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Introduction: Extramedullary disease (EMD) in patients (pts) with multiple myeloma (MM) is a poor prognostic feature which is not curable with currently approved treatments. Consequently, there is a significant unmet need for effective therapies with good safety profiles. Daratumumab with cyclophosphamide, bortezomib and dexamethasone (daraVCD) is a novel treatment combination with a good efficacy profile in pts with EMD based on preclinical synergistic data. Methods: EMN19 is a phase 2, open-label, multicenter study which aims to enroll 40 MM pts presenting with EMD either at diagnosis or following one line of treatment but not refractory to bortezomib-based regimens, from 8 sites in Turkey, Greece and Italy. Pts with bortezomib or daratumumab hypersensitivity, who received previous autologous stem cell transplant (ASCT) ≤12 weeks before Day 1 of treatment Cycle 1, or with previous allogenic stem cell transplant were excluded. Daratumumab was initially administered intravenously at 16 mg/mL, and since July 2020 has been administered subcutaneously at a fixed dose of 1800 mg, weekly during Cycles 1-2, every 2 weeks for Cycles 3-6, and every 4 weeks thereafter. Intravenous bortezomib 1.5 mg/m 2 and oral/intravenous cyclophosphamide 300 mg/m 2 is administered weekly, and oral/intravenous dexamethasone 20 mg is administered on Days 1, 2, 8, 9, 15, 16, 22 and 23. DaraVCD will be administered until progression or unacceptable toxicity unless refractory disease is detected by the end of Cycle 3 (progressive disease [PD] or failure to achieve a confirmed partial response [PR] or better). The present analysis includes pts who initiated study treatment ≥3 months prior to the cut-off date (01 June 2021). Results: In total, 34 patients were screened, 27 patients were enrolled, 2 relapsing patients died during the screening phase due to severe COVID-19 infection, 22 pts were analyzed (59% female; median age 56 years, range 44-77); 14 pts (64%) were still on treatment and 8 (36%) discontinued; due to inadequate response after 3 cycles of treatment (n=3, 38%), PD (n=4, 50%), death (n=1, 13%). Fourteen pts (64%) were newly diagnosed and 8 (36%) first relapsed. International Staging System stage at baseline was I, II and III for 8 (36%), 9 (41%) and 5 (23%) pts, respectively. Eastern Cooperative Oncology Group performance status was 0, 1 and 2 for 14 (64%), 7 (32%), and 1 patient (5%), respectively. On average, 3.0 (range 1-20) extramedullary plasmacytomas were observed per patient; most commonly reported sites were thorax (6 pts, 27%), brain, head and lower extremities (4 pts, 18% each). Twenty (91%) pts had ≥1 serious or non-serious treatment-emergent adverse event (TEAE); 8 pts (36.4%) experienced ≥1 sTEAEs; COVID-19 infection (n=3, 14%) urinary tract infection (n=2, 9%), infectious myocarditis, hip arthroplasty, pneumonia, cytomegaloviral pneumonia and thrombocytopenia (n=1 each, 4.5%). Thirteen (59%) pts ≥1 Grade 3/4 TEAE; neutropenia observed in 8 pts (36%), followed by thrombocytopenia (n=4, 18%) and COVID-19 infection (n=3, 14%). Overall, 16 (73%) pts missed ≥1 dose of any of the study drugs; 2 (9%) pts missed ≥1 dose due to COVID-19 infection (7 doses), 8 (36%) due to COVID-19 vaccination (37 doses), 2 (9%) due to other COVID-19-related issues (65 doses), 10 (46%) due to other safety events (104 doses) and 9 (41%) due to other reasons (25 doses). Overall, 20 cycle delays were observed in 13 (59%) pts, with median (range) delay of 12.0 (4-133) days. Two pts (9%) had a cycle delay due to COVID-19 infection (2 cycles), 1 (5%) due to COVID-19 vaccination (1 cycle), 5 (23%) due to adverse events (8 cycles), 2 (9%) due to ASCT (2 cycles) and 7 (32%) due to other reasons (7 cycles). Total number of missed doses (missed doses due to COVID-19-related issues) were 17 (3) for daratumumab, 53 (11) for bortezomib, 45 (9) for cyclophosphamide and 123 (86) for dexamethasone; 238 doses missed in total. No fatalities occurred due to any infection. Conclusions: DaraVCD was associated with a good safety profile in this high risk MM with EMD patient population. The COVID-19 impact on missed doses was greater for dexamethasone (>60% of missed doses) than other components (~20%), however overall, the pandemic did not significantly affect the patients' safety and data integrity of the study. The enrollment in the study is ongoing, and more safety and efficacy data will become available with the inclusion of additional pts in an updated analysis. Disclosures Beksac: Amgen,Celgene,Janssen,Takeda,Oncopeptides,Sanofi: Consultancy, Speakers Bureau. Tuglular: GSK: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Karyopharm: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Janssen-Cilag: Honoraria, Research Funding; Genesis Pharma: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding. Cavo: Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Accommodations, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES, Speakers Bureau; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GlaxoSmithKline: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squib: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Adaptive Biotechnologies: Consultancy, Honoraria. Gay: GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Bluebird bio: Membership on an entity's Board of Directors or advisory committees. Katodritou: GSK, Amgen, Karyopharm, Abbvie, Janssen-Cilag, Genesis Pharma, Sanofi: Honoraria, Research Funding. Merante: EMN Italy Medical Monitor: Research Funding. Manousou: Health Data Specialists: Current Employment. Sonneveld: Karyopharm: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Celgene/BMS: Consultancy, Honoraria, Research Funding; SkylineDx: Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding. Terpos: GSK: Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Genesis: Consultancy, Honoraria, Research Funding; Novartis: Honoraria; Janssen-Cilag: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; BMS: Honoraria; Takeda: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding.

Background and Aims: The term Systemic Mastocytosis (SM) identifies a poorly understood group of rare and clinically heterogenous myeloproliferative neoplasms characterized by abnormal growth and activation of mast cells (MCs) and their precursors in the bone marrow and in various tissues and organs. Based on phenotype and extent of organ infiltration/dysfunction, a spectrum of disease variants can be recognized ranging from indolent SM (ISM) to aggressive SM (ASM) and mast cell leukemia (MCL). The fact that in all cases, including ISM who have a (near) normal life expectancy, neoplastic MCs display the same D816V KIT gene mutation points to additional mechanisms and molecular defects as responsible for ASM and MCL. So far, however, this issue has mainly been addressed with targeted resequencing studies of candidate gene panels. We thus decided to undertake an integrated molecular characterization study of ASM and MCL to identify novel, functionally relevant molecular lesions and/or clinically actionable signaling pathways. Methods: A discovery panel including 6 patients with ASM and 6 patients with MCL was studied using whole exome sequencing (WES) and copy number variation (CNV) analysis. WES (80x) was performed on a Hiseq 2500 (Illumina). CNV was done using Cytoscan HD Arrays (Affymetrix). Paired normal/MC DNA was analyzed in all but 2 archival MCL cases for whom germline DNA was not available. A validation panel of 30 ISM, 5 smoldering SM and 20 additional ASM was also included in this study. Results: In the discovery panel, WES identified a total of 1554 point mutations, small insertions and deletions. Seven hundred and eighty-five were non-silent mutations in 698 genes, with an average of 51 (range, 30-186) non-silent mutations per patient. Non-silent mutations included 354 missense mutations, 188 nonsense mutations, 145 frameshift insertions/deletions, 98 non-frameshift insertions/deletions. C to T transitions were by far the most frequent. Orthogonal validation estimated the accuracy of mutation calls at >95%. Interrogation of the COSMIC and OMIM databases revealed 42 known cancer genes. Among the missense mutations, 87 were predicted to have a high probability of being deleterious by Condel. MCL cases were found not to harbour a higher mutation load as compared to ASM cases. High resolution CN analysis showed that focal amplifications/deletions/loss-of-heterozygosity (LOH) were prevalent over arm-level alterations (found in 3 patients only). Genes were selected for further assessment when recurrently mutated in ≥2 patients or concurrently identified in WES and CNV analyses or previously associated with leukemogenesis or cancer pathogenesis. Among these, genes already reported to be affected by mutations in SM included TET2, NRAS, ASXL1, CBL, IDH1, SRSF2, SF3B1, RUNX1. We also identified genetic alterations in genes not previously implicated in SM pathogenesis including TP53BP1, RUNX3, NCOR2, CDC27, CCND3, EI24, MLL3, ARID1B, ARID3B, ARID4A, SETD1A, SETD1B, KDM1B, PRDM1, ATM, WRN. A long tail of infrequently mutated genes dominated, resulting in significant intertumoural heterogeneity. However, when genes were assigned to functional pathways to discern patterns of mutations across different patients, we found that PI3K/Akt and MAPK pathways, calcium pathway, chromatin modification, DNA methylation, and DNA damage repair were consistently affected (Figure 1). Further assessment of the mutation frequency of selected genes within each pathway and functional validation at the protein level are currently ongoing in the validation panel. Preliminary findings on a tumor suppressor selected among those identified by WES show transcript and/or protein downmodulation due to inactivating mutations, transcriptional silencing or enhanced degradation in 17/20 ASM. Detailed results will be presented at the meeting. Conclusions: WES and CNV analyses of ASM and MCL revealed a complex landscape, not unexpected when considering the clinical heterogeneity of these patients. Nonetheless, key pathways were found to be recurrently altered. Further investigation of selected candidate genes and pathways is warranted and will cast light on the cooperative genetic (and epigenetic?) events underlying the more aggressive forms of SM - paving the way to a better prognostic stratification and more effective treatment. <>This study was supported by ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Soverini: Ariad: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy. Valent:Novartis: Consultancy, Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Celgene: Honoraria. Cavo:Janssen-Cilag, Celgene, Amgen, BMS: Honoraria. Martinelli:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; ROCHE: Consultancy; Pfizer: Consultancy; Ariad: Consultancy; AMGEN: Consultancy; MSD: Consultancy.

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.

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

Imatinib (IM) is a cornerstone in the treatment of chronic myeloid leukemia (CML). Dose change or discontinuation of IM in patients who experience sustained molecular response is a subject of debate. We retrospectively studied 142 CML patients in chronic phase (Table 1) treated with IM and followed-up during 2000-2013 at our institution. Dose changes, discontinuation of therapy, cytogenetic and molecular analyses were regularly recorded during follow-up. Patients’ history was subdivided into 483 treatment time-periods at constant dosage. Response was evaluated at the end of each treatment period. We assessed whether the probability of observing a complete cytogenetic response (CCyR) or a molecular response (MR: complete, CMR or major, MMR) or a progression were influenced by treatment dose and/or duration. We applied generalized estimating equation (GEE) logistic models for the analysis of longitudinal panel data. These models are designed to account for individual patient variation due to repeated measurements during each patient’s follow-up. Out of 483 time periods at constant IM dose, 236 were followed by dose modification, 116 by IM discontinuation, 29 by a change to other treatments due to tolerability issues or non-response; 102 were still ongoing without dose changes. Treatment response: 74% of time periods resulted in a CCyR and 2.3% showed no response; 31.9% showed a CMR, 29.6% showed a MMR (MR3, MR4, MR4.5), 35.6% a suboptimal response, 2.9% no MR. CMR+MMR was observed in 61.5% time periods. Periods at standard dose showed a higher response rate, both when considering CCyR (response rate after low, standard, high dose: 69.3%, 79.6%, 68%, respectively, P=0.023) and when considering MR (CMR after low, standard, high dose: 27%, 40.5%, 15.4%, respectively, P After adjusting for length of treatment period in a multivariate GEE model, dose lost significance and treatment duration was the only significant predictor of CCyR (P In a GEE analysis of MR accounting for treatment duration, reduction in CMR rate after periods at high dose compared with periods at standard dose remained significant (P=0.025). Treatment dose lost significance when considering CMR+MMR. In 32 patients who discontinued IM therapy for >1 mos and then re-started IM, the CR rate after a period preceded by a treatment suspension was significantly higher than in other periods (CCyR: 85.7% vs. 73%, P=0.058; CMR 55.1% vs. 29.3%, P=0.001). In a GEE model with dose category, duration of treatment and previous suspension as covariates, both duration and previous suspension maintained a positive significant association to CCyR (P Cytogenetic progression rate at the end of a period of IM treatment was 7.1% while molecular progression rate was 12.8%. Treatment dose was not associated to progression: cytogenetic progression after low, standard, high dose: 6.1%, 6.7%, 10.5%, respectively, P=0.428; molecular progression after low, standard, high dose: 14.8%, 11.3%, 13%, respectively, P=0.584. In a multivariate GEE analysis, neither dose nor duration of treatment predicted progression. After adding type of previous treatment (IM, other drug, no treatment) as a covariate, treatment periods preceded by at least 1 month of discontinuation had a significantly lower molecular progression rate (P=0.011). The possibility of varying dosage is often considered in “real-life” patient clinical management. In our study, 62% of time periods were followed by a dose change, but this did not affect response and progression rate after accounting for length of treatment period. The prompt response to resumed IM therapy suggests that patients may be candidates for intermittent therapy. Future studies could use the same statistical model to pick up individual patient variation in longitudinal data collected over time, including trials at reduced IM dose or of “on-off” therapy or on new TKI. Table 1. Patients’ characteristic Variable Description N. of patients 142 Sex M/F 58%/42% Age at diagnosis (years), median (range) 52 (18-78) N. of IM treatment periods 483 IM dosage, N (%) Low dose (100-300 mg/day) 163 (34%) Standard dose (400 mg/day) 242 (50%) High dose (450-800 mg/day) 78 (16%) Duration of IM at constant dosage (days), median (range) 258 (7-4526) Disclosures No relevant conflicts of interest to declare.

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

Abstract 786 Interferon alpha (INFα) either alone or in combination with Ara-C was the frontline therapy of Ph+ chronic myeloid leukaemia (Ph+ CML) between 1980 and 2000. INFα prolonged survival as compared to conventional chemotherapy and it was the first drug able to induce complete cytogenetic responses (CCgRs). Patients achieving a CCgR by INFα ± Ara C were less than 10–15%, but they represent fascinating elite of patients who are the most likely candidates for prolonged survival and possibly for cure. The last update of the largest European cohort of 317 CML patients who had obtained a CCgR on IFNα was reported in 2001 (Bonifazi et al., Blood, 2001). Briefly, the median time to first CCgR was 19 months, the 10 year survival rate from first CCgR was 72% and the survival probability for patients with low Sokal risk was 84%. In this study, the contribution of the Italian Cooperative Study Group on CML was of 119 cases. Although INFα was used more than 20 years ago, obtaining information on this selected category of CCgR–INFα responding patients in the Imatinib era may be interesting both from the biological and clinical point of view. We report here the preliminary results of an observational study aimed to update the overall survival (OS), the progression free survival (PFS) to accelerated-blastic phase (ABP) and the CCgR duration in 92 Ph+ CML patients who were treated in Italy with an INFα based therapy between 1986 and 2001 and who obtained a CCgR at least once. In this selected cohort of patients, the median time to first CCgR was 24 months (range, 3–143), and the median duration of the first CCgR was 87 months (3-252). The overall survival (OS) calculated from diagnosis, from start of IFNα and from the time of the first CCgR was 185 months (range, 74–334), 179 months (range, 74–287) and 154 months (range, 51–274), respectively. This is the longest follow-up of Ph+ CML patients who obtained a CCgR with an IFNα-based therapy. Out of 92 patients in CCgR, 71 (77%) cases stopped IFNα and 21 (23%) continued to be treated with IFNα. Out of the 71 cases who stopped IFNα, 38 (53%) cases lost CCgR and 3 (4%) cases died because of progression to ABP; 15 (21%) maintained CCgR without any other therapy and 18 (25%) maintained CCgR but shifted to Imatinib. Among the latter 33 patients maintaining the CCgR, 4 cases died because of CML unrelated causes. Out of the 21 cases who continued to be treated with IFNα, 18 (86%) currently maintain the CCgR and are alive and well, while 3 lost CCgR and died (2 cases for ABP). These data show that there are at least 33/92 (36%) patients who are alive and well and are maintaining a CCgR, either with continued IFNa treament (18 cases) or after IFNα treatment discontinuation (15 cases) but who have never been treated with Imatinib or any other tyrosine kinase inhibitor (TKI). We are now analyzing molecular data and we are collecting peripheral blood and bone marrow samples for correlative biological studies aimed to characterize the peculiar genetic and epigenetic features of these patients Work supported by European LeukemiaNet and Cofin 2009 Disclosures: Castagnetti: Bristol Myers Squibb: Honoraria; Novartis: Honoraria. Rosti:Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Research Funding; Novartis: Honoraria; Bristol Myers Squibb: Honoraria. Baccarani:Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Honoraria; Bristol Myers Squibb: Honoraria; Pfizer: Honoraria; Ariad: Honoraria.

Abstract 1986 Introduction: Recent molecular studies of atypical Philadelphia negative chronic myeloproliferative disorders (CMPDs) have produced the identification of more than 40 fusion genes. The presence of FIP1L1-PDGFRA (F/P, cytogenetically invisible) have been associated more frequently with diagnosis of CEL. As this is the second most common fusion gene targeted by imatinib (IM) after BCR-ABL1, it is expected to monitoring response at molecular level as in CML, but from the analysis of major series of cases, it emerges that the diversity of F/P fusion transcripts is much more complex. We conducted a prospective phase II multicenter study of HES to explore the activity and safety of IM 400 mg in the treatment of patients affected by HES, apart from molecular status. A total of 33 patients with F/P were enrolled and now we report on the clinical and molecular follow up of 32 of them. We also report about the genomic aspects of the variability of fusion transcripts with some statistical correlations between molecular characteristics and phenotype of disease. Methods: The primary end-point of the study was to assess the clinical anti-proliferative activity of IM in HES. Patients received IM 400 mg once daily for the first year, than the dose could be modified. In patients with F/P rearrangement, the presence of the transcripts was assessed and monitored by nested RT-PCR every 3 months until negativity, and then every 6 months. After RNA extraction, RT-PCR was performed and direct sequencing was done using the Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems) and an ABI PRISM 377 DNA Analyzer. Results: All 32 patients with F/P rearrangement had prompt responses to IM. Molecular and hematologic responses (MR, HR) were maintained with continuous therapy, molecular relapse were detected when IM was stopped for different reasons, but second and third MR was obtained with IM resumption. Deep molecular analysis revealed that all the 32 fusion transcripts were different at genomic level. Eight different FIP1L1 exons (exons 9 to 15 and 18) were found to be fused to PDGFRA exon 12 that was truncated in all cases to a variable degree. In 5 cases more than 1 band was evident for the same sample, and in these cases sequencing of c-DNA was performed for each band, identifying genomic variability also in the same patient, at different time point. Following the molecular classification proposed by Walz et al, we identified transcript type A in 4/27 cases(44%), type B in 11/27 cases (41%) and type C in 3/27 cases (15%). The distribution of genomic breakpoint reflects those reported by Walz et al, with the majority of breakpoints located in FIP1L1 intron 10, 11 and 13. Others location were rare. All genomic PDGFRA breakpoints were found in exon 12 (55% in position 83/84, 30% in 100/101, 11% in 43/44, 4% in 25/26, where position +1 represents the first base in exon 12). Conclusions: After 5 years of follow-up, with this large series of patients we can confirm very high sensibility of F/P to IM, the necessity of continuous therapy and absence of acquired resistance. Our molecular data strictly confirm the data previously reported by others. Clinical correlation between this heterogeneity and phenotype of disease and response to imatinib therapy is not clear with present data and require largest studies. Primers and probes for quantitative RT-PCR have to be designed for every single patients for the complexity and variability in F/P transcripts. Disclosures: Pane: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Saglio:Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria. Baccarani:Novartis, Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.

Abstract 4478 Introduction: Imatinib mesylate (IM) therapy is effective in patients with chronic myeloid leukemia (CML). However, its discontinuation or dose variation in patients who experience sustained molecular response is debated. The possibility of treating patients with an intermittent therapy could also be applied to the second-generation TK-inhibitors. We describe our single institute experience in patients with undetectable levels or in major molecular response of BCR-ABL transcript who reduced or were discontinued from IM therapy. We applied a model for the analysis of longitudinal data to study the BCR/ABL variation according to dose change or discontinuation. Methods: One hundred forty CML patients came to our observation between 1985 and 2009. Among these, 89 patients in chronic phase were eventually treated with IM. Fifty-five patients were treated with IM as naive patients. Each patient's treatment history was subdivided into time periods at constant dosage. Fifty-nine patients were followed-up for a total of 288 periods at constant dosage. At the end of each period, cytogenetic and/or molecular responses were evaluated. Thirty-eight progressions were recorded: 22 molecular, 6 cytogenetic and 10 of both types. The association between progression (molecular, cytogenetic or either) and treatment dose was assessed with the aid of generalized estimating equations (GEE) models, i.e. regression models designed to account for correlation due to repeated measurements over time on the same subject. Ten patients discontinued IM therapy for a period which ranged from three to 60 months, after the patient's individual request and informed consent. Results: We found no association between dose and progression, not even after accounting for period length. Discontinuation of treatment was not associated to an increased risk of progression. No association with a higher risk of progression was found for periods at reduced dosage ( Conclusions: It is unclear whether IM can “cure” chronic myeloid leukemia but according to our data this therapy can be safely stopped or its dose varied in patients with complete cytogenetic and major molecular response of up to 18 months. Our experience suggests that withdrawal of IM therapy in CP-CML patients after achievement of a complete molecular response may result in divergent molecular outcomes. The prompt improvement seen after the restart of therapy argues against the development of resistance. The selection of resistant clones after IM exposure, and the emergence of Ph negative clones with secondary cytogenetic abnormalities, are matters of concern, particularly in patients receiving long-term IM. The improved quality of life while off therapy, and the prompt response to restart of IM therapy, suggest that the subset of patients who have sustained complete molecular response may be candidates for a tailored approach to intermittent therapy. We suggest that the same statistical analysis can also be used for other TK-inhibitors that are under study for both retrospective or prospective trials in CML. Disclosures: No relevant conflicts of interest to declare.

Abstract 3894 Poster Board III-830 Background An interstitial deletion in the long arm of chromosome 4 leads to the formation of the fusion gene FIP1L1-PDGFRalpha (F/P) coding for a constitutively activated form of PDGFRalpha. The fusion gene represents a marker of CEL, and it predicts a dramatic response to imatinib mesylate (IM). Different F/P transcripts have been described, but the correlation with kinetic of molecular response to IM and with the clinical presentation is unknown. Remission duration is also still undefined. Aim The aim of this study was to evaluate the duration of IM response in F/P positive (F/P+) CEL patients and the correlation between their clinical behaviour and molecular characteristics of the transcripts. Design and methods A prospective phase 2 multicentre study of the use of IM 400 mg/daily in patients with hypereosinophilic syndrome, independently of F/P status was established in 2001. Hypereosinophilic syndrome was defined according to Chusid criteria (Blood, 1994; 83: 2759-2779). The presence of F/P transcript was investigated on bone marrow cells using a nested reverse transcriptase polymerase chain reaction (RT-PCR), as reported by Cools J (N Engl J Med. 2003; 348:1199-200). The samples were also purified and sequenced using the Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems). Patients at diagnosis were systematically screened for organ damage with instrumental evaluation (chest radiography, echocardiogram, abdomen ultrasonography) and for the presence of symptoms. 72 patients were treated with IM 100 to 400 mg daily; the 33 F/P+ were monitored every three months for two years then every six months using nested RT-PCR. We will present the result of the 33 F/P+ patients. Results Median follow-up was of 51 months (range 30-92 months). There were 32 males and one female patients. Organ involvement was recorded in 42% of patients, with notably no skin involvement, splenomegaly reported in 7, cardiac involvement in 5, and in 2 cases the peculiar site of localization of soft tissue. After imatinib therapy all patients achieved a complete hematologic response (CHR) in less than one month, and PCR negativity in a median time of 3 months (range 1-9). They became negative for organ localizations and free of symptoms. No patient experienced cardiac failure. All patients who continue imatinib therapy remained in CHR and RT-PCR negative, with a dose of 100 to 400 mg daily. From September 2007 all patients except one (late responder) were treated with 100 mg daily. In six patients IM treatment was discontinued for variable period for different reasons, and in 5 cases the fusion transcript became rapidly detectable; CHR was maintained, other than in one case and the transcript was again undetectable upon treatment resumption, other than in one case. Twenty-eight samples were evaluable for sequencing. Molecular analysis demonstrate an extreme variability of FIP1L1-PDGFRA junction sequences, with FIP1L1 breackpoints scattered between exon 9 to 18, with several splicing variants, whereas all breackpoints in PDGFRA are located within exon 12. Fusion gene sequencing demonstrate an extreme variability, with lack of whole exons of FIP1L1, deletion of exons, with the presence of introns in a minority of cases. Region of FIP1L1 varies in length from 109 to more than 500 nucleotides. Transcript of the only female patient is the same of one of the males. No evidence of correlation was noted with kinetic of molecular response or with the presence at diagnosis of peculiar organ involvement. More complexity of transcript is noted in patients with longer history of disease prior to imatinib therapy. Interpretation and conclusion with this large series of patients we can confirm that the response to imatinib therapy is durable, is not influenced by the biologic features, but depends on continuous therapy. More complexity and variability in FIP1L1-PDGFRalpha transcripts does not lead to differences in terms of response to the therapy or phenotype of disease at diagnosis. Disclosures: Pane: Novartis: Research Funding; Ministero dell'Università/PRIN: Research Funding; Regione Campania: Research Funding; Ministero della Salute/Progetto integrato Oncologia: Research Funding. Saglio:Novartis: Honoraria; Celgene: Honoraria. Baccarani:Novartis Pharma: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Mayer Squibb: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Background. The presence of an interstitial deletion in the long arm of chromosome 4 leads to the formation of the fusion gene FIP1L1-PDGFRalpha (F/P) coding for a constitutively activated form of PDGFRalpha. F/P has become the molecular marker of clonal hypereosinophilic syndrome (CEL) and it predicts a dramatic response to imatinib mesylate (IM). Different F/P transcripts have been described, but the correlation with kinetic of molecular response to IM and with the clinical presentation is unknown. Aims. The aim of this study was to evaluate the duration of IM response in FIP1L1-PDGFRalpha positive CEL patients and the correlation between their clinical behaviour and molecular characteristics of the transcripts. Methods. A prospective multicenter study of the HES was established in 2001. The 33 FIP1L1-PDGFRalpha positive patients (F/P+) were monitored every three months for two years then every six months using a nested retrospective reverse transcriptase polymerase chain reaction (RT-PCR). The RNA was subsequently reverse transcribed in cDNA and then amplified by reverse transcription-PCR. The samples were purified and sequenced using the Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems). Patients were systematically screened for organ damage with instrumental evaluation and for the presence of symptoms. Patients were treated with IM 100 to 400 mg daily. The observation period ranges between 15 and 72 months (median 32 months). Results. There were 32 males and one female patients. Organ involvement was recorded in 42% of F/P+. After imatinib therapy all patients achieved a complete hematologic response (CHR) in less than one month, and PCR negativity in a median time of 3 months (range 1–9). They became negative for organ localizations and free of symptoms. All 29 patients who continue imatinib therapy remain in CHR and RT-PCR negative, with a dose of 100 to 400 mg daily. In four patients IM treatment was discontinued and the fusion transcript became rapidly detectable. CHR was maintained. The transcript was again undetectable upon treatment resumption. 28 samples of the totality were valuable for molecular analysis. All deletions of 4q12 generates in-frame chimeric fusion gene. FIP1L1 breakpoints scattered between exon 9 to 18, with several splicing variants. All breakpoints in PDGFRA are located within exon 12. Fusion gene sequencing demonstrate an extreme variability. Region of FIP1L1 varies in length from 109 to more than 500 nucleotides. The more conserved regions are exon 10 and exon 11 of FIP1L1, that are repeated together in 13 out of 28 analyzed transcripts. Transcript of the only female patient is the same of one of the males. No evidence of correlation was noted with kinetic of molecular response or with the presence at diagnosis of peculiar organ involvement. More complexity of transcript is noted in patients with longer history of disease prior to imatinib therapy. Interpretation and conclusion. with this large series of patients we can confirm more complexity and variability in FIP1L1-PDGFRalpha transcripts than data reported in other series, but no evidence of clinical correlation between this heterogeneity and phenotype of disease. Moreover, the response to the imatinib therapy is not influenced by the biologic features, but it depends on continuous therapy.

Idiopathic hypereosinophilic syndrome (HES) is a rare hematological disorder characterized by persistent eosinophilia presenting often with organ involvement. The increased blood and tissue levels of eosinophils (eos) appear to produce tissue damage via local release of toxic substances producing inflammation and fibrosis. Data from the literature are fragmentary and deduced from several historical series. The most commonly involved organs reported are cardiovascular, pulmonary, cutaneous and neurologic systems. Cardiac disease is described as the major cause of morbidity and mortality. Due to the new knowledge about myeloproliferative variant of HES and PDGFRA rearrangement, we tried to identify different clinical picture. The aim of this retrospective analysis is to evaluate the prevalence of HES-related organ damage, its relation to F/P status, and response to imatinib therapy. A prospective multicenter study of the HES began in 2001. Patient enrolled had HES diagnosis, independently of presence of FIP1L1-PDGFRA (F/P) rearrangement. 72 patients were treated with IM 100 to 400 mg daily with a median observation time of 28 months (r 12–68). 33 patients (46%) were positive for the F/P rearrangement (F/P+), while 39 (54%) were negative (F/P−). At the time of enrolment HES patients were systematically screened for organ damage with instrumental evaluation (chest radiography, echocardiogram, abdomen ultrasonography). Symptoms were considered too, so if respiratory symptoms were presented or a reduction of ejection fraction was found organ involvement was established. Results. Organ involvement was recorded in 42% of F/P+ and in 51% of F/P−. Skin involvement was only recorded in F/P−, whereas splenomegaly was reported in 7 F/P+ and only in one case of F/P−. To date, soft tissue was peculiar site of F/P+ patients. All the 33 F/P+ patients achieved a complete hematologic remission (CHR) and molecular negativity, and together they became negative for organ localization and free of symptoms. In the two patients with soft tissue involvement (temporal/parietal and retro-orbital masses) imaging response was documented with positron emission tomography (PET) and with CT scan one month after the beginning of the therapy. Favourable response was recorded also in patients with cardiac involvement, apiece to grade of fibrosis. 5 out of 39 F/P− patients achieved a transitory CHR, but no durable effects on organ involvement. All these patients are alive. Conclusion. Organ involvement do not seems to be a constant characteristic of HES, irrespective to F/P status, but there are differences between F/P+ and F/P− patients. Organ damage in F/P+ subset is reversible before fibrosis development. In the whole population observed, no deaths were recorded in more than five years. F/P+(33 pt) F/P−(39 pt) Statistic Organ damage 42% 51% p=NS More than 1 organ involvement 18% 3% p=0,01 Heart 15% 5% p=NS Lung 18% 26% p=NS Spleen 21% 3% 0,001 Skin 0 15% p=0,001 Liver 3% 3% p=NS Soft tissue 6% 0 p=0,1 Skeleton 3% 0 p=NS Gut 0 3% p=NS

Resistance to the Bcr-Abl tyrosine kinase inhibitor (TKI) imatinib mesylate (IM) in patients (pts) with chronic myeloid leukemia (CML) and Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) is often caused by selection of point mutations in the Abl kinase domain (KD) altering residues that are directly or indirectly critical for IM binding. Several novel TKIs are now available for IM-resistant pts. In vitro studies have postulated that each of them is likely to retain its own distinct set of insensitive mutations, including novel inhibitor-specific mutations. To assess how Abl KD sequences evolve under the selective pressure of sequential therapy with one or more novel TKIs, we have monitored the mutation status of 101 IM-resistant pts before and during treatment with up to three consecutive novel TKIs (dasatinib, nilotinib, bosutinib). Forty-seven pts had CML in chronic phase; 54 pts had CML in accelerated/blastic phase (AP/BP) or Ph+ ALL. Overall, presence or emergence of TKI-resistant mutations accounted for 90% of cases of dasatinib, nilotinib or bosutinib failure. At the time of IM failure, 55/101 (54%) pts had KD mutations. After switching to a 2nd TKI (n=101 pts), 21/55 (38%) pts who had mutations at baseline as against 8/46 (17%; p=.02) pts who did not have mutations at baseline subsequently relapsed with newly acquired mutations. Median time to relapse was 8 months (range, 3-18). Cloning showed that these mutations could either be acquired by the pre-existing mutated subclone or arise in an independent one. In addition, 15/55 mutated pts did not respond to the 2nd TKI because of the mutation they were harboring at baseline. After switching to a 3rd TKI (n=20 pts), 11/16 mutated pts as against 0/4 non-mutated pts relapsed with newly acquired mutations. Median time to relapse was 3 months (range, 1–5). Switch to a 4th TKI has so far been attempted in 3 mutated pts, but observation time is still too short. Dasatinib failure was associated with the following mutations: T315I, F317L, V299L, T315A, F317I/S/V. Nilotinib failure was associated with the following mutations: Y253H, E255V, T315I, F359C. Bosutinib failure was associated with the following mutations: E255K, T315I, V299L. More detailed analyses will be presented. In summary, the Bcr-Abl kinase is a moving target and pts already harboring mutations, especially those with CML in AP/BP or with Ph+ ALL, have a higher likelihood of developing further mutations under the selective pressure of novel TKIs. It can be hypothesized that in these pts a higher genetic instability may foster rapid emergence of multiple mutations over time within the same or different Bcr-Abl-positive subclones, which are selected or de-selected depending on the sensitivity profile of the specific TKI employed. In this clinical setting combination therapies would probably be more effective than single-agent treatment for long-term disease control. Supported by European LeukemiaNet, AIL, FIRB, COFIN, Fondazione del Monte di Bologna e Ravenna.

An interstitial deletion in the long arm of chromosome 4 leads to the formation of a novel fusion gene (FIP1L1-PDGFRalpha) coding for a constitutively activated form of PDGFRalpha. The fusion gene has been identified only in patients with the hypereosinophilic syndrome and has become a marker of clonal HES. The PDGFRalpha transcript can be neutralized by several protein tyrosine kinase inhibitors (PTKI). Recently, imatinib mesylate (IM) has been shown to induce remissions (Cools J, N Engl J Med. 2003; 348:1199–200, Pardanani A, Blood2003;102:3093–6, Cortes J, Blood2003; 102:4714–6) but remission duration is still undefined. The aim of this study was to evaluate the duration of IM in FIP1L1-PDGFRalpha positive HES patients. Hypereosinophilic syndrome was defined according to Chusid criteria (Blood, 1994; 83: 2759–2779). The FIP1L1-PDGFRalpha transcript was identified and monitored every six months on bone marrow cells using a nested retrospective reverse transcriptase polymerase chain reaction (RT-PCR), as reported by Cools J (N Engl J Med.2003; 348:1199–200). Twenty-one FIP1L1-PDGFRalpha positive HES patients have been enrolled in a prospective national study of treatment with IM at a daily dose of 400 mg. All patients were male. Median age was 48 years ( range 25–72). All 21 patients achieved a complete hematologic response (CHR) in less than one month. Currently, the median follow-up of all 21 patients is 28 months ( range 13– 67), with 13 patients with a follow-up longer than 2 years. The longest follow-up are 42, 46 and 67 months. All these patients remained in complete hematologic remission and all became PCR negative and have remained PCR negative as of today. All patients are continuing on imatinib, but in several cases the dose has been reduced to 300, 200 or 100 mg daily. No patients developed heart failure. We conclude that in FIP1L1-PDGFRalpha positive HES patients the therapeutic effect of IM at doses ranging from 400 to 100 mg daily is sustained over time, up to five years, without detectable adverse effects.

SM is characterized by activating mutations of the Kit tyrosine kinase receptor. While the so-called ‘enzimatic site’ (ES) type mutation D816V renders Kit resistant to imatinib, ‘regulatory’ type mutations are sensitive to inhibition. Kit mutation screening with sensitive methods is important for appropriate therapeutic management of SM. Our aims were: to set up and optimize a D-HPLC-based screening method for mutations in different critical regions of the kit receptor; to assess the sensitivity and reliability of our D-HPLC assay as compared to RFLP analysis; to characterize additional mutations/polymorphisms. The analysis was performed on 37 SM pts. For each sample, a RT-PCR product spanning the catalytic and activation loops in the ES was screened in parallel by D-HPLC, followed by sequencing of D-HPLC-positive cases, and by RFLP according to an already reported method for the detection of the D816V. By RFLP analysis, 24/37 (65%) pts were positive for the D816V. By D-HPLC analysis, an abnormal eluition profile was seen in 26/37 (70%) pts - all the 24 pts scored as mutated by RFLP as well as two additional pts. Direct sequencing confirmed the presence of the D816V in all the 24 RFLP-positive cases, and showed that in two of these cases a I798I polymorphism was also present. The two pts scored positive by D-HPLC but negative by RFLP were found to have the same I798I polymorphism. The 11 pts who did not harbour ES type mutations were further investigated by D-HPLC analysis of a RT-PCR product corresponding to the transmembrane (TM) and juxtamembrane (JM) domains. D-HPLC showed an abnormal elution profile in 4 pts. Direct sequencing confirmed the presence of a point mutation in all cases. One patient showed a silent mutation at codon 546. Three pts showed a novel point mutation at codon 541 in the TM domain, resulting in a Met to Leu amino acid substitution. This is the second Kit TM domain mutation reported in a human disease and further supports the hypothesis of a role of the TM domain in regulating the enzymatic activity of an otherwise normal catalytic site. Further characterization of this novel mutant is ongoing. Morphologic and cytofluorimetric analyses of bone marrow biopsies and aspirates will be compared in order to assess whether the pts share any peculiar pathologic feature. Cos-7 cells are currently being transfected with M541L, D816V and wild-type kit in order to evaluate the effects of the M541L on kit enzymatic activity by western blot analysis of total and phosphorylated kit. Patient mast cells will be cultured in the presence or absence of kit ligand or imatinib, dasatinib and nilotinib in order to assess the sensitivity of the M541L to different kit inhibitors. Our novel D-HPLC-based assay proved a straightforward, reliable and sensitive method for kit mutation analysis. It also highlighted the importance of screening for mutations other than the D816V. D-HPLC analysis allowed us to find a novel M541L mutation which is now under characterization.

Hypereosinophilia is a common biological finding, arising in a number of different clinical situations. Hypereosinophilia can result from the presence of a defect in the hematopoietic stem cell giving rise to a primary eosinophilia, it can be present in many myeloproliferative disorders or alternatively it may be a reactive form, secondary to many clinical conditions. The hybrid gene FIP1L1-PDGRFa was identified in a subset of patients presenting with primary HES. In spite of this, the majority of HES patients do not present detectable molecular lesions. So far, for the majority of them, the diagnosis is based on exclusion criteria and in same cases it may remain doubt. Since many therapeutic approaches now available may change the prognosis of primary HES patients, it is actually of great clinical importance to identify these patients. The aim of the study was to explore the possibility to distinguish between primary and secondary hypereosinophia basing on WT1 expression. BM and PB samples from 292 patients affected by hypereosynophilia and 50 BM and PB from healthy subjects were studied. In 202 patients (69%) clinical and/or laboratory findings were consistent with other eosinophilic disorders, including Churg-Strauss vasculitis, chronic eosinophilic pneumonia, eosinophilic gastroenteritis and episodic angioedema. 90 patients (31%) were diagnosed as primary HES or chronic eosinophilic leukaemia (CEL). Conventional cytogenetic analysis and FISH were performed to study the involvement of the PDGFRalpha and beta and FGFR1. All the patients were characterized at the molecular level for the presence of the known fusion transcript associated with eosinophilia: BCR-ABL, CBF-MHY11, AML-ETO, BCR-FGFR, TEL-PDGFR and for FIP1L1-PDGFRa transcript. In primary HES patients treated with Imatinib (70 out of 90) or with other conventional therapies (hydroxiurea in 8 pts, IFNalpha in 4 pts) the BM and PB samples were analyzed at regular time intervals during treatment (after 1, 3, 4, 6,12 months). 32 patients were positive for the presence FIP1L1-PDGFRa. Real Time PCR for the detection of WT1 transcript amount was performed at diagnosis and during follow-up in all the 292 patients. We found that WT1 amount in secondary HES is not significantly different compared to healthy controls with a mean value of WT1 copies/104 ABL copies of 32 (range 0–75) in BM and 5 (range 0–17) in PB vs 35 (range 3–90) in normal BM and 4 (range 1–20) in normal PB. By contrast, primary HES showed significantly higher values compared to normal controls or to secondary conditions with a mean of 651 WT1 copies in BM (p=0,0003) and 151 in PB (p=0,00002). WT1 is not significantly different in FIP1L1-PDGFRa when compared to HES with normal karyotype (p=0,3). Moreover, WT1 transcript follows the behaviour of FIP1L1-PDGFRa or of the specific cytogenetic marker during follow-up. In conclusion the quantitative assessment of WT1 is a useful tool for the diagnosis and follow-up of primary HES patients with normal karyotype.

Idiopathic hyper-eosinophilic syndrome (HES) is a rare haematological disorder characterized by persistent peripheral blood greater than 1,500 cells/μL lasting for more than 6 months, in the absence of other apparent aetiologies for eosinophilia with signs and symptoms of organ involvement. HES may be a reactive condition or a chronic myeloproliferative disorder with evidence of clonal proliferation, in which latter case it is usually referred to as chronic eosinophilic leukemia (CEL). Patients with HES generally have a poor prognosis, but the course of the disease may be variable. Severe visceral complications, including cardiopathies, are common and are often fatal illness. Treatment of HES includes corticosteroids, chemotherapeutic agents (such as cyclophosphamide, vincristine, and hydroxyurea), and, more recently, interferon-alpha (IFN-alpha). Recently, Cools et al. reported the involvement of PDGFRA, fused with FIP1L1, in a number of HES patients responsive to imatinib therapy. We treated with imatinib mesylate (100 to 400 mg daily) 59 patients affected by Hyper-Eosinophilic Syndrome (HES) enrolled in a multicentric Italian phase 2 clinical trial. All the patients were studied by molecular analysis for expression of FIP1L1-PDGFRA, TEL-PDGFRB, FGFR1-BCR and BCR-ABL chimerical transcripts. 32 patients (45%) were positive for the FIP1L1-PDGFRA rearrangement. Rapid, haematological complete responses (HCR) were recorded after one month of therapy in all FIP1L1-PDGFRA positive. In 36 patients resulted negative for FIP1L1-PDGFRA rearrangement we observed 8 (22%) hematological improvement (HI) and one HCR (HI+HCR 25%). Furthermore, a molecular complete remission (defined as the disappearance of FIP1L1-PDGFRA at qualitative RT-PCR evaluation) was recorded in 13 (68%) patients of the 19 valuable after three months of therapy, and all but one (94%) patients resulted negative for the presence of the rearrangement after six months of therapy. No significant toxicity was seen during the treatment. The median follow up was 4 months (range: 2–39). This is the largest series of HES patients treated with Imatinib with strong evidence of hematological and molecular effectiveness and absence of long term significant toxicity. This phase II study supports the use of Imatinib as first line therapy in FIP1L1-PDGFRA rearrangements positive HES patients. Acknowledgments: COFIN 2003, by FIRB 2001, by the University of Bologna (60%), by the Italian Association for Cancer Research (A.I.R.C.), by the Italian National Research Council (C.N.R), by Fondazione Del Monte of Bologna and Ravenna (Italy), A.I.L. grants, European LeukemiaNet grants.

We studied 141 patients with HES, of which 55 primary HES (39%) defined as a peripheral blood eosinophilia greater than 1,500 cells/μL for longer than 6 months, absence of other apparent aetiologies for eosinophilia and symptoms of organ involvement. All patients were studied by molecular analysis for PDGFRB-TEL, FGFR1-BCR and BCR-ABL transcripts, frequently associated with HES/CMML/MDS syndrome: all these transcripts were absent in our series. We also sought for the recently reported involvement of PDGFRα, cryptically translocated with FIP1L1 in some HES pts responsive to Imatinib therapy: 13 pts (23%) were positive for the FIP1L1-PDGFRA rearrangement and all of them showed previously unreported, abnormal-sized fusion transcripts. Curiously, all FIP1L1-PDGFRA positive pts were male. We enrolled in a national clinical trial 31 (55%) primary HES pts, including all 13 (23%) FIP1L1-PDGFRA positive, with imatinib mesylate (100 to 400 mg/day). Median follow up of treatment was 4,5 moths (range 2–28). Rapid and complete haematological responses to imatinib therapy were recorded only in all FIP1L1-PDGFRA positive pts (100%) after one months of therapy and partial response in only one cases with HES without FIP1L1-PDGFRA fusion transcript. Complete molecular response without evidence of FIP1L1-PDGFRA transcript by qualitative RT-PCR was also recorded in all responding pts after median 2 months of therapy. We conclude that FIP1L1-PDGFRA rearrangement may be useful molecular marker of myeloproliferative HES, a predictor of imatinib-responsiveness and as a means to follow therapy in this subgroup of pts.