ALBERT

Description: Key Points UDS demonstrated that BCR-ABL KD mutations detectable with conventional methods may just be the tip of the iceberg. The information provided by conventional Sanger sequencing may not always be sufficient to predict responsiveness to a given TKI.

Description: Introduction. Autologous Bone Marrow Transplantation (Auto-BMT) is currently rarely used in the treatment of Acute Myeloid Leukemia (AML). However, it may represent a good therapeutic option in a specific subset of patients, mainly in consolidation of both low risk (LR) and MRD negative AML without an available HLA matched donor. Aims. To review our database of AML patients who received Auto-BMT from 2005 to 2014 and who were referred to Bologna Institution, in order to assess the efficacy of the procedure in terms of Overall Survival (OS) and Disease Free Survival (DFS). Patients and methods: From 2005 to 2014, 98 AML patients underwent Auto-BMT in several Italian Institutions. 89/98 patients are evaluable for survival and outcome data. The 89 patients considered (42 female, 47 male), had a median age of 49 years (range 15-70). Cytogenetics was performed in all patients by conventional karyotype (22 patients were also analyzed by Single Nucleotide Polymorphisms Array); molecular analysis (FLT3 TKD and ITD, and NPM1 mutational analysis) was available for 51/89 patients. Molecular monitoring by specific fusion transcripts (CBF-MYH11 and AML1-ETO) was performed in CBF positive leukemias (inv(16) and t(8;21)) at the time of diagnosis, after induction, consolidation courses, and every 3 months in the first 2 years of follow-up. Based on this data, and according to ELN guidelines, a risk stratification identified 41 patients with a LR AML (t(8:21), inv(16) or NPM1+/FLT3- with normal karyotype), 4 patients with a high risk (HR) AML (complex karyotype or FLT3 ITD mutated or inv(3) or t(6;9)) and 44 patients with a standard risk (SR) AML (normal karyotype, other alterations). Results. All the patients received an induction chemotherapy treatment, as follows: a "3+7-like" course in 48 cases, a Fludarabine-based regimen in 20 patients and a Gemtuzumab-ozogamicin (GO)-based regimen in 21. 83/89 (93.3%) patients received a median of 2 consolidation courses of chemotherapy (range 1-4) before proceeding to Auto-BMT, performed in 1st CR. 6/89 (6.7%) patients received Auto-BMT in first relapse. 41 patients relapsed after auto-BMT and were treated with a re-induction chemotherapy, or were enrolled in clinical trials. 24 patients reached a 2nd complete remission, and 12 patients underwent an allogeneic BMT in 2nd CR. With a median follow up of 6 years, the median Overall Survival (OS) of the entire population was 64.3 months (range 5.8-294.2 months); the 1 year OS and the 5 years OS were, 97.1%, and 67.9%, respectively. The median Disease Free Survival (DFS) of the 83 patients treated with Auto-BMT in 1st CR was 36 months (range 1.3-293 months). The 1-year DFS and the 5-years DFS were 85% and 56.7%, respectively. Transplant related mortality (TRM, death in 100 days after BMT) was 1.2% for auto-BMT and 6.5% for allogeneic BMT. First, to assess the role of the number of consolidation courses we compared patients who received none or 1 consolidation course with patients who received 2 or more cycles, who showed a better OS (p= 0.0061, Figure 1). There was no statistical difference in terms of OS between young and elderly patients (cut off=65 years). Second, we compared patients who achieved a negative minimal residual disease status before auto-BMT (n=37) with patients who did not (n=9). MRD negativity offered a significantly better outcome in terms of 5-years OS (83.4% and 50% respectively); the median OS of MRD neg was not yet reached; the median OS of MRD pos was 27 months (p= 0.0130) (Figure 2). Conclusions: Auto-BMT offers a chance to achieve long-term DFS and OS if used as a consolidation therapy both in patients with LR and SR AML. The major role could be played in MRD negative patients, offering the best chances to achieve a long-term OS. Auto-BMT can be also a good choice as consolidation therapy for elderly patients, in which allo-BMT could induce high morbidity and mortality rates. The small patients cohort and the retrospective analysis don't allow us to define the best induction therapy to be used before auto-BMT. However, based on our findings we suggest a therapy schedule including two or more consolidation courses in patients who obtain a first CR, and to proceed then to auto-BMT. Acknowledgments: work supported by ELN, AIL, AIRC, Progetto Regione-Università 2010-12 (L.Bolondi), Fondazione del Monte di Bologna e Ravenna, FP7 NGS-PTL project. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Rodeghiero:Celgene Corporation: Honoraria, Research Funding. Cavo:Janssen-Cilag, Celgene, Amgen, BMS: Honoraria. Martinelli:AMGEN: Consultancy; Novartis: Consultancy, Speakers Bureau; Ariad: Consultancy; BMS: Consultancy, Speakers Bureau; ROCHE: Consultancy; Pfizer: Consultancy; MSD: Consultancy.

Description: Abstract 2522 Introduction: Although p53 gene mutations are relatively infrequent in cases of B-ALL, the CDKN2A locus is deleted or inactivated in nearly half of all cases, especially Ph+ B-ALL (Mullighan et al., 2008; Iacobucci et al., 2011), contributing to a worse prognosis. In testing novel therapeutic approaches activating p53, we investigated the preclinical activity of the MDM2 antagonist Nutlin-3a in leukemic cell line models and primary B-ALL patient samples. Methods: TP53 mutation screening was performed by Sanger sequencing of exons 4 to 11; copy number status of CDKN2A was determined by MLPA kit P335-A2 ALL-IKZF1 (MRC Holland); cellular viability was assessed by using a colorimetric assay based on mitochondrial dehydrogenase cleavage of WST-1 reagent (Roche); apoptosis was assessed by use of Annexin V/Propidium Iodide (PI); gene expression profile was performed using Affymetrix GeneChip Human Gene 1.0 ST platform (Affymetrix). Mdm2 inhibitor (Mdm2i) Nutlin-3a was provided by Roche. Results: BCR-ABL1-positive (BV-173, SUPB-15) and negative (NALM19, REH) ALL cell lines were investigated for TP53 mutations and CDKN2A deletion. A p53 mutation (R181C) was identified in REH cells, whereas all the remaining cell lines resulted p53 wild-type but they were deleted in the locus containing CDKN2A. Leukemia cell lines were incubated with increasing concentrations of Nutlin-3a (0.005–2 μM) for 24, 48 and 72 hours (hrs). Mdm2 inhibition resulted in a dose and time-dependent cytotoxicity with IC50 at 24 hrs ranging from around 1.5 μM for BV-173 and SUPB-15 to 3.7 μM for NALM-19. By contrast, no significant changes in cell viability were observed in RHE p53-mutated cells after incubation with Mdm2i. The time and dose-dependent reduction in cell viability were confirmed in primary blast cells from a Ph+ ALL patient with the T315I Bcr-Abl kinase domain mutation found to be insensitive to the available tyrosine kinase inhibitors and from a t(4;11)-positive ALL patient (IC50 at 24 hrs equal to 2 μM). Consistent with the results of cell viability, Annexin V/PI analysis showed a significant increase in apoptosis after 24 hrs in sensitive cell lines and in primary leukemia blasts, whereas no apoptosis was observed in REH cells. To examine the possible mechanisms underlying Mdm2i-mediated cell death, western blot analysis was performed. Protein levels of p53, p21 (an important mediator of p53-dependent cell cycle arrest), cleaved caspase-3 and caspase-9 proteins increased as soon as 24 hrs of incubation with Mdm2i. In order to better elucidate the implications of p53 activation and to identify biomarkers of clinical activity, gene expression profiling analysis was next performed, comparing sensitive cell lines at 24 hrs of incubation with concentrations equal to the IC50 and their untreated counterparts (DMSO 0.1%). A total of 621 genes (48% down-regulated vs 52% up-regulated) were differentially expressed (p 〈 0.05). We found a strong down-regulation of GAS41 (growth-arrest specific 1 gene) and BMI1 (a polycomb ring-finger oncogene) (fold-change −1.35 and −1.11, respectively; p-value 0.02 and 0.03, respectively) after in vitro treatment as compared to control cells. Both genes are repressors of INK4/ARF and p21 and their aberrant expression has found to contribute to stem cell state in tumor cells. Additionally, experimental reduction of BMI1 protein levels results in apoptosis in tumor cells and increases susceptibility to cytotoxic agents and radiation therapy (Wu et al., 2011). Given the importance of BMI in the control of apoptosis, we investigated by western blot its pattern in treated and untreated cells, confirming a marked decrease as soon as 24 hrs of exposure to MDM2i both in leukemia cell lines and primary blast samples. Noteworthy, the BMI-1 levels remained constant in resistant cells. Conclusions: Inhibition of Mdm2 efficiently activates the p53 pathway promoting apoptosis. BMI-1 expression is markedly reduced in sensitive cells and it may be used as a biomarker of response. Evaluation of its expression before and after treatment in clinical settings will better gain insight into its role. Supported by: ELN, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, Ateneo RFO grants, Project of integrated program, Programma di Ricerca Regione – Università 2007 – 2009, INPDAP. Disclosures: Soverini: Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:BMS: Consultancy, Honoraria, Speakers Bureau; NOVARTIS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Description: Background: AML is a heterogeneous disease with various chromosomal aberrations. The karyotype at diagnosis provides important prognostic information that influences therapy and outcome, and patients (pts) with complex karyotype (CK) have generally a poor outcome. TP53 is the most frequently mutated gene in human tumors. The reported TP53 mutation rate in AML is low (2.1%). In contrast, the incidence of TP53 mutations in AML with a complex aberrant karyotype is higher (69-78%). Aims: To investigate the frequency, the types of mutations, the associated cytogenetic abnormalities and the prognostic role of TP53 mutations in adult AML pts, we focused the screening on subgroups of AML with chromosome abnormalities. Patients and Methods: 886 AML patients were analysed at the Seràgnoli Institute of Bologna between 2002 and 2013 for morphology, immunophenotype, cytogenetic and for a panel of genetic alterations (FLT3, NPM, WT1, CBF fusion transcripts, DNMT3A, IDH1, IDH2, etc). Of these, 172 adult AML pts were also examined for TP53 mutations using several methods, including Sanger sequencing, Next-Generation deep-Sequencing (Roche) and HiSeq 2000 (Illumina) platform (35/172 pts). 40 samples were genotyped with Genome-Wide Human SNP 6.0 arrays or with CytoScan HD Array (Affymetrix) and analysed by Nexus Copy Number™ v7.5 (BioDiscovery). Results: Of the 886 AML patients beforehand analysed, 172 pts were screened for TP53 mutations and were correlated with cytogenetic analysis (excluding 15 pts where the karyotype was not available). 1. Fifty-two pts (30,2%) have 3 or more chromosome abnormalities, i.e. complex karyotype; 2. 71 (41,3%) presented one or two cytogenetic abnormalities (other-AML) and 3. 34 pts (19,8%) have normal karyotype. Sanger sequencing analysis detected TP53 mutations on 29 patients with 36 different types of mutations; seven pts (4%) have 2 mutations. Mostly (23/29) of the TP53 mutated pts (79.3%) had complex karyotype while only 6/29 mutated pts have “no CK” (21% and 3% of the entire screened population). Overall, between pts with complex karyotype, TP53 frequency is 44.2%. Regarding the types of the TP53 alterations, 32 were deleterious point mutations (http://p53.iarc.fr/TP53GeneVariations.aspx) and 4 deletions. Forty pts were also analysed for Copy Number Alterations (CNAs) by Affymetrix SNP arrays: several CNAs were found ranged from loss or gain of complete chromosome (chr) arms to focal deletions and gains targeting one or few genes involving macroscopic (〉1.5 Mbps), submicroscopic genomic intervals (50 Kbps - 1.5 Mbps) and LOH (〉5 Mbps) events. Of relevance, gains located on chr 8 were statistically associated with TP53 mutations (p = 0.001). Seven genes are included in these regions (RGS20, TCEA1, LINC01299, ARMC1, MTFR1, RAD54B, KIAA1429). In addition to the trisomy of the chr 8, others CNAs, located on other chromosomes are significantly associated (p = 0.05) with TP53 mutations: loss of chr 5q, chr 3 (p22.3), chr 12 (p12.3) and the gain of chr 17 (p11.2), chr 16 (p11.2-11.3) and chr 14 (q32.33). The zinc finger gene ZNF705B, implicated in the regulation of transcription was the most differentially associated gene (gain). WES analysis was done in 37 pts, 32 TP53 were wt while 5 pts were TP53 mutated: of importance, CDC27, PLIN4 and MUC4 were found also mutated in 3 out of 5 TP53 mutated (60%). Clinical outcome: as previously reported, alterations of TP53 were significantly associated with poor outcome in terms of both overall survival and disease free-survival (P 〈 0.0001). Conclusions: Our data demonstrated that TP53 mutations occur in 16.86% of AML with a higher frequency in the subgroup of complex karyotype AML (p〈 0.0001–Fischer’s exact test). Since TP53 mutations have predicted to be deleterious and significantly correlated with prognosis, TP53 mutation screening should be recommended at least in complex karyotype AML pts. Furthermore, although further studies in larger numbers of patients are needed, the gain of chromosome 8 was observed to be significantly associated to TP53 mutations pts. Supported by: ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Martinelli: Novartis: Speakers Bureau; Bristol Mayers Squibb: Speakers Bureau; Pfizer: Speakers Bureau.

Description: Background Ponatinib, a potent third generation pan BCR-ABL inhibitor, has recently shown relevant activity against native and mutant forms of BCR-ABL, including the TKI resistant T315I mutant. The aim of this compassionate protocol was to confirm and evaluate the efficacy and the safety of the compound in patients with advanced Ph+ ALL and CML. Design and Methods Ponatinib was obtained through a compassionate use named patient program, approved by ARIAD Pharmaceuticals and by the Bologna Ethical Committee. After informed consent was signed, 17 patients (M/F: 8/9) have been treated with Ponatinib (45 mg orally, once daily) between February 2012 and July 2013, including 14 Ph+ ALL (10 p190, 4 p210) and 3 blast phases of CML (2 Myeloid and 1 Lymphoid, p210). The median age of the patients was 64 years (range 23 -77). The median time from diagnosis was 754 days (range 46-2264). All the patients were resistant or intolerant to previous TKIs (median number of previous TKIs: 2; range 1-3). Standard chemotherapy was previously performed in 7/17 patients (41%). Four patients (23%) had previously received allogeneic stem cell transplantation. At the time of enrolment, median Hb, PLTs and WBC values were 10,9 g/dl (range 8.6-13.9), 139000/mmc (range 14000-325000) and 4300/mmc (range 1700-17000), respectively. In 6 out of 17 patients, additional cytogenetic alterations were revealed. Mutational analysis showed the presence of T315I mutation (9 pts), G250E (1 pt), T315I and Y253H (1 pt), T315I and Y253A (1 pt), V299L (1 pt). No mutations were detected in 4 patients. Results The median treatment duration was 139 days (range 14-540+). Causes of treatment stop were: progression disease (5 patients), savage allogenic stem cell transplantation (6 patients), drug intolerance (1 patient), consisting in grade III headache. With a median follow up of 284 days (range 8-540+), a maHR was obtained in 13/17 patients (76%). After one month of treatment, a reduction of BCR-ABL fusion transcript level was observed in 9/15 patients (60%). For two patients the follow up is too short to be evaluable. The level became undetectable in 4 patients (3 presenting with T315I mutation). After treatment, T315I mutation disappeared in 6 out of the 9 patients who showed this molecular alteration at the beginning of therapy. At the time of this report, 6/17 patients are still on study (35%). Five patients died due to progression disease. As expected, the drug was well tolerated. Non-hematologic adverse events were described in 7/17 patients (grade 〉III skin rash in 3 patients; grade〉II serum lipase increase in 2 patients; grade〉II myalgia in 1 patient; grade III headache). Conclusion In our experience, the activity of Ponatinib in advanced Ph+ leukemias, mainly in T315I mutated patients, was confirmed. No treatment-related deaths occurred. The understanding of molecular mechanisms responsible for resistance or lack of response to the drug will be necessary in order to identify patients early on who could take advantage of this treatment. Acknowledgments Work supported by European LeukemiaNet, AIRC, PRIN 2010-2011, University of Bologna and BolognAIL. Disclosures: Soverini: Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Martinelli:NOVARTIS: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Description: Background: The European LeukemiaNet (ELN) response criteria are widely used to decide, at given time points, when the treatment with tyrosine-kinase inhibitors (TKIs) of CML patients should be continued (optimal response, OR), when a careful monitoring is required (warning, W) or when the therapy should be changed (failure, F). The 2013 ELN response criteria are the same for all chronic phase CML patients, irrespective of the prescribed TKI, but the time to response is influenced by the first-line TKI. Despite faster responses, a clear survival advantage of 2nd generation TKIs over imatinib (IM) has not been demonstrated yet. A validation of the 2013 ELN response definitions and an analysis of their prognostic impact in IM-treated patients may provide important information. Aims: The aim of our study was to assess the significance of 2013 ELN response criteria in CML patients treated frontline with IM, investigating whether or not optimal responders, warnings or failures at 3, at 6 and at 12 months have a different long-term outcome. Methods: 559 patients enrolled within 3 prospective clinical trials (NCT00514488, NCT00510926, observational trial CML/023) were analyzed (ITT population of each study). The 3-month response according to 2013 ELN criteria was not fully evaluable due to missing cytogenetic analysis in 452/559 patients, so we focused on the early molecular response (EMR, BCR-ABL 〈 10% at 3 months), corresponding to OR. The responses at 6 and 12 months were retrospectively defined according to 2013 ELN criteria: F, BCR-ABL 〉 10% and/or Ph+ 〉 35% at 6 months, BCR-ABL 〉 1% and/or Ph+ 〉 0 at 12 months; OR, BCR-ABL 〈 1% and/or Ph+ 0 at 6 months, BCR-ABL 〈 0.1% at 12 months; W: intermediate conditions. As the ELN criteria changed over time, not all the failures switched to alternative treatment. Progression: transformation to advanced phases (2013 ELN definitions) at any time, including after treatment discontinuation. Overall survival (OS): all the deaths at any time (in-study or off-study) were included. Leukemia-unrelated death: known cause of death, no progression, CCyR and/or MMR 〈 6 months prior to death; all other deaths were classified as leukemia-related (LRD). The cumulative incidence of LRD was estimated considering the competing risk of leukemia-unrelated death. Results: The median follow-up was 76 months (66-99 months). The patients with OR at 3 months were 82%; the patients with OR-W-F at 6 months were 76%, 14% and 10%, respectively; the patients with OR-W-F at 12 months were 65%, 20% and 14%, respectively. The OS, the progression-free survival (PFS) and the cumulative incidence of LRD according to the presence-absence of EMR were 87-81% (p=0.015), 85-81% (p=0.035) and 11-5% (p=0.019), respectively. Combining the Sokal score and the EMR, the patients were divided into 4 groups, low and intermediate risk/responders, low and intermediate risk/not responders, high risk/responders, high risk/not responders: the OS and the cumulative incidence of LRD across the 4 groups were 88%, 84%, 86% and 70% (p=0.005, Figure 1) and 3%, 9%, 10% and 20% (p

Description: Background and Aims- In Ph+ ALL pts treated with tyrosine kinase inhibitors (TKIs), the likelihood of acquiring TKI-insensitive mutations and the striking incidence of highly resistant T315I and compound mutants underscore the importance of BCR-ABL1 kinase domain (KD) sequence surveillance for timely and rational therapeutic reassessment. We used an amplicon DS strategy of the BCR-ABL1 KD to assess the following issues: i) whether DS allows earlier detection of emerging TKI-insensitive mutations in pts undergoing BCR-ABL1 KD mutation screening for minimal residual disease (MRD) persistence; ii) whether TKI-insensitive low burden mutations can be identified in relapsed pts with negative conventional sequencing results; iii) whether TKI-insensitive low burden mutations are necessary and sufficient to predict for treatment failure in all cases. Methods- This study was conducted in a total of 56 Ph+ ALL pts who received TKI-based therapies at our or collaborating institutions and were referred to our laboratory for MRD follow-up monitoring by RQ-PCR and for BCR-ABL1 KD mutation analysis in case of MRD positivity. These pts were divided into three different cohorts: i) 10 de novo and 24 advanced Ph+ ALL pts who relapsed and developed BCR-ABL1 KD mutations on TKI-based therapy administered 1st-line or for recurrent disease, respectively. To reconstruct the dynamics of mutation emergence, longitudinal re-analysis of monthly-collected samples from the time of hematologic relapse backwards was performed by DS. Whenever samples were available, the analysis was done back to the time of diagnosis (n=10/10) or back to the time of first or former relapse (n=15/24), respectively. Two to 6 samples were analyzed for each pt, for a total of 109 samples. ii) 14 Ph+ ALL pts who were known to be negative for mutations at the time of hematologic relapse as assessed by conventional sequencing. Relapse samples were reanalyzed by DS. iii) 8 Ph+ ALL pts with long-term relapse-free survival despite persistent or intermittent MRD positivity at multiple timepoints. Up to 5 samples were analyzed for each pt, for a total of 28 samples. DS was performed on a Roche GS Junior. Lower mutation detection limit of DS was 1%. Results- In the 34 de novo or advanced Ph+ ALL pts who were known to have acquired TKI-insensitive mutations at the time of relapse on tyrosine kinase inhibitor (TKI) therapy, longitudinal retrospective reanalysis by DS allowed mutation backtracking in 13 (41%) cases. One patient was found to harbour a low burden Y253H at diagnosis. In 3 imatinib (IM)-resistant pts who switched to dasatinib (DAS), a low burden T315I mutation was already detectable at baseline. In the 14 pts with no mutations detectable by conventional sequencing at the time of relapse on IM or DAS, low burden TKI-insensitive mutations were detected by DS in 6 (43%) cases. In 2 cases who had relapsed on DAS, a T315I and an F317L mutation, respectively, were present just below the lower detection limit of conventional sequencing (15.9% and 12.4%, respectively); in the remaining 4 pts, DS identified multiple (2 to 3) low burden mutations, all of which known to confer a moderate to high degree of insensitivity to the ongoing TKI. In the 8 pts with persistently or transiently detectable BCR-ABL1 transcripts at multiple timepoints despite stable hematologic remission, DS detected low burden mutations in 9 samples from 4 pts. However, no mutation known to be truly insensitive to the ongoing TKI could be recognized. Conclusions MRD persistence in Ph+ ALL pts may hide emerging TKI-insensitive BCR-ABL1 KD mutations that DS may identify earlier than conventional sequencing - allowing a greater leeway before overt hematologic relapse occurs; polyclonal resistance sustained by multiple TKI-insensitive low burden mutations may explain relapse in a proportion of cases with unmutated BCR-ABL1 KD sequences as assessed by conventional sequencing; the type of mutation matters: detection of low burden mutations insensitive to the ongoing TKI was always found to predict/correlate with treatment failure. Detection of low burden mutations with low/unknown IC50 might explain low level MRD but does not predict for an impending relapse; MRD-triggered, BCR-ABL1 KD mutation screening by DS may be precious for earlier and more effective use of preemptive rescue therapies. Supported by ELN, AIL, AIRC, FP7 NGS-PTL project, Progetto Regione-Università 2010-12 (L. Bolondi) Disclosures Soverini: Ariad: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy. Abruzzese:BMS, Novartis, Pfizer, Ariad: Consultancy. Baccarani:ARIAD Pharmaceuticals, Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; NOVARTIS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Cavo:Onyx: Honoraria; BMS: Honoraria; Novartis: Consultancy, Honoraria; Millenium Pharmaceuticals: Honoraria; Celgene: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Jansenn: Consultancy, Honoraria. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Martinelli:Pfizer: Consultancy; BMS: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; ROCHE: Consultancy; AMGEN: Consultancy; Ariad: Consultancy; MSD: Consultancy.

Description: Abstract 284 Background and Aims: Selection of drug-resistant mutations in the Bcr-Abl kinase domain (KD) is a critical problem undermining the long-term efficacy of tyrosine kinase inhibitor (TKI)-based therapies in Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) patients. Bcr-Abl KD mutation screening is routinely performed by Sanger sequencing (SS). Before the advent of ultra-deep sequencing (UDS) technologies, no method was available that could conjugate the possibility to scan the KD for the so many mutations known to be associated with TKI resistance with a sensitivity higher than that of SS. UDS technologies also allow high throughputness and accurate quantitation of mutated clones and their application in a diagnostic setting is not far to come. We used an UDS strategy for Bcr-Abl KD mutation screening in order to study the dynamics of expansion of mutated clones in Ph+ ALL patients receiving TKI-based therapies and to test the ability of UDS to highlight emerging clones harboring critical mutations. Methods: 72 samples from 25 Ph+ ALL patients who had developed resistance to one or multiple lines of TKI (imatinib, dasatinib, nilotinib, bosutinib, ponatinib) therapy were selected for this retrospective analysis. All the patients had previously been analyzed by Sanger sequencing (SS) and were known to have developed one or more TKI-resistant Bcr-Abl KD mutations on treatment. In order to reconstruct the dynamics of mutation emergence, longitudinal re-analysis of monthly collected samples was perfomed with UDS on a Roche GS Junior. UDS allowed to achieve a lower detection limit of at least 0.1% (by generating a minimum of 5,000 sequence reads/patient), as compared to 20% of SS. Results: 39 samples were known to harbor one (n=27 samples) or more (n=12 samples) TKI-resistant mutations with 〉20% abundance, as assessed by SS. UDS could successfully detect all the 54 mutations previously identified by SS. In addition, UDS detected one or multiple lower-level (20% abundance. The type of lower-level mutations detected by UDS could easily be accounted for by TKI exposure history, since the majority were known to be poorly sensitive either to the TKI being administered or to the previous TKI received. Overall, 44 samples turned out to carry multiple (two to five) mutations at any level, distributed in the same and/or in different subpopulations with a complex clonal architecture that UDS allowed to reconstruct. Of note, in 14/25 (56%) patients with molecularly detectable disease but not yet evidence of cytogenetic or hematologic relapse, UDS could identify emerging TKI-resistant mutations 1 to 2 months before they became detectable by SS. These outgrowing mutations were detected at 1–19% abundance in 12 patients and at 0.1–1% abundance in 2 patients. In the remaining 11 patients, dynamics of outgrow of the TKI-resistant mutations (five T315I, two Y253H, two E255K, one E255V and one F317L) was so rapid that not even strict monthly monitoring could allow to pick them up before they became dominant. Conclusions: Now that multiple options are available, Bcr-Abl KD mutation monitoring has become a precious tool for rational decision-making in order to maximize the efficacy of TKI-based regimens as induction or salvage therapy for Ph+ ALL patients. UDS proved as reliable as SS for the detection of mutations with 〉20% abundance and to have comparable costs. As a key advantage, UDS added precious quantitative and qualitative information on the full repertoire of mutated populations, that SS failed to appreciate in more than half of the samples analyzed. TKI-resistant mutations leading to patient relapse were not necessarily preexisting at low levels at diagnosis or at the time of switchover to another TKI, underlining the importance of regular monitoring of patients. Although TKI-resistant populations may arise and take over very rapidly, in approximately half of the patients monthly monitoring with UDS would have allowed to identify them earlier than SS and well in advance of clinical relapse, thus allowing a more timely therapeutic intervention. Disclosures: Soverini: Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Luppi:CELGENE CORPORATION: Research Funding. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Description: Abstract 1678 Background. Imatinib mesylate (IM) is the therapeutic standard for chronic myeloid leukemia (CML), but nilotinib and dasatinib, at least in selected patients, have the potential to replace it. The early prediction of poor outcome is important to optimize the treatment strategy. In IM-treated patients, BCR-ABL transcript levels according to the International Scale (IS) 〉 10% at 3 and 〉 1% at 6 months were able to identify high-risk groups (Marin et al, JClinOncol 2011; Hanfstein et al, Leukemia 2012). Similar analysis were performed within the IM arms of the ENESTnd trial (Hochhaus et al, EHA 2012) and the DASISION trial (Jabbour et al, EHA 2012). Methods. To investigate the prognostic impact of BCR-ABLIS levels at 3 and 6 months on the future response status and the long-term outcome of CML patients treated frontline with IM, we analyzed 559 patients enrolled within 3 trials of the GIMEMA CML WP (ClinTrialsGov NCT00514488/NCT00510926, observational trial CML023). Patients with evaluable QPCR sample at 3 and 6 months: 487/559 (87%) and 492/559 (88%), respectively. Definitions: major molecular response (MMR): BCR-ABLIS ratio 1%) at 3 and 6 months were analyzed. Patients with events or censored within 3 or 6 months were excluded from the respective analysis. Receiver operating characteristic (ROC) curves were used for descriptive purposes. Results. Median age: 52 years (range 18–84). IM dose: 76% 400mg, 24% 800mg. Sokal score: 39% low, 39% intermediate, 22% high; EUTOS score: 93% low, 7% high. Median follow-up: 76 months (range: 7–99); 95% of patients had at least 5-year observation. BCR-ABLIS at 3 months: ≤1% in 336/487 (69%), 〉1% to ≤10% in 120/487 (25%) and 〉10% in 31/487 (6%). BCR-ABLIS at 6 months: ≤1% in 425/492 (86%), 〉1% to ≤10% in 54/492 (11%) and 〉10% in 13/492 (3%). Responses and outcomes according to transcript levels are presented in table 1. Patients with BCR-ABLIS 〉10% at 3 months achieved inferior CCgR and MMR rates at 1 year and inferior MR4.0 rate at 2 years, but the long-term outcome was comparable to patients with transcript levels 〈 10%. On the contrary, a BCR-ABLIS 〉1% at 3 months was associated, not only to lower subsequent response rates, but also to significantly inferior FFS, PFS and OS. The BCR-ABLIS levels able to predict for FFS, PFS and OS with maximal sensitivity and specificity (ROC curves) were 1.9%, 0.8% and 0.8%, respectively. Results were similar, with small differences, in the 6-month analysis. Conclusions. In a multicentric nationwide experience, the proportion of patients with BCR-ABLIS transcript levels 〉10% at 3 and 6 months was low. The risk distribution and the proportion of patients treated with high-dose IM may explain, at least in part, the differences with other published reports. At 3 and 6 months, a BCR-ABLIS cutoff of 1% was a reliable surrogate marker of response and outcome. A transcript level 〉10% identified a smaller cohort with inferior responses, but failed to predict the long-term outcome. A BCR-ABLIS level 〉1% at 3 and 6 months represents a warning, requiring a close monitoring. A switch to 2nd generation tyrosine kinase inhibitors should be considered. Acknowledgments. University of Bologna, BolognaAIL, COFIN, Fondazione Carisbo. Disclosures: Castagnetti: Bristol Myers Squibb: Consultancy, Honoraria, Speakers Bureau; Novartis Pharma: Consultancy, Honoraria, Speakers Bureau. Gugliotta:Novartis: Consultancy, Honoraria; Bristol-Myers-Squibb: Consultancy, Honoraria. Breccia:Bristol Myers Squibb: Consultancy; Novartis: Consultancy. Abruzzese:Bristol Myers-Squibb and Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Cavazzini:Novartis Pharma: Honoraria; Bristol Myers Squibb: Honoraria. Soverini:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Saglio:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy. Martinelli:Bristol-Myers-Squibb: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Rosti:Novartis Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Speakers Bureau; Pfizer: Speakers Bureau.

Description: Abstract 1680 Background. Chronic myeloid leukemia (CML) is characterized by the presence of the BCR-ABL1 hybrid gene. Different types of BCR-ABL1 fusion transcripts can be found, but the most frequent are the e13a2 (b2a2) and the e14a2 (b3a2). In the tyrosine kinase inhibitors (TKIs) era, few data about the prognostic significance of the transcript type in early chronic phase (ECP) CML are available. Three larger studies suggested that the e13a2 transcript may have an adverse prognostic impact in ECP CML patients treated with imatinib (IM): Vega-Ruiz et al. (251 patients, ASH 2007) reported inferior molecular responses; Lucas et al. (71 patients, Haematologica 2009) reported lower cytogenetic response rates and lower event-free survival (EFS); the GIMEMA CML WP (493 patients, EHA 2011) reported a slower time to major molecular response (MMR) with inferior EFS and progression-free survival (PFS). To our knowledge this is the first evaluation of the prognostic influence of the BCR-ABL1 transcript type on the responses and the outcome of ECP CML treated frontline with nilotinib (NIL). Methods. The CML Italian Registry of Nilotinib includes 215 patients treated with NIL-based regimens. The patients were enrolled within 2 multicenter phase II studies conducted by the GIMEMA CML WP (ClinicalTrials.gov. NCT00481052 and NCT00769327) or treated at the “S. Orsola-Malpighi” University Hospital (Bologna, Italy), with NIL 300 mg BID or 400 mg BID as initial treatment. All the registered patients were analyzed. Patients expressing rare transcripts and patients with both b2a2 and b3a2 transcripts were excluded: 201 out of 215 patients were evaluable, 81 (40%) with e13a2 transcript and 120 (60%) with e14a2 transcript. Differences between groups were tested using χ2 test, Fisher exact test or t-test, as appropriate. Response monitoring: conventional cytogenetic examination (bone marrow) and QPCR (peripheral blood). Definitions: MMR: BCR-ABLIS ratio