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The impact of splenectomy on health-related quality of life (HRQoL) in patients with immune thrombocytopenia (ITP) remains scarcely explored. Therefore, we evaluated HRQoL with the 36-Item Short-Form Health Survey (SF-36) in an internal cohort of 69 chronic ITP patients, overall and by type of treatment. Of these patients, 26 patients were splenectomized, while other patients were treated medically with thrombopoietin-receptor agonists (TPO-RAs) or immunosuppressive treatment. We also compared HRQoL of the splenectomized patients (internal cohort) with an external cohort of 63 splenectomized ITP patients and the general population. The median follow-up was 10 years (range 1-20). Splenectomized patients had a worse overall HRQoL profile than those who received medical therapy either with TPO-RAs or other treatments (OT), with clinically meaningful differences registered in several domains. These included physical functioning (Δ = - 17.0 and Δ = - 15.2, for TPO-Ras and OT, respectively, p = 0.065), role physical (Δ = - 9.7 and Δ = - 13.8, p = 0.483), and bodily pain (Δ = - 14.2 and Δ = - 18.8, p = 0.053). Compared to the general population, both internal and external splenectomized cohorts had an impaired HRQoL profile. Further studies on HRQoL in splenectomized ITP patients are needed to better understand the long-term impact of this surgical procedure.

Background Recent investigations in chronic myeloid leukemia (CML) have focused on the identification and characterization of leukemic stem cells (LSCs). These cells reside within the CD34+ /CD38─ /Lin─ fraction and score positive for CD26 (dipeptidylpeptidase IV) a marker, expressed in both bone marrow (BM) and peripheral blood (PB) samples, that discriminates CML cells from normal hematopoietic stem cells (HSCs) or from LSCs of other myeloid neoplasms. CD26 evaluation could be a useful tool to improve the identification of CML LCSs by using flow-cytometry assay. Methods CD26+ LSCs have been isolated from EDTA PB and BM samples of patients with leucocytosis suspected for CML. Analysis of LSCs CML has been performed by using custom-made lyophilized pre-titrated antibody mixture test and control tube and a CD45+ /CD34+ /CD38- /CD26+ panel as a strict flow cytometric gating strategy. Results The expression of CD26 on CD34+ /CD38- population was detectable in 211/211 PB and 84/84 BM samples of subsequently confirmed BCR-ABL+ CP-CML patients. None of the 32 samples suspicious for CML but scoring negative for circulating CD26+ LSCs were diagnosed as CML after conventional cytogenetic and molecular testing. To validate our results, we checked for PB CD26+ LSCs in patients affected by other hematological disorders and they all scored negative for CD26 expression. Conclusions We propose flow cytometry evaluation of CD26 expression on PB CD34+ /CD38- population as a new rapid, reproducible, and powerful diagnostic tool for the diagnosis of CML. © 2019 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals, Inc. on behalf of International Clinical Cytometry Society.

Transfusion-associated iron overload may lead to increased risk of infection, but its role in myelofibrosis (MF) has been scarcely explored. We evaluated 106 consecutive patients with primary or secondary MF. Up to 38% of patients were transfusion-dependent (TD) with a median of 14 RBC units received. Median observation time was 36 months (range 3–203). Forty-five percent of patients experienced one or more infectious episodes for a total of 69 infectious events, 13 (19%) of which were severe. The 60-month cumulative incidence of infection was 64.1 ± 6.5%. TD patients showed a higher incidence of infection (HR = 2.13, p = 0.019). Transfusion burden was markedly greater in TD patients with infectious complication (median 24 RBC units vs 15 RBC units; p = 0.012). The 60-month overall survival was 40 ± 5.9%. Lower International Prognostic Scoring System (IPSS) risk (p

SETBP1 variants occur as somatic mutations in several hematological malignancies such as atypical chronic myeloid leukemia and as de novo germline mutations in the Schinzel–Giedion syndrome. Here we show that SETBP1 binds to gDNA in AT-rich promoter regions, causing activation of gene expression through recruitment of a HCF1/KMT2A/PHF8 epigenetic complex. Deletion of two AT-hooks abrogates the binding of SETBP1 to gDNA and impairs target gene upregulation. Genes controlled by SETBP1 such as MECOM are significantly upregulated in leukemias containing SETBP1 mutations. Gene ontology analysis of deregulated SETBP1 target genes indicates that they are also key controllers of visceral organ development and brain morphogenesis. In line with these findings, in utero brain electroporation of mutated SETBP1 causes impairment of mouse neurogenesis with a profound delay in neuronal migration. In summary, this work unveils a SETBP1 function that directly affects gene transcription and clarifies the mechanism operating in myeloid malignancies and in the Schinzel–Giedion syndrome caused by SETBP1 mutations., SETBP1 variants occur as somatic mutations in several malignancies and as de novo germline mutations in developmental disorders. Here the authors provide evidence that SETBP1 binds to gDNA in AT-rich promoter regions to promote target gene upregulation, indicating SETBP1 functions directly to regulate transcription.

The introduction of second-generation tyrosine-kinase inhibitors (TKIs) has generated a lively debate on the choice of first-line TKI in chronic phase, chronic myeloid leukemia (CML). Despite the TKIs have different efficacy and toxicity profiles, the planned use of two TKIs has never been investigated. We report on a phase 2 study that was designed to evaluate efficacy and safety of a treatment alternating nilotinib and imatinib, in newly diagnosed BCR-ABL1 positive, chronic phase, CML patients. One hundred twenty-three patients were enrolled. Median age was 56 years. The probabilities of achieving a complete cytogenetic response, a major molecular response, and a deep molecular response (MR 4.0) by 2 years were 93%, 87%, and 61%, respectively. The 5-year overall survival and progression-free survival were 89%. Response rates and survival are in the range of those reported with nilotinib alone. Moreover, we observed a relatively low rate of cardiovascular adverse events (5%). These data show that the different efficacy and toxicity profiles of TKIs could be favorably exploited by alternating their use. Am. J. Hematol. 91:617-622, 2016. © 2016 Wiley Periodicals, Inc.

Supplemental Digital Content is available for this article., Atypical chronic myeloid leukemia (aCML) is a BCR-ABL1-negative clonal disorder, which belongs to the myelodysplastic/myeloproliferative group. This disease is characterized by recurrent somatic mutations in SETBP1, ASXL1 and ETNK1 genes, as well as high genetic heterogeneity, thus posing a great therapeutic challenge. To provide a comprehensive genomic characterization of aCML we applied a high-throughput sequencing strategy to 43 aCML samples, including both whole-exome and RNA-sequencing data. Our dataset identifies ASXL1, SETBP1, and ETNK1 as the most frequently mutated genes with a total of 43.2%, 29.7 and 16.2%, respectively. We characterized the clonal architecture of 7 aCML patients by means of colony assays and targeted resequencing. The results indicate that ETNK1 variants occur early in the clonal evolution history of aCML, while SETBP1 mutations often represent a late event. The presence of actionable mutations conferred both ex vivo and in vivo sensitivity to specific inhibitors with evidence of strong in vitro synergism in case of multiple targeting. In one patient, a clinical response was obtained. Stratification based on RNA-sequencing identified two different populations in terms of overall survival, and differential gene expression analysis identified 38 significantly overexpressed genes in the worse outcome group. Three genes correctly classified patients for overall survival.

Therapy with Tyrosine Kinase Inhibitors (TKIs) changed the fate of Philadelphia-positive Chronic Myeloid Leukemia (CML). At present, the therapeutic strategy aims to improve the management of the disease and the quality of life of the patients. In July 2015, we started a prospective multicentric randomized trial with the aim to validate the policy of the intermittent de-escalation treatment and to explore the impact of this strategy on the Quality of Life. To this purpose, CML patients older than 60 years in stable (≥2 years) MR3.0 or MR4.0 molecular response were randomized to receive a FIXED intermittent TKIs regimen (one month ON and one month OFF), as previously published (Russo D, Blood 2013; Russo D, BCJ 2015), versus a PROGRESSIVE intermittent TKIs regimen (one month ON and one month OFF for the 1st year; one month ON and two months OFF for the 2nd year; one month ON and three months OFF from the 3rd year) (OPTkIMA study, ClinicalTrials.gov: NCT02326311). Molecular monitoring was performed according to the 2015 ELN guidelines, every 3 months by RT-PCR on peripheral blood (Baccarani M,Blood 2013). In case of MR3.0 (MMR) loss, checked in two monthly consecutive RT-PCR analysis, patients were planned to exit the study and to resume TKIs daily. This first interim report have been focused on the patients who, by intention to treat, have completed the first year of the study for an historical comparison with the previous INTERIM trial (Russo D, Blood 2013; Russo D, BCJ 2015). During the first year, both the patients randomized in the FIXED and in the PROGRESSIVE arms were given the TKIs treatment one month ON and one month OFF. Up to June 2018, 177 patients have been enrolled by 26 Italian Hematological Centers (first patient randomized in July 2015) and 121/177 patients (68%) completed the first year of OPTkIMA study. The median age was 71 years (range 60-89) and 64% of the patients were belonging to the Sokal intermediate/high risk goup. 96/121 (79%), 14/121 (12%) and 11/121 (9%) patients were receiving imatinib (IMA), nilotinib (NILO) and dasatinib (DAS), at the time of enrollment. Overall, 59/121 (49%) and 62/121 (51%) patients have been randomized in the FIXED and PROGRESSIVE arm, respectively. 41/62 patients (66%) randomly assigned to the PROGRESSIVE arm have entered the second year of therapy. 34/121 patients (28%) went out of the study during the first year. The reasons for protocol discontinuation were: informed consent withdrawn (2 cases), second cancer (4 cases), loss of MR3.0 (28 cases). (Table 1). Among the 28 patients who lost the MMR, 17 and 11 were in MR4.0 and MR3.0, respectively, when they were enrolled into the study. Thus the probability of loosing the MR3.0 while on OPTkIMA was 21,7% at one year (Figure 1). All the 28 patients resumed TKIs continuously and all obtained at least the MR3.0 response, within 6 months and are currently included in the study follow up. The intermittent treatment was well tolerated, with 4 serious adverse events (1 appendicitis, 1 atrial fibrillation, 1 cardiac failure, 1 hip fracture) and 3 adverse events (1 diarrohea, 1 pruritus and 1 fever), none of which have been considered treatement-related. None of the patients experienced the TKI withdrawn syndrome. According to this first interim report, we found that a policy of intermittent TKIs administration in elderly patients is safe and well tolerated. Analysis of patient-reported QoL outcomes is ongoing and will further add information on the overall treatment effectivness of the new PROGRESSIVE intermittent TKI administration. After the 1st year, 28/121 patients (23%) lost MR3.0 and all of them re-gained the major molecular response within 6 months from resumption of continuous treatment. The probability of MR3.0 loss while on OPTkIMA at 1 year was 21,7% and this is quite comparable with the 20% MR3.0 loss observed in the previous INTERIM trial (Russo D, Blood 2013; Russo D, BCJ 2015). Disclosures Efficace: Bristol Meyers Squibb: Consultancy; Seattle Genetics: Consultancy; Lundbeck: Research Funding; TEVA: Research Funding; AMGEN: Research Funding; Incyte: Consultancy; Amgen: Consultancy; TEVA: Consultancy; Orsenix: Consultancy. Abruzzese:Novartis: Consultancy; BMS: Consultancy; Pfizer: Consultancy; Ariad: Consultancy. Bonifacio:Incyte: Consultancy; Pfizer: Consultancy; Amgen: Consultancy; Novartis: Research Funding; Bristol Myers Squibb: Consultancy. Castagnetti:Incyte: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Bristol Meyers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Giai:Novartis: Consultancy; Pfizer: Consultancy.

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Chronic myeloid leukemia (CML) patients in sustained “deep molecular response” may stop TKI treatment without disease recurrence; however, half of them lose molecular response shortly after TKI withdrawing. Well-defined eligibility criteria to predict a safe discontinuation up-front are still missing. Relapse is probably due to residual quiescent TKI-resistant leukemic stem cells (LSCs) supposedly transcriptionally low/silent and not easily detectable by BCR-ABL1 qRT-PCR. Bone marrow Ph+ CML CD34+/CD38− LSCs were found to specifically co-express CD26 (dipeptidylpeptidase-IV). We explored feasibility of detecting and quantifying CD26+ LSCs by flow cytometry in peripheral blood (PB). Over 400 CML patients (at diagnosis and during/after therapy) entered this cross-sectional study in which CD26 expression was evaluated by a standardized multiparametric flow cytometry analysis on PB CD45+/CD34+/CD38− stem cell population. All 120 CP-CML patients at diagnosis showed measurable PB CD26+ LSCs (median 19.20/μL, range 0.27–698.6). PB CD26+ LSCs were also detectable in 169/236 (71.6%) CP-CML patients in first-line TKI treatment (median 0.014 cells/μL; range 0.0012–0.66) and in 74/112 (66%), additional patients studied on treatment-free remission (TFR) (median 0.015/μL; range 0.006–0.76). Notably, no correlation between BCR-ABL/ABLIS ratio and number of residual LSCs was found both in patients on or off TKIs. This is the first evidence that “circulating” CML LSCs persist in the majority of CML patients in molecular response while on TKI treatment and even after TKI discontinuation. Prospective studies evaluating the dynamics of PB CD26+ LSCs during TKI treatment and the role of a “stem cell response” threshold to achieve and maintain TFR are ongoing.

Determination of liver iron concentration is essential to predict iron related tissue damage and to guide chelation therapy. To assess the reliability of a single biopsy iron concentration determination in representing the whole liver iron concentration, we conducted a prospective study performing two immediately successive liver biopsies from 61 noncirrhotic, iron overloaded thalassemia patients, directing the needle to different direction from the same skin cut. The correlation among sample biopsies was determined by both regression analysis and the Bland–Altman method. The results showed that overall correlation between the two samples was high (Pearson's coefficient of correlation r = 0.970, P < 0.0001; 95% CI 0.951–0.981; R2 = 0.941). To evaluate if sample dimension had an impact on the analysis we analyzed separately biopsy couples were both sample gross weight were ≥1 mg dry weight (n = 16) from the others [one or both had a specimen gross weight

Determination of liver iron concentration is essential to predict iron related tissue damage and to guide chelation therapy. To assess the reliability of a single biopsy iron concentration determination in representing the whole liver iron concentration, we conducted a prospective study performing two immediately successive liver biopsies from 61 noncirrhotic, iron overloaded thalassemia patients, directing the needle to different direction from the same skin cut. The correlation among sample biopsies was determined by both regression analysis and the Bland-Altman method. The results showed that overall correlation between the two samples was high (Pearson's coefficient of correlation r = 0.970, P < 0.0001; 95% CI 0.951-0.981; R(2) = 0.941). To evaluate if sample dimension had an impact on the analysis we analyzed separately biopsy couples were both sample gross weight were ≥1 mg dry weight (n = 16) from the others [one or both had a specimen gross weight

Despite the recent identification of recurrent SETBP1 mutations in atypical chronic myeloid leukemia (aCML), a complete description of the somatic lesions responsible for the onset of this disorder is still lacking. To find additional somatic abnormalities in aCML, we performed whole-exome sequencing on 15 aCML cases. In 2 cases (13.3%), we identified somatic missense mutations in the ETNK1 gene. Targeted resequencing on 515 hematological clonal disorders revealed the presence of ETNK1 variants in 6 (8.8%) of 68 aCML and 2 (2.6%) of 77 chronic myelomonocytic leukemia samples. These mutations clustered in a small region of the kinase domain, encoding for H243Y and N244S (1/8 H243Y; 7/8 N244S). They were all heterozygous and present in the dominant clone. The intracellular phosphoethanolamine/phosphocholine ratio was, on average, 5.2-fold lower in ETNK1-mutated samples ( P < .05). Similar results were obtained using myeloid TF1 cells transduced with ETNK1 wild type, ETNK1-N244S, and ETNK1-H243Y, where the intracellular phosphoethanolamine/phosphocholine ratio was significantly lower in ETNK1-N244S (0.76 ± 0.07) and ETNK1-H243Y (0.37 ± 0.02) than in ETNK1-WT (1.37 ± 0.32; P = .01 and P = .0008, respectively), suggesting that ETNK1 mutations may inhibit the catalytic activity of the enzyme. In summary, our study shows for the first time the evidence of recurrent somatic ETNK1 mutations in the context of myeloproliferative/myelodysplastic disorders.

The aim of this study was to investigate the effects of a non-standard, intermittent imatinib treatment in elderly patients with Philadelphia-positive chronic myeloid leukaemia and to answer the question on which dose should be used once a stable optimal response has been achieved. Seventy-six patients aged ⩾65 years in optimal and stable response with ⩾2 years of standard imatinib treatment were enrolled in a study testing a regimen of intermittent imatinib (INTERIM; 1-month on and 1-month off). With a minimum follow-up of 6 years, 16/76 patients (21%) have lost complete cytogenetic response (CCyR) and major molecular response (MMR), and 16 patients (21%) have lost MMR only. All these patients were given imatinib again, the same dose, on the standard schedule and achieved again CCyR and MMR or an even deeper molecular response. The probability of remaining on INTERIM at 6 years was 48% (95% confidence interval 35–59%). Nine patients died in remission. No progressions were recorded. Side effects of continuous treatment were reduced by 50%. In optimal and stable responders, a policy of intermittent imatinib treatment is feasible, is successful in about 50% of patients and is safe, as all the patients who relapsed could be brought back to optimal response.

Therapeutic approach for chronic myeloid leukemia (CML) patients has undergone a revolutionary change with the introduction of tyrosine kinase inhibitors, which improved overall survival and quality of life. Optimal therapy adherence has become of paramount importance to maximize the benefits in the long-term outcome. Several evidences have been reported that personal factors, such as social support, psychological and subjective perceptions about the drug used and the future, could influence adherence. We here report the results of a questionnaire specifically designed to evaluate factors influencing adherence and perceptions about the future, distributed to patients during regional Italian meetings. Overall, 1133 patients compiled the questionnaire: median age was 57 years. High rate of adherence was reported, but 42% of interviewed patients admitted that they had occasionally postponed a dose and 58% had discontinued therapy mainly for forgetfulness. The majority of patients discussed with personal physician about the importance of adherence and received sufficient information about illness and treatment, but would like to have discussed more about discomfort, anxiety and fear of the future. Summarizing personal drug compliance and estimating how many days a month, on average, the patients did not take the drug, the majority answered that it was less than 3 days (55%) and only a minority (4%) admitted that it was more than 7 days. Interviewed about discontinuation, 49% of patients answered that wouldn't interrupt because of fear of losing all the results achieved so far. This study suggests a higher level of satisfaction with more information received but the need of improving communication about possible future treatment free remission.

BACKGROUND: The incidence of chronic myeloid leukemia (CML) increases with age, but it is unclear how the characteristics of the disease vary with age. In children, where CML is very rare, it presents with more aggressive features, including huge splenomegaly, higher cell count and higher blast cell percentage. PATIENTS AND METHODS: To investigate if after childhood the disease maintains or loses these characteristics of aggressiveness, we analyzed 2784 adult patients, at least 18 years old, registered by GIMEMA CML WP over a 40-year period. RESULTS: Young adults (YAs: 18-29 years old) significantly differed from adults (30-59 years old) and elderly patients (at least 60 years old) particularly for the frequency of splenomegaly (71%, 63% and 55%, P < 0.001), and the greater spleen size (median value: 4.5, 3.0 and 1.0 cm, P < 0.001). According to the EUTOS score, that is age-independent, high-risk patients were more frequent among YAs, than among adult and elderly patients (18%, 9% and 6%, P < 0.001). In tyrosine kinase inhibitors-treated patients, the rates of complete cytogenetic and major molecular response were lower in YAs, and the probability of transformation was higher (16%, 5% and 7%, P = 0.011). CONCLUSIONS: The characteristics of CML or the host response to leukemia differ with age. The knowledge of these differences and of their causes may help to refine the treatment and to improve the outcome. CLINICAL TRIAL NUMBERS: NCT00510926, NCT00514488, NCT00769327, NCT00481052. © The Author 2014. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

BACKGROUND: The INTERIM study (ClinicalTrials.gov NCT 00858806) showed that in elderly (> 65 years) Ph+ CML patients selected for a stable complete cytogenetic response (CCgR) lasting > 2 years, the policy of intermittent imatinib treatment (one month on/one month off) may affect the markers of residual disease (CCgR and major molecular response, MMR or MR3.0), but not the clinical outcomes (overall survival and progression-free survival) (Russo D et al, Blood 2013; 121(26):5138-44). AIMS: To update the results of the INTERIM Study, with a follow up ≥ 5 years. METHODS: After 4 years of follow up, patients continouing INTERIM treatment were monitored with peripheral blood RT-Q-PCR every 3 months according to the ELN-2013 guidelines. RESULTS: At 48thmonth, out of 76 patients enrolled in the INTERIM study, 13 (17%) had lost CCgR and MMR, 14 (18%) had lost MMR only and 50 patients (75%) continued INTERIM. The patients who had lost CCgR and/or MMR resumed imatinib continuously and all of them regained the CCgR and the MMR, within 3 to 12 months. No patient progressed to accelerated or blastic phase, or developed clonal chromosomal abnormalities in Ph+ cells, or BCR-ABL mutations. No patient complained of new or more severe side effects during the months “on”. After a follow up ≥ 5 years, 45/76 (59%) enrolled patients are on INTERIM, with a probability of maintaining intermittent administration of 59% (95% CI: 46-69). No patient lost the CCgR and only 9 additional patients lost the MMR while on intermittent treatment. All these patients resumed continuous imatinib treatment and regained the MMR. Thus, at ≥ 5 years, the probability of maintaing CCgR is 80% (95% CI 68-87) and the probability of maintaining the MMR is 61% (95% CI: 48-71). From start of INTERIM, 6 patients died but no deaths were related to CML progression (3 cases of other non haematological neoplasms, 1 stroke, 1 myocardial infarction, 1 chronic obstructive pulmonary disease).The PFS at ≥ 5 years is 94% (95% CI: 89-100) CONCLUSIONS: In summary, with a follow up ≥ 5 years, intermittent imatinib administration (INTERIM) confirmed to be safe, to produce a reversible increase of residual molecular disease in about one third of patients, but not to affect the long-term outcome. Aknowledgments: This work was supported in part by EuropeanLeukemiaNet (contract LSHC-CT-2004-503216) through the European Treatment and Outcome Study (EUTOS), supported by Novartis Oncology Europe, and COFIN 2009 Disclosures Russo: Celgene: Research Funding; Gilead: Research Funding; Novartis: Consultancy. Martinelli:Novartis: Speakers Bureau; Bristol-Meyers and Squibb: Speakers Bureau; Pfizer: Speakers Bureau. Soverini:Novartis: Consultancy, Honoraria; Bristol-Meyers Squibb: Consultancy, Honoraria; Ariad: Consultancy, Speakers Bureau. Turri:Novartis: Consultancy, Honoraria; Bristol-Meyers Squibb: Consultancy, Honoraria. Castagnetti:Novartis: Consultancy, Honoraria; Bristol-Meyers Squibb: Consultancy, Honoraria. Breccia:novartis: Consultancy; BMS: Consultancy; Celgene: Consultancy. Abruzzese:Novartis: Consultancy. Tiribelli:Novartis: Consultancy, Honoraria; Bristol-Meyers and Squibb: Consultancy, Honoraria. Rosti:Consultant: Consultancy, Speakers Bureau; Bristol-Meiers Squibb: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau.

Atypical Chronic Myeloid Leukemia (aCML) is a clonal disorder belonging to the Myeloproliferative/Myelodysplastic (MPN/MDS) group. The molecular lesions responsible for the onset of aCML remained unknown until 2013 when recurrent somatic mutations of SETBP1 were identified. However, the frequency of SETBP1 mutations in aCML does not exceed 25-30%, which suggests that other lesions may play a role in the remaining cases. To gain further insight into the somatic variants responsible for the onset of aCML, we generated whole-exome and transcriptome sequencing data on 15 matched case/control aCML samples. A total of 151 non-synonymous and 42 synonymous single-nucleotide somatic variants were identified. Of these, 140 were transitions and 53 transversions. Of the non-synonymous mutations, 141 were missense and 10 nonsense mutations. In 2/15 (13.3%) samples we identified the presence of missense, single-nucleotide somatic variants occurring in the ETNK1 gene affecting two contiguous residues: H243Y and N244S. Sanger sequencing confirmed the presence and the somatic nature of the variants. Targeted resequencing of 383 clonal hematological disorders showed evidence of mutated ETNK1 in 7/70 aCML (10.0%, 95% C.I. 4.6-19.5%) and in 2/77 chronic myelomonocytic leukemia samples (CMML; 2.6%, 95% C.I. 0.2-9.5%) %), while no ETNK1 mutations were identified in the remaining hematological disorders. All the variants were heterozygous and clustered in the same, highly conserved region within the kinase domain (1/9 H243Y and 8/9 N244S). Somatic, heterozygous ETNK1 variants have been also recently reported in 10% of Systemic Mastocytosis (SM) cases and in 22% of SM with associated hypereosinophilia (Lasho T et al., Abstract 4062, EHA2014); strikingly, there is a large overlap between the variants that we identified in aCML and CMML and those described for SM (3 N244S and 2 G245A), which suggests that the common hotspot region may play a critical and yet unknown functional role. The hitherto described data suggest that ETNK1 variants are restricted to a limited subset of hematological disorders. This is further supported by the lack of somatic ETNK1 mutations in 60 paired whole-genome and over 600 exomes, comprising 276 paired tumor/germline primary samples and 344 cancer cell lines (http://cancer.sanger.ac.uk/cancergenome/projects/cell_lines/). In 2/6 ETNK1 mutated aCML cases (33%, 95% C.I. 9%-70%), we detected the presence of a coexisting somatic SETBP1 variant. The fraction of SETBP1 mutations identified in this group is perfectly in line with the overall frequency of SETBP1 mutations in aCML, suggesting that mutations occurring in ETNK1 and SETBP1 are not mutually exclusive. To discriminate if ETNK1 and SETBP1 mutations occur in different or in the same clone, we performed colony assay experiments, revealing the coexistence of the two somatic mutations within the same clone. Liquid Chromatography – Mass Spectrometry experiments revealed that in ETNK1 mutated cells the intracellular levels of phosphoethanolamine are over 5-fold lower than in the wild-type counterpart (p < 0.05), suggesting that ETNK1 mutations may impair the physiological catalytic activity of the kinase. Taken globally these data identify ETNK1 somatic mutations as a new oncogenic lesion in aCML and CMML, two overlapping MDS/MPN neoplasms. They also show that ETNK1 variants apparently cause a loss-of-function effect, leading to a decrease in the intracellular levels of phosphoethanolamine. Disclosures Campbell: 14M Genomics Limited: Consultancy, Equity Ownership.

Background: the Registro Italiano Trombocitemie (RIT) was activated mainly to evaluate the diagnosis and therapy appropriateness in the thrombocythemic patients with Ph-negative myeloproliferative neoplasm (MPN) observed in the adhering centers. Objective: to evaluate how the diagnostic and therapeutic approach changed in the RIT patients diagnosed with thrombocythemic MPN in the last two decades. Methods: the RIT centers registered by a web-based system during the years 2005-2014 their thrombocythemic MPN patients, and semesterly updated their follow-up data. For patients diagnosed before 2005, the data on diagnosis and prior follow-up were retrospectively collected. The diagnostic process and the initial treatment (started into the first year after diagnosis) were comparatively analyzed in the patients diagnosed before and after 2005. Results: the RIT centers registered 2629 patients. 2388 of them, object of this analysis, were diagnosed between1995 and 2014: 1098 (46%) in the decade 1995-2004 (Group I), and 1290 (54%) in the decade 2005-2014 (Group II). The diagnostic process in the patients of Group II and Group I included bone marrow biopsy (BMB, performed into 1 year and before any cytoreduction): 85% vs 80%, p The patients of Group II, as compared with those of Group I, showed a similar gender distribution (M/F ratio 0.61 vs 0.65, p 0.452), and had at diagnosis: a higher age (median 60 vs 57 years, p 60 years in 50% vs 45% of cases, p Moreover, they had: a lower platelet (PLT) count (median 737 vs 775 x 109/L, p 10 x 109/L in 28% vs 26% cases); a similar median levels of hematocrit (HCT %, in females 41.4 vs 41.0; in males 44.7 vs 44.6) and hemoglobin (Hb g/dL, in females 13.8 vs 13.6; in males 15.0 vs 14.9). The BMB, revised according to the WHO 2008 criteria, showed a not different distribution (p 0.21) of ET (64% vs 61%), ep-PMF (16% vs 17%), PMF (3% vs 2%), PV (4% vs 4%), and U-MPN (13% vs 16%. The JAK2 V617F mutation in patients of Group II (at diagnosis) and of Group I (after diagnosis) was found in 62% and in 58% of tested cases (p 0.054), respectively. The patients at high standard thrombotic risk were 58% vs 52%, p 0.004. In the patients of Group II and Group I the distribution of the treatment started into the first year was significantly different (p The treatment CYT ± AntiPLT was started in the patients at high standard thrombotic risk with a rate of 81% vs 80%, respectively, and in the patients at low standard thrombotic risk in 37% vs 43% of cases, respectively. The initial treatment CYT±AntiPLT was related, in multivariate analysis, both in patients of Group II and Group I, with older age (>60 and 40-60 vs 1000 and 700-1000 vs Conclusion: in the studied thrombocythemic MPN patients the real-life diagnostic approach was improved after 2005 not only due to the routine use of JAK2 tests, but also due to the higher rate of BMB done (85% vs 80%). The appropriateness of the cytoreductive treatment (CYT±AntiPLT started into one year from diagnosis) remained high in patients at high standard thrombotic risk (over 80% of cases were treated). Concomitantly, the inappropriate use of the cytoreductive drugs in patients at low standard thrombotic risk appreciably decreased (from 43% to 37% of cases). Moreover, it has to be remarked that the therapeutic approach was significantly influenced not only by older age and prior thrombosis, but also by thrombocytosis (PLT count >700 x 109/L), disease-related symptoms, and inconstantly by leukocytosis and CVRFs. Table Table. Disclosures Gugliotta: SHIRE Co.: Honoraria. Gugliotta:Bristol Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria.

Background: In chronic phase (CP) chronic myeloid leukemia (CML) the disease characteristics at diagnosis, risk distribution, probability of transformation to accelerated/blast phase (AP/BP), and toxicities may vary according to age. Nilotinib has shown better efficacy compared to imatinib, but it has been associated to a higher incidence of arterial thrombotic events (ATEs). Little is known on the efficacy, safety and long-term outcome of nilotinib treated patients in different age groups. Aims: To investigate the efficacy and safety, particularly the cardiovascular safety, of first-line treatment with nilotinib according to age distribution. Methods: We analyzed 345 patients ≥ 18 years of age with CP CML enrolled in clinical trials of the GIMEMA CML WP investigating nilotinib as first-line treatment. Patients were treated with: nilotinib 400 mg BID (n=73); rotation of nilotinib 400 mg BID / imatinib 400 mg OD (3-month periods for each drug)(n=123); nilotinib 300 mg BID (n=149). The median follow-up was 58 months. We divided the patients in 3 groups of age (table): 18-49 years (group A, 147 pts, median age: 39 years); 50-64 years (group B, 109 pts, median age 58 years); and ≥ 65 years (group C, 89 pts, median age 71 years). We analyzed in detail the response rates, events and outcome. Definitions: Major molecular response (MMR): BCR-ABL≤0.1% (IS), with > 10.000 ABL copies; MR4: BCR-ABL≤0.01% (IS), with > 10.000 ABL copies. Events: permanent discontinuation of nilotinib for any reason, including adverse events, progression to AP/BP, or deaths. ATEs: peripheral arterial obstructive disease (PAOD), acute coronary syndrome, chronic ischemic heart disease, significant carotid stenosis and ischemic stroke, or other significant ischemic events. Results: EUTOS high risk patients were: 8.8, 5.5, 1.1% in group A, B, and C, respectively (p=0.048). We did not observe significant differences in molecular response rates (table), including BCR-ABL/ABL 65 years (p=0.015). Progressions to AP/BP were significantly more frequent in younger (18 - 49 years) patients (6.8%) compared to older (> 50 years) patients (1.5%), p= 0.019. Overall, 19 patients died (group A: 4.8%; B: 2.8%; C: 10%). In patients < 65 years all deaths (10) were leukemia-related, while in patients >65 years, all but one (8/9) deaths were leukemia-unrelated. The 6-year overall-survival was 94%, 97%, and 84% in pts 18-49 years, 50-64 years, and > 65 years of age (p=0.12) Summary/Conclusion: Nilotinib as first-line treatment of newly diagnosed CP CML patients showed high rates of molecular responses in all age groups. However, compared to older patients, significant more progressions occurred in younger (18-49 years) ones; the causes of death in this group were all leukemia-related. In contrast, in elderly patients (>65 years) the causes of death were almost all leukemia-unrelated. ATEs were rare (2%) in patients of 18-49 years, while high rates (> 10%) of ATEs were recorded in those of 50-64 years; as expected, ATEs occurred even more frequently in patients > 65 years. These data suggest that while in patients > 50 years more attention should be focused on the prevention of ATEs, in younger ones more efforts are needed to avoid the progression of CML to accelerated/blast phase. Disclosures Gugliotta: Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria. Castagnetti:Bristol-Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; ARIAD Pharmaceuticals: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Breccia:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Honoraria; Celgene: Honoraria; Ariad: Honoraria; Pfizer: Honoraria. Carella:Millenium: Speakers Bureau; Genentech: Speakers Bureau. Tiribelli:Ariad Pharmaceuticals: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau. Soverini:Ariad: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy. Cavo:Janssen-Cilag: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Millennium: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Martinelli:Pfizer: Consultancy, Speakers Bureau; ARIAD: Consultancy; Roche: Consultancy; MSD: Consultancy; Novartis: Speakers Bureau; Amgen: Consultancy; Genentech: Consultancy; BMS: Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; ARIAD: Consultancy; Roche: Consultancy; MSD: Consultancy; Amgen: Consultancy; Genentech: Consultancy. Saglio:Ariad: Consultancy; Pfizer: Consultancy; Bristol Myers Squibb: Consultancy. Baccarani:Bristol Myers Squibb: Consultancy, Honoraria, Speakers Bureau; Pfizer: Honoraria, Other: personal fees, Speakers Bureau; Ariad: Honoraria, Other: personal fees, Speakers Bureau; Novartis: Honoraria, Speakers Bureau. Rosti:Roche: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau; Incyte: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau.

Abstract 4 Introduction Current treatment recommendations in polycythemia vera (PV) have emphasized to maintain the hematocrit (HCT) values Aim In a large scale randomized clinical trial (Cyto-PV) we prospectively determined the efficacy and safety of maintaining the recommended HCT target versus HCT levels in the range of 0.45–0.50 to prevent thrombotic events in PV patients. Methods Patients were eligible if they met WHO-2008 diagnostic criteria for PV. Both cases with newly diagnosed disease and previous treatment were centrally randomized to Arm A (HCT Results Arm A and Arm B included 182 and 183 patients respectively. At randomization, there were no significant differences between the two groups with respect to age, gender, years from diagnosis to recruitment, previous history of major thrombosis, bleeding, concomitant cardiovascular risk factors, and hematological presentation. Treatments were equally distributed with regard to phlebotomy, antiplatelet drugs, warfarin and hydroxyurea or their combination. After randomization, median HCT levels in arm A and Arm B during follow-up (median 31.0 months) were 0.44 and 0.48 respectively. A quarter of patients of arm A and Arm B failed to maintain the assigned HCT levels during the study period. Noticeable was that leukocyte levels remained higher in arm B than Arm A while no difference was revealed concerning the platelet count. Additionally, no difference in the safety profile was recognizable. As compared with arm B, the more intensive treatment aimed at maintaining the HCT Conclusion In this randomized clinical trial, the incidence of major cardiovascular events was 4 fold higher in patients who maintained HCT levels >0.45. Therefore, an HCT level 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 Tyrosine Kinase Inhibitors (TKI) have completely changed the scenario of CML and dramatically improved the outcomes. Thus, early identification of patients expecting poor outcome is crucial to offer alternative TKI regimens or in some selected cases stem cell transplantation before disease progression may occur. The Evaluating Nilotinib Efficacy and Safety in Trial as First-Line Treatment (ENEST1st) is a phase 3b is an open-label study of nilotinib 300 mg twice daily (BID) in adults with newly diagnosed BCR-ABL positive CP-CML. Aim of the ENEST1st sub-study N10 was to investigate BM microenvironment markers that regulate leukemic stem cells in the bone marrow (BM) niche of Nilotinib-treated patients. Methods The study enrolled patients in 21 Italian ENEST1st participating centers. Response was based on ELN recommendations (Baccarani M, et al. Blood 2013 122:872-884). In an interim analysis, molecular and cytogenetic response by 24 months was assessed. Mononuclear cells were collected from BM and PB samples at the screening visit (V0) and after 3 months of treatment (V4). RT-qPCR for the expression of 10 genes (ARF, KIT, CXCR4, FLT3, LIF, NANOg, PML, PRAME, SET and TIE), involved in the stemness and hematopoietic stem cells survival signaling regulation was conducted. RT-qPCR data were normalized by the expression of GUS mRNA (normalized copy number, NCN). Plasma samples were collected at different time points from both BM or PB samples. Concentrations of 20 different analytes, including IL-1a, IL-3, M-CSF, SCF, SDF1-a, TRAIL, HGF, PDGF-bb, IL1b, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, G-CSF, GM-CSF, MIP-1a, TNF-a, and VEGF, were simultaneously evaluated using commercially available multiplex bead-based sandwich immunoassay kits. Results 33 out of 37 patients enrolled were available for an interim molecular analysis at 24 months: an optimal response was achieved in 25 patients, a warning response in 5 patients and a failure response in 3 patients. We observed a significant correlation between the expression of two genes involved in the regulation of stem cell pluripotency (NANOg) or cytokine signaling (SET) and patient outcome. Indeed, NANOg and SET mRNA were significantly down-regulated in PB samples at diagnosis of patients with optimal response compared to patients with warning/failure response (NANOg mRNA: 0.3±0.25 NCN vs 0.6±0.7 NCN, respectively; p=0.05; SET mRNA: 0.2±0.3 NCN vs 2.3±4.2 NCN, respectively; p=0.03). We also investigated the plasma level of several factors involved in the hematopoietic stem cells (HSCs). Some of these markers showed a significant correlation with patient's outcome when evaluated at diagnosis in either PB or BM samples. Indeed, high level of IL12 (in the BM samples), or HGF, mCSF and SCF (in the PB samples) were associated to a worst prognosis markers, since significantly correlating with no MMR@12months (IL12, p=0.03), intermediate/high Socal score (mCSF, p=0.03; SCF, p=0.03), no reduction of MMR below to 1 at 3 month (SCF, p=0.04) or warning/failure response to Nilotinib treatment (HGF, p=0.03; SCF, p=0.04). Indeed, we find a lower levels of PDGFb, SDF1, TNFa, TRAIL (in the BM samples), and HGF, SDF1, TRAIL (in the PB samples) in those patients with intermediate/high Hasford or Sokal score (PDGFb, p=0.0007; SDF1, p=0.02), warning/failure response to Nilotinib treatment (HGF, p=0.03) or lacking of MMR4.0 (SDF1, p=0.01; TNFa, p=0.02; TRAIL, p=0.05). Conclusion/Summary Taken together, our results suggest that the expression analysis of genes involved in cell pluripotency (NANOg) and/or cell signaling (SET) at baseline, may indicate early achievement of deep molecular response in shown CML-CP patients treated with nilotinib. In addition, in patients with optimal response to Nilotinib, high concentration of SDF-1, TRAIL (inversely correlated with BCR-ABL, and associated to an higher susceptibility to apoptosis in the leukemic blasts) were observed as well as BM TNF (cell-extrinsic and potent endogenous suppressor of HSC activity). A lower concentration of several factors associated to hematopoietic progenitor cell growth and survival (including HGF, SCF and IL12) were observed compared to patients failing to achieve response to Nilotinib. These data strongly suggest that stromal microenvironment supports the viability of BCR-ABL cells in BM niches through direct feeding, or environment releasing of survival factors. Disclosures Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Martinelli:MSD: Consultancy; BMS: Speakers Bureau; Roche: Consultancy; ARIAD: Consultancy; Novartis: Speakers Bureau; Pfizer: Consultancy. Saglio:Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; Novartis Pharmaceutical Corporation: Consultancy, Honoraria. Galimberti:Novartis: Employment. Giles:Novartis: Consultancy, Honoraria, Research Funding. Hochhaus:Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

In addition to dysregulated JAK/STAT signaling, activation of the AKT/mTOR pathway occurs in myelofibrosis, a myeloproliferative neoplasm with no approved therapies. We conducted a phase 1/2 study with everolimus, an mTOR inhibitor, in 39 high- or intermediate-risk primary or postpolycythemia vera/postessential thrombocythemia myelofibrosis subjects. Responses were evaluated in 30 patients of phase 2. No dose-limiting toxicity was observed in phase 1 up to 10 mg/d. When this dose was used in phase 2, grade ≥ 3 toxicities were infrequent; the commonest toxicity was grade 1-2 stomatitis. Rapid and sustained splenomegaly reduction of > 50% and > 30% occurred in 20% and 44% of subjects, respectively. A total of 69% and 80% experienced complete resolution of systemic symptoms and pruritus. Response in leukocytosis, anemia, and thrombocytosis occurred in 15%-25%. Clinical responses were not associated with reduced JAK2V617F burden, circulating CD34+ cells, or cytokine levels, whereas CCDN1 mRNA and phospho-p70S6K level, known targets of mTOR, and WT1 mRNA were identified as possible biomarkers associated with response. Response rate was 60% when European Network for Myelofibrosis criteria were used (8 major, 7 moderate, 3 minor responses) or 23% when IWG-MRT criteria (1 partial response, 6 clinical improvements) were used. These results provide proof-of-concept that targeting mTOR pathway in myelofibrosis may be clinically relevant.

Chronic myelogenous leukemia (CML) is characterized by the expression of BCR-ABL tyrosine kinase, which results in increased cell proliferation and inhibition of apoptosis. In this study, we show that BCR-ABL-positive CML cell lines treated with imatinib (STI571) undergo G₁ cell cycle arrest associated with the accumulation of p57(Kip)², a cyclin-dependent kinase inhibitor (CKI). Interestingly, p57(Kip)² increase precedes the reported STI571-dependent upregulation of p27(Kip)¹. A number of complementary approaches allow the demonstration that p57(Kip)² buildup is due to the transcriptional activation of CDKN1C, the p57(Kip)²-encoding gene, while neither p57(Kip)² half-life elongation nor its cell relocalization were observed. We also identified a heretofore undescribed pattern of p57(Kip)² phosphorylated isoforms which, however, did not change in response to STI571 cell treatment. The imatinib-dependent p57(Kip)² upregulation occurs only in STI571-responsive cells, while the CKI accumulation was not evidenced in an imatinib-resistant clone. Nilotinib and dasatinib (second-generation BCR-ABL inhibitors), at concentrations comparable to those used in therapy, increase the CKI but do not affect p27(Kip)¹ level. Finally, CD34(+) cells from CML patients display a clear imatinib-dependent p57(Kip)² upregulation, which was not observed in CD34(+) cells from control subjects. In conclusion, our study points to p57(Kip)² as a novel and precocious effector of BCR-ABL targeting drugs.

Purpose Dasatinib is remarkably effective in treating patients with chronic myeloid leukemia (CML) resistant or intolerant to front-line imatinib. To date, most of the available data come from multicenter study in which the patients were censored for diverse reasons. Thus, further studies based on non selected populations are indeed opportune. Patients and Methods A total of 124 unselected patients with CML resistant or intolerant to imatinib received Dasatinib as second line therapy outside from clinical trials. Response (hematologic, cytogenetic and molecular), toxicities according to NCI-CTC, discontinuation/dose reduction of Dasatinib, event-free survival (EFS) and overall survival (OS) were evaluated. Results At 12 months, cumulative incidences of complete hematologic response (CHR), complete cytogenetic response (CCyR) and major molecular response (MMR) were 94%, 51% and 32% respectively. Cumulative EFS and OS were 91 and 93% respectively. These data are in line with those of clinical trials, but the population of the present study was not selected. Intriguingly, neither the dose nor the duration nor the response to prior imatinib affected the probability of achieving CCyR at 1 year with dasatinib (p= 0.7, 0.9 and 0.8 respectively). Moreover, we observed a statistically significant better outcome in term of both DFS and OS for patients not discontinuing dasatinib, even at low doses (p=0.05 and p=0.04), with respect to those who discontinued the drug due to grade III-IV toxicity. Conclusions Dasatinib as second line therapy is safe and highly effective for unselected patients with CML either resistant or refractory to IM even outside from clinical trials. Neither the dose nor the duration nor the response to imatinib affects the probability to respond to dasatinib. These findings indicate an increase possibility of cure for unselected patients resistant or intolerant to imatinib submitted to second line therapy with dasatinib in everyday clinical practice.

Previous observational studies suggest that rituximab may be useful in the treatment of primary immune thrombocytopenia (ITP). This randomized trial investigated rituximab efficacy in previously untreated adult ITP patients with a platelet count of 20 × 109/L or less. One hundred three patients were randomly assigned to receive 40 mg/d dexamethasone for 4 days with or without 375 mg/m2 rituximab weekly for 4 weeks. Patients who were refractory to dexamethasone alone received salvage therapy with dexamethasone plus rituximab. Sustained response (ie, platelet count ≥ 50 × 109/L at month 6 after treatment initiation), evaluable in 101 patients, was greater in patients treated with dexamethasone plus rituximab (n = 49) than in those treated with dexamethasone alone (n = 52; 63% vs 36%, P = .004, 95% confidence interval [95% CI], 0.079-0.455). Patients in the experimental arm showed increased incidences of grade 3 to 4 adverse events (10% vs 2%, P = .082, 95% CI, −0.010 to 0.175), but incidences of serious adverse events were similar in both arms (6% vs 2%, P = .284, 95% CI, −0.035 to 0.119). Dexamethasone plus rituximab was an effective salvage therapy in 56% of patients refractory to dexamethasone. The combination of dexamethasone and rituximab improved platelet counts compared with dexamethasone alone. Thus, combination therapy may represent an effective treatment option before splenectomy. This study is registered at http://clinicaltrials.gov as NCT00770562.

Abstract 1124 Poster Board I-146 Background Dasatinib (BMS-354825) is a multi-targeted kinase inhibitor of BCR-ABL and SRC with significant activity in patients with CML resistant or intolerant to front-line Imatinib (IM). However, the information so far available are to great extent limited to controlled clinical trials, which usually recruited patients with acceptable performance status, and no major organ dysfunction. Noteworthy, this fact, although essential for ethical reasons in clinical trials, may somehow determine an overestimation of the clinical benefit of new compounds. Thus, further studies based on non selected populations are indeed opportune. Patients and Methods A total of 124 patients (61 male and 63 females) with CML resistant or intolerant to IM received Dasatinib as second line therapy outside from clinical trials. 91 patients had a resistant disease, 19 were intolerant to IM and 14 patients were both resistant and intolerant to IM. Median age at switching to Dasatinib was 56.5 years (range 21-82). 62/124 patients escalated IM before switching from 400 to 600 mg/day and 26/62 patients received an additional dose escalation from 600 to 800 mg/day. Median time on IM prior to the switch to Dasatinib was 36 months (range 2-99), whereas median time on IM after dose escalation was 9 months (range: 1-43). Dasatinib was given at 100 mg once daily in 54/124 patients (43%), at 70 mg twice daily in 43/124 patients (35%) and at 50 mg once daily in the remaining patients (22%). Response (hematologic, cytogenetic and molecular), toxicities according to NCI-CTC, discontinuation/dose reduction of Dasatinib, event-free survival (EFS) and overall survival (OS) were evaluated. Results With a median follow-up of 12 months, 94% of patients attained a complete hematological response (CHR), whereas 69% and 51% achieved a major (MCyR) or a complete cytogenetic response and 32% attained a major molecular response (MMR). Cumulative EFS and OS at 12 months were 91 and 93% respectively. These data are in line with those of clinical trials, but the population of the present study was not selected. The commonest grade III/IV adverse events were thrombocytopenia and neutropenia, occurring in around half of patients. Only 3% of the patients suffered from a grade III-IV pleural effusion or dyspnea. The achievement of CHR, MCyR, CCyR and MMR significantly influenced EFS (p=0.006; 0.04; 0.008 and 0.003 respectively) and OS (p=0.002; 0.02; 0.004 and 0.001 respectively) both in univariate and in multivariate analysis. Intriguingly, neither the dose nor the duration of prior IM affected the response to Dasatinib, neither before switching to Dasatinib (p= 0.9 and p=0.4) nor after it (p=0.4 and 0.7). Moreover, dose escalation to IM 600 or 800 mg/day did not influence negatively the subsequent response to Dasatinib (p=0.8). In addition, we did not observe any difference in the response rate (CHR, MCyR, CCyR, MMR) between patients who received prior IM for more than 3 years with respect to those patients who received it for less than 3 years (p=0.9). Regarding Dasatinib therapy, we observed a statistically significant better outcome in term of both DFS and OS for those patients receiving lower doses continuously without interruption (p=0.05 and p=0.04) with respect to patients who received higher doses but were forced to stop the treatment due to grade III-IV toxicity. This observation clearly indicate the necessity of administer dasatinib continuously to patients in order to override possible mechanisms of resistance due to the reduction of the plasma level of the drug. Conclusions We confirm the safety and efficacy of Dasatinib as second line therapy for unselected patients with CML either resistant or refractory to IM even outside from clinical trials. Neither the dose nor the duration of Imatinib affects the response to Dasatinib. As well, patients receiving continuously Dasatinib, even at low doses, perform better. In conclusion, these findings show an increase possibility of cure for unselected patients resistant or intolerant to Imatinib submitted to second line therapy with Dasatinib in everyday clinical practice. Disclosures No relevant conflicts of interest to declare.

Abstract 860 Background: Elderly CML patients treated with Imatinib (IM) in early chronic phase (CP) have similar cytogenetic response and survival compared with younger patients, but they show a lower compliance to standard IM therapy (400 mg/day). Aims: The aim of the study is to investigate if CCgR that has been achieved with standard (daily administration) IM therapy can be maintained with the same dose of IM given intermittently (INTERIM). Methods: The study population is represented by elderly patients (≥ 65 years old) with Ph+ CML and with stable CCgR after at least 2 years of standard IM therapy (daily administration). IM is given at the same dose that was given at the time of enrollment by the following intermittent schedule: 1 week on / 1 week off for the 1st month; 2 weeks on / 2 weeks off for the 2nd and 3rd month; 1 month on / 1 month off from the 4th month thereafter. In cases of loss of CCgR INTERIM was stopped and standard therapy (daily administration) was resumed. After 12 months, the patients who are in continuous CCgR are advised to continue the intermittent study schedule and to be followed indefinitely. The CgR status was evaluated at baseline (by conventional cytogenetics on bone marrow and FISH on peripheral-blood) and every 3 months during the study (only by FISH on peripheral-blood). If FISH (% of Ph+ cells) increased more than 1% in two consecutive examinations, evaluation of marrow cells metaphases was performed to confirm the loss of CCgR and to check for additional cytogenetic abnormalities. Quantitative molecular assessment of BCR-ABL transcript by RQ-PCR on peripheral blood was due at baseline and every 3 months during the study and mutational analysis of ABL was performed in case of loss of CCgR. Results: One-hundred and fourteen patients have been considered eligible, but 17 (15%) refused to enter into the protocol. Out of 97 enrolled patients, 87 started INTERIM, 5 patients (5%) went off the study for major protocol violation before the 3rd month and, at present, 82 patients are ongoing. Of these 82 patients, 52, 30 and 11 completed the 3rd, 6th and 9th month, respectively. The preliminary results of the first 6 months are here reported. The distribution of patients according to FISH results is shown in Fig. 1. Only 1/68 pts (at 6th month) showed an increased >1% in Ph+ cells by FISH but he maintained a CCgR when checked by conventional cytogenetic. As showed in Fig. 2, 96 to 87% of patients maintained a major molecular response MMR (≤0,1) according to International Scale (IS). Conclusions: This study is trying to test the minimum effective dose of Imatinib to maintain the CCgR in elderly CML patients with stable CCgR. The preliminary results at 6 months do not show negative trends both for cytogenetic and molecular response. Therefore, the study is ongoing and all patients are expected to complete the trial time (12 months). Disclosures: No relevant conflicts of interest to declare.

Imatinib therapy for chronic myeloid leukemia patients who relapse after allogeneic stem cell transplantation: a molecular analysis

Previous uncontrolled studies have highlighted the potential activity of Rituximab in patients with idiopathic thrombocytopenic purpura (ITP) relapsed or refractory to standard treatments. To better address this effect, a prospective randomized, multicenter, phase III study comparing treatment with Dexamethasone alone (arm A) vs Dexamethasone plus Rituximab (arm B) was started in July 2005 for adult patients with ITP according to the ASH guidelines. Main inclusion criteria were: age ≥ 18 years, untreated ITP, platelet (PLT) count ≤ 20 x109/L, HIV- HCV-HbsAg negativity, informed consent. Patients randomized to arm A received a single course of Dexamethasone 40 mg po on days +1, +2, +3, +4, while patients randomized to arm B received Dexamethasone (as in arm A) in association with Rituximab 375 mg/m2 iv on days +7, +14, +21, +28. Patients in arm A who failed to achieve a sustained response (SR) could be rescued with arm B treatment. The primary objective of the study was to compare SR, i.e. PLT ≥ 50 x 109/L at month + 6 of treatment. The secondary objectives were: the initial overall (OR= PLT ≥ 50 x109/L) and complete response (CR= PLT ≥ 100 x 109/L) by day 30 after starting treatment, respectively; the toxic profile. The statistical plan considered three interim analyses, after the first 50, 100 and 150 enrolled patients, with an estimated sample size of 198 patients (99 per arm). Table 1 summarizes the main demographic data and the results of efficacy and toxicity according to an intention to treat analysis of the first interim analysis. The toxic profile was characterized by only grade 3 adverse events (AE); no patient died during the study period. 16 patients of arm A were rescued with arm B. For this group SR was 81% and no patient experienced SAE or ≥ grade 3 AE. In accordance with the initial statistical plan of the study, which stated that patients’ recruitment would ceased if a ≥ 50% difference in sustained response was demonstrated, enrolment has been stopped in June 2007 with a total number of 103 randomized patients. This preliminary report indicates a significantly higher SR for arm B of treatment with no difference in toxicity profile. A final report will be prepared when the results on the entire study group will be available. Table 1 Therapy Arm A Arm B Statistics Patients 24 26 Male/female 11/13 10/16 p = NS Age (median ± SD) 54.54 ± 18.78 48.65 ± 15.10 p = NS Initial OR 15 (62.5%) 18 (69%) p = NS Initial CR 10 (42%) 16 (61%) p = NS SR 7 (29%) 21 (81%) p = 0.0001 SAE or grade 3 AE 3 (12.5%) 2 (8%) p = NS

Background. Nilotinib (NIL) is a potent and selective BCR-ABL inhibitor approved for the frontline treatment of chronic myeloid leukemia (CML) based on the results of the ENESTnd study. The sustained superiority of NIL vs. imatinib (IM) was confirmed after 5 years of follow-up (Hughes et al, abs. 677, EHA 2014). However, few data are available on patients (pts) treated frontline with NIL outside of Company-initiated trials. Objectives. To analyze the long-term outcome in a large, independent cohort of newly diagnosed CML pts treated frontline with NIL-based regimens. Methods. We analyzed 215 pts, enrolled in 2 multicenter phase II studies conducted by the GIMEMA CML WP (ClinicalTrials.gov. NCT00481052 and NCT00769327) or treated at the Bologna University Hospital, with NIL 300 mg or 400 mg BID as initial treatment; 123 pts received a sequential treatment with NIL and IM (NIL-IM), with a 3-months (mos) rotation period (all patients received NIL in the first 3 mos). The median age was 53 years (range 18–86). Ten out of 215 pts (5%) had a high EUTOS score. The median follow-up was 57 mos (range 36–81 mos). We assessed: the rates of Complete Cytogenetic Response (CCyR) and Major Molecular Response (MMR); the rates of optimal responders at each milestone according to ELN 2013 recommendations; the overall survival (OS; any death included), progression-free survival (PFS; progression to accelerated/blast phase [AP/BP] and deaths for any cause), failure-free survival (FFS; failures according to ELN 2013 recommendations and deaths for any cause), and event-free survival (EFS; events: failures, permanent discontinuation of NIL for any cause, including deaths). All analysis was made according to the intention-to-treat principle. Results. The cumulative rates of CCyR and MMR were 93% and 88%, respectively. At 3 mos, 82% of the pts were in Partial Cytogenetic Response and 90% had a BCR-ABL/ABL (IS) < 10%; at 6 mos, 86% were in CCyR and 83% had a BCR-ABL/ABL (IS) < 1%; at 12 mos, 72% were in MMR; all these pts were optimal responders according to ELN 2013 recommendations. Overall, 80 (37%) pts permanently discontinued NIL: 45 (21%) for adverse events or intolerance; 25 (12%) for failures; 7 (3%) while in stable MR4; 3 (1%) for other reasons. Cardiovascular adverse events (CVAE) were cause of permanent NIL discontinuation, after a median time of 37 mos, in 13 (6%) pts, and included 4 peripheral arterial occlusive diseases and 3 ischemic coronary diseases; only one pt died for CVAE. Nine (4.1%) pts progressed to AP/BP, 8/9 during the 1st year of therapy and one after 25 mos; all pts subsequently died (after a median of 13 mos, range 1-34 mos). NIL-resistant mutations were identified in 6 of these pts (4 T315I; 1 Y253H; 1 F359V); 7/9 progressions occurred in patients receiving NIL-IM. In addition, 6 pts were classified as failures at 3,6, or 12 mos according to ELN 2013 recommendations; afterwards, 10 pts developed a secondary resistance (3 loss of CHR, 3 loss of CCyR, and 4 confirmed loss of MMR). Overall, 17 (8%) pts died, in 7 cases for reasons unrelated to CML progression. The estimated 6-year OS, PFS, FFS, and EFS were 91%, 91%, 83%, and 59%, respectively. Conclusions. Our National experience showed that most pts treated frontline with NIL-based regimens were optimal responders according to ELN recommendations and that 91% of the patients were estimated to be alive and progression-free at 6 years. In particular, NIL alone was highly effective in the prevention of AP/BP. Considering that AP/BP had in most cases an early onset and an extremely poor prognosis, its prevention should be the priority of CML treatment, especially in the firsts 2-3 years. However, afterwards, the relatively high number of CVAE observed, suggests to focus, at least in selected patients, on strategies aimed at the prevention of CVAE (NIL dose reduction? switch to IM?). Acknowledgments. European LeukemiaNet, COFIN, Bologna University, BolognAIL Disclosures Gugliotta: Novartis: Consultancy; Bristol-Myers-Squibb: Consultancy. Castagnetti:Novartis: Consultancy; Bristol-Myers-Squibb: Consultancy. Breccia:novartis: Consultancy; BMS: Consultancy; Celgene: Consultancy. Abruzzese:Novartis: Consultancy. Martinelli:ARIAD: Consultancy; Pfizer: Consultancy; BMS: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau. Saglio:BMS: Consultancy, Fees for occasional speeches Other; Novartis: Consultancy, Fees for occasional speeches, Fees for occasional speeches Other; Pfizer: Consultancy, Fees for occasional speeches, Fees for occasional speeches Other; ARIAD: Consultancy, Fees for occasional speeches, Fees for occasional speeches Other. Baccarani:Novartis, BMS, Pfizer, Ariad: Consultancy, Honoraria, Speakers Bureau. Rosti:Novartis: Consultancy; Bristol-Myers-Squibb: Consultancy; Ariad: Consultancy; Pfizer: Consultancy.

Background Nilotinib is a potent and selective BCR-ABL inhibitor approved for the frontline treatment of CML. The latest update (4-yr follow-up) of the ENESTnd study demonstrated sustained superiority of nilotinib vs. imatinib (Hochhaus et al, EHA 2013, abstract 712). The CML Italian Registry of Nilotinib is the largest series of patients treated frontline with nilotinib-based regimens, outside of Company-sponsored trials. Therefore, it represents an important resource for an independent evaluation of the outcome of such patients. Aims to analyze the response rates and outcome in an independent cohort of patients treated frontline with nilotinib-based regimens in Italy. Methods The CML Italian Registry of Nilotinib includes 215 patients, enrolled in 2 multicenter phase II studies conducted by the GIMEMA CML WP (ClinicalTrials.gov. NCT00481052 and NCT00769327) with nilotinib 300 mg or 400 mg BID as initial treatment; 123 patients received a sequential treatment with nilotinib and imatinib, with a 3-mo rotation period. The median age was 53 years (range 18–86). Ten out of 215 patients (5%) had a high EUTOS score. The median follow-up was 43 months (range 18–69 months). We analyzed: the rates of Complete Cytogenetic Response (CCyR) and Major Molecular Response (MMR); the overall survival (OS; any death included), progression-free survival (PFS; progression to accelerated/blast phase [AP/BP] and deaths for any cause), failure-free survival (FFS; failures according to ELN 2013 recommendations and deaths for any cause), and event-free survival (EFS; events: failures, permanent discontinuation of nilotinib for any cause, including deaths). Results Rates of CCyR were 72% and 92% by 3 months and 12 months, respectively. Rates of MMR were 56% and 83% by 3 months and 12 months, respectively. The cumulative rates of CCyR and MMR were 93% and 92%, respectively. Overall, events were recorded in 64 (30%) patients: 31 (14%) patients permanently discontinued nilotinib for adverse events or intolerance; 22 (10%) patients failed therapy according to ELN 2013 recommendations, including 8 (3.7%) patients that progressed to AP/BP; 11 (5%) patients permanently discontinued nilotinib for other reasons. All progressions to AP/BP occurred within the first year of therapy, and all patients subsequently died. Nilotinib-resistant mutations were identified in 5 of these patients (4 T315I; 1 Y253H). No difference in the rate of progression to AP/BP was observed between patients receiving nilotinib alone or nilotinib and imatinib in sequential schedule. Overall, 15 (7%) patients died, in 7 cases for reasons unrelated to CML progression. The 4-year OS, PFS, FFS, and EFS were 93%, 93%, 86%, and 69%, respectively. Conclusion These Italian nilotinib registry data provide an independent and unbiased overview of the therapeutic effects of nilotinib, with high and early rates of complete cytogenetic and major molecular response. All progressions (3.7%) to AP or BP occurred within the 1st year of therapy; however, all cases were fatal, emphasizing how crucial is the prevention of AP/BP. With a median follow-up of 43 months, 69% of patients were still on nilotinib, and 93% were alive and progression-free. Acknowledgments European LeukemiaNet, COFIN, Bologna University, BolognAIL Disclosures: Gugliotta: Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria. Castagnetti:Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria. Bocchia:Novartis and Bristol Mayer Squibb: Honoraria. Soverini:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Saglio:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Cavo:Celgene: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Millennium: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Onyx: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees. Baccarani:Novartis: Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Pfizer: Honoraria; Ariad: Honoraria. Rosti:Novartis: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Speakers Bureau; Ariad: Consultancy; Roche: Speakers Bureau.

Abstract 1748 Introduction: Polycythemia vera (PV) is a chronic myeloproliferative neoplasms characterized by erythrocytosis, vasomotor disturbances, pruritus, risk of disease progression into acute myeloid leukemia or myelofibrosis and cardiovascular events, the last representing the main cause of morbidity and mortality. Since 2005 the V617F point mutation in Janus Kinase 2 (JAK2) gene gained a dominant role in determining the molecular basis and the diagnosis of PV. We compared the clinical epidemiology of the 1638 patients included in the ECLAP trial in the years 1997 to 2001, with that of a “modern” cohort of 365 PV, JAK2-positive patients included in the Italian CYTO-PV randomized clinical trial and followed from the year 2008 to 2012. Methods: Patients were eligible in CYTO-PV trial and in ECLAP study if they met WHO-2008 diagnostic criteria and the criteria established by the PVSG or Pearson/Messinezy respectively. Clinical characteristics have been compared. The incidence of major cardiovascular events (CV death plus major thrombosis [stroke, acute coronary syndrome, transient ischemic cerebral attack, peripheral arterial thrombosis, pulmonary embolism, abdominal thrombosis, deep vein thrombosis) and total CV events incidence has been evaluated. The median follow up was 31.0 months (range 0– 48.13 months) and 33.1 months (range 0–63.6) for patients included CYTO-PV and in ECLAP respectively. Results: In CYTO-PV 49.3% patients with recent PV diagnosis were included (within 2 years prior inclusion) while in ECLAP the proportion was 35.5%. Mean age at recruitment was similar for patients in CYTO-PV (64.5 yrs) and ECLAP (65.4 yrs). History of thrombosis was reported in 28.9 % vs 38.6% patients in the CYTO-PV and in ECLAP, respectively (p 65 and no PT, age 65 and PT at randomization was 2.2, 4.8, 3.5 and 3.4 per 100 person/years, respectively. Conclusions: The comparison of these two cohorts of PV patients followed 10 years apart suggests that JAK-2 PV patients are currently better managed for the control of classical CV risk factors, are more frequently administered aspirin, and HU with better control of their disease, and eventually have a risk of thrombosis approximately half than in the past. Disclosures: No relevant conflicts of interest to declare.

Abstract 2797 Background: Nilotinib is a second-generation tyrosine kinase inhibitor that demonstrated higher and faster responses and lower progression rates compared to imatinib in ECP CML patients. Early cytogenetic and molecular responses have been associated with a better outcome in patients treated with imatinib, dasatinib, or nilotinib frontline. In particular, BCR-ABL transcript level < 10% at 3 months has been associated with: increased 2-year cumulative incidence of complete cytogenetic response (CCyR), major molecular response (MMR), and MR4.5 (>4.5 log reduction in BCR-ABL transcript level) with dasatinib (Marin D et al, Blood 2012); increased CCyR rate by 1 year, MMR rate by 2 years, and better progression-free survival (PFS) at 2 years, with dasatinib (DASISION; Hochhaus A et al, ASH 2011, abs. 2767); increased MMR rate by 1 year, and better PFS and overall survival (OS), with nilotinib (ENESTnd; Hochhaus A et al, EHA 2012, abs. 584). Here we report a landmark analyses based on BCR-ABL transcript level and cytogenetic response at 3 months to evaluate their impact on subsequent response and outcome in an independent cohort of patients treated with nilotinib-based regimens in Italy (CML Italian Registry of Nilotinib). Methods: The CML Italian Registry of Nilotinib includes 215 patients, enrolled in 2 multicenter phase II studies conducted by the GIMEMA CML WP (ClinicalTrials.gov. NCT00481052 and NCT00769327) or treated at the Dpt. of Hematology and Oncology, Bologna University Hospital, with nilotinib 300 mg BID or 400 mg BID as initial treatment. The median age was 53 years (range 18–86). The median follow-up was 29 months (range: 18–47 months). At 3 months 196/215 (91%) and 189/215 (88%) patients were evaluable for the molecular and cytogenetic response, respectively. BCR-ABL transcript levels were: 21% in 88%; > 1% to 2 10% in 11%; > 10% in 1%; given the very low proportion of patients with a BCR-ABL > 10% (n. 2), to the purpose of these analyses, patients have been divided into 2 groups, with a transcript level 21% (n. 173, 88%), or > 1% (n. 23, 12%). Cytogenetic response was: CCyR 84%; partial cytogenetic response (PCyR) 9%; less than PCyR 7%. We analyzed the rate of MMR at 1 year, and the failure-free survival (FFS, according to ELN 2009 definitions), PFS (transformation to accelerated or blastic phase), and OS (any death included) according to the BCR-ABL transcript levels and to the cytogenetic response at 3 months. Results: Of the 2 patients with a BCR-ABL transcript level > 10% at 3 months, 1 progressed to blastic phase at 1 year, while the other one obtained a CCyR at 6 months and a MMR at 24 months. Patients with BCR-ABL 2 1% at 3 months had a higher rate of MMR at 12 months with respect to those with a transcript level > 1% (79% vs. 35%, p Conclusion: In our national experience, where 42 centers are represented with at least 1 patient, a very small proportion of patients treated frontline with nilotinib failed to achieve a reduction to < 10% level of BCR-ABL transcript (2/196 or 1%) if compared to other reports: in the frame of the ENESTnd trial, 24/258 or 9% (Hochhaus et al, EHA 2012, abs. 584). The cut-off of 1% of BCR-ABL at 3 months in our experience is a reliable surrogate marker of response at 1 year (MMR) and outcome (PFS and FFS), along with the level of cytogenetic response achieved. These patients with > 1% of BCR-ABL at 3 months are characterized by a higher propensity to fail the treatment and to progress. Consequently, they deserve a close monitoring and, for those not achieving the expected cytogenetic and molecular responses later on (at 6–12 months), an optimization of the dose of nilotinib or a treatment modification is advised. Acknowledgments: European LeukemiaNet, University of Bologna, BolognAIL, COFIN. Disclosures: Gugliotta: Novartis: Consultancy, Honoraria; Bristol-Myers-Squibb: Consultancy, Honoraria. Castagnetti:Novartis: Consultancy, Honoraria; Bristol-Myers-Squibb: Consultancy, Honoraria. Breccia:Bristol Myers Squibb: Consultancy; Novartis: Consultancy. Abruzzese:Bristol Myers-Squibb and Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Soverini:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Saglio:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy. Martinelli:Novartis: Consultancy, Honoraria, Speakers Bureau; Bristol-Myers-Squibb: Consultancy, Honoraria, Speakers Bureau. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Rosti:Novartis Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Speakers Bureau; Pfizer: Speakers Bureau.

Abstract 1682 Background: The introduction of Imatinib has significantly improved the outcome for patients with Ph+ CML. The complete cytogenetic response (CCgR) is a strong and confirmed predictor of improved long-term outcome. According to current recommendations, Imatinib (IM) should be continued indefinitely. However, optimal responders can be eligible for investigational trials of treatment discontinuation. AIMS: This study (ClinicalTrials.gov NCT 00858806) describes the effects of a policy of intermittent Imatinib (INTERIM) treatment (one month on/one month off) on cytogenetic and molecular responses in a selected population of patients ≥ 65 years old who were receiving treatment with Imatinib for > 2 years and were in stable complete cytogenetic response (CCgR). The primary endpoint of the study was the proportion of patients who maintained CCgR after 1 year of INTERIM. The secondary endpoint was the level of BCR-ABL transcripts during INTERIM METHODS: Cytogenetic and molecular responses were monitored by FISH and RT-Q-PCR every 3 months. The definition of CCgR, and of CCgR loss was based on CBA of marrow metaphases which was performed at baseline and in all the patients who became FISH positive (BCR-ABL–positive nuclei > 1%). Major molecular response (MMR), corresponding to a 3-log reduction in BCR-ABL transcript level from the standardized baseline, was defined as BCR-ABL ≤0.1%IS and was indicated as MR3.0. For the purposes of this study, complete molecular response (CMR) was defined as a 4-log reduction in BCR-ABL transcript level ( RESULTS: Seventy-six patients have been enrolled. Six patients (8%) lost CCgR (CBA positive), and 3 other patients became FISH positive while remaining CBA negative. At 12 months, the probability of maintaining CBA negativity was 92% (95% CI 86–98%), while the probability of maintaining FISH negativity was 87% (95% CI 79–94%). None of the factors that were examined by univariate and multivariate analysis were found to be associated with an higher probability of either loosing the CCgR (CBA) or showing a FISH positivity (> 1%), with the exception of the duration of imatinib therapy (HR = 0.23, 95% CI 0.008–0.73, P =.01). Among patients with prior Imatinib treatment longer or shorter than 48 months, the probability of maintaining FISH negativity was 94% (95% CI 88–100%) vs 71% (95% CI 53–89%), respectively, HR = 0.23 (P =.007). All the 6 patients who lost CCgR regained the CCgR with daily Imatinib, at the same dose, defined by FISH negativity after 3 to 9 months (4/6 also CBA negative, 2 patients refused bone marrow aspiration). At baseline, all but one patient (99%) were in MMR (MR3.0, BCR-ABL ≤0.1%IS), and 63 patients (83%) were in MR4.0 ( CONCLUSIONS: The intermittent use of imatinib in older patients in stable CCgR after continuous imatinib treatment results in the transient loss of the CCgR in a minority (8%) of the patients. However, the disease burden at the molecular level significantly increased. A policy of intermittent treatment may be an alternative both to chronic continuous treatment and to treatment discontinuation, particularly in the elderly. However, a longer follow up is required before drawing final conclusions. Acknowledgments: This work was supported in part by EuropeanLeukemiaNet through the European Treatment and Outcome Study (EUTOS) and by Cofin 2009. Disclosures: Baccarani: Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Honoraria; Bristol Myers Squibb: Honoraria; Pfizer: Honoraria; Ariad: Honoraria. Castagnetti:Novartis: Honoraria; Bristol Myers Squibb: Honoraria. Di Raimondo:celgene: Honoraria. Rosti:Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Research Funding; Novartis: Honoraria; Bristol Myers Squibb: Honoraria.

Abstract 2415 Poster Board II-392 We previously reported that in adults with primary immune thrombocytopenia (ITP) the addition of four weekly administrations of rituximab 375 mg/m2 to a single course of dexamethasone (40 mg daily for four days) improves the rate of sustained response (SR, i.e. platelet count 3 50 × 109/L at month 6 from the beginning of treatment) if compared with dexamethasone alone (63% vs 36%, P= 0.004, 95% C.I.: [0.079-0.455]) (ML18542 study, Zaja et al. Blood 2008; 112:Abstract 1). Moreover, the rate of SR in 27 patients receiving dexamethasone plus rituximab salvage therapy because of primary refractoriness or relapse after dexamethasone monotherapy was 56%. In order to better address the impact of therapy beyond month 6, a subsequent observational follow-up study up which lasted from month 6 up to month 36 was planned. Participating centers were asked to continue to monitor the patients enrolled in the ML18542 study at least every 4 months up to the end of year 3 from accrual, documenting the occurrence of delayed side effects, the response status and the need for further treatment. We considered as efficacy parameters the duration of response (RD), i.e. the interval between achievement of SR and relapse, the relapse rate (number of patients who lost their SR status) and the need of further specific anti ITP therapy after month 6 (TFS). Eighty out of the original 101 patients enrolled in the ML18542 study, 15 treated with the dexamethasone monotherapy arm, 41 with the dexamethasone plus rituximab arm and 24 who received dexamethasone plus rituximab salvage therapy, were systematically followed-up beyond month 6 for a median overall period of observation of 20 months (range: 4-40 months). Twenty-one patients could not be valuable, most of them because lost at follow up during the study period. As far as the safety profile, we documented one single case of Herpes Zoster reactivation at month 22 in a patient initially allocated to the dexamethasone arm who further received dexamethasone plus rituximab salvage therapy. No other infectious, delayed toxic events or deaths were registered. Fifty-three out 80 patients who achieved a SR according to the study protocol could be evaluated for long term efficacy. This group included 13 patients from the dexamethasone monotherapy arm who were observed after month 6 for a median period of 20 months (range: 12-28 months), 26 of dexamethasone plus rituximab arm observed for a median period of 24 months (range: 8-40 months), and 14 from the dexamethasone plus rituximab salvage therapy group observed for a median period of 20 months (range: 12-34). The rate of relapse in the three groups was 23% (3/13), 23% (6/26), and 14% (2/14) respectively (Fisher test, P= 0.824). The 30 months estimated probability of RD within each of the three treatment groups was 77%, 71%, and 85% (Log-rank test, P= 0.755), respectively. The time of SR loss was comprised between month 4 and 30 in the dexamethasone monotherapy arm, month 4 and 40 in the experimental arm, and month 8 and 34 in the salvage therapy arm. In these three groups, the use of further specific anti ITP therapy after month 6 was registered in 33%, 29% and 36% of patients (Fisher test, P= 1), respectively. Overall, in this period of observation 4 out 80 (5%) valuable patients underwent splenectomy. In conclusion the results of this study indicate: i) similar and very low pattern of long term toxicity between ITP patients treated either with single course of dexamethasone or dexamethasone plus rituximab; ii) no significant differences in the relapse rate, RD and TFS among patients who achieved SR after dexamethasone monotherapy or dexamethasone plus rituximab, either front line or salvage therapy, suggesting that relapse rate may be a possible surrogate of SR, rather than of the initial therapeutic choice. Disclosures: Zaja: Roche: Honoraria, Research Funding, financial support for the costs of travel to the ASH annual meeting. Off Label Use: Rituximab in ITP.

Introduction. Previous uncontrolled studies have highlighted the activity of rituximab in patients with idiopathic thrombocytopenic purpura (ITP) relapsed or refractory to standard treatments. To better address this effect, a prospective randomized (1:1), multicenter, phase III study comparing treatment with dexamethasone alone (arm A) vs dexamethasone plus rituximab (arm B) was conducted from July 2005 through June 2007 for adult patients with previously untreated ITP and a platelet (PLT) count ≤20 × 109/L. Material and methods. Patients randomized to arm A received a single course of oral dexamethasone 40 mg on days +1, +2, +3, +4, while patients randomized to arm B received dexamethasone (as in arm A) in association with rituximab 375 mg/m2 iv on days +7, +14, +21, +28. Patients in arm A who failed to achieve a sustained response and had a platelet count ≤20 × 109/L (from day +30 up to the end of 6 months) could receive salvage treatment with the experimental arm (dexamethasone plus rituximab). The primary objective of the study was to compare the sustained response (SR), i.e. PLT count 50 × 109/L at month + 6 of treatment. Secondary objectives were: evaluation of the safety, the initial response (PLT count 50 × 109/L) by day 30 after the initiation of treatment, the activity of salvage therapy with dexamethasone plus rituximab in patients non responding to dexamethasone monotherapy, the definition of clinical and laboratory factors predictive of response and to explore the pharmacokinetics parameters of rituximab and their potential relation with response. Results were analyzed by an intention to treat (ITT) and by a per-protocol (PP) analysis. Results. One-hundred-one patients (52 for arm A and 49 for arm B) and 64 patients (38 for arm A and 26 for arm B) represented the ITT and PP population, respectively. Demographic baseline data were in accordance to what expected for a population of ITP patients. No significant differences among the two groups of randomization were present. There was a female prevalence and the mean age was 47 and 49 years in arm A and B, respectively. Table 1 summarizes the ITT and PP efficacy results considering 3 different levels of response (i.e. PLT count 50 × 109/L, 100 × 109/L and 150 × 109/L). A significant advantage for arm B patients was documented. Table 1 Initial response Initial response Sustained response Sustained response Analysis Intention-to Treat Per-Protocol Intention-to Treat Per-Protocol Treatment Arm A Arm B P value Arm A Arm B P value Arm A Arm B P value Arm A Arm B P value Valuable patients 44 25 32 13 52 49 38 26 PLT 3 50×109/L 27% 68% .001 31% 69% .009 36% 63% .004 39% 85% Twenty-seven patients initially allocated to arm A and who failed to achieve initial response or SR received salvage treatment with the dexamethasone plus rituximab. In this group, ITT and PP SR rate were 56% and 59%, respectively. No clinical or laboratory factors predictive of SR were identified. In arm B patients the serum concentrations of rituximab levels did not correlate with the rate of response. Twelve SR patients of arm A, 27 of arm B and 19 of salvage therapy group were systematically followed up beyond month 6 for a median period of observation of 18 months (range 10–34 months). The rate of SR loss (platelets < 50 × 109/L) in these three groups was 25 % (3/12), 11% (3/27) and 10.5% (2/19). The safety profile was good with no substantial difference between the two arms of randomization. No patient died during the study period. Conclusion. The results of this study indicate that the association of dexamethasone plus rituximab improves patients outcome without worsening of the safety profile. This effect is characterized by prolongation of SR and reduction in relapse rate. The long period of relapse free survival registered in some patients suggests a possible curative effect. This treatment can be offered as an option before splenectomy, particularly in those patients where the surgical option is not well accepted or have higher risk of complications.

Imatinib 400 mg (SD) is the established first line treatment of chronic myeloid leukemia (CML) in chronic phase. The efficacy of imatinib in early chronic phase has been demonstrated by phase 2 and 3 controlled trials like the IRIS study (O’ Brien et al NEJM 348:11, 2004). Large multicentric studies aimed to evaluate the impact of imatinib 400 mg outside strictly monitored frameworks are not yet available. The GIMEMA (Gruppo Italiano Malattie Ematologiche dell’Adulto) CML Working Party opened in January, 2004, an observational study (serial n. CML/023) to investigate the efficacy of imatinib SD in newly diagnosed CML pts. Clinical and anagraphical data were collected through a web-based system. Peripheral blood samples for quantitative molecular analysis (RT-Q-PCR, Bcr-Abl/Abl × 100 - Taqman) were centralized in Bologna. Overall, 55 italian centers enrolled 367 (359 evaluable) newly diagnosed CML pts in chronic phase between Jan 2004 and Jan 2006. Median age was 50 yrs (range 18–84), 220 male and 139 females. Sokal risk at diagnosis was low, intermediate and high in 221 (62%), 123 (34%), 15 (4%) pts, respectively. 359 pts are evaluable for response at 3 months, 310 at 6 months and 187 at 12 months. The median observation time is 12 months. At 3 months, 94% of the pts reached a stable CHR. At 6 months, 80% of evaluable cases obtained a complete cytogenetic response (100% Ph-neg, CCgR). A major molecular response (MMolR) defined as a Bcr-Abl/Abl × 100 ratio < 0.1%, was shown in 52% of CCgR pts. At 12 months, the CCgR rate was 87% and the MMolR rate in CCgR pts was 63%. At 12 months, 3% of CCgR cases showed an undetectable level of transcript (ratio Bcr-Abl/Abl × 100 < 0,0001). With this short observation period, only 4 pts (1,1%) progressed to accelerated/blastic phase. Limiting the observation to low Sokal risk, at 12 months 221 such pts got a CCgR and MMol rates of 88% and 62%, respectively; 201 low Sokal risk pts were enrolled in the IRIS trial: at 12 months CCgR and MMolR (reduction of Bcr-Abl/Bcr ratio level > 3 logs) rates were 76% and 66%, respectively ( (T Hughes et al, NEJM 349:15, 2003). This study confirns that imatinib is efficacious and manageable, confirming and improving the results of the IRIS trial.

We analyzed clinical and molecular follow-up of 16 patients with chronic myeloid leukemia (CML) relapsing after allogeneic stem cell transplantation (SCT). The median interval between allogeneic SCT and relapse was 26 months (7–162). Two patients had failed treatment with donor lymphocyte infusions prior to Imatinib; four patients had received therapy with IFN alpha. All patients were treated with Imatinib (400 or 600 mg/daily). One patient had received Imatinib before allogeneic SCT. The overall complete hematological (CHR) and cytogenetic responses (CCyR) were 100% for all patients either relapsed in CP or AP. All patients achieved complete molecular response (CMR), intended as 3 logs reduction of BCR-ABL/B2M within 18 months; 8/16 patients obtained a CMR within three months, independently of the phase of the disease at of relapse. Median follow-up after start of Imatinib therapy was 24 months (range 6–36). Chimerism status evaluated in 9 patients showed conversion to full donor chimerism after therapy in all but one of them. Imatinib has significant activity against CML in relapse after allogeneic bone marrow transplantation with durable cytogenetic and molecular remissions obtainable in all patients.

Anagrelide treatment is considered potentially responsible of cardiovascular toxicity and side effects. For this reason, we have retrospectively evaluated 130 patients (pts) with Essential Thrombocythemia (ET) treated with Anagrelide in 10 Italian Hematological Centres belonging to the GIMEMA Group. The pts, 55 males and 75 females, had at diagnosis a mean age of 43 years (range 13–82) and a mean platelet (PLT) count of 1086x10(e)9/L. The Anagrelide treatment was started after a mean of 47 months from diagnosis(range 0–196). A previous treatment with cytoreductive drugs (mainly HU and IFN) and antiplatelet agents (mainly ASA), was performed in 65% and 98% of cases, respectively. The mean Anagrelide dose was 1 mg/day at start and 1.7 mg/day in the maintenance phase (mean PLT count below 500x10(e)9/L). Twenty-five patients (19%) discontinued the treatment after a mean of 27 months (range 1–85) for the following reasons: compliance loss (n8), no satisfactory response (n7), palpitations and tachycardia (n6), other side effects (n4), anemia (n1), renal toxicity (n1), thromboembolism (n1). During the follow-up (median 28 months) 4 thrombotic complications (1.15/100 pt-yrs) and 2 minor hemorrhages (0.57/100 pt-yrs) were registered. Moreover, 15 pts showed non lethal cardiovascular events: angina (n6), cardiomiopathy (n4), arrhythmia (n3), AMI (n2). An instrumental cardiovascular evaluation with Echocardiography (ECHO) and ECG was performed at Anagrelide start in 58 pts: the Ejection Fraction (EF) was abnormal in only one pt (EF 47%). The instrumental monitoring was done in 28 pts every 6–12 months. Four out of these 28 pts showed a decrease of the EF below the level of 50% (43%,45%,47%, and 49%), and only 1 pt discontinued Anagrelide for this reason (see table). The clinical and instrumental cardiovascular evaluation in ET patients receiving Anagrelide is becaming of routinary application and an observational prospective study of the Registro Italiano Trombocitemia (RIT) is now in progress. | CASES | AGE | EF % BASELINE | EF % 1ST YEAR | EF% 2ND YEAR | |:---------------------------------------------------------- | --- | ------------- | ------------- | ------------ | | *Anagrelide discontinuation because tachycardia and EF

Abstract 1118 Poster Board I-140 Background The median age of an unselected population of Ph+ CML patients is close to 60 years. In the prognostic classifications (Sokal, Blood 1984; Hasford, JNCI 1998) that were elaborated before the introduction of IM, age was a significant and important prognostic factor. The most recent IM studies have not clarified the prognostic importance of age and IM therapy is still denied to several elderly patients. Aim to asses the relationship between age (less and more than 65 years) and outcome, in CML patients treated front-line in early chronic phase (ECP). Methods We analyzed the data of 559 previously untreated ECP patients who were assigned to receive IM 400 mg daily (76%) or 800 mg daily (24%) in three controlled, prospective studies of GIMEMA (Clin Trials Gov. NCT00514488 and NCT00510926; and an observational study of IM 400 mg). The median follow-up is currently 42 (extremes 1 – 64) months. There were 115 patients more than 65 years old (median age 71 years), while 444 (79%) were less than 65 years (median age 46 years). The proportion of patients who were treated with IM 800 mg daily was the same in both age groups. Results The cumulative complete cytogenetic and major molecular response rates were identical in the two age groups (88% vs 88% and 82% vs 83%, respectively). However, overall survival (86% vs 93%, p = 0.01), failure-free survival (72% vs 81%, p=0.03) and particularly event-free survival (calculated based on the intention-to-treat principle, where events were any failure [according to the European LeukemiaNet criteria – Baccarani, Blood 2006] and treatment discontinuation for any cause) (60% vs 71%, p=0.006) were significantly inferior in the older age group. All these difference were mainly due to comorbidities leading to more deaths in CP (table). Conclusions/Methods These data show that response to IM was not affected by old age. Survival curves were affected because of age-related complications and comorbidities. Age should never be a contraindication to IM treatment. Acknowledgments: European LeukemiaNet, COFIN, University of Bologna and BolognAIL. Disclosures Saglio: Novartis: Honoraria. Baccarani:Novartis Pharma: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Mayer Squibb: Consultancy, Honoraria, Research Funding, Speakers Bureau.