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Introduction Chronic myeloid leukemia (CML) is a stem cell disease characterized by the constitutive activity of the oncoprotein BCR-ABL that activates multiple signal transduction pathways. Tyrosine-kinase inhibitor (TKI) nilotinib successfully inhibits the activation and the proliferative function of BCR-ABL in patients with CP-CML. Despite the success of nilotinib, some patients become refractory suggesting the presence of a population of Philadelphia positive (Ph+) quiescent stem cells escaping the drug activity. Thus, the molecular mechanisms underlying CML remain poorly understood. In this study, we enrolled 87 CP-CML patients (Pungolino et. al. Am J Hematol. 2018). Samples were collected on the behalf of the Rete Ematologica Lombarda (REL) the PhilosoPhi34 study (EudraCT: 2012-005062-34), which included 15 centers from Italy. We undertook gene expression profiling (GEP) of selected bone marrow (BM) CD34+/lin- cells of 80 patients at diagnosis vs. the same patients after 12 months of nilotinib to investigate gene expression changes induced by the treatment. Methods We isolated CD34+/lin- cells from BM samples in 87 patients at diagnosis whereas the same cells were also selected from 80/87 patients after 3, 6 and 12 months of nilotinib (Trojani et. al. Cancer Biomark. 2017). BM mononuclear cells (MNCs) as well as BM CD34+/lin- cells of all 80 CML patients were counted at diagnosis and during the treatment with nilotinib (at 3, 6, and 12 months, respectively). Standard FISH tested isolated BM CD34/lin- cells for the 87 patients at diagnosis, and for 80/87 patients after 3, 6 and 12 months of nilotinib treatment, respectively. Therefore, we performed GEP analyses of selected BM CD34+/lin- cells of 80/87 patients at diagnosis vs. the same patients after 12 months of nilotinib treatment. Then, we executed bioinformatic preprocessing and correction for batch effects on raw microarray data. Finally, we conducted differential expression analysis and significantly perturbed genes were subjected to functional clustering. Results We observed a wide variability of the number of BM MNCs as well as the number of the BM CD34+/lin- cells among the 80 CP-CML patients at diagnosis and after 3, 6 and 12 months of nilotinib for each patient (Table 1). Figure 1 showed that the number of the BM CD34+/lin- cells dramatically decreased between the diagnosis and after 3 as well as 6 months of nilotinib. We noticed that the BM CD34+/lin- cells slightly increased between 6 and 12 months of nilotinib which might be caused by the gradual repopulation of the normal CD34+/lin- cells in the bone marrow as FISH results suggested. FISH analysis detected CD34+/lin- Ph+ cells in 87 CP-CML patients at diagnosis. No positive Ph+ nuclei were detected on CD34+/lin- cells of 79/80 patients after 12 months of treatment (to categorize a sample as negative, at least 200 nuclei were examined). All of these 79 patients achieved at least complete cytogenetic response. 1/80 patient relapsed at 12 months. We conducted GEP analyses on 78 subjects because, due to experimental issues, two patients were not considered for differential expression analyses, as the microarray CEL files of the 12 months' samples were corrupted and missed probe intensities for most of the probes. GEP analyses determined 2,959 significantly differently expressed probes between diagnosis and after 12 months of nilotinib treatment. Functional clustering identified some pathways significantly enriched between diagnosis and 12 months of nilotinib. Among these pathways, we found that ABCC4, ABCC5, and ABCD3 genes associated with ATP-binding cassette (ABC) transporters were up regulated at diagnosis. GEP results highlighted that 26 genes belonging to cell cycle, mitosis, DNA damage and repair were over expressed at diagnosis. Moreover, GEP data demonstrated that JAK-STAT signaling pathway was deregulated: JAK2, IL7, STAM, PIK3CA, PTPN11, RAF1, SOS1 were over expressed whereas IL22RA1 was under expressed at diagnosis vs. 12 months of nilotinib, respectively. Conclusions In summary, we reported that BM CD34+/lin- cells from CP-CML patients after 12 months of nilotinib were characterized by changes of expression of genes involved in cell cycle checkpoints and mitosis, ABC transporters genes that pump drugs outside form the cells, and JAK-STAT signaling pathway genes responsible for the proliferation, differentiation and cell survival in CML. Disclosures Rossi: Teva: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Mundipharma: Honoraria; BMS: Honoraria; Sandoz: Honoraria; Gilead: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees.

High-dose chemotherapy with autologous stem cell transplantation (ASCT) has been shown effective in the control of relapsed/refractory follicular lymphoma. We evaluate the long-term outcome of patients with relapsed or refractory follicular lymphoma treated with ASCT with in vivo purged progenitors cells. We report the long-term results of a prospective multicenter phase 2 trial on 124 relapsed/refractory follicular lymphoma patients treated with a program of anthracycline-based debulking chemotherapy, immunochemotherapy, mobilization of in vivo purged PBSC followed by ASCT. Median age was 52 years; 14% of patients had grade 3A histology. Debulking chemotherapy produced CR in 16% and PR in 71%, while 13% of patients progressed. After rituximab, cyclophosphamide, vincristine, prednisone (R-COP), CR was obtained in 60% and PR in 35%; 118 patients successfully mobilized PBSC and 117 proceeded to ASCT. The harvest in all the 32 molecularly informative patients was bcl-2 negative. TRM was 0%. The 5-year PFS was 54% and the 5-year OS was 83%. After a median f-up of 6.7 years (range 1.5-13.6), 54% are still in CR. These data show that prolonged PFS is achievable in relapsed/refractory patients with high dose autologous transplantation of in vivo purged progenitor cells.

Core-binding factor leukemia (CBFL) is a subgroup of acute myeloid leukemia (AML) characterized by genetic mutations involving the subunits of the core-binding factor (CBF). The leukemogenesis model for CBFL posits that one, or more, gene mutations inducing increased cell proliferation and/or inhibition of apoptosis cooperate with CBF mutations for leukemia development. One of the most common mutations associated with CBF mutations involves the KIT receptor. A high expression of KIT is a hallmark of a high proportion of CBFL. Previous studies indicate that microRNA (MIR) 222/221 targets the 3' untranslated region of the KIT messenger RNA and our observation that AML1 can bind the MIR-222/221 promoter, we hypothesized that MIR-222/221 represents the link between CBF and KIT. Here, we show that MIR-222/221 expression is upregulated after myeloid differentiation of normal bone marrow AC133(+) stem progenitor cells. CBFL blasts with either t(8;21) or inv(16) CBF rearrangements with high expression levels of KIT (CD117) display a significantly lower level of MIR-222/221 expression than non-CBFL blasts. Consistently, we found that the t(8;21) AML1-MTG8 fusion protein binds the MIR-222/221 promoter and induces transcriptional repression of a MIR-222/221-LUC reporter. Because of the highly conserved sequence homology, we demonstrated concomitant MIR-222/221 down-regulation and KIT up-regulation in the 32D/WT1 mouse cell model carrying the AML1-MTG16 fusion protein. This study provides the first hint that CBFL-associated fusion proteins may lead to up-regulation of the KIT receptor by down-regulating MIR-222/221, thus explaining the concomitant occurrence of CBF genetic rearrangements and overexpression of wild type or mutant KIT in AML.

Selecting units of rare blood for transfusion to patients with complex immunisation is one of the most critical processes of a Transfusion Centre. In January 2005 the 'Rare Blood Components Bank - Reference Centre of the Region of Lombardy' w as established with the following goals: 1) identifying regional rare blood donors; 2) creating a regional registry of rare donors; 3) organising a regional bank of liquid and frozen rare blood units; 4) setting up a regional Immunohaematology Reference Laboratory (IRL) to type donors and resolve complex cases.The key elements in establishing the Bank were periodic meetings organised by the directors and representatives of the regional Departments of Transfusion Medicine and Haematology (DTMH) and the institution of three working groups (informatics, regulations, finance).The regional IRL was set up, the relevant operating procedures were distributed region-wide, software features were defined and later validated upon activation, and the funds assigned were allocated to various cost items. The number and characteristics of the donors to be typed were identified and 14 regional DTMHs started to send samples. Overall, 20,714 donors were typed, for a total of 258,003 typings, and 2,880 rare donors were identified. Of these, 97% were rare donors because of combinations of antigens (2,139 negative for the S antigen and 659 negative for the s antigen) and 3% (n=82) because they were negative for high-frequency antigens. In the first 2 years of activity, the IRL carried out investigations of 140 complex cases referred from other Centres and distributed 2,024 units with rare phenotypes to 142 patients.The main goal achieved in the first 24 months from the start of the project was to set up a regional network able to meet the transfusion needs of patients with complex immunisation.

This report by an international consensus panel updates current recommendations for defining clinical response in Waldenstrom’s macroglobulinemia (WM). The previously published response criteria incorporated parameters for monoclonal protein reduction and/or improvement of marrow and nodal involvement, and included definitions of complete and partial remissions. The criteria have been updated to include minor response and stable disease categories. In addition, the criteria now recognize that delayed responses after treatment with nucleoside analogues and biologic agents and the time point for assessing response in patients with WM should be considered so as to not miss or miscategorize a response. The new criteria should therefore help in better delineating responses to therapy in patients with WM, particularly with the wide use of nucleoside analogues and biologically based agents for this disease.

Background. Several studies showed the effectiveness of fibrin glue (FG) in reducing bleeding from cut surface in liver resection. This study compares the efficacy in reducing bleeding of a homemade FG, in comparison with a commercial product. Methods. Thirthy-four patients submitted to liver resection for carcinoma were enrolled. Fourteen of them received homemade FG, while the others the commercial product (Tissucol, Baxter). Homemade FG was obtained from allogeneic fresh frozen plasma by using the CryoSeal CS-1 system (Thermogenesis Corp.) device. Fibrinogen concentration in homemade FG was assessed according to the Clauss method. In all the patients the same amount of FG (5 mL) was sprayed on liver cut surface, at the end of the liver resection. Results. Mean fibrinogen concentration in homemade FG was 23.16±5.93mg/mL. In homemade group the baseline haematocrit (Hct) was 40.3±4.1%, in the Tissucol group 40.1±5.4% (p NS). The Hct in 5thpostoperative day was 35±4.1 in group treated with homemade FG, and 32.8±3.8 in pts receiving Tissucol (p

Background. Polycythemia vera (PV) and essential thrombocythemia (ET) are chronic myeloproliferative neoplasms that can progress to secondary myelofibrosis (MF), named post-PV (PPV) MF and post-ET (PET) MF. While outcome of primary myelofibrosis is defined on the basis of the IPSS models, information on outcome of secondary myelofibrosis (SMF) is scant. The objective of this study is to develop a prognostic model to predict survival of SMF. Patients and Methods.A total of 718 patients from 16 international Centers were included in this study. Diagnosis of PPV-MF and PET-MF was according to the IWGMRT criteria. Since survival varied among centers, statistical analyses took into account this cluster effect. Survival was calculated utilizing Kaplan-Meier estimates for univariate categorical risk factors and Cox proportional hazard regression, when performing multivariate analysis. The log-rank test was applied to compare Kaplan-Meier curves. Survival was censored at date of stem cell transplant, splenectomy or trial enrollment. Results. Among 718 SMF, 364 (51%) were PET-MF and 354 (49%) PPV-MF. The median follow-up was 2.4 years (range, 0.6-20.1). Median age was 64 years (range, 25-96), 370 were males (52%). Median time from PV/ET diagnosis to SMF diagnosis was 10.6 years (range, 2.5-41). Median values of hemoglobin, white blood cell count and platelet count were 11.2 g/dL (range, 5-17), 10.2 x10(9)/L (range, 1.1-98.4) and 343 x10(9)/L (range, 16-1908), respectively. Splenomegaly was present in 606 patients (87%) and constitutional symptoms in 329 (47%). Cytogenetic profile (N= 338) was normal in 222 (66%), unfavorable in 40 (12%), abnormal but favorable in 76 (22%), and the JAK2 (V617F) mutation (N=679) in 73%. Anemia, leukocytosis, thrombocytopenia, advanced age, circulating blasts, JAK2(V617F), constitutional symptoms, time to SMF, history of thrombosis negatively affected survival in univariate analysis (maximum P values, 0.043), while type of diagnosis (PPV, PET), splenomegaly, spleen size, cytogenetics, and cardiovascular risk did not. Since survival did not differ between PPV and PET, multivariable analysis was carried out on a subset of 493 SMF patients randomly selected and stratified by diagnosis from the original cohort of 718 patients to generate the MYSEC-PM. The remaining 225 were used as validation cohort. By multivariable analysis, age over 65 years (Hazard Ratio –HR- 2.6, 95%CI: 1.5-4.4; p65 years (3 points), time to SMF >15 years (2 points), history of thrombosis (2 points), constitutional symptoms (2 points), hemoglobin = 1% (2 points). To facilitate the use in clinical practice, the MYSEC-PM was recoded into three broader categories: low-risk (score, 0 to 2), intermediate-risk (score, 3 to 6), high-risk (score >6). Survival was significantly different among the risk groups (Figure 1, p< 0.0001): median survival and its 95% CI was not reached in low-risk, 7.9 years (95% CI: 6.7-9.3) in intermediate and 3.8 (95% CI: 2.2-5) in high-risk. The predictive value of MYSEC-PM was confirmed in the validation cohort (N=190) with the maximum p value in pairwise comparisons of 0.01. In the validation cohort, by Akaike information criterion, MYSEC-PM model better predicted survival than the IPSS/DIPSS models (N=190, AIC, 330.5 vs. 336.5 vs. 334.3, respectively) and also compared to the DIPSS-plus (N=91, 103.8 vs. 106.5, respectively): lower score implies better prediction. Conclusions. The MYSEC prognostic model, based on age >65 years, time to SMF >15 years, previous thrombosis, constitutional symptoms, hemoglobin 1%, clearly distinguishes outcome of patients with SMF and outperforms PMF risk models among those patients. This model can be considered for tailoring treatment according to disease risk in SMF. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

CVC-related complications were retrospectively analysed for 373 CVCs inserted in 261 hematologic pts consecutively admitted to our Hematology Department between January 2002 and March 2004. Pts diagnosis comprised AML (96 pts, 36.8%), ALL (19 pts, 7.3%), lymphoproliferative disorders (74 pts, 28.3%), MM (51 pts, 19.5%), CML (13 pts, 5%), others (8 pts, 3.1%). The CVCs were polyurethane Plastimed three lumen 7 Fr (193 cases) for chemotherapy and polyurethane Arrow three lumen 12 Fr (173 cases) for chemotherapy and peripheral blood stem cell (PBSC) apheresis. CVCs were inserted, according to physician’s judgement, either at bedside (369 cases) or in the operating room (4 cases) and were used for drug infusion in 309 cases (82.8%), for total parenteral nutrition in 19 cases (5.1%), for blood transfusions in 226 cases (60.6%), for PBSC apheresis in 152 cases (40.7%). Fifty five CVCs were inserted in neutropenic pts ( N < 1 x 109/L); severe thrombocytopenia (Plts < 30 x 109/L) was present in 33/373 cases. Antithrombotic prophylaxis with low molecular weight heparin because of previous thrombosis was instituted in 33 cases (8.8%). At univariate and multivariate analysis the following risk factors for catheter-related bloodstream infections were considered: pt age, number of days/catheter, haematological disease, catheter lumen, administration of chemotherapy (standard- vs high-dose), presence of neutropenia. The median duration of CVCs after placement was 22.7 days (range 2–70) for the 7 Fr lumen, and 9 (range 1–39) for the 12 Fr. Major hemorrhagic complications related to the insertion procedure were observed in 4 cases of whom 2 were severely thrombocytopenic. CVC occlusion were observed in 24 cases (6.4%). Thrombotic complications developed in 7 cases (1.87% of inserted CVCs) and in 2/7 were complicated by pulmonary embolism; thrombophilia tests were negative in all 7 pts who developed thrombosis. Among febrile pts the frequency of bacteriemias was 19.6% (73/373 cases) of which 52/373 were CVC-related (13.9%). Approximately 6% of cases (23/373 CVCs) had CVC exit point infection with or without associated bacteriemia. Gram positive bacteria were isolated in 49 cases (67.1%), 38 of which were CVC-related (19 S. epidermidis, 4 S. pneumoniae, 3 S. aureus, 3 Enterococci species, 9 others). Gram negative bacteria were isolated in 23 cases, of which 13 were CVC-related (5 E.coli, 4 E. cloacae, 2 P. aeruginosa, 2 K pneumoniae). Candida was isolated in one case. Overall mortality in our patient population was 4.2% (11/261 pts); one patient only died of infection (P. aeruginosa sepsis) not CVC-related. At univariate and multivariate analysis significant risk factors for infection (p

Polycythemia vera (PV) is a chronic myeloproliferative disorder with a propensity to develop myelofibrosis, a condition named post polycythemia vera myelofibrosis (post-PV MF). Survival and prognostic factors after transition to MF remain to be defined. We studied 68 patients with post-PV MF to define survival and prognostic factors for survival at diagnosis of post-PV MF. We also developed a dynamic prognostic model to predict survival at any time from diagnosis of post-PV MF. The median interval between the diagnosis of PV and that of post-PV MF was 13 years (range, 4–29.6 years). Patients with post-PV MF were observed for 181 person-years of follow-up. At diagnosis of post-PV MF, 43 (63%) of 68 patients had less than 65 years. During the follow-up, the incidence of thrombosis was 42 × 1000 person-years (95% CI: 19–93.5) and the incidence of leukemia was 50.3 × 1000 person-years (95% CI: 26–115). The median survival was 5.7 years. Multivariable Cox proportional hazard regression including age, hemoglobin value, platelet count, leukocyte count, and spleen size, showed that hemoglobin < 10 g/dL (P < .001) and platelet count < 100 × 109/L (P= .026) were independent risk factors for survival. We stratified patients at diagnosis of post-PV MF, according to these factors, obtaining two risk groups with significantly different survival (P = .003): low risk (Hb > 10 g/dL and platelet count > 100 × 109/L) with a median survival of 7 years, and high risk (Hb < 10 g/dL or platelet count < 100 × 109/L) with a median survival of 2 years. The prognostic model retained significance after adjustment for age in a multivariable Cox proportional hazard regression (HR: 4.3, 95% CI: 1.6–11.4; P= .003). To assess whether this prognostic model may predict survival at any time from diagnosis of post-PV MF, we evaluated in a time-dependent analysis 64 patients who had longitudinal blood cell counts during follow-up. As first step, we evaluated univariate survival analysis with hemoglobin value < 10 g/dL and platelet count < 100 ×109/L as time-dependent covariates. Both time-dependent parameters affected survival (HR for hemoglobin 5.8, 95% CI: 2.2–15.2, P < 0.001; HR for platelets 4.5, 95% CI: 1.67-12, P=.002). As second step, we evaluated the prognostic model assessed at diagnosis as time-dependent covariate, to define whether the acquisition of one risk factor during follow-up may affect survival. The HR was 7.5 (95% CI: 2.4-23.4; P < .001). The time-dependent prognostic model retained statistical significance after adjustment for age (P < .001). In conclusion, in patients developing post-PV myelofibrosis, a prognostic model based on hemoglobin level < 10 g/dL and platelet count < 100 × 109/L may predict survival at diagnosis of post-PV MF and at any time thereafter.