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Signaling through the transforming growth factor–β (TGF-β) pathway results in growth inhibition and induction of apoptosis in various cell types. We show that this pathway is blocked in Kaposi sarcoma herpesvirus (KSHV)–infected primary effusion lymphoma through down-regulation of the TGF-β type II receptor (TβRII) by epigenetic mechanisms. Our data also suggest that KSHV infection may result in lower expression of TβRII in Kaposi sarcoma and multicentric Castleman disease. KSHV-encoded LANA associates with the promoter of TβRII and leads to its methylation and to the deacetylation of proximal histones. Reestablishment of signaling through this pathway reduces viability of these cells, inferring that KSHV-mediated blockage of TGF-β signaling plays a role in the establishment and progression of KSHV-associated neoplasia. These data suggest a mechanism whereby KSHV evades both the antiproliferative effects of TGF-β signaling by silencing TβRII gene expression and immune recognition by suppressing TGF-β–responsive immune cells through the elevated secretion of TGF-β1.

Signaling through the transforming growth factor–β (TGF-β) pathway results in growth inhibition and induction of apoptosis in various cell types. We show that this pathway is blocked in Kaposi sarcoma herpesvirus (KSHV)–infected primary effusion lymphoma through down-regulation of the TGF-β type II receptor (TβRII) by epigenetic mechanisms. Our data also suggest that KSHV infection may result in lower expression of TβRII in Kaposi sarcoma and multicentric Castleman disease. KSHV-encoded LANA associates with the promoter of TβRII and leads to its methylation and to the deacetylation of proximal histones. Reestablishment of signaling through this pathway reduces viability of these cells, inferring that KSHV-mediated blockage of TGF-β signaling plays a role in the establishment and progression of KSHV-associated neoplasia. These data suggest a mechanism whereby KSHV evades both the antiproliferative effects of TGF-β signaling by silencing TβRIIgene expression and immune recognition by suppressing TGF-β–responsive immune cells through the elevated secretion of TGF-β1.

Background.Patients with Essential Thrombocythemia (ET) may have thrombotic and bleeding complications with increased morbidity and mortality. Age, previous history of thrombosis, elevated white cell counts and JAK2 allele burden are considered risk factors for both arterial (ATE) and venous (VTE) thrombosis while elevated platelet counts and qualitative defects of the von Willebrand factor (VWF) are associated with bleeds. ADAMTS13 regulates the pro-thrombotic activities of the VWF: reduced levels of ADAMTS13 or VWF activities expose patients to increased risk of thrombosis or bleeds. Aims:to evaluate whether or not the reduced activities of ADAMTS13/VWF were associated with thrombotic/bleeding complications, 72 patients with ET diagnosed according to WHO criteria at the Hematology Center of the L. Sacco University Hospital of Milan have been observed prospectively during a 36-month follow-up. Methods:Patients were exposed to all the diagnostic tests required for ET diagnosis and signed an informed consent for these ADAMTS13/VWF evaluations. VWF (VWF:RCo, VWF:Ag) activities and FVIII were measured in citrated plasma by the automatic ACL-TOPsystems using commercially available reagent kits while ADAMTS-13 by TECHNOZYM activity ELISA. Statistical analyses among the cases with or without complications were performed with SPSS. Results:the cohort of ET patients was positive for JAK2(59%)/MPL(5%)/CALR(12%) with negative mutations in 17 cases. 3 different groups were found in this cohort: A)NOCOMPL=39/72(54.2%);B)ATE/VTE=22/72(30.5%);C)BLEEDS=11/72(15.2). The following clinical-lab data [median(range) according to these A/B/C groups are: M-F=11-28/5-17/3-8; Age=68(17-88)/73(33-89)/64(38-80);Plt-counts(/uL)=566(430-780)/559(398-551)/704(478 -1342);VWF:ACT(U/dL)=98(76-238)/163(86-282)/63(27-92); ADAMTS13(U/dL)=86(56-144)/45(31-105)/85(49/152);VWF:ACT/Ag:ratio=0.85(0.7-1.4)/0.75(0.6-1.2)/0.65(0.2/0.8); ADAMTS13/VWF:ACTratio=0.74(0.41-1.8)/0.54(0.25/1.2)/0.76(0.5-2.5). Liver and/or spleen enlargement was found in 23/72(32%) of all A/B/C patients but did not correlate with complications. A total of 30 ATE/VTE and 18 major/minor bleeds were observed in these 22 and 11 ET with complications while 6 patients had both VTE and BLEEDS during follow-up. Reduced levels of ADAMTS13 and of VWF activities correlated with the observed thrombotic and bleeding event. Conclusion:In a well characterized cohort of ET patients we could find that reduced levels of ADAMTS13 and VWF activities can be associated with their thrombotic and bleeding complications. These assays might be useful during the clinical follow-up to identify patient at risk for these complications.

Viviani: Takeda Italia SpA: Consultancy; Teva Italia SpA: Consultancy; Italfarmaco SpA: Consultancy; Takeda International: Consultancy. Corradini:Celgene: Consultancy; Novartis: Consultancy.

BackgroundThe majority of relapses in HL occur within the first three years from end of first-line treatment. Late relapses, occurring after more than five years, are rare events and there is no consensus on the optimal treatment.

We tested the efficacy and safety of fixed doses of Low-Molecular Weight Heparin (LMWH) in cancer patients requiring interruption of Vitamin-k Antagonist (VKA) because of invasive procedures (defined as major and non major surgery) or chemotherapy inducing platelets depletion. Methodology. Cancer patients were defined to be at high (atrial fibrillation [AF] with previous stroke, prosthetic mitralic valves and venous thromboembolism [VTE] lasting < 3months) or low risk of thrombosis (AF without previous stroke, VTE lasted > 3 months, and prosthetic aortic valves). They discontinued VKA 5 + 1days before surgery or chemotherapy; in those at low-risk for thrombosis, LMWH was given at a prophylactic dosage of 3.800 U.I. (nadroparin) or 4.000 U.I. (enoxaparin) anti-FXa once daily the night before the procedure or the beginning of chemotherapy. In patients at high-risk for thrombosis, LMWH was started early after VKA cessation (when an INR < 1.5) and given at fixed sub-therapeutic doses (3.800 or 4.000 UI anti-FXa twice daily) until procedure or beginning of chemotherapy. In patients undergoing procedures, LMWH was reinitiated 12 hours after procedure while VKA the day after; in patients receiving chemotherapy, LMWH was reinitiated 12 h after a stable platelet count > 30.000 mmc3 and VKA 12 h after a stable platelet count > 50.000 mmc3. Heparin was continued until a therapeutic INR value was reached. The primary efficacy endpoints were the incidence of thromboembolism and major bleeding from VKA suspension to 30 + 2 days post-procedure or next chemotherapy. Results. A total of 156 patients (68, 53.9% at low-risk and 58, 46.1% at high-risk for thrombosis) were enrolled (Table 1); 44 (28.2%) underwent major surgery, 53 (33.9%) non-major surgery and 29 (18.5%) chemotherapy. Overall, thromboembolic events occurred in 5 patients (3.2%, 95% confidence intervals 0.5–5.9), 4 belonging to high-risk and 1 to low-risk group. Overall, major bleeding occurred in 5 patients (3.2%, 95 CI 0.5–5.9), all belonging to high-risk group (3 during major surgery and 2 during chemotherapy). The mean time of anticoagulation with LMWH was 7.5 days in patients underwent procedures and 13.2 days in those who received chemotherapy. Conclusion. LMWH given at fixed sub-therapeutic doses appears to be a feasible and relatively safe approach for bridging therapy in chronic anticoagulated cancer patients requiring VKA interruption.No relevant conflicts of interest to declare.

Abstract 1244

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of cells of myeloid origin, and include immature macrophages, granulocytes, dendritic cells (DC) and other myeloid cells. In mice, are phenotypically characterized as CD11b+Gr-1+. Humans MDSC have an immature phenotype, including lineage negative (Lin-), CD14-, HLA-DR-, CD15+, CD34+, CD11b+, CD33+, and CD13+ cells. MDSC impair T-cell functions through secretion immunosuppressive cytokines, perturbation of the arginine metabolism by inducible nitric oxide synthase (iNOS), up-regulation of reactive oxygen species (ROS), therefore are considered an important tumor escape mechanism. These cells, also promote tumor-dependent angiogenesis as well as tumor metastasis, and to provide tumor resistance to antiangiogenic drugs. Their accumulation has been described in the peripheral blood of patients affected by breast, lung, renal and neck carcinoma, melanoma, chronic infections, inflammatory diseases, and traumatic stress.We investigate MSDC in patients with multiple myeloma (MM) and monoclonal gammopathy undetermined significance (MGUS) by flow cytometryWe studied 22 patients with MM at diagnosis, 20 patients with MGUS, and 10 healthy controls (HC).we observed that patients with MGUS showed the same number of MDSC (CD11b+,CD13+,CD34+,CD14-,CD45+) in the peripheral blood compared to HC (1,90±1,03/mmc, p=0,23). On the contrary, patients affected by MM showed a significant increase of MDSC vs HC (6,80±8,79/mmc vs 1,45± 0,98/mmc, p=0,003).Our results suggest that these myeloid-derived suppressor cells, through their mechanisms immunesuppressive and proangiogenesis, could be involved in the progression of MGUS towards overt MM.No relevant conflicts of interest to declare.

Immune suppression and angiogenesis are mechanisms key to tumour growth and progression. Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of cells of myeloid origin and include immature macrophages, dendritic cells (DC) and other myeloid cells. In mice are phenotypically characterized as CD11b+Gr-1+ cells, while in human they have an immature phenotype, including lineage negative (Lin-), CD14-, HLA-DR-, CD15+, CD34+, CD11b+, CD33+, and CD13+ cells. MDSC reduce activated T-cell number and inhibit their function through different mechanisms including: L-arginine metabolism, nitric oxide (NO), up-regulation of reactive oxygen species (ROS), and secretion of immunosuppressive cytokines. MDSC also promote tumor-dependent angiogenesis as well as tumor metastasis.Their accumulation has been described in patients affected by some solid tumors but information on haematological neoplasms are lacking. Our study investigated by flow cytometry the presence of MSDC in the peripheral blood of patients affected by Hodgkin Lymphoma (HL).We studied 14 patients with HL at diagnosis and 10 age-matched healthy controls (HC). Peripheral blood mononuclear cells were stained with the following monoclonal antibodies:CD11b, CD13, CD14, CD34, CD45, for 20 minutes at room temperature. After lysing red cells, cells were analyzed by flow cytometry.we observed a increased number of MDSC (CD11b+,CD13+,CD34+,CD14-, CD45+) in the peripheral blood of patients with HL compared to HC (13,37 ± 17,77 ×109/l vs 1,45± 0,98 ×109/l, p=0,0007). We also found that patients with advanced-stage Hodgkin disease (III and IV) have higher number of MDSC, compared to patients stage I and II (p= 0,04).These data suggest a role for myeloid-derived suppressor cells in promoting tumor cell proliferation in hodgkin lymphoma.No relevant conflicts of interest to declare.

Abstract 4890

Abstract 3662

Kaposi’s sarcoma-associated herpesvirus (KSHV) interferes with a number of cellular pathways involved in apoptosis and cell cycle regulation. Since TGFß is a potent inducer of cell cycle arrest and apoptosis in hematopoietic lineages, we were interested in examining whether TGFß signaling is blocked in primary effusion lymphoma (PEL) and if so whether KSHV plays a role in this repression. Treatment with TGFß1 had no effect on either cell cycle or apoptosis in several PEL cell lines. The defect in this pathway was found to be a lack of the TGFß type II receptor (TßRII), expression. Upon transfection of TßRII, PEL cell lines become responsive to TGFß1 treatment. We are currently examining how TßRII transcription is regulated in PEL. The lack of TßRII expression in some tumors has been attributed to altered chromatin structure due to hypoacetylation. Treatment with both a demethylating agent and a histone deacetylase inhibitor resulted in expression of this receptor in PEL. Several KSHV latent gene products are known to interact with or recruit p300, a histone acetyltransferase, for other cellular processes. It is possible that the interaction of one or more of these viral gene products with p300 may serve to prevent it from acetylating histones associated with the TßRII gene and thereby repress its transcription. We have also found by ELISA that PEL cells secrete a higher level of TGFß1 than other TGF ß responsive B cell lines. Increased secretion of TGFß1 in conjunction with downregulation of TßRII may be a mechanism by which KSHV creates an immunosuppressed environment while allowing infected cells to grow free from the constraints of this pathway.

Kaposi's sarcoma-associated herpesvirus (KSHV) interferes with a number of cellular pathways involved in apoptosis and cell cycle regulation. Since TGFβ is a potent inducer of cell cycle arrest and apoptosis in hematopoietic lineages, we were interested in examining whether TGFβ signaling is blocked in primary effusion lymphoma (PEL) and if so whether KSHV plays a role in this repression. Treatment with TGFβ1 had no effect on either cell cycle or apoptosis in several PEL cell lines. The defect in this pathway was found to be a lack of the TGFβ type II receptor (TβRII), expression. Upon transfection of TβRII, PEL cell lines become responsive to TGFβ1 treatment. We are currently examining how TβRII transcription is regulated in PEL. The lack of TβRII expression in some tumors has been attributed to altered chromatin structure due to hypoacetylation. Treatment with both a demethylating agent and a histone deacetylase inhibitor resulted in expression of this receptor in PEL. Several KSHV latent gene products are known to interact with or recruit p300, a histone acetyltransferase, for other cellular processes. It is possible that the interaction of one or more of these viral gene products with p300 may serve to prevent it from acetylating histones associated with the TβRII gene and thereby repress its transcription. We have also found by ELISA that PEL cells secrete a higher level of TGFβ1 than other TGF β responsive B cell lines. Increased secretion of TGFβ1 in conjunction with downregulation of TβRII may be a mechanism by which KSHV creates an immunosuppressed environment while allowing infected cells to grow free from the constraints of this pathway.