Search

When multiple haploidentical donors are available for transplantation, those of younger generations are generally selected over those of older generations. However, it is unclear who is the optimal donor when selecting candidates from within a generation, such as father versus mother, son versus daughter, or brother versus sister. Although traditionally male donors are favored over female donors, particularly for male recipients, and significant associations of individual HLA mis(matches) on outcomes are being increasingly recognized, the hierarchy of factors for donor selection is indeterminate. To assess whether HLA factors take precedence over non-HLA factors and to isolate the influence of specific characteristics on outcomes, we analyzed 412 patients stratified by donor relationship: child donor (son [n = 202] versus daughter [n = 96]), parent (father [n = 28] versus mother [n = 29]), and sibling (noninherited maternal [NIMA; n = 29] versus paternal [NIPA; n = 28] mismatched). Among siblings, NIMA mismatch was associated with a lower risk of acute graft-versus-host disease (aGVHD); B-leader mismatch was associated with high nonrelapse mortality (NRM), poor progression-free survival, and a trend toward poor overall survival (OS), whereas A-mismatch was associated with lower aGVHD. Among parent donors, the relationship did not impact any outcome; B-leader mismatch was associated with higher NRM and a trend toward poor OS, whereas A-mismatch was associated with lower NRM and improved progression-free survival and OS. Among child donors, no individual HLA mismatch was predictive of any outcome, and daughter donors were not associated with any adverse outcomes in multivariate analyses. Our data suggest that certain HLA factors may be more significant in some cases and should be given priority over simply selecting a donor based on relationship/sex.

Recent compelling evidence showed that innate immune effector cells could recognize allogeneic grafts and prime an adaptive immune response. Signal regulatory protein α (SIRPα) is an immunoglobulin superfamily receptor that is expressed on myeloid cells; the interaction between SIRPα and its ubiquitously expressed ligand CD47 elicits an inhibitory signal that suppresses macrophage phagocytic function. Additional studies showed that donor-recipient mismatch in SIRPα variants might activate monocytic allorecognition, possibly as the result of non-self SIRPα-CD47 interaction. However, the frequency of SIRPα variation and its role in hematopoietic stem cell transplantation (HSCT) remains unexplored. We studied 350 patients with acute myeloid leukemia/myelodysplastic syndrome who underwent HLA-matched related HSCT and found that SIRPα allelic mismatches were present in 39% of transplantation pairs. SIRPα variant mismatch was associated with a significantly higher rate of chronic graft-versus-host disease (GVHD; hazard ratio [HR], 1.5; P = .03), especially de novo chronic GVHD (HR, 2.0; P = .01), after adjusting for other predictors. Those with mismatched SIRPα had a lower relapse rate (HR, 0.6; P = .05) and significantly longer relapse-free survival (RFS; HR, 0.6; P = .04). Notably, the effect of SIRPα variant mismatch on relapse protection was most pronounced early after HSCT and in patients who were not in remission at HSCT (cumulative incidence, 73% vs 54%; HR, 0.5; P = .01). These findings show that SIRPα variant mismatch is associated with HSCT outcomes, possibly owing to innate allorecognition. SIRPα variant matching could provide valuable information for donor selection and risk stratification in HSCT.

With the continuous increase in the use of haploidentical donors for transplantation, the selection of donors becomes increasingly important. Haploidentical donors have been selected primarily based on clinical characteristics, while the effects of killer cell immunoglobulin-like receptors (KIRs) on outcomes of haploidentical-hematopoietic stem cell transplantation (haplo-HSCT) with post-transplant cyclophosphamide (PTCy) remain inconclusive. The present study aimed to thoroughly evaluate the effect of KIRs and binding ligands assessed by various models, in addition to other patient/donor variables, on clinical outcomes in haplo-HSCT. In a cohort of 354 patients undergoing their first haplo-HSCT, we found that a higher Count Functional inhibitory KIR score (CF-iKIR) was associated with improved progression-free survival (adjusted hazard ratio [HR], 0.71; P = .029) and overall survival (OS) (HR, 0.66; P = .016), while none of the other models predicted for survival in these patients. Moreover, using exploratory classification and regression tree analysis, we found that donor age 58 years was associated with the worst OS. In the rest of our cohort (80%), cytomegalovirus-reactive recipients with a donor P = .044), 44.5% (HR, 2.01; P = .003), and 18.5% (HR, 5.44; P

Haploidentical donors are increasingly used for patients requiring hematopoietic stem cell transplantation (HSCT). Although several factors have been associated with transplant outcomes, the impact of HLA disparity in haploidentical HSCT (haplo-HSCT) remains unclear. We investigated the impact of HLA disparity quantified by mismatched eplets (ME) load of each HLA locus on the clinical outcome of 278 consecutive haploidentical transplants. Here, we demonstrated that the degree of HLA molecular mismatches, at individual HLA loci, may be relevant to clinical outcome in the haplo-HSCT. A significantly better overall survival was associated with higher ME load from HLA-A (hazard ratio [HR], 0.97; 95% confidence interval [CI], 0.95-0.99; P = .003) and class I loci (HR, 0.99; 95% CI, 0.97-0.99; P = .045) in the host-versus-graft direction. The apparent survival advantage of HLA-A ME was primarily attributed to reduced risk in relapse associated with an increase in HLA-A ME load (subdistribution HR, 0.95; 95% CI, 0.92-0.98; P = .004). Furthermore, we have identified an association between the risk of grade 3-4 acute graft-versus-host disease (GVHD) and a higher ME load at HLA-B and class I loci in graft-versus-host (GVH) direction. Additionally, GVH nonpermissive HLA-DPB1 mismatch defined by T-cell epitope grouping was significantly associated with relapse protection (subdistribution HR, 0.19; 95% CI, 0.06-0.59; P = .004) without a concurrent increase in GVHD. These findings indicate that alloreactivity generated by HLA disparity at certain HLA loci is associated with transplant outcomes, and ME analysis of individual HLA loci might assist donor selection and risk stratification in haplo-HSCT.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for hematologic malignancies. Alloreactivity after HSCT is known to be mediated by adaptive immune cells expressing rearranging receptors. Recent studies demonstrated that the innate immune system could likewise sense the non-self signals and subsequently enhance the alloimmune response. We recently demonstrated that the donor/recipient mismatch of signal regulatory protein α (SIRPα), an immunoglobulin receptor exclusively expressed on innate cells, is associated with a higher risk of cGVHD and relapse protection in a cohort of acute myeloid leukemia patients who underwent allo-HSCT. Whether these effects also occur in other hematologic malignancies remains unclear. In the present study, we compared outcomes by SIRPα match status in a cohort of 310 patients who received allo-HSCT from an HLA matched-related donor for the treatment of lymphoid malignancies. Multivariable analysis showed that SIRPα mismatch was associated with a significantly higher rate of cGVHD (hazard ratio [HR] 1.8, P= .002), cGVHD requiring systemic immunosuppressive therapy (HR 1.9, P= .005), a lower rate of disease progression (HR 0.5, P= .003) and improved progression-free survival (HR 0.5, P= .001). Notably, the effects of SIRPα mismatch were observed only in the patients who achieved >95% of donor T-cell chimerism. The mismatch in SIRPα is associated with favorable relapse protection and concurrently increased risk of cGVHD in patients who undergo allo-HSCT for lymphoid malignancies, and the optimal donor could be selected based on the finding of the study to mitigate the risk of GVHD and relapse.

HLA-DPB1 mismatches between donor and recipient are commonly seen in allogeneic hematopoietic stem cell transplantation from an unrelated donor. HLA-DPB1 mismatch, conventionally determined by the similarity of the T-cell epitope (TCE), is associated with an increased risk of acute graft-versus-host disease (GVHD) and a decreased risk of disease relapse. We investigated the clinical impact of HLA-DPB1 molecular mismatch quantified by mismatched eplets (ME) and the Predicted Indirectly Recognizable HLA Epitopes Score (PS) in a cohort of 1,514 patients receiving hematopoietic stem cell transplants from unrelated donors matched at HLA-A, -B, -C, -DRB1/3/4/5, and - DQB1 loci. HLA-DPB1 alloimmunity in the graft-versus-host direction, determined by high graft-versus-host ME/PS, was associated with a reduced risk of relapse (hazard ratio [HR]=0.83, P=0.05 for ME) and increased risk of grade 2-4 acute GVHD (HR=1.44, P

We investigated the impact of donor-recipient HLA-DPB1 matching on outcomes of allogeneic hematopoietic stem cell transplantation with in vivo T-cell depletion using antithymocyte globulin (ATG) for patients with hematological malignancies. All donor-recipient pairs had high-resolution typing for HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DPB1, and HLA-DRB3/4/5 and were matched at HLA-A, HLA-B, HLA-C, and HLA-DRB1. HLA-DPB1 mismatches were categorized by immunogenicity of the DPB1 matching using the DPB T-cell epitope tool. Of 1004 donor-recipient pairs, 210 (21%) were DPB1 matched, 443 (44%) had permissive mismatches, 184 (18%) had nonpermissive mismatches, in graft-versus-host (GVH) direction, and 167 (17%) had nonpermissive mismatches in host-versus-graft (HVG) direction. Compared with HLA-DPB1 permissive mismatched pairs, nonpermissive GVH mismatched pairs had the highest risk for grade II to IV acute graft-versus-host disease (aGVHD) (hazard ratio [HR], 1.4; P = .01) whereas matched pairs had the lowest risk (HR, 0.5; P < .001). Grade III to IV aGVHD was only increased with HLA-DPB1 nonpermissive GVH mismatched pairs (HR, 2.3; P = .005). The risk for disease progression was lower with any HLA-DPB1 mismatches, permissive or nonpermissive. However, the favorable prognosis of HLA-DPB1 mismatches on disease progression was observed only in peripheral blood stem cell recipients who were in the intermediate-risk group by the Disease Risk Index (HR, 0.4; P = .001) but no other risk groups. Our results suggest avoidance of nonpermissive GVH HLA-DPB1 mismatches for lowering the risk for grade II to IV and III to IV aGVHD. Permissive or nonpermissive HVG HLA-DPB1 mismatches may be preferred over HLA-DPB1 matches in the intermediate-risk patients to decrease the risk for disease progression.

7049 Background: NK cells play a pivotal role in cancer immune vigilance. NK cell function is regulated by a balance between activating and inhibitory signals derived from various receptor-ligand interactions. Methods: 390 AML pts received 10/10 unrelated donor SCT. KIR genotype was performed by PCR with sequence specific primers. Hazard ratio (HR) for 2-year relapse was calculated using Fine-Gray regression and adjusted for disease risk index, remission status, pre-SCT MRD, conditioning regimen and presence of HLA-DP mismatch. KIR-ligand (K-L) match was defined as the presence a given KIR in the donor and the presence of its reported ligand in the patient (ex. 2DL1 and HLA-C2). KIRs that have no known ligands were not included in this analysis. The Table shows pt characteristics and K-L matches. Results: There was no correlation between the number of inhibitory or activating KIRs or KIR haplotype (A or B) and the probability of relapse after SCT. However, donor KIRs had a dramatic effect on relapse when they were considered together with the presence of the corresponding ligand in the pt. The 210 pts who had ≥3 inhibitory K-L matches had a significantly higher probability of relapse (HR=1.748, CI=1.147-2.667, p=0.009) than the remaining 180 pts. Similarly, the 96 pts who had at least one known activating K-L match had a lower probability of relapse (HR=0.581, CI=0.345-0.978, p=0.04). When we considered inhibitory and activating K-L matches together, we found that the 168 pts who had ≥3 inhibitory and no activating K-L matches had a significantly higher probability of relapse (HR 2.001, CI=1.376-2.908, p

Purpose C4, a cobalt dichloride-N-acetyl cysteine complex, is being developed as a positive-signal magnetic resonance imaging (MRI) marker to localize implanted radioactive seeds in prostate brachytherapy. We evaluated the toxicity and biodistribution of C4 in rats with the goal of simulating the systemic effects of potential leakage from C4 MRI markers within the prostate. Methods and Materials 9-μL doses (equivalent to leakage from 120 markers in a human) of control solution (0.9% sodium chloride), 1% (proposed for clinical use), and 10% C4 solution were injected into the prostates of male Sprague-Dawley rats via laparotomy. Organ toxicity and cobalt disposition in plasma, tissues, feces, and urine were evaluated. Results No C4-related morbidity or mortality was observed in the biodistribution arm (60 rats). Biodistribution was measurable after 10% C4 injection: cobalt was cleared rapidly from periprostatic tissue; mean concentrations in prostate were 163 μg/g and 268 μg/g at 5 and 30 minutes but were undetectable by 60 minutes. Expected dual renal-hepatic elimination was observed, with percentages of injected dose recovered in tissues of 39.0 ± 5.6% (liver), >11.8 ± 6.5% (prostate), and >5.3 ± 0.9% (kidney), with low plasma concentrations detected up to 1 hour (1.40 μg/mL at 5-60 minutes). Excretion in urine was 13.1 ± 4.6%, with 3.1 ± 0.54% recovered in feces by 24 hours. In the toxicity arm, 3 animals died in the control group and 1 each in the 1% and 10% groups from surgical or anesthesia-related complications; all others survived to scheduled termination at 14 days. No C4-related adverse clinical signs or organ toxicity were observed. Conclusion C4-related toxicity was not observed at exposures at least 10-fold the exposure proposed for use in humans. These data demonstrating lack of systemic toxicity with dual routes of elimination in the event of in situ rupture suggest that C4 warrants further investigation as an MRI marker for prostate brachytherapy.

Cord blood transplant requires less stringent human leukocyte antigen matching than unrelated donors. In 133 patients with hematologic malignancies who engrafted after double cord blood transplantation with a dominant unit, we studied the effect of high resolution testing at 4 loci (-A, -B, -C, -DRB1) for its impact on 2-year transplant-related mortality. Ten percent of the dominant cord blood units were matched at 7-8/8 alleles using HLA-A, -B, -C, and -DRB1; 25% were matched at 6/8, 40% at 5/8, and 25% at 4/8 or less allele. High resolution typing at 4 loci showed that there was no 2-year transplant-related mortality in 7-8/8 matched patients. Patients with 5-6/8 matched dominant cord blood units had 2-year transplant-related mortality of 39% while patients with 4/8 or less matched units had 60%. Multivariate regression analyses confirmed the independent effect of high resolution typing on the outcome when adjusted for age, diagnosis, CD34(+) cell dose infused, graft manipulation and cord to cord matching. The worst prognostic group included patients aged over 32 years with 4/8 or less matched cord blood units compared with patients who were either younger than 32 years old independent of allele-level matching, or aged over 32 years but with 5-6/8 matched cord blood units (Hazard Ratio 2.2; 95% confidence interval: 1.3-3.7; P

Aim Studies have shown that natural killer cells play an important role in mediating a graft-versus-leukemia effect through the interaction of NK cell receptors that can recognize ligands on their target cells. Interactions between the killer-cell immunoglobulin-like receptors (KIR) and their HLA ligands could influence outcomes in several clinical conditions including infections, autoimmunity and cancer. Patients with certain leukemia (e.g., AML) could benefit from hematopoietic stem cell transplant (HSCT) with NK-alloreactive donors resulting in significantly lower relapse rate. The aim of the study was to investigate first the KIR matching and KIR haplotype frequencies among HSCT patients and their HLA matched donors in order to further study the NK impact on transplant outcomes. Methods 149 pairs of HSC transplanted patients and their HLA matched related donors (7 with 1 DP mismatch) were included in the study. Patients and donors were high resolution typed at all HLA class I and class II loci. KIR genotyping were performed using fluorescently labelled beads conjugated to oligonucleotide probes (reverse SSO) and detected in a Luminex platform. KIR haplotype A or B were assigned per KIR genes content. Results Out of the 149 HLA matched pairs, 50 (33.6%) were also KIR matched and 99 (66.4%) KIR not matched for at least one KIR gene. KIR haplotypes were found A/A 31.5%, A/B 60.4%, and B/B 8.1% in patients; and KIR A/A 38.9%, A/B 57.1%, and B/B 4.0% in donors. Conclusions Because HLA genes and KIR genes are encoded on different chromosomes (6 and 19, respectively), one would expect KIR mismatch between HLA full matched family members. KIR mismatching could be beneficial to transplant patients from the NK alloreactivity of the donor. We have observed about one third HLA and KIR matched pairs. Further study on transplant outcomes between those HLA/KIR matched and HLA matched/KIR not matched groups would be warranted aiming for better donor selection to improve outcome. Better outcomes have been shown from donor with two or more KIR B motifs especially centromeric ones. Further outcome study from these transplants pairs would provide important information and understanding of the impact of these KIR haplotypes especially those donors with KIR A/A which could be disadvantageous to the patient for a better outcome.

Introduction Recombinant meiotic event(s) can occur within human families. Chromosomal crossover is the exchange of genetic material between homologous chromosomes that results in recombinant chromosomes. Even though crossover at HLA region is a rare event, the resulting haplotype would introduce linkage disequilibrium at HLA loci which could decrease the chance for hematopoietic stem cell transplant (HSCT) patient to find a suitable matched donor. The aim of the study was to investigate the frequency of recombination and the crossover location within the HLA class I and II regions. Methods HSCT patients and related family members as potential donors with clearly defined haplotypes within family were included in the study. All the patients and majority of family members were HLA typed at class I-A, B, C and class II-DR, DQ, DP using molecular methods (SSO and/or SBT) with some related donors serologically typed. Crossover haplotypes in patients ( N = 5219) or related donors ( N = 15,224) were analyzed and the crossover location between HLA loci was identified for each case. Results Crossover between HLA loci was identified in 202 individuals. The frequency of recombination was found to be 0.73% (38/5219) in HSCT patients and 1.08% (164/15224) in related potential donors. The overall crossover rate was 0.99% in the study population. There were two potential donors with recombination on both haplotypes. Five individuals in four families inherited the recombinant haplotype from a parent. Seven families had two members with recombination haplotype. The rate of recombination was 50.5% between HLA-B x DR, 36.63% between HLA-A x C, 9.41% between HLA-DQ x DP, and 3.47% between HLA-C x B loci. Conclusion Crossover events were observed between nearly all the neighboring HLA loci (A-C, C-B, B-DR, DQ-DP) except for between HLA-DR and DQ. We didn’t observe any recombination event between these loci in the study. The recombination rate is overall about 1% in the study population. The crossover hot spots appear to be between HLA-B and DR, HLA-A and HLA-C. It’s interesting to note that crossover event was lower in patients with different leukemia malignancies than in healthy family members. The finding suggests that leukemia patients were not disadvantaged from recombination in HLA regions.

Introduction Unambiguous, high resolution HLA typing plays a central role in hematopoietic stem cell transplantation. Current methodologies, primarily Sanger SBT, often fails to resolve all ambiguities creating the need for additional testing and longer turnaround times. Next generation sequencing (NGS) has the potential to address these shortcomings by providing full length, phase resolved sequences of HLA alleles with a single test. The Immucor Mia Fora NGS TM is an HLA typing kit and analysis software package that allows for typing of multiple samples per run in multiplex using the Illumina platform. In this study, we evaluated the NGS system against Sanger SBT methods. Methods NGS was performed on 11 HLA genes (HLA -A, B, C, DQA1/B1, DPA1/B1, and DRB1/3/4/5) for 69 subjects (1221 alleles) in alpha test, 69 (1223 alleles) in beta, and 35 (618 alleles) in further test. Automated liquid handling instruments were used for long range PCR and library preparation. Size selection was performed using the Pippin Prep followed by real-time PCR quantification on the ABI 7500. The final library was sequenced on the Illumina using the 300-cycle v2 kit. FASTQ files were analyzed with the Mia Fora software. NGS alleles to the 3rd fields were compared to the typing obtained by SBT, SSO, and SSP. Results A panel of 173 subjects carrying 3062 HLA class I and II alleles (total of 369 unique HLA alleles including novel alleles) was selected and typed by NGS. The overall concordance between NGS (automatic allele assigned by the software without editing) and Sanger SBT typing ranged from 98.5–99.7% in class I and 96.7–100.0% in class II. After manual review using the software’s tools for flagged alleles, the overall concordance will reach even higher. Conclusions The current NGS HLA typing product is able to produce robust and reliable allelic level HLA typing at all 11 HLA class I and II loci with no or very few ambiguities. Discordance was primarily observed in class II, most notably certain DRB1 ∗ 04 and DQB1 ∗ 03 alleles which often suffered from low read coverage in the presence of other alleles, and the ability to assign novel alleles or ambiguous results (e.g. DPB1) by the software. Improvements on test condition to prevent potential preferential amplification of certain HLA alleles would improve even more the accurate assignment of alleles at all HLA loci.

The use of unrelated donors matched in all alleles of HLA-A, -B, -C, and -DRB1 loci has been associated with superior outcomes compared with those having 1 or more mismatches. Recent studies showed increased transplant-related mortality (TRM) with the use of HLA-DPB1 mismatched donors supporting the notion that the ideal volunteer unrelated donor should fully match at HLA-A, -B, -C, and -DRB1 and lack -DPB1 mismatches. The issue of the effect of HLA-DPB1 mismatch on the disease progression rate is still controversial and we aimed to investigate the impact of HLA-DPB1 mismatch in the graft versus host direction on transplant outcomes in patients categorized according to the recently defined disease risk index (DRI) for disease risk classification. Our study cohort included 1,211 transplant patients with hematological malignancies whohave received an hematopoietic stem cell transplant (HSCT) from an unrelated HLA-A, -B, -C,-DRB1 matched donor by high resolution typing (8/8 matched) after 2005 through 2014. The study cohort had a median age of 55 (range, 19-77); the hematopoietic stem cell source was peripheral blood (PB) in 698 and bone marrow (BM) in 513 patients. Disease risk index (DRI) at HSCT was high or very high in 382 (33%), intermediate in 598 (51%), low in 185 (16%) patients. Of the pairs, 1,154 (95%) were matched atHLA-DQB1 and 1,116 (92%) at HLA- DRB3/4/5 by high resolution testing. However, 633 (52%) had mismatch at one of the DPB1 alleles and 208 (17%) had two mismatches. There was association between matching for DPB1and matching for DRB3/4/5 (p=0.002) but not with DQB1. In PB recipients, there was a highly significant decreaseof disease progression in DPB1 mismatched pairs (one and two allele; HR=0.7, p=0.01 and HR=0.6, p=0.01 respectively) as compared tothose pairs with DPB1 matched. The impact of mismatches at one or two alleles were not different on disease progression (HR=1.2, p=0.4). However, the impact of DPB1 mismatch on disease progression was not uniform in different disease risk groups by DRI. Mismatch at DPB1 significantly decreased disease progression only in the intermediate risk group (HR=0.5, p=0.002) but not in low risk and high/very high disease groups by DRI (HR=0.9, p=0.8 and HR=0.7, p=0.1 respectively) (Figure 1a-c). In BM recipients, increasing number of DPB1 incompatibilities decreased disease progression (HR=0.9, p=0.4 and HR=0.6, p=0.1 for 1 and 2 allele mismatches respectively) but did not reach significance. Mismatches at HLA-DQB1 and -DRB3/4/5 had no impact on disease progression in both PB and BM recipients. Pairs with one or two allele-level DPB1 mismatches increased TRM compared with DPB1 matched pairs in PB (HR=1.5, p=0.04 and HR=1.9, p=0.006 respectively) and BM recipients (HR=1.8, p=0.03 and HR=1.9, p=0.05). There was no difference between two and one allele DPB1 mismatched for TRM in PB and BM recipients. Multivariate analyses revealed that the negative impact of DPB1 mismatch on TRM was not uniform in younger or (?) older patients. Interestingly, DPB1 mismatches increased TRM only in younger (aged We next analyzed the impact of DPB1 matching on progression free survival (PFS) and did not observe any impact of DPB1 mismatches on PFS in PB (HR=0.9, p=0.9) and BM (HR=1.12, p=0.6) recipients. Subgroup analyses by DRI to identify a specific risk group that the use of HLA-A, -B, -C and -DRB1 matched but DPB1 mismatched unrelated donor might lead to improved PFS did not reveal any particular risk group in both PB and BM recipients. Thus, in recipients of HLA-A, -B-C and DRB1 allele-level matched unrelated donors a mismatch for DPB1 is associated with a significantlydecreased risk of disease progression with no impact on PFS in intermediate risk group by DRI. Further analysis permissive vs. non-permissive DPB1 mismatches would be warranted. Figure 1. The cumulative incidence of disease progression by DPB1 mismatch and Disease Risk Index in peripheral blood recipients. Figure 1. The cumulative incidence of disease progression by DPB1 mismatch and Disease Risk Index in peripheral blood recipients. Disclosures No relevant conflicts of interest to declare.

The cytolytic function of natural killer (NK) cells is controlled by a balance of activatory and inhibitory signals transduced by specific NK-cell receptors. NKG2 family is one of the superfamilies of these receptor genes located on chromosome 12 in the NKC gene complex. NKG2C is expressed as heterodimers with CD94 and they deliver activating signals upon contacting HLA-E via adaptor molecules. NKG2C genes are not only polymorphic and also NKG2C gene deletion was identified. The aim of the study was to develop a genotyping assay for the detection of the wild type and the deletion of the NKG2C gene. Method Copy numbers of the NKG2C genes were genotyped and assessed in 352 subjects by PCR-SSP method using a set of primer pairs described previously (Mararu M & Miyashita R, etc.). Two pairs of primers are used to detect the NKG2C genotypes: one pairs to amplify a 201 bp fragment from NKG2C wild type (wt) carrier; and the second pairs to amplify a 411 bp fragment from NKG2C deletion (del) carrier. A single-tube PCR-SSP genotyping strategy combining the two sets of primers was modified from Moraru M, etc. (Tissue Antigens, 2012). Results The presence or absence of NKG2C gene was determined by the single-tube PCR-SSP assay. Three genotypes were observed in the study subjects: wild type homozygous carrier (wt/wt), wild type and deletion heterozygous carrier (wt/del), and deletion homozygous carrier (del/del). Copy number of NKG2C in 3 International Histocompatibility Workshop (IHW) cells was assessed and confirmed: IHW 9043 as NKG2C wt/wt , IHW 9047 as NKG2C wt/del , and IHW 9044 as NKG2C del/del . Of the 352 subjects, wt/wt genotype accounted for 62.82% ( n = 221), wt/del for 32.88% ( n = 116), and del/del for 4.3% ( n = 15). The allele frequency of NKG2C deletion was 20.74% in this study group. Conclusion Studies have shown that NK cells expressing the activating receptor NKG2C preferentially expand after coculture with cytomegalovirus (CMV) infected fibroblasts and CMV-induced innate memory cells may contribute to malignant disease relapse protection and infectious disease control after transplantation. NKG2C deletion was shown to be a risk factor of HIV infection. Further study of the impact of the NKG2C CNV on outcomes of stem cell transplants with umbilical cord blood, related or unrelated donors would be warranted.

Cord blood (CB) as a hematopoietic stem cell source has the advantage of decreased stringency of human leukocyte antigen (HLA) matching requirements. However, recent studies showed better outcomes in single CB transplant (CBT) with improved allele-level matching for 4 HLA loci (-A, -B, -C, and -DRB1). We retrospectively analyzed 146 patients with hematological malignancies who engrafted after dCBT between 2003 through 2013 to compare the prognostic value of the current standard HLA matching criteria (intermediate resolution HLA typing at A and B and allele-level typing at DRB1) versus that of allele level matching at –A, -B, -DRB1 (6/6 allele) and at -A, -B, -C, and -DRB1 (8/8 allele). The effect of HLA match was based on the HLA type of the predominant long term engrafting unit. The primary endpoint was 1-year transplant related mortality (TRM). In this cohort, median age was 43 (range; 1 –73). Disease diagnoses were AML in 56%, ALL in 21%, other hematological malignancies in the rest of the group. Disease status at dCBT was first or second complete remission in 55% and third remission or active disease in 45% of the patients. Conditioning was myeloablative in 62% of the patients consisting of mostly thiotepa, fludarabine and melphalan (86%). Reduced intensity conditioning was given to older and/or frail patients (38%) and consisted of cytoxan, fludarabine and TBI 200 cGy in 64% and melphalan with fludarabine in 36% of the patients. Immunosuppressive were mycophenolate mofetil (MMF) with tacrolimus and all patients received ATG as a part of the conditioning. Per protocol requirements over the years, 63% of the patients had ex-vivo expansion of one of the CB units with mesenchymal-cell coculture and 2% had one of the CB unit fucoylated. Median cell dose infused was 2.2 x 10e7 TNC/kg (range 0.5-291). Of 146 patients, 54% had 4/6, 40% 5/6 and only 6% 6/6 matched CB units by the current standard HLA matching criteria. Patients who had 6/6 matched units have the lowest TRM compared with 4/6 group but this did not reach statistical significance (p=0.2). By 6/6 allele-level matching criteria; 20% of the patients had ≤3/6; 51% 4/6 and 29% were 5-6/6 matched CB units. Per this criteria, patients with 5-6/6 allele-level matched units had lower TRM compared with ≤4/6 allele-level matched unit recipients (HR=0.4, p=0.04). More importantly, of 65 patients with 5-6/6 matched units by standard criteria, 37% were found to have ≤4/6 allele-level matched units that would lead to high TRM. By 8/8 allele-level matching criteria; 26% of the patients had 3-4/8; 39% 5/8, 26% 6/8 and 10% 7-8/8 matched CB units. Based on this criteria, 3 different groups emerged; 7-8/8, 5-6/8, and ≤4/8. Recipients of 7-8/8 allele-level matched units did not experience TRM at one year. Patients with 5-6/8 allele-level matched units also had less TRM compared with ≤4/8 matched recipients (HR=0.5, p=0.02). Among other variables analyzed; including age, disease, disease status, conditioning regimen, CB unit modification, unit TNC dose; only age >30 was a poor risk factor for 1-year TRM (HR=2.5, p=0.03). When we simultaneously accounted for age and 8/8 allele-level matching, we were able to identify 3 distinct risk groups for 1-year TRM (Table and Figure): 1) Low risk; 7-8/8 allele-level matched unit recipients with 1 year TRM of 0%. 2) High risk; age>30 and ≤4/8 allele-level matched CB units with 1-year TRM of 60% 3) Intermediate risk; all remaining patients with TRM of 30%. This risk stratification was independent of TNC cell dose infused. The respective 1-year cumulative incidence of TRM in the low-, high-, and intermediate-risk groups was 0%, 64% and 30% for patients who received TNC dose ≤ median (0.22X10e7); and 0%, 54% and 31% for patients who received TNC dose > median. Abstract 2532. Table 1TRMProgressionOSnHR95%CIpHR95%CIpHR95%CIpHigh risk27RefRefRefIntermediate risk930.40.1-0.70.0041.50.4-4.40.40.50.3-0.80.005Low risk13NE0.0032.30.6-8.50.20.40.1-0.90.04 Figure 1 Figure 1. These results show that, compared with the current standard matching, allele-level matching for HLA -A, -B, -C, and –DR may have a stronger prognostic value as it identifies subgroups of patients likely to have very high or very low rate of TRM after dCBT. We suggest that allele-level matching for 4 HLA loci should be used for the selection of dCB units. Disclosures No relevant conflicts of interest to declare.

Aim HLA-C∗04:09 N is characterized by a point deletion at nucleotide position 1095 in exon 7. This mutation results in a frame shift and causes the addition of 32 extra amino acids and the loss of cell surface expression (Balas A., et al. Tissue Antigens 2002;59:95–100). The goal of this study was to determine the frequency of the B∗44:03-C∗04:09 N bearing haplotype in leukemia patients treated at MDACC. Method Patients were typed for both HLA Class I and Class II using PCR-SSOP and PCR-SBT methodologies. The HLA-C∗04:09 N allele was resolved using PCR-SSP method. Results We identified and evaluated 300 consecutive patients with various forms of leukemia treated at our center carrying the HLA-B∗44:03-C∗04:01:01G typing with the inclusion of C∗04:09 N allele (2.2% of the total patient population). Of the 300 B∗44:03-C∗04:01:01G bearing patients, 21 were identified as HLA-C∗04:09 N (7%), which accounted for 0.15% in the entire patient population. Discussion Of the 21 patients bearing HLA-B∗44:03-C∗04:09 N alleles, 9 had haplotype assigned. Five of them (55.6%) carrying the haplotype of HLA-A∗23:01-C∗04:09 N-B∗44:03:01-DRB1∗07:01-DRB4∗01:01-DQB1∗02:02 and 2 others carrying the same haplotype but with different HLA-A alleles (Table 1). The study explored the frequency of HLA-C∗04:09 N in the presence of B∗44:03 in leukemia patients. The identification of this null allele would be important in HLA matching for patients in need of stem cell transplant. Misidentification of a patients null allele could impact graft outcome with a mismatched donor carrying the expressed allele or visa versa.

Aim Killer cell immunoglobulin-like receptor (KIR) genes are receptors expressed on the surface of natural killer cells. KIR genes recognize polymorphic epitopes of HLA class I-A, B & C, called KIR HLA ligands (HLA-L). After binding specific ligands the KIR may transmit inhibitory or activating signals. NKC sense & kill target cells missing MHC class I molecules. Studies showed that hematopoietic stem cells (HSC) transplant patients may benefit from NK alloreactivity of the donor or HLA-C alleles (C1/x) of the recipients. Previously we reported the diversification of KIR HLA-L in CBU from MD Anderson Cord Bank. The goal of this study was to further explore the frequency (Freq) of HLA-L in selected CBU from the bank and their transplant recipients aimed at providing information for finding the best donor and improving graft outcome. Methods 640 CBU selected from MD Anderson Cord Bank and 589 recipients were included. HLA typing of class I & II loci were obtained using PCR/probe based methods & sequence-based typing as needed. Freq of Bw4, C1, and C2 group of HLA-C alleles were examined. Results KIR3DL1 recognizes allotypes with HLA-Bw4 motif; KIR2DL1 recognizes HLA-C epitope with lysine at codon 80 (group 2-C2); while KIR2DL2 and KIR2DL3 recognize C alleles with asparagine at residue 80 (group 1-C1). Among the 640 CBU and 589 recipients, 70.0% and 72.0%, respectively, carry Bw4 bearing phenotypes. HLA-C1 bearing phenotypes (C1/x) were found to be predominated in both CBU (85.2%) and their transplant recipients(81.5%). Missing HLA-C1 was lower (C2/C2, 18.5%) than missing C2 (C1/C1, 33.1%) in recipients (Table 1). Conclusion Here we show that CBU can be classified based on their KIR repertoire (licensed versus unlicensed) and the presence of KIR-ligand mismatch with the recipient. Analysis of graft outcome in these HSC transplant recipients is underway. The ultimate goal of our study is to develop algorithms based on the KIR profile of CB units to select the units with maximum antileukemic activity to improve the outcome of CBU transplant.

Aim Natural killer cells (NKC) express many different cell surface receptors including KIR. KIR genes recognize polymorphic HLA class I including HLA-A, B & C allotypes. The KIR genes after binding specific ligands may transmit inhibitory or activating signals. NKC sense & kill target cells lacking MHC class I molecules & release various cytokines on activation. UCB is an increasingly used source of HSC for pts without matched adult donor for Tx. The goal of the study was to attain frequencies (freq) of HLA-L in UCBU aimed at providing information (info) for finding UCB with NK benefit for HSC Tx pts. Methods Total of 21,002 UCBU from Cord Bank at MD Anderson was included. HLA typing of class I & II loci were obtained using PCR/probe based methods & sequence-based typing as needed. Freq of Bw4 alleles, C1 and C2 HLA-C alleles were obtained. Results KIR3DL1 recognizes allotypes with HLA-Bw4 serological motif; KIR2DL1 recognizes HLA-C allotypes with lysine at codon 80 (group 2 - C2); & KIR2DL2/KIR2DL3 recognize C alleles with asparagine at residue 80 (group 1 - C1). Among the 21,002 UCBU, 70.7% carry Bw4 alleles. UCB bearing C1/C2 heterozygous genotype (GT) accounted for 45.6%, C1/C1 homozygous GT for 39.6%, & C2/C2 GT for 14.7% only. Conclusions Incompatibility of KIR HLA-L between pt & donor has been shown to impact graft outcome (GO) in HSC Tx. NKC from the donor greatly contribute to eradication of leukemia blasts escaping the preparative regimen & to clearance of residual host dendritic cells & T lymphocytes thus preventing graft-versus-host disease & graft rejection. This study showed high freq of Bw4-bearing GT, increased freq of C1/C2, & C1/C1 but not C2/C2 GT in UCBU. These freq info could be useful for prediction of the possibility for HSC Tx pts, especially those with Bw6 or C2/C2 GT, to find suitable UCB donor(s) with beneficial NK reactivity, thereby improving GO. Further study of diversification of KIR HLA-ligands in pts would be warranted to assess the importance of these findings.

Aim Identification of novel HLA alleles has been invaluable contributions to the literature of HLA genes. New HLA sequences will be imperative in population study & in transplantation. The goal of the study was to describe the characterization of newly identified HLA alleles. Methods Unusual or potential new variants of HLA alleles were identified using PCR/probe based methods, and characterized by SBT using group/allele specific primers & then sequenced. Results Novel HLA class I sequences have been identified and characterized including three new HLA-A alleles in patients and one in a healthy individual, one HLA-B allele in a patient, and one HLA-C allele in a donor. The profiles of these novel alleles are shown in Table 1. Conclusions Two new HLA-A alleles, A∗02:397 & A∗11:129, have amino acid (AA) substitution from their potential progenitor alleles in exon 4, meaning these changes are not likely to affect peptide binding or T cell receptors. The other four alleles, A∗01:114, A∗32:46, B∗44:160, and C∗07:307, had AA substitutions in either exon 2 or exon 3. These changes could affect peptide binding and/or T cell receptors. Therefore, mismatch for these alleles could result in an increased risk for graft versus host disease or graft rejection in stem cell transplant, or possibly elicit anti-HLA antibodies to these alleles in patients with stem cell or solid organ transplantation to their donors, thereby impacting graft outcome. Further investigation of graft outcome of patients need ingof transplant bearing these new alleles would be warranted. [ Table 1 ]