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The development of next-generation sequencing (NGS) methods for HLA genotyping has already had an impact on the scope and precision of HLA research. In this study, allelic resolution HLA typing was obtained for 402 individuals from Cape Town, South Africa. The data were produced by high-throughput NGS sequencing as part of a study of T-cell responses to Mycobacterium tuberculosis in collaboration with the University of Cape Town and Stanford University. All samples were genotyped for 11 HLA loci, namely HLA-A, -B, -C, -DPA1, -DPB1, -DQA1, -DQB1, -DRB1, -DRB3, -DRB4, and -DRB5. NGS HLA typing of samples from Cape Town inhabitants revealed a unique cohort, including unusual haplotypes, and 22 novel alleles not previously reported in the IPD-IMGT/HLA Database. Eight novel alleles were in Class I loci and 14 were in Class II. There were 62 different alleles of HLA-A, 72 of HLA-B, and 47 of HLA-C. Alleles A∗23:17, A∗43:01, A∗29:11, A∗68:27:01, A∗01:23, B∗14:01:01, B∗15:10:01, B∗39:10:01, B∗45:07, B∗82:02:01 and C∗08:04:01 were notably more frequent in Cape Town compared to other populations reported in the literature. Class II loci had 21 different alleles of DPA1, 46 of DPB1, 27 of DQA1, 26 of DQB1, 41 of DRB1, 5 of DRB3, 4 of DRB4 and 6 of DRB5. The Cape Town cohort exhibited high degrees of HLA diversity and relatively high heterozygosity at most loci. Genetic distances between Cape Town and five other sub-Saharan African populations were also calculated and compared to European Americans.

The human leukocyte antigen (HLA) genes are highly variable and are known to play an important role in disease outcomes, including infectious diseases. Prior knowledge of HLA polymorphisms in a population usually forms the basis for an effective case-control study design. As a prelude to future disease association analyses, we report HLA class I and II diversity in 334 unrelated donors from a Dengue vaccine efficacy trial conducted in Thailand. Long-range PCR amplification of six HLA loci was performed on DNA extracted from saliva samples. HLA-A, -B, -C, -DPB1, -DQB1 and -DRB1 were genotyped using a next-generation sequencing method presented at the 17th International HLA and Immunogenetics Workshop. In total, we identified 201 HLA alleles, including 35 HLA-A, 57 HLA-B, 28 HLA-C, 24 HLA-DPB1, 21 HLA-DQB1 and 36 HLA-DRB1 alleles. Very common HLA alleles with frequencies greater than 10 percent were A∗11:01:01, A∗33:03:01, A∗24:02:01, B∗46:01:01, C∗07:02:01, C∗01:02:01, C∗08:01:01, DPB1∗05:01:01, DPB1∗13:01:01, DPB1∗04:01:01, DPB1∗02:01:02, DQB1∗03:01:01, DQB1∗05:02:01, DQB1∗03:03:02, DRB1∗12:02:01, DRB1∗09:01:02, and DRB1∗15:02:01. A novel HLA allele, B∗15:450, had a non-synonymous substitution and occurred in more than one donor. Population-based full-length NGS HLA typing is more conclusive and provides a sound foundation for exploring disease association in a given population.

Background The association of HLA mismatching with kidney allograft survival has been well established. We examined whether amino acid (AA) mismatches (MMs) at the antigen recognition site of HLA molecules represent independent and incremental risk factors for kidney graft failure (GF) beyond those MMs assessed at the antigenic (2-digit) specificity. Methods Data on 240 024 kidney transplants performed between 1987 and 2009 were obtained from the Scientific Registry of Transplant Recipients. We imputed HLA-A, -B, and -DRB1 alleles and corresponding AA polymorphisms from antigenic specificity through the application of statistical and population genetics inferences. GF risk was evaluated using Cox proportional-hazards regression models adjusted for covariates including patient and donor risk factors and HLA antigen MMs. Results We show that estimated AA MMs at particular positions in the peptide-binding pockets of HLA-DRB1 molecule account for a significant incremental risk that was independent of the well-known association of HLA antigen MMs with graft survival. A statistically significant linear relationship between the estimated number of AA MMs and risk of GF was observed for HLA-DRB1 in deceased donor and living donor transplants. This relationship was strongest during the first 12 months after transplantation (hazard ratio, 1.30 per 15 DRB1 AA MM; P < 0.0001). Conclusions This study shows that independent of the well-known association of HLA antigen (2-digit specificity) MMs with kidney graft survival, estimated AA MMs at peptide-binding sites of the HLA-DRB1 molecule account for an important incremental risk of GF., In a population of 240 024 kidney transplant recipients using the data of the Scientific Registry of Transplant recipients, the authors demonstrate that, independently of HLA antigen mismatches, estimated amino-acid mismatches at peptide-binding sites of the HLA-DRB1 molecule, accounts for an increased graft failure risk. Supplemental digital content is available in the text.

Preclinical studies have shown that persistent mixed chimerism is linked to acceptance of organ allografts without immunosuppressive (IS) drugs. Mixed chimerism refers to continued mixing of donor and recipient hematopoietic cells in recipient tissues after transplantation of donor cells. To determine whether persistent mixed chimerism and tolerance can be established in patients undergoing living donor kidney transplantation, we infused allograft recipients with donor T cells and hematopoietic progenitors after posttransplant lymphoid irradiation. In 24 of 29 fully human leukocyte antigen (HLA)–matched patients who had persistent mixed chimerism for at least 6 months, complete IS drug withdrawal was achieved without subsequent evidence of rejection for at least 2 years. In 10 of 22 HLA haplotype–matched patients with persistent mixed chimerism for at least 12 months, reduction of IS drugs to tacrolimus monotherapy was achieved. Withdrawal of tacrolimus during the second year resulted in loss of detectable chimerism and subsequent rejection episodes, unless tacrolimus therapy was reinstituted. Posttransplant immune reconstitution of naïve B cells and B cell precursors was more rapid than the reconstitution of naïve T cells and thymic T cell precursors. Robust chimerism was observed only among naïve T and B cells but not among memory T cells. No evidence of rejection was observed in all surveillance graft biopsies obtained from mixed chimeric patients withdrawn from IS drugs, and none developed graft-versus-host disease. In conclusion, persistent mixed chimerism established in fully HLA- or haplotype-matched patients allowed for complete or partial IS drug withdrawal without rejection.

Introduction Chimerism testing of peripheral blood and bone marrow aspirates following allogeneic hematopoietic cell transplantation (HCT) is important to evaluate engraftment kinetics and potentially early detection of disease relapse1. NGS-based chimerism assays use SNP panels and provide standardized workflow with automated data analyses. Objectives We aim to evaluate performance of an NGS-based chimerism assay and compare it to existing laboratory method based on Short Tandem Repeat (STR) analysis. Methods Genomic DNA (gDNA) derived from PBMCs of unrelated donors were mixed at fractions ranging from 0.1% - 50% to evaluate sensitivity and specificity of the NGS-based chimerism assay in artificial genomic chimeras. 3-10 ng gDNA was used to prepare libraries with the assay kit and sequenced on Illumina MiSeq. Results Linear regression analyses of gDNA fractions (expected, observed) detected a linear signal at a minimum of 0.1% of minor gDNA in a two-gDNA chimeric mix (R2=1, average CV = 4.5%, range = 0.2-12.4). Chimeric samples with three distinct gDNAs gave linear signal as low as 0.5% of minor gDNA fraction (CV Conclusions Using the test NGS-based chimerism assay on two and three gDNA mixes, we observe 10X greater sensitivity of minor fraction quantitation compared to current STR method. In our experience, high sensitivity, precision over a wide range of detection and robust performance with low input DNA may qualify this NGS-based chimerism assay as a potential method of choice for routine engraftment monitoring and potential early detection of disease relapse.

We examined current outcomes of unrelated donor allogeneic hematopoietic cell transplantation (HCT) to determine the clinical implications of donor-recipient HLA matching. Adult and pediatric patients who had first undergone myeloablative-unrelated bone marrow or peripheral blood HCT for acute myelogenous leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, and myelodysplastic syndrome between 1999 and 2011 were included. All had high-resolution typing for HLA-A, -B, -C, and -DRB1. Of the total (n = 8003), cases were 8/8 (n = 5449), 7/8 (n = 2071), or 6/8 (n = 483) matched. HLA mismatch (6-7/8) conferred significantly increased risk for grades II to IV and III to IV acute graft vs host disease (GVHD), chronic GVHD, transplant-related mortality (TRM), and overall mortality compared with HLA-matched cases (8/8). Type (allele/antigen) and locus (HLA-A, -B, -C, and -DRB1) of mismatch were not associated with overall mortality. Among 8/8 matched cases, HLA-DPB1 and -DQB1 mismatch resulted in increased acute GVHD, and HLA-DPB1 mismatch had decreased relapse. Nonpermissive HLA-DPB1 allele mismatch was associated with higher TRM compared with permissive HLA-DPB1 mismatch or HLA-DPB1 match and increased overall mortality compared with permissive HLA-DPB1 mismatch in 8/8 (and 10/10) matched cases. Full matching at HLA-A, -B, -C, and -DRB1 is required for optimal unrelated donor HCT survival, and avoidance of nonpermissive HLA-DPB1 mismatches in otherwise HLA-matched pairs is indicated.

Study Objectives: To identify rare allelic variants and HLA alleles in narcolepsy patients with hypocretin (orexin, HCRT) deficiency but lacking DQB1*06:02. Settings: China (Peking University People's Hospital), Czech Republic (Charles University), Denmark (Golstrup Hospital), Italy (University of Bologna), Korea (Catholic University), and USA (Stanford University). Design: CSF hypocretin-1, DQB1*06:02, clinical and polysomnographic data were collected in narcolepsy patients (552 with and 144 without cataplexy) from 6 sites. Numbers of cases with and without DQB1*06:02 and low CSF hypocretin-1 were compiled. HLA class I (A, B, C), class II (DRBs, DQA1, DQB1, DPA1, and DPB1), and whole exome sequencing were conducted in 9 DQB1*06:02 negative cases with low CSF hypocretin-1. Sanger sequencing of selected exons in DNMT1, HCRT, and MOG was performed to exclude mutations in known narcolepsy-associated genes. Measurements and Results: Classic narcolepsy markers DQB1*06:02 and low CSF hypocretin-1 were found in 87.4% of cases with cataplexy, and in 20.0% without cataplexy. Nine cases (all with cataplexy) were DQB1*06:02 negative with low CSF hypocretin-1, constituting 1.7% [0.8%-3.4%] of all cases with cataplexy and 1.8% [0.8%-3.4%] of cases with low CSF hypocretin independent of cataplexy across sites. Five HLA negative subjects had severe cataplexy, often occurring without clear triggers. Subjects had diverse ethnic backgrounds and HLA alleles at all loci, suggesting no single secondary HLA association. The rare subtype DPB1*0901, and homologous DPB1*10:01 subtype, were present in 5 subjects, suggesting a secondary association with HLA-DP. Preprohypocretin sequencing revealed no mutations beyond one previously reported in a very early onset case. No new MOG or DNMT1 mutations were found, nor were suspicious or private variants in novel genes identified through exome sequencing. Conclusions: Hypocretin, MOG, or DNMT1 mutations are exceptional findings in DQB1*06:02 negative cases with hypocretin deficiency. A secondary HLA-DP association may be present in these cases. These represent particularly difficult diagnostic challenges.

Cytotoxic T-lymphocyte antigen-4 (CTLA-4) plays an essential role in T cell homeostasis by restraining immune responses. AG and GG genotypes of donor CTLA-4 SNP rs4553808 in patients after unrelated donor hematopoietic stem cell transplantations (HSCT) have been shown to be an independent predictor of inferior relapse-free survival (RFS) and overall survival (OS) compared with those with the AA genotype, in single-center studies. We tested the hypothesis that SNP rs4553808 is associated with RFS, OS, nonrelapse mortality (NRM) and the cumulative incidence of acute graft-versus-host disease (GVHD) and chronic GVHD in adults with acute myeloid leukemia and advanced myelodysplastic syndrome undergoing a first 8/8 or 7/8 HLA-matched unrelated donor HSCT. Multivariable analysis adjusting for relevant donor and recipient characteristics showed no significant association between SNP rs4553808 and OS, RFS, NRM, and incidence of acute and chronic GVHD. An exploratory analysis of other CTLA-4 SNPs, as well as studying the interaction with antithymocyte globulin, also demonstrated no significant associations. Our results indicate that CTLA-4 SNPs are not associated with HSCT outcomes.

The human leucocyte antigen (HLA) system shows extensive variation in the number and function of loci and the number of alleles present at any one locus. Allele distribution has been analysed in many populations through the course of several decades, and the implementation of molecular typing has significantly increased the level of diversity revealing that many serotypes have multiple functional variants. While the degree of diversity in many populations is equivalent and may result from functional polymorphism(s) in peptide presentation, homogeneous and heterogeneous populations present contrasting numbers of alleles and lineages at the loci with high-density expression products. In spite of these differences, the homozygosity levels are comparable in almost all of them. The balanced distribution of HLA alleles is consistent with overdominant selection. The genetic distances between outbred populations correlate with their geographical locations; the formal genetic distance measurements are larger than expected between inbred populations in the same region. The latter present many unique alleles grouped in a few lineages consistent with limited founder polymorphism in which any novel allele may have been positively selected to enlarge the communal peptide-binding repertoire of a given population. On the other hand, it has been observed that some alleles are found in multiple populations with distinctive haplotypic associations suggesting that convergent evolution events may have taken place as well. It appears that the HLA system has been under strong selection, probably owing to its fundamental role in varying immune responses. Therefore, allelic diversity in HLA should be analysed in conjunction with other genetic markers to accurately track the migrations of modern humans.

Hepatitis C virus (HCV) is the most common blood-borne infection in the USA, though seroprevalence is elevated in certain high-risk groups such as inmates. Correctional facility screening protocols vary from universal testing to opt-in risk-based testing. This project assessed the success of a risk-based HCV screening strategy in the Philadelphia Prison System (PPS) by comparing results from current testing practices during 2011–2012 (Risk-Based Screening Group) to a September 2012 blinded seroprevalence study (Philadelphia Department of Public Health (PDPH) Study Cohort). PPS processed 51,562 inmates in 2011–2012; 2,727 were identified as high-risk and screened for HCV, of whom 57 % tested HCV antibody positive. Twelve percent (n = 154) of the 1,289 inmates in the PDPH Study Cohort were anti-HCV positive. Inmates ≥30 years of age had higher rates of seropositivity in both groups. Since only 5.3 % of the prison population was included in the Risk-Based Screening Group, an additional 4,877 HCV-positive inmates are projected to have not been identified in 2011–2012. Gaps in case identification exist when risk-based testing is utilized by PPS. A more comprehensive screening model such as opt-out universal testing should be considered to identify HCV-positive inmates. Identification of these individuals is an important opportunity to aid underserved high-risk populations and to provide medical care and secondary prevention.

Introduction: Non-myeloablative conditioning with total lymphoid irradiation and antithymocyte globulin (TLI-ATG) is associated with a low risk of graft-versus-host disease (GVHD), attributed to skewing of residual host T-cell subsets to favor regulatory IL-4 producing natural killer T cells that polarize the infused donor T cells to a Th2 phenotype, while retaining graft-versus-tumor (GVT) reactions in lymphoid and myeloid malignancies. We hypothesized that the safety of TLI-ATG conditioning with its protection from GVHD would extend beyond matched donors, yet with mismatching there would be at least as potent GVT reactions as with matched donors. We therefore compared outcomes in patients who received TLI-ATG conditioning and allogeneic hematopoietic cell transplantation (HCT) from HLA-mismatched unrelated donors to those with HLA-matched unrelated donors. Methods: Patients: We included all consecutively transplanted patients at Stanford University Hospital from December 2001 through May 2015 who received TLI-ATG conditioning and allogeneic HCT from 10/10 HLA-matched unrelated donors (MUD, N=193) or HLA-mismatched unrelated donors (mmUD, N=72), including 70 patients matching in 9/10 alleles or antigens (55 mismatched at HLA-A, -B, -C or -DRB1 loci and 15 mismatched at HLA-DQB1) and 2 patients matched in 8/10 alleles. All patients received G-CSF-mobilized peripheral blood hematopoietic cells and cyclosporine and mycophenolate mofetil for GVHD prophylaxis. Statistical methods: For time-to-event analyses, Kaplan-Meier curves were used to estimate survival and cumulative incidence of acute and chronic GVHD, non-relapse mortality, and relapse. Log-rank tests were used to detect differences between groups. Results: Median age at the time of transplant was 60 years in both groups (range 21-76) with a median follow up of 677 days (range 53-3682) in the mmUD cohort and 762 days (range 37-4551) in the MUD cohort. There were no significant differences between the mmUD and MUD cohorts in underlying diagnosis (acute myeloid leukemia 32% vs 31%, non-Hodgkin lymphoma 22% vs 29%, chronic lymphocytic leukemia 17% vs 17%, myelodysplastic syndrome and myeloproliferative neoplasms 11% vs 14%, Hodgkin lymphoma 8% vs 5%; chronic myeloid leukemia 7% vs 2%, and acute lymphoblastic leukemia 3% vs 3%).The majority of patients in both mmUD and MUD cohorts had advanced stage lymphoid disease (75% vs 72%) and advanced stage myeloid disease (53% vs 54%), defined as disease status beyond second complete remission at the time of transplant, relapse following prior autologous or allogeneic transplant, high-risk cytogenetic abnormality and age over 55, myelodysplastic syndrome with progression to acute myeloid leukemia, or therapy-related myeloid neoplasm. Durable chimerism was achieved in 90% of patients in the mmUD group and 94% of patients in the MUD group. There were no significant differences between the mmUD and MUD cohorts in the cumulative incidence of acute GVHD at day +100 (grades II-IV: 21% vs 15%, p=0.78 and grades III-IV: 6% vs 3%, p=0.61). Non-relapse mortality (NRM) was not significantly different (14% at 1 year in mmUD group vs 6% in MUD group, p=0.34). There was no significant difference in the cumulative incidence of chronic GVHD (31% vs 27% at 2 years and 39% vs 35% at 5 years, p=0.49). There was a trend towards a lower rate of relapse in the mmUD group (40% vs 48% at 2 years, 45% vs 60% at 5 years, hazard ratio 0.71, p=0.094). There was no significant difference in overall survival (58% vs 60% at 2 years and 46% vs 46% at 5 years) or event-free survival (46% vs 44% at 2 years and 38% vs 28% at 5 years, p=0.25). In both groups, sustained remissions occurred among patients with active disease at the time of transplantation. Conclusions: TLI-ATG conditioning is associated with low rates of acute and chronic GVHD and NRM even in the setting of HLA-mismatched unrelated donor transplantation. There was a particularly low incidence of severe acute GVHD grades III-IV (3-6%) in this high-risk cohort. The trend towards lower relapse in recipients of HLA-mismatched grafts may reflect enhanced GVT reactions. TLI-ATG conditioning may overcome the traditionally poorer outcome and high NRM associated with HLA mismatch and should be considered in patients with advanced lymphoid and myeloid malignancies who cannot tolerate myeloablative preparatory regimens and who lack a fully HLA-matched donor. Figure 1 Figure 1. Disclosures Meyer: Stanford University: Patents & Royalties. Negrin:Stanford University: Patents & Royalties.

HLA alleles are observed in specific haplotypes, due to linkage disequilibrium (LD) between particular alleles. Haplotype frequencies for alleles have been established for specific ethnic groups and racial categories from population studies. HLA typing using Next Generation Sequencing (NGS) platforms allows generating full-length gene sequences, obtaining four-field HLA types and detecting novel alleles. NGS technologies also permit high-throughput HLA typing for large numbers of samples in a cost effective manner. Aim To establish HLA haplotypes with four-field resolution from quartet (two children and two parents) families. Methods We selected quartet families from our clinical database. Extended family members, aunt, uncle, cousin and grandparents, were also included if available. HLA types generated using SBT, SSP and SSO were reviewed for selecting subjects. Two children were selected if they did not share two HLA haplotypes. DNA was processed using MIA FORA NGS kits, and sequenced on either MiSeq or NextSeq sequencer. HLA types were obtained using MIA FORA 2.0 software. HLA haplotypes were built by comparing children’s and parents’ HLA types. The HLA haplotypes were reported according to the order of genes along the chromosome to identify potential meiotic recombination, and were also compared to the previously established haplotypes. Results Of 100 selected families, we sequenced 50 families. We built 200 haplotypes consisting of four-field HLA types from these. We developed automated haplotype building software. It was feasible to resolve almost all ambiguities including phase ambiguities and to generate completely phased full-length gene sequences from the consensus sequences generated from these studies. Conclusions The haplotypes generated from quartet families will be important resources for hematopoietic stem cell donor match prediction and play a role as reference haplotypes for expanding a collection of HLA haplotypes.

Next generation sequencing (NGS) technology allows genotyping for Human Leukocyte Antigen (HLA) genes with full resolution for multiple samples in a single run. Compared to traditional methods of SSO, SSP and Sanger sequencing, NGS delivers more complete coverage of the genome and phased contig sequences to resolve ambiguities. Despite the advantages of the NGS technology, validation at 3 and 4 field resolution of the HLA typing results is challenging because other technologies provide only up to 3 field resolution. We first introduce a HLA typing method based on NGS developed for high throughput applications and then the accuracy is assessed through pedigree analysis on a large cohort of family trios from a disease association study. This high throughput typing method can generate accurate genotype calls for all 11 major HLA genes with 4 field resolution based on NGS technology. For data analysis, three orthogonal algorithms are combined to rank the genotype candidates and generate consensus sequences for individual alleles. Ambiguity is resolved for heterozygous sample by phasing analysis except for certain allele combinations in DPB1. The method can be used to type 96–384 samples in a single sequencing run for high throughput HLA typing applications, including registry typing, disease association and population studies. For family trios, the accuracy of genotyping results at 3 and 4 field resolution is assessed through pedigree analysis. Approximately 1000 family trios are typed by the above method. At 3 field resolution, the validated genotyping accuracies for automatic calls are above 98.8% and 98.3% for class I and class II genes respectively. For reviewed calls, the validated accuracies are around 99.9% and 99.8%. The concordance between automatic and reviewed calls is above 99.4%. The validation based on pedigree analysis provides a way to assess the accuracy of HLA genotyping methods with 3 and 4 field resolution. The high concordance rate between automatic and reviewed calls showed that the software provides high quality calls and the review process is made effortless for trained technicians. H. Ollila : Grant/Research Support; Company/Organization; 5R01MH096262 . D. Levinson : Grant/Research Support; Company/Organization; 5R01MH096262.

In myeloablative unrelated donor allogeneic hematopoietic cell transplantation (HCT) a 1-locus HLA-mismatch (-A, -B, -C, -DRB1) is associated with lower survival compared to fully matched pairs. However data in reduced-intensity and non-myeloablative conditioning (together called RIC) HCT are limited. We analyzed adult AML/ALL/CML/MDS recipients of first 8/8 HLA-matched or 1-locus mismatched unrelated donor (MUD, MMUD) RIC HCT performed in the period 1999-2011 and registered in the CIBMTR. HLA-A, -B, -C and -DRB1 loci were typed in all pairs at high resolution; -DQB1 and -DPB1 loci could not be evaluated in all pairs. Transplants involving ex-vivo T-cell depletion, CD34+ selection, or post-transplant cyclophosphamide were excluded. Overall survival (OS) was the primary outcome. Secondary outcomes included non-relapse mortality (NRM), relapse, disease-free survival (DFS) and acute and chronic GVHD. Individual locus mismatch was also assessed. Apart from HLA matching, variables related to patient (age, race, sex, KPS, diagnosis, disease-risk), donor (age, parity), both (sex match/ABO match/CMV match) treatment (conditioning intensity, TBI use, in-vivo T-cell depletion (ATG), graft source (PB, BM) and GVHD prophylaxis (CyA-, Tac-based)) were considered. 2588 RIC HCT (8/8 MUD: 2025; 7/8 MMUD: 563) from 144 centers and 12 countries were analyzed. Median follow up in 8/8 MUD and 7/8 MMUD was 38 and 48 months respectively. Diagnoses were AML (65%), ALL (8%), CML (7%), MDS (20%). Conditioning intensity was RIC (79%), NMA (21%). 58% received in-vivo T-cell depletion. Graft source was PBSC (85%), BM (15%). GVHD prophylaxis was Tac-based (70%), CyA-based (27%). Mismatches involved HLA-A (188), -B (81), -C (219), and -DRB1 (75); with -DPB1 and -DQB1 typing available in 1382 and 2502 cases respectively. Compared to 8/8 MUD, 7/8 MMUD recipients were more likely to be younger and ethnic minorities and to have older and parous donors. In univariate analyses DQB1- and -DPB1 mismatch was not associated with worse OS, DFS, or NRM and was not further evaluated. There was a trend toward more grade II-IV acute GVHD in -DPB1 double (p=0.02) but not single mismatches. In multivariate models 7/8 MMUD RIC HCT had worse grade II-IV and III-IV acute GVHD, NRM, DFS and OS, but not relapse or chronic GVHD (Table). No significant interactions were identified between degree of HLA matching and other clinical variables. Adjusted 1- and 3-year NRM for 8/8 MUD vs. 7/8 MMUD was 20.4% vs. 28.9% (p Table 1 7/8 vs. 8/8 HLA HR (95% CI) p-value Acute GVHD II-IV 1.29 (1.09-1.53) 0.003 Acute GVHD III-IV 1.69 (1.00-3.36) 0.05 Chronic GVHD 1.11 (0.96-1.28) 0.15 Relapse 1.01 (0.87-1.17) 0.92 NRM 1.52 (1.29-1.79) Compared to 8/8 MUD, both 7/8 allele and antigen MMUD RIC HCT have greater treatment toxicity and worse survival, of a magnitude similar to that seen in myeloablative transplantation. An isolated mismatch at HLA-A, -B, -C, or -DRB1 was associated with 1 or more adverse outcomes. In unrelated donor RIC HCT, matching for all alleles of HLA-A, -B, -C and -DRB1 loci results in superior outcomes. Disclosures: No relevant conflicts of interest to declare.

Disease relapse is a significant cause of treatment failure after allogeneic hematopoietic stem cell transplantation (HCT). In the setting of reduced-intensity conditioning (RIC), a graft-versus-leukemia (GVL) effect is critical for successful outcomes in patients with advanced myeloid malignancies. A GVL effect has been attributed in part to donor-derived alloreactive natural killer (NK) cells, which are regulated by interaction of KIRs with their HLA-class I ligands. Several models of NK reactivity (missing KIR ligand, centromeric haplotype-B content, non-tolerized KIR2DS1) have been associated with improved outcomes following HLA-matched, HLA-mismatched, related, and unrelated donor HCT, particularly for AML patients given myeloablative conditioning. The effect of KIR-HLA combinations on outcomes after RIC HCT, however, is not known. We retrospectively analyzed donor-recipient KIR/HLA genotypes from 929 URD HCTs facilitated by the National Marrow Donor Program for patients with AML (n=624) or MDS (n=305) treated with RIC between 1990 and 2007. 664 donor-recipient pairs were 10/10 HLA-matched and 265 were 9/10 HLA-matched. 332 (37%) patients received ATG and 73 (8%) received alemtuzumab. P-values less than 0.01 were considered significant. We hypothesized that donor-recipient KIR-HLA interactions may be associated with improved post-transplant outcomes following RIC HCT for AML and MDS. Patients lacking the HLA-Bw4 ligand for donor inhibitory KIR3DL1 experienced lower relapse following HCT at 1 year (27 vs 36%, p=0.002) and 5 years (31 vs 42%, p=0.003) compared to patients with the Bw4 ligand (Figure). The lower risk for relapse was confirmed in multivariate analysis (HR 0.71, p=0.005). However, there was no significant association of Bw4 ligand with disease-free survival (HR 0.84, p=0.05) and overall survival (HR=0.97, p=0.70). Risk for acute GVHD was higher among patients lacking KIR ligands after adjusting for other clinical factors. In particular, patients lacking HLA-C2 for donor KIR2DL1 experienced higher grade 2-4 (HR 1.3, p=0.005) and 3-4 acute GVHD (HR 1.5, p=0.002), and patients lacking multiple KIR ligands experienced higher grade 3-4 acute GVHD (HR 1.5, p=0.007). The analysis was then restricted to AML patients, the patient population with greatest reported KIR-HLA effects. Patients whose donors were KIR2DS1+ and HLA-C2C2 (n=33) had higher transplant-related mortality (TRM) (HR, 2.4, p=0.002) compared to all other patients. There was no significant effect of KIR2DS1 with HLA on relapse. In a multivariate analysis, lack of HLA-C2 in AML patients was associated with higher grade 2-4 (HR 1.4, p=0.002) and 3-4 acute GVHD (HR 1.5, p=0.01), and risk for grade 3-4 acute GVHD was higher in patients lacking multiple KIR ligands (HR 1.6, p=0.005). There were no significant associations between donor homozygosity for the centromeric B-haplotype (cenBB) or overall B-haplotype KIR content and RIC HCT outcomes. Overall, these results suggest that in the RIC HCT setting, lack of Bw4 ligand for KIR3DL1 is associated with a lower risk of relapse for AML/MDS. This observation corroborates previous findings in myeloablative HCT transplants. Furthermore, tolerance of donor KIR2DS1 by HLA-C2C2 was associated with worse outcome, as manifested by higher TRM, in AML patients. In contrast, it appears that in RIC HCT homozygosity for the centromeric B-haplotype does not have a significant role in leukemia relapse. The associations of KIR ligands with acute GVHD were not previously observed suggesting that NK cell alloreactivity depends on multiple variables, including RIC. Further elucidation of the biology of NK cell alloreactivity in the RIC setting may provide guidance for future approaches to help optimize conditions for generating GVL reactions without GVHD and less TRM in this transplant population.FigureFigure. Disclosures: No relevant conflicts of interest to declare.

Aim The Arab Bone Marrow Registry in Hadassah Medical Center collected HLA typings for 18,000 Arab adult donors. We used this information to estimate population allele and haplotype frequencies (HFs). Methods We calculated HLA A ∼ B ∼ DRB1 HFs from 1,541 Christian Arab (CA) and 16,129 Muslim Arab (MA) donors typed by SBT, resolving phase and allelic ambiguity using Expectation Maximization. HFs were estimated at the four-digit level. Results The most common CA haplotype was A∗33:01 ∼ B∗14:02 ∼ DRB1∗01:02 at a frequency of 1.6%, which is the fifth most common MA haplotype at 0.9%. This haplotype was the second ranked in the US Middle Eastern/North Coast of Africa (MENAFC) at 0.8% and is also a common Jewish haplotype. The most common MA haplotype was A∗01:01 ∼ B∗52:01 ∼ DRB1∗15:02 at 1.4%, which is ranked 41st in CA at 0.04%. This haplotype was ranked 12th in MENAFC at 0.26%. The fourth ranked haplotype for both CA and MA, A∗02:05 ∼ B∗50:01 ∼ DRB1∗07:01, is the most common in Saudi Arabia, Kuwait and Qatar and the fourth most common among Jewish populations. Average homozygosity for CA and MA for HLA-A, -B and -DRB1 was estimated at 6.7% and 6.2% while the top 30 haplotypes had a cumulative frequency of 25% and 19% respectively indicating higher MA genetic diversity. Interestingly, eight DRB1 alleles: 01:02, 03:01, 04:03, 07:01, 11:01, 11:04, 13:03 and 15:02 constituted over 63% of the allele frequencies for both CA and MA while 97% and 30% of patients served by the Arab registry had one or two of these alleles respectively. Additionally, DRB1∗03:01 occurred without the common Caucasian B∗08:01 association in about 85% of CA and MA donors. Clinical data showed that 40% of transplants were for Arab patients while others were for patients worldwide. Conclusions Comparing CA and MA to other published HFs, it is clear that some haplotypes have migrated across populations while others remain isolated, thus adding these donors to the world registry is vital to increase genetic diversity for the benefits of all patients.