ALBERT

Description: Abstract 4459 Chronic myeloid leukemia (CML) is a myeloproliferative stem cell disorder characterized by the translocation t(9,22)(q34;q11), resulting in the Philadelphia chromosome (Ph) and the bcr-abl fusion gene. A small proportion of Ph+ CML patients (5–10%) show variant Ph translocations involving one or more chromosomes in addition to chromosomes 9 and 22. Its generation mechanism and clinical significance remains unclear. We report a novel four-way translocation detected in a newly diagnosed chronic phase CML patient. Case presentation A 51 year-old white women with no significant past medical history presented with a two-week upper left abdominal pain. She denied weight loss, fever, night sweats, fatigue or thrombosis. She has 11 siblings. On physical examination an enlarged spleen was detected, 7 cm below left costal margin. No hepatomegaly was found. Peripheral blood counts on admission showed WBC 286 × 109/l, with immature myeloid forms (21%bands, 31%neutrophils, 3%lymphocytes, 5%basophiles, 1%eosinophils, 16%metamyelocites, 14%myelocytes, 6% promyelocytes, 1% blast cells), hemoglobin 11,8 g/dl and platelet count of 986 ×109/l. LDH= 1094 IU/L. Uric acid, renal and hepatic functions were normal. Abdominal ultrasonography showed homogeneous splenomegaly (185 mm) with no focal lesions. Bone marrow aspirate demonstrated increased cellularity with myeloid hyperplasia without remarkable basophilia. Trephine demonstrated hypercellularity without fibrosis and 1% blast cells. Conventional cytogenetic analysis by GTG-banding revealed a variant Ph chromosome positive in 100 % of metaphases analysed involving chromosomes 1 and 17 inaddition to chromosomes 9 and 22: 46 XX, t (1;17; 9; 22) (p?35;q24;q34;q11)[20]. No normal cells nor additional numerical and structural chromosomal changes were detected at diagnosis. A fragment of 1605 bp was amplified with primers BCR-P1F and ABL-P1R. The sequence analysis of the amplified PCR product showed a complex BCR-ABL rearrangement formed by exon 8 of the BCR (NM_004327) and exon 2 of the ABL genes (NM_005157), with a 148 bp insertion corresponding to the whole exon 2 of the MAST2 (NM_015112, Homo sapiens microtubule associated serine/threonine kinase 2) gene. Bone marrow cytogenetical evaluation at four months do not show Philadelphia chromosome on 20 cells analysed. Interphase FISH with DUAL Colour Dual Fusion probe has a normal pattern on 300 nuclei: 2R2G. It corresponds to a complete cytogenetic response. Chronic phase CML was diagnosed and cytoreductive treatment with hydroxyurea 1,5 g/day, fluids, thromboprophilaxis and allopurinol was initiated. A good control of blood counts was obtained, as well as a complete reduction of the enlarged spleen and regression of left abdominal pain. Once cytogenetic/molecular confirmed diagnosis, treatment with Imatinib 400 mg/day was started, and hidroxiurea was discontinued. She achieved complete hematologic response at 1 month. Splenomegaly was no longer detected. At 2 months she developed neutropenia (1100/mm3) and thrombocytopenia (55000/mm3) so Imatinib was held. Two weeks later, after hematologic recovery, Imatinib was re initiated but in the following control neutropenia and thrombocytopenia were again detected. Discussion About 90–95% of CML have the characteristic t(9;22)(q34;q11.2) reciprocal translocation that results in the Ph chromosome [der(22q)], the cytogenetical hallmark of this entity. The remaining cases can have variant translocations that involve 3, 4 or more chromosomes and its occurrence is not associated with disease evolution. This patient shows a four way translocation involving chromosomes 1 and 17 in addition to chromosome 9 an d 22. It is controversial whether variant translocations result in a different disease phenotype. There is no impact on the cytogenetic or molecular response or outcome, so a special approach is not recommended. Conclusion This is a novel four way translocation described in a chronic phase CML patient, with a good response to standard therapy. Complete haematological and cytogenetic response at 4 months of treatment with Imatinib was achieved. Up to now, a differential approach does not appear to be mandatory in CML with variant translocations. However, in this patient alternative tyrosine kinase inhibitor may be of benefit due to hematologic toxicity attributable to Imatinib. Disclosures: No relevant conflicts of interest to declare.

Description: Posttransplantation human herpesvirus-8 (HHV8)/Kaposi sarcoma herpesvirus (KSHV) primary infection and/or reactivations are associated with uncommon and sometimes fatal, neoplastic, and non-neoplastic diseases. HHV8-related clinical manifestations notably range from Kaposi sarcoma (KS) to either primary effusion lymphoma or multicentric Castleman disease B-cell malignancies, and from polyclonal HHV8-positive plasmacytic lymphoproliferative disorders to bone marrow failure and peripheral cytopenias, associated or not with hemophagocytic syndromes, and to acute hepatitis syndromes. We reviewed the patient series reported in the literature and summarized clinical management aspects, in terms of diagnosis, follow-up, and treatment. We described typical clinical presentations and histopathologic diagnostic features of these diseases, and we discussed the role of HHV8-specific serologic, molecular, and immunologic assays, particularly focusing on recent data from HHV8-specific T-cell monitoring in posttransplantation KS patients. We finally discussed actual therapeutic options, namely, the reduction or discontinuation of immunosuppressive therapy or the switch from calcineurin inhibitors to mTOR inhibitors, as alternatives to antineoplastic chemotherapy, along with the use of antiherpesvirus agents as prophylactic or therapeutic measures, and treatment with rituximab in posttrans-plantation multicentric Castleman disease patients and non-neoplastic HHV8-associated syndromes.

Description: Abstract 1898 Background: In mice, graft-versus-host disease (GvHD) can be abrogated by ex vivo expanded, bone marrow derived myeloid-derived suppressor cells (MDSCs) generated in the presence of GM-CSF, G-CSF and IL-13 (Highfill et al). Whether MDSCs play a role in human allogeneic hematopoietic stem cell transplantation (allo-HSCT) is still unclear. We hypothesized that G-CSF stem cell mobilization may be protective from acute GvHD (aGvHD) by increasing MDSC frequencies. Methods: G-CSF-mobilized peripheral blood (PB) samples were collected from 40 healthy unrelated donors (median age 34, range 20–43, male/female 31/9) who received G-CSF (Filgrastim) at 10 μg/kg/day for 5 days. Donor selection had been performed according to standard criteria, including molecular typing for HLA-A,-B,-C, DRB1, and DQB1. Donors were 10/10 HLA-matched (MUD) in 20 cases, 9/10 in 13 cases and 8/10 in the remaining 7 cases (MMUD). Patients (median age 46, range 18–67) received reduced intensity conditioning based or low-dose total body irradiation (TBI 2Gy) (8), Fludarabine-Rabbit Antithymocyte Globulin (ATG) (9) or Thiotepa-ATG (23). Diagnosis were lymphomas (29), myelomas (8), acute myeloid leukemia (3). GvHD prophylaxis was cyclosporine plus either methotrexate (36) or mycophenolate mofetil (4). As controls, PB samples were collected from 10 healthy adults. Informed consent was obtained from all subjects. Cells were characterized using flow cytometry with Abs against CD3;CD14;CD16;CD19;CD20;CD56;CD11b;HLADR;CD33. The frequencies of MDSCs and T regulatory cells (Tregs, CD4+CD25+CD127-FoxP3+) in the grafts were correlated with the clinical characteristics and outcome of the 40 patients. To verify that G-CSF treatment increases the number of MDSCs that are transferred with the graft and prevent GvHD, a major histocompatibility complex (MHC) mismatched HSCT mouse model was also used. After lethal irradiation, recipient BALB/c mice received spleen and bone marrow (BM) cells from C57BL/6 (B6) donors (GvHD cohort, n=5) or BM cells only (negative control, n=3). To generate MDSC enriched allografts, donor mice were treated with G-CSF (5 μg/d for 5 days) and thereafter BM cells and spleen cells were transferred in recipient mice (MDSC cohort, n=5). Results: Expansion of MDSCs (Lin-/LoHLADR−CD33+CD11b+) in the PB of G-CSF-treated unrelated donors was found with respect to steady state control individuals (p〈 0.03, Mann Whitney-U). Acute GvHD occurred in 16 of 40 patients (40%). There was no significant correlation between the incidence of aGvHD and the degree of HLA incompatibility or the presence of donor-recipient sex mismatches. Neither the conditioning regimens nor the GvHD prophylaxis had effect on risk of aGvHD. There was no correlation between the number of Tregs infused and the occurrence of GvHD.Conversely, aGvHD patients received grafts containing significantly lower number of MDSCs when compared to non- aGvHD patients (p 〈 0.006, Mann Whitney-U). The ability of MDSC levels in the graft to predict the occurrence of aGvHD was determined by the receiver operating characteristic (ROC) curve: sensitivity was 100%, specificity 60%, AUC=0.768. The immunosuppressive activity of the MDSCs on activated T lymphocytes was confirmed in vitro. To confirm these results, we set up a MHC mismatched HSCT mouse model. G-CSF treatment induced a significant increase in MDSCs (up to four fold, p

Description: Abstract 499 Background: Treatment of splanchnic vein thrombosis (SVT) is a clinical challenge due to heterogeneity of clinical presentations, increased bleeding risk and lack of evidences from clinical trials. We carried out an international registry aimed to describe current treatment strategies and factors associated with therapeutic decisions in a large prospective cohort of SVT patients. Methods: Between May 2008 and January 2012, consecutive SVT patients were enrolled in the registry and information on clinical presentation, risk factors, and therapeutic strategies was collected in an electronic database. Clinical outcomes during the first month of treatment were documented. A two-year follow up is ongoing. Results: 613 patients from 12 countries were enrolled in the registry. Mean age was 53.1 (SD ± 14.8) years (range 16–85); 62.6% were males, 74.4% Caucasians. SVT occurred in the portal vein in 470 patients, in the mesenteric vein in 266, in the splenic vein in 139, and in the supra-hepatic veins in 56; 38.8% of patients had multiple vein segments involved. In 29.8% of patients SVT diagnosis was incidental. Most common risk factors included cirrhosis (27.8%), solid cancer (22.3%), intra-abdominal inflammation/infection (11.5%), surgery (8.9%), and myeloproliferative neoplasm (MPN)(8.2%); in 27.6% of patients SVT was idiopathic. During the acute phase, 471 (76.8%) patients were treated with anticoagulant drugs: unfractionated heparin (10.4%), low molecular weight heparin or fondaparinux (66.4%), vitamin K antagonists (VKA) (48.5%). Four patients received aspirin, 9 received thrombolysis. A total of 135 patients (22.0%) remained untreated. Of patients with incidentally diagnosed SVT, 61.1% received anticoagulant treatment. Incidental diagnosis (p

Description: Abstract 4945 Background: bone marrow blast count is a crucial parameter to determinate the prognostic score according to IPSS in myelodysplastic syndromes. However even among the major experts the agreement about detection of a blast cells by optical microscopy is less than 90% (Mufti et al: Haematologica 2008). The aim of this study is to evaluate prognostic significance of immunocytometric count of medullar blasts compared to cytological count. Method: in a retrospective analysis of 104 patients with a minimum follow-up of three years, IPSS was calculated replacing cytological count of blasts with the flow cytometry one. Blasts were expressed as percentages of total cellularity, and identified by the combination of CD45/SSC with/without CD33, CD34 and CD117 antigens. The monoclonal antibodies used were CD34 FITC, CD117 PE, CD45 PerCp, CD33 APC. Acquisition of information on 1×105 stained cells corresponding to the whole bone marrow cellularity was assessed on a dual-laser FACSCalibur flow cytometer using the CellQUEST software (Becton Dickinson, San José CA USA). Survival of “low + intermediate-1 risk” (“IPSS1+2”) patients was compared to “intermediate-2 + high risk” ones (“IPSS3+4”) using Kaplan Meier method followed by logrank test. The comparison of ability among all blast count models to distinguish the probability of survival of the two groups (“IPSS 1+2” vs “IPSS3+4”) was based on χ2 value obtained by the logrank test. Results: considering IPSS obtained from any of the immunocytometric markers considered (CD33, CD34, CD117 and CD45dim/SSC), “IPSS1+2” group had always an extremely better survival than “IPSS 3+4” group with a level of statistical significance similar to survival study of “standard IPSS”. Of all markers, CD45dim/SSC was the most capable to distinguish survival of “IPSS1+2” group versus “IPSS3+4” group (χ2 = 42. 5). Among others, CD117 and CD33 had a better discriminating power than cytological count (respectively χ2 = 38, 1 and χ2 = 22, 3 versus χ2 18, 2); only CD34 was worse than cytological count (χ2 =14, 2). Conclusion: the immunocytometric markers considered are demonstrated to be even better than cytological count as parameter in IPSS score. Their use has to be encouraged, especially where there is little experience in optical microscopy. Disclosures: No relevant conflicts of interest to declare.