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: Systemic mastocytosis (SM) is a rare myeloproliferative neoplasm characterized by proliferation and hyperactivation of clonal mast cells. Clinical manifestations are heterogeneous and encompass cutaneous lesions, gastrointestinal alterations, osteoporosis, anaphylaxis and involvement of bone marrow and other organs due to neoplastic mast cells (MC) infiltration. As consequence, diagnosis may be difficult and patients (pts) are often evaluated by different specialists before the disease is recognized. To date, only few studies (Lim 2009, Escribano 2009, Cohen 2014) described relatively large series of pts with SM. We performed a multicentre retrospective study to evaluate clinical and biological features and therapeutic management in a large series of pts from 10 Italian centres experienced in management of SM and organized in multidisciplinary groups of specialists. We collected 455 pts diagnosed with SM according to WHO criteria. Additionally 26 pts with mastocytosis in the skin (MIS) evaluated with BM examination did not fulfil criteria for SM, leading to diagnosis of Cutaneous Mastocytosis (CM); however 2/26 pts with CM had both cKITD816V mutation and CD2/CD25 expression on MC in BM, additional 3 showed either cKITD816V or CD2/CD25. Moreover, we found 22 pts without MIS but with features of monoclonal mast cell activation syndrome. Of the 455 pts with WHO-SM (male 56%), 252 (55%) had MIS: median age at MIS diagnosis (dg) was 37 years (y) (range 0-79), while at SM dg it was 46.5 (range 18-82). Time from onset of MIS to dg of SM was 9 y (range 0-43). In 18/252 pts (7%) MIS occurred before age of 18 y (median 9, range 0-17) and persisted over childhood. Median age at dg of SM without MIS (203/455 pts, 45%) was older: 54 y, range 19-79 (p

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 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: Imatinib (IM) targets the constitutively active BCR-ABL1 tyrosine kinase (TK) in chronic myeloid leukaemia (CML) and has become standard treatment based on good responses achieved in clinical trials. However, IM resistance can occur through several mechanisms including BCR-ABL1 kinase domain mutations, amplification, overexpression and clonal evolution. The discovery that Aurora kinases (AK) were abnormally expressed in malignancies including leukaemia prompted the development of agents that inhibit their activity. Aurora kinase A (AKA) is a central mitotic regulator necessary for mitotic entry, mitotic spindle assembly and accurate chromosome separation. The therapeutic potential of specifically targeting AKA activity as an anticancer strategy has not been rigorously investigated because all of the agents previously designed to target AKs have significant off-target effects on other family members and/or BCR-ABL1 kinase activity. However, the role of AKA in chemoresistance of CML is not well understood. In this study we investigated the mechanism of action of AKA in CML in vitro and ex vivo models (i.e. K562 cell lines and CD34+ cells derived from bone marrow aspirates of both CML at the clinical diagnosis and in mononuclear cell fraction of IM-resistant patients). Moreover, the role of two major players (i.e. FOXM1 and Polo-like kinase 1 (PLK1) a ser-thr kinase involved in G2/M progression) in the AKA signalling pathway has been investigated. First, we investigated AKA expression and activity by Western Blot and immunoprecipitation analyses. CD34+ cells from peripheral blood aphereses of hematologically healthy donors, pooled to avoid individual differences, were used as normal controls. Informed consent was obtained by all participants before their admittance to the study. Our data showed an over-expression and hyper-phosphorylation of AKA, FOXM1 and PLK1 in CD34+ cells of CML patients at clinical diagnosis compared to mononuclear cell fractions. Our results also showed a FOXM1 increment associated with IM resistance. An IM-resistant K562 cell line (LD50 0.25 mM vs 0.026 mM of parental cells) generated in our lab exhibited FOXM1 over-expression and hyper-phosphorylation contingent upon the upstream activation of AKA and PLK1. AKA, FOXM1 and PLK1 involvement in IM resistance was observed also in clonal progenitors from bone marrow samples of CML patients who developed IM resistance independent from BCR-ABL1 point mutations. Interestingly, the putative BCR-ABL1+/CD34+ LSC compartment, which is neither dependent on BCR-ABL1 TK for proliferation and survival nor killed by IM or second generation inhibitors, showed a hyper-phosphorylation of AKA and a consequent overexpression and hyper-activation of FOXM1 and PLK1. Taken together, these evidences may pave the way to the set up a new strategy to overcome drug resistance in CML. Moreover, our data identify a new signaling pathway involved both in drug resistance and in CD34+ cells survival, suggesting that AKA, FOXM1 and PLK1 are three very interesting druggable targets to eradicate the TKIs resistant CD34+ progenitors. Acknowledgments: Manuela Mancini was supported by a Fondazione Umberto Veronesi Fellowship. University of Bologna (RFO funds), Ministero della Pubblica Istruzione e della Ricerca (PRIN funds), Umberto Veronesi Foundation, AIRC, FP7 NCS-PLT project, Progetto Regione-Università 2010-12 (L. Bolondi) and BolognaAIL are acknowledged for financial support. Disclosures Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Rosti:Bristol Myers Squibb: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau. Cavo:Janssen-Cilag, Celgene, Amgen, BMS: Honoraria. Martinelli:BMS: Consultancy, Speakers Bureau; ROCHE: Consultancy; Ariad: Consultancy; Pfizer: Consultancy; AMGEN: Consultancy; MSD: Consultancy; Novartis: Consultancy, Speakers Bureau.

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 1749 Introduction. Mastocytosis is a myeloid neoplasm characterized by abnormal accumulation and frequent activation of mast cells (MCs) in various organs, mostly bone marrow, skin, liver and gastrointestinal tract. In most adult patients, the systemic form of mastocytosis (SM) is diagnosed, which includes an indolent, an aggressive and a leukemic subvariant. The c-kit mutation D816V is detectable in most adult patients with SM. Treatment of SM usually focuses on symptom relief by histamine receptor antagonists and other supportive therapy. However, in aggressive and leukemic variants, cytoreductive and targeted drugs must be applied. Methods. From 2008, 11 patients (male/female=3/8) affected by Mastocytosis, have been referred to our Institution. The median age was 53 years. All the patients underwent a bone marrow biopsy, with flow citometry and molecular biology analysis, in order to identify the presence of D816V mutation of c-Kit gene. Serum tryptase level was tested, resulting elevated in all cases. According to 2008 WHO diagnostic criteria, 9 patients presented with SM, whereas in the other cases a skin isolated involvement was detected. Systemic symptoms were characterized by nausea, diarrhoea, asthenia, weight loss, pruritus and serotine fever, identifying an aggressive form of the disease in 5/11 patients, due to skeletal involvement in three cases, ascitis and liver function impairment in another one and bone marrow disfunction in the fifth one. Therefore, since a first line therapy with supportive care and histamine receptor antagonists wasn't followed by a significant benefit, a personalized use of PKC412 was asked and obtained for all these five patients. Results. From March 2011 three out of the five patients with aggressive SM have received a prolonged period of treatment with PKC412, which was administered orally, at the dosage of 100 mg twice daily, without rest periods. The drug was well tolerated. No serious adverse events were observed. All the patients obtained a quick and prolonged improvement of clinical symptoms, in terms of weight gain, bowel function and skeletal pain. At the bone marrow evaluation, the persistence of the D816V c-kit mutation was observed, despite a significant decrease of mast cell marrow involvement. In one case we observed, after a first good response, a disease progression, characterized by the sudden reoccurrence of the same symptoms detected at diagnosis, confirmed by a relevant expansion of a pathologic mast cell population in the bone marrow. After a rest period, the drug was readministered, and a second remission was obtained. Conclusions. PKC412 is safe and effective in patients with SM, being able to significantly improve not only the gastrointestinal and systemic symptoms, but also the haematological profile. The persistence of the D816V c-kit mutation, despite a morphologic remission, suggests that many other oncogenic factors may be responsible for the pathogenesis of the disease. Acknowledgments. Work supported by European LeukemiaNet, AIRC, AIL, PRIN, Fondazione del Monte di Bologna e Ravenna, University of Bologna. Disclosures: Baccarani: Novartis: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy, Honoraria, Speakers Bureau; ARIAD: Consultancy, Honoraria, Speakers Bureau. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Description: Background and Aims: Next generation amplicon-based deep sequencing (DS) on the Roche, Illumina or Ion Torrent instruments is becoming accessible to a wider and wider number of diagnostic laboratories. Although conventional sequencing is still the gold standard, DS has been hailed by many as the future of diagnostic BCR-ABL1 kinase domain (KD) mutation screening. BCR-ABL1 KD mutations are infrequent in newly diagnosed chronic myeloid leukemia (CML) patients (pts) receiving 1st-line TKI therapy, but remain a challenge in relapsed pts, who usually display a greater genetic instability. Indeed, pts already harboring BCR-ABL1 KD mutations have a higher likelihood of developing additional, dasatinib (DAS)- or nilotinib (NIL)-resistant mutations – which is defined as a ‘failure’ by the 2013 European LeukemiaNet (ELN) recommendations. Taking advantage of a next-generation amplicon sequencing design and protocol set up and validated in the framework of the IRON-II international study, we aimed to assess whether DS may allow a larger window of detection of emerging BCR-ABL1 KD mutants predicting for an impending relapse. Methods: among the imatinib (IM)-resistant CML pts who switched to 2nd-line TKI therapy and were referred to our laboratory for routine BCR-ABL1 transcript level monitoring and KD mutation screening by conventional sequencing, 51 acquired DAS- or NIL-resistant mutations after a median of 9 months (range, 3-27 months) of therapy and had leftover cDNA available at previous timepoints. To reconstruct the dynamics of mutation emergence, resequencing on a Roche GS Junior instrument was performed from the time of failure and mutation detection by conventional sequencing backwards. Runs were designed to achieve high sequencing depth, allowing reliable detection of variants down to 1% abundance. BCR-ABL1/ABL1%IS transcript levels and/or cytogenetic response, whichever available, were used to define whether the patient had an ‘optimal response’, ‘warning’ or ‘failure’ at the time of first mutation detection by DS. Results: baseline mutation status, as assessed by conventional sequencing, was available for all the 51 CML pts included in this retrospective study; 29/51 pts were positive for BCR-ABL1 KD mutations, with switch to NIL or DAS selected accordingly. Twenty-six pts were later found to have acquired DAS-resistant mutations (T315I, n=13; F317L/V, n=10; V299L, n=3) and 25 pts were later found to have acquired NIL-resistant mutations (T315I, n=4; F359V/I/C, n=7; Y253H, n=6; E255K, n=9; one patient acquired two mutations). DS was able to backtrack the DAS- or NIL-resistant mutations to the previous sample(s) in 23/51 (45%) pts. Median mutation burden at the time of first detection by DS was 5% (range, 1-17%); median interval between detection by DS and detection by conventional sequencing was 3 months (range, 3-9 months). In 5 cases, the mutations were traceable at baseline; in the remaining cases, correlation with response at the time mutations were first detected by DS revealed a ‘warning’ according to the 2013 ELN definitions of response to 2nd-line therapy in 13 cases; an ‘optimal response’ in one case; a ‘failure’ in 4 cases. As a control, we used DS to explore BCR-ABL1 KD mutation status in 10 randomly selected pts with ‘warning’ at various timepoints, that later turned into optimal responses; no DAS- or NIL-resistant mutations were detected. Conclusions: the 2011 ELN recommendations for mutation analysis suggest BCR-ABL1 KD to be screened by conventional sequencing in case of ‘failure’ of 2nd-line TKI therapy – according to the provisional definitions available at the time. Earlier detection of emerging BCR-ABL1 KD mutations allows a greater leeway in tackling drug resistance and enhancing therapeutic efficacy. Data presented herein indicate that: 1) DS may reliably pick TKI-resistant mutations earlier than conventional sequencing in a proportion of pts, and that 2) the recently introduced definitions of ‘warning’ may provide a rational trigger, besides ‘failure’, for DS-based BCR-ABL1 KD mutation screening in CML pts on 2nd-line TKI therapy. A prospective cost-benefits evaluation of using DS in this and other settings is warranted, and will contribute useful information to the revision of the ELN recommendations for BCR-ABL1 KD mutation analysis. Supported by: European LeukemiaNet, AIL, AIRC, FP7 NGS-PTL project, Progetto Regione-Università 2010-12 (L. Bolondi). Disclosures Soverini: Novartis: Consultancy; Bristol-Meyers Squibb: Consultancy; Ariad: Consultancy. Castagnetti:Novartis Farma: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy. Gugliotta:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria. Bonifacio:Amgen Inc.: Consultancy. Rosti:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy. Baccarani:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Ariad: Consultancy; Pfizer: Consultancy. Martinelli:NOVARTIS: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Description: Abstract 1396 Introduction. Mutations in the kinase domain (KD) of BCR-ABL are a known mechanism of resistance to the tyrosine kinase inhibitor (TKI) treatment of patients with chronic myeloid leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL). The gold standard for the routine examination of mutations in patients with therapy failure is the Sanger sequencing of amplified cDNA region of BCR-ABL KD with the sensitivity of ∼15–20%. More than 100 BCR-ABL KD mutations were described in association with development of resistance to imatinib. It was suggested that early ultra-sensitive detection of KD BCR-ABL mutations, at the time of diagnosis or shortly after imatinib start and before switch to 2nd generation TKI, may predict their subsequent expansion and failure of the treatment if the patient is treated with TKI against which is the type of mutation resistant. Ultradeep amplicon pyrosequencing represents a promising tool to study BCR-ABL mutation development and their expansion under TKI treatment. In our study, the detection of mutations reached the sensitivity of 0.03-0.01%. At this level we may expect detection of artifacts introduced during amplicon library preparation and pyrosequencing. Aims. The aim is to define the threshold for relevant detection of published BCR-ABL mutations (n= 124). Methods. We evaluated 155 CML or Ph+ALL patient samples. As the non-mutated control, the cDNA of KD c-ABL of healthy individuals (n= 9) and cDNA of wild type BCR-ABL from Ph+ cell lines (n=3) were used. To cover cDNA region of kinase domain (950 bp), multiple amplicons with the length up to 400 bp should be prepared to apply 454 technology. The experiments consist of: 1) comparison of two amplicon designs with 3 and 4 fragments covering KD to follow the amplicon-related artifacts; 2) the reproducibility test of 5 samples using 3 and 4 PCR fragments for amplicon library preparation; 3) tests with 2 different reverse transcriptases and Taq polymerases to detect artifactual nucleotide substitutions. Results. Using 454 technology we detected high number of ultralow-level single nucleotide substitutions (∼ 200) with the frequency 〈 1 in 100 in all samples analyzed including non-mutated controls using 3 and also 4 amplicon design. Surprisingly, we found that almost all low-level variants were T#x2610;#x0025;C or A#x2610;#x0025;G substitutions (Fig. 1), including those that may result in clinically relevant mutations (V288A, M244V, V289A, F317L, F359L, M351T, E355G, E453G). Evaluation of pyrograms suggested that they were unlikely to be errors of pyrosequencing, because they were not necessarily associated with hompolymer regions. The frequency of T#x2610;#x0025;C or A#x2610;#x0025;G low-level variants was lower when using proof-reading Taq polymerase. The pattern of these nucleotide substitutions is similar to that reported in IGH hypermutation analysis in CLL (Campbell et al. PNAS 2008) showing that T#x2610;#x0025;C or A#x2610;#x0025;G substitutions are errors caused by Taq polymerases. This is supported by the studies on PCR fidelity suggesting that Taq polymerase caused this pattern of base substitution even when using a proof-reading enzyme. Thus, the T#x2610;#x0025;C or A#x2610;#x0025;G substitution in BCR-ABL KD should not be evaluated below the level of 1%. The other but less frequent transitions caused by Taq polymerase are G#x2610;#x0025;A or C#x2610;#x0025;T substitutions that were detected in our study with the frequency 〈 1 in 1000, thus, the variants detected below 0.1% should not be examined as the true-positive result. Conclusion. Mutation data from ultradeep amplicon sequencing of BCR-ABL KD below 1% must be evaluated cautiously to avoid misleading interpretations. Attention must be paid to the fact that T#x2610;#x0025;C or A#x2610;#x0025;G substitution may arise spontaneously in vivo, but there is no way to differentiate in vivo spontaneous mutations from Taq polymerase errors in vitro. Disclosures: Soverini: Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. 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. Machova Polakova:Novartis: Honoraria, Research Funding; BMS: Honoraria.

Description: Systemic mastocytosis (SM) includes a heterogeneous group of disorders ranging from indolent SM to mast cell leukemia (MCL). Somatic mutations in the Kit receptor tyrosine kinase (most frequently, D816V) can be detected in 〉90% of patients with SM and are thought to play a key pathogenetic role. Nevertheless, morphological and clinical diversity, as well as the fact that some patients are negative for KIT mutations, suggest that the underlying molecular picture is far from being fully elucidated. To shed further light on this issue, we undertook an integrated molecular genetic study of a KIT gene mutation-negative MCL patient who came to our attention in 2012 – a 63 year-old woman diagnosed with MCL, aleukemic variant (50-60% atypical mast-cells in the bone marrow [BM] smear; CD117+/CD2+/CD13+-/CD33+/CD59+ immunophenotype; serum tryptase, 2500 µg/L; no C-findings). The patient had received imatinib for 6 months, with no clinical benefit. The disease, however, had had an overall chronic clinical course for 6 more months until severe anemia occurred. The patient rapidly progressed and died after 21 months from diagnosis. After having obtained written informed consent, we extracted genomic DNA and total RNA from purified MCs isolated from BM at diagnosis and at progression, as well as DNA from saliva, and performed whole exome sequencing (WES) and RNA sequencing on an HiSeq1000 (Illumina, San Diego CA). Cytoscan HD arrays (Affymetrix, Santa Clara CA) were also used to scan for chromosomal gains and losses as well as for loss of heterozigosity (LOH). Among the mutated genes detected by WES, SETD2 stood out among others because two distinct putatively inactivating heterozygous mutations were identified, a frameshift insertion of a C in exon 20 (NM_014159:c.7595_7596insC: p.Gly2515ArgfsTer5) and a nonsense mutation in exon 15 (NM_014159:c.G6753T:p.Glu2234Ter). The two mutations were found to hit distinct alleles, pointing to a loss-of-function event. Western Blotting (WB), however, showed that only the 2234 a.a. Setd2 truncated isoform resulting from the nonsense mutation, losing the highly conserved WW and SRI functional domains, was detectable in the sample. The SETD2 gene encodes a histone methyltransferase nonredundantly responsible for trimethylation of lysine 36 of histone H3, a key hystone mark associated not only with active chromatin but also with transcriptional elongation, alternative splicing, DNA replication and repair. SETD2 gene mutations have been described in a variety of cancers and, more recently, have been found to be cooperating events in acute leukemia initiation and progression. In yeast, deletion of the SRI domain abolishes Set2-RNA polymerase II (PolII) interaction causing transcription elongation defects and abolishes K36 methylation. The truncated SETD2 isoform was actually found to lose the ability to bind RNAPolII, as shown by co-immunoprecipitation. Accordingly, RNA-sequencing showed evidence of spurious transcripts initiated from cryptic promoter-like sequences within genes rather than from canonical promoters. More importantly, WB confirmed that H3K36Me3 was completely abrogated. In line with the recently reported role of SETD2-dependent H3K36Me3 in homologous recombination (HR) repair and genome stability, Cytoscan HD arrays showed that LOH and several gains and losses at many chromosomal loci, undetectable at diagnosis, had been acquired at the time of progression. Haploinsufficiency of PSIP1 (recruiting HR machinery at double strand breaks) at 9p24.3 might have represented a cooperating event. Downmodulation of the Setd2 protein (in the presence of LOH but in the apparent absence of sequence variations other than polymorphisms) and reduced H3K36Me3 levels were detected in two more MCL cases, in which putative cooperative lesions were also identified. Results of WES and high resolution karyotyping of additional SM cases will be presented. Our findings point to epigenetic regulation and/or DNA repair as two candidate pathways deserving further investigation in an attempt to identify novel actors or mechanisms contributing to the pathogenesis and progression of SM, or novel modulators of disease phenotype. They also extend the recent observation that the molecular landscape of SM is much more complex than the initial finding of KIT mutations allowed to imagine. Supported by FP7 NGS-PTL project and Progetto Regione-Università 2010-12 (L. Bolondi) Disclosures Soverini: Novartis: Consultancy, Honoraria; Bristol-Meyers Squibb: Consultancy, Honoraria; Ariad: Consultancy, Speakers Bureau.