Human Leukocyte Antigen Typing Research Articles

Correlation of patient HLA class I genotypes with severe immune-related adverse events: A retrospective study in Chinese population.

10586 Background: Immune checkpoint inhibitors (ICIs) are effective in multiple tumors. However, about 10% of immune-related adverse events (irAEs) are severe and can have serious or even life-threatening consequences for patients. We investigated whether Human leukocyte antigen (HLA) genes predispose to and thus hold a predictive role of developing of severe irAEs in Chinese population. Methods: In a case/control study, we collected data from patients with metastatic cancer treated with ICIs and developed severe irAEs (Grade ≥3 and required hospitalization). Patients treated with ICIs without any irAEs were used as controls. Data was collected from15th Nov, 2019 until 10th Oct, 2023. HLA typing was performed via next generation sequencing. Results: We enrolled 83 patients with severe irAEs and 286 patients treated with ICIs without any irAEs. We found HLA-B*15:27 (OR=9.1, X21,95 = 7.2, P = 0.008) and HLA-C*04:01 (OR=2.5, X21,95 = 7.0, P =0.008) were significantly associated with severe overall irAEs risk. When assessing irAE individually, a significant association between HLA-B*13:02 and serious myocarditis (OR = 3.4, X21,95 = 6.2, P =0.01) as well as striking enrichment for HLA-A*02:03 haplotypes in severe adrenal insufficiency (OR = 10.0, X21,95 = 19.1, P<0.0001) were observed. Conclusions: Specific HLA alleles were associated with severe irAEs. Consideration of pre-prescription HLA typing and vigilance for serious reactions to ICIs are warranted.

Novel HLA-Ilo/HLA-IIhi phenotype and immune evasion in triple-negative breast cancer.

1095 Background: Immune checkpoint inhibitors (ICIs) are a therapeutic option for patients with triple-negative breast cancer (TNBC), however resistance and non-response remain an unmet clinical need. Human leukocyte antigens (HLAs) play an important role in induction and regulation of host immune responses. Downregulation of HLA class I expression is one mechanism by which cancer cells evade the immune control. HLA typing and understanding the role of HLAs can provide valuable insight into optimizing immunotherapy efficacy in TNBC. In this study, we evaluate the association of HLA I/II phenotypes with biomarkers of tumor immune interaction and overall survival (OS) in a real-world breast cancer cohort. Methods: A targeted RNA-seq assay was performed on 142 breast cancer samples. HLA I and HLA II expressions were estimated as average expression of HLA-A, HLA-B, HLA-C and HLA-DP, HLA-DQ, HLA-DR genes, respectively, by a percentile rank values [0-100]. Cohen D index was used to estimate ratio of HLA-I and II in each specimen. We particularly focused on the HLA-Ilow(lo)/HLA-IIhigh(hi) ratio given a proposed mechanism of immune escape by tumor cells (low expression of HLA-I) in response to a highly immune infiltrated state where there is increased expression of HLA-II by the immune cells. We studied the co-expression of HLA-Ilo/HLA-IIhi with other checkpoint targets using Spearman correlation(rs). The impact of HLA phenotypes on OS were analyzed using Kaplan-Meier analysis and Chi-squared assessed associations. Results: The cohort consisted of 106 non-TNBC (ER+/HER2- or HER2+) and 36 TNBC tumors. Most samples originated from metastatic visceral sites (96, 68%) followed by primary breast (33, 23%), and lymph nodes (13, 9%). Among TNBC, HLA-Ilo/HLA-IIhi phenotype displayed a significantly higher co-expression of immune checkpoint targets such as PD-1, PD-L1, TIM3 and LAG-3 than HLA-Ihi/HLA-IIlo phenotype (p<0.001). However, this difference was not observed between HLA-Ilo/HLA-IIhi and HLA-Ihi/HLA-IIlo phenotypes in non-TNBC tumors. In TNBC, there was no significant difference in OS between HLA-Ilo/HLA-IIhi when compared to HLA-Ihi/HLA-IIlo (43 vs. 37 months, p=0.21). Similarly, in non-TNBC, there were no significant differences in OS between HLA-Ilo/HLA-IIhi when compared to HLA-Ihi/HLA-IIlo (107 vs. 66 months, p=0.5). Conclusions: Concurrent loss of HLA I with increased HLA II expression was associated with co-expression of immune checkpoint molecules indicative of immune escape mechanism but not survival outcomes in TNBC. This same effect was not seen in non-TNBC with a more immunologically silent environment. Characterization of HLA phenotypes by immune profiling provides insight into the complex interplay of tumor immune microenvironment and offers an opportunity for developing novel approaches to overcome the immune evasion with cancer vaccines and adoptive cell therapies in TNBC.

The role and novel use of natural killer cells in graft-versus-leukemia reactions after allogeneic transplantation.

Allogeneic hematopoietic cell transplantation (HCT) has transformed over the past several decades through enhanced supportive care, reduced intensity conditioning (RIC), improved human leukocyte antigen (HLA) typing, and novel graft-versus-host disease (GVHD)-prevention and treatment strategies. Most notably, the implementation of post-transplantation cyclophosphamide (PTCy) has dramatically increased the safety and availability of this life-saving therapy. Given reductions in nonrelapse mortality (NRM) with these advances, the HCT community has placed even greater emphasis on developing ways to reduce relapse - the leading cause of death after HCT. When using RIC HCT, protection from relapse relies predominantly on graft-versus-leukemia (GVL) reactions. Donor lymphocyte infusion (DLI), adoptive cellular therapy, checkpoint inhibition, and post-HCT maintenance strategies represent approaches under study that aim to augment or synergize with the GVL effects of HCT. Optimizing donor selection algorithms to leverage GVL represents another active area of research. Many of these strategies seek to harness the effects of T cells, which for decades were felt to be the primary mediators of GVL and the focus of investigation in relapse reduction. However, there is growing interest in capitalizing on the ability of natural killer (NK) cells to yield potent anti-tumor effects. A potential advantage of NK cell-based approaches over T cell-mediated is the potential to reduce NRM in addition to relapse. By decreasing infection, without increasing the risk of GVHD, NK cells may mitigate NRM, while still yielding relapse reduction through identification and clearance of cancer cells. Most T cell-focused relapse-prevention strategies must weigh the benefits of relapse reduction against the increased risk of NRM from GVHD. In contrast, NK cells have the potential to reduce both, potentially tipping the scales significantly in favor of survival. Here, we will review the role of NK cells in GVL, optimization of NK cell match or mismatch, and burgeoning areas of research in NK cell therapy such as adoptive transfer and chimeric antigen receptor (CAR) NK cells.

High-resolution African HLA resource uncovers HLA-DRB1 expression effects underlying vaccine response.

How human genetic variation contributes to vaccine effectiveness in infants is unclear, and data are limited on these relationships in populations with African ancestries. We undertook genetic analyses of vaccine antibody responses in infants from Uganda (n = 1391), Burkina Faso (n = 353) and South Africa (n = 755), identifying associations between human leukocyte antigen (HLA) and antibody response for five of eight tested antigens spanning pertussis, diphtheria and hepatitis B vaccines. In addition, through HLA typing 1,702 individuals from 11 populations of African ancestry derived predominantly from the 1000 Genomes Project, we constructed an imputation resource, fine-mapping class II HLA-DR and DQ associations explaining up to 10% of antibody response variance in our infant cohorts. We observed differences in the genetic architecture of pertussis antibody response between the cohorts with African ancestries and an independent cohort with European ancestry, but found no in silico evidence of differences in HLA peptide binding affinity or breadth. Using immune cell expression quantitative trait loci datasets derived from African-ancestry samples from the 1000 Genomes Project, we found evidence of differential HLA-DRB1 expression correlating with inferred protection from pertussis following vaccination. This work suggests that HLA-DRB1 expression may play a role in vaccine response and should be considered alongside peptide selection to improve vaccine design.

HLA variations in patients with diffuse large B-cell lymphoma and association with disease risk and prognosis: a case-control study.

Human leukocyte antigen (HLA) polymorphisms have been associated with the development of various autoimmune diseases, as well as malignant neoplasms. Non-Hodgkin lymphomas (NHLs) are a heterogenous group of lymphoid malignancies in which a genetic substrate has been established and is deemed to play a crucial role in disease pathogenesis. This study aimed to identify whether variations in the HLA gene region were associated with diffuse large B-cell lymphoma (DLBCL) risk and prognosis. We defined HLA class I (HLA-A, HLA-B, HLA-C) and class II (HLA-DRB1, HLA-DQB1) alleles in 60 patients with DLBCL and compared the results to those found by 236 healthy adult donors from the bone marrow bank of Northern Greece. HLA typing was performed by two molecular methods, Sequence - Specific Oligonucleotide HLA typing (SSO) and Sequence - Specific Primer HLA typing (SSP), from white blood cells recovered from peripheral blood. The phenotypic frequencies of HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 between patients and controls were compared with the 2-sided Fisher's exact test. Results with p-value <0.05 were considered statistically significant. Odds Ratios with 95% Confidence Intervals were calculated to further strengthen the results. The 2-sided Fisher's exact test was also applied to alleles found only in one of the two groups, while the odds ratios together with the confidence intervals were corrected with Haldane-Anscombe method. Among the studied HLA polymorphisms, the frequency HLA-C*12 allele was significantly lower in patients with DLBCL compared with control subjects (6.7% vs. 34.7%, OR = 0.16, 95% CI: 0.04-0.44). Frequency of HLA-B*39 was significantly lower in patients with DLBCL compared with controls, but due to the low frequency of this polymorphism in the studied population and small sample size, determinations regarding the significance of this findings were limited. Survival analysis revealed that the presence of HLA-C*12 was not associated with improved or worsened overall and progression-free survival. No statistically significant associations were observed in the phenotypic frequencies of HLA-A, HLA-DQB1, HLA-DRB1 and the rest of HLA-B alleles between the control and DLBCL groups. Collectively, our results provide valuable insight regarding the role of HLA variations on DLBCL risk. Further studies are required to consolidate our findings and ascertain the clinical implications of these genetic variations on DLBCL management and prognosis.

A novel framework for human leukocyte antigen (HLA) genotyping using probe capture-based targeted next-generation sequencing and computational analysis

Human leukocyte antigen (HLA) genes play pivotal roles in numerous immunological applications. Given the immense number of polymorphisms, achieving accurate high-throughput HLA typing remains challenging. This study aimed to harness the human pan-genome reference consortium (HPRC) resources as a potential benchmark for HLA reference materials. We meticulously annotated specific four field-resolution alleles for 11 HLA genes (HLA-A, -B, -C, -DPA1, -DPB1, -DQA1, -DQB1, -DRB1, -DRB3, -DRB4 and -DRB5) from 44 high-quality HPRC personal genome assemblies. For sequencing, we crafted HLA-specific probes and conducted capture-based targeted sequencing of the genomic DNA of the HPRC cohort, ensuring focused and comprehensive coverage of the HLA region of interest. We used publicly available short-read whole-genome sequencing (WGS) data from identical samples to offer a comparative perspective. To decipher the vast amount of sequencing data, we employed seven distinct software tools: OptiType, HLA-VBseq, HISAT genotype, SpecHLA, T1K, QzType, and DRAGEN. Each tool offers unique capabilities and algorithms for HLA genotyping, allowing comprehensive analysis and validation of the results. We then compared these results with benchmarks derived from personal genome assemblies. Our findings present a comprehensive four-field-resolution HLA allele annotation for 44 HPRC samples. Significantly, our innovative targeted next-generation sequencing (NGS) approach for HLA genes showed superior accuracy compared with conventional short-read WGS. An integrated analysis involving QzType, T1K, and DRAGEN was developed, achieving 100% accuracy for all 11 HLA genes. In conclusion, our study highlighted the combination of targeted short-read sequencing and astute computational analysis as a robust approach for HLA genotyping. Furthermore, the HPRC cohort has emerged as a valuable assembly-based reference in this realm.

Abstract 5377: Investigating human leukocyte antigen (HLA) class 1 leukemogenic pathways in acute myelogenous leukemia

Abstract Background: Aging, smoking and germline predisposition are known to accelerate leukemogenesis. However, the the ability for myeloid clonal pathogenic variants (MCPV) to elicit an antigen-driven host antileukemia immune response is not well characterized. A better understanding on how cellular immunity selectively discriminates leukemia-related “neopeptides” could uncover mechanisms supporting tumor genome evolution. Methods: 122 patients with acute myelogenous leukemia (AML) were included and 111 had available protein coding sequences. A neopeptide library was created using protein FASTA coding extracted for individual myeloid clonal mutations via National Center for Biotechnology Information (NCBI). Patient class 1 HLA typing was obtained through Tempus, and Genomic Testing Cooperative laboratories. Prediction of number and strength of AML neopeptide bindings to MHC class I molecules were obtained via NetMHCPan-1. Analysis was performed with K-means algorithm assigning age at diagnosis, detected MCPV, number of neopeptides binding (as continuous variable), overall survival (OS) and event-free survival (EFS) as variables. Unsupervised data input allowed 3 cluster partitions for differential survivals allowing HLA class 1 peptide binding aggregation with potential roles in leukemogenesis and patient outcome. Descriptive statistics were performed with SAS. Results: Sixty-six different MCPV were detected. Of 111 patients, 70 (63%) with available MCPV produced at least 1 MHC I neopeptide binding. Frequently observed variants were P53 (13%), FLT3 (9.2%), WT1 (7%), DNMT3A (6.5%), RAS (5%), ASXL1 (4.3%), RUNX1 (4.2%), NPM1 (4.2%) and IDH1/2 (2.6%). Cluster 2 demonstrated a lack of favorable and higher proportion of adverse ELN 2022 subgroups. Cluster 3 exhibited a higher of P53 [p=0.03] and MDS-related mutations. OS was 79, 239, and 1551 days for cluster 2,1 and 3, respectively [p=0.0001]. EFS was 134, 363 and 451 days for cluster 2,1 and 3, respectively [p=0.0001]. Interestingly, Cluster 2 and 3 assembled the highest probability for peptide binding [1 v 2 v 3 = 1.76% v 36.6% v 62%, p=0.01]. Lastly, albeit in low frequencies, higher diversity of DNA repairing enzymes was seen in cluster 2. Conclusions: Different MCPV arising in AML genome are variably immunogenic and leukemia-related neopeptide binding’s ability is differentially expressed in our 3 clusters. Frequent neopeptide bindings in clusters 2 and 3 may reflect higher AML immunogenicity resulting in genomic instability. It is possible that less “edited” genomes in cluster 1 preserve higher chemosensitivity resulting in better OS, while highly edited genomes in cluster 2 and 3 are less chemo-sensitive and circumvent host immunosurveillance. Further expansion of cluster 2 is necessary to better understand the role of DNA repairing enzymes in younger patients’ outcomes, especially when genomic instability is observed. Citation Format: Paul Sackstein, Sarah Mudra, Lacey Williams, Garrett Diltz, Rachel Zemel, Sravanti Teegavarapu, Martha Mims, Catherine Lai, Kimberley Doucette, Anne Renteria, Olga Timofeeva, Gustavo A. Rivero. Investigating human leukocyte antigen (HLA) class 1 leukemogenic pathways in acute myelogenous leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5377.

Abstract 5378: Spatially resolved human leukocyte antigen (HLA) expression correlates with motif neoepitope expression and durable response to immune checkpoint blockade (ICB) in non-small cell lung cancer (NSCLC)

Abstract Background: We previously showed electrostatic interactions enhance binding between “motif” neoepitopes and HLA and predict advanced NSCLC ICB benefit, suggesting optimal neoantigen presentation drives effective anti-tumor immune response (Cummings 2020). However, not all cases with motif neoepitopes exhibit favorable ICB outcomes. Methods: As proof-of-concept, participants from our published NSCLC ICB cohort with available pre-treatment archival tissue were selected based on HLA supertype B44, predicted motif neoepitopes, and best response of progressive disease (PD) vs partial/complete response (PR/CR). Whole exome sequencing, HLA typing and neoepitope prediction were previously performed (Cummings 2020). GeoMx slides were prepared, hybridized with RNA probes and stained with antibodies to cytokeratin (CK), nuclear material and CD45 per manufacture instructions (Geiss 2008). Antibody to CD8 (Novus) was optimized at 1:50 dilution. Twelve regions of interest (ROI) were selected per sample including the following compartments: CK+CD8+, CK+CD8-, CK-CD8+ and CK-CD8- (negative control). Each ROI included ~150 nuclei selected using GeoMx Digital Spatial Profiler. Sequencing was performed on Illumina Novaseq 6000 (50bpx2) demultiplexed using Illumina Bcl2fastq v2.19.1.403. Raw reads were normalized and compared using paired student’s T-tests with Tukey’s correction via R v4.1. Neoepitope (non/motif) comparisons used matched numbers per sample selected by highest expression. Results: Seven participants (3 PD, 3 PR, 1 CR) were included, each exhibiting 1-7 motif neoepitopes without HLA loss of heterozygosity. 84 ROI included 24 CK+CD8+, 24 CK+CD8-, 24 CK-CD8+ and 12 CK-CD8- with at least one ROI per category per participant. PD samples had fewer CD8+ ROI (12 vs 36) and lower CXCL9 (p=0.011) and CD274 (p=0.014). HLA-A/B/C expression followed a gradient with highest expression in CK+CD8+ ROI with limited detection in CK-CD8- ROI. Among HLA, HLA-B had highest median expression (A: 226, B: 650, C: 202; p&amp;lt;0.001). In CK+ ROI, motif neoepitope expression was detectable for all participants but not statistically different from non-motif expression (p=0.23). HLA-A/B/C all exhibited lower expression in PD CK+ ROI (p&amp;lt;0.001). Conclusion: HLA expression may modify prediction of clinically meaningful neoepitopes and response to ICB. Validation in a larger, prospective cohort is planned. Citation Format: Lucas Goldman, Sarah Gorbatov, Collin Shen, Alexa Berezowitz, Edward Garon, Amy Lauren Cummings. Spatially resolved human leukocyte antigen (HLA) expression correlates with motif neoepitope expression and durable response to immune checkpoint blockade (ICB) in non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5378.

Abstract 5001: Personalized neoantigen/cancer-testis antigen nanovaccine for advanced solid tumors, a single-arm, open-label pilot study

Abstract Background: Neoantigens, derived from somatic mutations in tumor cells, have been identified as promising targets of immunotherapy. A personalized neoantigen/cancer-testis antigen (CTA) nanovaccine (PNVAC) platform has been established by us previously, and demonstrated its feasibility, safety and efficacy in preventing recurrence of high-risk resected gastric/gastroesophageal junction (G/GEJ) cancer in both preclinical and clinical studies. This study aims to explore the universality of PNVAC monotherapy or combined with anti-angiogenesis drugs or anti-programmed death 1 (PD-1) in patients with multiple advanced solid malignancies. Methods: Patient-specific neoantigens were selected based on tumor-specific mutations identified by whole-exome sequencing (WES) and RNA sequencing of paired blood and tumor tissues. Bioinformatic analysis for neoantigen prediction, including sequencing read filtering, human leukocyte antigen (HLA) typing and neoantigen filtering was performed. PNVAC is an amphiphiles nanovaccine loaded with multiple personalized neoantigens designed to induce specific T cell responses. PNVAC is administrated to patients with metastatic solid tumors on days 1, 4, 8, 15, 22, 43 (prime phase) and 64, 85 and 169 (boost phase) alone, or combined with anti-angiogenesis or anti-PD-1 drugs. Safety, immunogenicity and clinical efficacy are evaluated. Results: Of the 30 enrolled patients, no treatment-related severe adverse events (AEs) occurred and the vast majority of AEs were limited to grade 1-2, only 1 patient developed grade 3 thrombocytopenia. The objective response rate (ORR) was 26.7% (8/30), including 2 cases of complete response (CR) and 6 cases of partial response (PR), and a disease control rate (DCR) of 66.7% achieved. The median progression-free survival (PFS) was 9.90 months (95% CI, 3.46-16.34 months), while the median overall survival (OS) was not reached (range, 0.80-43.53 months), and the estimated 1- and 2-year survival rates were 86.2% and 60.6%. Notably, among the 21 patients who had relapsed disease after previous ICB and/or anti-angiogenesis, disease control was asserted in 14 (66.7%) of them (7 of SD, 5 of PR, 2 of CR). ORR of this set was 33.3% (7/21), higher than that of all the enrolled patients (26.7%), and the set without prior ICB and/or anti-angiogenesis treatment (11.1%). For immune analysis, PNVAC elicited robust and persistent immune responses against neoantigens/CTAs. Additionally, peripheral T cells with a cytotoxic phenotype tended to increase after vaccination, and immune memory was also detected in some representative patients. Conclusions: These data supported the safety, immunogenicity, and efficacy of this regimen in patients with advanced solid malignancies, thus broadening the application and combined strategies of neoantigen-based vaccines. Clinical trial information: ChiCTR1800017319. Citation Format: Jia Wei, Qin Liu, Lijing Zhu, Jie Shao, Ju Yang, Guanghui Xu, Nandie Wu, Hanbing Wang, Rutian Li, Huizi Sha, Qiuping Xu, Jie Shen, Li Xie, Lifeng Wang, Juan Du, Lanqi Cen, Manman Tian, Lixia Yu, Baorui Liu. Personalized neoantigen/cancer-testis antigen nanovaccine for advanced solid tumors, a single-arm, open-label pilot study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5001.

Abstract 6911: Expanding reported molecular characteristics for the NCI Patient-Derived Models Repository

Abstract The National Cancer Institute’s Patient-Derived Models Repository (NCI PDMR; https://pdmr.cancer.gov) has developed a national repository of Patient-Derived Models (PDMs) comprised of patient-derived xenografts (PDXs), in vitro patient-derived tumor cell cultures (PDCs) and cancer associated fibroblasts (CAFs) as well as patient-derived organoids (PDOrg). These PDMs are clinically annotated with molecular information available in an easily accessible database for the extramural community. To ensure the data and models remains pertinent to current translational efforts, NCI actively reviews and adds new molecular features to the public database for researchers to use. While these data could be obtained through analysis of the available NextGen sequencing files in the PDMR database, many laboratories do not have the bioinformatics support to run these assessments for models that are of research interest. Therefore, new pipelines for an array of additional model and molecular characteristics present in PDX, PDOrg, and PDC models have been validated and are reported in the public database in a searchable format. Current model characteristics include addition of OncoTree Codes as a second histologic classifier, use of the PDXNet Consortium’s inferred ancestry caller to better represent Native/Latin American populations, human leukocyte antigen (HLA) typing, molecular classification of tumors (e.g. CMS subtyping of colorectal adenocarcinoma, PAM50 signature based subtyping of breast carcinoma), microsatellite instability (MSI) assessment, and identification of clinically relevant fusions. SOPs for the pipelines used are available on the public website to allow for independent validation and use by other laboratories. Funded by NCI Contract No. HHSN261200800001E Citation Format: Cindy R. Timme, Yvonne A. Evrard, Biswajit Das, Ting-Chia Chang, Alice Chen, Li Chen, Michelle A. Crespo-Eugeni, Shahanawaz Jiwani, Chris A. Karlovich, Rini Pauly, Peter I-Fan Wu, Dianne L. Newton, P. Mickey Williams, Melinda G. Hollingshead, James H. Doroshow. Expanding reported molecular characteristics for the NCI Patient-Derived Models Repository [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6911.

Abstract 6570: HLA genotyping and HLA-based clinical trial matching using MSK-IMPACT, a targeted next-generation sequencing assay

Abstract Purpose: Performing tumor next-generation sequencing (NGS) and human leukocyte antigen (HLA) typing in a single test can maximize the likelihood of matching patients with cancer to clinical trials. We investigated the utility of a clinical-grade NGS assay for this purpose. Methods: HLA class I genotypes were determined on an investigational basis by MSK-IMPACT (FDA-authorized hybrid-capture, tumor and matched normal DNA-based NGS) using POLYSOLVER for patients with cancers of any histology between March 2014 and September 2023. Patient HLA genotype matches for phase I clinical trials were identified; for a subset of patients, concordance between HLA genotyping was compared to a CLIA-certified laboratory test. Results: 60,565 patients successfully underwent complete class I HLA genotyping (two alleles each of HLA-A, HLA-B, and HLA-C). The median age at diagnosis was 64 years old and most patients were female (55%) and white (76%). The most common genotypes were HLA-A*02:01 (42%, n=25,457), HLA-C*04:01 (28%, n=17,274), HLA-A*01:01 (25%, n=14,867), HLA-C*07:01 (24%, n=14,538), HLA-A*03:01 (21%, n=12,668), HLA-C*07:02 (21%, n=12,493), and HLA-B*07:02 (16%, n=9,787). Most patients (96%, n=58,287) were HLA genotype eligible [HLA-A*02:01 (n=28), HLA-A*11:01 (n=3), HLA-A*01:01 (n=1), and HLA-C*07:02 (n=1)] for ≥1 of 33 active early phase adoptive T cell therapy or T cell receptor bispecific trials on Clinicialtrials.gov. 280 patients with HLA class I matches were screened for phase I trial candidacy by the MSK phase I group. 117 were candidates for confirmatory HLA and neoantigen testing, of whom 43 patients were enrolled on a trial after additional screening. 72 patients had HLA genotyping both by MSK-IMPACT and an outside CLIA-certified laboratory; 100% (72/72) of these subjects had concordant genotypes. Conclusions: MSK-IMPACT successfully determined class I HLA genotypes in a large, multi-ethnic population of patients. A substantial proportion of patients were identified as potential candidates for HLA-matched trials, supporting local trial enrollment. Citation Format: Monica F. Chen, Michael V. Gormally, Anne Marie Noronha, Kristalla Panageas, Maggie Reynolds, Kaelyn Kohlasch, Bryan Novak, Tatiana Kee-Velez, Nikeysha Clarke, Chrisann Kyi, Claire Friedman, Sandra D'Angelo, Mark G. Kris, Christopher A. Klebanoff, Ronak Shah, Michael Berger, Chaitanya Bandlamudi, Alexander Drilon, Mark T. Donoghue. HLA genotyping and HLA-based clinical trial matching using MSK-IMPACT, a targeted next-generation sequencing assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6570.

Ready for polygenic risk scores? An analysis of regulation of preimplantation genetic testing in European countries.

Would the different regulatory approaches for preimplantation genetic testing (PGT) in Europe permit the implementation of preimplantation genetic testing using polygenic risk scores (PGT-P)? While the regulatory approaches for PGT differ between countries, the space provided for potential implementation of PGT-P seems limited in all three regulatory models. PGT is a reproductive genetic technology that allows the testing for hereditary genetic disorders and chromosome abnormalities in embryos before implantation. Throughout its history, PGT has largely been regarded as an ethically sensitive technology. For example, ethical questions have been raised regarding the use of PGT for adult-onset conditions, non-medical sex selection, and human leukocyte antigen typing for the benefit of existing siblings. Countries in which PGT is offered each have their own approach of regulating the clinical application of PGT, and a clear overview of legal and practical regulation of PGT in Europe is lacking. An emerging development within the field of PGT, namely PGT-P, is currently bringing new ethical tensions to the forefront. It is unclear whether PGT-P may be applied within the current regulatory frameworks in Europe. Therefore, it is important to investigate current regulatory frameworks in Europe and determine whether PGT-P fits within these frameworks. The aim of this study was to provide an overview of the legal and practical regulation of the use of PGT in seven selected European countries (Belgium, France, Germany, Italy, the Netherlands, Spain, and the UK) and critically analyse the different approaches with regards to regulatory possibilities for PGT-P. Between July and September 2023, we performed a thorough and extensive search of websites of governments and governmental agencies, websites of scientific and professional organizations, and academic articles in which laws and regulations are described. We investigated the legal and regulatory aspects of PGT by analysing legal documents, regulatory frameworks, scientific articles, and guidelines from scientific organizations and regulatory bodies to gather relevant information about each included country. The main sources of information were national laws relating to PGT. We divided the PGT regulation approaches into three models. The regulation of PGT differs per country, with some countries requiring central approval of PGT for each new indication (the medical indication model: the UK, the Netherlands), other countries evaluating each individual PGT request at the local level (the individual requests model: France, Germany), and countries largely leaving decision-making about clinical application of PGT to healthcare professionals (the clinical assessment model: Belgium, Italy, Spain). In the countries surveyed that use the medical indication model and the individual requests model, current legal frameworks and PGT criteria seem to exclude PGT-P. In countries using the clinical assessment model, the fact that healthcare professionals and scientific organizations in Europe are generally negative about implementation of PGT-P due to scientific and socio-ethical concerns, implies that, even if it were legally possible, the chance that PGT-P would be offered in the near future might be low. The results are based on our interpretation of publicly available written information and documents, therefore not all potential discrepancies between law and practice might have been identified. In addition, our analysis focuses on seven-and not all-European countries. However, since these countries are relevant players within PGT in Europe and since they have distinct PGT regulations, the insights gathered give relevant insights into diverse ways of PGT regulation. To the best of our knowledge, this is the first paper that provides a thorough overview of the legal and practical regulation of PGT in Europe. Our analysis of how PGT-P fits within current regulation models provides guidance for healthcare professionals and policymakers in navigating the possible future implementation of PGT-P within Europe. This project has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement no. 813707. The authors declare no conflict of interest. N/A.

Pathogenetic analysis of transfusion-related acute lung injury caused by human leukocytes antigen antibody against human leukocyte antigen

From September 2019 to October 2020, pathogenetic analysis of three patients clinically diagnosed as transfusion-related acute lung injury (TRALI) caused by human leukocyte antibodies was conducted by Guangzhou Blood Centre, including 2 males and 1 female, aged 56, 50 and 20 years old, respectively. Solid phase agglutination, anti-human globulin test and flow cytometry method were used to detect the presence of antibodies against patients. Sequencing-based human leukocyte antigen (HLA-SBT) typing technique was used to detect the human leukocyte antigen (HLA) genotypes of patients. Lifecodes single antigen class Ⅰ/Ⅱ kit (LSA-Ⅰ/Ⅱ) were used to detect the specificity of HLA-class Ⅰ and class Ⅱ antibodies in donor blood by Luminex 200 liquid suspension chip system. The HLA specific antibodies and corresponding epitopes in donors were also analyzed. The results showed that HLA class Ⅰ or class Ⅱ specific antibodies against TRALI patients were detected in the blood donors. The plasma of donor 3 received by patient 1 contained antibodies against the patient's HLA-DRB1*09∶01 antigen, and the epitopes mediating the antibody reaction of the donor and recipient were 70R, 31I, 70QA. There were antibodies against the HLA-A*11∶02, HLA-A*11∶01, DRB1*12∶02, and DRB1*09∶01 antigens of patient 2 in the plasma of donor 4, and the associated antigenic epitopes were 151AHA, 57V, and 16Y. Antibodies against the HLA-DRB1*14∶04, DRB1*11∶01, and DPB1*05∶01 antigens of patient 3 were present in the plasma of donor 6 and donor 7, and the associated epitopes were 96HK, 140TV, 13SE, and 111K. Three cases of TRALI were confirmed to be caused by HLA antibodies through laboratory analysis, and human leukocyte antibody detection should be paid attention in clinically suspected cases of TRALI, and targeted diagnosis and treatment should be given.

Frequency distribution of HLA class I and II alleles in Greek population and their significance in orchestrating the National Donor Registry Program.

Human leukocyte antigens (HLA) represent one of the most polymorphic systems in humans, responsible for the identification of foreign antigens and the presentation of immune responses. Therefore, HLA is considered to play a major role in human disorders, donor-recipient matching and transplantation outcomes. This study aimed to determine the HLA class I and II alleles and haplotypes in the Greek population. Moreover, a comparative analysis of HLA alleles and haplotype frequencies found in Greek and pooled European populations was also performed to acquire a better knowledge about the HLA alleles distribution. A total number of 1896 healthy individuals were typed for their HLA alleles in the National Tissue Typing Center of Greece. High-resolution HLA typing for the HLA-A, -B, -C and -DR, -DQ, -DP with the use of the next-generation sequencing analysis was performed, followed by data analysis for establishing the HLA allele and haplotype differences. The results of this study showed that the most frequent alleles for the HLA-A were the A*02:01:01 (27.1%), *24:02:01 (14.4%), *01:01:01 (9.3%), for the HLA-B were the B*51:01:01 (15.3%), *18:01:01 (9.7%), *35:01:01 (6.8%) and for the HLA-C were the C*04:01:01 (15.4%), *07:01:01 (13.1%), *12:03:01 (9.6%). For the HLA class II, the most frequent alleles for the HLA-DRB1 were the DRB1*11:04:01 (16.4%), *16:01:01 (11.3%), *11:01:01 (9.5%), for the HLA-DQB1 were the DQB1*03:01:01 (30.5%), *05:02:01 (15.1%), *05:01:01 (10.6%) and for the HLA-DPB1 were the DPB1*04:01:01 (34.8%), *02:01:01 (11.6%), *04:02:01 (7.3%). Additionally, the most frequent haplotypes were the A*02:01:01∼C*07:01:01-B*18:01:01∼DRB1*11:04:01 (2.3%), followed by the A*01:01:01∼C*07:01:01∼B*08:01:01∼DRB1*03:01:01 (2.2%), A*24:02:01∼C*04:01:01∼B*35:02:01∼DRB1*11:04:01 (1.4%) and A*02:01:01∼C*04:01:01∼B*35:01:01-DRB1*14:01:01 (1.2%). The results herein were comparable to those obtained from the pooled European populations. Moreover, these results can be used for the improvement of the donor-recipient matching procedure and to understand better the disease association in Greece.

Genome-wide association study identifies human genetic variants associated with fatal outcome from Lassa fever

Infection with Lassa virus (LASV) can cause Lassa fever, a haemorrhagic illness with an estimated fatality rate of 29.7%, but causes no or mild symptoms in many individuals. Here, to investigate whether human genetic variation underlies the heterogeneity of LASV infection, we carried out genome-wide association studies (GWAS) as well as seroprevalence surveys, human leukocyte antigen typing and high-throughput variant functional characterization assays. We analysed Lassa fever susceptibility and fatal outcomes in 533 cases of Lassa fever and 1,986 population controls recruited over a 7 year period in Nigeria and Sierra Leone. We detected genome-wide significant variant associations with Lassa fever fatal outcomes near GRM7 and LIF in the Nigerian cohort. We also show that a haplotype bearing signatures of positive selection and overlapping LARGE1, a required LASV entry factor, is associated with decreased risk of Lassa fever in the Nigerian cohort but not in the Sierra Leone cohort. Overall, we identified variants and genes that may impact the risk of severe Lassa fever, demonstrating how GWAS can provide insight into viral pathogenesis.

Role of Complement-dependent Cytotoxicity Crossmatch and HLA Typing in Solid Organ Transplant.

Solid organ transplantation is a life-saving medical operation that has progressed greatly because of developments in diagnostic tools and histocompatibility tests. Crossmatching for complement-dependent cytotoxicity (CDC) and human leukocyte antigen (HLA) typing are two important methods for checking graft compatibility and reducing the risk of graft rejection. HLA typing and CDC crossmatching are critical in kidney, heart, lung, liver, pancreas, intestine, and multi-organ transplantation. A systematic literature search was conducted on the internet, using PubMed, Scopus, and Google Scholar databases, to identify peer-reviewed publications about solid organ transplants, HLA typing, and CDC crossmatching. Recent advances in HLA typing have allowed for high-resolution evaluation, epitope matching, and personalized therapy methods. Genomic profiling, next-generation sequencing, and artificial intelligence have improved HLA typing precision, resulting in better patient outcomes. Artificial intelligence (AI) driven virtual crossmatching and predictive algorithms have eliminated the requirement for physical crossmatching in the context of CDC crossmatching, boosting organ allocation and transplant efficiency. This review elaborates on the importance of HLA typing and CDC crossmatching in solid organ transplantation.

Utilizing Massively Parallel Sequencing (MPS) of Human Leukocyte Antigen (HLA) Gene Polymorphism to Assess Relatedness in Deficiency Parentage Testing.

In the realm of DNA testing with legal implications, the reliability and precision of genetic markers play a pivotal role in confirming or negating paternity claims. This study aimed to assess the potential utility of human leukocyte antigen (HLA) gene polymorphism through massively parallel sequencing (MPS) technology as robust forensic markers for parentage testing involving genetic deficiencies. It sought to redefine the significance of HLA genes in this context. Data on autosomal short tandem repeat (aSTR) mutational events across 18 paternity cases involving 16 commonly employed microsatellite loci were presented. In instances where traditional aSTR analysis failed to establish statistical certainty, kinship determination was pursued via HLA genotyping, encompassing the amplification of 17 linked HLA loci. Within the framework of this investigation, phase-resolved genotypes for HLA genes were meticulously generated, resulting in the definition of 34 inherited HLA haplotypes. An impressive total of 274 unique HLA alleles, which were classified at either the field 3 or 4 level, were identified, including the discovery of four novel HLA alleles. Likelihood ratio (LR) values, which indicated the likelihood of the observed data under a true biological relationship versus no relationship, were subsequently calculated. The analysis of the LR values demonstrated that the HLA genes significantly enhanced kinship determination compared with the aSTR analysis. Combining LR values from aSTR markers and HLA loci yielded conclusive outcomes in duo paternity cases, showcasing the potential of HLA genes and MPS technology for deeper insights and diversity in genetic testing. Comprehensive reference databases and high-resolution HLA typing across diverse populations are essential. Reintegrating HLA alleles into forensic identification complements existing markers, creating a potent method for future forensic analysis.

DQB1 antigen matching improves rejection-free survival in pediatric heart transplant recipients

IntroductionPresence of donor-specific antibodies (DSAs), particularly to class II antigens, remains a major challenge in pediatric heart transplantation. Donor-recipient human leukocyte antigen (HLA) matching is a potential strategy to mitigate poor outcomes associated with DSAs. We evaluated the hypothesis that antigen mismatching at the DQB1 locus is associated with worse rejection-free survival. MethodsData was collected from Scientific Registry of Transplant Recipients for all pediatric heart transplant recipients 2010-2021. Only transplants with complete HLA typing at the DQB1 locus for recipient and donor were included. Primary outcome was rejection-free graft survival through 5 years. ResultsOf 5,115 children, 4,135 had complete DQB1 typing and were included. Of those, 503 (12%) had 0 DQB1 donor-recipient mismatches, 2,203 (53%) had 1, and 1,429 (35%) had 2. Rejection-free survival through 5 years trended higher for children with 0 DQB1 mismatches (68%), compared to those with 1 (62%) or 2 (63%) mismatches (pairwise p=0.08 for both). In multivariable analysis, 0 DQB1 mismatches remained significantly associated with improved rejection-free graft survival compared to 2 mismatches, while 1 DQB1 mismatch was not. Subgroup analysis showed the strongest effect in non-Hispanic Black children and those undergoing retransplant. ConclusionMatching at the DQB1 locus is associated with improved rejection-free survival after pediatric heart transplant, particularly in Black children, and those undergoing retransplant. Assessing high-resolution donor typing at the time of allocation may further corroborate and refine this association. DQB1 matching may improve long-term outcomes in children stabilized either with optimal pharmacotherapy or supported with durable devices able to await ideal donors.

Non-invasive molecular biomarkers for monitoring solid organ transplantation: A comprehensive overview.

Solid organ transplantation is a life-saving intervention for individuals with end-stage organ failure. Despite the effectiveness of immunosuppressive therapy, the risk of graft rejection persists in all viable transplants between individuals. The risk of rejection may vary depending on the degree of compatibility between the donor and recipient for both human leucocyte antigen (HLA) and non-HLA gene-encoded products. Monitoring the status of the allograft is a critical aspect of post-transplant management, with invasive biopsies being the standard of care for detecting rejection. Non-invasive biomarkers are increasingly being recognized as valuable tools for aiding in the detection of graft rejection, monitoring graft status and evaluating the efficacy of immunosuppressive therapy. Here, we focus on the importance of molecular biomarkers in solid organ transplantation and their potential role in clinical practice. Conventional molecular biomarkers used in transplantation include HLA typing, detection of anti-HLA antibodies, killer cell immunoglobulin-like receptor genotypes, and anti-MHC class 1-related chain A antibodies, which are important for assessing the compatibility of the donor and recipient. Emerging molecular biomarkers include the detection of donor-derived cell-free DNA, microRNAs (regulation of gene expression), exosomes (small vesicles secreted by cells), and kidney solid organ response test, in the recipient's blood for early signs of rejection. This review highlights the strengths and limitations of these molecular biomarkers and their potential role in improving transplant outcomes.

HLA inherence as a potential parameter in checkpoint inhibitor-associated autoimmune adverse event assessment.

The success of immunotherapy has made it a lifesaving treatment, but not without side effects. Currently, the risk factors for developing immune-related adverse events (irAEs) in patients who receive immunotherapy are poorly understood, and there is no risk-stratifying mechanism for potentially fatal irAEs. It is postulated that oncology patients with preexisting autoimmune diseases are likely to have flares on immunotherapy. However, some patients develop de novo autoimmune conditions on immunotherapy without a prior history. Literature reports have postulated that human leukocyte antigen (HLA) inherence may play a role in irAEs. However, this potential remains underexplored. The oncology patients who developed autoimmune adverse events on immunotherapy for whom the continuation of treatment was prudent or lifesaving were selected. Of note, all nine patients received checkpoint inhibitors (CIs). Of the nine selected patients, only one had a prior history of an autoimmune condition. None of the nine selected patients had an active autoimmune condition at the time of CI initiation. Their HLA was typed, and the results were cross-referenced with the literature reports in PubMed and Google search with the corresponding autoimmune condition of each patient. Herein, we report nine patients with irAEs for whom retrospective HLA typing revealed the inherence of multiple related HLA alleles that may correspond to the autoimmune condition that they had developed on immunotherapy. It is to be mentioned that the inherence of enriched disease-related HLA alleles was shared among patients with the same irAEs. These patients developed a range of irAEs including bullous pemphigoid, pemphigus foliaceus/vulgaris, thyroiditis, vitiligo, and hepatitis on immunotherapy. Although some combinations of disease-related HLA were well reported in otherwise idiopathic autoimmune diseases, a frequently repeated HLA allele combination in our patient population was found to be rarely seen in the general population. The authors suggest that an enriched inherence of disease-related HLA alleles may play a role in the genetic propensity for the development of irAEs in oncology patients, who receive immunotherapy, including CIs. Inherence of more than one or a cluster of particular autoimmune disease-related HLA alleles in patients who receive immunotherapy may unmask the corresponding autoimmune disease as the genotype inherence presents with the phenotype of the corresponding condition. It is suggested that enriched linked HLA genotypes, which are otherwise rare in the general population, may present as the corresponding phenotype of the autoimmune condition. Such clinical presentation, enhanced by immunotherapy, such as CIs, can play a role in risk stratifying patients for precision medicine and improve the outcome.