ALBERT

Description: Decitabine is a pyrimidine nucleoside analogue of cytidine and with azacytidine has been approved for treatment of MDS and non-proliferative CMML. Anyhow, few CMML cases were included in the registrative studies and CMML treatment still remains very arduous, being CMML an heterogenous disease by itself, with characteristics of both MDS and MPN and with a variable prognosis. Decitabine has been suggested to be particularly effective in CMML in a few studies. We evaluated efficacy and safety of decitabine 20mg/m2/day for 5 days every 28 days for a minimum of 6 cycles in a group of pretreated CMML patients. Evaluation of response was performed after 4 and 6 cycles of therapy, according to IWG 2006 criteria. Morphological diagnosis, evaluation of dysplastic signs and monocytic component was performed centrally after first on-site diagnosis. The mononuclear cell expression of decitabine metabolizing enzymes CDA, hENT1, DCK, and CN-II was determined. Between April 2010 and October 2011, 44 patients were enrolled from 15 Italian Hematology centers, 43 had a confirmed centralized diagnosis of CMML (M/F 30/13), and 42 were finally evaluable. According to WHO 27/42 pts were classified CMML-I with organomegaly and 15/42 CMML-II. Among these patients, 46% had received previous therapy. Proliferative CMML was diagnosed in 32/42 cases. Median age was 71 (42-84) yrs, median number of cycles 6 (1-28). Eleven patients received 〈 4 cycles, 16 from 4 to 6 cycles and 15 〉 6 cycles. Eighty one percent of patients with CMML-I received 〉 4 cycles. All cycles were administered in an out-patient basis. Overall response rate (ORR) was 33%, mCR in 19%, CR in 14.3 %, PR 2.3% and SD in 34.9% and at 3 year-observation time 31% of patients are still alive (13/42). Median duration of response was 9 months. At present, 16.3% (7/42) of patients are still in treatment. Splenomegaly was significantly reduced in 28% of cases. No significative difference in response rate was present in CMML I vs CMML II, nor in proliferative CMML vs dysplastic ones. At analysis of karyotype, 57% of cases had no cytogenetic abnormality and there was no difference in RR between normal and abnormal cases, among which only 5% had complex karyotype. Median OS is 19 months (2-39). For CMML I cases OS is 26 months vs 15 months for CMML II (p

Description: Alemtuzumab is effective for CLL patients previously treated with purine analogues and/or alkylating agents. Currently, pretreated or refractory CLL receive iv alemtuzumab 30 mg 3 times/week for 12 wk. During alemtuzumab an increase of infections (particularly CMV) and a long term reduction of lymphoid subset were described. Some authors have used alemtuzumab 10 mg (4–8 weeks) for minimal residual disease or in combination with fludarabine. Primary endpoint was to assess the efficacy of alemtuzumab, iv at the targeted dose of 10 mg. Secondary endpoints were duration of remission, infectious complications and immune recovery. Twelve CLL patients either in relapse or refractory were treated with alemtuzumab 10 mg for 10 wks (total dose 300 mg). All patients were previously treated with at least two lines of chemotherapy. Median age of the patients was 61.5 years. At the time of treatment 5 patients were in stage B/II, 6 in stage C/IV and 1 in stage A/progressive, the median of lymphocytes in peripheral blood (PB) was 45,030/ μl and 80 % in bone marrow (BM). Six patients showed unmutated VH status, 3 patients had mutated VH status and 3 patients were not evaluable. FISH analysis was evaluable in 11 patients and detected in 1 patient 17p- and 13q-, in 1 patient 11q-, in 1 patient 17 p-, in1 patient 13q-. Trisomy 12 was not detected in any samples. Patients were weekly monitored for CMVAg and CMV PCR and monthly physical examination and BM until the end of the study. Immunological subset (CD3, CD4, CD8, CD16/56, IgG, IgM, IgA) were studied on day 60, 120, 180 and 240 after the end of treatment. Two months after the end of treatment 2 patients obtained CR, and 3 patients PR (OR 41%). At this time 2 patients are in continuous CR at 10 and 15 months. One patient died in continuous PR at 11 months from pneumonia with persistent lymphopenia (200/mmc). One patient in PR showed progression at 5 months and is alive at 21 months, the other is in continuos PR at 11 months. Among 7 patients not responsive to alemtuzumab 2 patients died for progression at 4 and 15 months, while 5 patients are alive at a median of 9 months. Eight patients (66%) showed CMV reactivation by antigenemia and/or CMV DNA. No patient showed CMV disease. Patients were immediately treated, after discontinuation of alemtuzumab, with oral ganciclovir 1000 mg tid. After a median of 14 days all patients achieved negativization of CMV PCR and/or antigenemia. Until the end of the study no patients showed further CMV reactivation. Before treatment all lymphoid subsets, except for total lymphocytes and NK cells, showed median values into the normal range. Total T lymphocytes and CD4 subset rapidly decreased achieving minimum level on day +60; thereafter a mild recovery was noticed but until day +240 median values were constantly below the normal range. CD8 subset showed a trend to normalization even if at the end of the study the median values were below the normal range. NK cells (CD16/56) showed median values above the normal range during the study period, IgA, IgG, IgM showed normal values before and after treatment. Low-dose alemtuzumab in this heavily pre-treated group of patients, was well tolerated and induced good response rate (OR 41%) similarly to standard dose but high incidence of CMV reactivation and the delay of immune reconstitution, still remain a maior issue.

Description: Death-associated protein kinase (DAP-kinase), a proapoptotic serine/threonine kinase, is a candidate tumor suppressor gene. We studied the methylation status of DAP-kinase of 194 bone marrow samples from 160 patients with acute myeloid leukemia (AML) and 34 with a myelodysplastic syndrome (MDS) at the time of initial diagnosis by polymerase chain reaction (PCR). Hypermethylation of DAP-kinase was present in 27.5% (44 of 160) of AML and in 47% (16 of 34) of MDS specimens and significantly correlated to loss of DAP-kinase expression (P = .008). It was significantly more frequent in AML secondary to therapy for other malignancies (s-AML; 14 of 29, 48.3%), as compared to de novo AML (30 of 131, 22.9%, P = .01). DAP-kinase hypermethylation in AML was associated with myelodysplastic changes in the bone marrow at the time of the initial diagnosis (P = .002) and with the presence of cytogenetic abnormalities (P = .02). Alteration in the apoptotic response due to the loss of DAP-kinase function may be an early event in the transformation pathway to secondary leukemia via myelodysplasia.

Description: Hemovigilance studies report that from 6.7% to 15% of overall transfusion-related acute lung injury (TRALI) events occur among obstetrics-gynecological patients (Chapman et al, 2009, Transfusion, 49:440; Ozier et al, 2011, Transfusion, 51: 2102). Severe postpartum hemorrhage (sPPH, i.e. the blood loss in excess of 1000 ml), complicates from 1 to 5% of all deliveries and requires massive transfusion, which is a well acknowledged TRALI risk factor. In this retrospective study we evaluate the incidence and risk factors for TRALI among patients with sPPH. We identified in the blood bank database EmoNet those patients admitted to the delivery room of our hospital from January 2005 to December 2011, necessitating the urgent transfusion of a minimum of 3 red blood cell (RBC) units, with or without fresh frozen plasma (FFP) and platelet (PLT) concentrates. Clinical records of identified patients were then retrieved and demographics, clinical and obstetric data and laboratory and radiological findings were collected. Two anesthesiologists blinded to all information of transfused units independently examined clinical records. Suspected or possible TRALI were diagnosed according to the 2004 consensus criteria; (Kleinman et al. 2004; Transfusion 44: 1774; Toy et al. 2005; Crit Care Med 33:721). In total 71 patients received at least 3 RBC units for sPPH; suspected or possible TRALI was identified in 14 cases (overall incidence 21,6%). The 2 patients with possible TRALI had pneumonia and pulmonary embolism, respectively. On the whole, patients with TRALI were more frequently admitted to the intensive care unit and had a longer hospitalization (p=0.021 and p=0.001, respectively). At univariate analysis, patients with TRALI received a higher number of RBC (p=0,008), PLT (p =0,008,) and FFP units (p =0,034). No difference were found between TRALI and no TRALI groups according to the number of PLT and FFP units from female donors, the storage time of RBC and PLT units or the number of transfused RBC units with a storage time longer than 14 days. Relatively to patient-related factors, TRALI was not associated with age or smoke habit, or with the presence of co-morbidities pre-existing to pregnancy. In contrast, the presence of pregnancy-related hypertensive disorders (PR-HD, including gestational hypertension and preeclampsia /eclampsia) (8 patients, p=0.006) was an important risk factor for TRALI. The poor adverse role was confirmed also in gestational hypertension (6 patients, p=0.012 ) or preeclampsia/eclampsia (4 patients, p=0.022) separately evaluated. In a multivariate model combining both transfusion- and patient-related factors with significance level equal or inferior to 10% at univariate analysis, only PR-HD, gestational hypertension and preeclampsia/eclampsia maintained their significance. In particular, we found that the odds ratio for TRALI was 15.98 (95% IC 2.5-103.5, p=0.004) in PR-HD in total, 24.8 (95% IC 2.5 - 248.4, p=0.006) in gestational hypertension and 33.8 (95% IC 2.2-484.1, p=0.011) in preeclampsia/eclampsia. The strong association between PR-HD and TRALI has never been reported before and it is probably due to the widespread endothelial cell activation occurring in PR-HD. These results impose a careful observation when PR-HD patients receive transfusions. The study was supported by the “Gruppo Donatori Sangue, Francesco Olgiati” Disclosures: No relevant conflicts of interest to declare.

Description: Laboratory workup of the cause of anemia requires clinical staff to develop a differential diagnosis based on routine CBC parameters and to subsequently order confirmatory tests. The number of parameters to be reviewed and the complexity of calculations which are performed by individuals on a routine basis is necessarily limited. The wealth of information in the many novel parameters and the complex patterns provided by modern hematology analyzers are frequently not utilized in routine clinical care. The use of computers for pre-classification of common RBC disorders would provide immediate information to order reflex confirmatory tests on the first sample, thereby improving patient care and allowing significant cost savings. Eleven European sites collected 2,303 data files from hematologically normal patients and individuals diagnosed with at least one of 36 RBC disorders. Samples were run on the ADVIA 120 Hematology System (Bayer HealthCare LLC, Diagnostics Division, Tarrytown, NY), an automated cell counter used in routine clinical hematology laboratories worldwide. Based on their representation within this database, a subset of 5 diseases (β-thalassemia heterozygote, β-thalassemia homozygote, Hb S homozygote, Hb SC, and hereditary spherocytosis; n=779 samples) and 123 normal cases were selected and used to develop a neural-network based computer program, the Computer Assisted RBC Disorder (CARD) Classification tool. The CARD utilizes hundreds of routine and novel CBC, differential and reticulocyte parameters available from the ADVIA 120 and 2120 Hematology Systems to determine possible causes of a patient’s anemia. We evaluated the CARD by using it to classify 273 new cases from 9 worldwide centers. The program correctly identified 93% of the cases. The majority of misidentifications were due to normal cases being classified as abnormal. 2 Hb S homozygote and one β-thalassemia heterozygote samples were misidentified as Hb SC. Only 2 of 137 abnormal cases, which were β-thalassemia heterozygote, were misclassified as normal. The performance of the tool for the presence of any hemoglobinopathy/thalassemia investigated was: sensitivity: 99%; specificity: 90%; PPV: 90%, NPV: 98%. This neural network-based computer program has demonstrated excellent performance with a validation set of samples and demonstrates the potential for using information from automated hematology analyzers to screen for the presence of certain hemoglobinopathies and to provide real-time information to direct an anemia workup. CARD TOOL ACCURACY # Correct # Incorrect Normal 122 14 β-Thalassemia Heterozygote 99 3 β-Thalassemia Homozygote 18 0 Hb S Homozygote 11 2 Hb SC 2 0 Hereditary Spherocytosis 2 0 TOTAL 254 (93%) 19 (7%)

Description: Abstract 1718 In the embryo, hematopoiesis and angiogenesis occur concurrently, since endothelial and hematopoietic cells derive from a common cell precursor, the hemangioblast. In the post natal life, the bone marrow (BM) vascular niche has an important role in supporting hematopoietic stem cells (HSC) proliferation and differentiation. Moreover, during the processes of homing and mobilization, BM vascular niche regulates HSC migration. In BM failure, these functions are promoted by vascular niches located outside of the BM, such as in the spleen, thus confirming that endothelial cells are relevant to the normal hematopoietic differentiation. The dominant feature of myelodyspslatic syndromes (MDS) is the impaired capacity of HSC to give rise to a terminally differentiated normal cell population in one or more lineages. We hypothesized that in MDS patients the vascular niches within and eventually outside of the BM might be altered and that they do not support adequately the physiologic hematopoietic differentiation, thus contributing to the pathogenesis of the disease. To investigate this hypothesis, we isolated and expanded endothelial colony forming cells (ECFCs) (Ingram et al. Blood. 2004;104: 2752) from the peripheral blood of patients with MDS (RCMD) and we used ECFCs as a surrogate of the vascular niche to differentiate hematopoietic cells. Normal CD34+ were seeded over a layer of endothelial cells and cultured for 8 days in different cytokine- containing media able to induce selective granulocytic-macrophage, erythroid or megakaryocyte differentiation. At baseline and after 5 and 8 days of co-culture, hematopoietic cells were recovered by gentle repeated washings, and then they were analyzed with RT-PCR for the expression of lineage specific genes. For each lineage, two genes, mainly involved either in the early or in the late phase of the differentiation, respectively, were analyzed. Basically, we found that co-culturing CD34+ cells in contact with a normal endothelial layer the gene expression observed in the corresponding no-layer cultures was amplified. This effect was strictly dependent on the physical contact between hematopoietic and endothelial cells, since it was abolished when cells were grown separated from a porous membrane. We observed that in co-cultures with normal hematopoietic cells and MDS ECFCs, the modulatory effect exerted by the endothelial layer was partially or completely lost. For example, CD34+ cells undergoing granulocyte differentiation showed a progressive decline of PU.1 gene expression, followed by the rise of the expression of MPO gene; these effects were much more evident if cells were cultured over normal ECFCs. In contrast, this pattern of gene expression was deeply perturbed when normal CD34+ cells were cultured with MDS ECFCs: actually, at day 7, the PU.1 RNA level was six folds higher and the MPO RNA level was three folds lower than that observed in co-cultures with normal ECFCs. Similar figures were observed for RUNX1 and GP1b gene expression in cells undergoing megakaryocyte differentiation. Moreover, CD34+ cells undergoing erythroid differentiation on normal ECFCs exhibited the progressive up-regulation of FOG-1 and of transferrin receptor (TFR) gene expression: interestingly, the co-culture with MDS ECFCs resulted in a lower expression of FOG -1 gene but also in a surprisingly higher expression of the TFR gene. Overall, these data suggest that in MDS does exist an impairment of talking between endothelial and hematopoietic cells. To understand which molecular pathways are involved could represent the basis for a novel therapy approach. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2814 Purpose: ESA are generally the first line treatment of anemia of IPSS low and int1 (lower) risk MDS. While endogenous EPO level and RBC transfusion requirement are well defined prognostic factors of response to ESA (Hellstrom, BJH 2003), many patients (pts) are treated before they require RBC transfusions and, in a GFM large cohort of lower risk MDS with anemia, baseline EPO level was below 500U/l in 88% of the cases (Kelaidi, Haematologica 2010). In non transfusion dependent (non TD) lower risk MDS, it is therefore important to find additional simple prognostic factors of response to ESA. In a joint study of the French (GFM) and Italian (FISM) MDS groups, we retrospectively assessed prognostic factors of response to ESA in non TD lower risk MDS. Particular emphasis was put on the prognostic value of marrow dysplastic features on ESA response, suggested in a previous work (Howe, Blood, 2004). Methods: We are performing a cooperative (GFM and FISM) retrospective study in IPSS low and int 1 (lower risk) MDS diagnosed between 1994 and 2010. The present analysis focused on lower risk MDS with Hb level

Description: Introduction. The granulocyte transfusions (GTXs) are used to booster antimicrobial drugs in severely neutropenic hematological patients. However, the optimal GTX dose and the actual efficacy of this practice are debated. Methods. We retrospectively evaluated the infection-attributable mortality (IAM, i.e. the mortality at 30 days after the last GTX) in 84 consecutive patients with hematological malignancies receiving GTXs (January 2009- December 2014). The indications for GTXs were i) presence of absolute neutrophil count (ANC)

Description: Background: About 10% of low risk patients with myelodysplastic syndromes (MDS) experience severe thrombocytopenia. Bleeding and the scarce efficacy of platelet (PLT) transfusions drive research in novel treatments. Eltrombopag is an oral agonist of the thrombopoetin-receptor (TPO-R). Its potential in increasing platelet (PLT) counts in low risk MDS has not been evaluated. We present interim results on the efficacy and safety of eltrombopag in inducing PLT responses in patients with low and intermediate-1 International Prognostic Scoring System (IPSS) risk MDS with severe thrombocytopenia in a Phase II, multicentre, prospective, placebo-controlled, single-blind study (EQoL-MDS). Methods: Primary endpoints are safety and efficacy of eltrombopag. Secondary endpoints include changes in quality of life (QoL), PLT transfusion requirement, incidence and severity of bleeding, and survival. Inclusion criteria are adult age; PLT200 Gi/L or adverse events. Study design is shown in the figure. PLT response, assessed at each visit, is defined as Response if: 1) baseline PLT〉20 Gi/L: absence of bleeding and increase by at least 30 Gi/L from baseline; 2) baseline PLT20 Gi/L and increase by at least 100%, not due to PLT transfusions; and Complete Response if PLT≥100 Gi/L and absence of bleeding. QoL scores are analysed by MDS-specific instrument, QOL-E v. 3. Results: Seventy patients (46 on eltrombopag - Arm A, 24 on placebo -Arm B) have been randomized at the time of this report. Mean age is 68.3 (SD 13.0) years, M/F 38/32. ECOG performance status was 0 in 47 cases, 1 in 16 cases, 2 in 7 cases. Ten patients had comorbidities. According to the WHO 2008 classification, 22 patients had refractory cytopenia with unilineage dysplasia, 9 had refractory anemia with ringed sideroblasts, 31 had refractory cytopenia with multilineage dysplasia (of which 15 with ringed sideroblasts), 6 had refractory anemia with excess blasts-1 and 2 were unclassified. IPSS score was low in 48 cases. Mean baseline platelet (PLT) count was 17.1 (SD 8.2) Gi/L, mean hemoglobin level 10.8 (SD 2.5) g/dL and mean white blood cell count was 5.0 (SD 3.8) Gi/L. Twenty-five (36%) patients were red blood cell transfusion-dependent. Thirty-three had a WHO bleeding scale of 1, 2 experienced mild blood loss, 4 a gross blood loss and 1 a debilitating blood loss. Fourteen patients in Arm A and 8 in Arm B had required PLT transfusions in the 8 weeks prior to randomization. Twenty-three cases (50%) in Arm A have responded versus 2 (8%) in Arm B (p=0.001). Thirty-three patients have completed at least 24 weeks of study. Median time to response was 14 days (IQR 7-46 days) at a median daily dose of 75 (IQR 50-162.5 mg). PLT count increased by mean 53.2 (SD 68.1) Gi/L (p=0.001) in Arm A versus no significant changes in Arm B by week 24. QOL-E scores at baseline and 12 weeks in 47 cases in both arms are shown in the table. There was an increase in treatment outcome index,mainly experienced in the first 3 weeks (p=0.034). Fatigue improved from baseline to 12 weeks associated with response (p=0.016). Related Grade III-IV adverse events (AE) occurred in 10 patients (22%) in Arm A and consisted in: nausea (4), hypertransaminasemia (3), hyperbilirubinemia (1), sepsis (1), pruritis (1), heart failure (1), asthenia (1), vomit (1), while in Arm B 1 patient (4 %) experienced grade 3 bone marrow fibrosis. MDS disease progression occurred in 5 (11%) in Arm A versus 2 (8%) in Arm B, p=ns. Conclusions: Preliminary data indicate that lower risk MDS patients with severe thrombocytopenia undergoing treatment with eltrombopag experience significant improvements in PLT counts accompanied by improvements in fatigue. The drug appears to be well-tolerated and not associated with MDS progression. Further follow-up is required to evaluate the impact on survival. Figure 1. Study design Figure 1. Study design Figure 2. QOL-E domains at baseline and 12 weeks between Arm A (eltrombopag) and Arm B (placebo) Figure 2. QOL-E domains at baseline and 12 weeks between Arm A (eltrombopag) and Arm B (placebo) Disclosures Oliva: Novartis: Speakers Bureau; Celgene: Other: Advisory Board, Speakers Bureau; Amgen: Consultancy. Santini:celgene, Janssen, Novartis, Onconova: Honoraria, Research Funding. Palumbo:Novartis: Honoraria, Other: Advisory Board. Fenaux:Novartis: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding; Amgen: Honoraria, Research Funding.

Description: Introduction. POEMS syndrome is a rare paraneoplastic condition associated to an underlying plasmacellular dyscrasia. The use of alkylating agents and autologous peripheral blood stem cell transplantation (aPBSCT) seem to be the best strategy. At present aPBSCT should be considered the first line therapy in young patients with POEMS, eligible for high-dose Melphalan (HD-Mel), in absence of organ dysfunction. The best protocol to collect PBSC in patients affected by POEMS remains to be defined, because of the disease rarity and the heterogeneity of published case series. We therefore decided to combine the case series of our two institutions to describe and compare results and outcomes in order to contribute to the definition of the best CD34+ cell mobilization strategy. Patients and methods. We collected clinical and laboratory data of patients affected by POEMS syndrome undergoing hematopoietic stem cells (HSC) mobilization for aPBSCT from 2003 to 2018. Data were organized in order to perform a statistical analysis using "GraphPad Prism" GraphPad Software Inc., (5755 Oberlin Drive, #110, San Diego, CA 92121, USA). The COBE Spectra continuous flow cell separator (Terumo BCT, Shinagawa, Tokyo) was used for leukapheresis. Results. Our data set consisted of 25 patients, of whom 11 were mobilized using cyclophosphamide (CY) 4 g/m2 followed by granulocyte colony-stimulating factor (G-CSF) 5 μg/kg and 14 patients were mobilized using G-CSF 10 μg/kg for 5 days. All patients submitted to mobilization underwent collection procedure. Three patients, because of low CD34+ cells after the administration of G-CSF alone, were submitted to plerixafor infusion achieving a median pre-apheresis CD34+cell count of 28 cells collecting a median of 4.5 CD34+cell/kg body weight. All patient underwent aPBSCT after HD-Mel conditioning regimen receiving an infusion of 4.7 CD34+cell/kg body weight (range 1.5-8.4) and achieved a successful engraftment. At present all the patients are alive and in remission. In order to compare mobilization schedule we performed a comparison analysis between 11 patients receiving chemotherapy as mobilizing regimen versus 14 patients receiving only G-CSF. Data analysis according to mobilization schedule was reported in Table 1. Analysing mobilization efficacy, chemo-mobilized patients achieved a higher pre-apheresis CD34+ cell count (57 vs 33 cells/µl, p