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We have examined the alleles of eleven minor histocompatibility antigens (MiHAs) and investigated the occurrence of immunogenic MiHA disparities in 62 recipients of allogeneic hematopoietic cell transplantation (allo-HCT) with myeloablative conditioning performed between 2000 and 2008 and in their HLA-matched sibling donors. Immunogenic MiHA mismatches were detected in 42 donor-recipient pairs: in 29% MiHA was mismatched in HVG direction, in another 29% in GVH direction; bidirectional MiHA disparity was detected in 10% and no MiHA mismatches in 32%. Patients with GVH-directed HY mismatches had lower both overall survival and disease-free survival at 3 years than patients with compatible HY; also higher incidence of both severe acute GvHD and extensive chronic GVHD was observed in patients with GVH-directed HY mismatch. On contrary, GVH-directed mismatches of autosomally encoded MiHAs had no negative effect on overall survival. Results of our study help to understand why posttransplant courses of allo-HCT from siblings may vary despite the complete high-resolution HLA matching of a donor and a recipient.

Although anti-human leukocyte antigen antibodies (anti-HLA Abs) are important factors responsible for graft rejection in solid organ transplantation and play a role in post-transfusion complications, their role in allogeneic hematopoietic stem cell transplantation (allo-HSCT) has not been finally defined. Enormous polymorphism of HLA-genes, their immunogenicity and heterogeneity of antibodies, as well as the growing number of allo-HSCTs from partially HLA-mismatched donors, increase the probability that anti-HLA antibodies could be important factors responsible for the treatment outcomes. We have examined the incidence of anti-HLA antibodies in a group of 30 allo-HSCT recipients from HLA-mismatched unrelated donors. Anti-HLA Abs were identified in sera collected before and after allo-HSCT. We have used automated DynaChip assay utilizing microchips bearing purified class I and II HLA antigens for detection of anti-HLA Abs. We have detected anit-HLA antibodies against HLA-A, B, C, DR, DQ and DP, but no donor or recipient-specific anti-HLA Abs were detected in the studied group. The preliminary results indicate that anti-HLA antibodies are present before and after allo-HSCT in HLA-mismatched recipients.

Streszczenie Przeciwciala skierowane przeciw glownemu ukladowi zgodności tkankowej są istotnym czynnikiem odpowiedzialnym za odrzucenie przeszczepu w przypadku transplantacji narządow litych. Ich wplyw na wyniki allogenicznego przeszczepienia komorek krwiotworczych, szczegolnie od dawcy niezgodnego w ukladzie HLA, nie zostal dotychczas poznany. Wobec wzrastającej liczby allotransplantacji komorek krwiotworczych od dawcow niespokrewnionych nie w pelni zgodnych w ukladzie HLA, zaistniala potrzeba zbadania wystepowania oraz wplywu przeciwcial anty-HLA na wyniki procedury przeszczepowej. Celem naszej pracy bylo zbadanie wystepowania, swoistości oraz wplywu przeciwcial anty-HLA na wyniki przeszczepienia macierzystych komorek krwiotworczych od dawcow niespokrewnionych nie w pelni zgodnych w ukladzie HLA z biorcą przeszczepu. Do badania wlączono 30 pacjentow poddanych transplantacji komorek krwiotworczych w Klinice Hematologii i Transplantacji Szpiku SUM w Katowicach w latach 2001–2007. Przeciwciala anty-HLA byly wykrywane przy uzyciu techniki DynaChip w surowicach pobranych od chorych w roznym czasie od przeprowadzonej procedury przeszczepowej. Technika DynaChip lączy w sobie zmodyfikowaną metode ELISA z techniką microchipow wykorzystującą rozpuszczalne, oczyszczone glikoproteiny HLA zarowno klasy I, jak i II, oplaszczone na powierzchni studzienek. Wstepne obserwacje wskazują, ze po allotransplantacji są wytwarzane przeciwciala skierowane przeciw glownemu ukladowi zgodności tkankowej i mogą one miec potencjalny wplyw na wyniki procedury transplantacyjnej.

We searched for immunogenic mismatches of H-Y minor histocompatibility antigens among unrelated HLA-matched donor-recipient pairs and for their association with transplant outcomes. We included 92 patients who were treated with 10/10 HLA allele–matched, unrelated allogeneic hematopoietic stem cell transplantation (alloHSCT). H-Y genotyping was performed in the Regional Blood Center with use of the Dynal Minor Histocompatibility Antigen Typing Kit. H-Y mismatches in the graft-versus-host direction of female donor to male recipient decreased the relapse rate (6% vs 23%; P = .046) and tended to improve disease-free survival (79% vs 44%; P = .067), but it also increased the incidence of chronic graft-versus-host disease (66% vs 38%; P = .02). Thus it influenced the results of alloHSCT from HLA-matched unrelated donors. The results of this study may help to explain the impact of gender differences between donor and recipient in alloHSCT.

Purpose To analyze the graft survival rate and stability of the corneal surface in patients who underwent limbal stem cell transplantation. Three surgical techniques were performed based on the origin of the ocular surface lesion: conjunctival limbal autograft (CLAU), living-related conjunctival limbal autograft (lr-CLAL), and keratolimbal allograft (KLAL) transplantations. Methods Nonrandomized consecutive comparative case series study. Eighty-four patients (90 eyes; 31 women and 53 men; age range: 11-78 years) were included in the study. Mean follow-up was 31.2 months (range: 6-72 months). Patients were divided into three groups: CLAU, lr-CLAL, and KLAL, comprising 21, 26, and 43 eyes, respectively. Graft survival rate and clinical success of the stem cell transplantation was confirmed by impression cytology. The Kaplan Meier survival curve and generalized Peto tests were used for the analyses. Results Graft survival rate and the regularity of the corneal surface differed significantly between the allo- and autografts. The 3-year and 6-year graft survival rates were 76.1% and 61.9%, respectively, for the autologous transplantation group, and 59.4% and 46.3%, respectively, for the allogeneic transplantation group. Corneal surface restoration correlated with positive staining for corneal epithelial cells in impression cytology. Conclusions Significantly better long-term outcomes were achieved with autotransplantation of the limbus compared with allogeneic limbal grafts from living-related and cadaveric donors.

The effect of minor H antigen mismatching on the occurrence of graft-versus-host disease (GvHD) and graft-versus-leukemia (GvL) after HLA-matched hematopoietic stem cell transplantation (HSCT) has mainly been demonstrated in single-center studies. Yet, the International Histocompatibility and Immunogenetics Workshops (IHIW) provide a collaborative platform to execute crucial large studies. In collaboration with 20 laboratories of the IHIW, the roles of 10 autosomal and 10 Y chromosome-encoded minor H antigens were investigated on GvHD and relapse incidence in 639 HLA-identical related donor (IRD) and 210 HLA-matched unrelated donor (MUD) HSCT recipients. Donor and recipient DNA samples were genotyped for the minor H antigens HA-1, HA-2, HA-3, HA-8, HB-1, ACC-1, ACC-2, SP110, PANE1, UGT2B17, and HY. The correlations with the primary outcomes GvHD (acute or chronic GvHD), survival, and relapse were statistically analyzed. The results of these multicenter analyses show that none of the HLA class I-restricted HY antigens were found to be associated with any of the primary outcomes. Interestingly, of the HLA class II-restricted HY antigens analyzed, HLA-DQ5 positive recipients showed a significantly increased GvHD-free survival in female-to-male HSCT compared with male-to-female HSCT (P= .013). Yet, analysis of the overall gender effect, thus independent of the known HY antigens, between the gender groups demonstrated an increased GvHD incidence in the female-to-male transplantations (P < .005) and a decreased GvHD-free survival in the female-to-male transplantations (P < .001). Of all autosomally encoded minor H antigens, only mismatching for the broadly expressed minor H antigen HA-8 increased the GvHD incidence in IRD HSCT (Hazard ratio [HR]= 5.28, P < .005), but not in MUD HSCT. Most striking was the influence of hematopoietic restricted minor H antigens on GvL as mismatching for hematopoietic minor H antigens correlated with lower relapse rates (P= .078), higher relapse-free survival (P= .029), and higher overall survival (P= .032) in recipients with GvHD, but not in those without GvHD. In conclusion, the significant GvHD effect of the broadly expressed minor H antigen HA-8 favors matching for HA-8 in IRD, but not in MUD, patient/donor pairs. The GvHD-GvL association demonstrating a significant lower relapse in hematopoietic minor H antigen mismatched patient/donor pairs underlines their clinical applicability for adoptive immunotherapy, enhancing the GvL effect in a GvHD controllable manner. © 2013 American Society for Blood and Marrow Transplantation.

We determined the alleles of 11 mHAs and investigated the association of immunogenic mHA mismatches between a donor and a recipient with a course of allogeneic hematopoietic SCT (allo-HSCT) from 10/10 alleles HLA-matched unrelated donors in 92 recipients after myeloablative conditioning between 2004 and 2006. The frequency analysis of mHA alleles, genotypes and phenotypes accompanied by appropriate restriction HLA Ags allowed for an estimation of the probability of immunogenic mismatches, which was the highest for HA-1, HA-8 and HY. GVH-directed disparity of mHAs with broad tissue distribution, especially of the sex-related HY Ag, influenced the results of allo-HSCT from HLA-matched unrelated donors by not only increasing the probability of chronic GVHD (cGVHD) but also by decreasing the relapse rate.

This study aims to assess the autoimmunological status and forms of celiac disease (CD) among children with type 1 diabetes mellitus (T1DM). The study group comprises 27 patients at the mean age of 12.30 years (±SD3.12). The measurement of the level of diabetes-specific antibodies and organ-specific antibodies was gained at the T1DM-onset and repeated annually. The following risk factors influencing time of CD diagnosis were analyzed: age, sex, T1DM duration, autoantibodies, and HLA-haplotype. The prevalence of antibodies was GADA-74%, IAA-63%, IA2A-67%, ATA-11%, and ATG-4%. The intestinal biopsy revealed in 19% no changes and in 77% stage 3 (Marsh scale). In most cases, no clinical manifestation of CD was observed. The diagnosis of Hashimoto's disease was made twice. The negative correlation between the age at T1DM-onset and the interval between onset of T1DM and CD (r=‐0.35,p<.05) was noted. The high-comorbidity ratio of CD and thyroiditis with T1DM demands regular screening tests especially in the first years after T1DM-onset.

The case of a rare coexistence of systemic lupus erythematosus (SLE) with coeliac disease (CD) is described. Systemic lupus erythematosus was diagnosed prior to CD and initially SLE treatment was administered. After several years, when CD symptoms developed, the diagnosis was corrected and additional treatment with a gluten-free diet was applied with beneficial effects.

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.

The aim of the study was to assess chosen factors of genetic susceptibility to DMT1: DRB1, DQB1, and TNF-αpolymorphisms-308 (G/A) in children with DMT1 and their up-to-now healthy siblings. Then we tested whether the association between TNF-αgenes and DMT1 is independent of HLA. 87 diabetic children, their 78 siblings, and 85 persons from healthy control group were followed up. The highest risk of DMT1 was connected with alleles: DRB1*0401 (OR = 3.39; CI: 1.55–7.41), DRB1*0301 (OR = 2.72; CI: 1.48–5.01), DQB1*0201 (OR = 4.04; CI: 2.17–7.52), DQB1*0302 (OR = 5.08; CI: 2.54–10.14), and TNF-α-308 A allele (OR = 2.59; CI: 1.23–5.44). Moreover linkage disequilibrium for TNF-α-308 A allele with DRB1*0301 and DQB1*0201 was observed in both diabetic children and their siblings. Diabetic children and their siblings present similar genetic risk factors for DMT1. The association between TNF-α-308 A allele and DMT1 is dependent of HLA-DRB1 and DQB1 alleles.

In this study a genetic determination of some cytokines synthesis is presented in group of 23 children with various kinds of nephrotic syndrome (NS). The gene polymorphisms of TNF-alpha, TGF-beta, IL-6, IL-10, INF-gamma were identified using PCR-SSP method combined with the measurement of levels of TNF-alpha, TGF-beta, IL-6, IL-10, INF-gamma synthesis. The differences in occurring frequency of high, middle and low genotypes TNF-alpha, TGF-beta and IL-6 synthesis between children with NS and control group were revealed. Significantly more frequently high TGF-beta and high IL-6 synthesis genotype in NS group were found. Because of high variability of cytokine level in blood in duration of NS and methodic difficulties of their measurement, identification of cytokines genes polymorphisms seems to be a method that can objectively describe the cytokine participation in NS pathophysiology.

The cornea is an immune privileged site, but failure of corneal limbal stem cells is the cause of vascularisation, increasing the risk of immune response activity. The outcome after conjuctival limbal stem cells transplantation depends mainly on HLA system compatibility. HLA class I typing is one of the important factors considered in donor/recipient par matching. The aim of our study was the analysis of post transplant period in 16 related donor/recipient pars after limbal stem cells transplantation. We conclude that HLA class I and other immunogenetic factors compatibility (inside and out of the HLA region), may play the role in effective treatment.

The objective of this study was to evaluate the genetic profiles of selected cytokines (transforming growth factor beta 1, tumor necrosis factor alpha, interleukin-6, interferon gamma, and interleukin-10) in systemic lupus erythematosus and the contributions of human leukocyte antigen (HLA)-DRB1 and -DQB1 alleles to susceptibility for this disease. The study was carried out in 24 SLE patients and 36 healthy controls (from Upper Silesia) using polymerase chain reaction methods. All persons were of Caucasoid origin. Standard association analysis was used to compare the HLA alleles and frequency of cytokine gene polymorphisms between these groups. Only the frequency of HLA-DRB1*07 allele was higher in SLE patients than controls (odds ratio 2.92, 95% confidence interval 1.16–7.33), but the difference did not reach statistical significance when Bonferroni’s adjustment procedure was performed. No other significant associations were noted between class II alleles (DR1-DR6, DR8-DR10, DQ1-DQ4) and SLE. The frequency of the interleukin-6 GG and GC genotypes was significantly higher in SLE patients than in controls, and a significantly higher percentage of the G vs C alleles between patients and controls was revealed (odds ratio 2.53, 95% confidence interval 1.37–4.65, chi-squared test 8.16, P

Type 1 diabetes is a genetically conditioned autoimmune disease. Genes that account for strong clustering of the disease susceptibility are located within the HLA region. There is also considerable individual variation in the immune response and role of cytokine genes in the disease predisposition.The aim of our research was identification of the genetically controlled TNF-alpha, IL-10, IL-6, IFN-gamma secretion profile in children with diabetes type 1.We have examined 36 children with diabetes type 1 and 36 healthy individuals. DNA was extracted from mononuclear peripheral blood cells. For identification of the cytokine polymorphism PCR-SSP method was used.Patients with diabetes type 1 differ from the group of healthy persons in the cytokine synthesis level and in the cytokine genotypes distribution. Genotype TNF-alpha (A/G) as well as IL-10 (ATA/ATA) was found only in group of children with diabetes but not in the control group. Genotypes IL-10 (GCC/GCC), IL-6 (C/C), IFN-gamma (T/T) were observed with decreased frequency in children with diabetes type 1. No differences between patients and control group in the frequency of IL-10 (GCC/ACC) (GCC/ATA), (ACC/ACC) (ACC/ATA) IL-6 (G/G), (G/C) and IFN-gamma (T/A), (A/A) genotypes were observed. Children with diabetes type 1 were more frequent "high producers" of TNF-alpha and IL-6.It is possible to us molecular method to estimate the genetically controlled immune reactivity. It is a very important immunogenetic factor of the disease predisposition.

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.

Abstract 4475 Introduction: Anti-HLA Antibodies (Abs) are considered an important factor in solid organ transplants and transfusion medicine, but role of humoral arm of immunological response to HLA antigens in allogeneic hematopoietic stem cell transplantation (allo-HSCT) is unknown. 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 presence and profiles of anti-HLA Abs detected before or after allo-HSCT from HLA-mismatched unrelated donors and their impact on allo-HSCT results. Material and methods: 35 HLA-mismatched donor/recipient pairs entered the study. Indication for allo-HSCT was: ALL (7pts), AML(18pts), CML(5pts), SAA(2pts), CLL(1pt), MDS(1pt) and PNH (1pt). Preparative regimen was myeloablative in 33pts (94.3%) and reduced in 2pts (5.7%). Standard GVHD prophylaxis consisted of cyclosporine, methotrexate and pre-transplant anti-thymocyte globulin (34pts) or Alemtuzumab (1pt). HLA A, B, C, DR, DQ alleles were PCR-typed. 21(60%) pts had mismatch of single HLA-antigen: A-4(11.4%), B-1(2.8%), C-13(37%), DQ-3(8,5%); 10(28.5%) pts had mismatch of single HLA-allele: A-3(8.5%), B(11.4%), DQ-3(8.5%); 4 pts had double antigenic (A+C and A+DQ) or combined antigenic/allelic (A/B and C/A) HLA mismatches. Anti-HLA A, B, C, DR, DQ, DP Abs were identified in sera collected before start of the conditioning treatment and +30 days, +100 days and 1 year after allo-HSCT with use of automated DynaChip assay utilizing microchips bearing purified class I and class II HLA antigens. Results: Anti-HLA Abs pre-formed before allo-HSCT were detected in 17(48.5%) pts: against class I, II or both in 6(35%), 4(24%) and 7(41%) pts. Anti-HLA Abs were detected after allo-HSCT in 25(71.4%) pts, against class I, II or both in 9(36%), 3(12%) and 13(52%) pts, respectively. In 7 pts anti-HLA Abs were not detected neither before nor after allo-HSCT. Anti-HLA Abs directed against the mismatched HLA antigens were observed in 4 pts before and in 10 pts after allo-HSCT, no anti-HLA Abs specific against mismatched alleles were detected. Allo-HSCT results obtained in studied subgroups are presented in the Table below: Conclusions: Our preliminary results indicate that anti-HLA Abs are present pre- or post-transplant in mismatched allo-HSCT recipients and may be potentially responsible for the occurrence of complications, what needs to be further investigated and analyzed. Disclosures: No relevant conflicts of interest to declare.

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.

Aim Biology of antibody production and function of immunologically competent cells is closely related to transplant process.This kind of research gives a possibility to observe and estimate patient and donor matching. The most interesting is to appoint some common denominator of HLA mismatches and antibody specificity detected in a serum of mismatched patient. We collected sera from patients both pre and post allo-HSCT transplantation. All the donor/recipient pair mismatches were known. Methods We tested 130 sera collected from patients there were three periods:before transplantation, 6 month and 12 months after transplantation. DynaChip and LabScreen Luminex methods were used to identify anti-HLA antibody against HLA-A,B,C,DR,DQ and DP. We were able to define specificity of antibodies on the low and high resolution level. Software DynaChip and Fusion were used to perform immunogenetic analyses of our research. Results In a group of patients 57% people expressed anti-HLA antibodies. We identified anti-HLA antibodies against HLA class I and HLA class II. We were able to define specificity of antibodies on low and high resolution level. In a group of 43% of patients we did not identify anti-HLA antibodies. We devided a patient into two groups mismatched in HLA class I antigen and mismatched in HLA class II antigen. We analysed people produced antibody against one or many antigens. As a second step of our research we analyse donor recipient mismatches on the level of allele. Donor recipient mismatches were connected with HLA allele locus C (6,6%), HLA allele locus B (13,3%), HLA allele locus A (3,3%), HLA allele locus DQB1 ∗ (10%). When we look at the PRA positive patient 20% of them express presence of antibodies against many specificities, 16,7% against no more than 3 specificity, 20% against single antigen. Conclusions As a continuation of our research we are going to use the Immport Database. We would like to try compare specificity of anti-HLA antibodies and molecular structure of mismatched alleles. Siekiera: Blood Center: Other: HLA Lab Director. Dobrowolska: Blood Center: Employee. Markiewicz: Medical University of Silesia: Other: Clinician.

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.

Abstract 4337 Introduction Donor and recipient sex is an important factor that may influence outcomes of HLA-identical allogeneic hematopoietic stem cell transplantation. The rationale of this study was to evaluate the impact of sex-related HY antigen mismatch on results of allo-HSCT from HLA-matched unrelated donors. Patients and Methods 92 patients treated with 10/10 HLA alleles-matched unrelated allo-HSCT performed in Hematology and BMT center in Katowice in years 2004-2006 entered the study. HY genotyping was performed in the Regional Blood Center with use of Dynal Minor Histocompatibility Antigen Typing Kit. Only immunogenic HY disparities were analysed. Results The estimated probability of immunogenic HY mismatches corrected by the frequency of restrictive HLA molecules was 34%. When HY was mismatched in GVH direction (female donor, male recipient), the incidence of cGVHD was increased (66% vs 38%, p=0.02), the relapse rate was decreased (6% vs 23%, p=0.046) and DFS tended to improve (79% vs 44%, p=0.067). Conclusions The sex-related HY antigen mismatches in GVH direction influence the results of allo-HSCT from HLA-matched unrelated donors. Results of this study may help to understand why sex difference between the donor and the recipient plays role in the allo-HSCT. Disclosures: No relevant conflicts of interest to declare.

Abstract 4324 Introduction Albeit it is generally presumed that monozygotic twins are genetically identical and that phenotypic differences between twins are mainly due to environmental factors, large-scale variation in copy number of DNA segments recently evidenced by Bruder et al. (AJHG, 2008) showed presence of genotypic diversity in monozygotic twins. The rationale of this study was to test whether monozygotic twins display disparities of minor Histocompatibility antigens (mHags) which may play role in syngenic HCT. We and others have previously shown that mHags constitute an important immunogenetic factor influencing immune responses following transplantation from HLA-matched allogeneic donors. Patients and Methods mHags HA-1, HA-2, HA-3, HA-8, HB-1, ACC-1, ACC-2, HwA-9, HwA-10, UGT2B17, HY genotypes were defined with use of Dynal AllSet kits by PCR-SSP method in secured DNA samples from 3 monozygotic twins pairs aged 34, 24 and 28, who underwent syngenic allo-HCTs due to different hematological malignancies (NHL, CML, AML) in the Department of Hematology and BMT in Katowice, Poland in years 2000-2004. Results In 2 out of 3 syngenic pairs we have found differences in genes encoding mHags: different allele of EB-1 was present in one pair (NHL) (recipient HH, donor HY), and two different alleles of HwA-9 (RR, RG) and HwA-10 (**, R*) were present in second pair (CML). No differences in mHags were observed in the third pair (AML). Conclusions Our results question the long-standing belief that monozygotic twins are genetically identical and open up a possibility to further study the role of disparate mHags in disease and transplantation. Disclosures: No relevant conflicts of interest to declare.

Disparities of minor Histocompatibility antigens (mHag) have been considered as an important immunogenetic factor influencing immune responses following transplantation despite fully matched HLA of donor and recipient. The aim of our study was to answer whether mHag incompatibility may influence the outcome of allo-HCT. DNA samples from 92 unrelated donor/recipient pairs completely matched in HLA-A*,B*,C*,DRB1*,DQB1* alleles were collected in order to define minor Histocompatibility HA-1, HA-2, HA-3, HA-8, HB-1, ACC-1, ACC-2, HwA-9, HwA-10, UGT2B17, HY genotypes with use of Dynal AllSet kits by PCR-SSP method. Unrelated allo-HCTs were performed in the Department of Hematology and BMT in Katowice, Poland, with use the same standard operating procedure from January 2004 until December 2006. Host versus Graft or Graft versus Host direction of immune responses in donor/recipient pairs was analyzed with use of the minor Histocompatibility Database of Leiden University Medical Center. We observed the impact of mHAg mismatches upon chronic GVHD, incidence of leukemia relapse and graft failure. Patients transplanted from donors with mHAg’s incompatibility in GVH direction had higher probability of chronic GVHD in comparison to other pairs (54% vs 31%, p=0.07). This impact was most prominent for mismatched mHAgs with broad tissue distribution (62% vs 36%, p=0.04), especially HY (66% vs 38%, p=0.02). Opposite influence of HY mismatches in GVH direction was observed upon the probability of relapse, which was lower in the HY-mismatched than in HY-matched group (6% vs 23%, p=0.046). Two mHAgs with tissue distribution restricted to hematopoietic cells mismatched in HVG direction were associated with higher incidence of graft failure than in matched pairs: HA-1 (38% vs 12%, p=0.09) and HA-2 (50% vs 13%, p=0.09). Minor Histocompatibility typing of completely HLA matched patients helps to identify individuals who are at higher risk of cGVHD, leukemia relapse and graft failure, and thus supports precise matching of donor/recipient pairs for unrelated allo-HCT.

Factors contributing to relapses and GVHD following allogeneic Hematopoietic Stem Cell Transplantations (HSCT) from HLA-matched unrelated donors are not well established. We analyzed 35 patients (pts) transplanted from HLA 10/10 alleles (A, B, C, DRB1, DQB1) matched unrelated donors (URD) in the Dept. of Hematology and BMT, Katowice, Poland, with use of the same standard operating procedure from January 2004 until March 2006. Indication for HSCT was AML (13 pts), ALL (8), CML (7), MDS (2), PNH (3), CLL (1), myeloid sarcoma (1). Preparative regimen was Treosulfan+Fludarabine (23 pts), TBI+Cy (9) and Bu+Cy (3). Alleles encoding 11 minor Histocompatibility Antigens (mHA: HA-1, HA-2, HA-3, HA-8, HB-1, ACC-1, ACC-2, HwA-9, HwA-10, UGT2B17, HY) were analyzed for each donor-recipient pair with use of Dynal AllSet mHA typing kit and PCR-SSP method. Only immunogenic mHA mismatches were analyzed. Information on whether mHA mismatches might result in Host-versus-Graft or Graft-versus-Host responses was established with use of the minor Histocompatibility Knowledge Database of Leiden University Medical Center’s minor Histocompatibility Workshop. Patients transplanted from donors with mHA mismatched in HVG direction had significantly higher probability of relapse (64+/− 8% versus 10+/− 9%, p=0.003) when compared to pairs without mHA HVG immonogenicity. Patients transplanted from donors with mHA mismatched in GVH direction had higher probability of aGVHD (88+/− 8% versus 53+/− 11%, p=0.14) and cGVHD (41+/− 16% versus 37+/− 17%, p=0.65) when compared to pairs without GVH immunogenicity. GVHD was also observed more often on day +100 (33% versus 6%, p=0.06) when mHA mismatch in GVH direction was present. Observed influence of HVG and GVH immonogenicity on probability of relapse and GVHD did not lead to significantly different survival in observed groups of pts. These results indicate that mHA immunogenic mismatches in HVG and GVH direction may be responsible for relapse and GVHD, respectively, in HSCT recipients from HLA-matched unrelated donors. The study is being continued to confirm these primary observations and to establish the associations of specific mHA mismatches with HSCT outcomes. | Immunogenicity of mHA mismatches | HVG | no HVG | p | |:-------------------------------- | -------- | -------- | ----- | | # of patients | 18 | 17 | | | Relapse at 1 year | 64%+/−8 | 10%+/−9 | 0.003 | | Immunogenicity of mHA mismatches | GVH | no GVH | p | | # of patients | 16 | 19 | | | aGVHD | 88%+/− 8 | 53%+/−11 | 0.14 | | cGVHD at 1 year | 41%+/−16 | 37%+/−17 | 0.65 | Impact of mHA mismatch on probability of relapse and GVHD | Immunogenicity of mHA mismatches | HVG GVH | only HVG | only GVH | no HVG no GVH | p | |:-------------------------------- | -------- | -------- | -------- | ------------- | ---- | | # of patients | 9 | 9 | 7 | 10 | | | Survival at 9 months | 63%+/−17 | 57%+/−25 | 83%+/−15 | 69%+/−15 | 0.86 | Impact of mHA mismatch on probability of survival