KIR3DL1/HLA-B Subtypes Govern Acute Myelogenous Leukemia Relapse After Hematopoietic Cell Transplantation.

Purpose Disease relapse remains a major challenge to successful outcomes in patients who undergo allogeneic hematopoietic cell transplantation (HCT). Donor natural killer (NK) cell alloreactivity in HCT can control leukemic relapse, but capturing alloreactivity in HLA-matched HCT has been elusive. HLA expression on leukemia cells-upregulated in the post-HCT environment-signals for NK cell inhibition via inhibitory killer immunoglobulin-like (KIR) receptors and interrupts their antitumor activity. We hypothesized that varied strengths of inhibition among subtypes of the ubiquitous KIR3DL1 and its cognate ligand, HLA-B, would titrate NK reactivity against acute myelogenous leukemia (AML). Patients and Methods By using an algorithm that was based on polymorphism-driven expression levels and specificities, we predicted and tested inhibitory and cytotoxic NK potential on the basis of KIR3DL1/HLA-B subtype combinations in vitro and evaluated their impact in 1,328 patients with AML who underwent HCT from 9/10 or 10/10 HLA-matched unrelated donors. Results Segregated by KIR3DL1 subtype, NK cells demonstrated reproducible patterns of strong, weak, or noninhibition by target cells with defined HLA-B subtypes, which translated into discrete cytotoxic hierarchies against AML. In patients, KIR3DL1 and HLA-B subtype combinations that were predictive of weak inhibition or noninhibition were associated with significantly lower relapse (hazard ratio [HR], 0.72; P = .004) and overall mortality (HR, 0.84; P = .030) compared with strong inhibition combinations. The greatest effects were evident in the high-risk group of patients with all KIR ligands (relapse: HR, 0.54; P < .001; and mortality: HR, 0.74; P < .008). Beneficial effects of weak and noninhibiting KIR3DL1 and HLA-B subtype combinations were separate from and additive to the benefit of donor activating KIR2DS1. Conclusion Consideration of KIR3DL1-mediated inhibition in donor selection for HLA-matched HCT may achieve superior graft versus leukemia effects, lower risk for relapse, and an increase in survival among patients with AML.

Impact of Allele-Level HLA Mismatch on Outcomes in Recipients of Double Umbilical Cord Blood Transplantation.

The impact of allele-level HLA mismatch is uncertain in recipients of double umbilical cord blood (UCB) transplantation. We report a single-center retrospective study of the clinical effect of using allele-level HLA mismatch HLA-A, -B, -C, -DRB1, and -DQB1 of the 2 UCB units. We studied 342 patients with hematologic malignancy. Donor-recipient pairs were grouped according to the number of matched HLA alleles, with 32 matched at 9-10/10, 202 at 6-8/10, and 108 at 2-5/10 alleles. The incidence of hematopoietic recovery, acute and chronic graft-versus-host disease, and nonrelapse mortality and treatment failure was similar between groups. In an exploratory analysis of 174 patients with acute leukemia, after adjusting for length of first remission and cytogenetic risk group, a 2-5/10 HLA match was associated with lower risk of relapse and treatment failure. These data indicate that a high degree of allele-level HLA mismatch does not adversely affect transplant outcomes and may be associated with reduced relapse risk in patients with acute leukemia.

Multiple mismatches at the low expression HLA loci DP, DQ, and DRB3/4/5 associate with adverse outcomes in hematopoietic stem cell transplantation.

A single mismatch in highly expressed HLA-A, -B, -C, and -DRB1 loci (HEL) is associated with worse outcomes in hematopoietic stem cell transplantation, while less is known about the cumulative impact of mismatches in the lesser expressed HLA loci DRB3/4/5, DQ, and DP (LEL). We studied whether accumulation of LEL mismatches is associated with deleterious effects in 3853 unrelated donor transplants stratified according to number of matches in the HEL. In the 8/8 matched HEL group, LEL mismatches were not associated with any adverse outcome. Mismatches at HLA-DRB1 were associated with occurrence of multiple LEL mismatches. In the 7/8 HEL group, patients with 3 or more LEL mismatches scored in the graft-versus-host vector had a significantly higher risk of mortality (1.45 and 1.43) and transplant-related mortality (1.68 and 1.54) than the subgroups with 0 or 1 LEL mismatches. No single LEL locus had a more pronounced effect on clinical outcome. Three or more LEL mismatches are associated with lower survival after 7/8 HEL matched transplantation. Prospective evaluation of matching for HLA-DRB3/4/5, -DQ, and -DP loci is warranted to reduce posttransplant risks in donor-recipient pairs matched for 7/8 HEL.

Inter and intra laboratory concordance of HLA antibody results obtained by single antigen bead based assay.

Single antigen bead based assays (SAB) to identify antibodies to HLA are marketed as a qualitative test, however often used as a quantitative test as the results provide mean fluorescence intensity (MFI) which is found to correlate with the strength/avidity of the antibody. We studied the between and within laboratory variability in performing the SAB from one manufacturer. Ten samples were tested at four laboratories according to the manufacturer's suggested protocol. Additionally same samples were tested on four consecutive days in one laboratory. All tests were performed using the same lot of beads and secondary antibody. Results were classified as positive at four different MFI cutoffs: 1000, 5000, 8000 and 10,000. MFI values across and within the laboratory were compared in a pair-wise fashion with Pearson's correlations. Overall concordance for Class-I was 97% between laboratories and 98% within laboratory at all cutoffs. Pair-wise Pearson correlation between laboratories was 0.989-0.99, while within laboratory it was 0.998-0.999. For Class-II, overall concordance between and within laboratory was 98%. Pair-wise Pearson correlation between laboratories was 0.991-0.997, while within laboratory it was 0.997-0.999. There is good correlation between laboratories and within laboratory using the same manufacturer, same lot and same protocol while performing SAB.

Allele-level haplotype frequencies and pairwise linkage disequilibrium for 14 KIR loci in 506 European-American individuals.

The immune responses of natural killer cells are regulated, in part, by killer cell immunoglobulin-like receptors (KIR). The 16 closely-related genes in the KIR gene system have been diversified by gene duplication and unequal crossing over, thereby generating haplotypes with variation in gene copy number. Allelic variation also contributes to diversity within the complex. In this study, we estimated allele-level haplotype frequencies and pairwise linkage disequilibrium statistics for 14 KIR loci. The typing utilized multiple methodologies by four laboratories to provide at least 2x coverage for each allele. The computational methods generated maximum-likelihood estimates of allele-level haplotypes. Our results indicate the most extensive allele diversity was observed for the KIR framework genes and for the genes localized to the telomeric region of the KIR A haplotype. Particular alleles of the stimulatory loci appear to be nearly fixed on specific, common haplotypes while many of the less frequent alleles of the inhibitory loci appeared on multiple haplotypes, some with common haplotype structures. Haplotype structures cA01 and/or tA01 predominate in this cohort, as has been observed in most populations worldwide. Linkage disequilibrium is high within the centromeric and telomeric haplotype regions but not between them and is particularly strong between centromeric gene pairs KIR2DL5∼KIR2DS3S5 and KIR2DS3S5∼KIR2DL1, and telomeric KIR3DL1∼KIR2DS4. Although 93% of the individuals have unique pairs of full-length allelic haplotypes, large genomic blocks sharing specific sets of alleles are seen in the most frequent haplotypes. These high-resolution, high-quality haplotypes extend our basic knowledge of the KIR gene system and may be used to support clinical studies beyond single gene analysis.

A combined DPA1~DPB1 amino acid epitope is the primary unit of selection on the HLA-DP heterodimer.

Here, we present results for DPA1 and DPB1 four-digit allele-level typing in a large (n = 5,944) sample of unrelated European American stem cell donors previously characterized for other class I and class II loci. Examination of genetic data for both chains of the DP heterodimer in the largest cohort to date, at the amino acid epitope, allele, genotype, and haplotype level, allows new insights into the functional units of selection and association for the DP heterodimer. The data in this study suggest that for the DPA1-DPB1 heterodimer, the unit of selection is the combined amino acid epitope contributed by both the DPA1 and DPB1 genes, rather than the allele, and that patterns of LD are driven primarily by dimer stability and conformation of the P1 pocket. This may help explain the differential pattern of allele frequency distribution observed for this locus relative to the other class II loci. These findings further support the notion that allele-level associations in disease and transplantation may not be the most important unit of analysis, and that they should be considered instead in the molecular context.

Definitions of histocompatibility typing terms.

Histocompatibility testing for stem cell and solid organ transplantation has become increasingly complex as newly discovered HLA alleles are described. HLA typing assignments reported by laboratories are used by physicians and donor registries for matching donors and recipients. To communicate effectively, a common language for histocompatibility terms should be established. In early 2010, representatives from Clinical, Registry, and Histocompatibility organizations joined together as the Harmonization of Histocompatibility Typing Terms Working Group to define a consensual language for laboratories, physicians, and registries to communicate histocompatibility typing information. The Working Group defined terms for HLA typing resolution, HLA matching, and a format for reporting HLA assignments. In addition, definitions of verification typing and extended typing were addressed. The original draft of the Definitions of Histocompatibility Typing Terms was disseminated to colleagues from each organization to gain feedback and create a collaborative document. Commentary gathered during this 90-day review period were discussed and implemented for preparation of this report. Histocompatibility testing continues to evolve; thus, the definitions agreed on today probably will require refinement and perhaps additional terminology in the future.

Definitions of histocompatibility typing terms: Harmonization of Histocompatibility Typing Terms Working Group.

Histocompatibility testing for stem cell and solid organ transplantation has become increasingly complex as newly discovered human leukocyte antigen (HLA) alleles are described. HLA typing assignments reported by laboratories are used by physicians and donor registries for matching donors and recipients. To communicate effectively, a common language for histocompatibility terms should be established. In early 2010, representatives from clinical, registry, and histocompatibility organizations joined together as the Harmonization of Histocompatibility Typing Terms Working Group to define a consensual language for laboratories, physicians and registries to communicate histocompatibility typing information. The Working Group defined terms for HLA typing resolution, HLA matching and a format for reporting HLA assignments. In addition, definitions of verification typing and extended typing were addressed. The original draft of the Definitions of Histocompatibility Typing Terms was disseminated to colleagues from each organization to gain feedback and create a collaborative document. Commentary gathered during this 90-day review period were discussed and implemented for preparation of this report. Histocompatibility testing continues to evolve thus, the definitions agreed upon today, likely will require refinement and perhaps additional terminology in the future.

Anti-HLA antibodies in double umbilical cord blood transplantation.

Recent registry data suggest that host-versus-graft alloreactions mediated by anti-donor HLA antibodies in recipients of adult allogeneic hematopoietic stem cells or single-unit umbilical cord blood (UCB) contribute to the risk of graft failure. The present study evaluated the impact of anti-HLA antibodies on engraftment and unit predominance in 126 double-UCB (dUCB) recipients. Eighteen dUCB recipients were identified with at least 1 of 2 UCB units recognized by anti-HLA antibodies directed against donor-directed HLA-specific antibodies (DSAs). Overall, 9 of 12 patients who had DSAs against 1 of the 2 UCB units composing the graft and 5 of 6 patients who had DSAs against both units engrafted. The cumulative incidence of engraftment was similar in patients with and without DSAs (83% vs 78%). Thus, our data do not support a negative effect of anti-HLA antibodies on engraftment, at least in the setting of cyclosporine and mycophenolate mofetil and the conditioning regimens used at the University of Minnesota, and argue against routine screening for use in graft selection before dUCB transplantation. Further studies are needed to fully understand the value of anti-HLA antibody testing in dUCB graft selection and its impact on transplantation outcomes.

High immunologic risk living donor kidney transplant using bortezomib in a novel induction regimen without acute antibody mediated rejection.

Desensitization therapies have been used with modest success in kidney transplantation. Some candidates, however, have such great breadth and depth of anti-HLA antibodies that they remain incompatible with potential donors. Bortezomib has been used without much success in desensitization regimens, but we hypothesized that its use during induction may be helpful in targeting antibody production by long-lived plasma cells. This report describes a high-risk positive crossmatch son-to-mother transplant that was performed after desensitization. The induction immunosuppression was supplemented with bortezomib. Pre- and post-transplant immunosuppression, antibody monitoring, biopsy data, and the clinical course are described in detail. Following transplant, the patient had excellent early graft function. Serial biopsies did not reveal acute antibody mediated rejection. Despite excellent graft function, the patient underwent withdrawal of care and died due to complications of calciphylaxis and deconditioning. This case details the first report of bortezomib used as part of induction therapy in solid organ transplant. Donor specific antibody production remained stable after transplant, with near complete abrogation of class I specificities. There were no bortezomib-related complications.

Negative effect of KIR alloreactivity in recipients of umbilical cord blood transplant depends on transplantation conditioning intensity.

We examined the clinical impact of killer-immunoglobulin receptor-ligand (KIR-L) mismatch in 257 recipients of single (n = 91) or double (n = 166) unit umbilical cord blood (UCB) grafts after myeloablative (n = 155) or reduced intensity (n = 102) conditioning regimens. Analyses of double unit grafts considered the KIR-L match status of the dominant engrafting unit. After myeloablative conditioning, KIR-L mismatch had no effect on grade III-IV acute graft-versus-host disease (GVHD), transplantation-related mortality (TRM), relapse, and survival. In contrast, after reduced intensity conditioning, KIR-L mismatch between the engrafted unit and the recipient resulted in significantly higher rates of grade III-IV acute GVHD (42% [CI, 27-59] vs 13% [CI, 5-21], P < .01) and TRM (27% [CI, 12%-42%] vs 12% [CI, 5%-19%], P = .03) with inferior survival (32% [CI, 15%-59%] vs 52% [CI, 47%-67%], P = .03). Multivariate analysis identified KIR-L mismatch as the only predictive factor associated with the development of grade III-IV acute GVHD (RR, 1.8 [CI, 1.1-2.9]; P = .02) and demonstrated a significant association between KIR-L mismatch and increased risk of death (RR, 1.8; 95% CI, 1.0-3.1; P = .05). Our results do not support the selection of UCB units based on KIR-L status and suggest that KIR-L mismatching should be avoided in reduced intensity UCB transplantation.

Organ procurement and transplantation network/united network for organ sharing histocompatibility committee collaborative study to evaluate prediction of crossmatch results in highly sensitized patients.

BACKGROUND: The requirement for a prospective crossmatch limits some organ allocation to local areas. The delay necessitated by the crossmatch restricts the distance across which offers can be made without unduly increasing the ischemia time. A collaborative study involving 14 transplant centers was undertaken by the Organ Procurement and Transplantation Network/United Network for Organ Sharing (OPTN/UNOS) Histocompatibility Committee to evaluate the accuracy with which the detection of unacceptable human leukocyte antigen (HLA) antigens by most advanced solid phase immunoassays can predict crossmatch results. In addition, using actual patients' unacceptable HLA antigens, the number of compatible donors that would have been available from the OPTN deceased kidney donors during 2002 to 2004 were investigated. METHODS: Panel reactive antibodies were performed by conventional or solid phase assays, and crossmatches were performed by cytotoxicity or flow cytometry. Analyses were stratified for T and B cell and by method of identifying unacceptable HLA antigens and crossmatch techniques. RESULTS: Combination of solid phase immunoassays and flow cytometry crossmatches resulted in a higher prediction rates of positive T cell (86.1%-93.5%) and B-cell crossmatches (91%-97.8%). Prediction of negative crossmatches based on different combination of panel reactive antibodies and crossmatch techniques varied from 14.3% to 57.1%. Furthermore, numerous potential compatible donors were identified for each patient, regardless of their ethnicity, in the OPTN database, when predicted incompatible ones were excluded. CONCLUSIONS: The above results showed that with the advent of solid phase immunoassays, HLA antibodies can now be accurately detected resulting in prediction of crossmatch outcome. This should facilitate organ allocation and prevents shipment of organs to distant incompatible recipients.

High-resolution donor-recipient HLA matching contributes to the success of unrelated donor marrow transplantation.

The relative importance of various human leukocyte antigen (HLA) loci and the resolution level at which they are matched has not been fully defined for unrelated donor transplantation. To address this question, National Marrow Donor Program data from 3857 transplantations performed from 1988 to 2003 in the United States were analyzed. Patient-donor pairs were fully typed for HLA-A, -B, -C, -DRB1, -DQB1, -DQA1, -DPB1, and -DPA1 alleles. High-resolution DNA matching for HLA-A, -B, -C, and -DRB1 (8/8 match) was the minimum level of matching associated with the highest survival. A single mismatch detected by low- or high-resolution DNA testing at HLA-A, -B, -C or -DRB1 (7/8 match) was associated with higher mortality (relative risk, 1.25; 95% CI, 1.13-1.38; P < .001) and 1-year survival of 43% compared with 52% for 8/8 matched pairs. Single mismatches at HLA-B or HLA-C appear better tolerated than mismatches at HLA-A or HLA-DRB1. Mismatching at 2 or more loci compounded the risk. Mismatching at HLA-DP or -DQ loci and donor factors other than HLA type were not associated with survival. In multivariate modeling, patient age, race, disease stage, and cytomegalovirus status were as predictive of survival as donor HLA matching. High-resolution DNA matching for HLA-A, -B, -C, and -DRB1 alleles is associated with higher rates of survival.

A novel method for KIR-ligand typing by pyrosequencing to predict NK cell alloreactivity.

Studies have shown that KIR-ligand mismatching to predict NK cell alloreactivity may result in less relapse and better survival in patients with AML. KIR-ligands are distinguished by single nucleotide polymorphisms (SNPs) from HLA-B and HLA-C sequences. We hypothesized that pyrosequencing to determine KIR-ligand status by direct sequencing of the ligand epitope can be done as an alternative to high-resolution HLA-typing. Pyrosequencing is rapid and would be particularly useful in analysis of retrospective cohorts where high-resolution HLA-typing is unavailable or too expensive. To validate this assay, RNA and DNA from 70 clinical samples were tested for KIR-ligand by pyrosequencing. Primer binding to invariant regions without known SNPs was critical for KIR-ligand assignment by pyrosequencing to be in full concordance with high-resolution HLA-typing. Pyrosequencing is sensitive, specific, high-throughput, inexpensive, and can rapidly screen KIR-ligand status to evaluate potential alloreactive NK cell or transplant donors.

Common and well-documented HLA alleles: report of the Ad-Hoc committee of the american society for histocompatiblity and immunogenetics.

In histocompatibility testing some genotype ambiguities are almost always resolved into the genotype with the most common alleles. To achieve unambiguous assignments additional unwieldy tests are performed. The American Society for Histocompatibility and Immunogenetics formed a committee to define what human leukocyte antigen (HLA) genotypes do not need to be resolved in external proficiency testing. The tasks included detailed analysis of large datasets of high-resolution typing and thorough review of the pertinent scientific literature. Strict criteria were used to create a catalogue of common and well-documented (CWD) alleles. In total, 130, 245, 81, and 143 of the highly polymorphic HLA-A, -B, -C, and DRB1 loci fell into the CWD category; these represent 27%-30% of all alleles recognized. For the loci DRB3/4/5, DQA1, DQB1, and DPB1, a total of 29, 16, 26, and 52 CWD alleles were identified. A recommendation indicated that an acceptable report should only include one possible genotype; multiple genotypes can only be reported if only one of these includes two alleles of the CWD group. Exceptions in which resolution is not necessary are ambiguities involving functional alleles with identical sequences in the antigen recognition site. The criteria were established for proficiency testing, which could be a valuable tool when making clinical histocompatibility decisions.

HLA mismatching within or outside of cross-reactive groups (CREGs) is associated with similar outcomes after unrelated hematopoietic stem cell transplantation.

The National Marrow Donor Program maintains a registry of volunteer donors for patients in need of a hematopoietic stem cell transplantation. Strategies for selecting a partially HLA-mismatched donor vary when a full match cannot be identified. Some transplantation centers limit the selection of mismatched donors to those sharing mismatched antigens within HLA-A and HLA-B cross-reactive groups (CREGs). To assess whether an HLA mismatch within a CREG group ("minor") may result in better outcome than a mismatch outside CREG groups ("major"), we analyzed validated outcomes data from 2709 bone marrow and peripheral blood stem cell transplantations. Three-hundred and ninety-six pairs (15%) were HLA-DRB1 allele matched but had an antigen-level mismatch at HLA-A or HLA-B. Univariate and multivariate analyses of engraftment, graft-versus-host disease, and survival showed that outcome is not significantly different between minor and major mismatches (P = .47, from the log-rank test for Kaplan-Meier survival). However, HLA-A, HLA-B, and HLA-DRB1 allele-matched cases had significantly better outcome than mismatched cases (P < .001). For patients without an HLA match, the selection of a CREG-compatible donor as tested does not improve outcome.

A high degree of HLA disparity arises from limited allelic diversity: analysis of 1775 unrelated bone marrow transplant donor-recipient pairs.

The allelic diversity and associated human leukocyte antigen (HLA) disparity of 1775 bone marrow recipients and their unrelated donors, matched for six of six (1361/1775,77%), five of six (397/1775, 22%), or four of six (17/1775, 1%) HLA-A, -B, -DR antigens, were retrospectively evaluated. The comprehensive HLA analysis included the class I (A, B, C) and II (DRB1, DQA1, DQB1, DPA1, DPB1) loci. Most (>66%) of the predominantly Caucasian study population carried one or two of five to seven common alleles at each HLA locus. In spite of this limited diversity, 29% of the six of six antigen-matched transplants carried allele mismatches at HLA-A, -B, and/or -DRB1, and 92% carried at least one allele mismatch at one of the eight HLA loci tested. Of the 968 HLA-A,-B,-DRB1 allele-matched pairs, 89% carried mismatches at other HLA loci, predominantly at DP loci. The substantially greater than expected HLA allelic disparity between donor and recipient suggests extensive haplotypic diversity and underscores the importance of enhancing approaches to mitigate the deleterious effect of HLA mismatches.

HLA dictionary 2004: summary of HLA-A, -B, -C, -DRB1/3/4/5, -DQB1 alleles and their association with serologically defined HLA-A, -B, -C, -DR, and -DQ antigens.

This report presents serologic equivalents of human leukocyte antigen (HLA)-A, -B, -C, -DRB1, -DRB3, -DRB4, -DRB5, and -DQB1 alleles. The dictionary is an update of the one published in 2001. The data summarize equivalents obtained by the World Health Organization (WHO) Nomenclature Committee for Factors of the HLA System, the International Cell Exchange (UCLA), the National Marrow Donor Program, recent publications, and individual laboratories. This latest update of the dictionary is enhanced by the inclusion of results from studies performed during the 13th International Histocompatibility Workshop and from neural network analyses. A summary of the data as recommended serologic equivalents is presented as expert assigned types. The tables include remarks for alleles, which are or may be expressed as antigens with serologic reaction patterns that differ from the well-established HLA specificities. The equivalents provided will be useful in guiding searches for unrelated hematopoietic stem cell donors in which patients and/or potential donors are typed by either serology or DNA-based methods. The serological-DNA equivalent dictionary will also aid in typing and matching procedures for organ transplant programs whose waiting lists of potential donors and recipients are comprised of mixtures of serologic and DNA-based typings. The tables with HLA equivalents and a questionnaire for submission of serologic reaction patterns for poorly identified allelic products will be made available through the World Marrow Donor Association Web page (www.worldmarrow.org).

Impact of HLA class I and class II high-resolution matching on outcomes of unrelated donor bone marrow transplantation: HLA-C mismatching is associated with a strong adverse effect on transplantation outcome.

Outcome of unrelated donor marrow transplantation is influenced by donor-recipient matching for HLA. Prior studies assessing the effects of mismatches at specific HLA loci have yielded conflicting results. The importance of high-resolution matching for all HLA loci has also not been established. We therefore examined the effects of HLA matching (low or high resolution or both) on engraftment, graft-versus-host disease (GVHD), and mortality in 1874 donor-recipient pairs retrospectively typed at high resolution for HLA-A, -B, -C, -DRB1, -DQ, and -DP. Mismatches at HLA-A, -B, -C, and -DRB1 each had similar adverse effects on mortality. Only HLA-A mismatches demonstrated significant adverse effects on GVHD. These adverse effects on outcome were more evident in transplants with low-resolution versus only high-resolution mismatches. Mismatches for HLA-DQ or -DP did not significantly affect outcome. When high-resolution mismatches at HLA-A, -B, -C, and -DRB1 were considered together, adverse effects on survival and GVHD were observed. We therefore conclude that matching for HLA-C should be incorporated into algorithms for unrelated donor selection. High-resolution mismatches at HLA-A, -B, -C, and -DRB1 adversely affect outcome, but less so than low-resolution mismatches. When clinical circumstances allow, high-resolution class I typing may help optimize donor selection and improve outcome.