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Restriction endonuclease (RE) in situ digestion (REISD) of human metaphase chromosomes and interphase nuclei may uncover cryptic polymorphisms. This technique can be applied to identify the individual origin of cells and thus analyze the hemopoietic chimerism that eventually results in leukemic patients after allogeneic bone marrow transplantation (BMT). In the current study, results of REISD with different REs are shown. In particular, the use of Sau 3A reveals a polymorphism for constitutive heterochromatin of chromosome 3 and may differentiate BMT donor (D) and recipient (R) cells. Once pre-BMT characterization shows a different Sau 3A digestion pattern of D and R cells, it is possible to monitor the development of hematopoietic cell populations in the R bone marrow after BMT. A panel of 24 patients who underwent BMT and their Ds were analyzed. The method presented here allowed cells from D and R to be distinguished, and therefore to quantify the post-BMT hemopoletic chimerism, in 6 (25%) of the cases. This quantitative and sex-independent genetic approach to the study of hemopoietic chimerism has already shown itself to be useful in patients with leukemia who require a BMT, but could also be extended to other transplant situations.

The efficiency of restriction endonuclease in situ digestion (REISD) with Sau3A to analyze chimerism and residual disease (RD) has been tested before and after an allogenic bone marrow transplant (BMT) in an acute lymphoblastic leukemia (ALL) patient. The combined results obtained with REISD and FISH using the appropriate probes for detecting chromosome rearrangements have proven to be useful for the identification and quantification of both the hemopoietic chimerism achieved after BMT and the RD persistent in the patient. The sensitivity of REISD has been determined to be around 95%, i.e., similar to that obtained by FISH. REISD with Sau3A was particularly useful in the analysis of chimerism since this enzyme revealed the polymorphic status of constitutive heterochromatin in human chromosome 3 and thus allowed discrimination of cells derived from donor and recipient. The method itself seems promising since neither a donor/recipient sex mismatch nor a cytogenetic disease marker are needed for its application.

Abstract 2510 Relapse remains the main cause of treatment failure in patients with acute myeloid leukemia (AML) in first remission (CR1) after allogeneic hematopoietic stem cell transplantation (SCT). The detection of minimal residual disease (MRD) in AML has improved in the past years with multiparametric flow cytometry (MFC) and molecular analysis (RT-PCR). However the prognostic impact of pre-transplantation MRD and the outcome after SCT has not been well studied. The aim of this study was to evaluate pre-transplantation MRD in patients in first remission undergoing myeloablative allogeneic SCT. We retrospectively studied 35 consecutive patients receiving myeloablative SCT for AML in first cytologic remission after intensive chemotherapy with available MRD determination before transplant. MRD was studied by 4-color MFC on bone marrow aspirates, and quantitative RT-PCR (NPM1, WT1, MLL) on bone marrow and/or peripheral blood samples obtained within thirty days before transplant. Thirty-five patients consecutively transplanted in our institution between 1999 and 2012, and for which pre-transplant MRD data was available were analyzed (Table 1). Eighteen showed negative MRD pre-transplant whereas 17 showed positive values. Characteristics of patients were homogeneous between both groups, including number of chemotherapy cycles received before transplantation (Table 1). Within the MRD-negative group, 17 patients showed negative MRD by MFC (12 of them showed negative values also by PCR) and 1 patient showed negative MRD by PCR (MFC not available). Within the MRD-positive group, 9/17 (52%) patients showed MRD-positive values by MFC: in 5 cases MRD was also detected by PCR, only 1 showed negative PCR and in the remaining 3 cases, PCR was not available. On the other hand, in 8/17 (47%) patients MRD was not detected by MFC, however, PCR detected MRD in all of the cases in bone marrow (2), peripheral blood (4) or both samples (2). With a median follow-up of the whole series of 23 months, 2-years estimates of overall survival were 82% (95% CI, 97–55) and 30% (95% CI, 3–71) for MRD-negative and MRD-positive patients (p=0.045), respectively. Cumulative incidence of relapse were 21% (95% CI, 5–48) and 56% (95% CI 10–73) for MRD-negative and MRD-positive patients (p=0.11). In the MRD-negative group, cause of death was toxicity in 11% of the cases and relapse in 11%, while in the MRD-positive group, 17% of patients died due to toxicity and 23% due to relapse. Conclusions: Our data shows that the presence of MRD before allogeneic SCT in patients with AML in CR1 is associated with a significant worse OS rate compared to patients with negative MRD, as well as a tendency towards a higher risk of relapse. The detection of MRD by MFC correlates with the detection by PCR in most of the cases. However, in a significant group of patients, MRD was detected only by PCR. This could be related to differences in sensitivity between both methods. Further studies including larger series are needed to confirm these observations. Table 1 Patient and Disease Characteristics by MRD status Pre-transplant MRD MRD-negative MRD-positive N=35 18 17 Age at transplant (median, R) 36 (19-62) 42 (19-68) Gender (male/female) 8/10 12/5 Cytogenetics and molecular markers Normal/Intermediate risk 77% 83% FLT3+ 28% 27% NPM1+/FLT3- 6% 7% FLT3-/NPM1- 28% 40% High-risk 22% 17% Secondary AML 16% 6% Cycles pre-transplantation (median, R) 3 (2-5) 3 (2-5) MRD detection MFC only 27% 18% RT-PCR only 5% 0% Both 68% 82% Donor* HLA-identical sibling 50% 35% HLA-matched unrelated 22% 29% SCU-dual 22% 24% HLA-haploidentical related 6% 12% acute GVHD II-IV (n°/patients at risk) 44% (8/18) 24% (4/17) chronic GVHD lim/ext (n°/patients at risk) 53% (8/15) 57% (8/14) MRD: minimal residual disease, MFC: multiparametric flow cytometry, RT-PCR: real time PCR, GVHD: graft vs host disease, SCU-dual: single cord blood with co-infusion of selected CD34+ cells from a third party HLA-mismatched donor. *The MRD negative group includes 2 MAC SCU-dual cases with primary graft failure rescued immediatly by a second graft (1 Dual and 1 Haplo) using a RIC regimen. The MRD positive group includes 1 MAC SCU-dual case rescued immediatly by a second graft (1 Haplo). Disclosures: No relevant conflicts of interest to declare.

Abstract 4545 Introduction: Allogeneic transplantation is the only curative option in the treatment of multiple high risk hematologic neoplasms. Only 25–30% of patients have an HLA identical sibling donor and searching for a compatible unrelated donor or cord blood renders satisfactory results in around 60–70%. Haploidentical transplantation (HAPLO) offers a therapeutic alternative to more than 95% of such patients with the advantages of quick availability, easy programming and a committed donor. Patients and Methods: We evaluate the results of HAPLO with a reduced intensity conditioning regimen (Fludarabine 30 mg/m2 ×5 days (-6 to -2), Cyclophosphamide 14,5 mg/kg ×2 days (-6 and -5), IV Busulfan 3,2 mg/kg × 1–3 days (BUX, days -4 to -2) employing high doses of Cyclophosphamide post graft infussion (50 mg/kg days +3 and +4) as GVHD prophylaxis together with standard doses of cyclosporine and mycophenolate from day +5. Results: From Dec-2007, we have done 26 HAPLO in 4 spanish centers. Median age was 38 years (16–57), 20 were male and all were in advanced phases of their diseases (12 Hodgkin′s, 6 AML, 3 ALL, 2 MM, 1 MDS, 1 MF y 1 NHL). Previous autologous HSCT has been employed in 13 and allogeneic HSCT in 6 (2 MURD and 4 UCB). Disease status at HAPLO was CR in 8, PR in 14 and refractory in 4. Bone marrow was used in 16 and unmodified peripheral blood in 10. The haploidentical donor was patient′s mother (8), father (3), siblings (11) or other relatives (4). BUX was used in 1 dose (15), 2 doses (8) or 3 doses (2) and TBI 200 cGy in 1 case. Mean neutrophils engraftment was achieved on day +18 (13–26) and platelets >50K on day +27 (17–150) in all but 2 cases of graft failure (7.7%) due to progression (MF) or relapse (M7-AML). Main toxicities were grade 1–2 mucositis in 50%, febrile neutropenia in 75% and CMV reactivations in 58% with a 100 days NRM of 3.8% (1/26, VOD and MOF) and 10% NRM at 6 months (2/20). Grade II-IV acute GVHD appeared in 10/23 patients at risk (43%) and grade III-IV in 4/23 (17%). Chronic GVHD affected to 4/15 (27%), being extensive in 1/15 (6.7%). With a median follow-up of 9 months (1–38), 13/22 (59%) are alive in CR, progression or relapse has ocurred in 6/24 (25%). Immune reconstitution seems fast and complete in those evaluated. Conclusions: HAPLO with high-dose cyclophosphamide as GVHD prophylaxis is a useful alternative in the treatment of high risk hematologic tumours, with low toxicity, acceptable GVHD incidence and severity, long lasting remissions, and fast immunological reconstitution. Disclosures: No relevant conflicts of interest to declare.

Abstract 4146 Introduction The success of allogeneic stem cell transplantation (allo-SCT) relies on the result of the immune reactions between donor and recipient lymphohemopoietic systems. Chimerism quantification and monitoring post allo-SCT allows to estimate such immune reactions. In recent years, there has been a growing interest in the analysis of circulating cell-free nucleic acids in plasma. Although the ultimate origin of such nucleic acids is unknown, the most plausible hypothesis proposes cell disruption as the cause for the presence of nucleic acids in plasma. Analysis of chimerism in cell-free plasma DNA would complement the results of cell chimerism for a better prediction of different complications (engraftment failure, rejection, graft versus host disease…etc.). Objective Our goal was to quantitatively monitor chimerism in cell-free plasma DNA and to evaluate its usefulness for the prediction/early diagnosis of complications after allo-SCT that would favour early therapeutic intervention. Patients and Methods The study comprised 270 plasma samples from 29 allo-SCT patients. With a median follow up of 45,8 months (14,6– 74,7). Whole blood (WB) (10 mL) was collected in EDTA containing tubes and was processed immediately after collection. Plasma was separated from the blood cells by centrifugation at 1600g for 10 min and cell-free plasma DNA was extracted according to the blood and body fluid protocol of the QIAamp DNA Blood Mini Kit (Qiagen). Quantitative chimerism analysis was performed by microsatellite PCR (STR; AmpFlSTR SGM plus, Applied Biosystems) in whole blood (WB), T-cells (CD3+) purified using immunomagnetic means (Miltenyi Biotec) and cell-free plasma DNA. Chimerism was monitored at regular intervals post-transplant (+30,+60, +90, +180, +365). Sensitivity of the technique is 1% in WB and cell-free plasma DNA, and 5% in T-cells (derived from a 95% purity of enriched samples). Results During the first year of allo-SCT, the number of patients with mixed chimerism in cell-free plasma DNA was higher than in WB and T-cells (Table 1). Cell-free plasma DNA mixed chimerism was observed for a longer timer after transplant than “cellular” mixed chimerism, with a median of 105 vs. 40 and 27 days for cell-free plasma DNA, T-cells and WB, respectively. Chimerism analysis revealed two groups of patients, one with mixed chimerism in both cell-free plasma DNA and “cellular DNA”, in which the percentage of recipient plasma DNA ranged between 10–30%, probably mostly derived from hematopoietic cells. The other group presented mixed chimerism in cell-free plasma DNA but complete “cellular” chimerism (100% donor) in which cells the percentage of recipient plasma DNA was lower than in the former group (1–10%). A greater number of patients with graft versus host disease (GVHD),mainly acute but also chronic, presented mixed chimerism in cell-free plasma DNA and complete chimerism in WB and T-cells (Table 2). The presence of recipient cell-free plasma DNA could be derived by cell disruption in GVHD target organs. Discussion Quantitative follow-up of chimerism in cell-free plasma DNA, does not reproduce the results of “cellular” chimerism, as reported previously, and therefore, could not replace it for post-SCT follow-up. On the other hand, chimerism analysis in cell-free plasma DNA could be combined with the conventional follow-up of “cellular” chimerism for an improved diagnosis of life-threatening complications such as acute and chronic GVHD. Disclosures: No relevant conflicts of interest to declare.

Abstract 474 BACKGROUND: Autologous stem cell transplantation (autoSCT) is considered as standard treatment for non-frail patients with mantle cell lymphoma (MCL). However, little is known about outcome of MCL recurrence after autoSCT. We therefore conducted a retrospective analysis of patients with MCL who failed autoSCT using the EBMT database. PRIMARY OBJECTIVE was to analyse outcome and prognostic factors after relapse following autoSCT for MCL in the rituximab era. PRIMARY ENDPOINT was overall survival (OS) from relapse. ELIGIBLE were patients aged 18 years or more who relapsed following an autoSCT for MCL performed between 2000 and 2010 and who were registered with the EBMT. Centres were contacted to provide additional information on relapse treatment. STATISTICAL ANALYSIS was based on log-rank comparisons and multivariable testing using Cox regression models. RESULTS: 1054 patients meeting the eligibility criteria could be identified in the EBMT registry. Of these, a full data set could be retrieved for 382 patients. Sixteen patients had to be excluded due to loss of follow up (n=7), wrong diagnosis (n=6), or falsely reported relapse (n=3). Median age at autoSCT of 366 evaluable patients was 59 years (range: 37 to 76), 290 patients (79%) were men. 64% had undergone autoSCT as part of 1st-line therapy; 68% and 49% had documented exposure to rituximab (RTX) and high-dose ara-C (HA) before autoSCT; and 12% had had refractory disease at autoSCT. Median time from autoSCT to relapse was 20 months (range: 0.4 to 117). 21 relapses (6%) occurred beyond 5 years after autoSCT. With a median observation time of 37 months (95% CI 32–43), median OS after relapse of the whole study group was 20 months. By univariate analysis, a long (>12mo) interval between autoSCT and relapse (p Salvage chemotherapy after relapse resulted in only 31% complete responses and 29% partial responses, whereas 40% of patients have been refractory to first salvage chemotherapy. 83 patients (23%) received an allogeneic SCT (alloSCT), whereas only 7 patients (2%) received a second autoSCT after relapse. Median time after relapse to second SCT was 7 months (range: 1 to 40). Survival after relapse for patients who received a second autoSCT was poor with no long-term survivor. AlloSCT performed for late relapse (>12mo) after autoSCT was associated with superior OS compared to patients who received an allograft upon a shorter remission duration after autoSCT (5-year OS from alloSCT 50% vs 0%; p=0.001; Figure 1B). Achievement of CR before alloSCT (p=0.05 HR=0.5), but not donor source, T-cell depletion or conditioning intensity affected OS after alloSCT. CONCLUSIONS: Patients with MCL who relapse within one year after autoSCT have an extremely dismal outcome even with alloSCT. In contrast, about half of the patients who have MCL recurrence beyond one year after autoSCT and can undergo salvage alloSCT enjoy long-term survival. It remains to be shown if a similarly good outcome can be achieved without alloSCT in this favourable selection of patients. A 2nd autoSCT does not appear to be a promising option in patients with MCL failing a 1st autoSCT. Disclosures: Walewski: Mundipharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Roche: Honoraria, Speakers Bureau; GSK: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees; Cephalon: Research Funding.

Abstract 4665 Introduction: Little is known about immune reconstitution (IR) of HIV+ adult patients treated with HAART and high-dose chemotherapy followed by autologous stem cell transplantation (ASCT). Aims: To compare the IR in HIV+ and HIV- patients treated with ASCT, and to correlate it with clinical outcome. Material and Methods: From January 2007 to January 2011 all HIV+ patients with lymphoma or multiple myeloma treated with ASCT where prospectively included. HIV- patients with lymphoma or multiple myeloma treated with ASCT were included as control. IR lymphocytes (Ly) subsets in peripheral blood were quantified by flow cytometry (FACScan, Becton-Dickinson Immunocytometry Systems, San Jose, CA). Naïve ly, memory ly, memory-activated ly, effector ly, NK cytotoxic ly, B naïve ly and B memory ly were quantified at the time of transplantation as well as at 3, 6, 12 and 18 months after ASCT. Additionally, immunoglobulin value, neutrophil and platelet engraftment (>0.5×109/L and >20×109/L respectively) and infectious complications were recorded in both groups. The study was approved by the local Ethics Committee. Results: We included 14 patients, 6 HIV+ and 8 HIV-. All this patients were in complete remission at the time of the ASCT. All patients received BEAM conditioning regimen, except for Melphalan 200 in the case of MM. The median infused CD34 was 4,08×10e6 in HIV+ patients and 5.2 x10e6 in control patients. All HIV+ patients had undetectable viral load at the time of the ASCT. Values of ly populations are shown in Table 1. Median neutrophils days of engraftment: 11.5 days (11–13.75) in HIV- and 14 in HIV+ (12.75–17), platelets: 15 (13–28) and 30 (17–78) (p:≤0.05) respectively. Both groups reached IR at third month, while HIV+ group showed lower values especially for the T CD4 ly subgroup (not statistically significant). HIV+ patients had statistically significant lower values of CD4 memory ly and activated CD4 ly at 6th month, and lower values of NK ly at 3th month. On the other hand, we found lower values of B naïve Ly in HIV- patients at 6th month. There was no difference in the median value of immunoglobulin at 3, 6 and 12 months between both groups. Infections were more frequents in HIV+ patients: positive Aspergillus antigenemia (3/6 vs. 1/8), positive CMV antigenemia (3/6 vs. 1/6), fungal infection (2/6 vs. 0/8) and bacterial infections (5/ 6 vs. 2/8), nevertheless severe episodes didn't shown differences, as well as disease free survival and mortality. Conclusions: In this series, IR was reached at the third month in both HIV + and HIV- patients, with non-significant lower values for HIV+ patients. Despite infections were more frequent in HIV+ patients, this was not associated with a higher mortality. Median values of lymphocytes population studied. Yellow box remarked corresponds To the statistically significant results (p:≤0,05) Disclosures: No relevant conflicts of interest to declare.

Abstract 4676 INTRODUCTION: Bernard Soulier Syndrome (BSS) is a rare qualitative and quantitative autosomal recessive platelet disorder molecularly characterized by defective GPIb-IX-V membrane glycoprotein that is related to platelet adhesion and interaction with both Von Willebrand factor and endothelium. It is usually expressed by an enlarged bleeding time and decreased yet giant sized platelets, and can cause severe hemorrhagic complications during pregnancy. CASE REPORT: We present two successful pregnancies and labour management on a 33-year-old woman diagnosed in her childhood with BSS. Ristocetin induced platelet agglutination test and membrane glycoprotein study were performed to get the diagnosis. Furthermore, in the last year we have completed the diagnosis with platelet receptor expression flow citometry analysis, finding out depleted antiGPIbα and antiGPIX antibodies. Changes which prevent GPIbα for correct expression and processing were observed. Also integrin αIIbβ3 levels were risen due to the big sized platelets. A PCR product was obtained from genomic DNA using the GPIbα oligonucleotide pair; sense and antisense, and direct DNA sequencing of the amplification was performed in a model ABIprism 377 DNA sequencer (Perkin-Elmer Cetus). The direct sequencing of the sense strand of the 5x fragment shows in the patient the simultaneous presence of G and C nucleotids in 688 position, and the T and A nucleotids in 715 position. These punctual mutations give Ala200Pro substitutions and Cys209Ser in heterozygosis in mature peptide, respectively, Figure 1. The Ala200Pro mutation has not been reported in others cases of Bernard-Soullier, previously. The Cys209Ser has been described in other two Spanish patients: one in homozygosis and the other in heterozygosis associated to the mutation. Our patient presented hemorrhagic symptoms from 18 month old with an average of 40×103/μ l platelet count and increased platelet volume who required multiple platelet transfusions during her childhood. In the menarche main hemorrhagic manifestations consisted on methrorragia and epistaxis which were controlled with tranexamic acid. At the age of 26 a spontaneous ovarian cyst rupture required urgent hospital admission and two platelet pool transfusion. On December 2007 she consulted for epistaxis, and on September 2008 cesarean delivery was planned at 38th week of pregnancy; platelet count was 40×103/μ l, antiplatelet antibodies were negative for membrane glycoproteins and antiHLA-1 was positive against 98% donors tested. As a result, an HLA-compatible donor was searched, and two platelet apheresis were transfused one hour before surgery and every 12 hours after the procedure. The next 4 days one apheresis every 48 hours were transfused with no hemorrhagic symptoms. On January 2010, she presented with her second pregnancy; the patient had severe anemia, with high transfusion requirements; neither hemorrhage or hemolysis was detected, and even the possibility of a Munchausen syndrome was questioned. Unplanned caesarean was performed at 32th week because of the clinical instability of the patient, and this time we could not find on time an HLA-compatible donor, so two random platelet pools were transfused one hour before surgery and one platelet apheresis every day during 4 days after caesarean deliver, without any hemorrhagic complication. The newborn was asymptomatic with normal platelet count. The patientxs haemoglobin level returned to normal after five days of delivery. CONCLUSIONS: We present the successful labour management of two consecutive pregnancies on a young woman diagnosed with BSS. The low prevalence of this syndrome (less than one case per million population) explains the absence of well-established protocols for the management of pregnancy in this disease. Up to date there are only twelve reported cases of pregnancy and delivery in BSS patients, most of them presenting with severe hemorrhagic complications. Correct platelet transfusion is the mainstay of treatment, and screening for anti-platelet antibodies study is mandatory due to its high prevalence in these patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 3358 INTRODUCTION: Non-neoplastic chronic portal vein thrombosis (PVT) is a frecuent diagnosis in the course of liver cirrhosis, with reported prevalences of 0.6% to 15,8%. PVT can motivate life-threatening complications due to worsening portal hypertension, so anticoagulation therapy is challenging in these patients. OBJECTIVE: To analyze the response to antithrombotic therapy and changes in liver function tests in 28 patients with chronic PVT associated with cirrhosis. PATIENTS AND METHODS: 28 consecutive patients with liver cirrhosis and chronic PVT were treated with antithrombotic therapy from 2004 to 2009. Hepatocellular carcinoma and known thrombophilic risks were ruled out. Therapy consisted in 15 days of therapeutic doses of low molecular weight heparin (LMWH) (enoxaparin) adjusted according to baseline coagulability (Table 1), followed by either prophylactic doses (40mg/day) of LMWH or acenocoumarol (target INR 2–3), during 6 months. Response was evaluated after 6 months. If recanalization was complete, therapy was suspended. If recanalization was partial or no recanalization was observed, therapy was continued until response. RESULTS: From the 28 patients studied, 19 (68%) were males with a median age of 53 years (range 35–77). Cirrhosis was due to alcoholism (25%), virus (54%), mixed in 1 patient and other causes in 3 patients. PVT involved the portal trunk and/or branches in 19/28 (68%) patients, mesenteric vein in 2 patients and portal trunk and/or branches, mesenteric and/or splenic vein thrombosis coexisted in 7 patients. 19/28 (68%) of the patients had moderate or moderate-severe hypocoagulability range. Complete and partial thrombosis was seen in 18 and 10 patients at diagnosis, respectively. From the 28 patients, 18 (64%) responded to antithrombotic therapy after 6 months, with a complete recanalization in 13 patients 13/18 (72%) and partial in 5/18 patients (28%). None of the 28 patients presented hemorrhagic complications and none showed platelets counts below baseline values. 17 from the 18 patients who responded, showed altered liver function tests before therapy. After 6 months, 8/17 (47%) improved liver function (only one patient had received antiviral therapy). After a median follow up of 42 months (range 7–67), 15/18 (83%) patients continued showing complete or partial response while 3 patients progressed. Of note, 3 patients of this group could proceed to further liver transplantation. CONCLUSIONS: Antithrombotic therapy in chronic PVT in cirrhotic patients resulted in a high response rate (64%) in our study, with a complete recanalization in 72% of the cases. Adjusted dose scheme according to level of hypocoagulability seems to be effective and safe, since 63% of the subgroups of moderate and moderate-severe hypocoagulability responded with no haemorrhagic complications. Disclosures: No relevant conflicts of interest to declare.

Abstract 3051 INTRODUCTION The FOXP3 gene encodes for a protein (Foxp3) involved in the development and functional activity of regulatory T cells (CD4+/CD25+/Foxp3+, Tregs) which exert regulatory and suppressive roles over the immune system. After allogeneic stem cell transplantation (allo-SCT), Tregs are known to mitigate graft versus host disease (GVHD) while maintaining a graft versus leukemia effect (GVL). Allele (GT)15 for the functional (GT)n polymorphism in the promoter/enhancer of the FOXP3 gene is associated with a higher expression of FOXP3 and production of a greater amount of Tregs. However, its impact in the allo-SCT setting has not been analyzed. OBJECTIVE To analyze the impact of the (GT)n polymorphism in the promoter/enhancer of the FOXP3 gene on the development of complications and ultimately on the success of conventional HLA-identical allo-SCT. MATERIALS AND METHODS The study includes 33 patients with hematological malignancies, treated with myeloablative HLA-identical peripheral blood allo-SCT (Table 1). Diagnosis, classification and grading of GVHD were made by clinical criteria and confirmed when necessary by pathological examination of histological samples from gut, skin, liver or lung, according to international consensus criteria. Donor and recipient genomic DNA was purified from EDTA anticoagulated peripheral blood before allo-SCT and using QIAamp Blood DNA extraction kit (Qiagen). Genotyping of the (GT)n microsatellite polymorphism in the FOXP3 gene was performed by a fluorescence-based short tandem repeat-polymerase chain reaction (STR-PCR) method (GeneAmp 7900; Applied Biosystems) and sized by capillary electrophoresis (POP7 - ABI PRISM 3130 xL Genetic Analyzer; Applied Biosystems) followed by fragment analysis (GeneMapper 4.0 Software; Applied Biosystems) as previously described [Bassuny WM, et al. Immunogenetics. 2003;55 :149–56]. RESULTS The median follow-up time for the cohort was 34 months (range 9.5–110). Allelic frequencies observed were similar to those previously reported (50.5% (GT)15, 41% (GT)16 and 7% (GT)17; no (GT)14 or (GT)18 alleles were found). Patients transplanted from donors harboring allele (GT)15 showed a lower incidence of grades II-IV acute GVHD (29% vs 67%; p =0.049). These patients also showed a trend to a lower incidence of severe (grades III-IV) GVHD (12% vs 33%; p =0.167) as well as chronic GVHD (75% vs 100%; p =0.143; Table 1, Figure 1). No statistically differences were found between patients transplanted from (GT)15 and non-(GT)15 donors in terms of relapse rate (38% vs 33%; p =0.825; Table 1) or cumulative incidence of relapse (CIR at 2 years 35.3% vs 37.5%, Figure 2). Finally, survival analysis did not show statistically significant differences between the two groups of patients in terms of median event (relapse) free survival (EFS, 15.6 months vs 4.5 months, p =0.686) or overall survival (OS, 29 months vs not reached, p =0.610). CONCLUSIONS Tregs are known to modulate the allotolerance-alloreactivity balance between donor and recipient in the allo-SCT setting, mitigating GVHD while preserving the anti-tumor effect (GVL) of the donor graft. In the present study, the presence of allele (GT)15 in the donor, which promotes a higher expression of FOXP3 and greater amount of Tregs, affected allo-SCT outcome by decreasing grades II-IV acute GVHD and chronic GVHD, without affecting GVL (no differences in CIR and OS). Analysis of this polymorphism can help in appropriate donor selection and, more importantly, drive a tailored management of patients submitted to allo-SCT. Disclosures: No relevant conflicts of interest to declare.

Abstract 4557 BACKGROUND: Natural killer (NK) cells are innate immune effectors that directly lyse virally infected or malignant cells. There are 2 different subsets of NK cells with distinct phenotypic and functional characteristics: the CD56dim subset, which composes 90% of peripheral blood NK cells and has a cytotoxic function, and the CD56bright subset, which cooperates with dendritic cells and T cells in lymph nodes to secrete interferon and promote adaptive immune responses. NK cells are the first donor-derived lymphocyte subset to reconstitute after hematopoietic stem cell transplantation, reaching normal levels after 1 month. Nearly all phenotyping studies of NK subsets after haploidentical hematopoietic stem cell transplantation (HHSCT) reveal a rapid reconstitution of NK cells towards the CD56bright subset. In addition, Y.-J. Chang et al found the highest 2-year survival in patients with a high number of CD56bright NK cells after unmanipulated HHSCT. We analyzed reconstitution of the NK compartment between days 90 and 180 after unmanipulated bone marrow HHSCT with reduced intensity conditioning (RIC). METHODS: Six adults received unmanipulated bone marrow HHSCT after RIC (fludarabine 30 mg/m2 [day –6 to –2], cyclophosphamide 14.5 mg/kg [day –6 and –5], and busulfan i.v. 3.2mg/kg [day –3]) at our institution between July 2007 and July 2010. Prophylaxis for acute graft-versus-host disease (GvHD) consisted of cyclophosphamide 50mg/kg (days +3 and +4) and cyclosporine A and mycophenolate mofetil from day +5 onwards. We monitored the reconstitution kinetics of circulating NK cells (CD56+, CD3–), and the CD56bright and CD56dim subsets by multiparametric flow cytometry (FC 500 Beckman® Coulter) at day +90 and day +180 after transplantation. Patient characteristics and clinical outcomes are shown in Table 1. 6 patients who underwent allogeneic HLA-identical sibling HSCT with RIC during the same period were used as controls. RESULTS: After HHSCT, NK cells reached normal levels in all patients but one at day +90, with a median number of NK cells of 111/mm3 (range, 25–195/mm3). At day +180 the median number of NK cells was 92/mm3 (range, 4–272/mm3). When we analyzed the absolute number of CD56bright and CD56dim subsets at day +90, we observed 2 patterns: Two patients showed skewed NK cell reconstitution towards CD56bright (Patient no. 3: 54 CD56bright/mm3; 11 CD56dim/mm3. Patient no. 4: 70 CD56bright/mm3; 17 CD56dim/mm3). Three patients reconstituted with a CD56dim/CD56bright ratio towards the CD56dim cell subset, similar to that of healthy adults (Patient no. 1: 17 CD56bright/mm3; 178 CD56dim/mm3. Patient no. 5: 9 CD56brigh/mm3; 135 CD56dim/mm3. Patient no. 6: 20 CD56bright/mm3; 116 CD56dim/mm3). One patient did not achieve adequate NK cell reconstitution (Patient no. 2: 15 CD56bright/mm3; 10 CD56dim/mm3). In contrast, in the control group, an increase in the CD56bright NK cell subset was not observed in any of the patients at any point. It is worth noting that 2 of the 3 patients with better clinical outcome (no GvHD, no relapse), namely patients no. 3 and no. 4 were the ones with skewed NK cell reconstitution towards the CD56bright NK cell subset. The other patient with a better clinical outcome (patient no. 6) had a normal CD56dim/CD56bright ratio at day +90. However, he showed an early CD56bright reconstitution (363 CD56bright/mm3; 34 CD56dim/mm3) in an additional determination on day +30. NK cell subsets reconstitution kinetics is shown in Figure 1. CONCLUSIONS: In our experience, NK cell reconstitution is adequate after RIC unmanipulated bone marrow HHSCT. Some patients recovered with a high proportion of CD56bright NK cells, as previously reported in other studies on HHSCT. Although limited by the sample size, our results are consistent with the previously observed survival advantage of patients with high early levels of CD56bright NK cells after unmanipulated haploidentical transplantation. Disclosures: No relevant conflicts of interest to declare.

Abstract 4082 Intoduction. Cytokine gene polymorphisms are well know to be associated with functional differences in cytokine regulation and altered clinical performance in a variety of diseases. They seem to play an important role in allogeneic stem cell transplantation (allo-SCT), mostly in the incidence and severity of graft-versus -host disease (GVHD) which is one of the most common serious complications of allo-SCT. Interleukin-6 (IL-6) is a proinflamatory cytokine known to be implicated in the pathogenesis of aGVHD with increasing serum levels during the development. The IL6−174 (G/C) single nucleotide polymorphism (SNP), localized in the promoter region (7p21) has been linked to in vitro and in vivo productions, associating the presence of allele G with significantly higher levels of IL-6 and a trend to have higher grades of aGVHD. Objetive. To analyze the influence of Donor (D) and Recipient (R) genotype for the polymorphism IL6−174 (G/C) on the outcome of HLA-identical related stem cell transplantation. Patients and Methods. The study comprised 171 allo-SCT (342 D/R samples) included in the Spanish Group for Haematopoietic Stem cell Transplantation (GETH) DNA bank. Genomic DNA was purified from peripheral blood samples obtained pre-SCT from patients and donors, after written informed consent. The IL6−174 (G/C) SNP genotype was determined by allele-specific PCR (Cavet et al, Blood 98, 2001). Results were analyzed using the Pearson's Chi-square Test and survival estimation by Kaplan-Meier curves. Results. Genotypes for D and R as well as D/R combinations (table 1), were in accordance with previous reports. Homozygous recipients for IL6−174GG polymorphism showed a trend to higher incidence of grade III-IV aGVHD than those with other genotypes (19.9% vs 9.1%) p=0.14. No significant differences were found in terms of cGHVD, relapse or mortality rates. On the other hand, patients transplanted from homozygous GG Donors showed a higher incidence of extense cGVHD (20/52 (38%) vs 21/87 (24%) p=0.07), lower relapse rates (10/59 (23.8%) vs 32/105 (76.2%) p=0.041) and better global mortality rates (23/62 (36.9%) vs 51/106 (69%) p=0.11). Survival analysis with Kaplan-Meier curves in this group of patients (IL6−174GG homozygotes in the Donor) revealed a lower cumulative incidence of relapse (CIR) (NR vs 567 days p=0.028) a better event free survival (EFS) (732 days vs 380 days p=0.02) and showed a trend to a better overall survival (OS) (1235 days vs 836 days p=0.157); (Figure1). Conclusions. IL6 cytokine is known to be implicated in the pathogenesis of aGHVD with increasing serum levels during its development. The presence of allele G for the polymorphism of IL6−174(G/C) which is associated with higher levels of IL6, has shown to influence the outcome of our cohort of HLA-identical related stem cell transplantation. Since acute GHVD is mainly influenced by R genotype and chronic GHVD by D genotype, the present study revealed that homocigous presence in the R of allele G was associated with higher incidence of aGHVD. Moreover when this allele is homozigously present in the D, patients show greater extense cGHVD and better EFS and OS. Genotyping for this polymorphism could aid in D selection or even more interestingly drive a risk-adapted management of transplanted patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 4859 BACKGROUND: Most patients with classical Hodgkin's Lymphoma (CHL) are cured with primary treatment. However, a proportion of them fail to first line treatment needing to be rescued with subsequent lines of chemotherapy and/or autologous or allogeneic stem cell transplantation (auto-SCT and allo-SCT, respectively). The identification of clinical and biological characteristics of these patients at diagnosis is still a challenge and most prognostic systems fail to identify a proportion of patients with worse prognosis. In this context, different groups are currently analyzing several biological markers as determinants of clinical outcome. It has been reported that Bcl-2 immunohistochemical expression in Hodgkinxs Reed Sternberg cells (HRSC) might confer a worse prognosis. OBJETIVE: To analyze clinical outcomes following 1st line chemotherapy according to Bcl-2 expression at diagnosis of CHL. PATIENTS AND METHODS: CHL patients, older than 16 years old, receiving at least 1 line of treatment, were retrospectively studied for Bcl-2 expression in diagnostic samples. For this purpose, tissue sections were immunostained and semiquantitatively assessed for this marker. Cumulative incidence (CI) of treatment failure, treatment failure free survival (TfFS) and overall survival (OS) were defined as primary outcomes. Treatment failure was considered when a different treatment regimen was set up due to relapse after CR or failing to achieve CR following 1st line. RESULTS: 103 patients (55 Bcl-2 positive patients and 48 Bcl-2 negative patients) were analyzed. Main patient and clinical features are shown in Table 1. Both cohorts were well balanced for the main prognostic factors. At a median follow up of 36m (2-221), 34m (2-140) for Bcl-2 negative patients and 38m (4.5-221) for Bcl-2 positive patients, CI of 3 years treatment failure was 19% and 50% for the negative and positive cohorts, respectively (p=0.02). 3 years TfFS after diagnosis was 75% for Bcl-2 negative patients vs 47% for Bcl-2 positive patients (p=0.1). Within the cohort of Bcl-2 negative patients, 9/48 (19%) underwent auto-SCT as part of rescue treatment while 18/55 (33%) of Bcl-2 positive patients received an SCT (13 auto-SCT and 5 allo-SCT) and 3 of them, a second SCT (allo) for the treatment of post-auto-SCT relapse. 3 years OS was 84.5% for negative and 86% for positive patients (p=NS). CONCLUSIONS: According to these preliminary results, Bcl-2 expression in HRSC at diagnosis may constitute an independent biological prognostic marker in CHL patients, seemingly associated with worse outcome and need of second line chemotherapy. More studies and a longer follow up is needed in order to confirm that aggressive treatment strategies such as SCT may overcome the negative impact in survival of Bcl-2 expression in this population. Disclosures: No relevant conflicts of interest to declare.

Abstract 5218 INTRODUCTION: Diffuse large B-cell lymphoma (DLBCL) represents more than 80% of high grade lymphomas and is considered a heterogeneous entity regarding clinical presentation, morphology, molecular and cytogenetic features. The Revised International Prognostic Index (R-IPI) is a clinically useful prognostic index that may help guide treatment. In this context, BCL2 and MYC gene rearrangements, that have been associated to specific variants of lymphoma, seem to have a prognostic implication. Consideration of these genetic markers together with classical parameters could contribute to a better stratification of patients. OBJETIVE: To analyze the incidence and possible clinical relevance of BCL2 and MYC gene alterations in the outcome of patients with DLBCL. PATIENTS/METHODS: This retrospective analysis included 20 patients with DLBCL (January 2008-April 2011), 74% (20/27) males with a median age of 70 years (range 17–82). Median follow-up was 300 days (range 17–1134). Fluorescence in situ hybridization (FISH) was performed on paraffin-embedded (n=12) or fresh (“touch-down” preparation) samples (n=8) from different tissues (7 lymphadenopathies, 3 central nervous system, 3 biologic fluids and 7 others). Each sample was hybridized with break-apart probes for BCL2 (18q21) and MYC (8q24) genes (Vysis Inc). 200 nuclei were scored per slide to reach 7% sensitivity. RESULTS: Seventy percent (14/20) of patients analyzed showed BCL2 and/or MYC alterations at diagnosis (rearrangement and/or extra signals). BCL2 was present as unique alteration in 30% (6/20) of the samples, MYC was present as unique alteration in 30% (6/20) and 10% (2/20) showed both BCL2 and MYC rearrangement (Table 1). Two out of six patients with BCL2 as unique alteration showed rearragement, were treated with standard chemotherapy (R-CHOP or high dose of Citarabine/Methotrexate in central nervous system) and both patients relapsed. The other 4 patients presented extra signals as BCL2 alteration and also received standard chemotherapy. Fifty percent of these patients relapsed (Table 1). On the other hand, 3 out of 6 patients with MYC as unique alteration presented gene rearrangement, they were treated with standard chemotherapy, and all 3 relapsed. The other 3 patients with MYC alterations presented extra signals, received standard chemotherapy and 33.3% (1/3) relapsed. Finally, the 2 patients with simultaneous BCL2 and MYC rearrangement were treated with intensive chemotherapy and they reached and maintained complete remission. CONCLUSIONS: According to the results obtained from this study, FISH analysis on paraffin-embedded or fresh samples from lymphoma tissues is a feasible and useful technique at diagnosis in DLBCL patients. Although genetic markers are not included in the R-IPI score, the identification of BCL2 and/or MYC alterations at diagnosis, associated to other biological markers, could help to identify a subgroup of high risk patients who could benefit from more aggressive therapies. Further studies of larger series and genes are needed to confirm this observation. Disclosures: No relevant conflicts of interest to declare.

Abstract 4825 Introduction: The majority of patients with classical Hodgkinxs Lymphoma (cHL) are cured with primary treatment. However, a significant proportion of patients will not achieve a complete response (CR) or relapse after completion of initial therapy and need to be rescued with high-dose chemotherapy and stem cell transplantation (SCT): autologous (aSCT) and/or alogeneic (Alo-SCT). The identification of clinical and biological characteristics of these patients at diagnosis is still a challenge and the most prognostic systems used to date fail to identify a proportion of patients with worse prognosis. The best understanding of the biology of cHL could help to identify these patients and different groups works in the use of biological marker as determinants of clinical outcome. Bcl2 over-expression has been described in cHL and seems to be an independent marker associated to bad prognosis in probably relation with alterations in apoptosis regulatory molecules. Objetive: To retrospectively analyze the frequency of Bcl2 protein over-expression in tissue biopsies of patients diagnosed with cHL, which required intensive treatment in our centre. Patients and Methods: We revised clinical data and samples at diagnosis of patients with cHL who received SCT (aSCT or/and Allo-SCT) due to partial remission, relapse or progression and we determined Bcl2 expression protein by immunohistochemistry. All patients received at least 2 lines of treatment previous to intensive treatment with SCT. We analyzed the expression of Bcl2 according to each patient histology: Nodular Sclerosis (NE), Mixed cellularity (MC), Lymphocyte-rich (LR), and Lymphocyte-depleted (LD). Results: Between September 1997 and May 2010, 39 patients with cHL required intensive treatment in our center: 32 aSCT and 7 Allo-SCT due to partial remission, refractory, relapse or progression after first line of treatment. Average age was 32 years (range: 18–64), males: 25/females 14. The characteristics of patients and remission status at transplant are described in table 1. We had available samples at diagnosis from 31 out of 39 patients (80%): 20 NE, 5 MC, 6 LR and we found over-expression of Bcl2 in 17/20 (85%) NE; 5/5(100%) MC and 2/6 LR (33%). Conclusions: Our results suggest that over-expression of Bcl2 is frequent in patients with cHL which needed intensive treatment after failure of the first line of therapy (particularly with NE and MC). Further studies are needed to confirm the worse prognosis of Bcl2 expression in cHL and if may be useful at diagnosis in association with other clinical parameters to identify patients with poor prognosis. Table 1 . CLASSICAL HODGKIN LYMPHOMA (c HL) NODULAR SCLEROSIS cHL MIXED CELLULARITY cHL LYMPHOCYTE-RICH cHL LYMPHOCYTE-DEPLETED cHL N (39) 23 8 7 1 Sex (F/M) 11/12 8/0 5/2 1/0 Age, Years: Md(R) 30(18-64) 37(31-62) 36(19-64) 54 Stage: I, II/III, IV 11/12 3/5 5/2 0/1 Bulky disease 14YES/9NO 1YES/7NO 2YES/5NO 1NO B Symptoms: (N) YES/NO (13) YES/(10) NO (3) YES/(5) NO (6) YES/(1) NO (1) YES Intensive treatment 5Alo-SCT/18aSCT 2AloSCT/6aSCT 7aSCT 1aSCT N:type of trasplant (status at transplant) 18: aSCT(11CR/5PR/2RF); 5:AloSCT(2PR with previous aSCT/1PR/1CR/1RF) 6: aSCT (4CR/2PR) 7:aSCT (6CR/1PR) 1:aSCT (in CR) 2: Alo-SCT(1CR/1PR with previus aSCT) Sample revised: N 20 (23 total NE) 5 (8 total MC) 6 (7 total LR) 0/1 Bcl2 expression: P/Nv (%) 17P/3Nv (85%) 5P/0Nv (100%) 2P/4Nv (33%) Not aplicable Alo-SCT: Alogeneic stem cell transplant; aSCT: autologous stem cell transplant; CR: Complete Remission; F: female; LD: Lymphocyte-deplete; LR: Lymphocyte rich; M: male; MC: mixed cellularity Md: median; n: number; NE: nodular sclerosis; Nv: negative; P: positive; PR: Parcial remision R: range; RF: refractory Disclosures: No relevant conflicts of interest to declare.

Abstract 4397 OBJECTIVES Thrombotic events have been reported as a major cause of morbidity after the Fontan procedure. There is no consensus about the type and duration of postoperative anticoagulation prophylaxis, due to the high risk of bleeding complications, the difficulties in monitoring and the questionable therapeutic compliance in children. In spite of the lack of long term prospective studies in this situation, the ACCP has recommended in their guidelines OAT following Fontan or Glenn operation for at least 6 months. This has also been our practice in our institution during the period of study. AIM OF THE STUDY To analyze the efficacy and complications of OAT in our pediatric patients after undergoing the Fontan operation. METHODS Retrospective chart review of all the children treated with OAT in our institution between 1995 and 2009. All patients were treated initially with acenocumarol 0,2 mg/kg, except the Fontan patients, who received 0,1 mg/kg. Target INR was 2–3 for all patients. The Mann-Whitney test was used to compare the rate of complications, and the percentage of visits out of target INR between the Fontan patients and the rest of the cohort. RESULTS There were 61 children (26 female/35 males) aged between 1 month and 17 years, who received OAT with a range of follow up between 4 months and 14 years: 27 after Fontan operation (Group A), an 34 for other reasons (Group B: n=13 non prothetic valve cardiopathy, n=21 treatment of thromboembolic disease). The average follow-up was similar in both groups (median of 6.5 months in group A vs.7.5 months in group B). There were few complications: 1 mild epistaxis and 1 thrombotic event in group B, and none in group A. There were no differences in the proportion of controls in normal range between both groups; there was a moderate proportion of controls outside the target range of INR, with higher distribution below the range than above the range. The median dose used to achieve the target INR was 0.3 mg/kg/d in Group A and 0.4 mg/kg/d in Group B. CONCLUSIONS Oral Anticoagulant therapy is safe and effective in pediatric patients,with very low rates of thrombotic or hemorragic complications including those undergoing the Fontan surgery. There were not any differences between both groups in any of the analizyed parameters. Disclosures: No relevant conflicts of interest to declare.

Abstract 4973 Background: Comorbidity in older patients with AML or high risk MDS is frequent, and usually affects the therapeutic decision making. The hematopoietic cell transplantation specific comorbidity index (HCTCI) developed by Sorror et al, is widely used in predicting post-transplantation outcome in patients with AML and MDS. Recently, the HCTCI showed prognostic impact in a cohort of patients with high risk MDS treated only with supportive care. AZA have shown clinical efficacy in elderly patients with high risk MDS. However, it is not clear if AZA is safe and effective in patients with comorbidities. Purpose: To study the impact of comorbidity, determined by the HCTCI, in a retrospective cohort of patients with MDS or AML treated with AZA Patients: Between October 2007 and July 2010, 30 patients diagnosed with MDS or AML received AZA 75 mg/m2 /d × 7d/4 weeks, or less intensive schedules (7d/4w n=2, 5d/4 weeks n= 12, 5-2-2 n= 16). Median age was 71 years (range 44–88), 16M/14F. Regarding HCTCI, 17 patients were classified in low-intermediate risk, score 0–2, and 13 patients in high risk (score ≥ 3). Patients with HCTCI of 0–2 were classified as: 8 patients with MDS (High/ Int2 MDS n= 4, Intermediate-1 MDS n= 4), 8 patients with AML in first relapse after intensive chemotherapy (IC) or stem cell transplantation (SCT) and Response to treatment was estimated according to the International Working Group. Results: The median number of cycles of AZA was 7 (1-26); 5 cycles in the HCTCI of score 0–2 and 7 cycles in the HCTCI>= 3. There were three patients who received only 1 cycle due to progression (n=1) and early death, caused by infection (n=2). Both patients showed neutropenia before starting AZA, one showed HCTCI of low risk. Responses were evaluated when 3 or more cycles were administered. Overall response rate (ORR) was 48% (14/27), with 13% complete response (CR). In the group of patients with HCTCI of 0–2, ORR was 40% (6/15) including 2 CR, 1 marrow CR, 2 partial response (PR), and 1 patient with haematological improvement (HI). Median overall survival (OS) for responders with low risk by HCTCI was 18 months (4-28). Transformation to AML was seen in two responding patients, after 16 and 18 months. Allo SCT was performed in one patient. Regarding patients with HCTCI ≥3, ORR was 66% (8/12), including 2 CR, 2 PR, 4 HI, 3 of them with transfusion independence. Median OS for responders was 11 months (range 5–33 months). Transformation to AML was seen in a patient after 23 months of treatment, and relapse with 10% of blasts was observed in a patient with CR after six months of AZA. Grade 3–4 toxicity according to WHO was observed in 5 patients, 3 of them with HCTCI low (1 bleeding event, 4 respiratory tract infections). One patient with HCTCI ≥3 died after 8 months of HI, because no treatment-related event. One patient with HI and HCTCI≥3 did not continued AZA after 5 cycles because frequent respiratory tract infections. Conclusions In this preliminary cohort, AZA was well tolerated and active in patients with HCTCI≥3. The response rate was in accordance with results observed with a similar cohort of patients without significant comorbidities. These results might be confirmed with larger number of patients in further studies. Disclosures: Font: Celgene: Membership on an entity's Board of Directors or advisory committees.

Abstract 3532 In the absence of an HLA matched sibling donor, an unrelated adult donor matched (MUD) at 8 of 8 alleles for HLA A, B, C, DRB1 is considered as first alternative. However, only 50% of patients in Europe have an available suitable donor in a median time of 4 months. For those patients without a suitable MUD, we have adopted CB (cord blood) stem cell transplantation with the co-infusion of CD34+ cells from a third party non HLA-identical donor (Dual transplantation). The objective of this study was to analyze toxicity and overall survival (OS) rates of patients who underwent this procedure and compare them with a cohort of patients who received a myeloablative MUD transplantation in a single centre and during a similar period of time. Patients: Between 2005 and 2010, 16 consecutive patients with high risk disease underwent a total of 18 dual transplants and 17 patients, with similar pre-SCT characteristics recieved a myeloablative MUD transplantation with 12/12 HLA identity in a single centre (Table 1). Transplants performed before 2005, those with manipulated grafts and with HLA mismatches were excluded. Results: There were no significant differences in age, gender, pre-SCT disease status and previous therapy lines between both groups. Three cases among the dual group showed primary graft failure (2 of them showing third party donor cells engraftment) who were rescued by a second CB transplant in two cases and a MUD in one case (succesfull in 2). There were no graft failures in the MUD group. GVHD rates are shown in table 1. OS was 50% in the dual group and 53% in the MUD group with a median follow-up of 33 (1-67) y 13 months (1,1-48,5) respectively (Figure 1). The cumulative relapse incidence at 2 years was 54% in the dual group and 53% in the MUD group. There were no relpses after 2 years in both groups. All the transplant related deaths (TRM) took place within the first year after transplantation with no significant differences in cumulative incidence between both groups (p=0,69). Conclusions: In our experience, CB transplantion together with the co-infusion of CD34+ cells from a third party HLA-mismatched donor offers OS and DFS rates comparable to those from myeloablative HLA 12/12 MUD trasnplantation with an acceptable TRM. Therefore, dual transplantion is still our first choice for patients without an available MUD or for whom trasplant is urgent. Disclosures: No relevant conflicts of interest to declare.

Abstract 1328 Introduction: CD4+CD25+ regulatory T-cells (Treg) play an important role in inducing and maintaining allogeneic tolerance and can inhibit graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (SCT). However, the dynamics of donor and recipient Treg cell populations after Allo-SCT has not been studied yet. Objective: To analyze the dynamics of chimerism in Tregs and compare it to that of T lymphocytes (TL; CD3+) and whole blood leukocytes (WBL). Material and Methods: The study includes 53 patients subjected to Allo-SCT (myeloablative and non-myeloablative). PB samples were obtained weekly during the first month and every 14 days after day +30 until complete chimerism (CC) was achieved, and at fixed time-points (+30, +60, +90, +180, +365) thereafter. TL and Tregs were purified from PB until CC was achieved using inmunomagnetic technology (Miltenyi Biotec; CD3+ Microbeads and CD4+/CD25+ Treg Isolation Kit, respectively). Chimerism analysis was performed by microsatellite PCR (STR-PCR; AmpFlSTR SGM Plus; Applied Biosystems) on genomic DNA obtained from WBL as well as on cell lysates obtained from purified TL and Tregs. Complete chimerism was considered in samples with percentage of recipient cells Results: Median follow up for the whole cohort of patients was 338 days. CC was spontaneously achieved in WBL, TL and Tregs in 45/53 patients (85%) in a median time of 35.41, 38.8 and 42.5 days respectively. 5/3 patients (9%) suffered from graft failure/rejection showing mixed chimerism (MC) with increasing percentages of recipient cells both in WBL and leukocyte lineages. 3/53 patients (6%) maintained mixed chimerism (MC) in WBL and leukocyte lineages at day +180, with no signs of graft rejection or disease relapse. Analysis of the chimerism dynamics of those patients who spontaneously achieve CC revealed two different groups: Group 1 included 25/45 patients who achieved CC at the same time in WBL, TL and Tregs. Group 2 included 20/45 patients who achieved CC in WBL while maintaining MC in TL and Tregs. Interestingly enough, 9/20 patients from Group 2 maintained MC in Tregs 7–75 days after achieving CC in both WBL and TL (Figure 1). In a preliminary analysis, the small sample size precluded from obtaining statistically significant associations between the dynamics of chimerism in Tregs and the development of complications such as relapse or GVHD after SCT. Conclusions: To our knowledge, this is the first study dealing with Treg chimerism after SCT. We have shown it is feasible and can be performed on a routine basis together with standard lineage specific chimerism follow up. Although there is an association between chimerism dynamics in Tregs and TL, this is not absolute and a percentage of patients maintain residual Tregs of recipient origin after WTL and TL have become of complete donor origin. In this small cohort, Treg chimerism did not influence the development of post-SCT complications. Analysis of a larger and more homogeneous cohort would allow establishing the usefulness of Treg chimerism testing for the management of transplanted patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 2250 Background: Autologous stem cell (SC) transplantation (ASCT) is a potentially curative treatment for several hematologic malignancies and has been demostrated feasible and effective in HIV-related lymphoma (ARL). Peripheral blood SC collection could represent a major issue in the use of ASCT in HIV infected patients (pts). Aim: To evaluate the feasibility and efficacy of SC mobilization in HIV positive (pos) pts with lymphoma and identify factors influencing harvest results. Potential “ongoing” predictors of collection were also assessed. Patients and Methods: We retrospectively analysed 98 consecutive pts with ARL, candidates to ASCT, who underwent SC mobilization at 3 Italian and 2 Spanish centers from 2000 to 2010. A collection less than 2×106 CD34+ cells/kg was defined as “mobilization failure”, between 2–5 as “suboptimal collection” and more than 5 as “good collection”. Several parameters were evaluated for correlation with outcome: age, sex, lymphoma histopathology, disease status, WBC and Plt count at start of mobilization, type of mobilizing therapy, marrow disease, previous mobilization failure, n° of previous chemotherapy (CT) lines, months from first detection of HIV positivity, CD4 count and HIV-viremia. Moreover, circulating CD34+ and WBC count on the first day of CD34+ monitoring and their ratio (SC ratio = CD34/WBC) were assessed as “ongoing” outcome predictors. Results: A total of 127 attempts of SC harvest in 98 pts were analysed. Median age was 41.5 ys (28-65). Lymphoma diagnosis was DLBCL in 42% of cases, Burkitt 10%, plasmablastic 10%, HL 31%, anaplastic 5%, follicular lymphoma 1% and PEL 1%. Disease status was complete remission in 36%, chemosensitive disease in 53% and refractory disease in 10% of cases. In 3 cases bone marrow was involved and mobilizations failed. In 18% of cases pts received mobilizing therapy after 1 previous CT line, in 67% after 2 and in 16% after 3 or more. All pts but 2 were on antiretroviral therapy. Median CD4 count was 231/mcl (50-1146) and HIV-viremia was detectable in 22%. Median time from first HIV detection was 79.5 ms (3-295). In 24% of cases G-CSF alone (10-20 mcg/Kg) was used as mobilizing treatment, while CT + G-CSF (5-10 mcg/Kg) in 76%, including single-agent Cyclophosphamide (CTX) 1.5 gr/ms (13%), CTX >3 gr/ms (27%), platinum containing regimens (20%), ifosfamide containing regimens (11%) and others (5%). Mobilization failure occurred in 40% of procedures, a collection between 2–5 × 10^6 CD34/Kg in 24% and > 5 in 35%. Finally, of 98 pts who underwent SC mobilization, 22% failed to collect enough cell to perform ASCT, 12 pts even after repeated attempts, 33% had a suboptimal and 45% a good collection (4 and 5 pts respectively after repeated mobilizations). At univariate analysis failure was significantly associated with refractory disease, Plt < 150.000/cmm, CTX 1.5 gr/ms as mobilizing treatment, previous mobilization failure and circulating CD34+ cell < 7.4/mcl on the first day of monitoring; whereas CTX > 3 gr/ms, CD4 count and SC ratio > 0.002 were associated with a reduced risk of failure. In multivariate analysis refractory disease (p 0.002 (p 3 gr/ms, WBC count and circulating CD34+ cells >29,7/mcl at the first day of monitoring and SC ratio > 0,002, whereas G-CSF alone and previous mobilization failure were negative predictive factors. Multivariate analysis confirmed CTX > 3 (p 29,7 (p=0.0003) and SC ratio > 0,002 (0.0036) as indipendent factors for good collection. Conclusions: In this series of 98 ARL and 127 SC mobilization attempts, a substantial number of pts failed SC harvest (22%) whereas 33% had a suboptimal and 45% a good collection. Lymphoma status and mobilizing treatment seems the strongest predictors for outcome, with refractory disease and low CTX dose (1.5 gr/ms) significantly associated with failure and CTX > 3 gr/ms predictor for good collection. A high ratio between circulating CD34+ cells and WBC on the planned day of first apheresis might represent a useful “ongoing” parameter to predict the outcome. These data might help to decide the mobilizing strategy in ARL and could provide the framework to rationally explore the use of new mobilizing agents Disclosures: No relevant conflicts of interest to declare.

Abstract 1302 Introduction: Interleukin 17 (IL-17) is a pro-inflammatory cytokine secreted by CD4+ helper T (TH) cells, TH17 cells. IL-17F is the most recently discovered member of the IL-17 cytokine family. IL-17F induces several cell types to express cytokines, chemokines, growth factors, and adhesion molecules, which are crucial molecules in leukocyte recruitment and activation. On this basis, IL-17F has been associated with the pathogenesis of multiple autoimmune diseases. Allele G for the polymorphism A7488G seems to play a protective role for the development of such diseases. However, no data is available about the possible impact of this polymorphism on the development of complications after allogeneic stem cell transplantation (allo-SCT). Objective: To analyze the influence of the donor (Dn) and recipient (Rc) genotype for the polymorphism A7488G in the IL-17F gene on the outcome of HLA-identical related allo-SCT. Material and Methods: The study comprised 143 allo-SCT patients and their donors (286 samples) included in the Spanish Group for Hematopoietic Stem cell Transplantation (GETH) DNA bank. Genomic DNA was purified from peripheral blood samples obtained, after written informed consent, from patients pre-SCT and donors. The A7488G polymorphism of the IL-17F gene was analyzed by polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) following the procedure developed by Tahara et al. (J Clin Immunol 2008, 28:44-49). Results were analyzed using the Pearson's Chi-square Test. Results: In accordance with previous reports, allele G was present in a reduced number of individuals, 30/286 (10.5%) corresponding to 19/143 Allo-SCT (13.3%). The genotypes of the Dn were 129/143 (90.2%) AA and 14/143 (9.8%) AG, while those of the Rc were 127/143 (88.8%) AA and 16/143 (11.2%) AG. No Dn or Rc with GG genotype was found. Dn and Rc genotype combinations were as follows: DnAA-RcAA, 124/143 (86.7%); DnAG-RcAG, 11/143 (7.7%), DnAA-RcAG, 5/143 (3.5%), DnAG-RcAA, 3/143 (2.1%). The genotype of the donor influenced transplant outcome in terms of relapse (35/129 (27.1%) when the donor was AA vs. 1/14 (7.1%) when the donor was AG; p=0.119) and survival (exitus 64/129 (49.6%) when the donor was AA vs. 3/14 (21.4%) when the donor was AG; p=0,045). No association between Dn or Rc genotype and graft versus host disease (acute or chronic) or other complications was observed. The differences observed in relapse and survival according to the genotype of the donor, were especially true for AG genotype patients (no significant differences were observed in AA genotype patients). In fact, relapse (3/5 (60%) when the Dn was AA vs. 0/11 (0%) when the Dn was AG; p=0.004) and survival (exitus 5/5 (100%) when the Dn was AA vs. 1/11 (9,1%) when the Dn was AG; p Conclusions: The polymorphism A7488G of the IL-17F gene, which causes a His-to-Argsubstitution at amino acid 161 (H161R), influences the outcome of HLA-identical related allo-SCT. The presence of allele G in the Dn had a favourable effect on transplant outcome, in terms of lower relapse rate and better survival. Analysis of a larger set of Rc/Dn pairs is ongoing which will allow to increase the number of individuals harboring allele G and, ultimately, to confirm the results shown here. In such case, genotyping for the polymorphism A7488G of the IL-17F gene could aid in donor selection or drive a risk-adapted management of transplanted patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 3924 Poster Board III-860 Introduction The incidence of lymphomas is increased in HIV-infected individuals. Most of the cases are B-cell subtypes of non-Hodgkin lymphoma. Although the incidence of mature or peripheral T-cell lymphomas (PTCL) seems to be increased in HIV-positive cases, clinicopathological data is lacking. The objective of this study is to describe the characteristics of non-cutaneous PTCL in HIV-infected individuals and identify potential prognostic factors. Methods Institutions within and outside of the United States were invited to submit original data on cases of HIV-associated PTCL. Data on each case included country of origin, age, sex, ethnicity, CD4 count, use of highly active antiretroviral therapy (HAART), B symptoms, lymphoma subtype according to the WHO classification of lymphoid neoplasms, expression of ALK, EBV and Ki-67, T-cell gene rearrangement, number of extranodal sites, bone marrow involvement, clinical stage, performance status, lactate dehydrogenase (LDH) levels, frontline therapy, response, use of stem cell transplantation (HSCT), final outcome, survival in months and cause of death; these will be presented using descriptive statistics. Univariate analyses were performed using Kaplan-Meier survival estimates compared using the log-rank test. Cox proportional-hazard regression test was used for the multivariate analysis. P-values of less than 0.05 were considered significant. Results Thus far, data on 24 cases have been obtained from 7 institutions. From these cases, 13 (54%) are from South America, 5 (21%) from Europe, 3 (12.5%) from North America and 3 (12.5%) from Asia. Thirteen cases (54%) are Hispanic, 5 (21%) are Caucasian, 3 (12.5%) are Black and 3 (12.5%) are Asian. Median age is 39 years (range 26 to 58 years) and the male-to-female ratio is 7:1. Sixteen cases (70%) presented with B symptoms. Median CD4 count is 129 cells/mm3 (range 4 to 305 cells/mm3). Twelve cases (63%) reported use of HAART. Fourteen cases (58%) are PTCL, unspecified (PTCLU), 4 cases (17%) anaplastic large cell lymphoma (ALCL), 4 cases (17%) of NK/T-cell lymphoma (NKTCL) and 2 cases (8%) angioimmunoblastic lymphoma (AITL). All ALCL cases were ALK-negative; EBV was expressed in 50% of NKTCL cases but in none of the AITL cases. Four of 15 cases (27%) had involvement of more than 2 extranodal sites, 3 of 11 cases (27%) had bone marrow involvement, 19 of 24 cases (79%) presented with advanced stage, 5 of 12 cases (42%) had an elevated LDH level and 8 of 24 cases (33%) had a performance status higher than 2. Thirteen of 24 cases (54%) were treated with chemotherapy alone from which 8 cases (62%) received CHOP therapy; six of 24 cases (25%) did not receive any therapy. Nine of 14 cases (65%) responded to therapy (29% CR and 36% PR); 35% of cases did not respond to therapy. Five cases of 14 (36%) underwent HSCT; 4 cases (29%) in the frontline and 1 case (7%) in the salvage setting. At the time of this report, 63% of cases have died; 53% due to infectious complications and 40% due to lymphoma progression. The median survival for the group was 10 months. The median survival for treated (n=19) and untreated cases (n=5) were 10.5 and 1 month, respectively (p=0.005). Conclusions HIV-associated PTCL tends to affect younger men with CD4 counts of less than 200 cells/mm3. PTCLU is the most common subtype reported in HIV-infected individuals. HIV-associated ALCL cases do not appear to express ALK. The survival of treated HIV-associated PTCL cases is short at 10.5 months despite a 65% initial response to therapy. Accumulation of data continues. Disclosures: No relevant conflicts of interest to declare.

Abstract 1329 Poster Board I-351 The detection of minimal residual disease (MRD) in AML patients after allogeneic stem cell transplantation (SCT) is important for the early detection of relapse and the selection of patients who can benefit from immunomodulation. Up to 65% of patients with AML lack of molecular markers useful for follow-up. The Wims tumor gene (WT1) is overexpressed in more than 80% of AML patients at diagnosis, hence its utility as MRD marker in these patients. The objective of this study is to analyze the usefulness of WT1 expression for MRD study and relapse prediction in patients with AML after allogeneic SCT and to compare the results with those from flow cytometry and chimerism. Methods 149 samples were studied (63 BM and 86 PB) from 11 patients with AML (table 1) lacking of other molecular markers at diagnosis, who underwent allogeneic SCT in a single center from 2003 and 2008. Quantification of WT1 expression was performed by quantitative RT-PCR (LightCycler 1.2) using cDNA obtained from 1 ug of RNA (Trizol®, Invitrogen). K562 cell line was used as calibrator and ABL as reference genes (relative quantification). Cut-off values were defined at 0.5% in BM samples and 0.01% in PB. Results were correlated with those from flow cytometry and chimerism from the same samples. Results WT1 overexpression correlated with the presence of disease before transplantation (table 1). Post-transplantation, 5 of the 11 patients showed low and stable WT1 levels in BM and PB in the follow-up, remaining in CR (median follow-up 48 months, range 8-61). These 5 patients showed complete chimerism (CC) and negative MRD by flow cytomety (figure A). On the other hand, 6 from the 11 patients showed WT1 overexpression after SCT: 4 of them showed after initial low values, a significant increase in WT1 expression up to reaching positive levels in a median of 139 days (50-167), while 2 patients showed positive values continuously. Two patients showed positive levels only in PB samples, one only in BM and 3 both in BM and PB. From these 6 patients, 5 relapsed (1 with extramedullar disease who showed WT1 overexpression only in PB) in a median of 9 months after SCT (2.4-11). In one case, WT1 overexpression occured at the same time as relapse, while in the other 4 cases, relapse occured in a median of 137 days (134-170) after a significant increase in WT1 levels was seen in two consecutive samples. In these 5 relapsed patients, neither flow cytometry nor chimerism predicted relapse (figure B). Conclusions WT1 overexpression correlated with disease burden in AML patients after allogeneic SCT. In realapsed patients, relapse both medullar and extramedullar was anticipated sinificantly by WT1 overexpression compared to flow cytometry and chimerism. Quantification of WT1 overexpression by RT-PCR should be used for MRD detection in the follow-up after SCT in AML patients (not only in BM but also in PB for extramedullar disease detection) in order to facilitate immunossupresive therapy management and to select early DLI candidates. Disclosures No relevant conflicts of interest to declare.

Abstract 4355 Little is known about the additional benefit of Autologous Stem Cell Transplantation (ASCT) as consolidation treatment of NHL in 1st CR in HIV+ patients. We herein report a comparative analysis of HIV+ patients with a LBCL treated with chemotherapy (chemo) followed by ASCT and a matched cohort of HIV+ patients treated with chemo alone. Methodology Retrospective, registry-based, matched cohort study. ASCT cohort: patients with diffuse large B-cell (DLBC) or plasmablastic NHL treated with ASCT in 1st CR after standard chemo and reported to the EBMT Registry. Chemo cohort: For each patient within the ASCT cohort we selected two controls from the HIV+ patients with NHL GESIDA/PETHEMA registry. Patients in both cohorts were in 1st CR following front-line or rescue (for partially responding patients) chemo and were matched according to histology, IPI and the use of Rituximab. We compared overall survival (OS), disease free survival (DFS) and cumulative incidence (CI) of relapse between both cohorts. These primary outcomes were defined according to the EBMT. OS was computed from diagnosis while DFS and CI of relapse were computed from 3 weeks after the last standard chemo cycle administered (end of chemo). Results The ASCT cohort included 10 patients diagnosed between 1999 and 2005. The Chemo cohort included 20 patients, 16 diagnosed between 1999 and 2005. Both cohorts were comparable for the main clinical and patient features (Table 1). The median (range) follow-up (FU) time since the end of chemo for surviving patients was 56 months (mo) (24-106) in the ASCT cohort vs 37 mo (8-107.5) in the Chemo cohort; P=.28. Five years (yr) OS for the ASCT cohort and the Chemo cohort were 68.5% [CI95%: 39-98] and 46.5% [CI95%: 18-75], respectively; P=.6. Three yr DFS for the ASCT cohort and the Chemo cohort were 70% [CI95%: 41.5-98.5] and 59.5% [CI95%: 29-86]; respectively; P=.4. The CI of relapse in the ASCT cohort and the Chemo cohort were 21% [CI95%: 0-47] and 27% [CI95%: 2-51], respectively; P=.8 Conclusions In this retrospective registry-based, matched cohort study of HIV+ patients with large B-cell NHL we found a non-significant effect of ASCT as consolidation treatment in 1st CR patients, in terms of survival and relapse incidence. Nevertheless, due to the observed favorable tendency, future analysis including a higher number of patients and, eventually, randomized clinical studies, should be performed to further clarify these observations. Disclosures: No relevant conflicts of interest to declare.

Abstract 4307 Introduction Complications after Allogenic Stem Cell Transplantation (Allo-SCT) are influenced by the immunological fenomena associated to the alloreactivity/allotolerance between donor and recepient. In this context, the usefulness of chimerism quantification in T lymphocytes for the prediction of complications after Allo-SCT is well known. However, to our knowledge, there is no information on the utility of chimerism analysis in activated lymphocytes (CD25+). Objective To stablish an asociation betwen the more common complications post Allo-Tph (aGVHD, cGVHD, relapse, reject), and the chimerism dynamics in CD25+ activated lymphocytes in the early period post-transplantation, trying to predict the patients with more risk of suffering from this complications. Materials and Methods The study included 38 Allo-SCT (17 NMA, 21 MA; 7 Haploidentical, 13 non-related donor, 18 identical donor; 7 UCB, 5 BM, 26 PB). Chimerism analysis was performed every 2 weeks until complete chimerism (CC) was achieved, and every 3 months thereafter. Follow up was censored when patients received DLI or 2nd Allo-SCT.Chimerism quantification was performed by microsatellite PCR (STR-PCR; AmpFlSTR SGM Plus; Applied Biosystems) on DNA obtained from peripheral blood and purified cell lineages (95% putity) by immunomagnetic technology (Miltenyi Biotec). Results Table 1 shows the results at day +30 for T and activated lymphocytes (CD3+ and CD25+) allocating patients in two groups depending on the relative percentage of recipient cells (% rec) in both cell lineages. 20 patients were in CC in CD25+, maintening this CC during the study. 18 patients were found in MC in CD25+ (18/38 MC). 1/18 was in CC in CD3+ TL, and 10/18 were in CC in whole blood. 17/18 were in MC both in CD3+ and CD25+ lymphocytes, Although no statistical significance is achieved due to the reduced sample size, patients in the first group (% rec higher in activated that in T lymphocytes) tend to achieve CC later and to show higher risk of developing complications such as acute and chronic GVHD, relapse or rejection. Conclusions A higher incidence of complications post Allo-SCT (aGVHD, cGVHD, relapse, rejection) in patients with % of recipient in CD25+> % recipient in CD3 at day 30. Chimerism analysis in activated lymphocytes (CD25+) is of great utility after Allo-SCT since it allows beter prediction of complications than standard follow up of whole blood or T lymphocytes. Disclosures: No relevant conflicts of interest to declare.

WHO criteria defines platelet counts above 600×109/L as the threshold for essential thrombocythemia (ET) diagnosis. It has been argued that such threshold excludes a number of patients with ET with platelet counts below 600×109/L. Recently, a proposal for revision of the World Health Organization (WHO) diagnostic criteria for ET has been published, which includes the combination of histological bone marrow study and testing of JAK2 mutation. Design and methods : Retrospective analysis of 92 patients with ET diagnosis between 1989 and February 2008, isolating the subgroup of patients with platelet counts below 600×109/L. The aim of this study was to analyze the applicability of the 2008 WHO criteria in this subgroup. Results : Of the 92 patients, 30 patients did not fulfill the WHO criteria due to platelet counts 3.5 | 30/88 (34%) | 8/28 (29%) | | Fibrosis grade I | 2/90 (2%) | | | Compatible histology | 79/89 (86%) | 21/28 (75%) | | Abnormal Cytogenetics | 2/26 (8%) | | | Symptoms | 13 (14%) | 4 (13%) | | Thrombotic event | 16 (18%) | 5 (17%) | | Haemorrhagic event | 3 (4%) |

Introduction : The success of allogeneic stem cell transplantation (SCT) is conditioned by the dynamics and the result of the bidirectional interaction of immune reactions exerted by donor and recipient (alloreactivity/tolerance). Quantitative chimerism monitoring after SCT, mainly if performed on leukocyte lineages such as T lymphocytes (TL, CD3+), allows assessment of the immune interaction between donor and recipient. Objective : To evaluate the usefulness of chimerism quantification in activated lymphocytes (AL, CD25+) for the early detection of complications (graft failure/rejection, GVHD, relapse, etc.) which, eventually, would improve the efficacy of current therapeutic strategies. Material and methods: Chimerism quantification was done by microsatellite PCR (short tandem repeat, STR-PCR) on peripheral blood (PB) samples obtained every other week from 20 transplanted patients, as well as on leukocyte lineages, TL and AL, purified by immunomagnetic means (autoMACS, Miltenyi Biotec, purity>95%). Results : Three different patterns of chimerism dynamics were observed: 10 patients showed complete chimerism (CC) in all samples analyzed post-SCT. 6 patients showed mixed chimerism (MC) with lower percentage of recipient cells in AL than in TL. All this 6 patients spontaneously evolved to CC. 4 patients showed MC with higher percentage of recipient cells in AL than in TL. All these 4 patients showed decreasing MC (increasing percentage of recipient cells) in PB, TL and AL. Moreover, 3 of them showed complications involving the graft: graft failure (not recovered after two succesive transplants) and graft rejections (1 incipient), which recovered and achieved CC after donor leukocyte infusions. Concerning disease relapse, 1 case was observed in each of groups A and B, and three cases were observed in group C. Conclusions: Our results suggest that higher percentages of recipient cells in AL than in TL are associated with stronger host-versus-graft immune reactions and, therefore, with decreasing mixed chimerism and subsequently with various complications after SCT. On the contrary, lower percentages of recipient cells in AL than in TL predict the achievement of CC. Quantitative follow up of chimerism in AL (CD25+) increases the usefulness of the analysis of PB and TL (CD3+) samples, allowing to predict the dynamics of chimerism after SCT and favouring the early establishment of the appropriate therapeutic option in each transplanted patient.

Introduction: Myeloablative allogeneic stem cell transplantation in patients (pts) with MCL has been traditionally used only in young pts without co-morbidities due to its high toxicity. During the last 10 years, many different reduced intensity conditioning (RIC) regimens have been introduced into clinical practice trying to reduce this toxicity, preserving the establishment of a meaningful graft-tumor effect (GVT). However, few homogeneus pts series have been reported focused on MCL, with encouraging results in some studies. Thus, we report herein the outcome of 18 consecutive MCL pts who received an allo-PBSC-RIC from an HLA-identical sibling donor with a long follow-up. Patients and methods: The RIC consisted in iv fludarabine 125–150 mg/m2 from day-8 to -4 and iv melphalan 80 or 140 mg/m2 on day-3 to -2. Rituximab 375 mg/m2 was added to RIC in 8 pts on days-9, +1, +8 and +15. GVHD prophylaxis consisted in standard cyclosporine and methotrexathe. Gradual cyclosporine tappering from day +50 was initiated if mixed chimerism or persistent disease were present. We focus on engraftment kinetics, early toxicities, non-relapse mortality (NRM), chimerism kinetics, GVHD as well as estimated anti-tumoral efficacy, progression-free and overall survival (PFS and OS). Results: 18 MCL pts were included from 2000 to 2008, with a median follow-up of 50 months. Median age was 56 years (range: 43–68), 13 were males, with a median pre-RIC treatment lines of 2 (1–4) including 2 autoHSCT. Before alloRIC, 13 pts (72%) were in 1st or later CR, while 5 pts (28%) were in chemo-sensitive PR. Median CD34+/kg infused cells were 5,8 ×106 (4,3–10,8) and 2,0 ×108 (0,7–4,1) CD3+ lymphocytes/kg. All pts engrafted, reaching ANC>500 on day +16 (13–20) and platelets>20.000 on day +12 (8–32), with no graft failures. Early toxicity ( Conclusions: AlloRIC with fludarabine and melphalan in MCL pts offers a good toxicity profile, with high engraftment rate and good long term disease-free survival, especially in pts younger than 60. The very low long-term relapse rate seen in the context of a high incidence of chronic GVHD supports the continued sensitivity of MCL to a GVT effect. Figure Figure

INTRODUCTION: inherited antithrombin (AT) deficiency is the thrombophilia with the highest incidence of thrombotic events (TE). Homozygous AT deficiency is extremely rare. A variant of Budapest 3 (99Leu to Phe mutation) a subtype (type II deficiency), it is a defect at the heparin-binding site and carries an increased risk of severe venous and arterial thromboembolism from an early age. AT deficiency is also associated with a higher risk of adverse outcome during pregnancy (fetal loss, preeclampsia, intrauterine growth restriction) and its the mechanism is unclear. We describe the second case of a successful pregnancy in a woman who was homozygous for AT deficiency. PATIENT: A 32-year-old woman developed a spontaneous deep vein thrombosis in both femoral arteries, the iliac artery and inferior vena cava, at the age of 27 years. A severe AT deficiency was detected due to AT activity of 20% (normal 80–120%). The patient was put on long-term oral anticoagulation (OAC) with vitamin-K antagonists. Molecular analysis revealed homozygous for the 99Leu to Phe mutation, which was compatible with a type II defect at the heparin-binding site and, consequently, reduced affinity for heparin. When the patient was diagnosed to be pregnant in the 5th week of gestation, OAC was stopped due to the risk of embriopathy, low molecular weight heparin (LMWH) (enoxaparin) 1mg/kg/12h was started and 2,000 U per week of AT concentrates was administered. Anti-Xa activity was below the detection limit at all time and the AT level was below 40%. Therefore, the dose of enoxaparin was increased to 1.7mg/kg/12h without reaching anti-Xa therapeutic levels, despite a switch to others LMWH. The patient was hospitalized at week 12 of gestation to monitor anticoagulation and AT levels; 3,000 U/48h of AT concentrates was administered and AT levels of over 50% were achieved. Intravenous heparin (IH) was started and an acceptable anticoagulation range was reached. In the 14th week of gestation, OAC was introduced and LMWH and AT replacement were discontinued after an international normalized ratio (INR) of 2 to 3 had been reached. Around week 36, the patient was hospitalized to prepare for the birth, the dose of AT concentrate was increased to 3,000 U/d, and OAC was stopped and replaced by IH. Caesarean section was performed at week 38 with a successful outcome; the patient gave birth to a perfectly healthy boy, weighins 2,460g (Apgar score 8/9), and tubal ligation was performed at the patient’s request. On the 3rd day postpartum, OAC treatment was started. Therapeutic INR was reached on day 10, when AT replacement and IH were discontinued, and the patient and her baby were discharged in good health. Checkups during puerperium did not reveal evidence of acute thrombosis. CONCLUSIONS: There have only been six reports of homozygous AT deficiency (99Leu to Phe mutation) that led to severe, and in some cases, fatal TE during childhood. Interestingly, our patient developed TE in early adulthood. There have only been two reports (including the present case) of a successful pregnancy in patients with this deficiency. Only heparin is ineffective as an anticoagulant therapy and an AT replacement is needed. An efficient anticoagulant therapy with OAC during pregnancy has a positive impact on intrauterine growth and fetal outcome.

Introduction: Methylation of CpG dinucleotides is a fundamental mechanism of epigenetic regulation in eukaryotic genomes that controls the expression of certain genes. FOX-P3 is a member of the forkhead/winged-helix family of transcriptional regulators which expression is constitutive in regulatory T lymphocytes CD4/CD25 (Treg). Recent evidence suggests that human effector T cells express FOX-P3, albeit transiently and with significantly lower levels. Therefore, FOX-P3 mehtylation within the promoter region and gene expression could play a role in allogeneic stem cell transplantation (alloSCT). Objective: To analyze the association between the mehtylation status and the levels of expression of FOX-P3 with the dynamics of chimerism and the development of complications after alloSCT. Patients and methods: The degree of methylation was quantified in a 600 pb CpG island within the regulatory sequence of FOX-P3 by quantitative methylation-specific real time-PCR from 32 peripheral blood (PB) samples obtained within the first month post-alloSCT. Moreover, the expression level of FOX-P3 mRNA was analyzed by real-time quantitative PCR in 18 patients from which RNA samples were available. Results were analyzed using Fisher’s exact test due to the reduced sample size. Results: A statistically significant association (Table 1) was observed between a higher degree of methylation in the FOX-P3 promoter and a lower incidence of acute graft versus host disease (GVHD) grades II-IV (35.3% vs a 88.9% in low methylation patients; p=0.014). Moreover, patients with higher degree of methylation showed significantly higher incidence of disease relapse (41.2% vs a 0% in low methylation patients; p=0.05). The observation of a high degree of methylation would reflect a lower amount of FOX-P3 expressing cells, both Treg and effector cells. Since Treg comprises a minority population, such observation would result from relative low levels of effector T cells, which would explain the lower graft-versus-host and graft-versus-leukemia effect observed in high mehtylation group. Surprisingly, when FOX-P3 mRNA levels were analyzed, an association with statistical significance between a higher methylation status and a higher expression of FOX-P3 (90% vs 55.8%; p=0.047) was observed. It has been shown that FOX-P3 is mainly produced by Treg while effector T cells produce very low amounts of FOX-P3 mRNA. In this sense, a higher amount of Treg cells produces high FOX-P3 mRNA levels and reduces the number of effector T cells, resulting in a higher methylation status in PB samples, in which most cells would have methylated FOX-P3. Low FOX-P3 expression would, in turn, indicate a lower amount of Treg to suppress the immune activation, resulting in higher amount of effector T cells and therefore would be associated with a lower methylation. Additionally, a significant association between higher FOX-P3 expression and lower incidence of death (7.7% vs 80%; p=0.007) was observed. Moreover, the main cause of death (3/4) in the group with lower expression of FOX-P3 was acute GVHD. The lower amount of effector T cells in PB samples with high FOX-P3 expression would protect from the development of GVHD. Conclusions: Both FOX-P3 mRNA expression and promoter methylation are of prognostic value for the development of complications post-SCT. These determinations would favor an early establishment of the immunotherapeutic options for an improved management of transplanted patients. Table 1. Association between FOX-P3 promoter methylation and mRNA expression with chimerism and complications post-SCT. MC, mixed chimerism; PB, peripheral blood; Fail/Reject., graft failure/rejection. p, Fisher`s Test. Methylation MC (PB) Fail/Reject. aGVHD (II-IV) cGVHD Relapse Exitus High 6/19 (31.6%) 4/19 (21.01%) 6/17 (35.3%) 10/13 (76.9%) 7/17 (41.2%) 5/19 (26.3%) Low 2/9 (22.2%) 0/9 (0%) 8/9 (88.9%) 3/8 (37.5%) 0/9 (0%) 4/9 (44.4%) p NS NS 0.014 NS 0.05 NS Expression MC (PB) Fail/Reject. aGVHD (II-IV) cGVHD Relapse Exitus High 4/13 (30.8%) 2/13 (15.4%) 6/12 (50%) 10/11 (90.9%) 3/12 (25%) 1/13 (7.7%) Low 0/5 (0%) 0/5 (0%) 4/5 (80%) 3/4 (75%) 1/5 (20%) 4/5 (80%) p NS NS NS NS NS 0,007

We report two patients with cerebral haematomata who developed antibodies against bovine thrombin

Several authors have reported ASCT as a feasible, safe and effective treatment in HIV associated lymphoma patients (pts) receiving Highly Active Antiretroviral Therapy (HAART), particularly when being autografted with chemosensitive disease. To gain a better understanding of the usefulness of ASCT in HIV+ Lymphoma pts a retrospective comparative study (HIV+ vs HIV- lymphoma pts with ASCT) has been performed using the EBMT-LWP Registry. Methodology: Registry-based, retrospective, individually matched case-control analysis. Within the participating centres one or two HIV- controls for each HIV+ pt were selected from the registry with the following inclusion criteria: Known HIV serological status at ASCT, lymphoma (HL or NHL), ASCT performed between 1999 and 2006 and pts over 18 years of age. Two cohorts (HIV+ vs HIV-) were matched for histology, IPI at diagnosis (NHL), stage at diagnosis, disease status at ASCT, age at ASCT, year and country of ASCT. Results: 40 HIV+ lymphoma pts undergoing an ASCT were matched with 46 HIV- pts. Pts and transplant features are shown in table 1. With a median follow up of 36 mo, the differences regarding OS and PFS were not significant: 62% for HIV+ vs 69% for controls, and 56.5 vs 58%, respectively. No differences were seen regarding HL and NHL pts. An overall TRM of 10% was observed in the HIV cohort, mainly related to infections, while no cases of TRM were seen within the control arm. Since survival rates between the HIV+ and the matched HIV- lymphoma populations remained comparable, the HIV condition should not preclude these pts from being treated according to the same criteria as the HIV negative lymphoma population. Nevertheless, infectious complications should be cautiously followed within the HIV+ lymphoma pts undergoing an ASCT. Patients and transplant features HIV+ HIV- n=40 % n=46 % Age [Median (range)] in years 41.4 (29.2–62.5) 44 (16–62.4) p= NS Male sex 35 87.5 25 54.3 p=0.001 Histology DLBCL 24 60 25 54.3 p= NS Burkitt lymphoma 2 5 2 4.3 p= NS T-cell NHL 2 5 3 6.6 p= NS HL 12 30 16 34.8 p= NS Disease status at ASCT Complete remission (CR) 21 (12 in CR1) 52.5 (30 in CR1) 20 (11 in CR1) 43.5 (24 in CR1) p= NS Chemosensitive disease 16 40 25 54.3 p= NS Chemorefractory disease 3 7.5 1 2.2 p= NS CD34+ cells infused mill/kg [median (range)] 4.9 (1.6–21.2) 4.8 (0.9–21.2) p= NS G-CSF prior to engraftment 36 90 21 46 p= 0.0001 Neutrophil engraftment 39 98% 46 100 p= NS Cause of Death Relapse/progression 9 60% 10 84% p=0.08 Secondary malignancy 1 6.7% 1 8% p= NS Transplant-related deaths 4 26.7% 0 0% p=0.06 Other 1 6.7% 1 8% p= NS

Background: Genetic polymorphisms within the regulatory sequence of cytokine genes influence their expression and, ultimately, the immune reactivity. Such polymorphisms are therefore associated with the outcome of stem cell transplantation (SCT) supporting the hypothesis of a genetic predisposition towards certain complications post-SCT. Objective: To assess the association between donor (D) and recipient (R) genotypes for the IL-10 gene -1082 SNP (single nucleotide polymorphism) and -1064 STR (short tandem repeat) polymorphisms with the dynamics of chimerism and the development of complications after SCT. Patients and methods: The study included 39 conventional SCT from HLA-matched related donors. IL-10 genotypes were determined in an -1082 SNP allele-specific PCR (A vs G) including the -1064 (CA)n STR in the product which is revealed by capillary electrophoresis. Results were analyzed using Fisher’s exact test due to the reduced sample size. Results: The frequency of alloreactive genotypes (AA or A) for the SNP was 35,9% AA and 87,2% A in the D and 25,6% AA and 89,7% A in the R. No association was observed between the presence in the D or R of the alloreactive allele for the SNP genotype and the dynamics of chimerism or complications post-SCT. Alleles 4 (17 CA repeats) to 12 (25 CA repeats) for the STR genotype were found in the present series. The frequency (homozygous, +/+, or heterozygous, +) of the low IL-10 producer allele 7 (high alloreactivity allele) was 5,1% +/+ and 25,6% + in the D and 7,7% +/+ and 33,3% + in the R. The presence of allele 7 in the recipient was significantly associated with the development of graft failure/rejection (p=0,012; Table ). In these patients, the incidence of mixed chimerism (MC) in the CD3+ fraction (T lymphocytes) during the first month post-SCT was significantly higher (p=0,043; Table 1). Conclusions: The presence of the low producer allele 7 for the IL-10 -1064 STR polymorphism in the recipient would associate with a higher immune alloreactivity against donor cells. This would favour the establishment of mixed chimerism increasing the risk of graft failure/rejection. Therefore, our results suggest that recipient IL-10 -1064 STR polymorphism genotypes may influence SCT outcome in a chimerism mediated fashion. The analysis of a larger number of patients as well as further cytokines will eventually allow to confirm these observations and to establish this type of studies as a means for an improved management of transplanted patients. Table 1. Influence of the presence in the recipients of allele 7 for the IL-10 -1064 STR polymorphism on chimerism and complications post-STC. IL-10 -1064 allele 7 MC day+30 in T cells (CD3+) aGVHD II-IV cGVHD Graft failure/rejection Relapse Exitus Present 6/6 (100%) 6/12 (45,5%) 6/10 (60%) 5/13 (38,5%) 3/12 (25%) 6/13 (46,2%) Not present 6/13 (50%) 13/26 (50%) 16/18 (88,9%%) 1/26 (4%) 7/25 (28%) 12/26 (46,2%) p (Fisher’s Test) 0,043 NS NS 0,012 NS NS

Introduction: Myeloablative allogeneic stem cell transplant has been reserved for long time to young well-fitted patients (pts) due to its high toxicity. Lately, different non-myeloablative (NMA) conditioning regimens have been employed to reduce this toxicity preserving the graft-vs-tumor effect (GVT). Here we present our experience in advanced lymphoma pts. Patients & methods: Since year 2000, we used iv fludarabine 150 mg/m2 days −7 to −4 and iv melphalan 80 mg/m2 day −2 as NMA conditioning for heavily pretreated lymphoma pts, aged >55y or with comorbidities. GVHD prophylaxis consisted in cyclosporine and short course MTX. Stem cells were obtained from peripheral blood of an HLA-identical sibling donor. Gradual cyclosporine tappering from day +50 was initiated if mixed chimerism or persistent disease were present. We evaluated: engraftment rate, toxicities, transplant related mortality (TRM), chimerism kinetics, GVHD incidence and disease free and overall survival (DFS and OS). Results: 18 lymphoma pts (8 follicular, 4 mantle cell, 4 peripheral T and 2 DLBCL) were transplanted since 5/2000 to 1/2007 due to co-morbidities in 4, age>55y in 6 and heavily pretreated pts in 10. Median age was 52y (31–61), 10 were males, median treatment lines were 3 (1–5) including 4 auto-transplanted(22%). After the last treatment, 10 pts were in CR(56%), 4 pts had chemo-sensitive PR(22%) and 4 chemo-resistant disease(22%). Median CD34+/kg infused cells were 5,0 x106(3,7–10,8) and 2,0 x108(0,4–4,4) CD3+ lymphocytes/kg. All pts engrafted, reaching ANC>500 on day +14 (10–20) and platelets>20.000 on day +12 (9–33), except 1 early death. There weren’t any early or late engrafment failures. Early toxicity until day +100 included febrile neutropenia in 6, severe mucositis in 2, hepatic toxicity in 2, VOD in 1, hemorragic cystitis and arrythmia in 1. There were 2 deaths before day +100: 1 refractory congestive heart failure and 1 Pseudomonas’ sepsis(TRM 11,1%) and 2 late deaths beyond +1y due to infections during chronic GVHD treatment. Viral infections were the most prevalent(72%), mainly CMV reactivations (44%) that were relapsing in 17%. There were 7 bacterial infections (4 bacteriemias, 1 meningitis, 1 pneumonia and 1 urinary sepsis). Complete chimerism (CC) was present on day +30 in 38% pts and in 93% pts on day +90. Only 1 pt persisted on mixed chimera until day +180 and 10/10 evaluated pts were on CC after 1 year. Acute grade II-IV GVHD ocurred in 37%, with 25% grade III-IV. Chronic GVHD affected 10/15 pts (67%), limited in 27% and extensive in 40%. Only 1/10 CR pts at transplant relapsed after 95 days and 4/4 pts with chemo-sensitive PR reached CR that persisted after achieving CC on day +90. All chemo-refractory pts died (4/4): 2 early toxic deaths and 2 progressions. Median follow-up was 55 months(28–83) with DFS of 61% and 66,7% OS at 5y. Twelve pts persisted alive in CR with chronic GVHD in 4(33%). Predictors for DFS and/or OS were follicular and mantle cell histology vs others (p2 lines (p Conclusions: After long term follow-up, NMA conditioning with fludarabine and low melphalan dose in advanced lymphoma pts offers good toxicity profile, with high engraftment rate and good disease control if pts were in chemo-sensitive disease pretransplant.

INTRODUCTION: The myelodysplastic syndromes (MDS) are clonal hematopoietic disorders characterized by dysplasia and cytopenias, especially anemia. There is no widely accepted standard therapy for MDS. Exogenous erythropoietin (EPO) seems to be able to raise Hb levels and/or decrease transfusion requirements in patients with MDS, with reported responses rates of 25–30%. In our center, EPO has been used since 1999, based on the published recommendations, assessing the therapeutic response after 12 weeks of therapy. OBJECTIVE: To analyze retrospectively the therapeutic response to EPO in 45 patients with MDS and anemia. PATIENTS AND METHODS: 45 consecutive patients with MDS and calculable IPSS score, showing low or intermediate-1 risk, were treated with EPO from 1999 to 2006. From the initial 45 patients, 5 were excluded from the analysis because of lack of follow-up. The 40 selected patients belonged to the following FAB subgroups: AR 20 (50%), ARS 12 (30%), AREB 3 (7%) and LMMC 5 (12%). Other criteria for treatment were Hb levels 2 g/dL Hb increase or transfusion independence, and partial response (PR) with a 1–2 g/dL Hb increase or 50% decrease in transfusion requirements. Sixteen patients with no response after 12 weeks of therapy, continued with double EPO dose for a median of 10 weeks. RESULTS: From the 40 patients selected, 22 (55%) were female with a median age of 76 years old (47– 86). Mean baseline Hb level was 8,4 g/dl (5,8–11). Serum EPO levels were measured in 33 patients: 23 (70%) presented CONCLUSIONS: The response to EPO therapy of MDS-related anemia in our center was unexpectedly high (47,5%). Patients who do not show response after 12 weeks of initial therapy, seem to improve from switching to higher EPO doses since 18% of patients treated with this scheme responded. In our study, ARS subgroup showed an unexpected high rate of response (66%), although they were treated without association of granulocyte-stimulating colony factor. Remarkably, pretreatment serum EPO levels Table 1. Response rates in different FAB subgroups; N, number of patients; GR, global response; CR, complete response; PR, partial response FAB N GR (%) CR (%) PR (%) AR 20 35 20 15 ARS 12 66 16 50 AREB 3 33 33 0 LMMC 5 60 20 40

ASCT has been reported as a feasible, safe and effective treatment in HIV associated lymphoma patients (pts) receiving Highly Active Antiretroviral Therapy (HAART). Nevertheless comparative studies with HIV− lymphoma population have not yet been performed. We present a retrospective matched analysis comparing clinical outcomes in both HIV+ and HIV− auto-transplanted HL pts. Patients and Methods: 6 HIV+ HL pts who underwent ASCT since June 2000 were included. Twelve transplanted HIV− HL auto-transplanted pts were selected as controls. Both groups (HIV+ vs HIV−) were comparable for the most relevant clinical features (Mann-Whitney or chi-square tests p value > 0.05): median age (40 vs 36), Ann Arbor advanced stage (50% vs 25%), status at ASCT (CR>1 or PR, 83% vs 75%), all 18 pts received 2 lines of chemotherapy before ASCT. BEAM was used as conditioning regimen in all 18 pts. In the HIV+ group HAART was maintained during mobilization and ASCT, except during the conditioning in 1 Pt, in which it was needed to be resumed due to an increase in viral load. The median number of infused CD34+ cells/kg was 3,65×106 in HIV+ pts and 4,75×106 CD34+ in HIV− pts (p: NS). G-CSF was used in all HIV+ pt until engraftment starting at a median of 5 days (d) after ASCT. Results: All 18 pts engrafted, at a median of 13,5 (9–29) d after ASCT in HIV+ pts and 14 (11–18) d in HIV- pts. The incidence of acute infectious and toxic events was not different in both groups. All pts developed neutropenic fever. Documented infections were (HIV+ vs HIV−): Gram+ bacteraemia (2 vs 3), Gram− bacteraemia (1 vs 0), CMV antigenemia (2 vs 1). Pneumonia was documented in one pt from each group. One HIV+ pt showed grade II liver toxicity and grade II renal toxicity occurred in 1 HIV− pt. All events were succesfully resolved. In 4 out of 6 HIV+ pts, HAART was withdrawn due to gastrointestinal toxicity. The median follow-up time was 36,5 mo in HIV+ pts and 37,5 mo in HIV− pts. Two pts relapsed in each group. Within the HIV+ group 1 pt died, due to disease progression. Within the HIV− group 3 pts died, 2 of disease progression and 1 due to post-ASCT secondary acute leukaemia. The OS at 36 mo was 83% for HIV+ pts and 80% for HIV− pts (p=NS). EFS at 36 mo was 55% in HIV+ pts and 70% in HIV− pt (p=NS). Conclusions: Our results show that clinical outcomes are comparable in HIV+ and HIV− HL pts undergoing ASCT. Engraftment, complication events and survival were not different. ASCT may be applied with guarantees in HIV associated HL on HAART pts in a similar way that in the HIV− setting.

ASCT has been reported as a feasible, safe and effective treatment in HIV associated lymphoma patients (pts) receiving Highly Active Antiretroviral Therapy (HAART). For a better knowledge regarding the outcomes within this setting we have compared the clinical results of a multicentre cooperative cohort of auto-transplanted HIV+ NHL pts, with a matched cohort of HIV− NHL pts who underwent ASCT in our hospital. Patients and Methods: 17 HIV+ NHL pt auto-transplanted since 2001 have been included, as well as a similar number of matched HIV− NHL pts. Both cohorts were comparable for the most relevant clinical features (p value Mann-Whitney test or chi-square test > 0.05) (see table). HAART was maintained during mobilization and ASCT. The median number of CD34+ cells infused was 3,73x106 CD34+ cells/kg in HIV+ pts and 4,75x106 CD34+ cells/kg in HIV− pts (p: NS). G-CSF was used in 16/17 HIV+ pts until engraftment starting at a median of 7 days (d) after ASCT. Results: All but 1 HIV+ pt who died in relapse due to multiorgan failure on day +15 engrafted. The median time to engraftment was 12,5 (9–33) d in HIV+ pts, and 13 (9–18) d in HIV− pts. The incidence of acute infectious and toxic events was similar in both groups. All pts developed neutropenic fever. The documented infections were (HIV+ vs HIV−): Gram+ bacteriemia (5 vs 4), Gram- bacteriemia (1 vs 2), CMV antigenemia (1 vs 1). Aspergillus pneumonia was documented in one HIV+ pt and Candida parapsilosis was cultured in another HIV+ pt. One case of pneumonia ocurred in the HIV− group. Two toxic events were documented in the HIV+ group: 1 VOD and 1 multiorgan failure. All but one events (multiorgan failure) were succesfully resolved. The median time of follow up was 36,5 mo in HIV+ and 32,3 mo in HIV−. Within the HIV+ group 5 pt relapsed, 3 of them died and 2 remain in CR after rescue chemotherapy. Other 3 pts died (multiorgan failure, late HIV opportunistic infection and post-ASCT myelodysplasia). In the HIV− group 8 pts relapsed and all of them died. The median time to relapse/progression was 3,5 mo (HIV+) vs 3 mo (HIV−). The OS at 36 mo was (HIV+ vs HIV−) 67% vs 49% (p=NS) and EFS at 36 mo was 55% vs 52,3% (p=NS). Conclusions: Our results show that the clinical outcomes in NHL HIV+ and NHL HIV− pt undergoing ASCT are comparable. Engraftment, infectious and toxic events as well as survival were not different. ASCT may be applied in HIV+ NHL on HAART pts with similar guarantees as in the HIV-negative setting. Table: Clinical features of both series HIV+ NHL pt HIV− NHL pts Age 43 46 Adjusted IPI: >2 10 12 Status at ASCT: CR1 or PR 8 12 Pre-ASCT treatment lines: >1 12 13 HISTOLOGY Burkitt-Burkitt like 3 1 Peripheral T-cell 1 1 Anaplastic 2 1 DLBC 9 12 Plasmablastic 2 0 B or T Lymphoblastic 0 2 CONDITIONING BEAM/BEAC 16 11 TBI-based regimens 1 1 BuCy 0 4 CBV 0 1

INTRODUCTION: Non-neoplastic chronic portal vein thrombosis (PVT) has become a more frecuent diagnosis in the course of liver cirrhosis, with reported prevalences of 4,4% to 15,8%. PVT can motivate life-threatening complications due to worsening portal hypertension. The role of anticoagulation in patients with chronic PVT is not well established, and is considered challenging in cirrhotic patients. OBJECTIVE: To analyze the response to antithrombotic therapy in 12 patients with chronic PVT associated with cirrhosis, and to assess its tolerance and safety. PATIENTS AND METHODS: 12 consecutive patients with liver cirrhosis and chronic PVT were treated with antithrombotic therapy from 2005 to 2006. PVT was diagnosed by imaging procedures, and was classified as complete or partial according to absence or reduction of blood flow in the portal trunk, branches, superior mesenteric and splenic veins. Hepatocellular carcinoma and known thrombophilic risks were ruled out. Cirrhosis was scored according to Child-Pugh’s classification. History of hemorrhagic episodes was assessed. Therapy was started with 15 days of therapeutic doses of low molecular weight heparin (LMWH) (enoxaparin) adjusted according to baseline hipocoagulability (Table 1), followed by either prophylactic doses (0,5mg/kg/day) of LMWH or acenocoumarol (target INR 2–3), during 6 months. Response was evaluated after 3 and 6 months. RESULTS: From the 12 patients studied, 9 (75%) were males with a median age of 52 years old (range 35–72). Cirrhosis was due to alcoholism (41%), virus (33%), mixed in one patient and other causes in two patients. Two (16%) patients belonged to Child Pugh’s class A, 6 (50%) to class B and 4 (33%) to class C. Six patients (50%) had previous hemorrhagic episodes due to esophageal varices. PVT involved the portal trunk and/or branches in 91% of the cases, and in 1 patient coexisted with splenic and mesenteric vein thrombosis. Complete thrombosis was seen in 4 patients and partial in 8. From the 12 patients, 8 (66%) responded to antithrombotic therapy after 6 months, with a complete recanalization in 7 patients (87%). Interestingly, the moderate hipocoagulability level subgroup showed an 88% of response (Table2). After an overall follow-up of 6 months, none of the 12 patients presented hemorrhagic complications and none showed platelets counts below baseline values. Acenocoumarol and LMWH were well tolerated in all cases, with only one patient with intolerance to the subcutaneous injections. CONCLUSIONS: Antithrombotic therapy in chronic PVT in cirrhotic patients showed a high response rate (66%) in our study, with a complete recanalization in 87% of the cases. Adjusted dose scheme according to level of hypocoagulability seems to be effective and safe, since 80% of the subgroup of moderate hypocoagulability responded with no haemorrhagic complications. Table 1. Ranges of baseline coagulation study and adjusted therapeutic doses of LMWH. N, Normal range, LH, Low hypocoagulability; MH, Moderate hypocoagulability. Table 2. Responses rates according to baseline hypocoagulability.

Background: Allogeneic umbilical cord blood (UCB) stem cell transplantation (SCT) shows some advantages (HLA matching requirements or availability) respect to SCT using other sources of matched unrelated donor (MUD) stem cells. However, it is correlated with slower engraftment, increasing risk of infections and early mortality. It has been recently shown that co-infusion of third party donor (TPD) CD34+ cells (dual SCT) is useful to speed up engraftment. Objective: To evaluate the usefulness of lineage-specific chimerism quantification in the management of this transplant setting. Patients and methods: 8 dual SCT (Tables 1, 2) in 7 patients (1 CML-BC, 2 AML-M2, 1 AML-M4, 1 ALL-Ph+, 1 biphen. ALL, 1 NHL). Chimerism was analyzed by STR-PCR (AmpFlSTR SGM Plus, Applied Biosystems; sensitivity 1%) and quantitative real-time PCR (qrt-PCR) of null alleles and insertion/deletion polymorphisms (Light Cycler, Roche; sensitivity 0,01%). Peripheral blood (PB) and leukocyte lineages (T cells, CD3+, and myeloid cells, CD15+), isolated by positive selection using automated immunomagnetic technology (AutoMACS, Miltenyi Biotec), were analyzed weekly. Bone marrow (BM) was analyzed at days +30, +100, +180 and +365). Results: 7/8 cases showed initially a high proportion of TPD cells in PB which were progressively replaced by UCB cells. UCB complete chimerism (UCB-CC, absence of recipient or TPD cells even in qrt-PCR assays) was acquired in a median of 22.5 days (range 18–39). In one patient, fully HLA-mismatched with the TPD, no TPD cells were observed after dual SCT. 4/8 cases showed recipient cells in PB after dual SCT during a median period of 12 days (range 4–18 days). In 3/8 cases, recipient cells were found after CC had been acquired, which allowed early diagnosis of 1 graft rejection and 2 relapses. T cells (CD3+) are mainly of UCB origin early after dual SCT and reach UCB-CC a median of 7 days (range 0–21) before PB. However, myeloid cells (CD15+) derive primarily from the TPD and reach CC together with PB. TPD cellularity favoured early engraftment (before UCB-CC took place) in 4 cases. In this context, only one important infectious complication (hepatosplenic tuberculosis) was observed, which resolved with the appropriate treatment. Conclusions: Lineage-specific chimerism quantification allowed a close monitoring of the dynamics of engraftment of cells of both donors which is of key importance in this SCT setting. Moreover, lineage-specific chimerism analysis was useful to diagnose one graft rejection and two relapses (the patient with NHL showed a ganglionar relapse in CC). Table 1. Transplantation characteristics. Table 2. Transplantation results. Median (range).

Following the widespread use of Highly Active Antiretroviral Therapy (HAART), ASCT has been reported as a feasible and effective treatment in the setting of HIV-Ly patients (pts). Nevertheless just series including up to 20 patients have been published so far. We present a preliminary analysis of the outcomes regarding the experience within the EBMT-LWP. Methodology: A retrospective analysis including HIV-Ly patients undergoing peripheral blood ASCT from 1999 to 2005 and reported to the EBMT was performed. Main endpoints: OS, EFS and TRM. Results: 44 pts from 14 institutions/7 countries were included: 39 males, median age 43 (29–62) years old. One pt underwent more than 1 ASCT (follow up censored at time of 2nd ASCT). In addition to HIV infection, 11 pts were co-infected by HBV, 8/43 by HCV and 16/36 pt met AIDS criteria (other than lymphoma) at the time of lymphoma diagnosis. Histology: 34 NHL (20 DLBCL; 7 Burkitt/Burkitt-like; 4 plasmablastic; 3 other), 59% of them with IPI>2 at diagnosis; 10 HL (50% with Ann-Arbor stage>IIB at diagnosis). Patients received a median of 2 (1–5) treatment lines pre-ASCT. Status at ASCT: 22 CR (13 in CR1); 18 in PR/chemo-sensitive relapse and 4 in primary induction failure/chemo-resistant relapse. Conditioning: 41 pts received BEAM/variants and 3 TBI-based regimens. Post-ASCT anti-tumour pre-emptive treatment was used in 10 pts (8 radiotherapy; 2 Rituximab). The median count of T-CD4+ cells/μl was 162 (8–702) at the time of ASCT; 26/38 had undetectable HIV viral loads. HAART was given in 39/42 pts during conditioning but was withdrawn in 9 of them. The median number of CD34+ cells infused was 5 (1,6–21,2) x106/kg. G-CSF was used until engraftment in 39/43 pt during a median of 8 (2–29) days. All pts but one who died on day +15 reached neutrophils>500/μl at a median time of 11 (8–36) days. Platelet count >20.000/μl was reached in 39/44 pt at a median time of 14 (6–455) days. The pt engrafting platelets on day +455 was transfusion independent since day +49. Three new post-ASCT malignancies were reported: in situ epitelioma and myelodysplasia (+4 years) in 1 pt, and kidney adenocarcinoma (+3 years) in another one. Relapse occurred in 13 (29,5%) pts which status at SCT was CR in 4, PR/chemosensitive relapse in 5 and chemo-resistant disease in 4. The median time to relapse/progression was 5,2 (0,6–32,1) mo. Seventeen pts died (38,6%): disease relapse/progression (n=11), ASCT-related complication (n=4) -bacterial infection (n=3), secondary myelodysplasia (n=1)-, 1 pt died from an HIV-related complication and 1 pt died on day +15 of multi-organ failure within the context of a bacterial sepsis and pre-transplant renal dysfunction. With a median follow up time of 36,3 (3,2–72,3) mo, the OS and DFS probability at this point were 60,2% (67,5% when chemo-resistant pts were excluded from analysis) and 55% respectively. For those pts in CR1 at ASCT, OS at 36,3 mo was 85% vs 60% for those in CR>1/PR/chemosensitive relapse. Conclusion: The results from the EBMT-LWP experience, the largest reported so far, show that ASCT remains a useful treatment in terms of TRM, long-term OS, and DFS for high risk HIV-Ly patients. These outcomes are comparable to those observed in HIV negative lymphoma patients.

INTRODUCTION: Oligocloanality is a phenomenon shown in up to 40% of patients with childhood B-lineage acute lymphoblastic leukemia (ALL), and it might be associated with a poorer prognosis. OBJECTIVE: To present a case of oligoclonality based on the detection of IGH gene rearrangements in a patient with ALL and the correlation of the results with those of flow cytometry immunophenotyping and chimerism quantification. PATIENT AND METHODS: 26 years old female, pre-B ALL diagnosed in her childhood, in relapse after autologous and allogeneic HLA-identical unrelated stem cell transplantation (SCT). Immunophenotyping was done by multiparametric flow-cytometry, chimerism quantification was performed using fluorescent in situ hybridization (FISH) for the sex chromosomes, and IGH gene rearrangements were detected by nested-PCR revealed by capillary electrophoresis. Disease associated leukocyte lineages (CD19+, CD19+CD34+ and CD19+CD34−) were purified using immunomagnetic technology. RESULTUS: Flow-cytometry analysis in PB and BM samples at diagnosis showed expression of CD45 (weak), CD34, CD38, CD10, CD19, CD20, CD22, CD66, CD79acyt, TdT and aberrant expression of CD33, with a detectable clonal IGH rearrangement. At relapse, two distinct CD19+ cell populations were revealed from BM samples: one immature (CD34+, 15% of the total leukocytes) with similar phenotype at diagnosis, and another one mature (CD34−, 10% of the total leukocytes). FISH analysis showed mixed chimerism (MC)(31% recipient) in BM samples and complete chimerism (CC) in PB samples. Molecular analysis showed three different clones in BM and a polyclonal pattern in PB. In order to increase sensitivity, CD19+ cells were isolated from BM and PB. Cells selected from BM showed MC (98,5% recipient) and the three clonal IGH gene rearrangements while those from PB showed CC and a polyclonal pattern. Further studies exhibited persistence of the CD19+CD34+ population and significant reduction of the mature CD19+CD34− cells. A persistent three clone pattern was present in BM samples. In order to clarify the origin of the different IGH gene rearrangements, two consecutive immunomagnetic selections, one with CD19+ and other with CD34+ antibodies, were performed from BM samples to purify both CD19+CD34+ and CD19+CD34− populations. CD19+CD34+ cells showed all the three IGH clones and MC (88% recipient). Moreover, CD19+CD34− cells showed a monoclonal pattern with an unique peak, not detected in previous studies, and CC (Table 1). CONCLUSIONS: The clonal diversity of IGH gene rearrangements detected at relapse after SCT was generated from the original malignant CD34+ clone, possibly due to clonal evolution, and seems to be associated with the poor outcome of this case. After multiple therapy modalities, the CD19+CD34+ cells showed resistance to treatment. Surprisingly, the CD19+CD34− cells showed a new clonal rearrangement although its donor origin confirmed its reactive nature in the context of SCT. This observation has relevance in the further follow up and minimal residual disease detection in this case. Table 1. Correlation between clonality and chimerism analysis in the different samples studies. %R, % of recipient cells. MC, mixed chimerism. CC, complete chimerism.

Background: Polymorphisms in regulatory sequences of cytokine genes are known to influence their expression and, therefore, the intensity of the immune response. Some of such polymorphisms have been associated with the outcome of allogeneic stem cell transplantation (SCT). Objective: To evaluate the association between donor (D) and recipient (R) genotype for the IL-10 gene -1082 SNP (single nucleotide polymorphism) and -1064 STR (short tandem repeat) polymorphism with the dynamics of chimerism and the development of complications after SCT. Patients and methods: 24 HLA-matched conventional SCT. IL-10 genotypes were determined in an -1082 SNP allele-specific PCR (A vs G) including the -1064 STR in the product which is revealed by capillary electrophoresis. Results were analyzed using Fisher’s exact test due to the reduced sample size. Results: The frequency of alloreactive genotypes (AA or A) for the SNP was 37,5% AA and 79,2% A in the D and 33,3 AA and 95,8% A in the R. No association was observed between the homozygous or heterozygous presence in the D or R of the alloreactive allele for the SNP genotype and the dynamics of chimerism or complications post-SCT. Alleles 4 to 10 for the STR genotype were found in contrast to previous reports (alleles 7–16). The frequency of alloreactive alleles (homozygous, +/+, or heterozygous, +), with greater number of dinucleotide repeats (alleles 8–10 in the present series) for the STR was 8,3% +/+ and 62,5% + in the D and 8,3% +/+ and 50% + in the R. The presence of alloreactive alleles (heterozygous) in the donor was significantly associated with the development of extense chronic GVHD (EcGVHD, Table 1). In patients transplanted from such donors, the incidence of mixed chimerism (MC) in peripheral blood during the first month post-SCT was lower although without statistical significance (Table 1). The higher alloreactivity of these donors favors the establishment of complete chimerism and therefore, increases the incidence of EcGVHD. Conclusions: The present study shows an association between the presence in the donor of alloreactive alleles for the IL-10 -1064 STR polymorphism and the development of EcGVHD, in a chimerism mediated fashion, after HLA- identical SCT. The analysis of a larger number of patients as well as further cytokines will eventually allow to confirm these observations and to establish this type of studies as a means for an improved management of transplanted patients. Table 1. Influence of alloreactive alleles (8–10) for the IL-10-1064 STR polymorphism on chimerism and complications post-STC.

7612 Background: Auto-SCT is the preferred treatment modality for MM. From the results of IFM 9502 (Moreau et al. Blood, 2002) melphalan 200 mg/m2 was considered the new standard as conditioning regimen due to its lower toxicity. However, many chemotherapy regimens have been tested. We present our results with two cohorts of consecutive patients (pts) diagnosed of MM and treated with Auto-SCT between 1996 and 2005 in our BMT Unit. Methods: Following induction chemotherapy (median of 6 courses) 20 pts were conditioning with busulfan 16 mg/kg + melphalan 140 mg/m2 (BuMel) and 23 (since September-2002) with melphalan 200 mg/m2 (Mel-200). Clinical features: median age (BuMel vs Mel-200) 55 vs 58 (p=0.5); female sex 10 vs 10; Bence-Jones MM 5 vs 5; renal failure (B) 2/20 (10%) vs 7/23 (30%)(p=0.48); previous treatment with local irradiation 6/20 vs 3/23; status previous Auto-SCT (complete remission, CR) 7/20 (35%) vs 6/23 (26%), p=0.5. A median of 4.8 vs 4.4 x 10e6/kg CD34+ cells were infused, respectively (p=0.15). Results: Four toxic deaths: 2 pts by hemorrhagic cystitis-enterocolitis (at day +84 and +96), 1 pt with disseminated candidiasis (day +7), and 1 pt by pulmonary fibrosis (day +452). At 3 months following Auto-SCT, 13/19 (68%) treated with BuMel and 12/23 (52%) of those treated with Mel-200 achieved CR (p=0.28). At 24 months, probability of remaining disease free was 87% for BuMel group and 55% for Mel-200 (p=0.08). Conclusions: 1) Conditioning regimen BuMel was associated with a higher toxicity than Mel-200, as well as a higher mortality treatment related. 2) However, a trend for higher CR rates and probability of disease free progression were shown in BuMel group without statistical significance [Table: see text] No significant financial relationships to disclose.

Background: The dynamics of chimerism evolution determines the success of allogeneic stem cell transplantation (SCT). Several factors such as the intensity of the conditioning regimen, the T-cell content of the graft or the GVHD prophylaxis, influence the degree of chimerism after SCT. Objective: To evaluate the dynamics of chimerism after different SCT settings (ablative, reduced intensity conditioning -RIC- and T-cell depleted -TCD-) and its influence in the success of the procedure. Patients and Methods: The study includes 68 SCT: 32 ablative (including 7 from HLA matched unrelated donors -UD-), 19 RIC and 17 TCD (including 8 from haploidentical donors and 2 from UD with 1 HLA disparity). Chimerism analysis was performed by FISH for the sex chromosomes or STR-PCR (sensitivity 1 Samples obtained on days +30, +100, +180, +365 and once a year thereafter, included bone marrow (BM) and peripheral blood (PB). Moreover, chimerism was analyzed in PB and leukocyte lineages (T lymphocytes CD3+, B lymphocytes CD19+ and myeloid cells CD15+ isolated (purity >95%) by immunomagnetic means, AutoMACS, Miltenyi Biotec), every 2 weeks, starting on day +15 (except in ablative), and until complete chimerism (CC) was achieved. Results of chimerism follow-up were censored once the diagnosis of relapse or rejection was established. Results: The incidence (% patients) of mixed chimerism (MC) on day +30 (ablative: BM 27%, PB 15%; RIC: BM 40%, PB 41%, CD3 40%; TCD: BM 31%, PB 33%, CD3 54%), as well as its dynamics (MC on day +100: ablative BM 8%, PB 4%; RIC BM 8%, PB 15%, CD3 22%; TCD BM 20%, PB 40%, CD3 37%) were different in the three SCT settings. All except 2 CD15 samples analyzed showed CC. Moreover, the percentage of recipient cells (%R) was significantly higher after RIC and TCD than after ablative SCT, as well as in T lymphocytes than in BM or PB (7/8 cases with simultaneous studies showed MC in T lymphocytes and CC in PB). All RIC SCT evolved to CC by day +180 while TCD SCT showed persistent MC (2 patients with stable MC after one year). The incidence of rejection was greater after RIC (2/19) and TCD (4/17) than after ablative SCT (2/32). All these patients showed MC, mainly in T lymphocytes, which allowed early diagnosis and successful treatment with immunosuppression withdrawal and donor leukocyte infusion. Patients with CC in PB/T lymphocytes on day +30 had a higher incidence of GVHD>I than those with MC. In the present series, however, a relationship between chimerism and relapse, disease free survival or overall survival, was not observed. Conclusions: SCT with greater incidence of MC (RIC and TCD) favor immune tolerance between donor and recipient which reduces the risk/severity of GVHD at the expense of a higher incidence of graft rejection.

Background: Graft rejection constitutes a serious complication, infrequent after conventional (ablative) allogeneic SCT but with higher incidence after reduced intensity conditioning (RIC) or T-cell depleted (TCD) SCT. Objective: To evaluate the usefulness of chimerism monitoring for the early diagnosis of graft rejection in different SCT settings, as well as for the follow up of the response to treatment with immunosuppression withdrawal (ISW) and/or donor leukocyte infusions (DLI). Patients and Methods: The study includes 68 SCT (32 ablative, 19 RIC, 17 TCD -including 8 haploidentical-). Chimerism analysis was performed by FISH for the sex chromosomes or STR-PCR (sensitivity 1%). Samples obtained on days +30, +100, +180, +365 and once a year thereafter, included bone marrow (BM) and peripheral blood (PB). Moreover, chimerism was analyzed in PB and leukocyte lineages (T lymphocytes CD3+, B lymphocytes CD19+ and myeloid cells CD15+ isolated (purity >95%) by immunomagnetic means, AutoMACS, Miltenyi Biotec), every 2 weeks, starting on day +15 (except in ablative), and until complete chimerism (CC) was achieved. Results: After initial engraftment in all patients, graft rejection was diagnosed in 8 (2 (6%) ablative, 2 (10%) RIC, 4 (23%) TCD), either established (severe pancytopenia and BM aplasia) or incipient (progressive decrease in PB and BM cell counts) a median of 52.5 days (range 23–110) after SCT. All patients showed mixed chimerism (MC) in BM and PB with higher percentages of recipient cells (%R) in PB. In 7/7 patients studied, T cells showed persistent MC with high %R (>50% in 6/7; 25% in 1/7). B cells showed MC in 5/6 patients studied, with lower %R (50% in 1/5). Only 2 patients showed MC, transient in one of them, in myeloid cells. 2 patients were not treated due to concurrent multiorgan failure and subsequently died. Reduction of IS in 5 patients obtained 1 response (normal PB and BM counts, and CC). The other 4 patients underwent ISW but no further response was obtained. One of them received a second SCT while the other 3 were treated with DLI, and all of them responded. The last patient (transplanted from a haploidentical family donor) who was not receiving IS, responded to treatment with DLI. Time from therapeutic intervention to response was variable with a median of 2 months (range 1–7). 4 patients developed GVHD>I, which was the cause of death in one and was controlled in the other three. One patient died from sepsis in complete remission (CR) 9 months after the transplant. 4 patients are alive in CR a median of 50 months after SCT (range 16–72). Conclusions: The observation of MC, mainly in T lymphocytes, together with a decrease in PB and BM cell counts, allowed early diagnosis and successful treatment (6/6 patients) of graft rejection.

Background: Polymorphisms in regulatory sequences of cytokine genes are known to influence their expression and, therefore, the intensity of the immune response. Some of such polymorphisms have been associated with the outcome of stem cell transplantation (SCT), which supports the hypothesis of a genetic predisposition towards certain complications post-transplants. Objective: To evaluate the association between donor (D) and recipient (R) genotype in the IL-6 gene -174G/C SNP polymorphism with the development of complete or mixed chimerism (MC) and, consequently, with complications such as graft rejection, graft versus host disease (GVHD) and survival after SCT. Patients and methods: The study included 16 SCT (9 ablative, 3 T cell depleted, 4 reduced intensity conditioning). The IL-6 -174G/C SNP genotypes were determined by PCR on genomic DNA from Rs and Ds, using allele-specific oligonucleotides (ASO-PCR) as primers (forward: 5′-ccctagttgtgtcttgcc-3′ or 5-′ccctagttgtgtcttgcg-3′; reverse: 5′-gagcttctctttcgttcc-3′). Logistic regression was applied to identify associations between the polymorphism, the development of MC and of the mentioned complications. The reduced number of SCT analyzed, precluded the use of Pearson’s and/or Fisher’s p values or further statistics. Results: The frequency of the different genotypes was 44.4% CC, 33.3% GC and 22.2% GG for Rs, and 33.3% CC, 44.4% GC and 22.2% GG for Ds. 88.9% D/R pairs were matched for the polymorphism and 11,1% mismatched. None of the patients transplanted from matched donors showed MC at any time post-SCT while all mismatched patients developed MC (Table 1). MC in the D/R mismatched group was associated with a greater rate of graft rejection and with a reduced incidence of acute GVHD (aGVHD) when compared with the D/R matched group (Table 1). No association was observed in this sample between the polymorphism analyzed and the development of chronic GVHD (cGVHD). Although graft rejection was early diagnosed and successfully treated with immunosuppression withdrawal and donor leukocyte infusion, the D/R matched group showed higher mortality rate that the mismatched group (Table 1). Conclusions: The present study suggests an association between D/R matching for the IL-6 gene -174G/C SNP polymorphism and the development of MC and, consequently, of different complications after SCT. The analysis of a larger number of patients will eventually allow to confirm these observations as well as to establish this type of studies as a means for an improved management of transplanted patients. Table 1 IL6 -174G/C MC rejection aGVHD cGVHD Exitus D/R matched 0% 0% 75% 87.5% 50% D/R mismatched 100% 100% 0% 100% 100%

HAART has improved the outcome of AIDS patients (pts). Thus high-dose chemotherapy followed by ASCT has been used for the treatment of HIV+Ly pts, similarly to HIV-neg-Ly pts. In a recent report we found delayed engraftment after ASCT in HIV+Ly compared with HIV-neg Ly pts (Exp.Hem33;487–494,2005). To test the role played by HAART on engrafment we conducted this study, comparing engraftment results on HIV+Ly pts that received HAART with those in wich HAART was withdrawn after ASCT. Patients and Methods: From June 2000 a total of 19 HIV+Ly pts (17 male) received an ASCT in a multicenter cooperative study; 5 were Hodgkin disease (HD) and 14 non-Hodgkin high-grade malignant lymphoma (NHL). Median age was 43 years (range, 31–61); adjusted IPI (aIPI) was: 0–1 in ten pts and 2–3 in nine more pts; advanced Ann Arbor stage (III–IV) was present in 14 pts.Ten pts were treated with HAART during ASCT (HAART+) and in nine pts HAART was withdrawn due to gastrointestinal toxicity (HAART neg). Both groups were similar for the most relevant clinical characteristics (p value Mann-Whitney test or chi-square test > 0.05): HD/NHL, Ann Arbor stage, aIPI, status at ASCT (CR-1/more than CR-1), age, number of lines of chemotherapy before ASCT, number of cycles of mobilization to obtain an adequate amount of CD34+ cells, number of CD34+ cells infused, day of start of GCSF and duration of GCSF-treatment. Results: Median time to reach PMN>0.5 x 109/L was 12 days (9–17) for HAART neg group and 18 days (9–33) for HAART+ pts (p=0.069). Platelet engraftment (>20x109/L) was achieved after a median of 20 days (11–28) and 26 days (11–455) respectively (p=0.25). Nevertheless, a multivariate regression analysis using as covariates HAART, GCSF (starting day) and amount of CD34+ cells infused, was performed. Accordingly no statisticaly significant results were found to relate with myeloid engraftment. Conclusions: Despite the HAART+ group shows longer interval to reach PMN and platelets engraftment following ASCT, we can not definitively conclude that HAART plays a negative influence on engraftment in this clinical setting. In order to clarify these results further recruitment of patients is needed.

Introduction: The current gold standard for chimerism quantification after allo-stem cell transplantation (SCT) is based on the polymerase chain reaction of DNA short tandem repeats (STR-PCR). This methodology is sex-independent, highly informative and quantitatively accurate. However, this method has a moderate sensitivity (1–3%). Objective: To test the usefulness of increasing the assay sensitivity by 3 different approaches in order to detect recipient residual cells. Material and Methods: Two sets of 20 samples, belonging to artificial mixtures of male and female peripheral blood (PB) cells in different proportions (% male: 100, 75, 50, 25, 10, 5, 3, 1, 0.1, 0) were prepared to test the assay sensitivity and reproducibility. Additionally 58 samples from 10 selected patients showing different clinical evolution post-SCT were retrospectively studied. Chimerism was analyzed on genomic DNA (purified QIAamp DNA blood kit, Qiagen) by multiplex STR-PCR (AmpFlSTR SGM Plus revealed on capillary electrophoresis in an ABI Prism 3100 automated DNA sequencer; Applied Biosystems). Three different approaches aimed to increase the sensitivity of STR-PCR were tested: (1) Increasing DNA concentration in the PCR (1x, 2.5x, 5x), (2) Increasing the concentration of the PCR product for the capillary electrophoresis (1x, 2.5x, 5x) and (3) Increasing the sample injection time into the capillary for the electrophoresis (10s, standard time, 30s and 50s). Results: The standard STR-PCR showed a sensitivity of 1% in both sets of male-female PB artificial mixtures. Both approaches increasing DNA concentration in the PCR and the PCR product in the capillary electrophoresis did not achieved better sensitivity. Moreover, appropriate analysis of the samples was frequently hampered due to fluorescence saturation. However, longer injection times (30s, 50s) showed that currently underrepresented DNA fragments were conspicuously overestimated, giving a one-log increase in the sensitivity of the STR-PCR assay, which reached a 0.1%. This approach was used to re-analyze the 23 SCT patient samples previously identified as in complete chimerism by standard STR-PCR. The 0.1% sensitivity methodological approach allowed to detect residual/reappearing recipient cells in three of the 23 analyzed samples. Therefore this new approach would have allowed to anticipate the detection of graft rejection and disease relapse in 2/10 patients, 7 and 77 days respectively. Conclusion: Long injection times (30s and 50s) in the capillary electrophoresis after STR-PCR result in a one log sensitivity increase (from 1% to 0.1%). This would allow earlier diagnosis of adverse events after SCT favoring a prompt introduction of therapeutic interventions.