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Mastocytosis is a unique hematologic neoplasm with complex biology and pathology and a variable clinical course. The disease can essentially be divided into cutaneous mastocytosis (CM) and systemic mastocytosis (SM). In adults, SM is diagnosed in most cases and manifests as either indolent or advanced disease. Patients with advanced SM have an unfavorable prognosis with reduced survival. However, so far, little is known about the prevalence of various categories of SM and about prognostic factors. In an attempt to learn more about the behavior and evolution of various forms of CM and SM, the European Competence Network on Mastocytosis (ECNM) initiated a mastocytosis registry in 2012. In this article, the set up and start phase of this registry are described. Until 2018, more than 3000 patients from 12 countries and 25 centers have been enrolled. In a majority of all patients, robust follow-up data and relevant clinical end points are available. Using this data set, a series of registry projects have been launched, with the aim to validate previously identified diagnostic and prognostic variables and to identify new disease-related and patient-related parameters in various forms of mastocytosis. Moreover, the core data set of the registry will be useful to establish multiparametric scoring systems through which prognostication and individualized management of patients with mastocytosis should improve in the foreseeable future. (C) 2019 American Academy of Allergy, Asthma & Immunology

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]: The WHO classification separates mastocytosis into distinct variants, but prognostication remains a clinical challenge. The aim of this study was to improve prognostication for patients with mastocytosis., [Methods]: We analysed data of the registry of the European Competence Network on Mastocytosis including 1639 patients (age 17–90 years) diagnosed with mastocytosis according to WHO criteria between Jan 12, 1978, and March 16, 2017. Univariate and multivariate analyses with Cox regression were applied to identify prognostic variables predicting survival outcomes and to establish a prognostic score. We validated this International Prognostic Scoring System in Mastocytosis (IPSM) with data of 462 patients (age 17–79 years) from the Spanish network Red Española de Mastocitosis diagnosed between Jan 22, 1998, and Nov 2, 2017., [Findings]: The prognostic value of the WHO classification was confirmed in our study (p, [Interpretation]: The IPSM scores for patients with non-advanced and advanced mastocytosis can be used to predict survival outcomes and guide treatment decisions. However, the predictive value of the IPSM needs to be confirmed in forthcoming trials., This work was supported by the Austrian Science Fund (SFB grants F4701-B20 and F4704-B20, to PV), Deutsche Forschungsgemeinschaft (RA 2838, to AI), Koeln Fortune Program, Faculty of Medicine, University of Cologne (216/2016, to AI), Charles and Ann Johnson Foundation (to JG), and Instituto de Salud Carlos III and fondos FEDER (PI16/00642 and CB16/12/00400, to AO). VS is a senior clinical researcher of the Research Foundation Flanders/Fonds Wetenschappelijk Onderzoek (1804518N). CB is supported by the Clinical Research Fund of the University Hospitals Leuven.

BACKGROUND In mastocytosis, the skin is almost invariably involved, and cutaneous symptoms deeply affect patients' quality of life. METHODS A retrospective observational analysis of patients affected by cutaneous mastocytosis (CM) and indolent systemic mastocytosis (ISM) treated with phototherapy/photochemotherapy (PUVA or NB-UVB) has been conducted. For each patient, total numbers of PUVA or NB-UVB exposures, the cumulative UV dose (J/cm2 ), serum tryptase profile, and pruritus, before and after treatment, according to the visual analogue scale (VAS) were considered. Skin lesions of each patient were assessed, before and after treatment, according to a cutaneous scale score. RESULTS Twenty patients affected by CM and ISM were studied; in particular, 10 patients received NB-UVB therapy, and other 10 patients received PUVA. A statistically significant mean reduction of pruritus in both groups (P < 0.01) was observed. The number of treatments necessary to obtain symptom relief was significantly lower in the PUVA group, but the mean exposure dose was significantly higher, if compared to the NB-UVB group. Serum tryptase levels showed a downward trend. The cutaneous score improved in both groups. LIMITATIONS This study was a retrospective study with a small sample size and without a control group. CONCLUSION This work provides evidence that both NB-UVB and PUVA represent a safe and useful second-line therapy of the cutaneous symptoms in mastocytosis.

Systemic mastocytosis is a rare heterogeneous myeloproliferative neoplasm characterized by abnormal proliferation and activation of mast cells. We describe a large multicentre series of 460 adult patients with systemic mastocytosis, with a diagnosis based on WHO 2008 criteria, in a "real-life" setting of ten Italian centers with dedicated multidisciplinary programs. We included indolent forms with (n = 255) and without (n = 165) skin lesions, smouldering (n = 20), aggressive (n = 28), associated with other hematological diseases mastocytosis (n = 21) and mast cell leukemia (n = 1). This series was uniquely characterized by a substantial proportion of patients with low burden of neoplastic mast cells; notably, 38% of cases were diagnosed using only minor diagnostic criteria according to WHO 2008 classification, underlying the feasibility of early diagnosis where all diagnostic approaches are made available. This has particular clinical relevance for prevention of anaphylaxis manifestations, that were typically associated with indolent forms. In multivariate analysis, the most important features associated with shortened overall survival were disease subtype and age at diagnosis >60 years. Disease progression was correlated with mastocytosis subtype and thrombocytopenia. As many as 32% of patients with aggressive mastocytosis suffered from early evolution into acute leukemia. Overall, this study provides novel information about diagnostic approaches and current presentation of patients with SM and underlines the importance of networks and specialized centers to facilitate early diagnosis and prevent disease-associated manifestations. Am. J. Hematol. 91:692-699, 2016. © 2016 Wiley Periodicals, Inc.

The molecular basis of advanced systemic mastocytosis (SM) is not fully understood and despite novel therapies the prognosis remains dismal. Exome sequencing of an index-patient with mast cell leukemia (MCL) uncovered biallelic loss-of-function mutations in the SETD2 histone methyltransferase gene. Copy-neutral loss-of-heterozygosity at 3p21.3 (where SETD2 maps) was subsequently found in SM patients and prompted us to undertake an in-depth analysis of SETD2 copy number, mutation status, transcript expression and methylation levels, as well as functional studies in the HMC-1 cell line and in a validation cohort of 57 additional cases with SM, including MCL, aggressive SM and indolent SM. Reduced or no SETD2 protein expression-and consequently, H3K36 trimethylation-was found in all cases and inversely correlated with disease aggressiveness. Proteasome inhibition rescued SETD2 expression and H3K36 trimethylation and resulted in marked accumulation of ubiquitinated SETD2 in SETD2-deficient patients but not in patients with near-normal SETD2 expression. Bortezomib and, to a lesser extent, AZD1775 alone or in combination with midostaurin induced apoptosis and reduced clonogenic growth of HMC-1 cells and of neoplastic mast cells from advanced SM patients. Our findings may have implications for prognostication of SM patients and for the development of improved treatment approaches in advanced SM.Leukemia advance online publication, 30 June 2017; doi:10.1038/leu.2017.183.

Aim: We collected ‘real-life’ data on the management of patients with mastocytosis in the Italian Mastocytosis Registry. Methods: Six hundred patients diagnosed with mastocytosis between 1974 and 2014 were included from 19 centers. Results: Among adults (n = 401); 156 (38.9%) patients were diagnosed with systemic mastocytosis. In 212 adults, no bone marrow studies were performed resulting in a provisional diagnosis of mastocytosis of the skin. This diagnosis was most frequently established in nonhematologic centers. In total, 182/184 pediatric patients had cutaneous mastocytosis. We confirmed that in the most patients with systemic mastocytosis, serum tryptase levels were >20 ng/ml and KIT D816V was detectable. Conclusion: The Italian Mastocytosis Registry revealed some center-specific approaches for diagnosis and therapy. Epidemiological evidence on this condition is provided.

Objective As disruption of epigenetic control is a frequent event in solid tumors and leukemia, we investigated changes in DNA methylation (5mC) and hydroxymethylation (5hmC) in patients with systemic mastocytosis (SM), a rare myeloproliferative disease with a wide spectrum of severity, characterized by the accumulation of mast cells in various organs. Methods We measured overall genomic levels of 5hmC and 5mC in patients with SM by dot blot, as well as by quantitative immunofluorescence in samples of cutaneous mastocytosis. Results Overall 5hmC levels were reduced in all patients with SM, but to a greater extent in the presence of higher D816V mutational load in the KIT oncogene, which affects prognosis and therapeutic options in these patients. Loss of 5hmC was likely due to systemic effects of SM as it did not correlate with overall mast cell burden in these patients, nor it was due to inactivating mutations of TET2 or reduced TET2 expression. Conclusions The correlation between SM diagnosis and significantly low 5hmC levels suggests that reduction of 5hmC represents a systemic effect of SM that may be useful for patient stratification and that measurements of 5hmC levels may serve as a better prognostic marker than TET2 mutations.

Interferon a (IFNa) prolongs survival of CML patients achieving CCyR and potentially synergizes with TKIs. We report on the molecular status and long term outcome of 121 patients who were treated in Italy between 1986 and 2000 with IFNa based therapy and who obtained CCyR. After a median follow up of 16.5 years, 74 (61%) patients were switched to standard imatinib: 48 (65%) lost the CCyR on IFNa, and 36 (75%) are alive and in CCyR; 26 (35%) were switched to imatinib when they were still in CCyR on IFNa, and all 26 are alive and in CCyR. Forty-seven patients (39%) were never switched to imatinib: 24 (51%) continued and 23 (49%) discontinued IFNa, respectively, and 39/47 (83%) are alive and in CCyR. At last follow-up, the BCR-ABL transcripts level was available in 96/101 living patients (95%) The BCR-ABL:ABL ratio was between 0.1 and 0.01% (MR 3.0 ) in 17%, and less than 0.01% (MR 4.0 ) in 81% of patients. No patient was completely molecular negative (MR 4.5 or MR 5.0 ). The OS at 10 and 20 years is 92 and 84%, respectively. This study confirms that CCyR achieved with IFNa and maintained with or without imatinib or any other therapy significantly correlates with long term survival in CML patients who mostly have MR 4.0 . Complete molecular response (MR 4.5 or MR 5.0 ) seems to be unnecessary for such a long survival. This study further supports development of studies testing the clinical effect of the combinations of TKIs with IFNa. Am. J. Hematol. 89:119–124, 2014. V C 2013 Wiley Periodicals, Inc.

MicroRNAs (miRNAs) are a large class of small regulatory molecules able to control translation of target mRNAs and consequently to regulate various biological processes at a posttranscriptional level. Their importance is highlighted by the fact that altered miRNA expression is linked to a variety of human diseases, particularly cancer. Accordingly, miRNA biogenesis itself must be carefully regulated, both transcriptionally and posttranscriptionally. Here, we focus on the role of miRNAs in three lineages of myeloid cells important in both innate and acquired immunity: mast cells, macrophages, and dendritic cells. These three cell types are strategically located throughout the body tissues, where they can respond to foreign material, danger, and inflammatory signals. We discuss the role of miRNAs in these cell types, with a special focus on three of the most extensively studied miRNAs, namely miR-221, miR-146a, and miR-155. We also discuss the role of cell-to-cell transfer of miRNAs in dendritic cells, mast cells, and macrophages, and we speculate about possible future directions in the field.

Imatinib is a cornerstone of treatment of chronic myeloid leukemia. It remains unclear whether transient treatment discontinuation or dose changes affect outcome and this approach has not yet been approved for use outside clinical trials.We conducted a retrospective single-institution observational study to evaluate factors affecting response in 'real-life' clinical practice in 138 chronic myeloid leukemia patients in chronic phase treated with imatinib. We used a novel longitudinal data analytical model, with a generalized estimating equation model, to study BCR-ABL variation according to continuous standard dose, change in dose or discontinuation; BCR-ABL transcript levels were recorded. Treatment history was subdivided into time periods for which treatment was given at constant dosage (total 483 time periods). Molecular and cytogenetic complete response was observed after 154 (32%) and 358 (74%) time periods, respectively.After adjusting for length of time period, no association between dose and cytogenetic complete response rate was observed. There was a significantly lower molecular complete response rate after time periods at a high imatinib dosage.This statistical approach can identify individual patient variation in longitudinal data collected over time and suggests that changes in dose or discontinuation of therapy could be considered in patients with appropriate biological characteristics.

Objectives and background: According to the World Health Organization (WHO) classification, the diagnosis of Systemic Mastocytosis (SM) relies on bone marrow (BM) examination and is based on a major and four minor criteria. The somatic ‘autoactivating’ point mutation D816V in the KIT receptor gene is one of the minor criteria, founded in the great majority of patients (90%) and it plays a central role in the pathogenesis of the disease. Indolent Systemic Mastocytosis (ISM) is the most common variant of SM, characterized by a very low MC burden and associated with very different clinical pictures. A highly sensitive diagnostic methods for D816V detection are required to assure an appropriate diagnosis and to reduce false-negative results. The recent development of “ultra-deep amplicon sequencing” (UDS) technologies has opened the way to a more accurate characterization of molecular aberrations with higher sensitivity of screening for known and unknown mutations. Our aims were: i) to set-up and optimize a UDS-based mutation screening strategy of the KIT gene on the Roche GS Junior Instrument; ii) to test the sensitivity of our UDS assay to detect the D816V mutation; iii) to investigate the presence of additional KIT mutations in SM. Methods: We decided to take advantage of a next generation sequencing approach to perform an UDS KIT gene mutation analysis on 20 bone marrow (BM) samples from patients whit ISM that were negative for the D816V mutation by Sanger Sequencing which has a sensitivity of 20%. Fusion primers were designed to generate ten partially overlapping amplicon covering the whole KIT transcript (exons 1-21) by RT-PCR. To determine the lower detection limit of our UDS-assay, serial dilutions of the HMC-1 cell line (harboring the D816V mutation) into an unmutated K562 cell line in ratios such as to simulate the following mutation loads were sequenced: 50%, 37.5%, 25%, 12.5%, 5%; 2.5%, 1.25%, 0.5%, 0.25%. Results and significance: UDS of cell line dilutions showed a high accuracy of D816V mutation detection and linearity of mutation calling over the entire range down to 0.25%. The UDS technology allowed to detected the D816V mutation, below the lower detection limit of Sanger Sequencing, with an abundance from 0.5% to 11%, in 12/20 ISM patients. Two additional sequence variations were detected in a large proportion of patients. These two variations included a 3bp in-frame deletion in exon 15 (GenBank X06182.1: c.2164_2166delAGC; p.S715del) found in 11/20 patients and a 12bp in frame-deletion in exon 9 in all patients, whit an abundance ranging from 83% to 97% (GenBank X06182.1: c.1550_1561delGTAACAACAAAG; p.G510_K513del). Previously published studies indicate that the KIT Gly-Asn-Asn-Lys510-513+/- alternatively spliced located immediately downstream to the extracellular KIT domain and KIT Ser715+/-, an interkinase KIT domain, are expressed in normal human hematopoietic cell, leukemic cell lines, acute myeloid leukemia blast and GISTs and represent rather a splice variant of KIT transcript. Interestingly our results showed the presence of the transmembrane domain M541L (GenBank X06182.1: c.1642A>C; p.Met541Leu) KIT-activating mutation in exon 10, with an abundance of 50%, in addition to D816V, in 2/20 ISM. This mutation is known to retain sensitivity to imatinib mesylate. Conclusions: Our preliminary results suggest that our-UDS based KIT gene mutation screening assay might be a reliable and sensitive alternative to conventional sequencing methods for the detection of the D816V. We are now planning to investigate whether the greater sensitivity of UDS allows to detect the D816V mutation in peripheral blood mononuclear cells from patients with a suspected clonal mast cell disorder. These results could represent a starting point to plan other extensive studies to better understand the exact role of KIT receptor alterations in SM. Supported by ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Cavo: Celgene: Consultancy, Honoraria, Speakers Bureau. Martinelli:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.

Background Mastocytoses represent a heterogeneous group of stem cell disorders marked by an abnormal hyperplasia and accumulation of mast cells in one or more tissues, including bone marrow, gastrointestinal tract, liver, spleen, lymph nodes and skin. Indolent systemic mastocytosis (ISM) is characterized by red-brownish and pruriginous maculopapular lesions, a bone marrow infiltration without functional impairment and an indolent clinical course with a good prognosis. In particular, the most common cutaneous symptoms are urticarial rash and mild-to-high pruritus. Objectives This study analyses the clinical outcome of patients affected by ISM with prevalent pruriginous cutaneous symptoms and a scarce response to anti-histamines treated using narrowband ultraviolet B (NB-UVB) phototherapy. Methods Narrowband ultraviolet B phototherapy was administered in a UV-irradiation cabin equipped with fluorescent UVB lamps with a peak emission at 311–313 nm. The perception of pruritus severity was assessed using the Visual Analogue Scale (VAS) before starting the treatment and at each control. Results A complete remission of the cutaneous lesions and pruritus was documented in all patients after a median of 40.3 UV treatments and a median cumulative dose of 51.4 J/cm2, with a lasting remission over a 6-month follow-up. The median VAS score at the beginning of the treatment was 86.6 (SD = 6.64), whereas it decreased to 6.66 (SD = 3.75) after 3 months of therapy. Conclusions Our work provides evidence that NB-UVB phototherapy is useful for the treatment of the cutaneous symptoms and pruritus in ISM.

In a series of 172 patients with non-Hodgkin's lymphoma (NHL) classified according to the Working Formulation (WF) the overall incidence of bone marrow infiltration (BM +) at diagnosis was 39%: 59% for low-grade (LGML), 30% for intermediate-grade (IGML), and 25% for high-grade malignant lymphomas (HGML). The features most significantly correlated with the presence of BM + were a low grade of histological malignancy, the degree of splenomegaly and high values of LDH, while those correlated with the extent of BM+ were a non-focal pattern of BM disease, the presence of blood involvement at diagnosis, and the degree of BM fibrosis. Blood involvement was detected at diagnosis in 13% of patients, and a further 16% developed a leukemic phase during the course of the disease. Blood involvement correlated significantly with splenomegaly, bulky disease, advanced clinical stage, and extent of BM +. The presence of BM infiltration ‘per se’ at diagnosis did not significantly affect prognosis. However, the extent of BM disease was correlated with a poorer outcome in IGML and HGML patients. Regarding peripheral blood involvement, in LGML patients only late leukemic conversions were significantly associated with a worse prognosis. In patients with IGML and HGML, either initial or subsequent blood involvement was correlated with significantly poorer outcome.

We used intermediate doses of Ara-C (IDAra-C) in the treatment of 15 patients with chronic myelogenous leukemia (CML) in blast crisis and, combined with hydroxyurea, in 20 CML patients in accelerated phase. Patients with blastic CML received intensive 5-day courses of IDAra-C 600 mg/m2 every 12 h as a 2-h infusion. Of 15 patients, three achieved complete response (CR) and three partial response (PR), for an overall response rate of 40 per cent. All patients developed severe leukopenia and thrombocytopenia, and two died in hypoplasia. Except nausea and vomiting requiring medication, other nonhematologic toxicities were uncommon. Median response duration was 4 months (range 1 to 7 months). Survival was 5 months for responders and 1.5 months for nonresponders. Patients with CML in accelerated phase were treated with two-day courses of IDAra-C 600 mg/m2 every 12 h by 2-h infusion, every two-three weeks. Daily hydroxyurea 1-1.5 g/day was administered between courses. Of 20 patients, 15 (75 per cent) achieved a good PR with rapid improvement of the symptoms of disease acceleration. The median duration of response was 11 months (range 3 to 38 months); duration was over 24 months in five patients. The median survival from the start of IDAra-C was 13 months for responders and 3.5 months for nonresponders. We conclude that IDAra-C is an effective approach for CML in terminal phase. Its use in 5-day induction courses for blast crisis CML has a response rate comparable to that achieved with high-dose Ara-C. In patients in accelerated phase, the combination of short courses of IDAra-C with hydroxyurea is a well-tolerated treatment able to improve substantially the clinical and hematologic symptoms of disease progression.

Imatinib mesylate (STI571), a specific Bcr-Abl inhibitor, has shown a potent antileukemic activity in clinical studies of chronic myeloid leukemia (CML) patients. Early prediction of response to imatinib cannot be anticipated. We used a standardized quantitative reverse-transcriptase polymerase chain reaction (QRT-PCR) for BCR-ABL transcripts on 191 out of 200 late-chronic phase CML patients enrolled in a phase II clinical trial with imatinib 400 mg/day. Bone marrow samples were collected before treatment, after 12, 20 and at the end of study treatment (52 weeks) while peripheral blood samples were obtained after 2, 3, 6, 10, 14, 20 and 52 weeks of therapy. The amount of BCR-ABL transcript was expressed as the ratio of BCR-ABL to beta2-microglobulin (beta2M). We show that, following initiation of imatinib, the early BCR-ABL level trends in both bone marrow and peripheral blood samples made it possible to predict the subsequent cytogenetic outcome and response. We propose this method as the method of choice for monitoring patients on imatinib therapy. QRT-PCR studies may be able to identify degrees of molecular response that predict both complete cytogenetic response and long term stability, as well as patterns of response that provide an early indication of relapse and imatinib resistance.

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.

RAGE is a transmembrane receptor expressed on immune and endothelial cells, whose binding with its ligands, the S100 calgranulins, leads to chronic inflammation. Conversely, its soluble form (sRAGE) plays a protective role by acting as a decoy. We carried out a cross-sectional analysis of the sRAGE and S100A12 serum levels in patients with Crohn’s disease (CD) and ulcerative colitis (UC) and searched for a correlation with clinical and biological markers of activity. We enrolled 60 CD, 67 UC patients, and 66 controls (all adults). Disease activity was scored through the clinical, endoscopic, and histologic indexes of severity, whilst disease location and behaviour were assessed according to the Montreal classification. In all cases, the levels of serum sRAGE, S100A12, C-reactive protein, and faecal calprotectin were measured. sRAGE levels were significantly lower in UC, both active and inactive, than in controls and CD (817.35, range 437.3–1449; 1211, range 843.7–1618; 1207.5, range 743.15–1875.75; P

Hydroxyurea is an antimetabolite agent used in the treatment of myeloproliferative disorders and sickle cell anaemia. Although hydroxyurea is relatively well tolerated, adverse effects often involve skin and mucous membrane during long-term therapy. A group of 510 patients affected by chronic myeloid leukaemia from 1977 to 1998 has been considered. Only 158 patients were treated with hydroxyurea and fulfilled inclusion/exclusion criteria of this study. A spectrum of severe cutaneous and mucosal changes (inflammatory and neoplastic) was seen in about 13% of patients (21 patients out of 158) and was studied in detail. Cutaneous and mucosal atrophy were observed in all 21 patients. Skin atrophy was often characterized by numerous telangiectases, especially on legs and on sun-exposed sites (16/21). Cutaneous, mucosal and nail hyperpigmentation was evident, albeit with variable extent, in 10 of the 21 patients. Severe stomatitis and glossitis with flattening of papillae were another common finding. Five patients, who received a particularly long treatment with hydroxyurea, developed squamous-cell neoplasms on sun-exposed sites (both squamous-cell carcinomas and keratoacanthomas). Acral changes were characteristic and constant, including acral erythema (21/21), dermatomyositis-like changes on the dorsa of hands (7/21), ulcers localized on acral areas of legs, on genitalia and oral mucosae (20/21). The frequency and the variety of these muco-cutaneous changes are reported and the mechanisms by which hydroxyurea may induce this muco-cutaneous syndrome-like group of changes, are proposed.

We present the case of a 60-year-old woman who developed peripheral T-cell lymphoma following successful treatment for high-grade B-cell non-Hodgkin's lymphoma. We consider the possible aetiology of this unusual occurrence. We hypothesise that this case represents one of the undiagnosed adult-onset immunodeficiency, in which the pathogenesis of the patient's T-cell lymphoma may have been in part iatrogenic, namely related to previous immunotherapy with rituximab. We feel this case highlights the importance of rebiopsy in patients with recurrent lymphadenopathy and a history of haematological malignancy and hence acts as an important aide memoir in the investigation of such cases.

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.

Nilotinib restores long-term full-donor chimerism in Ph-positive acute lymphoblastic leukemia relapsed after allogeneic transplantation

Dasatinib and nilotinib are tyrosine kinase inhibitors (TKIs) developed to overcome imatinib resistance in Philadelphia-positive leukemias. To assess how Bcr-Abl kinase domain mutation status evolves during sequential therapy with these TKIs and which mutations may further develop and impair their efficacy, we monitored the mutation status of 95 imatinib-resistant patients before and during treatment with dasatinib and/or nilotinib as second or third TKI. We found that 83% of cases of relapse after an initial response are associated with emergence of newly acquired mutations. However, the spectra of mutants conferring resistance to dasatinib or nilotinib are small and nonoverlapping, except for T315I. Patients already harboring mutations had higher likelihood of relapse associated with development of further mutations compared with patients who did not harbor mutations (23 of 51 vs 8 of 44, respectively, for patients who relapsed on second TKI; 13 of 20 vs 1 of 6, respectively, for patients who relapsed on third TKI).

We have administered interferon alfa-2b, alone or in combination with chemotherapy, to 126 Ph1-positive chronic myelogenous leukaemia patients. Of 71 early chronic phase (CP) patients (less than 12 months from diagnosis), 41 (58%) obtained a complete haematological response (CHR). Daily interferon was more effective than intermittent administration. In previously untreated patients, the response was significantly influenced by risk status at diagnosis. Thirty-four out of 71 (48%) patients improved cytogenetically, the median of Ph1+ mitoses declining from 100% to 66% with complete Ph1-suppression in one case. Of 46 late CP patients (greater than 12 months from diagnosis), 32 (70%) achieved CHR with interferon alone or combined with chemotherapy. All 10 patients with disease well controlled by chemotherapy obtained stable CHR with interferon alone. Of 36 partial responders to conventional chemotherapy, 22 (61%) obtained CHR on interferon plus low-dose hydroxyurea. Ph1 mosaicism was reached by 16 (35%) late CP patients (median Ph1+ cells 75%). Of nine accelerated phase patients on interferon plus chemotherapy, one attained CHR, and two responded partially. At a median follow up of 36 months, of 41 CHR patients in early CP, 15 are controlled on interferon, 12 have had autologous bone marrow transplantation (BMT), and two allogeneic BMT. Blastic transformation (BT) has occurred in eight of 41 CHR patients (19%) versus 17 of 30 (57%) non-responders and partial responders to interferon. At a median follow up of 22 months, of 32 late CP patients obtaining CHR, 26 remain on interferon, one had allogeneic BMT, one had autologous BMT, and one developed BT (versus five out of 14 with less than CHR). These studies confirm the haematological and cytogenetic efficacy of interferon in CML and indicate that the disease status at the start of treatment is critical in determining the success of therapy.

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.

Idiopathic hypereosinophilic syndromes (HES) comprise a spectrum of indolent to aggressive diseases characterized by persistent hypereosinophilia. Hypereosinophilia can result from the presence of a defect in the hematopoietic stem cell giving rise to 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-PDGRFalpha was identified in a subset of patients presenting with HES or chronic eosinophilic leukemia (CEL). In spite of this, the majority of HES patients do not present detectable molecular lesions and for many of them the diagnosis is based on exclusion criteria and sometimes it remains doubt. In this study we explored the possibility to distinguish between HES/CEL and reactive hypereosinophilia based on WT1 transcript amount. For this purpose, 312 patients with hypereosinophilia were characterized at the molecular and cytogenetic level and analyzed for WT1 expression at diagnosis and during follow-up. This study clearly demonstrates that WT1 quantitative assessment allows to discriminate between HES/CEL and reactive eosinophilia and represents a useful tool for disease monitoring especially in the patients lacking a marker of clonality.

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.

A single nucleotide polymorphism at TP53 codon 72 means that two alleles exist: A1 (proline residue, Pro72) and A2 (arginine residue, Arg72). The Pro72 variant of p53 has a lower apoptotic potential. We found that allele A1 was more frequent in patients with chronic myeloid leukemia (CML) than in controls, and among CML patients who had no cytogenetic response than among responders.

Exposure to ionizing radiations (Rx) has been implicated as a causative factor of chronic myelogenous leukemia (CML). We performed a retrospective study of 443 consecutive CML patients, looking for a history of significant exposure to Rx, and evaluated the clinical and hematological characteristics in order to find any difference between radiation-related CML patients and those with de novo CML. We identified 406 patients without known exposure to mutagens (group I) and 37 patients with prior significant exposure to Rx (group II). In comparison to patients of group I, those of group II showed particular clinical and hematological features: significantly lower incidence of bulky splenomegaly (p0.05) and hyperleukocytosis (WBC100 x 10(9)/l; p0.05); significantly higher incidence of anemia (Hb10 g/dl; p0.01). Patients with radiation-related CML had a significantly better survival than those with de novo CML (median survival 61 months vs 42 months; p0.05). In conclusion, this study of a large cohort of CML patients indicates that the subgroup of patients with a history of significant exposure to ionizing radiation has particular clinical and hematological features at onset (lower tumor burden, higher frequency of anemia) and a better survival.

6531 Background: Advent of 2nd-generation TKIs has brought additional treatment options for Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) patients (pts). To analyze the changes they have determined in mutation frequency and type, we have reviewed the database recording the results of BCR-ABL mutation analyses done in our laboratory from 2004 through 2011. Methods: 781 tests on 258 pts were performed by direct sequencing. Results: 143 pts were analyzed because of imatinib resistance; 101 (71%) had one or more mutations (a single mutation in 91 pts; two mutations in 10 pts). Three mutation types were by far the most frequent: T315I (38 pts, 37%), E255K (19 pts, 18%) and Y253H (19 pts, 18%). Of 84 pts who had developed resistance to 2nd- or 3rd-line therapy with dasatinib, nilotinib or bosutinib after imatinib failure, 65 (77%) were positive for Bcr-Abl mutations; 30 (46%) carried multiple mutations (up to four) and in 19 of them (63%) this was consequence of multiple lines of therapy. The most frequent newly acquired mutation in this setting was the T315I, detected in 35/57 (61%) cases acquiring mutations on dasatinib. Mutation analysis was also performed in 15 resistant pts enrolled in a study of dasatinib as 1st-line treatment of Ph+ ALL; 12 pts were positive, 11 of them had a T315I. Taking advantage of a next-generation sequencer (Roche 454), allowing a high sensitive and quantitative mutation scanning of Bcr-Abl, serially collected samples from 24 selected cases who developed mutations and resistance to one or more TKIs were retrospectively analyzed to study the kinetics of expansion of mutant clones. Results will be presented. Conclusions: Although 2nd generation TKIs are more potent and have much fewer insensitive mutations, long-term disease control remains a problem and the T315I becomes an even tougher enemy. The high genetic instability fosters mutational events anytime during TKI treatment and some mutation types (T315I, Y253H) have been observed to emerge and take over very quickly (from

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.

7034 Background: The hypereosinophilic syndrome (HES) may be associated with the fusion of the PDGFRα gene with the FIP1L1 gene in chromosome 4 coding for a constitutively activated tyrosine kinase...

We analyzed the prognostic value of clinical, hematologic and bone marrow (BM) histologic findings at presentation in 94 patients with myelodysplastic syndromes (MDS) (28 RA; 2 RARS; 34 RAEB; 6 CMML; 24 RAEB-t). With survival as the dependent variable, stepwise multivariate analysis indicated as the prognostically most important factors among the MDS taken as a whole: latency from the first symptoms to diagnosis, age, and percentage of BM blasts. In each main MDS group the most unfavorable initial characteristics were: 1) low Hb, no macro-megaloblastosis, male sex for RA/RARS; 2) low Hb and low platelet levels for RAEB/CMML; 3) granuloblastic hyperplasia and high BM blastosis for RAEB-t. Of the BM histologic parameters, only the percentage of blasts had significant prognostic value. Histologic assessment of BM blastosis, however, did not differ statistically from that based on cytologic examination of BM smears, so that marrow histology seemed not essential for initial prognostic assessment in MDS patients. The finding of abnormal localization of immature precursors (ALIP) in BM biopsies was associated with a negative trend without reaching statistical significance. Using four objective parameters of proven significance (age, Hb, platelets, and BM blasts) we devised a staging system of immediate clinical utility for prognostic stratification and risk-adapted therapeutic choices.

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.

7085 Background: SM is characterized by activating mutations of Kit tyrosine kinase. While the enzimatic site (ES) type mutation D816V renders Kit resistant to imatinib, regulatory type mutations are sensitive to inhibition. Kit mutations screening with sensitive methods is important for an appropriate therapeutic management of SM. Methods: Our aims were: to set up and optimize a D-HPLC-based screening method for mutations in critical regions of Kit; to assess the sensitivity and reliability of our D-HPLC assay as compared to RFLP analysis; to characterize additional mutations. The analysis was performed on 51 SM pts. Results: For each sample, a RT-PCR product spanning the catalytic and activation loops (ES) was screened in parallel by D-HPLC, followed by sequencing of D-HPLC-positive cases, and by RFLP according to an reported method for the D816V detection. By RFLP analysis, 34/51 pts were positive for the D816V. By D- HPLC analysis, an abnormal eluition profile was seen in 36/51 pts - all the 34 RFLP-positive cases as well as two additional pts. Direct sequencing confirmed the presence of the D816V in all the 34 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 I798I polymorphism. The 15 pts who did not harbour ES type mutations were further investigated by D-HPLC analysis of a RT-PCR product spanning the transmembrane (TM) and juxtamembrane (JM) domains. D-HPLC showed an abnormal elution profile in 5 pts. By direct sequencing one patient showed the K546K mutation and 4 pts showed the M541L polymorphism. Conclusions: Our D-HPLC-based assay proved a straightforward, reliable and sensitive method for Kit mutation analysis and highlighted the importance of screening for mutations other than the D816V. No significant financial relationships to disclose.

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.