3 of 20 (15%) in the mixed clone group (P = 0.066). Conclusion:Analysis of serial samples from a same patient provided evidence of dynamic change of portion of compound mutation. In most case, portion of the clone containing compound mutation was increased as treatment went on, indicating the clone harboring compound mutation can take survival advantage over TKI treatment in comparison of the clone containing individual type of mutation. In addition, some patients showed change in individual mutation type comprising multiple mutations as treatment went on. Patients with compound clone showed poor outcomes compared with multiple clone group in our cohort, further investigation on a large patient cohort will be needed. Updated data with longer follow-up duration will be presented in the meeting. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Constitutively Activating Mutations At the FLT3 Activation Loop Residue D835 Are Associated with Clinical Resistance to AC220

Abstract 674 Background.Activating mutations in FLT3 occur in ∼30% of adult acute myeloid leukemia (AML) cases, including internal tandem duplication (ITD) mutations (∼25%) and point mutations in the tyrosine kinase domain (KD), primarily at the activation loop (AL) residue D835 (∼5%). FLT3-ITD and FLT3-AL mutations can co-occur in individual patients. AC220 (quizartinib) is a potent selective investigational FLT3/KIT inhibitor with encouraging preliminary clinical activity in FLT3-ITD+ AML, as evidenced by a composite complete remission rate of 45% in 53 chemotherapy refractory/relapsed patients in an interim analysis of a phase II study (Cortes et al, ASH 2011 abstract #614). While some patients without FLT3 mutations in the AC220 phase I dose escalation study responded, no objective response was observed in a small number of FLT3-AL mutant patients (Cortes et al, ASH 2008 abstract #767). We recently reported that secondary mutations at F691, D835 and Y842 in FLT3-ITD confer in vitro resistance to AC220, and that relapse in 8/8 AML patients who initially responded to AC220 was associated with evolution of secondary KD mutations in the FLT3-ITD+ allele (Smith et al., Nature, 2012). In 6 of 8 patients, relapse was associated with D835 substitutions, making this amino acid position the most commonly substituted residue in FLT3-ITD+ patients at the time of disease relapse. Given that D835 mutations in FLT3-ITD appear to confer a high degree of pre-clinical and clinical resistance to AC220, we hypothesized that D835 mutations in the absence of the ITD might also confer resistance to AC220 and could be associated with clinical relapse, especially in patients harboring a mixture of FLT3-WT, FLT3-ITD+, and FLT3-AL mutant blasts. Results.In vitro binding studies revealed that FLT3-D835V and FLT3-D835Y have decreased affinity for AC220 (Kd=4.8 and 7nM, respectively) compared to native FLT3 (Kd=1.8nM). In proliferation assays of BaF/3 cells, FLT3-D835V/Y mutants demonstrated increased relative AC220 resistance (IC50 24 and 6.8nM) compared to FLT3-ITD (IC50 0.13nM). To sensitively and precisely determine the frequency and phase (ITD+ vs ITD-) of mutations in clinical isolates, we utilized single molecule real-time (SMRT; Pacific Biosciences, Menlo Park, CA) sequencing, which can generate reads of sufficient length to enable focused interrogation of the KD of FLT3 ITD+ and ITD- alleles. We analyzed ITD- sequences from pretreatment and relapse samples obtained from the 8 FLT3-ITD-positive AML patients with acquired AC220 resistance reported in our previous analysis. Interestingly, all 6 patients with secondary mutations at D835 in ITD+ alleles at relapse also harbored D835 mutant ITD- alleles. All D835 mutations detected in ITD- alleles were also observed in ITD+ alleles from the same patient sample. In ITD+ alleles, we detected D835Y (n=6), D835V (n=3), and D835F (n=2) (range 2.7–50.6% of ITD+ alleles). In ITD- alleles, we found D835Y (n=6), D835V (n=2), and D835F (n=1) (range 3.8–50% of ITD- alleles). One patient had 2 D835 mutations (D835Y/F) in ITD- alleles at relapse. Although 2 of 8 patients evolved the F691L mutation in ITD+ alleles at relapse, this substitution was not detectable in ITD- sequences in any patient. In 7/8 patients, samples obtained immediately prior to AC220 administration were available for analysis. We were unable to document the presence of AC220-resistant mutations in either ITD+ or ITD- sequences in these pretreatment samples, suggesting that these mutants evolved under the selective pressure of AC220 treatment. We are currently performing single cell RT-PCR to determine if D835 mutations in ITD+ and ITD- alleles occur in distinct leukemic cells, which would suggest a polyclonal blast population at relapse. Conclusions.FLT3-AL mutations at D835 confer resistance to AC220 in vitro. The evolution of D835 substitutions in ITD- alleles in the majority of patients who respond and relapse on AC220 suggests that constitutively activating FLT3-AL mutations at residue D835 can confer acquired clinical resistance to AC220. AML patients with FLT3-D835 mutations may exhibit de novo resistance to AC220 and other FLT3 inhibitors. Our findings predict that the clinical activity of potent FLT3 inhibitors that are tolerant of D835 substitutions will mechanistically involve inhibition of D835 mutants in both the presence and absence of an ITD mutation. Disclosures:Chin:Pacific Biosciences: Employment. Levis:Astellas Pharma: Consultancy; Plexxikon: Consultancy; Symphogen: Consultancy; Ambit: Joined performance of clinical trial, Joined performance of clinical trial Other. Perl:Astellas Pharmaceuticals: Consultancy. Travers:Pacific Biosciences: Employment. Kasarskis:Pacific Biosciences: Equity Ownership. Radich:Ariad: Consultancy; Bristol-Myers Squibb: Consultancy; Pfizer: Consultancy; Novartis: Consultancy, Research Funding. Shah:Ariad: Consultancy, Research Funding.