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High coagulation factor VIII (FVIII) levels comprise a common risk factor for venous thromboembolism (VTE), but the underlying genetic determinants are largely unknown. We investigated the molecular bases of high FVIII levels in 2 Italian families with severe thrombophilia. The proband of the first family had a history of recurrent VTE before age 50 years, with extremely and persistently elevated FVIII antigen and activity levels (>400%) as the only thrombophilic defects. Genetic analysis revealed a 23.4-kb tandem duplication of the proximal portion of the F8 gene (promoter, exon 1, and a large part of intron 1), which cosegregated with high FVIII levels in the family and was absent in 103 normal controls. Targeted screening of 50 unrelated VTE patients with FVIII levels ≥250% identified a second thrombophilic family with the same F8 rearrangement on the same genetic background, suggesting a founder effect. Carriers of the duplication from both families showed a twofold or greater upregulation of F8 messenger RNA, consistent with the presence of open chromatin signatures and enhancer elements within the duplicated region. Testing of these sequences in a luciferase reporter assay pinpointed a 927-bp region of F8 intron 1 associated with >45-fold increased reporter activity in endothelial cells, potentially mediating the F8 transcriptional enhancement observed in carriers of the duplication. In summary, we report the first thrombophilic defect in the F8 gene (designated FVIII Padua) associated with markedly elevated FVIII levels and severe thrombophilia in 2 Italian families.

Antisense molecules are emerging as a powerful tool to correct splicing defects. Recently, we identified a homozygous deep-intronic mutation (F5 c.1296+268A>G) activating a cryptic donor splice site in a patient with severe coagulation factor V (FV) deficiency and life-threatening bleeding episodes. Here, we assessed the ability of 2 mutation-specific antisense molecules (a morpholino oligonucleotide [MO] and an engineered U7 small nuclear RNA [snRNA]) to correct this splicing defect. COS-1 and HepG2 cells transfected with a F5 minigene construct containing the patient's mutation expressed aberrant messenger RNA (mRNA) in excess of normal mRNA. Treatment with mutation-specific antisense MO (1-5 mu M) or a construct expressing antisense U7snRNA (0.25-2 mu g) dose-dependently increased the relative amount of correctly spliced mRNA by 1 to 2 orders of magnitude, whereas control MO and U7snRNA were ineffective. Patient-derived megakaryocytes obtained by differentiation of circulating progenitor cells did not express FV, but became positive for FV at immunofluorescence staining after administration of antisense MO or U7snRNA. However, treatment adversely affected cell viability, mainly because of the transfection reagents used to deliver the antisense molecules. Our data provide in vitro and ex vivo proof of principle for the efficacy of RNA therapy in severe FV deficiency, but additional cytotoxicity studies are warranted.

Severe factor V (FV) deficiency is associated with mild to severe bleeding diathesis, but many patients with FV levels lower than 1% bleed less than anticipated. We used calibrated automated thrombography to screen patients with severe FV deficiency for protective procoagulant defects. Thrombin generation in FV-deficient plasma was only measurable at high tissue factor concentrations. Upon reconstitution of FV-deficient plasma with purified FV, thrombin generation increased steeply with FV concentration, reaching a plateau at approximately 10% FV. FV-deficient plasma reconstituted with 100% FV generated severalfold more thrombin than normal plasma, especially at low tissue factor concentrations (1.36 pM) or in the presence of activated protein C, suggesting reduced tissue factor pathway inhibitor (TFPI) levels in FV-deficient plasma. Plasma TFPI antigen and activity levels were indeed lower (P < .001) in FV-deficient patients (n = 11; 4.0 ± 1.0 ng/mL free TFPI) than in controls (n = 20; 11.5 ± 4.8 ng/mL), while persons with partial FV deficiency had inter-mediate levels (n = 16; 7.9 ± 2.5 ng/mL). FV immunodepletion experiments in normal plasma and surface plasmon resonance analysis provided evidence for the existence of a FV/TFPI complex, possibly affecting TFPI stability/clearance in vivo. Low TFPI levels decreased the FV requirement for minimal thrombin generation in FV-deficient plasma to less than 1% and might therefore protect FV-deficient patients from severe bleeding.

Coagulation factor V (FV), present in plasma and platelets, is indispensable to thrombin formation, yet patients with undetectable plasma FV seldom experience major bleeding. We used thrombin generation assays to explore the role of platelet FV in 4 patients with severe congenital FV deficiency (3 with plasma FV clotting activity [FV:C] < 1%). When triggered with tissue factor (TF) concentrations up to 50pM, platelet-poor plasma (PPP) from the patients with undetectable plasma FV showed no thrombin generation, whereas platelet-rich plasma (PRP) formed thrombin already at 1 to 5pM of TF. Thrombin generation in PRP from the FV-deficient patients was enhanced to near-normal levels by platelet activators (collagen or Ca2+-ionophore) and could be completely suppressed by specific FV inhibitors, suggesting FV dependence. Accordingly, platelet FV antigen and activity were measurable in all FV-deficient patients and platelet FVa could be visualized by Western blotting. Normalization of the tissue factor pathway inhibitor (TFPI) level, which is physiologically low in FV-deficient plasma, almost completely abolished thrombin generation in PRP from the FV-deficient patients. In conclusion, patients with undetectable plasma FV may contain functional FV in their platelets. In combination with low TFPI level, residual platelet FV allows sufficient thrombin generation to rescue these patients from fatal bleeding.

Introduction We have evaluated Antithrombin (AT) values and Whole Blood Rotation Thromboelastometry (ROTEM) profiles in children affected by acute lymphoblastic leukemia (ALL), followed at the Clinic of Pediatric Hematology Oncology of Padua. L-Asparaginase, used in protocols for pediatric ALL, is an inhibitor of protein synthesis. Typical collateral effects are coagulation disturbances. Guidelines for identification of patients at risk of thrombosis and hemorrhages are not yet established. AT levels are the only reliable parameter for thrombotic risk. Very low levels of fibrinogen are not correctly detected by classical Clauss method; ROTEM test using FIBTEM is considered a valid tool to identify hypofibrinogenemia and hemorrhagic risk in surgical patients. The aim of the study was to analyze coagulation patterns during treatment with Pegilated L-Asparaginase (Peg-Asp). Methods Forty-two children (25 males, 17 females; 31 pB-ALL; 11 T-ALL; 23 at standard/medium risk; 19 at high risk) were consecutively diagnosed and treated with AIEOP BFM ALL 2009 protocol from June 2012 to March 2014. They received 3 (standard/medium risk) or 8 (high risk) doses of Peg-Asp (2500UI/mq/dose). ROTEM and AT determinations were performed at 5 fixed time-points before and after each Peg-Asp administration. Prothrombin time (PT), fibrinogen plasma levels and platelets counts were recorded. Maximum Clot Firmness (MCF; normal value 9-25 mm), measuring the maximum amplitude reached in FIBTEM thromboelastogram, assessed the specific role of fibrinogen in the whole blood clot formation following platelets inhibition by Cytocalasin D. Results: 798 AT and 706 FIBTEM tests were performed; details are reported in Table. We divided abnormal MCF in 3 grade levels. Only values of MCF >9 mm had a linear correlation with fibrinogen levels. Plasma fibrinogen values Twenty six children had a complete set of tests available during Induction phase (2 doses of Peg-Asp). They showed two different patterns of AT and MCF behavior. In 13 patients, mean AT levels decreased until 12-14 days after Peg-Asp, never being One child had a cerebral sinus thrombosis at the end of induction; one child had extensive thrombosis, strongly related to the central line used for dialysis. Thromboses were not related to fibrinogen concentrates administration or low AT levels. No bleedings were observed. Conclusions: Extensive analysis of coagulation parameters in children undergoing chemotherapy for ALL containing PEG Asp showed abnormalities of AT in 88% of patients and of MCF in 30%. These tests identified patients with significant abnormalities more closely than classical methods. Careful use of AT dosage and FIBTEM can help in managing coagulation disturbances, by identifying patients at risk and suggesting prevention measures. Table: AT and FIBTEM analysis in ALL patients Total N. pts / total N. tests AT 42/798 MCF 42/706 Pts with pathological tests AT Figure 1: Mean AT levels in ALL patients during induction (according to AT supplementation) Figure 1:. Mean AT levels in ALL patients during induction (according to AT supplementation) Figure 2: Mean MCF levels in ALL patients during induction (MCF>9 and MCF 9 and MCF Disclosures No relevant conflicts of interest to declare.

Introduction Consistent data show that the real goal in CML therapy is to obtain a very rapid and deep response because the cinetic of molecular response (MR) is important for quickly reducing the tumor burden by reducing the BCR/ABL level. The second generation TKIs Nilotinib was designed to have enhanced selectivity and potency toward BCR-ABL compared with imatinib and was able to obtain very rapid and deeper response as shown by many clinical trials. Nilotinib has been approved in multiple countries for use in first line therapy in CML-CP Pts. In order to verify the efficacy and safety of Nilotinib used in first line treatment, we collected the results of a real life cohort of Pts treated in some italian hematological Institutions. Patients and Methods 68 Pts affected by CML-CP were treated in 9 hematologic units with Nilotinib as first line therapy from 2010 to 2014. The median age was 47.7y with equal distribution by sex. Cytogenetic analysis at diagnosis showed in 66/68 Pts the presence of the typical Ph1 chromosome, 1 pt showed a Ph1 in a complex translocation and 1 pt showed the typical Ph1 chromosome plus der(17). The molecular analysis confirmed the presence of a BCR/ABL hybrid gene (55/68: b3a2 transcript; 12/68 b2a2 transcript; 1/68 both transcripts). The Sokal risk was: Low 24 Pts (35.3%), Intermediate 33 Pts (48.5%), High 11 Pts (16.2%); the Hasford risk was: Low 35 Pts (51.5%), Intermediate 27 Pts (39.7%), High 6 Pts (8.8%); the EUTOS risk was: Low 61 Pts (89.7%) and High 7 Pts (10.3%). All Pts have an ECOG 0/1 out of 1 with an ECOG 2. 45/68 pts received a cytoreduction by HU. All Pts received the Nilotinib standard dose of 600 mg/die except 1 pt who received 300 mg/die. At diagnosis some Pts showed comorbidities (21/68: hypertension; 6/68: diabetes; 5/68: dyslipidemia; 2/68: previous myocardial infarction; 1/68 was in treatment for HIV infection) Results 66/68 Pts received at least 3 months of treatment and were considered for analysis. At 3 months, all Pts obtained hematological response; cytogenetic response was complete for 65/68 and partial in 3/68. At 6, 12 and 18 months cytogenetic response was complete in all Pts except for 1 that showed a partial response (pt not compliant); at 24 mo the cytogenetic response was complete for all 25 evaluable Pts. The molecular results at the different time points are summarized in table 1. 6/68 Pts showed G1/G2 cutaneous side effects (pruritus, erithema, alopecia) , 6/68 a G1 increase of amylase and/or lipase, but only 1 developed clinical pancreatitis (at diagnosis: hypertension, hypercolesterolemia and cardiac disfunction). One pt underwent to leg amputation for PAOD (at diagnosis: hypertension and hypertriglyceridemia). He discontinued treatment in optimal response at 12mo. One pt had ischemic cerebral event (at diagnosis had hypertension and a previous heart attack). One pt discontinued treatment for pancitopenia in failure of response at 3mo and one pt died at 12mo in failure of response for IMF diagnosed during treatment. No pts developed mutations of BCR/ABL during treatment. In 24 months of observation no Pts lost the response. Conclusion The response to Nilotinib treatment at 3 months according to ELN revealed that all evaluable Pts were in optimal response; at 6 mo 51/55 (92,7 %) Pts were in optimal response, while at 12 mo 38/45 (86,7%) Pts were in optimal response. At 18 mo 94,1% reached at least MMR with a very deep response (MR>= 4) in 55,9% of Pts. This kind of response was mantained at 24 mo (92% of Pts had at least an MR3 and 15/25 had an MR 4). Notabily starting from 12 months, at least 40 % of pts showed a very deep response (MR >= 4) and the number increased at the subsequent time points. The side effects were few and of low grade. Only 3 Pts discontinued the treatment because side effects. Only a severe side effect (PAOD) was reported in a pt who already had a vascular risk because affected from hypertension and hypertriglyceridemia. It is recommended to check at diagnosis and carefully follow during the treatment for all comorbidities to prevent toxicity. These data collected in a real life treatment with Nilotinib in first line in CML -CP patients, confirmed the results of the clinical trials. The rapid and deep response with a tolerable toxicity profile allow to consider Nilotinib as a good choice for first line treatment also in consideration of a stop therapy in a future. Table 1 Time Pts MR1 MR2 MR3 MR4 MR4.5 MR5 (mo) (N) 3 60 10 31 13 4 2 - 6 55 4 16 20 4 11 - 12 45 1 6 20 4 13 1 18 34 0 2 13 6 12 1 24 25 0 2 8 6 8 1 Disclosures No relevant conflicts of interest to declare.

Backgrounds. Not pegilated liposomal doxorubicin (Myocet®) has a better pharmacokinetic profile with less myelosuppression, much lower gastroenterological and cardiological toxicity than conventional doxorubicin. In order to reduce toxicity of conventional doxorubicin, in our department from 2002 to 2007 we have treated patients with Multiple Myeloma (MM) and Non-Hodgkin’s Lymphoma (NHL) with therapeutical regimens replacing conventional anthracyclines with not pegilated liposomal doxorubicin. Methods. In our department, from 2002 to 2007, 35 patients (17M,18F) with a median age of 68.8±9.6(range 82–33) affected by Multiple Myeloma (MM) received one or more cycles according to chemiotherapic scheme: VCR 1mg iv at day 1 + Myocet® 25mg/sm iv at day 2 + CTX 100mg/sm os days 1–4 + PDN 60mg/sm os days 1–4. Diagnosis concerned patients with IgG Myeloma (n=26), IgA Myeloma (n=7), micromolecular Myeloma (n=1) and PL (n=1). 22 patients (16M,6F) with a median age of 63.6±9.6 (range 45–79) affected by NHL received chemoimmunotherapy COMP21-R (CTX, Myocet®, VCR and PDN plus Rituximab). Four patients were stage I, 7 stage II, 6 stage III and 5 stage IV. According to IPI score: 2 patients were low risk, 6 low-intermediate, 8 intermediate-high and 6 high risk. In all patients, chemoimmunotherapy induced cardiologic evaluation was made considering Left Ventricular Ejection Fraction (LVEF): cardiotoxicity was evaluated testing the null hypothesis that the LVEF delta is more negative than −4% of the baseline value before chemotherapy (which was felt to be a reasonable clinical threshold separating non-clinically-significant cardiac toxicity from clinically significant damage due to chemotherapy). Results. In MM patients evaluation of treatment was made for 31 patients: mean Monoclonal Component (MC) has been reduced from 3048.2±2296.9 to 2353,7±1912.4 and the one sided t-test on the variable delta resulted statistically significant (P = 0.038). Response of treatment was CR 43,3%, PR 23,3%, DP 16,7%, NR 16,7% but no statistically significant correlations were present with both the line of treatment and the diagnosis. For NHL evaluation was made in 20 patients: 17 (77.3%) obtained a Complete Remission (CR), 1 (4.5%) a Partial Remission (PR) and 2 (9.1%) Not Respond (NR) to therapy: no statistically significant correlations were present with both the line of treatment and the diagnosis. In all patients (21 NHL and 30MM, totally 51 subjects), Myocet® cardiotoxicity has been evaluated by LVEF assessment at baseline and monitored along the course of the treatment: baseline mean was 57.8±5.4% and slightly lowered to 55.6±5.2% at the end of treatment. For each sub-population and for the entire population a one-sided t-test was performed in order to determine whether therapy with not pegilated liposomal doxorubicin determines relatively low cardiotoxicity: the tests resulted significant (NHL: P = 0.022, MM: P = 0.0021, All: P = 0.00020), leading to reject the null hypothesis in favour of the alternative hypothesis that cardiotoxicity is actually less severe and that the decrement of LVEF is not as negative as −4%. Conclusion. Myocet® reduce cardiotoxicity and is effective in the treatment of MM and NHL patients.

Patients with hemophilia A usually develop inhibitory antibodies to factor VIII (fVIII) that are directed against epitopes in the A2 and C2 domains of the cofactor and result in increased morbidity and mortality because of an extended hemorrhagic syndrome. However, in some cases individuals spontaneously develop autoantibodies to fVIII resulting in acquired hemophilia A. While the etiology and mechanism of the disorder are still unknown, the study and determination of the properties of the autoantibodies is necessary to provide insights into their mechanism of generation. A 78-year-old woman was admitted to the Internal Medicine Department of the University-Hospital of Padua for severe anaemia and syncope. She developed an acute hemorrhagic syndrome characterized by the development of extended subcutaneous and mild intramuscular haematomas on both legs. The patient’s family history was negative for hemorrhagic manifestations. Total body computed tomography (CT) scan confirmed the presence of small intramuscular haematomas. Preliminary laboratory analyses revealed a significant prolongation of the aPTT (activated partial thromboplastin time) to 60 sec and a mild prolongation of the PT (prothrombin time) to 15 sec. Mixing experiments with normal plasma failed to correct the patient’s aPTT. Plasma fVIII levels were ~6%. Assay for fVIII inhibitor revealed a level of 6 Bethesda Units (BU). The patient was treated with high dose of steroids and recombinant fVIII (3,000 Units daily). Subsequently, she developed swelling to the neck in the sub maxillary region and symptoms consistent with an acute obstruction to the upper airways requiring oral-tracheal intubations and ventilation. These findings prompted the use of recombinant FVIIa to prevent progression of the hemorrhagic lesion. Three sessions of plasmapheresis were performed, which were followed by the administration of high dose immunoglobulin and the administration of immunosuppressive treatment. After 45 days, treatment was continued with steroids only. The patient recovered completely and no new bleeding episodes developed. The total immunoglobulin fraction was isolated from the patient’s plasma and found to induce a prolongation of the clotting time in an aPTT assay using purified reagents. The immunoglobulin fraction was also found to inhibit intrinsic tenase activity in an assay using purified reagents and a chromogenic substrate that detects factor X activation. In contrast, the total immunoglobulin fraction purified from normal pooled plasma didn’t prolong the aPTT nor inhibited intrinsic tenase activity. Immunoprecipitation experiments with the total immunoglobulins fraction purified from the patient’s plasma revealed that the antibody recognizes epitopes on the light chain of the cofactor. Immunobloting experiments performed with the same material demonstrated that the antibody recognizes fVIII heavy and light chains. Following activation by thrombin it was found that the antibody recognizes the Mr 73,000 light chain and the A2 domain of the cofactor. These data demonstrate that the immunoglobulin fraction isolated from the patient’s plasma has more than one epitope on the cofactor. Our findings provide the demonstration of a strong anti-fVIII acquired autoantibody with low titer which is not related to the presence of antiphospholipid antibodies and is responsible for a severe hemorrhagic syndrome.