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BACKGROUND Genital chronic graft-versus-host disease (GVHD) is a frequent but underdiagnosed complication of allogeneic stem-cell transplantation impairing quality of life. METHODS We identified 32 female patients with genital chronic GVHD (cGVHD) who underwent allogeneic hematopoietic stem-cell transplantation in our center between 2000 and 2010 and who were followed after transplantation in a specialized gynecological consultation. Pre- and posttransplantation clinical data and detailed acute and cGVHD data were collected. All patients received the same local treatment for genital lesions. RESULTS At presentation, most patients complained about vaginal dryness and dyspareunia with impairment in sexual activity. Fifty percent of patients had grade I genital lesions and 50% had grade II or III lesions. Patients seen later in gynecological consultation had more severe lesions than patients seen early after transplantation. At the time of diagnosis, most patients had other cutaneous or mucous localizations of cGVHD. In most cases, lesions were stabilized or decreased with local steroids and estrogen treatment, and most patients could resume sexual activity. Treatment was more efficient in patients with mild lesions than in others. CONCLUSIONS Genital cGVHD should be systematically searched for in women who have received allogeneic hematopoietic stem-cell transplantation in an early specialized consultation, especially in case of other cutaneous or mucous localizations of cGVHD. Local treatment associating steroids and estrogen seemed to prevent further evolution of grade I genital lesions and to avoid surgical treatment.

Background Voriconazole treatment increases early survival of allogeneic hematopoietic stem cell transplant recipients with invasive aspergillosis. We investigated whether this survival advantage translates into an increased long-term survival. Design and Methods This retrospective study involved all patients with an invasive aspergillosis diagnosis transplanted between September 1997 and December 2008, at the Saint-Louis Hospital, Paris, France. The primary end point was survival up to 36 months. Survival analysis before and after 12 weeks, as well as cumulative incidence analysis in a competing risk framework, were used to assess the effect of voriconazole treatment and other factors on mortality. Results Among 87 patients, 42 received first-line voriconazole and 45 received another antifungal agent. Median survival time was 2.6 months and survival rate at 36 months was 18%. Overall, there was a significant difference in the survival rates of the two groups. Specifically, there was a dramatic difference in survival rates up to ten months post-aspergillosis diagnosis but no significant difference after this time. Over the first 36 months as a whole, no significant difference in survival rate was observed between the two groups. First-line voriconazole significantly reduced aspergillosis-attributable mortality. However, first-line voriconazole patients experienced a significantly higher probability of death from a non-aspergillosis-attributable cause. Conclusions Although the prognosis for invasive aspergillosis after stem cell transplantation has dramatically improved with the use of voriconazole, this major advance in care does not translate into increased long-term survival for these severely immunocompromised patients.

PIGA mutations in paroxysmal nocturnal hemoglobinuria (PNH) patients lead to a glycosylphosphatidylinositol (GPI)-linked membrane proteins expression deficiency. Herein, we report the constitutive expression of the transmembrane CD160 (CD160-TM) activating receptor on non PIGA-mutated PNH patients circulating NK cells. In healthy individuals, only the GPI-anchored isoform of CD160 receptors is expressed on the circulating NK lymphocytes, while the transmembrane isoform appears after ex vivo activation. Similarly to CD160-GPI, we identified CD160-TM as a receptor for the MHC class I molecules. We demonstrate that PNH patients NK lymphocytes spontaneously produce significant amounts of IFN-γ that is inhibited by anti-CD160-TM or anti-MHC class I mAbs. These results indicate that circulating NK cells from PNH patients exhibit a self-MHC class I molecule reactive effector function, which could be mediated through the recruitment of CD160-TM receptor. Our data provide new insights regarding the possible role of CD160-TM on PNH patients NK lymphocytes and in the pathogenesis of the disease.

Background Due to increased rates of secondary solid organ cancer in patients with severe aplastic anemia who received an irradiation-based conditioning regimen, we decided some years ago to use the combination of cyclophosphamide and antithymocyte globulin. We report the long-term follow up of patients who underwent hematopoietic stem cell transplantation from an HLA-matched sibling donor after this conditioning regimen.Design and Methods We analyzed 61 consecutive patients transplanted from June 1991 to February 2010, following conditioning with cyclophosphamide (200 mg/kg) and antithymocyte globulin (2.5 mg/kg/day × 5 days).Results Median age was 21 years (range 4–43); 41 of the 61 patients were adults. Median duration of the disease before hematopoietic stem cell transplantation was 93 days. All but 2 patients received bone marrow as the source of stem cells and all but 2 engrafted. Cumulative incidence of acute grade II–IV graft-versus-host disease was 23% (95%CI 13–34) and 18 developed chronic graft-versus-host disease (cumulative incidence 32% at 72 months, 95% CI 20–46). In multivariate analysis, a higher number of infused CD3 cells was associated with an increased risk of developing chronic graft-versus-host disease (P=0.017). With a median follow up of 73 months (range 8–233), the estimated 6-year overall survival was 87% (95% CI 78–97). At 72 months, the cumulative incidence of avascular necrosis was 21% and 12 patients presented with endocrine dysfunction (cumulative incidence of 19%). Only one patient developed a secondary malignancy (Hodgkin’s lymphoma) during follow up.Conclusions Cyclophosphamide and antithymocyte globulin is an effective conditioning regimen for patients with severe aplastic anemia and is associated with low treatment-related mortality. Long-term complications include avascular necrosis and endocrine dysfunction.

Incidence on relapse and nonrelapse mortality (NRM) of chronic graft-versus-host disease (GVHD), per National Institutes of Health (NIH) criteria, is not well defined after reduced-intensity conditioning (RIC) regimens. We analyzed the association of chronic GVHD with the risk of relapse and NRM using Cox models in 177 consecutive patients who underwent transplantation for hematological malignancies after RIC. The cumulative incidence of chronic GVHD at 36 months was 74% when using Seattle's criteria compared with 54% with NIH consensus. In Cox model, NRM was significantly higher in patients with late-onset, persistent and recurrent acute GVHD (hazard ratio (HR): 6, 25 and 11; P = 0.014, P0.0001, P0.0001, respectively). The cumulative incidence of relapse was significantly decreased in patients with chronic GVHD compared with no GVHD group using either Seattle's or NIH criteria (HR 0.43 and 0.38; P = 0.022 and 0.016, respectively), whereas the presence of late-onset, persistent and recurrent acute GVHD was not associated with a decreased rate of relapse (HR: not significant, 0.70 and 0.71; P = not significant, P = 0.73 and P = 0.54, respectively). Chronic GVHD per NIH consensus definition is associated with the graft-versus-tumor effect, whereas all forms associated with acute features beyond day 100 are associated with NRM.

Hyper acute and acute vascular rejections represent the major problem in organ xeno-transplantation. According to the humoral theory of transplantation, acute and chronic rejections of allografts a...

Summary. Background: Low-molecular-weight heparins (LMWHs), derived from unfractionated heparin (UFH) by different depolymerization procedures, vary in both their relative abilities to enhance the inhibition of FXa (anti-FXa) and thrombin (anti-FIIa), and in their physicochemical properties. Objective: We aimed to profile the inhibition of thrombin generation induced by bemiparin, enoxaparin, nadroparin, dalteparin and tinzaparin in platelet-rich plasma (PRP), and to compare them with UFH and fondaparinux (a synthetic pentasaccharide that specifically enhances FXa inhibition). Methods: Different LMWHs, UFH or fondaparinux were added to normal PRP. Thereafter, tissue factor-triggered thrombin generation was assessed using the Thrombogram–Thrombinoscope® assay. Results: At equivalent anti-FIIa activity concentrations, LMWHs and UFH exhibited similar inhibitory effects upon thrombin generation. However, when used at equivalent anti-FXa activity concentrations, tinzaparin was significantly more active than the other LMWHs at inhibiting thrombin generation, and had similar activity to that of UFH. Enoxaparin, nadroparin and dalteparin all showed similar inhibitory activities. In these experiments, bemiparin exhibited the lowest inhibitory effect on thrombin generation of all the LMWHs. At 0.1 μg mL–1 (0.093 anti-FXa IU mL–1), fondaparinux inhibited the rate of thrombin generation by 50%. A 7-fold higher concentration of fondaparinux was required to inhibit the endogenous thrombin potential by 50%. Conclusions: LMWHs have a variable inhibitory effect on thrombin generation in vitro when compared by anti-FXa activity, but are similar when compared by their anti-FIIa activities. The rate of thrombin generation during the propagation phase, rather than the endogenous thrombin potential, is more sensitive to the anticoagulant activity of fondaparinux and the polysaccharide chains of LMWHs possessing only anti-FXa activity.

Bladder cancer is the seventh most common cancer in men worldwide and accounts for approximately 5–10% of all malignancies among men in Europe. Superficial transitional cell carcinoma (TCC) represe...

BACKGROUND Preemptive rituximab (R) treatment decreases the incidence of Epstein-Barr virus (EBV) posttransplantation lymphoproliferative disease, but the extent of immune deficiency related to R in patients who received allogeneic hematopoietic stem-cell transplantation is unclear. The aim of our study was to evaluate the incidence of late infections and immune reconstitution after preemptive R treatment of EBV infection. METHODS Seventy-eight patients receiving preemptive R between January 2005 and January 2010 were studied. Fifty-two of them could be matched with controls (not receiving R) according to administration of antithymoglobulin, stem-cell source and donor type, age and grade of acute graft-versus-host disease. RESULTS Among the 78 patients with EBV reactivation treated with R, the 36-month cumulative incidence of bacterial, viral, and fungal infections was 64%, 59%, and 23%, respectively. When compared with controls, bacterial infection incidence was significantly higher in R patients (55% vs. 35%), and a slower reconstitution of B cells was observed. R patients had modest but not significantly higher nonrelapse mortality (35% vs. 15%) than controls. CONCLUSION R has dramatically decreased risks of posttransplantation lymphoproliferative disease but is followed by a prolonged and profound B-cell deficiency associated with an excess risk of bacterial infection and higher mortality. R should be given with caution, and immunoglobulin replacement should be provided to limit these excess risks.

Bone complications after hematopoietic stem-cell transplantation (HSCT) are relatively frequent. Evaluation of biomarkers of bone turnover and dual energy x-ray absorptiometry (DEXA) are not known in this context.We prospectively evaluated bone mineral density, biomarkers of bone turnover, and the cumulative incidence of bone complications after allogeneic HSCT. One hundred forty-six patients were included. Bone mineral density was measured by DEXA 2-month and 1-year post-HSCT. The markers of bone turnover were serum C-telopeptide (C-TP), 5 tartrate-resistant acid phosphatase (bone resorption), and osteocalcin (bone formation) determined pre-HSCT and 2 months and 1 year thereafter. Potential association between osteoporosis at 2 months, osteoporotic fracture or avascular necrosis and, individual patient's characteristics and biologic markers were tested.C-TP was high before and 2 months after transplant. At 2 months, DEXA detected osteoporosis in more than half the patients tested. Male sex, median age less than or equal to 15 years, and abnormal C-TP before HSCT were risk factors significantly associated with osteoporosis. Three-year cumulative incidences of fractures and avascular necrosis were 8% and 11%, respectively. Children were at higher risk of fracture, whereas corticosteroid treatment duration was a significant risk factor for developing a clinical bone complication post-HSCT. Bone complications and osteoporosis are frequent after HSCT. Bone biologic markers and DEXA showed that subclinical bone abnormalities appeared early post-HSCT.The risk factors, age, gender, and C-TP easily available at the time of transplantation were identified. Biphosphonates should probably be given to patients with those risk factors.

Major ABO incompatibility between donor and recipient is not considered a barrier to successful allogeneic hematopoietic stem cell transplantation (HSCT), even if it can be associated with several immunohematologic complications. Nevertheless, conflicting data still exist as to its influence on graft-versus-host disease (GVHD) incidence, relapse rate, and survival. To further investigate the relevance of ABO major mismatch on transplantation outcome, we retrospectively analyzed results from 414 patients with major or major/minor ABO-mismatched bone marrow (BM), peripheral blood (PB), and cord blood (CB) allogeneic HSCT. Transplantation outcome was assessed by comparison with results from a 395-patient ABO-compatible population with similar characteristics. Median time to red cell transfusion independence was significantly longer in ABO-incompatible BM recipients (median time, 63 days vs 41 days; P =.001), with faster disappearance of antidonor IgM hemagglutinins in unrelated recipients (median time, 36 days vs 44 days; P = .03) and in patients with gradeor =II acute GVHD (aGVHD) (median time, 35 days vs 59 days ; P = .001). In PB stem cell (PBSC) and CB transplantation, erythroid reconstitution was not significantly delayed, regardless of donor type or presence of aGVHD. A slight correlation between ABO incompatibility and GVHD incidence was found in PBSC recipients when considering gradeor =II aGVHD incidence (63% in ABO-matched HSCT vs 83% in ABO-mismatched HSCT; P = .055), but this was not confirmed in multivariate analysis. In patients with acute leukemia, multivariate analysis revealed an association between major ABO mismatch and decreased relapse rate with borderline statistical significance (hazard ratio, 0.65; P = .04). Major ABO incompatibility mainly, if not exclusively, affects red blood cell engraftment after BM transplantation. Somewhat surprisingly, the graft-versus-plasma cell effect seems to be confined to this stem cell source.

Background. Current grading systems in acute graft-versus-host disease (GVHD) are not able to identify patients with poor prognosis at time of onset, because they take into account maximal grade. The aim of this study was to evaluate potential predictors for overall survival at disease onset. Methods. The study sample was composed of 142 allogeneic hematopoietic stem-cell transplant recipients who developed acute GVHD after a myeloablative conditioning regimen. Factors associated with the risk of nonrelapse mortality (NRM) were analyzed with proportional hazard models. Results. At time of diagnosis, GVHD involved a single organ in the majority of the patients (78%). Initial grade was I in 24%, II or III in 56% of patients. Significant risk factors for NRM were non-HLA matched sibling donor (68% vs. 55%, P=0.05), absence of methotrexate in GVHD prophylaxis (75% vs. 56%, P=0.02), initial liver involvement (80% vs. 56%, P

Diabetes is associated with multiple disorders including metabolic, cellular and blood disturbances leading to vascular complications. Increased circulating levels of platelet-leukocyte aggregates (PLA) have been described in several thrombotic diseases. In this study, we have evaluated circulating PLA in diabetic patients and we have investigated whether they may be a marker of vascular complications. Using flow cytometry assay, we have quantified PLA percentages in 65 diabetics including 20 patients with type I and 45 with type II diabetes, and 25 healthy subjects. Specific labelling identified platelet-polymorphonuclear aggregates (PPA) and platelet-monocyte aggregates (PMA). We have observed a significant increase of PPA and PMA levels in diabetics (22+/-12% and 45+/-18%, respectively) compared to controls (7+/-4% and 19+/-10%, respectively) (p0.01). However, both PPA and PMA values were similar in the two diabetes types. Circulating PPA and PMA were significantly enhanced in diabetics with vascular lesions (PPA: 24+/-13%; PMA: 50+/-18%) than in diabetics without vascular lesions (PPA: 18+/-8%; PMA: 38+/-15%) (p0.05 and p0.01). Patients with PPA18% and/or PMA38% showed a more important vascular injury (OR: 6; 95% CI: 1.6-23). Increased PMA circulating rate is particularly correlated to retinopathic injury (OR: 19; 95% CI: 2.3-154). Our findings established a relationship between increased circulating PLA levels, particularly PMA, and the incidence of microvascular complications in diabetes. They reinforce the concept of pro-inflammatory cells involvement in diabetic retinopathy pathogenesis and their link with thrombotic process.

Abstract 2397 Introduction: Paroxysmal nocturnal hemoglobinuria (PNH), a very rare disease, is characterized by hemolytic anemia, bone marrow failure, and venous thromboembolism. The disease is caused by somatic mutation of the X-linked gene PIG-A encoding a key enzyme responsible for the biosynthesis of the GPI-anchored proteins (GPI-APs), affecting hematopoietic stem cells (HSC). Venous thromboembolisms occur in unusual sites and the mechanism remains to be elucidated. A previous study showed an increase of endothelial cell activation markers in PNH patients (Helley et al., Haematologica 2010;95:574-581), however endothelial cells remain poorly studied in this disease. In polycythemia vera, associating JAK-2 mutation in HSC and thrombosis occurrence, Sozer and coworkers showed that liver endothelial cells bore the same JAK-2 mutation than the HSC (Sozer et al, Blood Cells, Molecules and Diseases, 2009;43:304-312). This suggests the existence of a common precursor between endothelial progenitor cells and HSC, the hemangioblast. In PNH, PIG-A gene mutations might be present in endothelial cells. To test this hypothesis we have used endothelial progenitor cells circulating in blood, also called endothelial-colony forming cells (ECFC), as witnesses of endothelial cells status. Methods: Peripheral blood mononuclear cells (PBMC) and neutrophiles (PMNL), from patients with classical PNH, were fractionated. PMNL were used to isolate DNA, while PBMC were plated on gelatine-coated plates to be cultured until appearance of ECFC colonies (usually 15–20 days), and then cultured to perform: (i) expression of GPI-anchored protein by flow cytometry; (ii) sequencing of the exons and flanking regions of the PIG-A gene from PMNL and ECFC. Results: Twelve PNH patients were enrolled (8 women, 4 men). Among them, 3 have venous thrombotic events (2 Budd-Chiari syndromes and one mesenteric veins thrombosis). ECFC colonies were obtained from 6 out of the 12 patients, a normal output due to the small number of ECFC in whole blood. Mutations identified by sequencing the PIG-A gene of PMNLs from these 6 patients are shown in Table 1, as well as clinical characteristics. In 5 patients only (two with previous Budd-Chiari syndrome), we obtained sufficient amounts of ECFC to isolate DNA. None of the ECFC colonies bore the identified mutations (one exhibited a very faint peak of mutated nucleotide on electrophoregram but mRNA transcripts analysis from this ECFC colony revealed traces amounts of CD45 and CD11b mRNA, suggesting a contamination by leukocytes). Conclusion: This is the first study reporting the absence of PIG-A gene mutation in ECFC from PNH patients, indicating that the mature endothelial cells are not bearing the PIG-A mutation. These cells are thus involved in thrombosis probably by free hemoglobin and thrombin cell activation, rather than by direct damage caused by PIG-A gene mutation. These results also indicate that, in PNH, the PIG-A gene mutation occurs most probably after the common precursor between endothelial progenitor cells and HSC. Disclosures: Peffault de Latour: Alexion: Consultancy, Research Funding. Fischer:Alexion: Consultancy. Helley:Alexion: Consultancy.

Abstract 4090 Background: Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) from an HLA- identical sibling is the treatment of choice for young patients with acquired severe aplastic anemia (SAA). Due to increased rates of secondary solid cancer in patients with SAA who received an irradiation-based conditioning regimen, we decided 2 decades ago to use the association of Cyclophosphamide (CY) and Antithymocyte globulin (ATG). We report here the long-term follow-up of patients who underwent HSCT from an HLA-identical related donor after this conditioning regimen. Patients and Methods: 61 consecutive patients with SAA who received a first transplantation from June 1991 to February 2010 in our center were included. Patients with Fanconi anemia or other congenital bone marrow failure were excluded. The conditioning regimen consisted in CY (200mg/Kg) and ATG (2.5 mg/kg/day × 5 days). The donors were HLA-identical siblings in 60 cases and family HLA-matched in 1 case. Graft-versus -host disease (GvHD) prophylaxis associated cyclosporine and methotrexate (days 1, 3, 6 and 11). Long-term clinical outcome, immune recovery and quality of life were assessed. Results: The median age was 21 years [range: 4–43], 41 being adults. Median duration of the disease before HSCT was 93 days. Most of the patients had idiopathic aplastic anemia (n=49, 80%). Median time from diagnosis to HSCT was 3 months (range, 1 to 140). All but 2 patients received bone marrow as source of stem cells and all but 2 engrafted (primary graft failure= 3.4%) with a neutrophils count > 0.5 G/L and a platelets count >20 G/L after a median of 23 (range, 19 to 43) and 21 days (range, 10 to 177), respectively. In patients who had achieved neutrophil recovery, no secondary graft failure was observed. Cumulative incidence (CI) of acute grade II-IV GvHD was 23% (95%CI, 13 to 34) and 18 patients developed chronic GvHD (CI: 32%, 95% CI, 20 to 46). In multivariate analysis, a higher number of infused CD3 cells was associated with an increased risk of developing chronic GvHD (p=0.017). With a median follow-up of 73 months (8 to 233), the estimated 6-year overall survival was 87% (95%CI, 78 to 97). At 72 months, the CI of secondary malignancies was 9%, 10 patients developed avascular necrosis (21% CI), 12 patients were diagnosed with endocrine dysfunctions (19% CI) and 5 presented cardiovascular complications (CI of 10%). The CI of bacterial, fungal and viral infections were 25% (95% CI, 15 to 36), 8% (95% CI, 3 to 17) and 61% (95% CI, 46 to 73) at 72 months, respectively. At 2 years post HSCT, the immune reconstitution was normal for CD3, CD8 T-cells, B-cell and NK-cell but still incomplete for CD4 T-cells. A FACT-BMT questionnaire of quality of life (QOL) was sent to all survivors (n= 53) of who 26 accepted to respond to the questionnaire. There was no evidence for a change in QOL perception with time after transplantation. Our data were compared with those obtained from HSCT recipients from a Swiss transplant center (n=125 patients), mainly transplanted for hematological malignancies. The perception of QOL in patients who were transplanted for SAA was similar to the group of patients who were transplanted for other reason than SAA. Conclusions: Our results confirm that HSCT from HLA-identical sibling donors after CY-ATG conditioning regimen is a curative treatment for patients with SAA, with an excellent long-term outcome. We found an increased risk of chronic GvHD associated with the number of infused CD3 cells. Furthermore, we also found non negligible late complications as well as a similar quality of life with patients transplanted for hematological malignancies. Improving long-term health conditions must thus be a priority field for research, exploring the use of new conditioning regimen as well as new GvHD prophylaxis to improve the quality of life post HSCT of such patients. Disclosures: Peffault de Latour: Alexion: Consultancy, Research Funding.

Abstract 5134 Aim: PNH is a rare disease with prevalence between 7.3 and 15.9 cases/million. PNH is a life-threatening disease that affects mostly young adults with median age at diagnosis being 33 years. Diagnosis is difficult and often delayed because of the clinical polymorphism of the disease. ECLIPSE is a French study which aimed to evaluate the delay between the onset of PNH symptoms and the date of diagnosis, to identify the clinical signs leading to diagnosis and to determine which medical specialists are seen first by PNH patients (pts). Patients and Methods: 4920 physicians were asked to participate in the study: 992 haematologists, 1638 internists, 1155 gastroenterologists, 697 nephrologists, 438 neurovascular physicians. Physicians were divided into 3 groups: (a) physicians having diagnosed PNH at least once, (b) having suspected a PNH without having confirmed the diagnosis, (c) neither having suspected nor diagnosed a PNH. The response rate was analysed by medical speciality and was defined by the number of physicians responding divided by the number of physicians invited to participate. Results: 528 physicians accepted to participate in the study (overall response rate: 10.7%). In total, 507 answers were analysed. Among the 507 physicians, 108 (21 %) had diagnosed PNH at least once (group a), 213 (42 %) had suspected PNH without confirming the diagnosis (group b) and 186 (37 %, CI95% [32.49 % - 41.05 %]) neither had suspected nor diagnosed PNH (group c). In group (a), the clinical signs and symptoms which led to diagnosis were: pancytopenia 44%, anaemia 37%, haemolysis 23%, peripheral venous thrombosis 18%, hepatic vein thrombosis 14% and hemoglobinuria 14%. The physicians of group (a) were also asked to describe the clinical situations which raised the suspicion for PNH diagnosis: unexplained thrombosis (86%), hemoglobinuria (84%), aplastic anaemia (83%), Coombs negative anaemia (80%), cytopenias (71%) were the most frequent symptoms triggering the test for PNH. Physicians of group (a) were also asked to describe the circumstances of their latest PNH diagnosis. The patient was referred to the physician most frequently by the Emergencies (23%), a haematologist (22%) or by an internist (21%). The most frequent functional symptoms of their latest diagnosed pts were: fatigue (39%), anaemia (24%), abdominal pain (20%) and thrombosis (14%). Seven percent of PNH pts did not report any functional symptom before diagnosis. PNH diagnosis was confirmed in a mean time of 9.32±11.46 months after the onset of symptoms, and a maximum delay between first symptoms and diagnosis being 60 months. Biological signs which raised the suspicion for a PNH were: anaemia (80%), increase of LDH (60%), increase of biluribin (44%), thrombocytopenia (41%) and/or neutropenia (28%). Confirmation of PNH diagnosis was made by flow cytometry in 87% of the cases. Two hundred and thirteen physicians belong to group (b), having at least once suspected PNH without confirming diagnosis. Half of them (50%) had suspected at least 5 times a PNH diagnosis, without confirmation. Clinical and biological signs which prompted physicians of group (b) to suspect PNH were: Coombs negative anaemia (48%), pancytopenia (42%) and/or aplastic anaemia (38.5%), myelodysplastic syndrome (18%), hemoglobinuria (15.5%), increase level of LDH associated with venous or arterial thrombosis (15%), abdominal pain (14%), dark urine (12%) or jaundice (11%). Finally, 186 physicians declared having neither suspected nor diagnosed any PNH pts. Furthermore, 6.5% of physicians have never heard about PNH. Conclusions: PNH diagnosis is usually difficult and delayed as its signs and symptoms are diverse and non-specific. The ECLIPSE study aimed to better understand the diagnostic procedures for PNH, to evaluate the delay between onset of symptoms and diagnosis and to determine the medical specialists involved in the diagnosis and management of PNH pts. PNH is mainly diagnosed by hematologists. Frequent symptoms leading to diagnosis were unexplained thrombosis, hemoglobinuria, Coombs negative anaemia, but also aplastic anaemia, unexplained cytopenias and myelodysplastic syndrome. Flow cytometry, the gold standard for PNH testing, was only used in 87% of cases. Diagnosis was usually delayed with a maximum of 5 years between onset of PNH symptoms and diagnosis. Fatigue and abdominal pain were commonly reported symptoms and should therefore be more routinely assessed. Disclosures: No relevant conflicts of interest to declare.

Abstract 3372 Poster Board III-260 Background: Although Hodgkin lymphoma is usually characterized by a high chemosensitivity and a good prognosis, a minority of patients with primary refractory disease or early relapse after intensive chemotherapy presents poor outcome (Brice. British Journal of Haematology 2008 ; 141 : 3-13. Moskowitz. British Journal of Haematology 2009 ; 146 : 158-163). AlloSCT is one of the treatment-options that can be proposed for these progressive patients but the optimal management remains controversial because comparative studies are lacking. However, several groups report encouraging survival results with acceptable non relapse mortality after alloSCT with reduced intensity conditioning. Method: We retrospectively studied 31 patients who received an alloSCT in Saint-Louis Hospital, Paris. The aim of the study was to compare their outcome with those of patients with similar characteristics who did not receive alloSCT. In order to have an homogenous group of patients, we only compared patients who relapsed after an autologous SCT (autoSCT) and were alive at least 12 months after autoSCT. To correct the recruitment bias, the propensity score (PS) methodology was used to determine the probability of receiving alloSCT in the group of patients who relapsed after autoSCT, according to a set of baseline characteristics (Rosenbaum. Biometrika 1983;70:41-55). Parameters used for this PS score were age, primary chemorefractory disease and stage III-IV at first relapse. This PS was estimated using logistic regression and used to match 1:1 patients with similar propensity to receive alloSCT, based on the nearest neighbor matching using calipers of width 0.2. AlloSCT recipients characteristics: Thirty-one patients (19 males and 12 females) received an alloSCT between 2000 and 2007. Median age at alloSCT was 26 years. Median number of previous lines of chemotherapy was 3.6 (range from 2 to 7) and 28 (90%) patients were previously autografted. 13 (42%) were considered refractory to primary treatment and 11 (35%) to secondary treatment. Status at alloSCT were: 20 (65%) patients in CR, 9 (29%) in PR and 2 (6%) in progressive disease. Pre-transplant co-morbidity score was 0 in 26 (84%), 1 in 3 (10%) and 2 in 2 (6%) patients. 26 (84%) patients received a reduced intensity conditioning regimen and TBI was performed in 23 (74%) recipients. Most of the patients (n=24, 77%) received peripheral stem cells and donors were HLA-identical siblings in 15 (48%) cases. Median follow-up after autoSCT and alloSCT was 62 and 51 months respectively. All patients engrafted. Grade II-IV acute GVHD occurred in 10 (32%) patients. Chronic GVHD occurred in 13 (42%). Three months after alloSCT, 19 patients were in remission, 5 presented stable disease and 7 were progressive. Eleven patients relapsed despite alloSCT and 11 patients died. The estimated 3-year overall and event free survival were respectively 69% (95% CI [48;82]) and 41% (95% CI [23;58]) and 3-year non relapse mortality was 21% (95% CI [13;29]). Causes of death after alloSCT were: progression in 6 (55%) and toxicity in 5 (45%) patients. From the 31 alloSCT recipients, 15 matched pairs were successfully constituted according to the PS. As expected, the distribution of age (p=0.97), stage III or IV at relapse (p=0.67) and primary chemorefractory disease (p=1) were not different between patients treated by alloSCT and untreated subjects. Based on these matched pairs, the estimated hazard ratio of death was 0.24 (95% CI [0.07;0.80]) for allografted patients compared with non allografted. In conclusion, alloSCT in poor-prognosis Hodgkin lymphoma gives promising survival outcomes although performed in advanced disease and provides survival benefit with acceptable non relapse mortality in comparison with non allograft treatment as suggested by PS method. Disclosures: No relevant conflicts of interest to declare.

Osteoporotic fractures (OF) and avascular necrosis (AN), are major late complications after allogeneic hematopoietic stem cell transplantation (HSCT). We prospectively studied bone complications (BC) after HSCT. Patients and Methods: Pts having received allogeneic HSCT in hospital Saint-Louis from February 2004 to May 2006 were included. The study was approved by the Institutional Ethical Board and patients’ informed consent was obtained. 121 adult pts, 72 males and 49 females, mean aged 37.4 ± 12.3 years old (y.o.) at the time of the transplantation and 25 children (18 males and 7 females, mean aged 11.6 ± 3 y.o.) were included. Pts presented acute leukemia (48 myeloid and 30 lymphoblastic), myelodysplastic syndromes (11pts), lymphomas (8pts Hodgkin and 9pts non-Hodgkin), myeloproliferative syndromes (14pts), aplastic anemia (13pts), multiple myeloma (10pts) or chronic lymphocytic leukemia (3pts). 71pts received bone marrow, 59pts peripheral blood stem cells and 16pts cord blood cells. 99pts were transplanted following a myeloablative conditioning, while 47pts following reduced intensity regimen. 37pts received 12Gy of total body irradiation (TBI), 31pts 2Gy and 78pts did not receive TBI. 69pts presented acute GVHD and 54 chronic GVHD. All pts received calcineurin inhibitors for a median of 356 ± 339 days. 15pts did not receive corticoids (CD) posttransplant, 11pts did not receive CD treatment at all (pre- and post-transplant), while 75pts had received CD therapy pre-transplant. Cumulative dose of CD was 164.5 ± 136.2 mg/kg and median duration of CD treatment was 350 ± 363.5 days (27–1501 days). Of the 131pts having received CD post-transplant, 60pts (46%) received at least one biphosphonate (BS) perfusion. Bone turnover was assessed by c-telopeptide (C-TP) and TRAP 5 (serum band 5 Tartrate Resistant Acid Phosphatase) (markers of bone resorption) and osteocalcin (OC-marker of bone formation) measurements, pre-transplant and on day 60 (D60) and at 1 year post-transplant. Bone mineral density (BMD) was evaluated by dual energy X-ray absorptiometry (DEXA) on D60 and at 1 year post-transplant, on the femoral site (FS) and lumbar spine (LS). Results: 136pts (93%) and 104pts (71%) were still alive on D60 and 1 year respectively. C-TP was increased pre-transplant and on D60 (8068 ± 6848 pmol/l and 8293 ± 6388 pmol/l respectively, normal= 920–4200 pmol/l), while it significantly decreased and reached normal values at 1 year post-transplant (5807 ± 4742 pmol/l, p=0.01 compared to pre-transplant and to D60). TRAP5 did not significantly change throughout the study period and remained within normal limits, while OC significantly decreased on D60 compared to pre-transplant values (4.4 ± 5.6 ng/ml versus 10 ± 6.4 ng/ ml, p

Background: Allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative treatment in MDS, and also in many cases of AML, 2 diseases where median age is higher than 50 years. Aim: Analyse the outcome of patients older than 50 years who received an allogeneic hematopoietic stem cell transplantation (HSCT) for MDS or AML, in Saint-Louis Hospital from January 1997 to December 2007. Method: 48 patients (pts) aged from 50 to 68 years (median: 56) received an HSCT for MDS (N=28) or AML (N=20) during this period. AML pts had intermediate (n=15) or high risk cytogenetics (n=3). 8 pts had failure to initial induction chemotherapy. FAB classification for MDS was: RAEB (n=8), RAEBt (n=6), secondary AML (n=2) or refractory anemia (n=2). Maximal IPSS score was high, intermediate-2 and intermediate-1 in 11, 14 and 4 pts. Ten pts with MDS received an “AML-like” chemotherapy and 5 received demethylating agents before transplant. 18 of the AML pts were in complete remission (CR) at time of transplant (CR1, n=15; CR2, n=3) and 2 were in relapse at time of HSCT. 10, 9 and 9 patients with MDS had < 5%, 5–10% and > 10% blasts in bone marrow at time of HSCT. Results: 19 patients with AML (95%) and 16 patients with MDS (57%) received a HSCT from an HLA-identical sibling donor. Other pts received a matched unrelated donor. Thirty-two pts, including 9/20 and 23/28 had at least one co-morbidity according to Sorror score. The conditioning regimen was myeloablative (MAC) in 14 pts (29%). Reduced intensity (RIC) was fludarabine based in 34 pts, associated with either 2 Gy TBI (n=25) or chemotherapy (n=9). All pts engrafted. 35 pts had acute graft-versus-host disease (GVHD): grade I in 11, grade II in 18 and grade III in 6. GVHD incidence did not differ between pts who received a MAC or a RIC regimen. Two-year overall survival (OS) was 41% (95% CI: 26–57). OS and relapse-free-survival (RFS) were similar after MAC or RIC regimen (OS: 29% vs 35% and RFS: 29% vs 30%). Short-term non-relapse mortality (NRM) was lower in patients who received a RIC as compared to patients who received a MAC regimen but was similar at long-term (6-month NRM = 21% vs 9% and 12-month NRM = 21 vs 19 %). Patients with AML or MDS had similar OS and RFS (OS: 42 vs 41%; RFS: 37 vs 26% at 2 years for AML and MDS, respectively). NRM was not significantly higher in pts with MDS (26 vs 10% at one year) whereas relapse rate was not significantly higher in pts with AML (13 vs 6%). Conclusion: HSCT for AML or MDS after 50 years is a curative option for pts with related or unrelated HLA-identical donor regardless kind of conditioning regimen and co-morbidity at time of transplantation.

In organ transplant, alloreactivity is supposed to induce modifications on the vascular endothelium. Complications occurring in the course of allogeneic haematopoietic stem cell (HSC) transplantation, such as acute or chronic graft-versus-host disease (GVHD), have been related to endothelial cell alterations. Moreover, the incidence of GVHD is significantly different following allogeneic bone marrow, peripheral blood stem cell or umbilical cord blood transplantation (BMT, PBSCT and UCBT respectively). Considering these different incidences of acute and chronic GVHD and taking into account the organ transplantation-related vasculopathy, we aimed to further clarify the vascular impact of the three aforementioned sources of HSC. We studied 21 patients (pts), 13 male and 8 female. Mean age was 42 years old (21–64). The underlying diseases were: acute leukaemia (myeloblastic 15pts; lymphoblastic 3pts), multiple myeloma (1pt), aplastic anemia (1pt) and chronic idiopathic myelofibrosis (1pt). Seven pts underwent BMT after myeloablative conditioning regimen (cyclophosphamide and busulphan or 12Gy total body irradiation-TBI), 6 pts PBSCT following non-myeloablative preparative regimen (fludarabine and 2Gy TBI) and 8 pts UCBT after reduced intensity conditioning regimen (cyclophosphamide, fludarabine and 2Gy TBI). We assessed the kinetics of plasma levels of 4 endothelial markers: soluble thrombomodulin (sTM), vWF-Ag, FVIII and the vascular endothelial growth factor (VEGF). Measurements were performed before the conditioning regimen and on days 0, 15, 30, 60, 90. Student paired t-test was used for statistical comparison. Differences were significant if p0.05). Similarly, vWF-Ag was significantly increased on day 0 in BMT and PBSCT (217±66% and 214±93% respectively, p

Thalidomide has emerged as a promising treatment for multiple myeloma (MM). Thrombosis is the most serious complication of thalidomide therapy, essentially when it is combined with dexamethasone. The pathogenesis of thrombosis in MM patients (pts) treated with thalidomide is not clear and probably of multi factorial origin. We used the Thrombin Generation test (TGT) and measured the plasma levels of soluble thrombomodulin (sTM) to better clarify the MM-related and thalidomide-related thrombogenicity. TGT was performed in citrated frozen platelet poor plasma (PPP). Blood was obtained from 26 MM pts, Salmon and Durie stage II and III, 62.5 years old (42–77), 9 males and 17 females, 10 treated with thalidomide (100–200mg/d orally) and dexamethasone (40mg/d for 4 days) (TD group) and 16 receiving no treatment (MM group). 13 healthy volunteers formed the control group. Thrombin Generation (TG) was initiated by adding the PPP reagent (Thrombogram-Thrombinoscope®) and the triggering solution (CaCl2 and fluorogenic substrate). We analyzed the endogenous thrombin potential (ETP), the Cmax and the velocity index of TG. The plasma levels of sTM in PPP were measured by a specific ELISA (Diagnostica Stago, France). In the MM group we observed an increase of the ETP, though not significant compared to the controls. The Cmax was almost equal to the control group value, while the velocity index of TG was statistically lower in the MM group compared to controls. In the TD group, a statistically significant increase of ETP was observed as compared to the control group. The Cmax was higher, compared to controls, though not significantly, whereas the velocity index of TG was almost equal to the control group value. There was no significant difference in the TG parameters between MM and TD groups. sTM in the control group was 45±14ng/ml. Both groups of pts had significantly increased sTM plasma levels as compared to the control but the difference between the two groups did not reach significance. Results are shown in Table 1. In patients with MM coexists an increase of sTM, a marker of endothelial cell damage, together with an increased TG capacity. The addition of thalidomide treatment is associated with a slight but not significant increase of ETP and Cmax. The co-existence of endothelial cell damage with increased TG capacity could be associated to the increased thrombotic risk in MM patients treated with thalidomide. This hypothesis will be controlled in a prospective study. Table 1: Thrombogram parameters and sTM plasma levels of studied pts. Control MM group TD group * Results significantly different between the MM and TD groups and the control group (p

Introduction: Low molecular weight heparins (LMWH) are derived from unfractioned heparin (UFH) by depolymerization. Thus, they present biochemical and pharmacological differences and the ratio of anti-Xa/anti-IIa activities varies from one product to another. In this study, we compared in vitro the Thrombin Generation (TG) inhibition potency of various LMWHs and UFH using the Thrombogram-Thrombinoscope® assay. Materials and Methods: TG was assessed after Tissue Factor (TF) pathway activation in Platelet Rich Plasma (PRP) (1.5x105 platelets/μl) using diluted thromboplastin (Dade Innovin®, 1:1000 final dilution). We studied five different LMWHs (Enoxaparin, Dalteparin, Nadroparin, Tinzaparin and Bemiparin), as well as UFH at five different prophylactic and therapeutic anti-Xa final concentrations. These agents were added to control plasma from 14 healthy volunteers with equivalent anti-Xa concentrations. TG was initiated by adding the triggering solution containing CaCl2 and the fluorogenic substrate. The analyzed TG parameters are the lag time, the maximal concentration of thrombin (Cmax), the time to reach Cmax (Tmax), the TG velocity and the endogenous thrombin potential (ETP). Results: Bemiparin had almost no effect on TG, with concentrations below 0.60 IUanti-Xa/ml. Dalteparin, Nadroparin and Enoxaparin showed a similar potency in inhibiting TG at equal anti-Xa concentrations. Tinzaparin proved to be the most active LMWH in inhibiting TG and had a similar potency to UFH. Tinzaparin and UFH, with the lowest anti-Xa/anti-IIa ratio, exerted their inhibitory effect mostly by prolonging lag time and Tmax and by reducing TG velocity, especially at concentration below 0.40 IU anti-Xa/ml. Besides, UFH totally inhibited TG, as expressed by ETP, at a concentration over 0.40 IU anti-Xa/ml. For a given anti-Xa/anti-IIa ratio characterizing each LMWH the IC50 for each parameter was different. The IC50 for the reduction of TG velocity was lower in comparison to the IC50 for the other parameters (Table 1). Conclusion: Our study reinforces the concept of LMWH heterogeneity and the important effect exerted by the additional anti-IIa activity combined with anti-Xa activity. Thus, their characterization can be made through their ability to inhibit TG and not only their anti-Xa/anti-IIa ratio. Nevertheless, in vitro study ignores pharmacokinetic characteristics which are important in clinical practice. Thus, Enoxaparin, at validated prophylactic concentrations (0.20–0.40 IU anti-Xa/ml) had a weak effect on TG parameters, whereas, at peak therapeutic concentrations, it showed an important inhibitory activity similar to Tinzaparin. (Table 2).The use of TG test for the biological monitoring of LMWH requires further evaluation. | | Lag time | Tmax | ETP | Cmax | Velocity | |:----------:| -------- | ---- | ---- | ---- | -------- | | Bemiparin | >1 | >1 | 0.98 | 0.85 | 0.68 | | Enoxaparin | 0.62 | 0.58 | 0.55 | 0.45 | 0.38 | | Nadroparin | 0.80 | 0.75 | 0.55 | 0.45 | 0.38 | | Dalteparin | 0.65 | 0.65 | 0.50 | 0.42 | 0.38 | | Tinzaparin | 0.35 | 0.28 | 0.35 | 0.25 | 0.18 | | UFH | 0.05 | 0.10 | 0.30 | 0.25 | 0.18 | Table 1. The IC50 for each parameter of TG (anti-Xa IU/ml) | | Therapeutic Concentration Peak (anti-Xa) | Lag Time | Tmax | ETP | Cmax | Velocity | |:---------- | ---------------------------------------- | -------- | ----- | --- | ---- | -------- | | Enoxaparin | 1IU/ml | 91% | 94% | 72% | 82% | 91% | | Tinzaparin | 0.85 IU/ml | >100% | >100% | 82% | 90% | 97% | Table 2. Effect of Enoxaparin and Tinzaparin on TG at therapeutic concentration peak in vitro

There is no consensus on the optimal treatment of steroid-refractory acute graft-versus-host disease (SR-aGVHD) after allogeneic hematopoietic stem cell transplantation. In our center, the treatment policy has changed over time with mycophenolate mofetil (MMF) being used from 1999 to 2003, and etanercept or inolimomab after 2004. An observational study compared survival and infection rates in all consecutive patients receiving 1 of these 3 treatments. Ninety-three patients were included. The main end point was overall survival (OS). Median age was 37 years. Acute GVHD developed at a median of 15 days after transplantation. Second-line treatment was initiated a median of 12 days after aGVHD diagnosis. Therapies were MMF in 56%, inolimomab in 22%, and etanercept in 23% of the patients. Overall, second-line treatment response rate was 45% (complete response: 28%), MMF: 55%, inolimomab: 35%, and etanercept: 28%. With 74 months median follow-up, the 2-year survival was 30% (95% confidence interval: 22-41). Risk factors significantly associated with OS in multivariate analysis were disease status at transplantation; grade III-IV aGVHD at second-line treatment institution; and liver involvement. None of the second-line therapy influenced this poor outcome. Viral and fungal infections were not statistically different among the 3 treatment options; however, bacterial infections were more frequent in patients treated with anticytokines. Over an 11-year period, 3 treatment strategies, including 2 anticytokines, give similar results in patients with SR-aGVHD.

Abstract 1244 Background. Voriconazole (V) treatment has been shown to improve the 12 week (W) survival rate of hematological patients (pts) with invasive aspergillosis (IA), including recipients of allogeneic hematopoietic stem cell transplants (HSCT). We investigated whether this early survival advantage could translate into a significant increase in overall survival. Methods. We retrospectively reviewed all consecutive pts who received a transplant between Sept. 1997 and Dec. 2008 at Saint-Louis Hospital and were diagnosed as having IA. The temporal origin of the study was the date of IA diagnosis for each patient. Factors associated with survival were analyzed using Cox proportional hazard models. Separate models were estimated for survival up to 12 W and for survival between 12 W and 24 months (M) in pts surviving longer than 12 W. The deaths of pts with and without IA were analyzed with a competing risk framework. Cumulative incidence curves were compared using Gray's tests. Results. Our study examined 89 IA pts. The median follow-up was 70 M (range, 11–130 M). Two pts did not receive any antifungal treatment and were excluded from subsequent analyses. Of the 87 pts, 42 received first-line V and 45 primarily received a lipid formulation of amphotericin B (n=25), amphotericin B deoxycholate (n=10), caspofungin (n=8) or itraconazole (n=2). The primary characteristics of pts with IA and their causes of death, separated by V as first-line treatment, are shown in the table below. The median survival was 2.6 M, and the overall survival at 24 M was 19% (95% CI 12–30 M) (see figure). Overall, the survival rates of the two groups were significantly different (P= 0.010). However, the differences in survival were quite dramatic prior to 10 M, whereas both survival curves became very close after one year. At 18 M, the numbers of surviving pts were almost identical in the two groups [19% (95% CI: 11–34%) in pts who did not receive V as first-line treatment vs. 21% (95% CI 11–38%) in pts who did]. Pts who did not receive V as a first-line treatment displayed a higher probability of dying from IA than those who did (P=0.004), whereas opposite results were found for mortality in pts without IA (P=0.006). The 24-M cumulative incidence of death from IA was 47% (95% CI 31–61%) in the no V group and 19% (95% CI 9–33%) in the group treated with V. The 24-M cumulative incidence of death in pts without IA was 4% (95% CI 7–14%) in the no V group and 27% (95% CI 14–42%) in pts treated with V. The probability of death from another cause, with IA, was similar in both groups (29% vs. 36% at 24 M; P=0.46). After adjusting for donor type, conditioning regimen, progressive GVHD at diagnosis of IA and cumulated steroid dose (mg/kg) in the W preceding IA diagnosis, administration of V as first-line treatment was found to decrease the risk of death during the first 12 W by approximately 70% [HR=0.31 (95% CI 0.16–0.60); P=0.0005]. Conversely, analysis of mortality between 12 W and 24 M failed to identify any significant predictor of risk of death; however, only 24 pts died during this period. Conclusions. The finding that first-line treatment with V, which is associated with a tremendous improvement in IA outcome, does not translate into an increase in overall survival (even in the context of early diagnosis) is striking. Diagnosis of IA following HSCT, whatever the outcome, appears to be a strong marker for poor long-term prognosis. Disclosures: Bergeron: Pfizer: Speakers Bureau, none; Merck: Speakers Bureau, none; Schering: Speakers Bureau, none. Sulahian:Pfizer: Research Funding, non; Merck: Research Funding, none. Ribaud:Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau, none; Schering: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau, none; Gilead: Speakers Bureau, none.