Killer Immunoglobulin-like Receptor Haplotypes Research Articles

The role of Killer immunoglobulin-like receptors (KIRs) in the genetic susceptibility to non-celiac wheat sensitivity (NCWS).

Non-celiac wheat sensitivity (NCWS) is an emerging clinical condition characterized by gastrointestinal and extraintestinal symptoms following the ingestion of gluten-containing foods in patients without celiac disease (CD) or wheat allergy. Despite the great interest for NCWS, the genetic risk factors still need to be fully clarified. In this study, we first assessed the possible contribution of KIR genes and KIR haplotypes on the genetic predisposition to NCWS. Fifty patients with NCWS, 50 patients with CD, and 50 healthy controls (HC) were included in this study. KIR genes and KIR genotyping were investigated in all subjects by polymerase chain reaction with the sequence oligonucleotide probe (PCR-SSOP) method using Luminex technology. We found a statistically different distribution of some KIR genes among NCWS, CD, and HC. Specifically, NCWS showed a decreased frequency of KIR2DL1, -2DL3, -2DL5, -2DS2, -2DS3, -2DS4, -2DS5, and -3DS1 genes, and an increased frequency of -3DL1 gene respect to both CD and HC. No difference was detected in the KIR haplotype expression. At the multivariate analysis, KIR2DL5, -2DS4, and -2DS5 were independent predictors of NCWS. Our findings suggest a role of KIR genes in NCWS susceptibility, with KIR2DL5, -2DS4, and -2DS5 having a protective effect. Further large-scale multicentric studies are required to validate these preliminary findings.

Centromeric AA motif in KIR as an optimal surrogate marker for precision definition of alloimmune reproductive failure

Throughout pregnancy, the decidua is predominantly populated by NK lymphocytes expressing Killer immunoglobulin-like receptors (KIR) that recognize human leukocyte antigen-C (HLA-C) ligands from trophoblast cells. This study aims to investigate the association of KIR-HLA-C phenotypes in couples facing infertility, particularly recurrent pregnancy loss (RPL) and recurrent implantation failure (RIF), in comparison to a reference population and fertile controls. This observational, non-interventional retrospective case–control study included patients consecutively referred to our Reproductive Immunology Unit from 2015 to 2019. We analyzed the frequencies of KIR and HLA-C genes. As control groups, we analyzed a reference Spanish population for KIR analysis and 29 fertile controls and their male partners for KIR and HLA-C combinations. We studied 397 consecutively referred women with infertility and their male partners. Among women with unexplained RPL (133 women) and RIF (176 women), the centromeric (cen)AA KIR genotype was significantly more prevalent compared to the reference Spanish population (p = 0.001 and 0.02, respectively). Furthermore, cenAA was associated with a 1.51-fold risk of RPL and a 1.2-fold risk of RIF. Conversely, the presence of BB KIR showed a lower risk of reproductive failure compared to non-BB KIR (OR: 0.12, p < 0.001). Women and their partners with HLA-C1C1/C1C1 were significantly less common in the RPL-Group (p < 0.001) and RIF-Group (p = 0.002) compared to the control group. Moreover, the combination of cenAA/C1C1 in women with C1C1 partners was significantly higher in the control group than in the RPL (p = 0.009) and RIF (p = 0.04) groups, associated with a 5-fold increase in successful pregnancy outcomes. In our cohort, the cenAA KIR haplotype proved to be a more accurate biomarker than the classic AA KIR haplotype for assessing the risk of RPL and RIF, and might be particularly useful to identify women at increased risk among the heterogeneous KIR AB or Bx population. The classification of centromeric KIR haplotypes outperforms classical KIR haplotypes, making it a better indicator of potential maternal–fetal KIR-HLA-C mismatch in patients.

Combined flow cytometry natural killer immunophenotyping and KIR/HLA-C genotyping reveal remarkable differences in acute myeloid leukemia patients, but suggest an overall impairment of the natural killer response.

Natural killer (NK) cells are key anti-tumor effectors of the innate immunity. Phenotypic differences allow us to discriminate in between three functional stages of maturation, named immature, mature and hypermature that are distinctive in terms of receptor expression, cytokine secretion, cytotoxic properties and organ trafficking. NKs display an impressive repertoire of highly polymorphic germline encoded receptors that can be either activating, triggering the effector's function, or inhibitory, limiting the immune response. In our study, we have investigated peripheral blood NK cells of acute myeloid leukemia (AML) patients. The Killer Immunoglobulin-like receptors (KIRs) and the HLA-C genotypes were assessed, as HLA-C molecules are cognate antigens for inhibitory KIRs. The AA mainly inhibitory KIR haplotype was found in a higher proportion in AML, while a striking low frequency of the 2DS3 characterized the mainly activating Bx haplotype. Flow cytometry immunophenotyping evidenced a lower overall count of NK cells in AML versus healthy controls, with lower percentages of the immature and mature subpopulations, but with a markedly increase of the hypermature NKs. The analysis of the KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, and NKG2A inhibitory receptors surface expression revealed a remarkable heterogeneity. However, an overall trend for a higher expression in AML patients could be noticed in all maturation subpopulations. Some of the AML patients with complex karyotypes or displaying a FLT3 gene mutation proved to be extreme outliers in terms of NK cells percentages or inhibitory receptors expression. We conclude that while the genetic background investigation in AML offers important pieces of information regarding susceptibility to disease or prognosis, it is flow cytometry that is able to offer details of finesse in terms of NK numbers and phenotypes, necessary for an adequate individual evaluation of these patients.

The Influence of Maternal KIR Haplotype on the Reproductive Outcomes after Single Embryo Transfer in IVF Cycles in Patients with Recurrent Pregnancy Loss and Implantation Failure—A Single Center Experience

(1) Background: Recurrent pregnancy loss (RPL) and recurrent implantation failure (RIF) have in common a deficient maternal adaptation to the semi-allogeneic fetus, in which killer immunoglobulin-like receptor (KIR) family expressed by natural killer (NK) cells play an important role. The aim of this study was to evaluate the influence of maternal KIR haplotype on the reproductive outcomes after single embryo transfer in IVF cycles in patients with RPL and RIF. (2) Methods: Patients with RIF and RPL who presented at Origyn Fertility Center from Iasi, Romania, were prospectively enrolled between January 2020 and December 2022. Clinical and paraclinical data was examined. Descriptive statistics and a conditional logistic regression model were used to analyze our data. (3) Results: Patients with a KIR AA haplotype had significantly more chances of miscarriage if they underwent an IVF procedure (aOR: 4.15, 95% CI: 1.39-6.50, p = 0.032) compared with those who spontaneously achieved a pregnancy. Moreover, it appeared that the same haplotype increased the chances of obtaining a pregnancy for patients who underwent an IVF procedure (aOR: 2.57, 95% CI: 0.85-6.75, p = 0.023). (4) Conclusions: Determination of KIR haplotype could be beneficial for patients with RPL or RIF in order to offer an individualized management.

Clinical impact of KIR haplotypes in 10/10 HLA-matched unrelated donor-recipient pairs undergoing allogeneic hematopoietic stem cell transplantation

To evaluate the impact of killer immunoglobulin-like receptor (KIR) genotyping in allogeneic hematopoietic stem cell transplantation for myeloid disorders at our institution, retrospective KIR genotyping was performed on 77 patients and their 10/10 matched unrelated donors. In a multivariate model including donor age, HLA-DPB1 permissiveness, and presence of donor KIR B/x, an association with overall survival was observed (p = .047). Within the model, increasing donor age increased risk (RR 1.03 [1.00–1.06]/year, p = .046), while donor KIR and HLA-DPB1 permissiveness were not associated with risk (RR 0.51 [0.26–1.03] and RR 0.68 [0.34–1.36]). Grouping recipients by conditioning regimen or limiting the analysis to recipients of peripheral blood stem cells, no association between donor KIR and survival or relapse was identified. No significant associations were observed between overall survival, relapse, grade III-IV acute, or chronic graft versus host disease and presence of KIR B (B/x), quantity of donor KIR B haplotype motifs, or centromeric KIR type (all p > .05).

Matched Unrelated Donor Hematopoietic Cell Transplant with Increased Donor Inhibitory KIR Improves Survival in a Pediatric Acute Leukemia: A CIBMTR Cohort Analysis

Natural killer (NK) cells potentially mediate innate immunity early after hematopoietic cell transplantation (HCT). NK cells posses an array of activating and inhibitory receptors on their surface, including killer immunoglobulin-like receptor (KIR) and which interact with KIR-ligands (KIRL) on target cells. The impact of NK cell mediated alloreactivity in pediatric HCT is not well characterized. In this study, we explored a mathematical approach to quantify KIR-KIRL combinations to assess clinical outcomes in a cohort of pediatric patients who underwent HLA-matched unrelated donor (MUD) HCT for acute leukemia including AML and ALL. This retrospective study was performed on deidentified donor and recipient demographic and clinical outcomes data provided by the Center for International Blood and Marrow Transplant Research (CIBMTR). KIR-KIRL interaction values were assigned as previously (Krieger et. al 2021, Bone Marrow Transplantation). Briefly donor inhibitory (iKIR) and activating (aKIR) KIR- recipient KIRL interactions, accounting for missing KIR-L (mKIR), were summed up, using a system of matrices. The study cohort was comprised of 686 pediatric donor-recipient pairs (DRP) who underwent MUD HCT for AML (N=355) and ALL (N=333) and had neutrophil engraftment (Table 1). This cohort of pediatric patients was aged 0-18 years (average 11). All patients received T cell replete grafts; 26% received in vivo T cell depletion, with either anti-thymocyte globulin (ATG) or alemtuzumab. The majority, 77% received a bone marrow graft and 90% received myeloablative conditioning. The effect of all 3 KIR-KIRL interaction scores (absolute values of iKIR, mKIR and aKIR) on clinical outcomes were evaluated. Higher donor iKIR score was associated with improvement in overall survival (OS) SHR 0.73 (multivariate P=0.003) and relapse-free survival (RFS), SHR 0.8 (P=0.019); grade 3-4 acute GVHD was lower in patients transplanted with higher iKIR or mKIR scores (iKIR; SHR 0.58 (P=0.013); mKIR SHR 0.6 (P=0.041)). Higher aKIR scores, were not significantly associated with any clinical outcomes examined. As in previous analysis, the iKIR and mKIR scores were added to give an IM-KIR score, which was dichotomized for analysis into those DRP with an IM KIR score of 5 (IM = 5), and those with an IM-KIR score of 2, 3 or 4 (IM < 5). Of note, KIR haplotype A patients always have an IM KIR score of 4, and are thus categorized among IM<5 donors. In this cohort of pediatric patients transplanted for both AML and ALL, those transplanted with IM=5 donors demonstrated improved OS, SHR 0.75 (P = 0.027), RFS, SHR 0.79 (P=0.05) and grade 3-4 aGVHD incidence, SHR 0.37 (P= 0.003) in multivariate analysis. We next examined outcomes in patients with ALL and AML to understand disease specific effects. Patients transplanted for AML with IM=5 donors had an improvement in OS, SHR 0.62 (P=0.01), as well as RFS, SHR 0.70 (p=0.033) (Fig. 1). There was a trend towards relapse risk reduction in this cohort SHR 0.67 (univariate P=0.048). Patients transplanted for ALL with IM=5 donors demonstrated a marked decrease in grade 3-4 acute GVHD incidence SHR, 0.31 (P=0.017). This large pediatric study confirms that unrelated DRPs with donor haplotype B genotypes and a higher inhibitory KIR content (regardless of KIR ligand status), confer significant improvement in overall survival and severe aGVHD after MUD HCT. Those patients transplanted for AML experienced an improvement in OS and RFS despite the lower event rate observed in pediatric-age patients, the majority of whom receive myeloablative preparatory regimens without the use of in vivo T cell depletion. This challenges the notion that KIR are irrelevant to pediatric donor selection. Future clinical trials evaluating donor selection for URD HCT should include this measure to evaluate its value prospectively. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Allelic Polymorphisms of Killer Immunoglobulin-Like Receptor Genes in Malay and Orang Asli Populations of Peninsular Malaysia

Next-generation DNA sequencing (NGS) technology advancements provide new insight into the level of variation in killer immunoglobulin-like receptor (KIR) genes. High resolution allele genotyping of seven KIR genes was conducted among 94 unrelated Malay and Orang Asli (OA) individuals of Peninsular Malaysia. A manual bioinformatics analysis is performed and optimised by Sanger sequencing method. The Malays expressed a total of 22 alleles, as compared to only 15 alleles in the OA population. In total, 12 centromeric and 9 telomeric allelic haplotypes were identified in the Malays, whereas 8 centromeric and 5 telomeric allelic haplotypes were identified in the OA. The KIR2DL1, KIR2DL3, and KIR2DS4 genes exhibited a high degree of variation and balanced distribution in the Malay and OA populations. On the other hand, KIR2DL4, KIR3DL1, KIR3DL2 and KIR3DL3 genes exhibited a high degree of conservation, with less number of alleles identified and the dominance of a single allele at high frequency. High-resolution KIR allele genotyping has revealed unique sequence variations and allelic haplotypes between individuals and populations. The distributions of KIR alleles and haplotypes are useful for genetic population studies and serve as a baseline for future transplantation matching and disease association research.

Microenvironment Profiling in Myelodysplastic Syndromes and Its Relationship to Clonal Hematopoiesis and Disease Progression

Microenvironment Profiling in Myelodysplastic Syndromes and Its Relationship to Clonal Hematopoiesis and Disease Progression

Genetic Association Analysis Between KIR2DS4 and Its Variant KIR1D With Pediatric Acute Lymphoblastic Leukemia

IntroductionThe killer immunoglobulin-like receptor (KIR) gene family encodes receptors of natural killer (NK) cells and some T cell subsets and is thereby involved in the regulation of anti leukemia effects. Every human being possesses an individual pattern of KIR genes, which can be assigned to either KIR haplotype A (which lacks KIR2DL2, KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS5 and KIR3DS1) or haplotype B (which contains at least one of the activating KIRs, KIR2DL2 or KIR2DL5). KIR2DS4 is the only activating receptor with a short cytoplasmic tail, which can be found in individuals with KIR haplotype A. Interestingly, besides the cell membrane-anchored receptor, a truncated soluble protein (designed as KIR1D) is produced once the 22 bp deletion in exon 5 of KIR2DS4 is present. Previously, Giebel et al., Leukemia 2008, reported the association of KIR1D with leukemia, in particular with chronic myelogenous leukemia (CML). The aim of our study was to clarify whether the KIR1D allelic variant plays any role for innate immunosurveillance against acute lymphoblastic leukemia (ALL) and whether KIR1D subclassification is helpful for donor selection in the case of pediatric ALL before undergoing haploidentical hematopoietic cell transplantation (HHCT). Patients and Methods312 pediatric patients with ALL and 222 healthy controls were analyzed for the presence of KIR2DS4 and its variant KIR1D and then further subdivided into the following groups: 2DS4-/1D+ (homozygous for the deletion variant KIR1D), 2DS4+/1D+ (heterozygous), 2DS4+/1D- (two intact KIR2DS4 alleles) and 2DS4-/1D- (no KIR2DS4 allele). Furthermore, we investigated the influence of the KIR2DS4 variants on event-free and relapse of 69 patients with ALL receiving HHCT. The 22 bp deletion of KIR2DS4 was determined by PCR and agarose gel electrophoresis using primers described by Alves et al., Tissue Antigens 2009. ResultsOf the 312 patients with ALL, n=160 (51.3%) were identified as 2DS4-/1D+, n=101 (32.4%) as 2DS4+/1D+, n=35 (11.2%) as 2DS4+/1D- and n=16 (5.1%) as 2DS4-/1D-. The frequencies in the control group were n=136 (61.3%) for 2DS4-/1D+, n=55 (24.8%) for 2DS4+/1D+, n=25 (11.3%) for 2DS4+/1D- and n=6 (2.7%) for 2DS4-/1D-. In our study, the occurrence of 2DS4+ alleles, as well as 1D+ alleles were similar for patient and control group (p>0.050, respectively).In order to discover any possible impact of the different KIR2DS4 variants on outcome after HHCT, a retrospective analysis using cumulative incidence adjusted for competing risk (CICR, Scrucca et al., Bone marrow transplantation 2007) was performed. As previously reported by our group, patients with ALL transplanted from a KIR B haplotype donor were found to have lower relapse rates (Oevermann et al., ASH 2011). In the presented study, n=34 (49.3%) stem cell donors showed 2DS4-/1D+, n=20 (29.0%) 2DS4+/1D+, n=5 (7.2%) 2DS4+/1D-, n=10 (14.5%) 2DS4-/1D-. We found that the various KIR2DS4 variants did not influence incidence of relapse rate (2DS4-/1D+ 42.5%, 2DS4+/1D+ 55.0%, 2DS4+/1D- 20.0%, 2DS4-/1D- 30.0%, p=0.514), as well as event-free survival of ALL patients (2DS4-/1D+ 45.7%, 2DS4+/1D+ 40.0%, 2DS4+/1D- 60.0%, 2DS4-/1D- 50.0%, p=0.870). ConclusionWe conclude from our results that KIR1D seems to have no meaningful influence on immunosurveillance against ALL. In contrast to KIR haplotype B donor selection, the preference of a KIR1D positive donor has no additional beneficial impact on the outcome of patients with ALL receiving HHCT. Disclosures:No relevant conflicts of interest to declare.

Increased donor inhibitory KIR with known HLA interactions provide protection from relapse following HLA matched unrelated donor HCT for AML.

Killer immunoglobulin-like receptor (KIR) and KIR-ligand (KIRL) interactions play an important role in natural killer cell-mediated graft versus leukemia effect (GVL) after hematopoietic cell transplant (HCT) for AML. Accounting for known KIR-KIRL interactions may identify donors with optimal NK cell-mediated alloreactivity and GVL. A retrospective study of 2359 donor-recipient pairs (DRP) who underwent unrelated donor (URD) HCT for AML was performed. KIR-KIRL combinations were determined and associations with clinical outcomes examined. Relapse risk was reduced in DRP with both higher inhibitory KIR-KIRL (iKIR) and missing KIRL (mKIR) scores, with HR 0.86 (P = 0.01) & HR 0.84 (P = 0.02) respectively. The iKIR and mKIR score components were summed to give a maximal inhibitory KIR ligand (IM-KIR) score for each donor, which if it was 5, as opposed to <5, was also associated with a lower relapse risk, SHR 0.8 (P = 0.004). All IM = 5 donors possess KIR Haplotype B/x. Transplant-related mortality was increased among those with IM-KIR = 5, HR, 1.32 (P = 0.01). In a subset analysis of those transplanted with 8/8 HLA-matched DRP, anti-thymocyte globulin recipients with IM-KIR = 5, had a lower relapse rate HR, 0.61 (p = 0.001). This study demonstrates that HLA-matched unrelated donors with the highest inhibitory KIR content confer relapse protection, albeit with increased TRM. These donors all have KIR haplotype B. Clinical trials utilizing donors with a higher iKIR content in conjunction with novel strategies to reduce TRM should be considered for URD HCT in recipients with AML to optimize clinical outcomes.

KIR Genotype and KIR Ligand Phenotypes That Improve the in Vitro Cord Blood or Autologous NK Cell Mediated Cytotoxicity Against Myeloma Marrow Plasma Cells

<h3>Instruction</h3> Killer Immunoglobulin-like Receptor (KIR) inhibition is known to influence the cytotoxic effects of natural killer (NK) cells against cancer cells. KIRs can be protective against relapse in myeloma (MM) (Gabriel et al Blood 2010, Kröger et al leukemia 2011) by NK cell licencing <i>or through</i> blocking of the inhibitory KIR receptors such as NKG2A (Mahaweni et al, 2018). <h3>Objectives</h3> In this study, the role of KIR geno/phenotypes and their ligands on autologous and cord blood (CB) derived NK cell mediated cytotoxicity against MM plasma cells (PC) were investigated. <h3>Methods</h3> Peripheral blood NK cells and marrow PCs were isolated from 20 patients with relapsed MM. PC were isolated from fresh BM aspirates using EasySep Human CD138 Positive Selection Kit (StemCell Technologies) and were subjected to cytotoxicity assays with either autologous or CB derived NK cells isolated (Human NK cell Enrichment Cocktail, Stemcell Technologies) and cultured in the presence of IL-15 (10ng/ml) for 2-8 days. KIR/KIR ligand genotyping were performed by Olerup SSP KIR Genotyping Kit (Olerup, Stockholm, Sweden) in order to detect KIR genotypes; KIR2DL1, 2DL2, 2DL3, 2DL4, 2DL5A/B, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1, 2DP1, 3DP1, and KIR ligands; HLA-ABw4+, HLA-BBw4+Thr80, HLA-BBw4+Ile80, HLA-BBw4+Asp77,Thr80, HLA-CAsn80, and HLA-CLys80. Effector cells were stained with antibodies against NKG2A (CD159a) and CD158i (KIR2DS4) and analyzed using a Beckman Coulter Flow Cytometer. <i>Cytotoxicity assays</i>: Using <i>3,3</i> (DiO) as an indicator and 1:10 target:effector cell ratio PC death was evaluated after 120 mins of incubation, using flow cytometry. <h3>Results</h3> 20 MM patients were tested in vitro. Following incubation of PC wo NK cells, spontaneous PC death ratio was found to be 22.0% (11.4%-51.2%). While autologous NK mediated cytotoxicity was median 17.3% (11.4%-43.6%), CB-NK cell mediated cytotoxicity was 23.2% (range: 11.5%-51.2%). Highest cytotoxicity was achieved with CB-NK cells (CB7) with a KIR AB haplotype, carrying 8 inhibitory KIR genes against PC from MM12. Effector cells with the iKIR gene number above 6 were found to cause higher cytotoxicity (13/19; p=0.031). Additionally, higher (>30%) expression of NKG2A on CB-NK cells were found to be associated with lack of cytotoxicity. <h3>Conclusion</h3> CB-derived NK cells were found to be able to induce higher PC death than autologous PB-NK cells. Also higher number of iKIR genotypes and lacking KIR2DS4 receptor was found to be associated with higher cytotoxicity. In our current study we are reporting KIR NKG2A which binds with HLA-E to play an additional role. Allogeneic NK cell mediated immunotherapies are promising due their clinical advantages ie no need for HLA matching. Furthermore certain KIR haplotypes (ie KIR2DS4⁺ or NKG2A⁻) may offer better therapeutic efficacy if confirmed by others as well.

Interactions between Donor Activating Killer Immunoglobulin-like Receptors (KIRs) and Somatic Mutations and Their Association with Outcomes after Allogeneic Hematopoietic Cell Transplant for Acute Myeloid Leukemia (AML)

Background: Graft-versus-leukemia (GVL) responses after allogeneic hematopoietic cell transplantation (alloHCT) for AML are mediated by alloreactive donor-derived immune effector cells including T lymphocytes and natural killer (NK) cells. The function of NK cells is regulated by inhibitory and activating signals mediated through cell-surface receptors, including KIRs. Various models of NK cell alloreactivity have been associated with post-transplant outcomes, including leukemia relapse. However, these results have varied widely between different investigators employing similar models of NK cell alloreactivity. Assessment of somatic mutations in AML on post-transplant outcomes has not been investigated in the context of KIR profiles. Methods: In this single-institution retrospective cohort study, we investigated KIR haplotypes (haplotype AA vs. Bx [associated with multiple activating KIRs]; Cooley S., et al. Blood. 113:726-732. 2009) in the context of somatic mutations. We included 34 adult patients with AML who underwent alloHCT from a matched related donor from 2006 to 2013. A targeted multi-amplicon deep NGS panel of 79 commonly mutated genes in myeloid neoplasia was performed. Post-HCT outcomes were assessed based on mutational status and KIR haplotype with Kaplan-Meier method and log-rank test. Results: Median age at transplant was 54 (range 31-73). Cytogenetic risk groups were 9% favorable, 56% intermediate, and 35% poor based on 2017 ELN classification. HCT-CI scores included 26% low, 32% intermediate, and 41% high. Disease risk group defined by ASTCT included 71% low, 26% intermediate, and 3% high. Disease status at HCT included 74% CR1 and 26% CR2. Frequencies of somatic mutations prior to HCT were: 21% DNMT3A, 18% IDH2, 9% each for STAG2 and NRAS, 6% each for ASXL1, JAK2, PHF6, RUNX1, TET2, and 3% each for CBL, FLT3, NPM1, and U2AF1. Overall, 53% of patients had at least 1 mutation: 24%, 18%, 9%, and 3% of patients had 1, 2, 3, and 4 mutations, respectively. 41% were carriers of KIR haplotype AA, and 59% were haplotype Bx. Relapse (p=0.40), relapse-free (p=0.33), and overall survival (p=0.30) between haplotypes AA and Bx were not statistically different. However, when considering somatic mutations in the context of KIR haplotypes, those with any somatic mutation (n= 18) present had inferior relapse-free (p=0.002) and overall survival (p=0.002; figures A-B) as compared to those with none. Further assessment of outcomes was then considered for those who had the following poor prognostic mutations (n=12): ASXL1, DNMT3A, FLT3, NRAS, RUNX1, and TET2. KIR haplotype AA with one or more of these mutations was associated with inferior relapse-free (p=0.05) and overall survival (p=0.008). At median follow-up of 83 (range 66-137) months, 38% were alive. Non-relapse mortality rates were 21% (9-36) at 1 year and 29% (15-39) at 3 years. The most common causes of death for all patients were relapse (48%) followed by infection (33%). Conclusion: In presence of somatic mutations, carrying KIR haplotypes Bx was associated with better survival in AML post-alloHCT. The presence of multiple activating KIRs may also help mitigate the worse prognosis associated with some of the more deleterious somatic mutations in AML. These observations may have implications for improving patient risk stratification prior to transplant and optimizing donor selection. Future investigation with larger cohorts interrogating KIR haplotypes in the context of pre-transplant AML somatic mutations on post-transplant outcomes may further elucidate which patients may benefit most from transplant. Disclosures Nazha: Tolero, Karyopharma: Honoraria; Abbvie: Consultancy; Daiichi Sankyo: Consultancy; Novartis: Speakers Bureau; Jazz Pharmacutical: Research Funding; Incyte: Speakers Bureau; MEI: Other: Data monitoring Committee. Mukherjee:Bristol-Myers Squibb: Speakers Bureau; Projects in Knowledge: Honoraria; Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Partnership for Health Analytic Research, LLC (PHAR, LLC): Consultancy; McGraw Hill Hematology Oncology Board Review: Other: Editor; Pfizer: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees. Advani:Pfizer: Honoraria, Research Funding; Macrogenics: Research Funding; Glycomimetics: Consultancy, Research Funding; Kite Pharmaceuticals: Consultancy; Amgen: Research Funding; Abbvie: Research Funding. Gerds:Sierra Oncology: Research Funding; Incyte: Consultancy, Research Funding; CTI Biopharma: Consultancy, Research Funding; Imago Biosciences: Research Funding; Celgene Corporation: Consultancy, Research Funding; Roche: Research Funding; Pfizer: Consultancy. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Majhail:Mallinckrodt: Honoraria; Atara Bio: Consultancy; Anthem, Inc.: Consultancy; Nkarta: Consultancy; Incyte: Consultancy. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.

Variability in Killler Immunglobulin like Receptor Gene Expression As a Periodic Function of Gene Position on Chromosome 19

Killer Immunoglobulin Like Receptors (KIR) are expressed on natural killer (NK) and T cell surface. KIR interactions with KIR-ligands have been implicated in outcomes of hematopoietic stem cell transplantation, placental implantation, autoimmune disease and viral infections. While these KIR interactions are clearly important our understanding of what governs their expression is lacking. The KIR Locus is made up of a highly polymorphic and homologous set of genes located on Chromosome 19q13.4 within the leukocyte receptor complex. Unique to the KIR Gene Cluster, KIR haplotypes not only vary allelically, but they also differ in the number of KIR genes present in different individuals, ranging from 7 to 12 genes. KIR haplotypes have been divided into 2 broad groups; Haplotypes A &amp; B, based on the number of activating and inhibitory KIR genes they possess; where haplotype A contains only one activating allele KIR2DS4 and haplotype B contains various combinations of activating alleles. Expression of KIR on the NK cell surface stochastic, with some KIR found more commonly then others. In this abstract we aim to explore the relationship of KIR gene expression and the organization of the KIR gene locus using available genomic and tissue expression data. KIR gene coordinates on chromosome 19 were collected using the UCSC Genome Browser GRCh38/hg38 gene assembly and the NCBI gene website gene assembly NC_000019.10. Initial KIR gene nucleotide position along chromosome 19 were obtained and converted to angular distance (A.D. in radians) from reference. This was done to account for the double helical nature of DNA molecules, using the following equation A.D. = 2px/10.4; where x is the initial KIR gene nucleotide coordinate. Haplotype A was utilized as an initial test case as gene expression data are available for the genes in this haplotype. GTEX Portal was used to collect mRNA expression for each of the haplotype A genes, KIR-2DL1, -2DL3, -2DL4, -2DP1, -2DS4, -3DL1, -3DL2, -3DL3 &amp; -3DP1, including total (median), splenic and whole blood expression in Reads/Kilobase of transcript/million mapped reads (RPKM). Gene expression was determined via RNA-seq of 53 tissues from 570 donors. KIR haplotype A is located along chromosome 19 between nucleotide position 55,236,713 and 55,370,584 which corresponded with an A.D. of 33,354,476 to 33,435,314 radians. When the KIR gene expression was aligned with the coordinates of the corresponding gene on the KIR locus a distinct periodic pattern of variation in expression levels was observed across the KIR genes comprising Haplotype A (Figure 1). This was true for total KIR expression from across various organs, as well as in blood and splenic tissue. This is borne out by expression data on several Haplotype A KIR genes reported in the literature (Figure 1, yellow curve). These observations suggesting that KIR gene expression may be a periodic function of the gene coordinates on the chromosome are analogous to the periodicity observed in T cell receptor VDJ recombination (Meier et al, BBMT 2019 25(5);868). This observation supports the idea that the spiral structure of the helical DNA molecules coiled around the histone molecules and their arrangement on chromosomes may have a fundamental influence on the expression of different genes, perhaps in conjunction with known epigenetic influence of DNA methylation and histone acetylation. This may also point to a role for the non-coding DNA in regulation of gene expression. We posit that the helical DNA chromosome structure may have a fundamental role in determining gene expression, as exemplified by the KIR and T cell receptor gene loci. This knowledge may allow an improved understanding of variability in global gene expression. Disclosures No relevant conflicts of interest to declare.

Clinical Significance of KIR Genotype in Patients with Methotrexate-Associated Lymphoproliferative Disorders

Background: Methotrexate-associated lymphoproliferative disorders (MTX-LPD) develops in patients with rheumatoid arthritis (RA) or other autoimmune disorders during low-dose MTX treatment. MTX-LPD includes wide variety disease spectrum, ranging from polymorphic proliferation to aggressive lymphoma. Although etiology of MTX-LPD has not been fully understood, approximately half of the MTX-LPD cases showed association with EB virus (EBV), suggesting that MTX treatment causes reduced immune response to EBV-positive cells, and results in MTX-LPD development. Natural killer (NK) cells play important roles in eradicating tumor and virus-infected cells. NK cell function is modulated by multiple cell surface receptors, including Killer immunoglobulin-like receptor (KIR). There are multiple KIR genes (inhibitory or activated), which are various in number and/or composition among individuals, on chromosome 19q. Previous reports demonstrated that combination of KIR genes affects NK cell function, and is associated with the risk of development of certain types of cancers, viral infections and collagen disease. There is no report about the association of KIR genotype and MTX-LPD. We consider that NK cells play a significant role in suppression of MTX-LPD development. In this study, we focused on examining genotype KIR and KIR-ligand (HLA class I). Methods: We retrospectively analyzed 35 MTX-LPD cases diagnosed between 2009 and 2019. Genomic DNA was extracted from mononuclear cells that were isolated from the bone marrow or peripheral blood samples of patients with MTX-LPD. KIR genotypes were analyzed using the KIR genotyping sequence-specific primers kit. The variations of KIR content and haplotype and their relationship with progression to malignant lymphoma (ML) and response to chemotherapy were investigated. HLA was analyzed using PCR-Luminex assay. The frequency of each HLA allele and each combination was determined by referring to the data base of an HLA laboratory. Chi-squared (χ2) tests and Wilcoxon rank sum tests were used to test associations between the variables. Results: Among the 35 patients, 25 were diagnosed with ML and 10 with polymorphic LPD. Diffuse large B cell lymphoma (DLBCL) was most common type in ML (57.7%). Table 1 showed characteristics of patients and summary of the results. All patients underwent MTX treatment for RA. The median duration of MTX administration at the time of MTX-LPD diagnosis is 11.5 years (range=0.8-27.2), and median MTX dose was 10mg/week (range=4-17.6). The duration and dose of MTX had no effect between ML and polymorphic LPD. Twenty-three patients required chemotherapy, and 12 patients had tumor regression after stopping MTX treatment. Relative patient populations requiring chemotherapy in ML or polymorphic LPD were 85% or 11%, respectively (P=0.0001). EBV-positive patients tended to regress tumors with MTX discontinuation alone (P=0.16). In KIR genotype analysis, patterns of number and combination of the KIR genes are mainly classified as haplotype "A" containing multiple inhibitory KIR genes with a KIR 2DS4 (an activated KIR [aKIR]) and haplotype "B" (other than haplotype "A"). Patients were classified in haplotype A (13 cases, 37%) and haplotype B (22 cases, 63%), respectively. ML patients showed higher ratio in haplotype A (ML 46.2% vs LPD 11.1% P=0.045). There was no difference in number of aKIR or iKIR between ML and polymorphic LPD patients. In HLA Class I analyses, there was significant difference in frequencies of HLA-C haplotype between lymphoma and polymorphic LPD patients (P=0.026). Furthermore, HLA- C1 / C1 patients were more relapsed or refractory to chemotherapy than C1 / C2 patients (P = 0.17). Conclusion: This is the first report showing clinical significance of KIR genotypes in MTX-LPD. Patients with haplotype A, a suppressive haplotype, seems to be at high risk for developing lymphomas that require chemotherapy during MTX treatment. HLA- C1/C1 patients are more likely to develop lymphomas that respond poorly to treatment, suggesting that the activity of NK cells may be lower because ligands can match with KIRs that are more restricted than C1/C2. Considering the potential NK functions with KIR genotype would improve the understanding of the prognosis and lead to prevention for MTX-LPD. Disclosures Hagiwara: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees. Harigai:Bristol Myers Squibb Co: Other: personal fees, Research Funding; Eisai Co: Other: personal fees, Research Funding; Ayumi Pharmaceutical Co: Other: personal fees, Research Funding; AbbVie Japan GK,: Other: personal fees, Research Funding; Eli Lilly Japan K.K: Other: personal fees; Kissei Pharmaceutical Co.: Other: personal fees; Teijin Pharma Ltd: Other: personal fees, Research Funding; Mitsubishi Tanabe Pharma Co: Research Funding; Nippon Kayaku Co.: Research Funding; Pfizer Japan Inc.: Other: personal fees; Chugai Pharmaceutical Co., Ltd.: Other: personal fees; Japan College of Rheumatology: Other: personal fees; Boehringer Ingelheim Japan, Inc: Other: personal fees; GlaxoSmithKline K.K: Other: personal fees; Oxford Immuotec,: Other: personal fees. Tanaka:Bristol-Myers Squibb: Research Funding.

56+ Cell Reconstitution Kinetics in HLA Matched Unrelated Donor Allografts: What Doesn't Kill You Makes You Stronger

Introduction Patients who receive HLA matched allogeneic hematopoietic cell transplant (HCT) from donors with killer immunoglobulin-like receptor (KIR) haplotype B have improved survival, decreased acute GVHD, and less CMV reactivation when compared with those transplanted with KIR haplotype A donors. However, the effect of NK cell (CD56+) reconstitution kinetics on clinical outcomes in patients with different KIR haplotypes is not well studied. Objectives Compare CD 56+ cell reconstitution kinetics in HCT patients with donor KIR Haplotypes A and B and its effect on clinical outcomes. Methods We assessed absolute NK and T cell counts at days +30, +60 and +100 in 10/10 allele level HLA matched unrelated HCT recipients with known donor KIR typing. KIR haplotype B was assigned when KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS5 or KIR3DS1 were present in the donor, and haplotype A in their absence. We also evaluated potential impact of NK cell and T cell count recovery on key post transplant outcomes in these patients. Results The study population consisted of 60 adult HCT patients with a median age of 52 years; 55% had lymphoid and 45% had myeloid disease; 57% received a reduced intensity conditioning regimen; 82% underwent T cell depletion with ATG; 88% received GCSF mobilized PBSCT. Recipients of donor haplotype A had a median absolute CD56+ cell count of 210mL−1 (±156) on day (d) +30; 189 (±225) on d+60, and 160 (±120) on d+100; recipients of haplotype B had median absolute CD56 cell counts of 286 (±276) on d+30, 196 (±194) on d+60, and 165 (±124) on d+100 (P=N.S. for haplotype B vs. A). As a whole, median CD56+ cell counts peaked at d+30 post HCT, a phenomenon more pronounced in haplotype B (fig. 1), and then declined at d+60 and d+100. There was no correlation between CD56 and CD3 at d+60. Survival was superior in patients who had CD56+ cell count > median for the entire cohort (227 µL−1) on d+60 (p= .039) (fig. 2); ROC-AUC analysis confirmed the deleterious effect of low NK cell counts, with a higher mortality rate observed in those with CD56+ cell count of Conclusions The magnitude of NK cells reconstitution at d+60 may override the effect of KIR Haplotype on survival in patients with myeloid and lymphoid malignancies, as well as play an important role in controlling viral reactivation. Our data demonstrates that poor NK cell recovery does not necessarily imply delayed T cell reconstitution; suggesting that the adverse survival effect may be NK cell mediated. Ongoing work is examining relationship between NK- and T-cell reconstitution as well as KIR-KIR ligand interactions, and clinical outcomes.

Donor Killer Cell Immunoglobulin-Like Receptor Genotype Does Not Improve Graft-versus-Leukemia Responses in Chronic Lymphocytic Leukemia after Unrelated Donor Transplant: A Center for International Blood and Marrow Transplant Research Analysis.

Allogeneic hematopoietic cell transplantation (alloHCT) remains the sole curative therapy for patients with chronic lymphocytic leukemia (CLL), leading to 40% to 45% long-term survival. The impact of donor killer immunoglobulin-like receptor (KIR) genotype on outcomes of unrelated donor (URD) alloHCT for CLL is unknown. We examined 573 adult URD CLL recipient pairs. KIR genotype (presence/absence) was determined for each donor, and comprehensive modeling of interactions with recipient HLA class I loci (KIR ligands) was used to evaluate their effect on relapse and survival. Recipients had a median age of 56 years, and most were not in remission (65%). Both 8/8 HLA-matched (81%) or 7/8 HLA matched grafts (19%) were studied. Factors associated with improved overall survival (OS) were reduced-intensity conditioning (hazard ratio [HR] of death, .76) and good performance status (HR, .46), whereas alloHCT in nonremission (HR, 1.96) and mismatched donors (HR, 2.01) increased mortality. No models demonstrated a relationship between donor KIR genotype and transplant outcomes. Cox regression models comparing donors with A/A versus B/x KIR haplotypes and those with KIR gene content scores of 0 versus 1 versus ≥2 yielded similar rates of nonrelapse mortality, relapse, acute graft-versus-host disease (GVHD), and chronic GVHD and the same progression-free survival and OS. Relapse risk was not different for grafts from donors with KIR3DL1 transplanted into HLA C1/1 versus C2 recipients. This large analysis failed to demonstrate an association between URD KIR genotype and transplant outcome for patients with CLL, and thus KIR genotyping should not be used as a donor selection criterion in this setting.

KIR 2DS4 May Influence Autologous and Cord Blood(CB) Natural Killer (NK)Cell Mediated in Vitro Cytotoxicity Against Freshly Isolated Human Bone Marrow Myeloma Plasma Cells and Cell Lines

Background and Aim: NK cell effects are mediated by Killer Immunoglobulin-like Receptor (KIR) inhibition. There are few reports on the protective role of KIRs against relapse in myeloma(MM) (Gabriel et al Blood 2010, Kröger et al leukemia 2011). However, which NK cells or for whom will be more effective and the role of individual differences in this mechanism is indefinite. In this study we aimed to accomplish cytotoxicity against both MM cells and human myeloma cell lines (HMCL) and to compare the cytotoxicity achieved with CB versus autologous NK cells as well as to investigate the influence of NK cell KIR geno/phenotypes.Patients and Methods: A total of 18 patients with refractory/relapsed MM were included in this study.Cell culturing: CD138+ PC isolated from fresh bone marrow aspirates of relapsed MM patients or HMCL (U266, RPMI8226 and H929) using RosetteSep™ Human Multiple Myeloma Cell Enrichment Cocktail (StemCell Technologies) were subjected to cytotoxicity assays with CB derived or autologous PB derived NK cells expanded in the presence of IL-15 (10ng/ml) for 2-8 days.Phenotyping: Effector cells (autologous or CB-NK) were analyzed by flow cytometry using CD45, CD3, CD16, CD56, CD158a (KIR2DL1), CD158i (KIR2DS4) and CD158b1/b2 (KIR 2DL2/2DL3) monoclonal antibodies.Cytotoxicity assays:3,3 dioctadecyloxacarbocyanine perchlorate (DiO) dye was used to distinguish target cells (PC) from effector cells (NK). PC and NK cells were mixed at an 1:10 target: effector cell ratio and inoculated with Propidium Iodide (PI) in order to counter stain the dead cells. Naive PC were used as controls. Following 120 mins of incubation, PC death was evaluated using flow cytometry. After incubation of target cells (PC) with effector cells (NK cells), cytotoxic effects were determined by subtraction of NK mediated PC death from spontaneous PC death.Results: Following IL-15 expansion NK cells were found to express the CD16+56+3- phenotype. Eighteen MM patient PCs and six different HMCL samples were used in cytotoxicity assays(Table 1). In vitro PC viability was less than 45% in four samples and could not be analyzed in the study. Following incubation of PC wo NK cells, spontaneous PC death ratio was found to be 22.6% (min-max: 3%-42.3%). Twelve cytotoxicity assays targeting six MM PC were performed with autologous and CB-derived NK cells. Autologous NK and CB NK mediated cytotoxicity median rates were found to be 19.0% (range: 1.4% - 43.6%) and 31.3% (range: 2.4% - 51.2%), respectively. Additionally, CB-NK cells were evaluated against HMCL samples (four U266, one RPMI8226 and one H929). Median cytotoxicity against HMCL was 27.4% (range: 5.6% - 52.1%). Highest cytotoxicity against MM PC (MM-9) and HMCL (H929) were achieved by CB NK cells (51.2% and 52.1 % respectively). There was no cytotoxic effect against PCs of patients MM6 and MM10 (Figure 1-2).Flow-cytometric phenotyping of effector NK cells showed consistency (10/13) with genotypes. Since only one (CB19) with 19% CD158i positivity was PCR negative, a %20 cut-off rate was chosen. Both CD158a (KIR2DL1) and CD158b1/b2 (KIR 2DL2/2DL3) were positive among all patients and no significant correlation with cytotoxic effects was detectable. However out of five high cytotoxic reactions three NK cells were 2DS4 (+). While out of eight assays which resulted with low cytotoxicity only three NK cells were positive for the activating KIR 2DS4. When we analyzed CB NK cells against HMCL U266, three CB NK cells two of which were 2DS4(+) showed high cytotoxicity, while the 2DS4(-) CB NKs did not. The only assay targeting RPMI-8226 which is known to be a NK resistant HMCL, KIR2DS4(+) (CB17) derived NK cells were not sufficiently cytotoxic.Conclusion: Allogeneic CB NK cells were able to induce higher cell kill than autologous PB-derived NK cells. KIR types of autologous NK cells were not predictive for cytotoxic efficacy. Lacking KIR2DS4 in CB NK cells was correlated with less cytotoxicity. Additionally, KIR2DS4(+) CB NK cells were found to be highly cytotoxic against both PC and HMCL. NK cells may be an effective alternative due to its profound clinical advantages such as matching for HLA or KIR ligand is not required. Furthermore, if confirmed by others, certain KIR haplotypes (ie Haplotype A and KIR2DS4 +) may offer better therapeutic potential.Acknowledgment: The study has been supported by Scientific and Research Council of Turkey (TUBITAK grant no: 115S579) and Turkish Academy of Sciences. [Display omitted] DisclosuresBeksac:Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen,Janssen-Cilag,Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

The impact of donor and recipient KIR genes and KIR ligands on the occurrence of acute graft-versus-host disease and graft survival after HLA-identical sibling hematopoietic stem cell transplantation

After allogeneic hematopoietic stem cell transplantation (allo-HSCT), donor natural killer (NK) cells trigger alloreactions against potential recipient cells by their killer immunoglobulin-like receptors (KIRs). This study investigated whether KIR/HLA genotypes and KIR haplotypes of donors and recipients exhibit a critical function in the prevalence of acute graft-versus-host disease (aGVHD) and persistence of the graft after HLA-identical sibling allo-HSCT for patients with hematological malignancies. We studied KIR and HLA genotypes in 115 related donors and recipients (56 patients with AML and 59 patients with ALL) who had received allo-HSCT from HLA-matched sibling donors. We evaluated 17 KIR genes and some alleles, including their ligands, using the PCR-SSP assay. KIR gene frequency results between donors and recipients showed that donors had more activating KIR than their recipients. Chi-square comparison of KIR genotype frequencies in donors versus recipients revealed a significant difference (P < 0.001). We found a survival association between the donor lacking and the recipient having group B KIR haplotypes, although this was not statistically significant. This study suggests that we could exploit NK cell alloreactivity as a part of the optimization of donor selection and potential immunotherapeutic regimens to help facilitate good engraftment and reduce the risk of aGVHD incidence after allo-HSCT.

Novel KIR genotypes and gene copy number variations in northeastern Thais.

KIR (Killer Immunoglobulin-like Receptor) variants influence immune responses and are genetic factors in disease susceptibility. Using sequence-specific priming PCR, we have previously described the diversity of KIR genes in term of presence/absence in northeastern Thais (NETs). To provide additional resolution beyond conventional methods, quantitative PCR was applied to determine KIR copy number profiles. Novel expanded and contracted KIR copy number profiles were identified at cumulatively high frequencies. These all comprise haplotypes with duplication (6·9%) or deletion (2·7%) of KIR3DL1/S1 along with adjacent genes. Five expanded KIR profiles comprised haplotypes with duplications of KIR2DP1, 2DL1, 3DP1, 2DL4, 3DL1/S1 and 2DS1/4, whereas two contracted profiles contained only a single copy of KIR3DP1, 3DL1/S1 and 2DL4. Using a KIR haplotype prediction program (KIR Haplotype Identifier), 14% of NET haplotypes carried atypical haplotypes based on the gene copy number data.

The effect of killer cell immunoglobulin-like receptor genotype on outcome of hematopoietic stem cell transplantation from matched sibling

The alloreactivity of natural killer (NK) cell after allogeneic hematopoietic stem cell transplantation (AHSCT) is regulated by the interaction between donor killer immunoglobulin-like receptors (KIRs) and recipient human leukocyte antigen (HLA)-class I molecules. The aim was to identify KIR genes, haplotypes and their HLA-class I ligands and to investigate their association with transplantation outcome. The study included 65 patient/donor pairs who received AHSCT from HLA-matched identical siblings. KIR genotyping was done for donors using reverse sequence specific oligonucleotide probes (rSSO) coupled with luminex technology, while HLA-C genotyping was performed in patients using rSSO strip assay. In multivariate analysis, KIR2DS4 was associated with significant reduced incidence of relapse (p = .002). A trend towards reduced incidence of relapse was also observed with more than two KIR B motifs (p = .09), whereas a significant increased relapse was associated with homozygous HLA-C2 ligand compared to combined C1/C2 and C1/C1 (p = .04). Activating KIR2DS3 was associated with rapid leukocyte engraftment (p = .02). While, KIR 2DL5 was associated with decreased CMV infection (p = .03) and better platelets engraftment (p = .05). KIR genes, haplotypes and HLA-C alleles have an impact on HSCT outcome. Better selection of donors with favorable KIR genotype can improve HLA-matched sibling HSCT outcome especially for AML patients.