Search

Introduction FLT3 receptor gene has been reported to be mutated in about 30% of AML, with two different kinds of mutations identified: in-frame duplications within the juxtamembrane region (FLT3-ITD) and point mutations in the tyrosine kinase domain (FLT3-TKD). In term of prognosis, the proper role of these mutations is still debated. Moreover, FLT3 mutations are often subjected to clonal evolution, thus how to properly monitor FLT3 mutated clones, evaluate minimal residual disease, manage FLT3 inhibitors (FLT3i) are only some of the open issues. FLAM is an observational study involving FLT3 positive AMLs to gain clinical and molecular data useful to ameliorate real-life physicians' management of this disease. Here we report the results of a preliminary analysis of the retrospective phase of the study. Methods The retrospective phase of FLAM multi-center observational study enrolled each AML patient treated in 33 participating Italian centers detected to carry a FLT3 mutation since 2012. Clinical and molecular data were collected in accordance with GCP and Helsinki declaration in electronic case report forms. Results At data cut-off , 1st of July 2020, 289 patients with FLT3 mutation at diagnosis were enrolled in the retrospective phase of FLAM study, being evaluable in this analysis, with a median age at diagnosis of 62 years (min-max: 18-94) and a M:F ratio of 141/148 (Patients' characteristics are summarized in table 1). 29 out 289 (10 %) patients had a low risk AML, 190/289 (65,7 %) had an intermediate risk AML and 27/289 (9,3 %) patients had a high risk AML, according to ELN 2017 risk stratification or ELN 2010 in case of allelic ratio unavailability (43 patients had no available ELN risk at baseline). A more frequent association between FLT3-ITD and normal karyotype and between FLT3-TKD mutation and other cytogenetic alterations not conferring a favorable/adverse risk has been observed (p = 0.045). Among the study population, 255/289 (88 %) patients carried a FLT3-ITD, 32/289 (11 %) a FLT3-TKD point mutation and 2/289 (1 %) patients both mutations. Capillary electrophoresis has been the technical method used to investigate FLT3-ITD in 163 of 226 (72 %) patients with information on the method used, while Sanger Sequencing in 47 out of 226 (21 %) patients and Next generation sequencing (NGS) in 16 out of 226 (7 %) patients. Overall, NGS has been adopted to investigate FLT3 gene status in 18 out of 259 patients (7 %). FLT3-ITD allelic ratio was available in 62 of 257 (24 %) ITD patients and was greater than or equal to 0.5 in 35/62 (56 %) patients. During patients' follow-up, 19/289 (7 %) patients affected by a FLT3 positive AML at diagnosis underwent a disease clonal evolution with a FLT3 negative AML progression or relapse. Regarding treatment options in FLT3-AML, in our cohort FLT3i were administered as first-line of therapy in only 36/289 (13 %) patients, always in a combination, of which in 26/36 (72.2 %) with intensive chemotherapy. As expected, intensive chemotherapy represented the induction regimen in the majority of the patients (211/289, 73 %). On the other hand, FLT3i were administered as rescue therapy in 62/171 (36 %) cases (47/62 single-agent and in 15/62 in combination) and as re-induction therapy in 22/80 (28 %) cases (10/22 single-agent and 12/22 in combination). Overall, a FLT3i has been administered as single-agent 81 times, of which Gilteritinib was the most frequently used (56/81, 69,1 %), followed by quizartinib (18/81, 22,2 %). Among the 52 documented combinations of a FLT3i with other drugs, particularly noteworthy is the administration of Sorafenib, in 20/52 (39 %) cases in this real-life study. Lastly, nine out of 289 (3 %) patients received a FLT3i as maintenance therapy after HSCT. Data regarding the correlation among the different regimens, with a special focus on FLT3i, other molecular features and response/survival are currently under analysis. Conclusions These data coming from a preliminary analysis portray the state of a large multi-center retrospective cohort of FLT3-positive AML patients treated in Italy between 2012 and 2020, including interesting insights regarding technical methods used to characterize the disease and the therapeutic scenario in which FLT3 inhibitors have been developed. Further safety and effectiveness data may reveal beneficial to ameliorate physicians' real-life clinical practice. Acknowledgements: work supported by Daiichi-Sankyo. Disclosures Martelli: Amgen: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Rigolin:Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Fracchiolla:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accommodations, expenses, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accommodations, expenses, Speakers Bureau; ABBVIE: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accommodations, expenses; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accommodations, expenses, Speakers Bureau. Bocchia:Incyte: Honoraria; CELGENE: Honoraria. Todisco:Jannsen, Abbvie, Jazz: Membership on an entity's Board of Directors or advisory committees. Papayannidis:Abbvie, Janssen, Novartis, Amgen, Pfizer: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. OffLabel Disclosure: Sorafenib is a registered drug for the treatment of hepatocellular carcinoma. Due to its multi-targeted tirosine kinase inhibitor effect it demonstrated efficacy in FLT3-AML when administered as an off-label prescription.

Fixed-duration treatment with venetoclax (Ven), a highly selective Bcl-2 inhibitor combined with an anti-CD20 monoclonal antibody, showed high efficacy inducing high rates of deep responses with undetectable minimal residual disease (uMRD) in patients with previously treated and untreated chronic lymphocytic leukemia (CLL). The efficacy and safety of the Ven and rituximab (VenR) combination have been investigated in a multicenter, prospective study of the GIMEMA group that included young patients with previously untreated CLL (LLC 1518, VERITAS, NCT03455517). The primary endpoint of this study was the CR rate assessed according to the iwCLL criteria. Inclusion criteria were: treatment requirement per iwCLL criteria, age ≤65 years, cumulative Illness rating scale score ≤6, creatinine clearance ≥30 mL/min, and an unfavorable biologic profile with IGHV unmutated and or TP53 disruption. Treatment consisted of the Ven dose ramp-up (from 20 to 400 mg daily, during 5-weeks) followed by Ven 400 mg daily, combined with R for six 28-day courses (375 mg/m2, course 1; 500 mg/m2, courses 2-6). Patients continued with Ven single agent, 400 mg daily, until month 13. Tumor lysis syndrome (TLS) prophylaxis measures included hydration, allopurinol, or rasburicase. All patients received PneumocystisJirovecii prophylaxis. G-CSF was given in patients with recurrent and severe granulocytopenia. Adverse events (AEs) were graded according to the CTCAE criteria v.5, TLS events were classified according to Howard's criteria. Response was assessed at months 7 and 15 and included clinical examination, PB evaluation, BM aspirate, BM biopsy, and CT scan. MRD was checked centrally in the PB and BM by a 6/4-color flow-cytometry assay with a sensitivity of at least 10-4 according to the internationally standardized European Research Initiative on CLL. Quantitative MRD results assessed by flow-cytometry were categorized as uMRD (uMRD4; Between October 2018 and May 2020, 77 patients with CLL were included in this study. Two patients were off study before the start of treatment (withdrawal of consent, 1; Covid-19 infection, 1) and were not included in the analysis. The median age was 53.5 years (range 38-65). Binet stage B/C was present in 84% of patients, increased beta-2 microglobulin in 41%. Seventy-one (96%) of patients were IGHV unmutated, while 3 (4%) were IGHV mutated and showed TP53 mutation (Table 1). At the data cutoff of June 30, 2020, 65 (87%) patients completed the ramp-up phase. The planned 400 mg dose of Ven was reached within 5 weeks in 78.5% of patients. Response was assessed in 34 patients at the end of the VenR combination therapy. A response was achieved by 32 (94%) patients. Responses included 20 (59%) CRs, 1 CRi (3%) and 11 (32%) PRs due to residual enlarged nodes (median maximum size, 1.9 cm). Treatment failure due to toxicity was recorded in 2 (6%) patients. Overall, a response with uMRD4 by flow-cytometry in the PB was observed in 26 (76.5%) cases, and in the PB and BM, in 17 (50.0%). The rates of patients with CR and uMRD4 by flow-cytometry in the PB, and both in the PB and BM, were 44%, and 35%, respectively (Table 2). No detectable disease by PCR, both in the PB and BM, was observed in 4 (12%) patients. With a median follow-up of 4.5 months from the start of therapy, no patient has progressed or died. Fifty-three percent of patients were hospitalized during the first seven days of the Ven ramp-up phase. A transient laboratory TLS was observed in 3 patients. Treatment was discontinued after the first dose of Ven in 1 patient with evidence of laboratory TLS associated with severe neurologic toxicity due to the concomitant administration of fentanyl. Selected grade ≥3 AEs included neutropenia in 10 patients (ramp-up phase, 5) and neutropenic fever in 4. Grade ≥3 infection was recorded in 3 patients and was the reason for treatment discontinuation in 1 who developed COVID-19 pneumonia. In conclusion, the preliminary results of this study demonstrate the high efficacy of the front-line VenR combination, which resulted in a high proportion of CRs and responses with uMRD4 in young patients with CLL and an unfavorable biologic profile. Disclosures Mauro: Astrazeneca: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jannsen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Octopharma: Consultancy. Reda:Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees. Trentin:Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Shire: Honoraria; Takeda: Membership on an entity's Board of Directors or advisory committees; Octapharma: Membership on an entity's Board of Directors or advisory committees. Coscia:Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm Therapeutics: Research Funding; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Sportoletti:Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Laurenti:Roche: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Gaidano:Astrazeneca: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sunesys: Membership on an entity's Board of Directors or advisory committees. Marasca:Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Honoraria; Roche: Membership on an entity's Board of Directors or advisory committees. Murru:Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Rigolin:Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Scarfo:Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AstraZeneca: Honoraria; Gilead: Membership on an entity's Board of Directors or advisory committees. Marchetti:Pfizer: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees. Levato:Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Galieni:Celgene: Honoraria; Takeda: Honoraria; AbbVie: Honoraria; Janssen: Honoraria. Liberati:Verastem: Research Funding; Onconova: Research Funding; Janssen: Honoraria, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Honoraria, Research Funding; Pfizer: Research Funding; Karyopharm: Research Funding; Morphosys: Research Funding; Novartis: Research Funding; GSK: Research Funding; Incyte: Honoraria; Oncopeptides: Research Funding; Takeda: Research Funding. Molica:Roche: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Visentin:Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Vitale:Janssen: Honoraria. Del Giudice:Janssen: Other: grant for meeting participation; Tolero: Membership on an entity's Board of Directors or advisory committees; Roche: Other: grant for meeting partecipation; AstraZeneca: Membership on an entity's Board of Directors or advisory committees. Cuneo:Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astra Zeneca: Honoraria; Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Foà:Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees.

In chronic myeloid leukemia (CML) patients, tyrosine kinase inhibitors (TKIs) may select for drug-resistant BCR-ABL1 kinase domain (KD) mutants. Although Sanger sequencing (SS) is considered the gold standard for BCR-ABL1 KD mutation screening, next-generation sequencing (NGS) has recently been assessed in retrospective studies. We conducted a prospective, multicenter study (NEXT-in-CML) to assess the frequency and clinical relevance of low-level mutations and the feasibility, cost, and turnaround times of NGS-based BCR-ABL1 mutation screening in a routine setting. A series of 236 consecutive CML patients with failure (n = 124) or warning (n = 112) response to TKI therapy were analyzed in parallel by SS and NGS in 1 of 4 reference laboratories. Fifty-one patients (22 failure, 29 warning) who were negative for mutations by SS had low-level mutations detectable by NGS. Moreover, 29 (27 failure, 2 warning) of 60 patients who were positive for mutations by SS showed additional low-level mutations. Thus, mutations undetectable by SS were identified in 80 out of 236 patients (34%), of whom 42 (18% of the total) had low-level mutations somehow relevant for clinical decision making. Prospective monitoring of mutation kinetics demonstrated that TKI-resistant low-level mutations are invariably selected if the patients are not switched to another TKI or if they are switched to a inappropriate TKI or TKI dose. The NEXT-in-CML study provides for the first time robust demonstration of the clinical relevance of low-level mutations, supporting the incorporation of NGS-based BCR-ABL1 KD mutation screening results in the clinical decision algorithms.

Introduction. Monoclonal Gammopathy of Undetermined Significance (MGUS) is a pre-malignant plasma cell disorder reported in approximately 3% of individuals aged > 50 years, characterized by a low risk (about 1% per year) of evolution into "overt" myeloma or other lymphoproliferative diseases. It is classified as IgM-MGUS (15%) and non-IgM-MGUS (80-85%). MGUS is usually asymptomatic, but a higher risk of deep venous thrombosis and infection has been reported. In March 2020, "Coronavirus Disease 2019" (COVID-19) outbreak has been declared a pandemic by the World Health Organization. Regarding outcome of COVID-19 in patients with plasma cell dyscrasia, many papers have been published about multiple myeloma (MM), reporting a higher fatality rate respect to general population, while few data are available about the outcome of SARS-CoV-2 infection in patients with MGUS. Methods. We collected clinical data on MGUS Apulian patients with SARS- CoV-2 infection, tested by RT-PCR on nasopharyngeal swabs between March 1st, 2020 and April 30st, 2021. Among 1454 MGUS patients followed at our center, 91 were found SARS-CoV-2 positive, enrolled in this observational, retrospective study and compared with 182 age and sex-matched normal controls. Clinical data collected regarded: symptoms, hospitalization, hospitalization in intensive care unit, death. Calculations were carried out using Stata MP17. Results. Mean age of whole group (n. 273) was 65,3+/-13,3 years (range: 29-89), with no statistically-significant differences (p=0,734) observed between MGUS-group (65,6+/-13,3; range: 29-89 years) and controls-group (65,2+/- 13,4; range: 29-89 years). Mean number of comorbidities in the whole group was 1,2+/-1,2 (range: 0-5) and no statistically-significant differences (p=0,844) were found between MGUS-group (1,3+/-1,3; range: 0-5) and control group (1,2+/- 0,9; range: 0-3). About MGUS-subtypes, the most frequent was IgG-kappa (n=36; 39,6%), followed by IgG-lambda (n=27; 29,7%) and IgM-kappa (n=6; 6,6%). Regarding MGUS risk-stratification, application of Mayo Clinic model identified 22 patients (24,2%) with low risk, 22 (24,2%) with low-intermediate risk, and 3 (3,3%) with high-intermediate risk; in 44 patients (48,3%) this data was missing. Immunoparesis was present in 13 cases (14,3%) and absent in 55 (60,4%), missing in 23 (25,3%). No patient developed MM or a lymphoproliferative disease progression during and immediately after COVID-19. Rates of symptoms (59,3% vs 56%), hospitalization (20,9% vs 14,3%), hospitalization in intensive care unit (11% vs 8,8%) and death (8,8% vs 5,5%) were slightly higher in MGUS group than controls (Table 1), but these differences were not statistically significant. A statistically significant association (p Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction Ibrutinib is the only once-daily Bruton's tyrosine kinase (BTK) inhibitor with significant survival benefit vs chemo- and /or immunotherapy in multiple phase 3 studies of patients (pts) with chronic lymphocytic leukemia (CLL). It has profoundly changed the treatment landscape of CLL with the longest follow-up. However, seven years (yrs) after ibrutinib was approved in Italy by regulatory agencies for CLL treatment, available data on the patterns of care of such pts in the setting of clinical practice is limited. Herein we present the first interim analysis (IA) of EVIdeNCE (ClinicalTrials.gov Identifier: NCT03720561), a multicenter, observational clinical study designed to describe the current management of pts receiving ibrutinib in real-world setting in Italy in terms of retention rate: the study's primary end point. Methods EVIDENCE 312 treatment-naïve (TN) 38% and relapsed/refractory (R/R) 62% pts with CLL according to the iwCLL diagnosis criteria observed at 39 Italian hematological institutions in the period between November 2018 and October 2019. Inclusion criteria were treatment with ibrutinib according to the European Summary of Product Characteristics as per routine clinical practice started within the previous 3 months. The purpose of this IA is to provide demographics and disease characteristics at baseline and a preliminary evaluation of ibrutinib retention rate after one year of follow-up, along with its safety profile. Results The median age of pts at the time of ibrutinib initiation was 71.0 yrs (range 41.0-89.0), with 60% ≥70 yrs, 63.2% male, and 90% with Eastern Cooperative Oncology Group (ECOG) performance 0-1. Baseline Rai stage 0-I, II, and III-IV accounted for 18.3%, 29.7% and 52.1% pts, respectively. Patients in stage IV were observed in 40% of the R/R and 27% in TN subgroup. Considering 120 pts with known mutational status, del(17p) and/or TP53 mutation were present in 50.0% of pts (TN=52.1%, R/R=48.6%), while IGHV was unmutated in 35.0% (TN=33.3% and R/R=36.15) and mutated in 15.0% (TN=14.6%, R/R=15.3%). At baseline, 62.9% of pts had comorbidities and 30.6% presented with a history of cardiovascular diseases (CVDs). A CIRS score ≥6 was observed in 28.5% of pts. The median time from CLL diagnosis to the start of ibrutinib was 5.1 yrs (TN 1.75 yrs vs R/R 7.27 yrs). At least 1 treatment-emergent adverse event (TEAE) of any grade was experienced by 70.7% of pts. Frequencies were as follows: infections (30.8%; COVID-19 infections 3.2%), arthralgia (10.8%), neutropenia (9.3%), fatigue (8.4%), diarrhea (7.7%), atrial fibrillation (7.4%; grade 3-4, 4.2%), fever (7.1%), rash (6.4%), anemia (6.1%) and hypertension (4.2%). Mild bleeding TEAEs were reported in 16.1% of pts with no major bleeding event. TEAEs were more frequent in the elderly (≥65 yrs) while no significant differences in the rate of TEAEs were recorded in TN and R/R pts (69.7% vs 71.4%, respectively). Serious TEAEs were reported in 21.9% of pts. Overall in intention to treat (ITT), 32 deaths (10%) were observed (TN=8, R/R=24). The most common causes of death were infections (3.5%) and progressive disease (PD) (1.9%). Permanent discontinuation was observed in 56 (18%) of the pts (TN=17.2%, R/R=18.7%) and it mostly occurred within the first 6 months. Main causes of discontinuation were toxicity (6.1%), PD (3.8%) or death (3.5%). Temporary interruptions (≤ 3 months without therapy and/or dose modifications) during the whole observation period occurred in 30.3% (TN=35.3%, R/R=27.2%) and 37.7% (TN=37.5%, R/R=37.8%) of pts, respectively, mainly determined by toxicity and clinical judgment. Finally, in this first IA after 17.3 months (range 1.1 - 27.0) median follow-up, the ibrutinib retention rate (calculated as the ratio between the number of patients who retained ibrutinib treatment over the total number of patients at risk) at 1-year was 81.9% [95% confidence interval (CI), 77.2% - 86.1%] with no difference between TN 83.2% (95% CI, 75.2% - 89.4%) and R/R 81.2% pts (95% CI, 74.9% - 86.4%). Conclusions EVIDENCE is the first real-world study of ibrutinib use in CLL clinical practice in Italy. Ibrutinib retention rate at one-year suggests a better knowledge and expertise of hematologists in the management of ibrutinib-related toxicities that may result in an improved long-term outcome of pts with CLL. Disclosures Molica: Janssen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Astrazeneca: Honoraria. Scarfo: Astra Zeneca: Honoraria; Abbvie: Honoraria; Janssen: Honoraria, Other: Travel grants. Murru: Abbvie: Consultancy, Honoraria, Other: travel and accommodation; Janssen: Consultancy, Honoraria. Sportoletti: AstraZeneca: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria. Frigeri: Celgene: Consultancy, Speakers Bureau; Abbvie: Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Amgen: Speakers Bureau. Sanna: Janssen: Consultancy; Abbvie: Consultancy; Astra Zeneca: Consultancy. Coscia: Janssen: Honoraria, Other, Research Funding; AbbVie: Honoraria, Other; AstraZeneca: Honoraria; Gilead: Honoraria. Reda: Abbvie: Consultancy; Astra Zeneca: Consultancy; Beigene: Consultancy; Janssen: Consultancy. Tafuri: Novartis: Research Funding; Roche: Research Funding; Celgene: Research Funding. Grugnetti: Janssen: Current Employment. Magarotto: Janssen: Current Employment. Mauro: Tskeda: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; Abbvie: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria; Astra Zeneca: Consultancy, Honoraria, Speakers Bureau.

Introduction Although plenty of data exists on efficacy and safety of CLL drugs, their impact on patients' Health-Related Quality of Life (HRQoL) is largely unknown (1-2). Documentation of drug safety via traditional use of adverse events (AE) in hematology is limited if not complemented with Patient-Reported Outcomes (PRO) measures (3-4). Incorporation of HRQoL PROs is now essential to better evaluate risk-benefit of new therapeutic approaches and it is also highly valued by regulatory stakeholders (5). Most PRO data currently available for CLL patients (pts) came from randomized controlled trial settings (6-7), hence limiting generalizability of findings to CLL real-life patients. CHOICE study was designed to investigate CLL patients' QoL and preference towards different treatment profiles through a Discrete Choice Experiment (DCE) methodology in Italy. Due to the timelines of the study, which started in February 2020, the related data offer an insight into patients' perception and worries during the first wave of the COVID-19 pandemic. Methods This cross sectional, multi-center, observational study included CLL patients, treatment naïve during the watch & wait period (W&W) or already TREATED (around 50% each, controlled at site level), who signed the informed consent for study participation. Exclusion criteria were inability to take oral drugs, cognitive disorders that could impair the comprehension of the questionnaires and concomitant treatment for other malignancies. Patients were asked to fill in the following HRQoL questionnaires: EQ-5D-5L, EORTC QLQ-C30 and QLQ CLL-16, as well as a DCE questionnaire, (described elsewhere). Each questionnaire was completed by the patient on a tablet - using an App specifically developed for the study. Results 401 pts were enrolled in Italy in 16 hematology centers (Feb - July 2020); 199 W&W and 196 TREATED pts completed the questionnaires and were included in the evaluable population. Main patients' characteristics are shown in Table 1. 73.7% of TREATED pts were ON-treatment (30.8% were in 1st-line, 69,2% in further lines) and 26.3% were OFF-treatment; the majority of pts (55,6%) were currently treated with a target therapy (Table1). The EQ-5D-5L questionnaire showed no significant differences between groups. In both groups more than 80% of pts reported low values (1 or 2, indicating no or small impact) on all items. Median VAS was 75 for the TREATED group and 80 for the W&W group (0-100; higher scores indicate higher QoL). QLQ C-30 / CLL-16 scores had very similar results between TREATED and W&W pts suggesting a limited impact of CLL on pts QoL. The median (IQR) QoL Scale was 83.3 (67- 83) for TREATED and 83.3 (67- 92) for W&W pts (0-100; all functional scales had high scores, that represent a better level of functioning; all symptoms' scales had low values, representing a less important symptomatology or problem, Figure 1). The main symptoms reported were fatigue, insomnia, pain, and dyspnea, while the main worry was for "future health" (Figure 1). Distribution of data was statistically different between the 2 groups only for the Role functioning Scale (p=0.024) and the Social Functioning Scale (p=0.003) of QLQ-C30 and for the Infection Scale (p Conclusions CHOICE study helps to understand the CLL patients' mindset and feeling in the light of the COVID-19 pandemic impact on health care for this category of pts, highlighting their preferences and worries in a large cohort of pts in Italy, allowing a comparison between TREATED and W&W pts. The main limitation of the study was its cross-sectional design, which does not allow us to evaluate any change in QoL neither with respect to the impact of the pandemic, nor to the effects of the treatment, if any. CLL pts showed a good QoL, as confirmed by both EQ-5D-5L and EORTC QLQ C-30 / CLL-16 scores, with very similar results between TREATED and W&W pts (although slightly better results in the W&W vs TREATED group). The results of the present study are consistent with previous reports, and fatigue was the most reported symptom, while worry for future health was the most relevant score in CLL-16 questionnaire. Hospital accesses reduction that was detected during the pandemic might have influenced patients' response, as well as the extreme attention towards the danger of infections, and might have impacted patients' perception on future health. Figure 1 Figure 1. Disclosures Tedeschi: Beigene: Honoraria, Speakers Bureau; AstraZeneca: Honoraria, Speakers Bureau; AbbVie: Honoraria, Speakers Bureau; Janssen: Honoraria, Speakers Bureau. Gozzetti: Janssen: Honoraria; AbbVie: Honoraria. Reda: Beigene: Consultancy; Astra Zeneca: Consultancy; Abbvie: Consultancy; Janssen: Consultancy. Gualberti: AbbVie: Current Employment. Malgieri: AbbVie: Current Employment. Finsinger: AbbVie: Current Employment.

Introduction. New guidelines for the management of dyslipidemia and lipid modification in order to reduce the risk of cardiovascular (CV) events have been recently published by the European Society of Cardiology (ESC) and the European Atherosclerosis Society (EAS). New recommendations regarding the target value of plasma lipids in very high and high CV risk patients have been provided, in addition to an estimate of the CV risk with a new Systematic Coronary Risk Evaluation (SCORE) chart. Few data have been reported on the management of dyslipidemia in chronic myeloid leukemia (CML) patients treated with nilotinib, and the association with arterial occlusive events (AOEs). We therefore analyzed a large real-life cohort of Italian patients with CML treated with nilotinib outside of clinical trials and evaluated the association between AOEs and plasma lipoproteins levels; moreover, we estimated the prognostic value of the new SCORE chart to predict AOEs. The secondary endpoint was to report the management of dyslipidemia in the clinical practice. Methods. We identified 233 adult patients with CML who were treated in 20 Italian centers with nilotinib. All patients were stratified into low to moderate (SCORE ≤ 5%) or high to very high (SCORE risk >5%) CV risk, according to the new version of the SCORE 2019. We recorded concentration levels of cholesterol, high-density lipoproteins (HDL), low-density lipoproteins (LDL) and triglycerides at diagnosis of CML, before starting ponatinib and therefore after 3, 6 and 12 months of treatment. All AOEs (cerebrovascular, peripheral vascular and CV events excluding hypertension) were considered. Results. The median age was 50 years (range 20-88) and the Sokal score was intermediate-high in 45.5% of patients. The median follow-up was 5 years (range 3.4-10.5). Nilotinib was administered as first line of therapy in (72%) of cases or second or subsequent lines of treatment for inefficacy (20.9%) or intolerance (7.1%). At baseline, nilotinib was administered at the following doses: 800 mg/day in 9.3% of patients, 600 mg/day in 87% of patients, 400 mg/day in 3.1% of patients and 300mg in 0.6% of patients, respectively. The median time of drug exposure was 60 months (range 2-155). The 48-month cumulative incidence rate of AOEs was 14.1±2.7%. Patients with cholesterol plasma levels > 200 mg/dL and LDL >70 mg/dL at baseline and 3 months after starting nilotinib, showed a significantly higher incidence of AOEs (24.5±7.3% vs 11±2.7%, P=0.02 and 22.3±4.9% vs 5.9±2.6, P=0.003, respectively) Figure 1. Patients with triglycerides levels > 200 mg/dL 3 months after starting nilotinib, showed a significantly higher incidence of AOEs (56±20.5% vs 13.3±2.7%, P=0.011) Patients belonging to the high and very high SCORE risk group showed a significant increase of AOEs (32.8.1±9% vs. 9±1%±2.6%, p=0.001). In multivariate analysis, statistical significance of cholesterol plasma levels > 200 mg/dL and LDL >70 mg/dL after 3 months and high-very-high SCORE was maintained (P=0.018, HR=3.4, 95% CI=1.2-9.4 and P=0.004, HR=3.5, 95% CI=1.5-8.2, respectively). Overall, 46 patients (20.5%) presented dyslipidemia at CML diagnosis and 65 (29%) at the start of treatment with nilotinib. Despite dyslipidemia, only 6 patients were taking statins during the treatment with nilotinib and only 5 started it after 3 months of nilotinib: 3 patients were treated with rosuvastatin and 2 with pravastatin. Conclusions. Our findings suggest that a proper control of dyslipidemia, keeping cholesterol and triglycerides plasma levels ≤ 200 mg/dL and LDL ≤70 mg/dL is associated with reduced risk of AOEs in CML patients treated with nilotinib. An under estimation of the clinical importance of elevated plasma lipids as a risk factor for AOEs events represents a possible issue in the real-life. Figure 1 Disclosures Abruzzese: Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bms: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Galimberti:Incyte: Honoraria; Novartis: Speakers Bureau. Castagnetti:Novartis: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria. Pregno:Incyte-Italy,: Membership on an entity's Board of Directors or advisory committees, Other: conference reports; Novartis-Italy: Membership on an entity's Board of Directors or advisory committees, Other: conference reports; Pfizer-Italy: Membership on an entity's Board of Directors or advisory committees, Other: conference reports. Bocchia:CELGENE: Honoraria; Incyte: Honoraria. Gugliotta:Novartis: Honoraria; Incyte: Honoraria; Pfizer: Honoraria. Foà:Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees; Incyte: Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees.

H3K36 tri-methylation by SETD2 has been linked to a plethora of pathways critical for the regulation of a multitude of cellular processes, including transcription, DNA replication and DNA repair after damage, yet the functional implications of perturbed H3K36 tri-methylation by SETD2 mutations or non-genomic loss of function in leukemia are still unclear. We have previously reported that the HMC-1.1 and -1.2 mast cell leukemia (MCL) cell lines and many patients (pts) with advanced systemic mastocytosis (advSM) display H3K36Me3 deficiency as a result of non-genomic loss of function of SETD2. Proteasome inhibition restored SETD2 protein expression and H3K36me3, suggesting that a functional protein is produced but rapidly degraded. To understand the mechanisms underlying this phenomenon, we used an in silico approach to identify candidate SETD2-interacting proteins, followed by experimental confirmation by co-immunoprecipitation. We found that, after proteasomal inhibition, SETD2 co-immunoprecipitates with Aurora Kinase A (AKA). We also found that AKA was overexpressed and hyper-activated in pts with advSM compared to ISM pts and to a pool of healthy donors, and that AKA can phosphorylate SETD2. Both pharmacological inhibition by Danusertib and siRNA-mediated knock-down of AKA rescued SETD2 expression and activity, raising the hypothesis that phosphorylation by AKA might be implicated in proteasome-mediated degradation of SETD2. The new standard of therapy in advSM is midostaurin, that inhibits the activity of both wild‐type and D816V mutant KIT, as well as of various other kinases including AKA. Therefore we investigated if midostaurin effects may be addressed to AKA inhibition and consequent SETD2/H3K36me3 rescue. To this purpose, HMC-1 cells were treated with 5 µM midostaurin for 24 h and AKA, SETD2 and H3K36me3 expression were evaluated by western blotting. Treatment with midostaurin was able to inhibit AKA activity by about 60%, partially restoring SETD2 expression and H3K36Me3. Moreover, midostaurin treatment of HMC-1 cells at micromolar doses induced cytostatic but not cytotoxic effects as shown by cell growth curves performed in liquid medium and as confirmed by annexin V/PI staining and subsequent cytofluorimetric analysis of apoptotic cell death. Our observations in cell lines were confirmed in neoplastic mast cells collected from six patients with advSM before and after three months of midostaurin treatment. Western blotting showed that midostaurin treatment in vivo indeed results in a rescue of SEDT2 expression and activity, associated with a partial de-phosphorylation of AKA. Our subsequent experimental step was therefore to hypothesize and test the possibility of a combined treatment between midostaurin and second generation TKIs to induce not only a cytostatic but also a cytotoxic effect both in our in vitro models and in primary cells obtained from bone marrow samples of advSM patients. We performed growth curves in liquid medium and clonogenic assays to evaluate the therapeutic potential of pharmacological combination of midostaurin with Nilotinib and Dasatinib in HMC-1 cells and in neoplastic mast cells from 3 patients with advSM and we observed in all cases synergistic effects at nanomolar doses. Moreover, cytofluorimetric analysis of apoptotic cell death in HMC-1 cells showed an important advantage in using the combination of the two drugs, compared to single agents, underlined by the significant reduction of drug doses used to obtain cytotoxic effects (Figure 1). Our results suggest that AKA-mediated post-translational modifications contribute to SETD2 non-genomic loss of function in advSM. Inhibiting AKA and c-Kit activity by midostaurin in combination with a second generation TKI is a promising therapeutic strategy in patients with SETD2/H3K36Me3 deficiency. Acknowledgments: study supported by AIRC (project code 16996) and AIL (Associazione Italiana contro le Leucemia, Linfomi e Mieloma). Disclosures Papayannidis: Incyte: Honoraria; Shire: Honoraria; Novartis: Honoraria; Teva: Honoraria; Amgen: Honoraria; Pfizer: Honoraria. Bonifacio:Pfizer: Honoraria; Amgen: Honoraria; Novartis: Honoraria; Incyte: Honoraria; BMS: Honoraria. Albano:Incyte: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Elena:Novartis: Consultancy; Pfizer: Consultancy. Valent:Deciphera: Honoraria, Research Funding; Blueprint: Research Funding; Pfizer: Honoraria; Celgene: Honoraria; Novartis: Consultancy, Honoraria, Research Funding. Cavo: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, Speakers Bureau; bms: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; novartis: Honoraria; takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Soverini:Incyte: Consultancy.

Background: Polycythemia vera (PV) and essential thrombocythemia (ET) are myeloproliferative neoplasms that can progress to post-PV (PPV) myelofibrosis (MF) and post-ET (PET) MF, also known as secondary myelofibrosis (SMF). The 2009 International Working Group for Myelofibrosis Research and Treatment group (IWG-MRT) diagnostic criteria for SMF require the presence of grade 2 or 3 bone marrow fibrosis (BMF) according to the European classification. The prognostic value of BMF in primary MF has been recently explored. Aim of this study is to describe the relevance of BMF grading in terms of presentation and outcome in SMF. Methods: The MYSEC (Myelofibrosis Secondary to PV and ET Collaboration) project is an international retrospective collaboration that collected 805 SMF cases. BMF grade at time of SMF evolution was defined as grade 2 (BMF2) in case of a diffuse increase in reticulin with extensive intersections, occasional bundles of collagen and/or osteosclerosis, or as grade 3 (BMF3) if presenting with coarse bundles of collagen, often associated with osteosclerosis. Chi-square or Fisher exact test for categorical variables, Wilcoxon rank-sum test for continuous ones and multiple logistic regression analysis were applied in order to explore pattern of association between BMF grading and patients' characteristics. A Poisson regression model was applied to estimate incidence rate of events, together with 95% Confidence Interval (CI). Survival was estimated using the Kaplan-Meier method. Differences between BMF groups were compared by a Log-rank test. The MYSEC study was approved by the Review Board of each Institution and conducted in accordance with the Declaration of Helsinki. Results: Detailed information about BMF grade was available in 675 patients: BMF2 was reported in 443 (65.6%) and BMF3 in 232 (34.4%). We did not find any correlation between BMF grade and advanced age, marrow blasts percentage, spleen and liver size, presence of constitutional symptoms, abnormal karyotype at the time of SMF diagnosis. No imbalance was evident for gender and for time to progression from PV/ET to SMF. Looking at genotype, BMF grading was not differently distributed among the 3 phenotypic driver mutations as well as the "triple negative" cases. On the contrary, in univariate analysis we found a significant association between BMF3 and previous diagnosis of ET vs. PV, decreased hemoglobin levels, reduced platelet and leukocyte counts, higher percentage of circulating blast cells and higher LDH value (Table 1). In a multivariate analysis that took into consideration SMF subtypes, complete blood count and circulating blast cells in 437 SMF patients, PET-MF, lower hemoglobin levels and reduced platelet count maintained their association with BMF3 (Table 2). The cumulative incidence of thrombosis after SMF was 3.2% (CI 95%: 2.4-4.2) person-year of follow up (PYFU) in BMF2, equivalent to BMF3 (2.7% PYFU, CI 95%: 1.2-5.7) (P = 0.44). Besides, BMF grade did not impact on the cumulative incidence of post-SMF blast phase, which resulted 2.4% (CI 95%: 1.8-3.3) PYFU in case of BMF2 and 3% (CI 95%: 1.3-6.9) PYFU in the BMF3 cohort (P = 0.68). Overall, MYSEC-PM (MYSEC-Prognostic Model) risk categories were differently distributed between BMF grades: patients with BMF2 were more frequently included in lower risk groups, while those with BMF3 were enriched in the higher categories (Table 1). Finally, we found a significantly higher mortality in patients with BMF3 vs. BMF2, mainly due to SMF progression (Table 1). Figure 1 shows the Kaplan-Meier estimate of survival in the 2 populations, resulting significantly different. Nevertheless, when considering the MYSEC-PM score in a multivariate analysis, BMF grade lost its prognostic impact on survival. Conclusions: This study provides evidence that BMF grade does correlate with specific clinical phenotype in SMF. BMF3 was associated with a more "cytopenic" phenotype (lower hemoglobin levels and reduced platelet count) and clustered with higher MYSEC-PM scores. Survival is significantly reduced in patients with BMF3. In conclusion, irrespectively of the PV/ET duration, progressive anemia and thrombocytopenia imply more frequently BMF3, finally resulting in a worse survival. This highlights the need of performing bone marrow biopsy earlier in disease evolution. Disclosures Rumi: novartis: Honoraria, Research Funding. Rambaldi:Omeros: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau, travel support; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Speakers Bureau; Italfarmaco: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding, Speakers Bureau. Komrokji:Incyte: Consultancy; pfizer: Consultancy; JAZZ: Speakers Bureau; celgene: Consultancy; JAZZ: Consultancy; Novartis: Speakers Bureau; Agios: Consultancy; DSI: Consultancy. Gotlib:Deceiphera: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Research Funding; Promedior: Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Blueprint Medicines: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Allakos: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Kiladjian:Novartis: Honoraria, Research Funding; AOP Orphan: Honoraria, Research Funding; Celgene: Consultancy. Cervantes:Novartis: Honoraria, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. Palandri:Novartis: Consultancy, Honoraria. Silver:PharmEssentia: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Benevolo:Novartis Pharmaceuticals: Consultancy. Albano:Incyte: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Iurlo:Novartis: Other: Speaker Honoraria; Incyte: Other: Speaker Honoraria; Pfizer: Other: Speaker Honoraria. Vannucchi:Incyte: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; Italfarmaco: Membership on an entity's Board of Directors or advisory committees; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Passamonti:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Celgene Corporation: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

BACKGROUND: In chronic phase (CP) chronic myeloid leukemia (CML) nilotinib (NIL) showed better efficacy compared to imatinib. The higher rates of deep molecular response with NIL may translate into more patients (pts) eligible for treatment discontinuation. On the other hand, cardiovascular toxicity may limit NIL use in selected groups of pts (e.g. elderly pts). Long follow-up is required for a proper evaluation of the benefit-risk ratio between efficacy (including treatment-free remission [TFR] rates) and toxicity. However, only few data are currently available on the very long-term outcome of NIL treated pts. AIM: To describe the very-long term outcome of patients treated with nilotinib first-line. METHODS: The GIMEMA CML WP initiated in 2007 a phase II study (NCT 00481052) with NIL first-line (400 mg BID). Seventy-three adult (≥18 years old) pts with newly diagnosed, CP-CML were enrolled at 18 GIMEMA Centers in Italy. Median age was 51 (18-83) years. ELTS risk score was low in 64.4% of pts, intermediate in 30.1% of pts, and high in 5.5% of pts. Early and mid-term results have been previously published. Here, we report the very long-term outcome of this study, focusing on survival measures, TFR rates and adverse events. The updated, final results (with 10-year follow-up) of the trial will be available for presentation at the meeting. RESULTS: After a median follow-up of 92 months, 70 (95.9%) pts are alive and progression-free. Three pts (4.1%) died (progression to blast phase, n=1; CML-unrelated death, n=2). Failures according to ELN 2013 recommendation occurred in 5 (6.8%) pts (progression to BP, n=1; BCR-ABL transcript level > 1% at 12 months, n=2; confirmed loss of major molecular response, n=2). At last contact, 45 (61.6%) pts were still on NIL (dose ≥ 600 mg/day in 29 pts; 400 or 450 mg/day in 12 pts; ≤ 300 mg in 4 pts). Reasons for permanent NIL discontinuation in the remaining 28 (38.4%) pts were: adverse events (n=17, 23.3%; two of these pts maintained a deep molecular response without treatment - TFR); successful TFR attempts (n=9, 12.3%); failures (n=2 pts, 2.7%). The median age at CML diagnosis of pts obtaining the TFR was 47 (21-70) years. Fifteen athero-thrombotic events (ATEs) occurred in 13 (17.8%) pts (peripheral arterial occlusive disease, n=5; acute myocardial infarction, n=5; others ATEs, n=5), after a total NIL exposure of 478 years (3.1 events/100 pt-years). The median age at CML diagnosis of these pts was 66 (43 - 83) years; 8/13 (61.5%) pts had at least a baseline cardiovascular risk factor (hypertension, diabetes, hypercholesterolemia, or BMI ≥ 30). SUMMARY: These data highlight the efficacy of first-line NIL in the long-term, with excellent overall survival. TFR was obtained in 15% of pts, and particularly in younger ones. Some safety concerns remain, with ATEs occurring in 17.8% of pts (mainly in elderly ones), although none of these events was fatal. Disclosures Gugliotta: Incyte: Honoraria; Pfizer: Honoraria; Novartis: Honoraria. Castagnetti:Incyte: Honoraria; Pfizer: Honoraria; Bristol Myers Squiib: Consultancy, Honoraria; Novartis: Honoraria. Breccia:Novartis: Honoraria; Incyte: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Celgene: Honoraria. Levato:BMS: Honoraria; Incyte: Honoraria; Pfizer: Honoraria; Novartis: Honoraria. Stagno:Pfizer: Honoraria; Novartis: Honoraria; BMS: Honoraria; Incyte: Honoraria. Tiribelli:Incyte: 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; Pfizer: Membership on an entity's Board of Directors or advisory committees. Bocchia:Incyte: Honoraria; BMS: Honoraria; Novartis: Honoraria. Cavazzini:Novartis: Honoraria; Incyte: Honoraria; Pfize: Honoraria. Caocci:Novartis: Honoraria; Celgene: Honoraria. Albano:Incyte: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Martinelli:Ariad: Consultancy, Other: trial grant; Amgen: Consultancy, Other: trial grant; Novartis: Consultancy, Other: trial grant; Pfizer: Consultancy, Other: trial grant; Janssen: Consultancy, Other: trial grant; Abbvie: Consultancy, Honoraria, Other: trial grant; Incyte: Consultancy, Other: trial grant; Daiichi Sankyo: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Other: trial grant; Roche: Consultancy, Other: trial grant. Soverini:Incyte: Consultancy. Foà:Abbvie: Consultancy, Speakers Bureau; Celltrion: Membership on an entity's Board of Directors or advisory committees; Amgen Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Consultancy, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celltrion: Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Pane:GSK: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: research founding; BMS: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. Cavo: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, Speakers Bureau; bms: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; novartis: Honoraria; takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Saglio:Celgene: Consultancy; BMS: Consultancy; Novartis: Consultancy; Ariad: Consultancy; Incyte: Consultancy; Pfizer: Consultancy; Jansen: Consultancy. Baccarani:Novartis: Consultancy, Speakers Bureau; Incyte: Consultancy, Speakers Bureau; Takeda: Consultancy. Rosti:BMS: Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

One of the hallmarks of chronic myeloid leukemia (CML) is genomic instability, that fosters the acquisition of tyrosine kinase inhibitor (TKI)-resistant BCR-ABL1 mutations and/or of additional chromosomal aberrations leading to progression to blast crisis (BC). Inactivating mutations in the SETD2 tumor suppressor occur in solid tumors and acute leukemias. SETD2 trimethylates histone H3 Lysine 36 (H3K36Me3) playing a key role in maintaining DNA integrity. We have recently demonstrated that, in CML, SETD2 loss of function may occur at the post-translational level. Reduced or null SETD2 and H3K36Me3 was detected in 83/96 (86%) patients (pts) with BC CML as compared to a pool of healthy donors and to chronic phase (CP) pts at diagnosis. Proteasome inhibition in primary cells from pts with undetectable SETD2 restored H3K36Me3 and led to accumulation of hyper-ubiquitinated SETD2. In K562 cells (SETD2/H3K36Me3low), we observed that after proteasome inhibition hyper-ubiquitinated SETD2 co-immunoprecipitates with MDM2. MDM2 inhibition rescued SETD2 expression and activity, suggesting that MDM2 is implicated in SETD2 reduced stability. Co-IP also showed that SETD2 interacts with Aurora Kinase A (AKA) a S/T kinase frequently overexpressed in CML. We found that AKA phosphorylates SETD2, and its inhibition rescued SETD2 expression and activity. To investigate whether SETD2/H3K36Me3 loss may be a druggable lesion, we performed clonogenic assays in LAMA84 (SETD2/H3K36Me3high) cells before and after SETD2 silencing, in imatinib-sensitive K562 (SETD2/H3K36Me3low) cells and in IM-resistant K562 cells, that are characterized by complete SETD2 loss. The extent of reduction of clonogenic growth after proteasomal, AKA or MDM2 inhibition was found to be inversely correlated to SETD2 residual expression. These observations were confirmed in cells from both CP (n=2) and BC (n=4) CML pts showing different levels of SETD2 expression and activity. Further experiments were performed in the aforementioned cell lines to verify if reduced clonogenic potential was due to cytostatic or cytotoxic effects. Apoptotic cell death was quantified by annexin V/propidium iodide staining and flow cytometry. Proteasomal inhibition by bortezomib, carfilzomib and ixazomib and AKA de-phosphorylation by Danusertib caused a time-dependent increase of annexin-V-positive cells by activating the mitochondrial apoptotic pathway as reflected by an increase in Bax expression and induction of the cleavage of caspase-3,-9 and PARP. Moreover, all drug treatments as single agent, at nanomolar doses (Bortezomib: 10 nM, Carfilzomib: 5 nM, Ixazomib: 10 nM and Danusertib: 500 nM) induced a significant increase of the DNA double-strand break marker γH2AX, suggesting that in a SETD2 knock-down context, proteasomal and AKA inhibition propagates genomic instability by forcing the cells through successive replication cycles, ultimately resulting in apoptosis from mitotic catastrophe. Reduced SETD2/H3K36Me3 levels, in association with MDM2 and AKA hyper-activation, were also detected when the CD34+ cell fraction of 10 CML-CP pts, was compared to the total mononuclear cell fraction or to the CD34+ compartment obtained from a pool of healthy donors. We thus hypothesized that leukemia progenitor cells, showing higher MDM2 and AKA activity and consequent SETD2 loss, accumulate genetic aberrations despite inhibition of BCR-ABL1 kinase. Studies are ongoing to verify if MDM2 or AKA inhibition may restore SETD2 expression and function and induce cell death. Finally, it has already been shown that alterations of epigenetic regulators such as the KDM4 family members control tumor cell proliferation in a variety of cancers including acute myeloid leukemia. Recent findings have identified KDM4 demethylases as putative therapeutic targets in a SETD2 mutated context and illustrated the efficacy of KDM4 inhibitors in AML therapy. Starting from these evidences, we will test the same approach in BC CML models. In conclusion, phosphorylation by AKA and ubiquitination by MDM2 contribute to SETD2 non-genomic loss of function in BC CML and in CD34+ leukemic progenitors. Restoring physiological H3K36Me3 may help to improve the outcome of this critical subset of pts. Acknowledgments: Study supported by AIRC (project code 16996), AIL (Associazione Italiana contro le Leucemia, Linfomi e Mieloma), Italian Ministry of Health, project GR-2016-02364880. Disclosures Gugliotta: Pfizer: Honoraria; Novartis: Honoraria; Incyte: Honoraria. Castagnetti:Novartis: Honoraria; Incyte: Honoraria; Pfizer: Honoraria; Bristol Myers Squiib: Consultancy, Honoraria. Rosti:BMS: Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Iurlo:Incyte: Other: Speaker Honoraria; Novartis: Other: Speaker Honoraria; Pfizer: Other: Speaker Honoraria. Abruzzese:Incyte: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; BMS: Consultancy. Pregno:Incyte: Consultancy, Honoraria; Pfizer: Honoraria; Novartis: Honoraria; Bristol Myers Squibb: Honoraria. Crugnola:Novartis: Honoraria; Incyte: Honoraria. Albano:Novartis: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees. Bonifacio:Novartis: Honoraria; Amgen: Honoraria; Pfizer: Honoraria; Incyte: Honoraria; BMS: Honoraria. Tiribelli:Pfizer: 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; Incyte: Membership on an entity's Board of Directors or advisory committees. Baccarani:Novartis: Consultancy, Speakers Bureau; Incyte: Consultancy, Speakers Bureau; Takeda: Consultancy. Martinelli:Roche: Consultancy; Pfizer: Consultancy; BMS: Consultancy; Novartis: Consultancy; ARIAD: Consultancy. Cavo:amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; novartis: Honoraria; takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; bms: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau. Soverini:Incyte: Consultancy.

Background and Rationale. Bosutinib (BOS), dasatinib (DAS) and nilotinib (NIL) are 2nd generation TKIs with similar second-line efficacy. The use of DAS and NIL may be burdened by pulmonary, infectious, cardiovascular and metabolic complications; these complications are more frequent and more clinically relevant in the elderly. BOS could represent an important therapeutic option in elderly patients intolerant to or failing a first-line TKI, but the dose of 500 mg OAD may be higher than necessary. Aims. All TKIs have been tested at a fixed initial dose, with dose adjustment in case of toxicity or treatment failure. On the contrary, the aim of our study was to evaluate in elderly CML patients if second-line BOS was effective and better tolerated at doses lower than 500 mg OAD, beginning with 200 mg OAD, then increasing the dose to 300 OAD or 400 mg OAD according to the molecular response, to find the minimum effective dose. Methods. A prospective phase 2 single-arm multicenter study has been designed by the GIMEMA CML Working Party (NCT02810990). Study design: all patients started with 200 mg OAD for 2 weeks ("run-in" period), then the dose was increased to 300 mg OAD; after 3 months, patients with BCR-ABLIS transcript ≤ 1% continued 300 mg OAD, while in patients with transcript > 1% the dose is furtherly increased to 400 mg OAD. In responsive patients, BOS dose was maintained, 300 mg or 400 mg OAD. Key inclusion criteria: > 60 yrs old, chronic phase CML, intolerance or failure of any first-line TKI (2013 ELN criteria), absence of T315I or V299L mutation. Sixty-three patients have been enrolled. The primary endpoint was the proportion of patients in MR3 at 1 year. Definitions: MR3, BCR-ABLIS < 0.1%; MR4, BCR-ABLIS < 0.01% with > 10.000 copies; MR4.5, BCR-ABLIS < 0.0032% with > 32.000 copies. Results. Median age: 73 yrs (range 60-90). Age distribution: 60-69 yrs, 18 pts (29%); 70-79 yrs, 31 pts (49%); > 80 yrs, 14 pts (22%). Sokal score at diagnosis: low 19%, intermediate 49%, high 32%. Reasons for switching to BOS: intolerance 63%, resistance 37%. First-line TKI: imatinib 83%, DAS 11%, NIL 6% (same TKI distribution in intolerant and resistant patients). Median follow-up: 9 mos (range 1-30). Overall, 10/63 patients had a dose-increase to 400 mg OAD, 49/63 to 300 mg OAD, while 4/63 continued on BOS 200 mg OAD without any dose increase. At baseline, 13 patients were already in MR3. The MR3 rates by 3 and 6 months were 43% and 56%, respectively. The cumulative rate of patients achieving or maintaining a MR3 by 12 months was 60% (65% in intolerant and 52% in resistant patients, p = 0.31). Interestingly, only 21% of patients > 80 yrs old achieved or maintained a MR3 (p < 0.001, compared to younger patients). Patients achieving MR4 or MR4.5 were 38% and 19%, respectively. Overall, 22%, 27% and 11% of patients had 1 log, 2 logs or > 3 logs reduction from baseline BCR-ABLIS transcript level. Selected adverse events: cardiac ischemia, 2 patients; pericardial effusion, 2 patients; no pleural effusions. Events leading to permanent treatment discontinuation: 2 unrelated deaths, 7 adverse events (3 hypertransaminasemia, 1 nephrotoxicity, 1 diarrhea, 1 skin rash, 1 myalgia/fatigue), 3 unsatisfactory responses (without progressions). Fifty-one out of 63 patients are still on BOS at the last contact: 6 on 400 mg OAD, 34 on 300 mg OAD, 11 on 200 mg OAD. Conclusions. A gradual dose increase, based on prospective molecular monitoring, allowed the great majority of enrolled patients (approximately 70%) to remain on treatment with BOS 300 mg OAD or less, achieving a major molecular response (MR3) in 60% of the cases. These results trial showed that, in elderly patients intolerant to or failing a first-line TKI, BOS may be highly effective and better tolerated at a dose lower than 500 mg OAD, namely at 300 mg OAD. Disclosures Castagnetti: Pfizer: Honoraria; Novartis: Honoraria; Incyte: Honoraria; Bristol Myers Squiib: Consultancy, Honoraria. Gugliotta:Pfizer: Honoraria; Incyte: Honoraria; Novartis: Honoraria. Bocchia:Novartis: Honoraria; Incyte: Honoraria; BMS: Honoraria. Trawinska:Novartis: Consultancy, Honoraria. Bonifacio:Novartis: Honoraria, Research Funding; Amgen: Honoraria; Pfizer: Honoraria; Incyte: Honoraria. Crugnola:Novartis: Honoraria; Incyte: Honoraria. Elena:Novartis: Consultancy; Pfizer: Consultancy. Lucchesi:Novartis: Honoraria; Incyte: Honoraria; Pfizer: Honoraria. Albano:Incyte: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Lunghi:Novartis: Honoraria; Incyte: Honoraria; Pfizer: Honoraria. Stagno:BMS: Honoraria; Incyte: Honoraria; Pfizer: Honoraria; Novartis: Honoraria. Pregno:Pfizer: Honoraria; Novartis: Honoraria; Bristol Myers Squibb: Honoraria; Incyte: Consultancy, Honoraria. Iurlo:Novartis: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Incyte: Honoraria. Ferrero:Novartis: Honoraria. Cavo:janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; novartis: Honoraria; takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; bms: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau. Saglio:BMS: Consultancy; Novartis: Consultancy; Ariad: Consultancy; Pfizer: Consultancy; Celgene: Consultancy; Jansen: Consultancy; Incyte: Consultancy. Pane:Novartis: Membership on an entity's Board of Directors or advisory committees, Other: research founding; Janssen: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Baccarani:Novartis: Consultancy, Speakers Bureau; Incyte: Consultancy, Speakers Bureau; Takeda: Consultancy. Rosti:BMS: Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. OffLabel Disclosure: Bosutinib is a second generation TKI already registered for the treatment of adult patients with chronic phase CML with resistance or intolerance to prior therapy. In the present study the initial dose is 200 mg instead of 500 mg OAD. A dose increase to 300 or 400 mg is scheduled according to molecular response and tolerabiliy

Background. Ponatinib is a third-generation tyrosine kinase inhibitor indicated for adult patients with resistant or intolerant chronic phase (CP), accelerated phase (AP) or blast phase (BP) chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia, or for those carrying the T315I mutation. In the real-world setting, there is a paucity of data regarding the use of ponatinib. The goal of the Observational study of Iclusig® (ponatinib) Treatment in patients with CML in Italy (OITI) is to evaluate treatment patterns and outcomes, including the safety and efficacy of ponatinib, in patients with CML treated in hematology centers in Italy since January 2015, when ponatinib became commercially available. Methods. This ongoing, non-interventional study includes patients aged ≥18 years with CP, AP or BP CML who initiated ponatinib treatment in routine clinical practice across 40 centers (academic and hospital settings) as of October 2018. The study population consists of a prospective cohort, including patients who started treatment with ponatinib after site activation during the ongoing enrolment period; a retrospective cohort, including patients who started treatment with ponatinib but who died or were lost to follow-up prior to site activation; and a retrospective/prospective cohort, including patients who started treatment with ponatinib prior to site activation and are still on treatment. Demographic, efficacy and safety data are collected from patient medical charts at study entry and at routine care visits. The primary endpoint is complete cytogenetic response (CCyR) rate in patients with CP CML 6 months after starting ponatinib treatment. Here, the first interim analysis after ≥6 months' observation is presented for the retrospective and retrospective/prospective cohorts. Results. At time of data analysis (02 July 2019), 56 patients (53 CP, 1 AP and 2 BP CML) had been enrolled across 21 Italian centers. Twenty-eight (50.0%) patients had received ponatinib as second-line (2L) treatment and 33.9% received ponatinib in third-line (3L). Twenty (35.7%) patients had a history of cardiovascular events and 23 (41.1%) had a history of hypertension. Among patients with CP, AP and BP CML, median age at study entry was 59.1, 33.7 and 48.5 years, respectively; among 37 evaluable patients, 12 (32.4%), 1 (2.7%) and 1 (2.7%) patient(s) had a confirmed BCR-ABL1 mutation. Of evaluable patients, 4 (10.8%) had the T315I mutation. The starting dose of ponatinib for patients with CP CML was 45 mg once daily in 41.5% of patients, 30 mg in 39.6% of patients and 15 mg in 17.0% of patients; 1 (1.9%) patient started ponatinib at another dose. Median treatment duration was 23.9 months (range, 3.3-49.9 months) at the time of analysis. At Month 6, 88.6% of patients with CP CML achieved a CCyR. Additionally, 37.5% and 15.0% of evaluable patients with CP CML achieved a major molecular response (MMR; MR3.0) and a deep molecular response (MR4.5), respectively (Table 1). Estimated progression-free survival rates for patients with CP CML at Months 12 and 24 were 86.6% (95% CI, 77.8-96.4%) and 83.7% (95% CI, 73.8-94.9%), respectively. Corresponding overall survival rates were 96.2% (95% CI, 91.1-100.0%) and 93.1% (95% CI, 85.6-100.0%), respectively. Thirty (53.6%) patients had treatment-related adverse events (TRAE). The most frequent TRAEs were hypertension (n=6), skin lesion (n=2), increased lipase (n=2), rash (n=2) and pain in extremity (n=2). The only hematologic TRAE reported was thrombocytopenia (n=1). Dose interruptions occurred in 13 patients: for TRAEs (n=5, 38.5%), medical decision (n=4, 30.8%) or other causes (n=4, 30.8%; comprising death in 3 cases and 1 attempt at treatment-free remission). Conclusions. Data show that ponatinib has a favorable efficacy and safety profile in patients with CML treated in standard clinical practice in Italy. By Month 6, most patients had achieved CCyR and 44% of patients achieved MMR in 2/3L. Furthermore, the probability of survival at 2 years was more than 90%. No new safety signals emerged with ponatinib treatment than those previously reported. The early use of ponatinib (84% of patients received it as 2/3L treatment) as well as careful dose selection appear key to the safety and efficacy outcomes observed in this preliminary study evaluation. Disclosures Iurlo: Incyte: Other: Speaker Honoraria; Pfizer: Other: Speaker Honoraria; Novartis: Other: Speaker Honoraria. Annunziata:Pfizer: Consultancy; Incyte: Consultancy; Novartis: Consultancy. Albano:Incyte: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Spadano:Incyte: Speakers Bureau; Pfizer: Speakers Bureau. Bonifacio:BMS: Honoraria; Amgen: Honoraria; Pfizer: Honoraria; Incyte: Honoraria; Novartis: Honoraria. Abruzzese:BMS: Consultancy; Incyte: Consultancy; Novartis: Consultancy; Pfizer: Consultancy. Pellegrino:Inycte Biosciences Italy S.R.L: Employment. Galimberti:Inycte Biosciences Italy S.R.L: Employment. Foà:Celltrion: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Consultancy, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celltrion: Membership on an entity's Board of Directors or advisory committees. Breccia:Celgene: Honoraria; BMS: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Incyte: Honoraria.

The SETD2 protein is a histone methyltransferase that specifically catalyzes the trimethylation of Lysine 36 on histone H3 (H3K36me3). SETD2/H3K36me3 are implicated in transcript elongation and splicing, DNA repair, chromosome segregation. SETD2 gene deletions and/or mutations (mostly frameshift or nonsense) have been reported in solid tumors (clear cell renal cell carcinoma, bladder cancer, lung cancer, melanoma, endometrial cancer) and in acute leukemias. Using a Western Blotting (WB) approach to screen for SETD2 protein expression and for H3K36me3 levels in a relatively large cohort of 80 advanced-phase chronic myeloid leukemia (CML) patients (pts), we could detect reduced or null SETD2 and H3K36me3 in 86% of pts as compared to a pool of healthy donors and to chronic phase (CP) pts at diagnosis who achieved optimal responses to TKI, but neither mutations/deletions nor transcriptional down-regulation were the underlying causes. Inhibition of proteasome-mediated degradation in primary cells from pts with undetectable SETD2 restored H3K36me3 and led to accumulation of hyper-ubiquitinated SETD2, suggesting that a functional protein is produced but rapidly degraded. Moreover, proteasome inhibition was found to induce apoptosis and to reduce clonogenic growth. In K562 cells (SETD2/H3K36me3low), co-immunoprecipitation (co-IP) performed before and after proteasome inhibition showed accumulation of the hyper-ubiquitinated form of SETD2 bound to MDM2. MDM2 inhibition by SP-141 resulted in cytostatic effects and restored SETD2 expression and activity. Superimposable results were achieved by siRNA-mediated silencing of MDM2, suggesting that MDM2 is implicated in SETD2 reduced stability. Co-IP also showed that SETD2 interacts with Aurora Kinase A a Ser-Thr kinase frequently overexpressed in CML. We found that Aurora Kinase A phosphorylates SETD2, and both pharmacological inhibition by Danusertib and siRNA-mediated silencing rescued SETD2 expression and activity. Next, to investigate whether SETD2/H3K36me3 loss may contribute to genetic instability, LAMA 84 (SETD2/H3K36Me3high) and K562 (SETD2/H3K36me3low) cells were studied by WB and immunofluorescence (IF) to assess phosphorylated histone 2A.X (γH2AX) and Rad51 foci in steady state conditions and after sub-lethal DNA damage by UV exposure. The same studies were performed after SETD2 silencing for 3 months. Cells with low or silenced SETD2 had significantly higher levels of γH2AX and were unable to induce homologous recombination (HR) repair after DNA damage. Clonogenic assays performed in LAMA 84 cells before and after SETD2 silencing, in K562 (SETD2/H3K36me3low) and in imatinib-resistant (IM-R) K562 cells which have lost SETD2 expression and activity, suggested that reduction of clonogenic growth after proteasomal or MDM2 inhibition is strictly dependent on SETD2 expression and functional status (Figure 1A). First and second generation proteasome inhibitors (bortezomib, carfilzomib and ixazomib) inhibited the clonogenic potential of the mononuclear cell fraction from both CP (n=2) and blast crisis (BC) (n=4) CML pts at subnanomolar concentrations, with the extent of anti-tumor activity clearly anti-correlated with SETD2 expression and H3K36me3 levels: pts with lower SETD2 expression showed lower LD50 when compared with pts with higher SETD2 expression and H3K36me3 levels (Figure 1B). Similarly, clonogenic assays performed by administrating increasing doses of SP-141 (from 0.25 to 1.25 µM) suggested that MDM2 specific inhibition had more significant effects in BC-CML pts showing low SETD2 levels and activity as compared to BC-CML pts showing intermediate SETD2 levels and activity and to CP CML pts. In conclusion, phosphorylation by Aurora Kinase A and ubiquitination by MDM2 contribute to SETD2 non-genomic loss of function in advanced-phase CML. Loss of SETD2/H3K36me3 is associated with increased DNA damage and impaired HR repair. Restoring physiological H3K36me3 levels may help improve the outcome of this critical subset of pts. Acknowledgments: study supported by AIRC (project code 16996) and AIL (Associazione Italiana contro le Leucemia, Linfomi e Mieloma). Figure 1. Figure 1. Disclosures Castagnetti: Incyte: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Bristol Meyers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Gugliotta:Novartis: Honoraria; Pfizer: Honoraria; Bristol-Myers Squibb: Honoraria; Incyte: Honoraria. Abruzzese:Pfizer: Consultancy; Novartis: Consultancy; BMS: Consultancy; Ariad: Consultancy. Bonifacio:Incyte: Consultancy; Pfizer: Consultancy; Amgen: Consultancy; Novartis: Research Funding; Bristol Myers Squibb: Consultancy. Martinelli:Ariad/incyte: Consultancy; Pfizer: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Roche: Consultancy. Cavo:Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: 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; Takeda: 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; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Soverini:Bristol Myers Squibb: Consultancy; Incyte Biosciences: Consultancy; Novartis: Consultancy.

In Philadelphia-positive (Ph+) Acute Lymphoblastic Leukemia (ALL) patients (pts), resistance to tyrosine kinase inhibitors (TKIs) is frequently associated with the selection of one or more mutations in the BCR-ABL1 kinase domain (KD). The swift emergence of mutant clones as early as during induction therapy supports the hypothesis that, at least in some cases, mutations may already be present at diagnosis. Next Generaton Sequencing (NGS) has been proposed as an alternative to Sanger sequencing (seq) for BCR-ABL1 KD mutation screening because of its greater sensitivity and accuracy, but no studies have so far evaluated its prospective use in Ph+ ALL. Between 2015 and 2018, we have used NGS in parallel to Sanger seq to analyze a consecutive series of 126 Ph+ ALL pts who were newly diagnosed (n=39) or who had relapsed/refractory disease (n=87) on TKI therapy. In 22 cases, both bone marrow and peripheral blood were analyzed and compared. NGS of ≈400bp amplicons generated by nested RT-PCR was performed on a Roche GS Junior (until April 2017) or on an Illumina MiSeq (from May 2017 on). Read alignment and variant calling (with a lower limit set to 3%) were done with the AmpSuite software (SmartSeq srl). When multiple mutations mapped within the same sequence reads, assessment of cis vs trans configuration was done correcting for the probability of PCR recombination. Three out of 39 (7.7%) de novo Ph+ ALL pts had low burden point mutations detectable by NGS: one had a V289A (variant frequency, 3.4%); one had a D276G (4.0%) and a F359V (3.5%); one had an E255K mutation (3.3%). The first pt was enrolled in the GIMEMA LAL1811 study of frontline ponatinib; the second and the third pts were enrolled in the GIMEMA D-ALBA study of frontline sequential treatment with dasatinib and blinatumomab. All pts achieved molecular remission, consistently with the mutations being sensitive to the TKIs received. The 35INS insertion/truncation mutant was detected in 27 (69%) pts, who all have so far achieved molecular remission. This is in line with the report by O'Hare et al (Blood 2011) suggesting that the 35INS variant is kinase-inactive and does not contribute to TKI resistance. For this reason, the 35INS was excluded from subsequent analyses. Relapsed/refractory pts positive for mutations by Sanger seq were 57 (65%); those positive for mutations by NGS were 69 (79%). Fifty-six out of 87 (49%) pts had >1 mutation (up to 13) detected by NGS. NGS identified low burden mutations (i.e., mutations present in a proportion of transcripts between 3 and 20%) in 12 pts who were negative for mutations by Sanger seq. Most importantly, NGS provided a more accurate picture of BCR-ABL1 mutations status in 40 (46%) pts who turned out to have one or more low burden mutations in addition to the dominant mutation(s) detectable by Sanger seq. In all cases, each low burden mutation detected by NGS could be recognized as poorly sensitive either to the TKI the pt was receiving at the time of testing, or to the previous TKI. The clonal nature of NGS-based analysis further proved its utility i) in 4 pts where Sanger seq had shown 2 base substitutions in the same codon so that the actual amino-acid change(s) were impossible to infer (a ponatinib-resistant pt with a T315M mutation, 2 dasatinib-resistant pts with various combinations of F317I, F317C and/or F1317L, a dasatinib-resistant pt with 2 different nucleotide substitutions both leading to the V299L), and ii) in 48/56 pts who had ≥2 mutations whose clonal configuration could not be resolved. Twenty-eight out of these 48 pts were found to carry one or more (up to 3) compound mutants. Compound mutants were more common in pts who had failed ≥2 lines of therapy, whereas polyclonality was more common in pts who had failed first line therapy. The most frequent compound mutants were T315I+E255K and T315I+E255V. Interestingly, the latter was associated with poor or no response to ponatinib. Our results in a relatively large series of Ph+ ALL pts suggest that an NGS-based approach provides a more accurate characterization of the complexity of BCR-ABL1 KD mutation status, including compound mutants some of whom may be poorly sensitive even to ponatinib. Mutations may already be detected at the time of diagnosis. It remains to be assessed whether more sensitive techniques like digital PCR may identify a greater number of pts with pre-therapy mutations and whether the detection of pre-therapy mutations may be used to guide 1st-line treatment selection. Disclosures Soverini: Incyte Biosciences: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Consultancy. Pagano:Gilead: Speakers Bureau; Basilea: Speakers Bureau; Merck: Speakers Bureau; Janssen: Speakers Bureau; Pfizer: Speakers Bureau. Abruzzese:Ariad: Consultancy; BMS: Consultancy; Novartis: Consultancy; Pfizer: Consultancy. Martinelli:Roche: Consultancy; Celgene: Consultancy, Speakers Bureau; Jazz Pharmaceuticals: Consultancy; Pfizer: Consultancy, Speakers Bureau; Novartis: Speakers Bureau; Abbvie: Consultancy; Janssen: Consultancy; Ariad/Incyte: Consultancy; Amgen: Consultancy. Cavo:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: 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, Research Funding, Speakers Bureau; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: 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; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Background: The advent of Tyrosine Kinase Inhibitors (TKI) totally changed the outcome of patients affected by Chronic Myeloid Leukemia (CML). Most of informations about the clinical characteristics of CML patients are based on data derived from clinical trials, that often exclude patients not fitting with strict inclusion criteria. In real life may be important to know the characteristics of the entire CML patients' population and this is better reflected by registries studies including all consecutive patients. Aim: To provide a robust and updated information on clinical, hematologic characteristics and treatment response in non-selected Italian patients with CML in each phase of the disease. Methods:We retrospectively and prospectively recorder in a web-based database clinical, hematological, cytogenetic and biological informations about all new diagnosis of CML patients referred to Italian Hematology Centers of the GIMEMA Study Group from January 2012 to June 2016. Results:Our study covered 1051 patients newly diagnosed with CML and treated between January 2012 and June 2016 in 66 Italian centers. Median age at diagnosis was 59 years (range 47-71) and 60.8% were males. At diagnosis among 908 patients 899 (99%) patients were in chronic phase, 7 (0.8%) patients in accelerate phase and 2 (0.2%) in blastic crisis. Among 1051 patients, 148 (14%), 351 (33%), 318 (30.3%) were high, intermediate and low risk by Sokal with 234 (22.2%) missing; 54 (5%), 418 (40%), 342 (32.5%) high, intermediate and low risk by EURO with 237 (22.6%) missing; 55 (5%) and 797 (76%) high and low risk by EUTOS with 199 (18.9%) missing; 96 (9%), 208 (20%), 533 (51%) high, intermediate and low risk by ELTS with 214 (20.4%) missing, respectively. The median follow-up was 36 months and 36 patients died (10 CML related). At cytogenetic analysis, there were 887 available informations: 809 (77%) without additional cytogenetic aberrations (ACA), 49 (5%) with major route and 29 (2.8%) with minor route ACA respectively. BCR-ABL transcripts among 873 data available were: b2a2 in 303 (34.7%), b3a2 in 493 (56.5%), b2a2+b3a2 in 61 (7%), e1a2 in 12 (1.4%), e19a2 in 2 (0.2%) b3a2+e1a2 in 2 (0.2%) patients respectively. ECOG performance status among 801 patients was 0, 1, or ≥ 2 in 585 (73%), 181 (22.6%) and 35 (4.4%) cases respectively. According to co-morbidity and excluding CML diagnosis as parameter, among 995 cases the Charlson comorbidity index, was 0, 1, 2 or ≥3 in 771 (73.4%), 115 (11%), 60 (5.7%) and 49 (4.7%) patients respectively; among 556 patients cardiovascular, lung, metabolic and oncologic diseases were observed in 283 (24%), 108 (10.3%), 77 (7.3%) and 88 (8.4%) patients respectively. Five hundred-ten (48.5%) and 541 (51.4%) patients were treated in first-line with imatinib (IMA) and with II generation TKI (IIGen-TKI) respectively. Three hundred twenty-one (30%) cases were pretreated with hydroxyurea. Molecular responses data at 3th month were available in 728 patients and of these 102 (14.01%) obtained at least Major Molecular Response IS (MR) MR3, 29 (3.98%) MR4, 27 (3.71%) MR4.5, 13 (1.79%) MR5 and 11 (1.5%) undetectable BCR-ABL1transcript, respectively. At 6thmonth, among 731 data available, at least 292 patients (39,57%) obtained MR3, 90 (12,19%) MR4, 75(10.16%) MR4.5, 35 (4.74%) MR5 and 29 (3.97%) undetectable BCR-ABL1transcript, respectively. At 12thmonth among 709 molecular responses available, 425 patients (59.94%) achieved at least MR3, 203 (28,63%) MR4, 171 (24.12%) MR4.5, 90 (12.69%) MR5 and 78 (11%) undetectable BCR-ABL1transcript, respectively. As showed in table 1, the IIGen-TKI were able to obtain a higher, earlier and deeper molecular response at 3th, 6thand 12thmonth than IMA. Data analysis about responses at 24thmonth are ongoing. Overall survival at 5 years was 93.4% (95% IC 90.1-95.6). Conclusions:Our preliminary results of this observational epidemiologic study suggest that collection of clinical data of CML patients treated out of strictly clinical trials represent an essential tool for long/term treatment, able to observe setting strategies based on the clinical characteristics, the degree of response obtained, and the toxicity related to the therapy in overall CML population. We are planning to continue to analyze all these endpoints to estimate the response and toxicity according to ELN guidelines, and feasibility of treatment sequence in a cohort of patients treated in real-life. Disclosures Castagnetti: Pfizer: Consultancy, Honoraria; Bristol Meyers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Incyte: Consultancy, Honoraria. Breccia:BMS: Honoraria; Incyte: Honoraria; Pfizer: Honoraria; Novartis: Honoraria. Pane:AMGEN: Speakers Bureau; Novartis: Research Funding, Speakers Bureau; BMS: Speakers Bureau.

The SETD2 gene encodes the only methyltransferase responsible for histone H3 lysine 36 trimethylation (H3K36Me3) in humans. H3K36me3 play a key role in preserving the fidelity of transcription elongation and splicing. In addition, SETD2/H3K36me3 have more recently been implicated in the maintenance of genomic integrity by regulating homologous recombination (HR) repair, Mismatch Repair (MMR) mitotic spindle assembly and chromosome segregation. SETD2 deletions and/or inactivating mutations occur in many solid tumors and have recently been found also in acute leukemias. We have reported that the HMC-1.1 and -1.2 mast cell leukemia (MCL) cell lines and many advanced systemic mastocytosis (SM) patients (pts) display H3K36Me3 deficiency as a result of non-genomic loss of function of SETD2. Proteasome inhibition restored SETD2 protein expression and H3K36me3, suggesting that a functional protein is produced but rapidly degraded. In an attempt to uncover the mechanisms underlying this phenomenon, we used an in silico approach to identify candidate SETD2-interacting proteins, followed by experimental confirmation by co-immunoprecipitation (co-IP). We found that, after proteasomal inhibition, SETD2 co-immunoprecipitates with the ubiquitin E3 ligase MDM2. Treatment with the MDM2 inhibitor SP-141 rescued SETD2 expression and H3K36Me3, suggesting that MDM2 may play a role in SETD2 degradation in ASM and MCL. Moreover, SP-141 treatment of HMC-1 cells at micromolar doses induced cytostatic but not cytotoxic effects as shown by cell growth curves. Clonogenic assays supported the cytostatic effects of SP141 in HMC-1.1 and -1.2 cells. siRNA-mediated knock-down of MDM2 also rescued SETD2 expression and activity, further supporting the hypothesis that SETD2 hyper-ubiquitination by MDM2 plays a role in SETD2 reduced stability and proteasomal degradation. Co-IP also showed that SETD2 interacts with Aurora Kinase A, as it was suggested in silico. We found that Aurora A is overexpressed in advanced SM and may target SETD2 for phosphorylation. Both pharmacological inhibition by Danusertib and siRNA-mediated silencing of Aurora A rescued SETD2 expression and activity, raising the hypothesis that phosphorylation by Aurora A might be the trigger for MDM-2 mediated degradation of SETD2. To evaluate whether increased DNA damage and reduced HR proficiency can be observed in SETD2/H3K36Me3-deficient SM, we used western blotting (WB) and immunofluorescence (IF) to assess phosphorylated histone 2A.X (γH2AX) and Rad51 foci. Compared to cells from healthy controls, SETD2- and H3K36Me3-deficient cell lines and pts had significantly higher levels of γH2AX and lower levels of Rad51. RNA-seq in SETD2-deficient pts showed evidence of transcription and splicing defects like transcription-induced chimeras, intron retention and non-canonical splicing patterns not observed in healthy donors. Next, the ROSAD816V cell line, which displays SETD2 and H3K36me3 levels superimposable to healthy donors, was studied by WB and IF to assess γH2AX and Rad51 in steady state and after sub-lethal DNA damage by UV exposure. The same experiments were carried out after SETD2 silencing for 2 months. Cells with silenced SETD2 had significantly higher levels of γH2AX and were unable to activate the HR repair. Interestingly, clonogenic assays in ROSAD816V cells before and after SETD2 silencing showed that reduction of clonogenic potential after proteasomal or MDM2 inhibition is indeed SETD2-dependent (Figure 1A). Finally, we performed clonogenic assays to evaluate the therapeutic potential of bortezomib, carfilzomib and ixazomib in neoplastic mast cells from 3 patients with advanced SM and we observed in all cases that both first and second generation inhibitors induced a significant reduction of clonogenic activity at nanomolar doses (Figure 1B). Taken together, our results suggest that AKA and MDM2-mediated post-translational modifications contribute to SETD2 non-genomic loss of function in advanced SM. Loss of SETD2 and H3K36me3 is associated with increased DNA damage and transcription and splicing defects in patients. Inhibiting AKA or MDM2 activity or proteasome-mediated degradation are promising therapeutic strategies in patients with low SETD2 expression levels. Acknowledgments: study supported by AIRC (project code 16996) and AIL (Associazione Italiana contro le Leucemia, Linfomi e Mieloma). Figure 1. Figure 1. Disclosures Bonifacio: Incyte: Consultancy; Pfizer: Consultancy; Amgen: Consultancy; Novartis: Research Funding; Bristol Myers Squibb: Consultancy. Pagano:Janssen: Speakers Bureau; Merck: Speakers Bureau; Gilead: Speakers Bureau; Basilea: Speakers Bureau; Pfizer: Speakers Bureau. Valent:Novartis: Honoraria; Pfizer: Honoraria; Incyte: Honoraria. Cavo:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; 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; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, 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, Research Funding, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Soverini:Novartis: Consultancy; Incyte Biosciences: Consultancy; Bristol Myers Squibb: Consultancy.

Background . Arterial occlusive events (AOEs) represent emerging complications in chronic myeloid leukemia (CML) patients treated with ponatinib, with a cumulative incidence correlated with the higher dose of the drug and longer treatment duration. Current recommendations highlight the importance of a careful evaluation of cardiovascular (CV) risk factors at baseline.Moreover, a preventive strategy with primary prophylaxis based on aspirin still remains under discussion and no data have been reported on secondary prophylaxis. Methods. We investigated a consecutive series of adult CML patients (mean age 50 years, range 24-81) who initiated ponatinib, between January 2012 and December 2016 at 15 Italian centers. Patients were stratified according to the Systematic Coronary Risk Evaluation (SCORE) assessment, based on gender, age, smoking habits, systolic blood pressure, and total cholesterol levels. Additional risk factors were considered the presence of diabetes, body mass index > 24.5 kg/m2, mild or severe renal insufficiency, and dyslipidemia. CML patients were also evaluated for both comorbidities and a positive anamnesis of CV diseases.Primary and secondary CV prophylaxis before starting ponatinib was also reported. We evaluated the cumulative incidence of AOEs (myocardial infarction, angina, ischemic cerebrovascular events and peripheral vascular disease) after initiating treatment with ponatinib, and their management. Results. A total of 71 patients were retrospectively identified. The reasons for treatment with ponatinib were inefficacy of previous tyrosine kinase inhibitors (TKIs) in 80.2% and intolerance in 19.8%. The median time of exposure to ponatinib was 16 months (range 3-69).The 60-month cumulative incidence of AOEs was 30.9±11.5%.Patients aged ≥60 years showed a higher incidence of AOEs (61.8±19.5% vs 19.5±12.0%, p=0.001) (Figure 1). The majority of patients (95%) were classified as at low-intermediate SCORE risk and 5% as at high-very high SCORE risk. Patients with a high-very high SCORE showed a significantly higher incidence of AOEs (100% vs. 25.8±11.5%; p Conclusions. This study confirms the increased risk of AOEs in CML patients treated with ponatinib in the real-life, particularly in patients aged ≥60 years. Our findings emphasize the need of personalized prevention strategies based on CV risk factors, in close collaboration with cardio-oncologists, angiologists and vascular surgeons. We suggest that patients treated with ponatinib should undergo prophylaxis with aspirin 100 mg. Data on the efficacy of primary prophylaxis need to be confirmed in larger cohorts of patients and in prospective randomised trials. Figure 1.Arterial Occlusive Events (AOEs) cumulative incidence according to age ≥ 60 years in 71 CML patients treated with ponatinib Figure. Figure. Disclosures Abruzzese: BMS: Consultancy; Novartis: Consultancy; Ariad: Consultancy; Pfizer: Consultancy. Bonifacio:Incyte: Consultancy; Pfizer: Consultancy; Amgen: Consultancy; Novartis: Research Funding; Bristol Myers Squibb: Consultancy. Foà:CELTRION: Other: ADVISORY BOARD; GILEAD: Speakers Bureau; ABBVIE: Other: ADVISORY BOARD, Speakers Bureau; CELGENE: Other: ADVISORY BOARD, Speakers Bureau; NOVARTIS: Speakers Bureau; ROCHE: Other: ADVISORY BOARD, Speakers Bureau; INCYTE: Other: ADVISORY BOARD; AMGEN: Other: ADVISORY BOARD; JANSSEN: Other: ADVISORY BOARD, Speakers Bureau. Breccia:Incyte: Honoraria; BMS: Honoraria; Novartis: Honoraria; Pfizer: Honoraria.

Introduction. Alu repeats, belonging to the Short Interspersed Repetitive Elements (SINEs) class, contain about 25% of CpG sites in the human genome. They are located in gene-rich regions, so their methylation is an important transcriptional regulation mechanism. Aberrant Alu repeats methylation has been associated with tumor aggressiveness and investigated in some solid tumors, but the global Alu methylation level has not yet been investigated in hematological malignancies. Moreover, today, some of the techniques designed to measure global DNA methylation are focused on the methylation level of specific genomic compartments, including repeat elements. In this work we propose a new method for investigating Alu differential methylation, employing droplet digital PCR (ddPCR) technology, applied in patients affected by chronic lymphocytic leukemia (CLL), myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML). Methods. The study included a total of 46 patients: 30 CLL patients, 7 patients with MDS at intermediate/high risk, and 9 CMML patients. The study also involved acute promyelocytic leukemia-derived NB4 cell line, either untreated or treated with azacytidine (AZA) 0.75 µM or decytabine (DEC) 0.75 µM. Four healthy donors (HD) were also included as controls. For each DNA sample two aliquots of 250ng of gDNA were simultaneously digested (with 1 unit of Alu-in/sensitive isoschizomers either MspI or HpaII) and ligated (to a previously prepared synthetic adaptor) in parallel in two separate tubes. Considering that the genomic DNA amount in a human diploid cell is about 6 pg/cell, for each sample we calculated the percentage of methylated consensus Alu sequences as the ratio between the sum of positive droplets obtained from the three wells of both HpaII (MH) and MspI (MM) final dilutions, according to the following formula: [1-(sumMH/sumMM)]x100. The significance level was set at p Results. Using our ddPCR assay, we observed a significant decrease of the global Alu methylation level in DNA extracted from NB4 cells treated with DEC, as compared to untreated cells, and a minor decrease with AZA (p=0.058). Moreover, comparing the global Alu methylation levels at diagnosis and after AZA treatment in MDS patients, we observed a statistically significant decrease of Alu sequences methylation after therapy as compared to diagnosis. We also extended the assessment of our assay in CLL patients at diagnosis. We observed a significant decrease of the Alu methylation level in CLL patients compared to HD. CLL patients were also classified in the following three cytogenetic risk groups according to the karyotypic alterations identified by Fluorescent In Situ Hybridization (FISH): low (with isolated 13q deletion), intermediate (without 11q, 13q and 17p deletions or with trisomy 12), and high risk (with 11q or, 17p deletions, or more than two chromosomal aberrations). Alu methylation status of the low and high-risk groups was more significantly reduced compared to HD, whereas considering intermediate-risk patients the difference was less evident. Finally, for CMML patients, a significant decrease of Alu sequences methylation was observed in patients harboring the main SRSF2 gene hotspot. However, these preliminary results should be confirmed by extending the analysis to other CMML patients. Conclusions. In our work, we propose a new method to investigate Alu differential methylation based on ddPCR technology. This assay represents an alternative to conventional quantitative-PCR (qPCR), introducing ddPCR as a more sensitive and immediate technique for Alu methylation analysis. Moreover, compared to qPCR, our ddPCR Alu assay may be carried out using very small amounts of digested gDNA (about 6 pg), and does not require a reference gene for the analysis of ddPCR data. To date, this is the first application of ddPCR to study global DNA methylation by inspecting DNA repeats. This approach may be useful to profile patients affected by hematologic malignancies for diagnostic/prognostic purpose. Disclosures No relevant conflicts of interest to declare.

Background: The European LeukemiaNet (ELN) response criteria are widely used to decide, at given time points, when the treatment with tyrosine-kinase inhibitors (TKIs) of CML patients should be continued (optimal response, OR), when a careful monitoring is required (warning, W) or when the therapy should be changed (failure, F). The 2013 ELN response criteria are the same for all chronic phase CML patients, irrespective of the prescribed TKI, but the time to response is influenced by the first-line TKI. Despite faster responses, a clear survival advantage of 2nd generation TKIs over imatinib (IM) has not been demonstrated yet. A validation of the 2013 ELN response definitions and an analysis of their prognostic impact in IM-treated patients may provide important information. Aims: The aim of our study was to assess the significance of 2013 ELN response criteria in CML patients treated frontline with IM, investigating whether or not optimal responders, warnings or failures at 3, at 6 and at 12 months have a different long-term outcome. Methods: 559 patients enrolled within 3 prospective clinical trials (NCT00514488, NCT00510926, observational trial CML/023) were analyzed (ITT population of each study). The 3-month response according to 2013 ELN criteria was not fully evaluable due to missing cytogenetic analysis in 452/559 patients, so we focused on the early molecular response (EMR, BCR-ABL < 10% at 3 months), corresponding to OR. The responses at 6 and 12 months were retrospectively defined according to 2013 ELN criteria: F, BCR-ABL > 10% and/or Ph+ > 35% at 6 months, BCR-ABL > 1% and/or Ph+ > 0 at 12 months; OR, BCR-ABL < 1% and/or Ph+ 0 at 6 months, BCR-ABL < 0.1% at 12 months; W: intermediate conditions. As the ELN criteria changed over time, not all the failures switched to alternative treatment. Progression: transformation to advanced phases (2013 ELN definitions) at any time, including after treatment discontinuation. Overall survival (OS): all the deaths at any time (in-study or off-study) were included. Leukemia-unrelated death: known cause of death, no progression, CCyR and/or MMR < 6 months prior to death; all other deaths were classified as leukemia-related (LRD). The cumulative incidence of LRD was estimated considering the competing risk of leukemia-unrelated death. Results: The median follow-up was 76 months (66-99 months). The patients with OR at 3 months were 82%; the patients with OR-W-F at 6 months were 76%, 14% and 10%, respectively; the patients with OR-W-F at 12 months were 65%, 20% and 14%, respectively. The OS, the progression-free survival (PFS) and the cumulative incidence of LRD according to the presence-absence of EMR were 87-81% (p=0.015), 85-81% (p=0.035) and 11-5% (p=0.019), respectively. Combining the Sokal score and the EMR, the patients were divided into 4 groups, low and intermediate risk/responders, low and intermediate risk/not responders, high risk/responders, high risk/not responders: the OS and the cumulative incidence of LRD across the 4 groups were 88%, 84%, 86% and 70% (p=0.005, Figure 1) and 3%, 9%, 10% and 20% (p Conclusions: The patients without OR at 3 months (particularly high Sokal score patients) had a significantly poorer outcome compared to patients with OR. The warnings at 6 and 12 months had a similar outcome to optimal responders at the same time points; both had a significantly better outcome than the failures. Compared to optimal responders, a larger proportion of patients classified as warnings was subsequently switched to alternative treatment; the subsequent treatment may explain, at least in part, the absence of outcome differences between these 2 groups. Disclosures Castagnetti: Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria. Gugliotta:BMS: Honoraria; Novartis: Honoraria. Abruzzese:BMS, Novartis, Pfizer, Ariad: Consultancy. Tiribelli:Ariad Pharmaceuticals: Consultancy, Speakers Bureau; Bristol Myers Squibb: Consultancy, Speakers Bureau; Novartis Farma: Consultancy, Speakers Bureau. Soverini:Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Cavo:Jansenn: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; BMS: Honoraria; Millenium Pharmaceuticals: Honoraria; Onyx: Honoraria; Celgene: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Martinelli:MSD: Consultancy; Roche: Consultancy; ARIAD: Consultancy; Pfizer: Consultancy, Speakers Bureau; BMS: Speakers Bureau; Novartis: Speakers Bureau. Saglio:Novartis: Consultancy, Honoraria; BMS: Consultancy. Baccarani:BMS: Honoraria, Speakers Bureau; Novartis: Honoraria, Other: Consulting or Advisory Role, Speakers Bureau; Pfizer: Honoraria, Other: Consulting or Advisory Role, Speakers Bureau; ARAID Pharmaceutical Inc.: Other: Consulting or Advisory Role, Speakers Bureau; Novartis: Other: Travel, Accommodations, Expenses; BMS: Other: Travel, Accommodations, Expenses; Pfizer: Other: Travel, Accommodations, Expenses; ARIAD Pharmaceutical Inc.: Other: Travel, Accommodations, Expenses. Rosti:Bristol Myers Squibb: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau.

Introduction. BCR-ABL1 tyrosine kinase inhibitors (TKIs) are considered an important component of treatment for adult patients affected by Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL). In fact, recent studies reported that treating Ph+ ALL with the combination of imatinib and multi-agent chemotherapy improved the overall outcome. Currently, no data are available on the impact of TKIs on minimal residual disease (MRD) in Ph+ ALL. In fact, although the real-time quantitative PCR (RQ-PCR) method, usually employed for monitoring the BCR-ABL1 residual transcript, is sensitive and easy to perform, it lacks a full standardization and international quality validation. Here, we describe a highly sensitive and reproducible droplet digital PCR (ddPCR) test to monitor BCR-ABL1 transcript level in Ph+ ALL. Methods. BCR-ABL1 expression analysis by ddPCR was performed in twenty-two newly diagnosed adult Ph+ ALL patients.The diagnosis was confirmed by qualitative RT-PCR specific for the BCR-ABL1 p190 fusion gene detection. ddPCR experiments were successfully performed in all twenty-two patients at the onset; several follow-up points were evaluated in thirteen patients. ddPCR experiments were performed using primers and probes specific for BCR-ABL1 p190. GUSB was used as control gene. Fifty ng and 750 ng of cDNA templates were used for the onset and for the post-treatment samples, respectively. To increase the limit of detection (LOD), three replicates were run for the post-treatment samples. ddPCR experiments were performed by Bio-Rad's QX200 system and ddPCR data were analyzed with QuantaSoft analysis software (version 1.7.4). Target concentration was expressed as BCR-ABL1 copies/mg. Results. First, we defined the LOD of the BCR-ABL1 p190 ddPCR system, a 10-fold dilution series (100, 10-1, 10-2, 10-3, 10-4, and 10-5) of a pool of p190 positive patients using a diluent-pool of healthy volunteers. This analysis showed remarkable linearity, trueness, and precision down to 10-5. After converting to log-log scale, linear regression showed no concentration-dependent bias, and R2 equaled 0.996. Because the negative samples showed no background, even the detection of a single droplet per well was considered a positive result. The median concentration of the BCR-ABL1 transcript at the onset was 233.8 (min 3.24 - max 1744) x 103BCR-ABL1 copies/mg. Concerning the analysis of follow-up samples, among the thirty-four points that were negative to qualitative nested RT-PCR, twenty-three (68%) resulted to be positive by ddPCR analysis, with a median concentration of 44.95 (min 0.27 - max 573.3) BCR-ABL1 copies/mg. Follow-up points that were negative in ddPCR remained negative even when the experiments were repeated increasing the depth of the analysis, evaluating a total quantity of 4.5 mg of RNA. Conclusions. This study indicates that, as compared to RQ-PCR, ddPCR increases the depth of the quantitative analysis of BCR-ABL1 p190 fusion transcript by allowing the evaluation of larger amounts of RNA. Moreover, our preliminary data revealed that the amount of the BCR-ABL1 fusion transcript at diagnosis is heterogeneous and that the ddPCR is much more sensitive than nested qualitative RT-PCR analysis, as the 68% of samples negative to nested PCR during the follow-up resulted to be positive by ddPCR. Therefore, we suggest that ddPCR represents a precise, sensitive and rapid method for both diagnosis and MRD monitoring of Ph+ ALL patients. Disclosures No relevant conflicts of interest to declare.

Introduction Lymphoid enhancer-binding factor 1 (LEF1) is a downstream effector of the Wnt/ β-catenin signaling pathway, which controls cell growth and differentiation. In normal hematopoiesis, LEF1 plays a pivotal role in the development of B- and T-lymphocytes as well as granulocyte progenitor cells. High LEF1 expression has been reported as a favorable prognostic marker in cytogenetically normal acute myeloid leukemia whereas it is associated with poor prognosis in adult B precursor acute lymphoblastic leukemia and in chronic lymphocytic leukemia. Moreover, marked downregulation of LEF1 is associated with disease progression in myelodysplastic syndromes. Given the functional role of LEF1 in hematopoiesis and its putative prognostic impact in several hematological malignancies, we evaluated the prognostic significance of LEF1 expression in adult acute promyelocytic leukemia (APL). Methods One hundred and three consecutive patients with newly diagnosed APL were observed and treated with ATRA-based regimens (AIDA04937 and AIDA2000 GIMEMA protocols), between January 1996 and December 2012. LEF1 expression was measured by real-time qPCR in 78 patients (median age 45 years, range 16 to 88 years) with sufficient available material from the primary cohort. Advanced relative quantification analysis was performed using LightCycler 480 Software 1.5.1, based on the ΔΔCt method. APL samples were dichotomized at the median value and divided into two expression groups: low LEF1 (39 patients) with LEF1 values below the median value (LEF1low) and high LEF1 (39 patients) with LEF1 values above the median value (LEF1high). Clinical and biological features of the two groups were compared. A p value Results Patients with LEF1high expression had lower white blood cell counts at baseline (1.8 vs 12.0 x109/L; p < 0 .0001), and were less likely to carry a FLT3-ITD than LEF1low patients (12.8% vs 35.9%, respectively, p=0.02). The association between LEF1low and the presence of FLT3-ITD was also confirmed when the 11 patients with FLT3-TKD were included among patients with FLT3 mutations (p=0.03), or the group of FLT3 wild type patients, as compared to those bearing FLT3-ITD (p=0.03). Early death (defined as death during induction treatment) occurred in nine cases (23%) of the LEF1low group versus no case in the LEF1high group (OR = 0.04; p= 0.002). LEF1low expression was associated with a high relapse risk score (53.9% vs 7.7%, OR=0.07; p < 0.0001). The LEF1high group showed a trend toward a statistically significant association with a lower median age (p = 0.08). No significant differences were observed regarding CD34, CD2, bcr3 positivity and LEF1 expression. Survival analysis of 61 APL patients < 60 years revealed that the LEF1high group had significantly longer overall survival (OS) (p = 0.03), whereas no differences were observed between the two groups in terms of relapse-free survival. Cox analysis for OS confirmed only LEF1 expression as an independent prognostic factor (HR=5.4; 95% CI, 1.0 – 27.5, p =0.04). Among the 17 patients over the age of 60, those with LEF1high expression showed a higher median survival than those in the LEF1low group (6.5 vs 0.04 y.rs, p = 0.05). In silico analysis of the differential expression of LEF1 in APL identified 9 differentially expressed, up-modulated genes associated with a high expression of LEF1 (ETS1, FAIM3, CCR7, IL7R, LCK, IL2RB, ITK, RASGRP1, TRBC1); the majority of these genes is involved in the regulation of apoptosis Conclusions Our study provides, for the first time, evidence that LEF1 expression is an independent prognostic factor in APL and that it could be used in patients risk stratification. The observation provided by in silico gene expression analysis that LEF1 expression in APL is associated with biologic changes, mostly in terms of apoptosis regulation, will need to be confirmed experimentally. Disclosures: No relevant conflicts of interest to declare.

Abstract 50623 Background: Bone marrow karyotype in myelodysplastic syndromes (MDS) is essential to define the prognosis and to guide treatment decisions, including targeted therapies. Due to the lack of an extensive national MDS registry in Italy, epidemiological data on MDS, including cytogenetics, throughout the territory is unknown. Objective: We evaluated the incidence of cytogenetic abnormalities amongst newly diagnosed MDS patients in in 2 southern Italian regions (Calabria and Puglia). Methods: A pilot project, denominated ANDROMEDA (ANalysis of cytOgenetics alteRatiOn in the MyEloDysplAstic syndromes) was developed in 17 centers to offer a service of conventional cytogenetic analysis for all consecutive patients undergoing diagnostic evaluation for cytopenia and suspected MDS between January 1 and December 31, 2011. The study conformed to the ethical standards set out in the Declaration of Helsinki and was approved by institutional review boards at each participating center. Patients were required to provide their written informed consent. Clinical characteristics of patients and bone marrow morphology, iron staining and histology were registered. Bone marrow samples were centralized for standard cytogenetic studies and fluorescence in situ hybridization to two dedicated genetics laboratories (one for each region), blind to patients' data. Results: Two hundred and thirty-five patients were evaluated and MDS diagnosis was confirmed in 220 cases (88. 3%), according to WHO criteria. The overall incidence of clonal chromosome abnormalities detected by conventional analysis was 36. 9%. Single abnormalities included +8 (13 cases, 5. 8%), del(5q) (12 cases, 5. 4%), –Y (11 cases, 5. 0%) and del(7)/-7 (4 cases, 1. 8%). Complex karyotypes were detected in 18 (8. 1%) cases. Among all cases only 10 (4. 5%) bone marrow samples were not evaluable for cytogenetic analysis. FISH revealed additional abnormalities not identified by conventional analysis only in 3 (1. 3%) out of 72 cases. Patients were classified in WHO subtypes: 39. 2% refractory cytopenia with unilineage dysplasia (RCUD), 1. 5% refractory anemia with ring sideroblasts (RARS), 32. 5% refractory cytopenia with multilineage (RCMD), 10. 8% refractory anemia with excess of blast-1 (RAEB-1), 9. 3% refractory anemia with excess of blast-2 (RAEB-2), 4. 1% MDS with deletion 5q (MDS 5q-) and 2. 6% MDS unclassifiable (MDS-U). Conclusions: These preliminary results demonstrate that the incidence of abnormal karyotype patterns and WHO subgroups in MDS patients in Southern Italy is comparable with that described in other geographical areas. It is confirmed that conventional cytogenetic analysis is a standard in the diagnostic workup of MDS of patients with a suspected myeloid malignancy in order to identify primary abnormalities and prognostic models. Disclosures: No relevant conflicts of interest to declare.

Abstract 5058 Leukemic transformation has been reported in 15–30% of primary myelofibrosis (PMF). However, very little is known about the molecular bases responsible for acute myeloid leukemia (AML) transformation. JAK2 status analysis of paired chronic myeloproliferative neoplasms (MPN) and AML samples showed that during AML transformation the JAK2 mutation could be lost. Two probable models have been proposed to explain the MPN evolution to AML: a) JAK2-mutated AML usually arose from PMF or myelofibrotic transformation of ET/PV as a consequence of genetic instability conferred by the presence of JAK2 mutations; b) JAK2 wild-type AML generally developed in patients with chronic-phase ET or PV, frequently as a consequence of a clone selection driven by the therapy (Thoennissen NH et al. Blood 2010, 115:2882; Beer PA et al., Blood 2010, 115:2891; Spivak JL, Blood 2010, 115:2727). In this report we describe two PMF cases with the JAK2V617F mutation associated with molecular/cytogenetic abnormalities who developed JAK2-wild type AML characterized by a leukemic clone bearing a new cytogenetic aberration. At the PMF diagnosis, Case #1 showed a normal karyotype 46, XY[20] and resulted JAK2V617F positive. Molecular analyses performed at the time of AML transformation revealed the presence of a JAK2V617F negative clone bearing a novel t(12;18)(p13;q12) rearrangement. As the t(12;18) breakpoints were located centromerically to SETBP1 (18q12. 3), quantitative real-time PCR (qRT-PCR) experiments were made, showing SETBP1 overexpression. To investigate the occurrence of SETBP1 dysregulation and the presence of t(12;18) at PMF onset, qRT-PCR and Fluorescence in situ hybridization were performed, revealing gene overexpression and absence of the chromosomal translocation, respectively. At PMF onset, Case #2 harbored a novel t(3;5)(q27. 1;q31. 1) in addition to the JAK2V617F mutation. At the time of AML evolution, disappearance of the t(3;5)(q27. 1;q31. 1) and leukemic clone expansion of t(3;3)(q21. 3;q26. 2) was associated with disappearance of the JAK2V617F mutation. In contrast to literature data showing that JAK2 mutation loss is commonly associated with AML transformation after PV and ET, our findings suggest that evolution to JAK2-wild type AML could also occur in JAK2V617F PMF patients. The presence of different cytogenetic abnormalities associated with PMF and AML allowed us to follow the sequence of molecular events that lead to JAK2V617F disappearance, indicating that MPN and AML are clonally unrelated and probably generated by the transformation of different stem cell levels. Moreover, the well-documented clonal heterogeneity landscape in our cases demonstrated that the genomic instability responsible for AML transformation already existed at PMF onset and was not generated either by JAK2V617F mutation expression or by the therapy. Disclosures: No relevant conflicts of interest to declare.

Abstract 1674 The t(9;22)(q34;q11) generating the Philadelphia chromosome and the BCR/ABL1 fusion gene represents the cytogenetic hallmark of chronic myeloid leukemia (CML). About 5–10% of CML cases show variant translocations with the involvement of other chromosomes in addition to chromosomes 9 and 22. The greater frequency of occurrence of genomic microdeletions proximally to ABL1 or distally to BCR has been reported in CML cases with variant translocations (30–40%) than in cases with a classic t(9;22) (10–18%). The prognostic significance of variant t(9;22) was unclear and debated in the pre-imatinib era, whereas recent studies of large CML series showed that the presence of variant translocations has no impact on the cytogenetic and molecular response or on prognosis (Marzocchi et al. Blood 2011,117:6793-800). However, the molecular bases of differences between CML patients with classic and variant t(9;22) have never been elucidated. Here we report a gene expression profile analysis of 8 CML cases with variant t(9;22) and 12 patients with a classic t(9;22). RNA samples were extracted from bone marrow cells and hybridized on the Agilent SurePrint G3 Human GE 8×60K Microarray slide (Agilent Technologies). Ingenuity Pathways Analysis (IPA, www.ingenuity.com) software was used to provide an accurate biological and statistical analysis of microarray experimental data revealing functional relationships among the identified genes. Gene expression analysis identified a 59 gene set able to distinguish the two CML subsets. These genes are mostly involved in the development of the hematological system and in the occurrence of hematological diseases. Forty-five out of 59 (76%) genes were up-regulated, causing the probable activation of different molecular mechanisms such as cellular responses to stimuli, protein degradation, DNA repair, cell cycle progression. IPA analysis revealed that most of the dysregulated genes are included in a network where they are functionally linked to MAPK p38, AKT, and NFKB. Moreover, several genes play a role in cytoskeleton organization (WIPF1), in signal transduction and cell cycle progression (TRIB1, PDE4B, PTK2B, PLK3), in regulation of apoptosis (ZFAND5, STK17B), and in protein degradation (ZFAND5, SNRPG). On the contrary, among the downregulated genes, 5 (BCDIN3D, TMEM68, HILPDA, TMEM68, and C17orf61) establish direct interactions with ubiquitin C (UBC), a crucial gene involved in different intracellular mechanisms such as protein degradation, DNA repair, cell cycle regulation, and the regulation of other signaling pathways. In conclusion, gene expression profiling in cases with variant t(9;22) revealed biological differences in this CML subset. Our data show an overall deregulation of genes involved in hematological system development and in cell proliferation signaling pathway. Disclosures: No relevant conflicts of interest to declare.

Abstract 4628 Background. Tribbles homolog gene 2 (TRIB2) is a pseudokinase gene belonging to a three member gene family. Tribbles gene was first identified in Drosophila melanogaster where is involved in the regulation of morphogenesis and mitosis. Likewise, mammalian homologous genes of Tribbles (TRIB1, TRIB2, TRIB3) promotes the degradation of specific transcription factors and interacts with several cell signaling mediators and modulators. Recently it has been demonstrated that TRIB2 was able to induce AML in bone marrow transplanted mouse by inducing C/EBPα proteasome-dependent degradation. Moreover, gene expression profiles data from AML patients revealed that patients carrying C/EBPα mutations clustered with those patients with up-regulated TRIB2.Aims. To test the hypothesis that TRIB2 expression was related to a differentiation degree in AML, we evaluated several AML cases based on FAB cytotype. Patients and Methods. We performed quantitative Real Time-PCR (qRT-PCR) analysis on AML patients (15 AML-M0, 14 AML-M2, 5 AML-M3, 2 M4 and 3 M5b bone marrow aspirate samples) to measure the expression level of TRIB2 in different FAB AML subtype. Four healthy bone marrows were used as reference samples. qRT-PCR was conduct using SYBR green chemistry and specific primers for TRIB2 transcript and for two housekeeping genes (B2M and IPO8) previously tested for their stability and efficiency. All AML sample were tested by conventional cytogenetic analysis and by FISH with TRIB2 specific probe. We performed, also, methylation analysis of a CpG island located in the TRIB2 promoter region by methylation sensitive restriction enzyme (MSRE) and qPCR. Briefly, DNA samples were digested with MSRE and were quantified by qPCR using specific primer pairs surrounding restriction enzymes recognition sites. Results. qRT-PCR experiments revealed that TRIB2 expression was higher (from 3 to 20 fold) in AML-M0 respect to the AML-M2 (p= 0.02), AML-M3 (p=0.01), AML-M5 (p=0.006) FAB subtypes, and references (p=0.003), respectively. Moreover, the AML-M0 cases showed a TRIB2 overexpression compared to that observed in the two AML-M4 cases (about 4 fold changes) but this difference there was not statistically significant probably because of M4 cases paucity in our series. Therefore qRT-PCR displayed a progressive decreasing TRIB2 expression along FAB subtypes. Conventional cytogenetic and FISH analysis did not show any kind of rearrangement involving TRIB2 gene. MSRE experiments revealed an higher methylation degree of a CpG island located in the TRIB2 promoter region in AML-M0 cases respect to the others FAB subtypes; moreover, methylation was significantly correlated with TRIB2 expression (r = 0.9; p = 0.0002). Conclusions. Our data showed that the TRIB2 gene overexpression was associated to AML-M0. In our cases TRIB2 dysregulation was not due to gene amplification as in other cancers. Surprisingly we observed that AML-M0 samples showed an higher methylation degree in the TRIB2 promoter region. Usually methylation cause gene silencing but our results showed that TRIB2 promoter region methylation was associated to the overexpression of the gene. Maybe methylation inhibits binding of some unknown transcriptional repressor as already seen for hTERT gene in others tumors cells. In conclusion, our data revealed that TRIB2 dysregulation seems to be linked to AML-M0 phenotype. Further studies are needed to clarify the reasons for this association. Disclosures: No relevant conflicts of interest to declare.

Abstract 2270 An update of the European LeukemiaNet criteria for monitoring response of chronic myeloid leukemia patients was recently published and provisional criteria to evaluate patients during second generation TKI therapy after resistance to imatinib were proposed. In our study we retrospectively tested these criteria in a large series of CML patients resistant to imatinib further treated with second generation TKIs with the aim to analyze the outcome of suboptimal response and failure patients compared to those with optimal response and to validate the provisional criteria for monitoring response. One hundred twenty-seven CML patients resistant to imatinib were collected from 6 different Italian hematologic centers. There were 66 males and 61 females, median age 54 years (range 25–80). Twenty-seven patients were in late chronic phase after IFN resistance. Ninety-seven patients received second-generation TKI after acquired resistance, whereas 30 patients had primary resistance. We found that at different time points (3, 6 and 12 months), patients classified as failure showed significantly worse 2-year overall survival (OS), progression-free survival (PFS) and event-free survival (EFS) than sub-optimal and optimal response patients. At 3 months, “failure” patients, had an OS of 83% compared to 86% of sub-optimal and 97% of optimal response patients (p=0.001); PFS was 77% for failure patients compared to 92% and 99% for sub-optimal and optimal response patients, respectively (p=0.001), whereas EFS was 41% for failure vs 59% for sub-optimal (p=0.001) and 85% and optimal response patients, respectively (sub-optimal vs optimal p Disclosures: No relevant conflicts of interest to declare.

Abstract 3261 Poster Board III-1 A crucial role of segmental duplications (SDs) of the human genome has been demonstrated in chromosomal rearrangements associated with several genomic disorders. Limited knowledge is yet available on the molecular processes resulting in chromosomal rearrangements in tumors. The t(9;22)(q34;q11) causing the 5'BCR/3'ABL gene formation has been detected in more than 90% of chronic myeloid leukemia (CML) cases. Some years ago, a 76-kb duplicon was reported, closely located to both ABL and BCR genes which are involved in the t(9;22)(q34;q11) translocation associated with CML. However, the exact role of this duplicon in mediating the t(9;22) rearrangement remained mostly speculative. In 10–18% of CML patients genomic deletions were detected on der(9) chromosome next to translocation breakpoints. The molecular mechanism triggering the t(9;22) and deletions on der(9) is still unknown. Our study presents an experimental evidence of the involvement of SDs in the genesis of the t(9;22) translocation in CML and in the occurrence of genomic deletions on der(9) chromosome. We identified 71 (17%) cases with der(9) deletions by FISH screening of 416 CML patients at diagnosis. Fine-mapping of deletions was performed using appropriate bacterial and Phage P1-derived artificial chromosome clones. The deletions sizes were heterogeneous, ranging from 230 Kb to 12.9 Mb on chromosome 22 and from 260 Kb to 41.8 Mb on chromosome 9. The mapping of all breakpoints revealed an evident breakpoints clustering, on both chromosomes 9 and 22, in two regions of about 2 Mb in size. Indeed, these regions contained the breakpoints detected in 54 out of 60 (90%) patients bearing chromosome 9 deletions and in all patients with chromosome 22 sequences loss. Bioinformatic analysis of chromosome 9 and chromosome 22 genomic regions involved in the deletions was performed to search for features that could correlate with the breakpoints clustering. To this aim, the breakpoint regions were subdivided into 250 Kb intervals. The most striking result was the fact that both clusters contain the above reported 76-kb duplicon, shared by chromosome 9 and 22 (SD_9/22). The SD_9/22 is the only duplication located inside the breakpoints clustering region on chromosome 9, whereas the chromosome 22 clustering region harbors several duplications. A remarkable feature of the chromosome 9 clustering region was the high frequency of Alu repeats. The mean Alu frequency overall on chromosome 9 is 10.8%, whereas the average Alu content on this cluster is 31.3%. Accordingly, as expected, the content in LINE sequences of the region was relatively low (average overall on chromosome 9: 21.2%, as opposed to 8.7% on the cluster region). Gene distribution analysis of chromosome 9 and 22 showed that both SD_9/22 map inside gene-poor regions, of about 460 Kb and 250 Kb in size, respectively. To corroborate the observations on the distribution of SDs and Alu/LINE repeats, the chromosome 9 and 22 regions surrounding the SD_9/22 were once more divided into 250 Kb segments, positioning the SD_9 and SD_22 as landmarks. A statistically significant negative association was observed between the number of breaks and the distance from SD_9/22, on both chromosomes 9 (p=0.01) and 22 (p=0.006), respectively. The relationship between the breaks and the interspersed repeats revealed, on chromosome 9, a positive linear regression with Alu repeats (p=0.04), and a negative one with LINEs (p=0.04). Very similar conclusions were obtained by comparing the distance from the SD_9 and the Alu (p= 0.03, positive) and LINE distribution (p=0.02, negative). No statistically significant relationship was observed on chromosome 22. In our study the involvement of SDs was proposed to explain the recurrent t(9;22) translocation in CML and the genomic deletions that could accompany the rearrangement. Although the chromosomes 9 and 22 breakpoints clustering regions are quite large, the strong non-randomness of SD_9/22 location and the genomic features identified in our work suggest that the chromosomal segments near the ABL and BCR genes are brought together by an active process, facilitating recombination. At the light of these findings, the analysis of secondary non-recurrent events could represent a new methodological approach able to identify architectural elements involved in the occurrence of recurrent primary rearrangements in human neoplasia. Disclosures: No relevant conflicts of interest to declare.

Abstract 2211 Poster Board II-188 Dasatinib is a 2nd generation tyrosine-kinase inhibitor active in CML patients resistant or intolerant to Imatinib; at present there is no data on its toxicity and efficacy in unselected elderly patients. To highlight this issue, 97 patients treated with Dasatinib when aged > 60 years were retrospectively evaluated from 16 Italian Centers on a “real-life” basis, including all patients treated at each Center independently from enrolment or not in controlled clinical trials.There were 52 males and 45 females, median age at Dasatinib start was 69.5 years (IR 65.0 – 73.3), Sokal Risk at diagnosis was low in 26 patients, intermediate in 37, high in 15 and not valuable in 19. Forthy-five patients (46.4%) were primarily resistant, 11 (11.4%) were intolerant and 41 (42.2%) had secondary resistance to Imatinib; all patients were in CP when Dasatinib was started. Median time from diagnosis to Dasatinib treatment was 85.0 months (IR 44.8 – 120.0); 53/97 patients (54.6%) had been pretreated with IFN ± Ara-C before Imatinib, all patients received Imatinib at standard dose (400 mg/day) followed in 50/97 (51.5%) by increased dose (600 – 800 mg/day) with an overall median period of Imatinib treatment of 48.6 months (IR 26.9 – 67.0). In addition, 28/97 patients (28.8%) received other 2nd line treatment (10 Nilotinib, 14 HU +/- other drugs, 3 Imatinib + HU or IFN and 1 allogeneic transplant) before Dasatinib. Starting dose of Dasatinib was 140 mg/day in 47 patients, 100 mg/day in 44 patients and ≥ 50 mg/day in 6 patients, respectively. After a median period of treatment of 15.6 months (IR 7.6 – 23.0) all patients were evaluable for toxicity; on the whole, grade 3 – 4 hematological and extra-hematological toxicities were reported in 36/97 (37.1%) and 27/97 (27.8%) patients, respectively. A grade 3 – 4 hematological toxicity occurred in 25/47 (53.1%) patients receiving 140 mg as compared to 10/44 (22.7%) patients receiving 100 mg (p 60 years resistant/intolerant to Imatinib; in particular, when employed at the current recommended dose of 100 mg/day, it is very effective and has a favourable safety profile also in heavily pretreated elderly subjects. Disclosures: Vitolo: Roche: . Pane:Novartis: Research Funding; Ministero dell'Università/PRIN: Research Funding; Regione Campania: Research Funding; Ministero della Salute/Progetto integrato Oncologia: Research Funding.

MicroRNAs (miRNAs) are small, single stranded non-coding RNAs, 19–24 nucleotides long, involved in crucial biological processes, including differentiation, apoptosis and proliferation. Recent evidence indicates that miRNAs may play an important role in tumorigenesis; changes in miRNAs expression level were identified in many types of human hematological and solid malignancies. To date, several papers have reported the occurrence of genomic deletions flanking the breakpoint on der(9)t(9;22) in 10%–18% of patients with chronic myeloid leukemia (CML). The most probable consequence of der(9) deletions is the loss of tumour suppressor genes, conferring a proliferative advantage to the Philadelphia-positive clone. On the other hand, two miRNAs, namely miR219-2 and miR-199b, are found to map centromeric to the ABL gene within the chromosomal region at 9q34 that is frequently lost in CML patients with der(9) deletions. In this study, we investigated the loss of miR-219-2 and miR-199b by fluorescence in situ hybridization (FISH) analysis with specific bacterial artificial chromosome (BAC) probes in 68 CML cases bearing der(9) deletions. We further evaluated miR-219-2 and miR- 199b expression levels by quantitative real-time polymerase chain reaction (qRT-PCR) experiments in cases showing deletions of at least one of these miRNAs. Depending on RNA sample availability, miRNAs expression level was evaluated in 7 and in 5 CML cases with miR-219-2 and miR-199b deletions, respectively. Statistical analysis of the relative expression results was performed by the Relative Expression Software Tool (REST). To explore the predicted miR-199b target genes, the miRGen targets database (http:// www.diana.pcbi.upenn.edu/cgi-bin/miRGen/v3/Targets.cgi) was queried; this interface provides integrated data of four widely used target prediction programs (miRanda, PicTar, TargetScan, DIANA-microT). FISH experiments revealed the loss of miR-219-2 and miR-199b in 17 (25%) and 10 (15%) out of 68 patients. The miR-199b expression study showed a downregulation in the analyzed group of 5 CML cases with miR-199b deletion as compared to a pool of 10 patients without deletions. The expression level of the miR-199b was 0.279 and the difference between the two groups was statistically significant (p= 0.028). On the contrary, the miR- 219-2 analysis did not reveal a detectable expression level in the examined patients. There were 26 predicted miR-199b target genes, involved in several biological processes such as signal transduction (Protein phosphatase inhibitor 2, PPP1R2), regulation of transcription (Hepatic leukaemia factor, HLF), chromosome organization and biogenesis (Zinc finger protein 238, ZNF238), cell proliferation (Mitogen-activated protein kinase 11, MAP3K11) and DNA repair (UV excision repair protein RAD23 homologue B, RAD23B). Among the CML patients evaluable for the response to the treatment, all cases with the miR-199b deletion were resistant to IFN-a and imatinib therapy. In conclusion, our data demonstrate a crucial role for miR-199b in CML cases bearing der(9) deletions. This miR-199b downregulation could influence the expression level of different target genes modifying important cellular pathways. Further analysis of miR-199b target genes will be needed to shed light on the link between miRNAs and CML.

Deletions on der(9) are associated with chronic myeloid leukemia(CML) in 15–18% of cases. To date, the biological significance of this genomic loss in the pathogenesis of CML is unknown. The most plausible hypothesis is that the loss of a tumor suppressor gene may confer a proliferative advantage to the Philadelphia-positive clone. On the other hand, it has now become evident that microRNAs (miRNAs) play an important regulatory role in some hematological malignancies. To investigate the presence of miRNAs within the genomic regions lost on der(9) we analyzed 60 CML patients with der(9) deletions. Methods. Genomic characterization of the deleted sequences was performed by fluorescence in situ hybridization (FISH) using a contig of DNA clones; the miRBase (http://microrna.sanger.ac.uk/) was queried to assess the presence of miRNAs in the der(9) deleted genomic regions. FISH experiments showed that the genomic loss on der(9) of the 9 (centromeric to ABL) and 22 (telomeric to BCR) chromosome sequences ranged from 260 Kb to 54 Mb and from 230 Kb to 12.9 Mb, respectively. Consultation of the miRBase revealed that in 16 (27%) patients there was loss of miRNAs mapping on chromosome 9 whereas no known miRNAs were mapped on the deleted genomic sequences belonging to chromosome 22. Moreover, 4 cases with a complex t(9;22) rearrangement and der(9) deletions showed loss of the miRNAs sequence also on the third derivative chromosome (4p16, 7p14, 13q14, and 11q13, respectively); among them, only in one case the loss of miRNAs on the third derivative was not associated with the miRNAs deletion mapped on chromosome 9. The most recurrent miRNAs deleted on der(9) were mir-219–2 (deleted in 100% of cases) and mir-199-b (lost in 67% of cases). It is noteworthy that mir-219–2 neighbors and overlaps CpG-islands, suggesting a potential role of this miRNA in CpG-island methylation. Experimental studies indicate that miRNAs can function as tumor suppressor genes or as oncogenes. In fact, in chronic lymphocytic leukemia associated with del(13)(q14) it has been demonstrated that the miRNAs loss can induce downregulation of the antiapoptotic BCL-2 protein. The novel evidence that deletions on der(9) in CML are associated with miRNAs loss may shed new light on the significance of genomic sequences loss. Further studies are needed since it is known that some microRNAs may have as many as a few thousand targets, so prediction algorithms and strategies allowing large-scale screening of multiple target genes are required.

It is known that the receptor activator of nuclear factor kB (RANK) is only identified on progenitor and mature osteoclasts and in dendritic cells. A recent report (Blood107:4334, 2006) described a dose-dependent decrease of RANK expression in osteoclasts when cultured in the presence of imatinib; these data were confirmed in adult mice treated with imatinib, in which a decrease in osteoclasts activity was demonstrated. Therefore, it is speculated that imatinib may have a potential antiresorptive effect. We evaluated bone marrow RANK molecular expression in patients affected by chronic myeloid leukemia (CML) receiving imatinib therapy, to verify the effect of the tyrosine kinases inhibitor on RANK expression. The study included 13 CML patients (7 males, 6 females, median age 37 yrs). Six were treated with Imatinib at standard dosage (400 mg) and the remaining 7 at higher doses (> 400 mg). The median duration of imatinib therapy was 28 months (range 13 – 44 months). The RANK gene expression level was determined by Real-Time PCR using 1X Platinum SYBR Green qPCR SuperMix-UDG. The RANK gene expression level variation was estimated by comparing the values of 2−Δ Δ Ct obtained in CML patients at diagnosis and during imatinib treatment. For each of the tested cases the sample at diagnosis was utilized as calibrator and 28S rRNA was used as reference gene. Eleven (85%) patients showed an upregulation of RANK, the gene expression fold increase ranging from 1.17 to 15.88 as compared to the value at diagnosis; all patients treated with high-dose imatinib showed a RANK overexpression, the fold increase ranging from 1.57 to 15.88. Although the median RANK expression fold increase observed in the group of patients treated with high-dose imatinib was higher than in the cases taking standard dosage (2.47 versus 1.34 fold increase, respectively), this difference was not statistically significant. There was no association between the RANK expression level and the duration of imatinib treatment. Our study reveals that imatinib mesylate is accountable for the molecular overexpression of the RANK gene in CML patients. These data seem to be in conflict with those previously reported (Blood2006;107: 4334). It is possible that the RANK overexpression which we observed was related to the immune (i.e. dendritic cells) and not the bone cells (i.e. osteoclasts). In this regard, a new type of cell has recently been described in mice, that may account for the immune effect of imatinib; the new cell, which seems to be a cross between a dendritic cell and a natural killer cell, has been called an “interferon-producing killer dendritic cell” (Nat Med2:214, 2006; Nat Med2:207, 2006). These studies suggest a new mechanism by which imatinib might exert an antitumor effect, apart from its direct action on chronic myeloid leukemia cells. Therefore, in this context, the RANK upregulation observed in our study could be an expression of the immune effect of imatinib. Further studies are needed to verify if the RANK gene overexpression mediated by imatinib could affect the molecular and cytogenetic response in CML patients.

The receptor activator of the nuclear factor kB ligand (RANKL) is a key molecule in the pathogenesis of malignant osteolytic lesions in multiple myeloma or bone metastases in prostate and breast cancer. Inhibition of RANKL is therefore a promising new therapy for controlling bone loss and pain in bone tumors and associated osteolysis. Changes in bone and mineral metabolism developing in a proportion of patients taking imatinib for either chronic myeloid leukemia (CML) or gastrointestinal stromal tumors have previously been described. The aim of our study was to verify in CML patients the effect of imatinib on RANKL bone marrow molecular expression. The study included 13 CML patients (7 males, 6 females, median age 37 yrs). Six were treated with imatinib at standard dosage (400 mg) and the remaining 7 at higher doses (> 400 mg). The median duration of imatinib therapy was 28 months (range 13 – 44 months). The RANKL gene expression level was determined by Real-Time PCR using 1X Platinum SYBR Green qPCR SuperMix-UDG. Variations in RANKL gene expression level were estimated by comparing the values of 2−Δ Δ Ct obtained in CML patients at diagnosis and during imatinib treatment. For each of the tested cases the sample at diagnosis was utilized as calibrator and 28S rRNA was used as reference gene. Eight (62%) patients showed a downregulation of RANKL gene expression, the fold decrease ranging from 0.01 to 0.47 as compared to the value at diagnosis; among these 8 patients, 6 (75%) were treated with imatinib at standard dosage. Instead, 5 (38%) patients showed an upregulation of RANKL, the gene expression fold increase ranging from 1.43 to 4.94 as compared to the gene level observed at CML onset; all these 5 patients were treated with high doses of imatinib. There was no association between RANKL expression and the duration of imatinib treatment. These findings suggest that imatinib mesylate could control RANKL expression. At standard dosage, imatinib can downregulate RANKL production. The RANKL upregulation observed in CML patients treated with high doses of imatinib could be explained by the possible activation of the immune system cells, such as T and B cells, induced by the tyrosine kinases inhibitor. Further studies are needed to verify the minimal effective dose of imatinib on RANKL expression, with the aim of administering it as an antiosteolytic agent in such diseases as osteoporosis, metastatic bone disease, and multiple myeloma.

Extramedullary infiltration (EMI) of malignant myeloid precursor cells in acute myeloid leukemia (AML) may occasionally be a presenting clinical symptom at onset and may develop at any site in the body but most commonly in the gum, skin, central nervous system (CNS) and soft tissue. There is controversy about the prognostic significance of extramedullary disease in AML. The present study examines the incidence, the biological features and prognostic significance of EMI at diagnosis in adult patients with AML. From January 1997 to December 2004, 213 untreated patients with de novo AML were studied. According to Grimwade et al, 14 patients were in the favorable-risk group, 159 in the intermediate-risk group, 25 in the poor risk group and in 15 the karyotype was not available. All patients had been treated with induction therapy according to the GIMEMA protocols including cytarabine, etoposide and idarubicin (15 pts), or mitoxantrone (15 pts) or daunorubicin (183 pts). Of 213 cases with de novo AML, 29 (14%) had EMI at diagnosis. Ten patients (34.8%) had skin infiltrates, 12 (41.4%) had gum hypertrophy, 5 (17.2%) had CNS involvement and 2 (6.9%) had soft tissue infiltration. No significant differences in terms of sex, age median Hb level and platelets count were found between patients with EMI and patients without EMI. The patients with EMI had higher median WBC counts (27 × 109/L) than patients without EMI (8.5 × 109/L) (p=0.05). The patients with EMI had a higher incidence of the M4/M5 FAB subtype (62%) than patients without EMI (27.4%) (p=0.005). Cytogenetic analysis was performed in patients with and without EMI; none of the abnormal cytogenetic findings was associated with EMI. We evaluated the relationship between the AML blasts surface antigen expression and EMI; the association between CD56/CD4 and CD56/CD14 was more significantly expressed in patients with EMI (35% and 29%, respectively) than without EMI (10.4% and 6.9%, respectively) (p=0.004, p=0.003). The overall CR rate was 65%; the CR rate was lower in patients with EMI (48.2%) than patients without EMI (76.1%) (p=0.001) and their disease free survival was also shorter (p=0.017); the median duration of CR was 10 and 25 months (range 2–96) in EMI and no EMI group, respectively. Our data show that a high WBC count, M4/M5 subtype, CD56/CD4 and CD56/CD14 expression are associated with extramedullary infiltrates of AML at diagnosis; the presence of EMI adversely affects the complete response rate to induction chemotherapy and the OS rate. Analysis of the clinical and biologic features in a larger series of adult AML patients is needed to evaluate the allocation of this subgroup in a different or more intensive treatment arm.

Chronic myeloid leukemia (CML) is characterized by a reciprocal translocation t(9;22)(q34;q11) that generates a BCR-ABL fusion gene on the Philadelphia (Ph) chromosome. Large deletions adjacent to the t(9;22) breakpoint on the derivative 9 chromosome have now been found, which result in genomic loss at both sides of the translocation breakpoint. We report a FISH study in CML cases bearing deletions on the der(9) chromosome. FISH analysis with specific BAC/PAC clones for the ABL1 and BCR genes was performed on bone marrow cells of 305 CML patients at diagnosis. A set of BAC/PAC probes, belonging to chromosomes 9, 22 was selected according to the University of Santa Cruz (UCSC) database and employed in FISH experiments. We detected der(9) deletions in 56 (18.4%) CML cases. Deletions of chromosome 9 sequences on the der(9) were found in 47 (84%) cases; they ranged from 350 Kb to 41.6 Mb. Chromosome 22 deletions on der(9) were found in 49 (87.5%) of the analysed cases; the deleted chromosome 22 sequences were shorter than the deleted chromosome 9 sequences (ranging from 400 Kb to 12.7 Mb). Although deletions size appeared to be heterogeneous, 2 main breakpoints cluster regions were identified proximally (at about 1.9 Mb) to the ABL and distally (at about 0.7 Mb) to the BCR genes, respectively. In fact, a cluster region of 1.1Mb, delimited by RP11-203J24 and RP11-247A12, was defined on chromosome 9 whereas a 0.8 Mb region, included between CTA-322B1 and RP5-930L11, was identified on chromosome 22. Bioinformatic analysis of breakpoints cluster regions was performed to identify sequence motifs that could mediate the chromosomal rearrangement. The presence of Matrix Association Regions (MARs) and the topoisomerase II consensus was investigated by MAR-Wiz software (http://www.futuresoft.org/modules/MarFinder/); the RepeatMasker program (http://www.repeatmasker.org/) was employed to detect interspersed repeats and low complexity DNA sequences (such as SINE, LINE, and LTR). Potential sequence similarities between breakpoints cluster regions were searched for using Genalyzer software. Sequence analysis revealed that the frequency distribution of MARs, topoisomerase II sites, and repeated elements was no different from the frequency observed in other genomic regions. Genalyzer software did not detect sequence similarities between the 2 clustering regions; however, a 76 Kb duplicon, previously reported in literature, was located at a distance of 500 kb and 550 Kb from the chromosomes 9 and 22 cluster regions, respectively. In conclusion, our analysis revealed that sequence motifs do not seem to be involved in the occurrence of der(9) deletions. We hypothesize that the presence of a duplicon could mediate the juxtaposition of ABL and BCR genes and that a probable open chromatin status of chromosomes 9 and 22 sequences could confer to DNA more vulnerability to double strand breaks, mediating the t(9;22) rearrangement and the occurrence of der(9) deletions.

Translocation t(8;21)(q22;q22) is a common karyotypic abnormality detected in about 15% of Acute Myeloid Leukemia (AML) cases. The rearrangement results in fusion of the RUNX1 (also known as AML1) and CBFA2T1 (also known as ETO) genes generating a 5′RUNX1/3′CBFA2T1 transcriptionally active fusion gene on derivative chromosome 8. In 1 to 8.5% of AML cases insertions events generating a 5′RUNX1/3′CBFA2T1 fusion gene have been reported, whereas the occurrence of inversions accompanying the t(8;21) has never been observed. We report a screening of 82 AML cases bearing the RUNX1/CBFA2T1 rearrangement detected by RT-PCR; all cases were tested by Fluorescence In Situ Hybridization (FISH) with BAC and PAC clones specific for CBFA2T1 and RUNX1 genes. This analysis allowed us to reveal five cases with ins(21;8), one with ins(8;21), and two with a pericentric chromosome 8 inversion followed by a t(8;21) translocation. A detailed molecular cytogenetic characterization of breakpoints has been performed in all cases. FISH co-hybridization experiments with CBFA2T1 and RUNX1 probes revealed the presence of a functional fusion gene on the der(21) instead of the der(8) chromosome in five cases with ins(21;8); a single fusion signal on the der(8) chromosome was detected in the case with ins(8;21). The use of the same clones in FISH studies showed the presence of a single unexpected fusion signal on the 8p derivative chromosome in addition to faint CBFA2T1 and RUNX1 signals on the long arm of der(8) and der(21) chromosomes, respectively. These results suggested that a pericentric chromosome 8 inversion involving CBFA2T1 gene occurred and that the chromosome 21 was rearranged with the 8p derivative chromosome. Appropriate chromosome 21 and 8 BAC clones were employed to precisely define the size of inserted regions in cases with insertions and the breakpoint on the 8p derivative chromosome in cases showing pericentric chromosome 8 inversion. The insertion size turned out to be very heterogeneous, ranging from a minimum of 2.4 Mb to a maximum of 44 Mb. In both cases with chromosome 8 inversion, the CBFA2T1 gene represents the breakpoint at the chromosome 8 long arm whereas the 8p breakpoint showed different mapping positions in 8p21.3 and 8p21.1, respectively. Our results illustrate that (1) heterogeneous mechanisms can lead to the generation of the 5′RUNX1/3′CBFA2T1 chimeric gene; (2) molecular cytogenetic techniques may identify cryptic chromosomal changes, not detected by conventional cytogenetic analysis; (3) the crucial role of the 5′RUNX1/3′CBFA2T1 fusion gene in leukemogenesis does not depend on its location.