ALBERT

Description: Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired clonal abnormality of hematopoietic stem cell leading to lack of phosphatidylinositol glycoproteins, sensitizing cells to complement-mediated lysis. Despite the efficient symptomatic treatment of hemolytic PNH with eculizumab, allo-HCT is the only curative treatment for the disease, although outcomes presented in the past were controversial. Material and methods: We report 41 allo-HCTs: 37 from MUD and 4 from MRD performed for PNH in 2004-2016. Median age of recipients was 29(20-62) years and donors 30(19-53), median time from diagnosis to allo-HCT was 16(2-307) months. Median size of PNH clone was 80% granulocytes (0.5%-100%). Indication for allo-HCT was PNH with aplastic/hypoplastic bone marrow (19 pts), MDS (2 pts), overlapping MDS/aplasia (3 pts), severe course of PNH with hemolytic crises and transfusion-dependency without access to eculizumab (17 pts). Additional risk factors were Budd-Chiari syndrome and hepatosplenomegaly (1 pt), history of renal insufficiency requiring hemodialyses (2 pts), chronic hepatitis B (1 pt) and C (1 pt). The preparative regimen consisted of treosulfan 3x14 g/m2 plus fludarabine 5x30 mg/m2 (31 pts) or treosulfan 2x10 g/m2 plus cyclophosphamide 4x40 mg/kg (10 pts). Standard GVHD prophylaxis consisted of cyclosporine-A, methotrexate and pre-transplant ATG in MUD-HCT. 2 pts instead of cyclosporine-A received mycophenolate mofetil and tacrolimus. Source of cells was bone marrow (13 pts) or peripheral blood (28 pts) with median 6.3x108NC/kg, 5.7x106CD34+cells/kg, 24.7x107CD3+cells/kg. Myeloablation was complete in all pts with median 9(1-20) days of absolute agranulocytosis

Description: Abstract 4575 Introduction: Anti-HLA antibodies constitute potentially important factor that may influence outcomes of HLA-mismatched allogeneic hematopoietic stem cell transplantation (allo-HSCT). The rationale of this study was to detect presence of anti-HLA antibodies in recipients of allo-HSCT from HLA-mismatched unrelated donors. Patients and Methods: Anti-HLA-A,B,C,DR,DQ,DP antibodies were identified in sera collected from 46 recipients of allo-HSCT from HLA-mismatched unrelated donors. Sera were collected between 1 month and 5.5 years after allo-HSCT, and additionally before allo-HSCT in 17 pts. We have used microchips spotted with purified HLA class I and HLA class II antigens to allow binding of anti-HLA antibodies present in tested sera to the surface of the microchip, pre-optimised reagents and DynaChip Processor (Dynal Invitrogen Corporation) for assay processing, data acquisition and analysis. Results: Antibodies against HLA class I, II or I and II were detected in 15%, 11% and 35% of pts whereas no antibodies were detected in 39% of patients. Antibodies were directed against HLA-A, B, C, DR and DQ in 37%, 46%, 35%, 48% and 35% of pts, respectively. Pre-transplant anti-HLA antibodies have been detected in 7 pts (41%) out of 17 tested before allo-HSCT. In this group percent of Panel Reactive Antibodies (% PRA) increased following allo-HSCT in 3 pts and decreased in 4. In 5 out of 10 remaining pts without pre-transplant antibodies, %PRA increased post-transplant. DynaChip software allowed to define specificities of HLA-A,B,C,DR and DQ antibodies on low and high resolution levels. The specificity of antigens that masked results of antibody identification has been also defined in 2 pts. At this stage we did not define exactly whether detected anti-HLA antibodies were donor-specific. Cross-reactive groups (CREG's) analysis has been also used to compare antibodies’ reactivity. Anti-HLA-DP antibodies were not detected in the examined group of transplanted patients. Conclusions: Presented preliminary study results indicate, that anti-HLA antibodies can appear post-transplant in mismatched allo-HSCT recipients. Further analysis aiming to evaluate their influence on transplant outcomes is ongoing. We intend to extend the search for anti-HLA antibodies with use of Luminex LabScreen method. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction Although anti-HLA Antibodies (Abs) are considered an important factor of graft failure in solid organ transplants, their role in allogeneic hematopoietic stem cell transplantation (allo-HSCT) is still undiscovered. Large polymorphism and immunogenicity of HLA-antigens and heterogeneity of anti-HLA Abs warrant the need of such investigation. The purpose of this study was to define the presence of anti-HLA Abs before allo-HSCT from HLA-mismatched unrelated donors and their impact on engraftment and post-transplant full donor’s chimerism. Material and Methods 70 HLA-mismatched donor/recipient pairs entered the study. Indication for allo-HSCT was: ALL, AML, CML, SAA, PNH, MDS and CLL. Preparative regimen was myeloablative in 68pts (97%) and reduced in 2pts (2.3%). Standard GVHD prophylaxis consisted of cyclosporine, methotrexate and pre-transplant anti-thymocyte globulin (69pts) or Alemtuzumab (1pt). HLA A,B,C,DR,DQ alleles were PCR-typed. Single HLA-antigen was mismatched in 46pts, single HLA-allele in 16pts, double antigens or alleles in 2 pts and another 2 pts had combined antigenic/allelic HLA mismatch. Anti-HLA A,B,C,DR,DQ,DP Abs were identified in sera collected prior to the conditioning treatment with use of automated DynaChip assay utilizing microchips bearing purified class I and class II HLA antigens. Post-transplant chimerism was analyzed using STR-PCR method at 30, 100-days and 1-year after allo-HSCT. Results Anti-HLA Abs pre-formed before allo-HSCT were detected in 32pts: against class I, II or both in 13(18.6%), 7(10%) and 12(17.1%) pts. Anti-HLA Abs were detected after allo-HSCT in 49pts: against class I, II or both in 22(32.4%), 7(10.3%) and 20(29.4%) pts, respectively. Anti-HLA Abs directed against the mismatched HLA antigens were observed in 4 pts before allo-HSCT. Although no Abs specific to mismatched HLA alleles were detected, Abs belonging to the same Cross-Reactive Groups (CREGs) were present in 5pts. No graft failure has been observed (graft failure was defined as absence of neutrophil recovery by day 30 after allo-HSCT or loss of donor’s chimerism). The detection of anti-HLA Abs before allo-HSCT was associated with decrease of post-transplant donor’s chimerism (18/31 vs 11/35, p=0.03). Anti-HLA Abs had no significant impact on engraftment of platelets and neutrophils. The median time to neutrophils engraftment was 16.9 days (range 7-31 days) in pts with and 18.9 days (range 13-30 days) in pts without anti-HLA Abs (p=0.188). The median time to platelets engraftment was 16.9 days (range 9-31 days) in patients with and 18.3 days (range 10-32 days) in pts without anti-HLA Abs (p=0.274). Conclusions Our preliminary results indicate, that anti-HLA Abs are present before transplantation in mismatched allo-HSCT recipients. They influence the post-transplant full donor’s chimerism, but they did not influence engraftment and graft failure. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: Although anti-HLA Antibodies (Abs) are considered an important factor of graft failure in solid organ transplants, their role in allogeneic hematopoietic stem cell transplantation (allo-HSCT) is still undiscovered. Large polymorphism and immunogenicity of HLA-antigens and heterogeneity of anti-HLA Abs warrant the need of such investigation. The purpose of this study was to define the presence of anti-HLA Abs after allo-HSCT from HLA-mismatched unrelated donors and their impact on outcomes of allo-HSCT. Material and methods: 68 HLA-mismatched donor/recipient pairs entered the study. Indication for allo-HSCT was: ALL, AML, CML, SAA, PNH, MDS and CLL. Preparative regimen was myeloablative in 66(97%)pts and reduced in 2(3%)pts. Standard GVHD prophylaxis consisted of cyclosporine, methotrexate and pre-transplant anti-thymocyte globulin (67pts) or Alemtuzumab (1pt). HLA A,B,C,DR,DQ alleles were PCR-typed. Single HLA-antigen was mismatched in 44pts, single HLA-allele in 16pts, double antigens or alleles in 2 pts and another 2 pts had combined antigenic/allelic HLA mismatches. Anti-HLA A,B,C,DR,DQ,DP Abs were identified in sera collected at +30, +100 days and 1 year post-transplant with use of automated DynaChip assay utilizing microchips bearing purified class I and class II HLA antigens. Post-transplant chimerism was analyzed using STR-PCR method at 30, 100-days and 1-year after allo-HSCT. Results: Anti-HLA Abs were detected post-transplant in 49(72.1%) patients at least at one of three examined time-points. They were directed against HLA class I, II or both in: 22(32.4%), 7(10.3%) or 20(29.4%) patients, respectively. In 3 (4.4%) patients antibodies for many specificities were detected. Anti-HLA antibodies detected during the first year after transplantation did not impact the donor's chimerism. Full donor's chimerism was observed in 22/48 (46%) patients without versus 7/18 (39%) patients with anti-HLA Abs, p=0.615). Anti-HLA Abs present after transplantation also did not impact the risk of developing aGVHD, grades neither I-IV (36/49, 73% in positive versus 17/19, 89% in negative group, p=0.270), nor II-IV (15/49, 31% in positive versus 8/19, 42% in negative group, p=0.372). Chronic GVHD and extensive cGVHD also were not influenced by anti-HLA Abs detected post-transplant (23/49, 47% versus 10/19, 53%, p=0.676) and (13/49, 27% versus 5/19, 26%, p=0.986), respectively. Post-transplant anti-HLA Abs did not influence the recurrence of the disease, which was observed in 9/49 (18.3%) patients with versus 1/19 (5.2%) patients without anti-HLA antibodies, p=0.323, nor the overall survival at 3-years (54% in anti-HLA Abs positive versus 46% in anti-HLA Abs negative patients, p=0.207). Conclusions: Our results indicate, that anti-HLA Abs can be detected post-transplant in HLA-mismatched allo-HSCT recipients. Presence of anti-HLA antibodies detected after allo-HSCT was not associated with occurrence of aGVHD, cGVHD, relapse nor overall survival. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Myelofibrosis (MF), chronic myeloid malignancy associated with shortened survival, in majority of patients develops de novo as Primary MF, but also polycythemia vera (PV) or essential thrombocythemia (ET) may progress into post-PV or post-ET MF. Although management of MF includes several treatment options, the only potentially curative treatment approach in MF is allogeneic hematopoietic stem cell transplantation (allo-HSCT). Aim of this study was to evaluate the results of allo-HSCT in patients with MF treated in Katowice, Poland. Material and Methods: 27 pts (14 male and 13 female) with median age 51 years (range 21–63) were treated with allo-HCT due to PMF (20), post-PV (4) or post-ET (3) MF. 11,7,11,26 and 41% of pts had DIPSS 0,1,2,3 and 4, respectively. Median bone marrow cellularity was 70% (10-100%), fibrosis was collagen-type (14 pts including 2 with osteosclerosis), reticulin (10) or it was not specified (3). Splenomegaly was present in all pts: 13-20 cm (14 pts), 〉 20 cm (13 pts). JAK2V617F point mutation was present in 18 pts. Karyotype was available in 14 pts: in 9 normal, in 5 with variable abnormalities. Median time from diagnosis to allo-HCT was 1.5 (0.4–9.5) years. 16 pts (59.3%) received cells from HLA-matched related donor (MRD), 11 pts (40.7%) from unrelated donor: 10/10 (9) or 9/10 (2) HLA-A,B,C,DR,DQ alleles matched. Reduced intensity conditioning (RIC) was used in 26 pts, 1 patient received myeloablative conditioning (MC). Sources of stem cells were: peripheral blood (21), bone marrow (4) and both (2). All pts but one had chronic phase of MF at time of transplantation. Results: 14/27 (52%) pts are alive at median 3.4 (0.4-5.4) years after allo-HSCT: 11/16(69%) from MRD and 3/11(27%) from MUD, p=0.032. Graft failure, graft loss or PRCA were observed in 3, 5 and 1 pt, respectively. Absolute neutrophil count 〉0.5×109/L and platelet count 〉50×109/L were achieved at median 16 and 28 days, respectively. 12/27 (44%) pts reached complete blood count of Hb〉10 g/dl, Plt〉100 G/l and WBC〉3.5 G/l; 11 of them (92%) are alive. 6/27 (22%) pts remained either RBC or PLT transfusions dependent post-transplant; 3 of them (50%) died. 9/27 (33%) pts remained both RBC and PLT transfusion dependent and all of them died. JAK2V617F mutation was completely eradicated in 11/16 evaluated previously positive patients (69%), decreased in 4 (25%) and stable in 1(6%) pt. Acute graft-versus-host disease (aGVHD) III-IV developed in 5/27 (19%) and extensive chronic GVHD in 5/19 (26%) pts. Relapse occurred in 4 pts and was treated with subsequent second transplant (in 1 pt thereafter by 3-rd allo-HSCT). Spleen length decreased at median by 5 (0.3-9.2) cm. Out of 7 pts with initial collagen fibrosis who were evaluated post-transplant, 1 had no fibrosis, 5 reticulin type and only in 1 pt collagen fibrosis was stable. Out of 3 pts with initial reticulin fibrosis it disappeared in 2 and progressed to collagen type in 1. Causes of death were GVHD (5 pts: 3 aGVHD, 2 cGVHD) and pancytopenia with either infection (7 pts) or CNS hemorrhage (1 pt). Conclusions: Allo-HSCT, the only curative treatment of myelofibrosis, provides chance of long survival, regression of the disease (lower stage of fibrosis, JAK2V617F eradication) and improved quality of life (transfusion independency, decreased splenomegaly). Transfusion independency may indicate good outcome. Favorable results are observed after allo-HSCT from MRD. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Despite the development of a variety of new investigational therapies for patients diagnosed with acute myeloblastic leukemia who either fail to achieve remission or who relapse thereafter, identifying suitable patients for allogeneic hematopoietic stem cell transplantation (allo-HSCT) and choosing a best tolerable therapy that is most likely to succeed remains a difficult clinical problem. The rationale of this study was to evaluate results of allo-HSCT in AML patients who have failed to achieve complete remission pre-transplant. Patients and Methods 33 pts with refractory or relapsed AML with no (18) or with partial remission (15), aged 35 (15-64) years received allo-HSCT from 10/10 (19) or 9/10 (14) HLA-matched Unrelated Donors (UD) in Hematology and BMT Center in Katowice, Poland, from Dec 2000 to Jan 2013. The myeloablative preparative regimen consisted of busulfan 4x4 mg/kg plus cyclophosphamide (Cy) 4x30 mg/kg (18 pts), TBI (12 Gy) plus Cy 3x40 mg/kg (6 pts), or treosulfan 3x14 g/m2 plus either fludarabine 5x30 mg/m2 (7 pts) or Cy 3x40 mg/kg (2 pts). Standard GVHD prophylaxis consisted of cyclosporine-A, methotrexate and pre-transplant ATG or thymoglobulin. Source of cells was peripheral blood (22 pts) or bone marrow (11 pts) with median 6.8 or 3.9 x10(6)CD34+cells/kg, respectively. Results All but 4 pts engrafted. The 2-year estimated overall survival rate was 47%. 17 pts died 4 (0.3-16) months following allo-HSCT due to relapse (7) infection (5), GVHD (1) GF (1) and unknown cause (3). Acute GVHD grade I, II, III and IV was observed in 15, 6, 2 and 1 pt (serious aGVHD grade III-IV in 3 pts (9%)), limited or extensive chronic GVHD was present each in 5 (20%) of 25 evaluable pts. Other complications included CMV reactivation (13), hemorrhagic cystitis (7), serious 3-4 grade mucositis (8), SOS (3) and renal failure (1). 16 pts (48.5%) are alive 45 months (4 months-10 yrs) post-transplant. No difference in survival has been observed between pts transplanted in PR or NR stages nor between pts treated with variable preparative regimens. Conclusions Allo-HSCT from UD with myeloablative conditioning is a feasible and effective curative therapy providing acceptable rates of long-term remission or cure in refractory or relapsed AML in patients who fail to achieve CR with conventional treatment. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired clonal abnormality of the hematopoietic stem cell caused by somatic mutation in the phosphatidylinositol glycan-class A (PIG-A) gene located on the short arm of the X chromosome. Cells with lack phosphatidylinositol glycoproteins are more sensitive to complement-mediated lysis. Despite the efficient symptomatic treatment of hemolytic PNH with eculizumab, allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment of the disease, although outcomes presented in the past were controversial. Material and methods: We report 32 allo-HSCTs: 31 from MUD and 1 from MRD performed for PNH in 2004-2014. Median age of recipients was 28 years (range 20-55) and donors 33(19-53), median time from diagnosis to allo-HSCT was 18(2-307) months. Median size of PNH clone was 80% granulocytes (0.41%-98%). Indication for allo-HSCT was aplastic/hypoplastic bone marrow (15 pts), overlapping MDS (2 pts), severe course of PNH with hemolytic crises and transfusion-dependency without access to eculizumab (15 pts). Additional risk factors were Budd-Chiari syndrome and hepatosplenomegaly (1 pt), history of renal insufficiency requiring hemodialyses (2 pts) and chronic hepatitis B (1 pt). The preparative regimen consisted of treosulfan 3x14 g/m2 plus fludarabine 5x30 mg/m2 (25 pts) or treosulfan 2x10 g/m2 plus cyclophosphamide 4x40 mg/kg (7 pts). Standard GVHD prophylaxis consisted of cyclosporine-A, methotrexate and pre-transplant ATG or thymoglobulin in MUD-HSCT. 2 pts instead cyclosporine-A received mycophenolate mofetil and tacrolimus. Source of cells was bone marrow (12 pts) or peripheral blood (20 pts) with median 7.7x10(8)NC/kg, 5.3x10(6)CD34+cells/kg, 24.2x10(6)CD3+cells/kg. Myeloablation was complete in all pts with median 9 days (6-13) of absolute agranulocytosis

Description: Abstract 4172 Introduction: Minor histocompatibility antigens (MiHA) are acknowledged non-HLA genetic factors which, in case of their incompatibility between the donor and the recipient, may contribute to post-transplant complications and impact the results of transplantation. The aim of this study was to investigate whether immunogenic MiHA disparities in either Graft-versus-Host or Host-versus-Graft direction influence Graft Versus Host Disease (GVHD) and overall survival after allogeneic hematopoietic cell transplantation (allo-HCT) from matched siblings. Methods: Alleles encoding 11 MiHAs: HA-1, HA-2, HA-3, HA-8, HB-1, ACC-1, ACC-2, HwA-9, HwA-10, UGT2B17 and HY were examined in 62 sibling donor/recipient pairs with use of Dynal AllSet mHA typing kit and PCR-SSP method. Only immunogenic MiHA disparities determined in accordance to dbMinor database, with regard to their GVH or HVG direction, were assessed. Median age of donors and recipients was 35(14–60) and 38(14–59) years, respectively. Median time from diagnosis to allo-HCT was 0.62(0.24–12.91) years. Allo-HCTs were performed between 2000–2008 for following indications: AML, ALL, MDS, CML and NHL. The conditioning treatment before allo-HCT was myeloablative (BuCy, TBI/Cy), reduced toxicity (Treo/Flu) or nonmyeloablative. Median follow-up was 3 (0.04–10) years. Results: Immunogenic MiHA mismatches were observed in 42 (68%) donor-recipient pairs: GVH- or HVG- directed in 18 pairs each and bidirectional in 6 pairs. Acute GVHD was observed in 27 patients, in 24 of whom it was severe (grade III or IV) and it was influenced by GVH-directed disparities of MiHA encoded by Y-chromosome (p=0.037), as shown in Fig. 1. Chronic GVHD was diagnosed in 25 patients, in 12 of them extensive, and it's incidence was influenced by the same kind of MiHA disparities (p=0.017), as shown in Fig. 2. Analysis of overall survival showed unfavorable impact of GVH-directed disparities of Y-chromosome encoded MiHAs (p=0.011), as presented in Fig. 3. Conclusions: Mismatches of Y-chromosome encoded MiHAs significantly impact the occurrence of severe acute and extensive chronic GVHD and decrease overall survival. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4494 Introduction: Viral hepatitis is, despite graft versus host disease (GVHD) and veno-occlusive disease (VOD), the third cause of hepatic complications after allogeneic hematopoietic stem cell transplantation (alloHSCT). The aim of this study was to analyze the effectiveness of molecular screening for hepatotropic viruses: hepatitis B virus (HBV) and hepatitis C virus (HCV), basing on a single centre experience. Materials and methods: We have evaluated 30 pts (15 women, 15 men), median age 33.5 years (18–61), who underwent alloHSCT from HLA-identical sibling (7) or unrelated donors (23) in years 2011–2012. The stem cell source was peripheral blood in all pts. Indication for allo-HSCT was: AML(11pts), ALL (10pts), OMF (4pts), SAA(2pts), PNH (2pt) and CML(1pt). Preparative regimen was myeloablative in 20pts (BuCy:10pts, Cy+TBI:10pts) and reduced in 10pts (Flu+Bu:5pts, Treo+Flu:3pts, Treo+Cy:1pt, Flu+Cy:1pt). Standard GVHD prophylaxis consisted of cyclosporine, methotrexate and pre-transplant anti-thymocyte globulin (in 28pts). Molecular quantitative polymerase chain reaction (QPCR) HBV-DNA and HCV-RNA tests and screening for serological markers of HBV (HBc-Ab, HBs-Ab, HBs-Ag, HBe-Ab, HBe-Ag) and HCV (HCV-Ab) infection were performed at four time-points (before the start of conditioning treatment and on days +28, +100 and +180 post-transplant). Results: HBc-Ab was initially positive in 2 pts, who presented negative results of other serological markers and molecular tests. In remaining 28 pts all serological and molecular markers were negative. No pt developed hepatitis infection and/or reactivated virus replication. Both pts who were initially HBc-positive did not develop any markers of HBV reactivation after one year observation, despite the intensive immunosuppression. Summary: Outcomes of our pilot study do not justify the routine molecular screening of hepatotropic viruses in patients undergoing alloHSCT, despite the profound immunosuppression. The routine molecular screening should be reserved only for patients with positive serological markers or/and those with symptoms of hepatitis. Disclosures: No relevant conflicts of interest to declare.