High Resolution HLA Typing Research Articles

Intrabone Transplantation of a Single Unwashed Umbilical Cord Blood Unit with Antithymocyte Globulin-Free and Sirolimus-Based Graft-versus-Host Disease Prophylaxis: Fast Immune Reconstitution and Long-Term Disease Control in Patients with High-Risk Diseases

Several strategies have been explored with the attempt of improving the safety and feasibility of umbilical cord blood transplantation (UCBT) in adults. The aim of this retrospective analysis was to examine the safety and efficacy of intrabone transplantation of a single unwashed cord blood unit in an antithymocyte globulin-free, sirolimus-based graft-versus-host disease prophylaxis platform. We collected data for all consecutive UCBTs infused intrabone (IB) and unwashed at San Raffaele Hospital in Milan between 2012 and 2021. Thirty-one consecutive UCBTs were identified. All but 3 UCB units had a high-resolution HLA typing on 8 loci at the time of selection. At the time of cryopreservation, the median CD34+ cell count was 1 × 105/kg (range, .6 to 12.0 × 105/kg) and the median total nucleated cell (TNC) count was 2.8 × 107/kg (range, 1.48 to 5.6 × 107/kg). Eighty-seven percent of patients received myeloablative conditioning, and 77% underwent transplantation for acute myeloid leukemia. The median duration of follow-up among survivors was 38.2 months (range, 10.4 to 123.6 months). No adverse events were related to the IB infusion at bedside under short-conscious periprocedural sedation or to the no wash technique. After thawing, median CD34+ cell and TNC counts were .8 × 105/kg (range, .1 to 2.3 × 105/kg) and 1.42 × 107/kg (range, .69 to 3.2 × 107/kg). The median time to engraftment was 27 days for neutrophils and 53 days for platelets. One patient experienced graft rejection and was subsequently rescued with a salvage transplantation. The median time to a CD3+ cell count >100/μL was 30 days. The 100-day cumulative incidence of grade III-IV acute graft-versus-host disease (GVHD) was 12.9% (95% confidence interval [CI], 4% to 27.3%), and the 2-year cumulative incidence of moderate-to-severe chronic GVHD (cGVHD) was 11.8% (95% CI, 2.7% to 28.3%). At 2 years, overall survival (OS) was 52.7% (95% CI, 33% to 69%), relapse incidence was 30.7% (95% CI, 13.7% to 49.6%), and transplantation-related mortality was 29% (95% CI, 14.3% to 45.6%). In univariate analysis, infused CD34+ cell count did not impact transplantation outcomes. In patients who underwent transplantation in first complete remission, relapse rate was 13%, with a 2-year OS >90%. In our cohort, IB infusion of a single cord blood unit was feasible, with no adverse reactions related to the no wash/IB infusion, low rates of cGVHD and disease relapse, and rapid immune reconstitution.

Development of a calculated panel reactive antibody calculator for the United Arab Emirates: a proof of concept study

Calculated panel reactive antibody (CPRA) is used to help increase sensitized patient’s access to transplantation. United Arab Emirates (UAE) has a diverse resident population hence we developed a UAE–CPRA calculator based on HLA antigen frequencies of the different ethnic groups that represent the UAE population. HLA antigen frequencies at serological split antigen level for HLA-A, -B, -C, -DRB1 and -DQB1 of 1002 healthy unrelated donors were performed. We subsequently compared the performance of the UAE CPRA calculator with the Organ Procurement and Transplantation Network (OPTN) and the Canadian CPRA calculators in 110 Kidney Transplant waitlist patients from January 2016 to December 2018. Lin’s concordance correlation coefficient showed a moderate agreement between the UAE and OPTN calculator (Rc = 0.949, 95% CI 0.929–0.963) and the UAE and Canadian calculators (Rc = 0.952, 95% CI 0.932–0.965). While there continued to be a moderate agreement (Rc = 0.937, UAE versus OPTN calculator) in the lower sensitized group, a poor agreement (Rc = 0.555, UAE versus OPTN calculator) was observed in the higher sensitized group. In this study, we provide a template for countries to develop their own population-specific CPRA calculator. Implementation of the CPRA algorithm based on HLA frequencies of the multi-ethnic UAE population will be more fitting to increase access to transplantation and improve transplant outcomes. Our study demonstrates that the CPRA calculators developed using the data from the western population had poor correlation in our higher sensitized patients disadvantaging them in potential organ allocations systems. We plan to further refine this calculator by using high resolution HLA typing to address the problem of a genetically diverse population.

HLA-B leader genotypes in a clinical population.

The -21 dimorphism in the leader sequences of HLA-B exon 1 is associated with risk of graft-versus-host disease (GVHD), relapse and overall survival after unrelated donor hematopoietic cell transplantation (HCT), haploidentical HCT and cord blood transplantation. Consideration of the leader dimorphism in the prospective selection of allogeneic donors for HCT may help to lower risks for patients, but requires understanding of the frequencies of the leader in patients and candidate transplant donors. We defined the frequencies of the HLA-B leader, and its association to HLA-B Bw4/Bw6 and C1/C2 KIR epitopes. Sequence variants of rs1050458 of exon 1 position -21 for 11,126 haplotypes were analyzed from high resolution HLA typing of over 5500 study subjects. HLA typing was performed by TruSight/AlloSeq NGS and analyzed using TruSight/AlloSeq Assign software. HLA-B Bw4/Bw6 and C1/C2 KIR epitopes were defined based on established sequence alignments and nomenclature. Alleles at rs1050458 of HLA-B exon 1 were validated as dimorphic: rs1050458-C or -T variants encoding threonine (T) or methionine (M) at anchor position 2 (P2) of nonameric HLA-B leader peptides, respectfully. No additional variants were observed. Among study subjects, 70% of HLA-B haplotypes encoded T-leader and 30% encoded M-leader sequences. The genotype frequencies of TT, MT, and MM were consistent among patient, related, and unrelated donor groups. The associations of M/T leader, Bw4/Bw6, and C1/C2 enhanced understanding of the Class I features involved in the innate immune response. A population of patients and transplant donors confirms the rs1050458 leader dimorphism and its association with HLA-B Bw4/Bw6 and C1/C2 KIR features.

Using Next-Generation Sequencing Technology to Confirm the HLA Rare Alleles Detected by PCR-SSOP

To confirm the HLA genotypes of the samples including 4 cases of magnetic bead probe HLA genotyping result pattern abnormality and 3 cases of ambiguous result detected by PCR sequence-specific oligonudeotide probe (SSOP) method. All samples derived from HLA high-resolution typing laboratory were detected by PCR-SSOP. A total of 4 samples of magnetic bead probe HLA genotyping result pattern abnormality and 3 samples of ambiguous result were further confirmed by PCR sequence-based typing (SBT) technology and next-generation sequencing (NGS) technology. A total of 4 samples of magnetic bead probe HLA genotyping result pattern abnormality were detected by PCR-SSOP method. The results of SBT and NGS showed that the HLA-A genotype of sample 1 did not match any known genotypes. NGS analysis revealed that the novel allele was different from the closest matching allele A*31:01:02:01at position 154 with G>A in exon 2, which resulting in one amino acid substitution at codon 28 from Valine to Methionine (p.Val28Met). The HLA-C genotype of sample 2 was C*03:119, 06:02, sample 3 was C*03:03, 07:137, and sample 4 was B*55:02, 55:12. A total of 3 samples with ambiguous result were initially detected by PCR-SSOP method. The re-examination results of SBT and NGS showed that the HLA-B genotype of sample 5 was B*15:58, 38:02, sample 6 was DRB1*04:05, 14:101, and sample 7 was DQB1*03:34, 05:02. Among them, alleles C*03:119, C*07:137 and DRB1*14:101 were not included in the Common and Well-documented Alleles (CWD) v2.4 of the Chinese Hematopoietic Stem Cell Donor Database. The abnormal pattern of HLA genotyping results of magnetic probe by PCR-SSOP method suggests that it may be a rare allele or a novel allele, which needs to be verified by sequencing.

The role of the HLA allelic repertoire on the clinical severity of COVID-19 in Canadians, living in the Saskatchewan province

AimsThe HLA system has been implicated as an underlying determinant for modulating the immune response to SARS-CoV-2. In this study, we aimed to determine the association of patients’ HLA genetic profiles with the disease severity of COVID-19 infection. MethodsProspective study was conducted on COVID-19 patients (n = 40) admitted to hospitals in Saskatoon, Canada, between March and December 2020. Next-generation sequencing was performed on the patient samples to obtain high-resolution HLA typing profiles. The statistical association between HLA allelic frequency and disease severity was examined. The disease severity was categorized based on the length of hospital stay and intensive care needs or demise during the hospital stay. ResultsHLA allelic frequencies of the high and low-severity cohorts were normalized against corresponding background allelic frequencies. In the high-severity cohort, A*02:06 (11.8-fold), B*51:01 (2.4-fold), B*15:01(3.1-fold), C*01:02 (3.3-fold), DRB1*08:02 (31.2-fold), DQ*06:09 (11-fold), and DPB1*04:02(4-fold) were significantly overrepresented (p < 0.05) making these deleterious alleles. In the low-severity cohort, A*24:02 (2.8-fold), B*35:01 (2.8-fold), DRB1*04:07 (5.3-fold), and DRB1*08:11 (22-fold) were found to be significantly overrepresented (p < 0.05) making these protective alleles. These above alleles interact with NK cell antiviral activity via the killer immunoglobulin-like receptors (KIR). The high-severity cohort had a higher predilection for HLA alleles associated with KIR subgroups; Bw4-80I (1.1-fold), and C1 (1.6-fold) which promotes NK cell inhibition, while the low-severity cohort had a higher predilection for Bw4-80T (1.6-fold), and C2 (1.6-fold) which promote NK cell activation. ConclusionIn this study, the HLA allelic repository with the distribution of deleterious and protective alleles was found to correlate with the severity of the clinical course in COVID-19. Moreover, the interaction of specific HLA alleles with the KIR-associated subfamily modulates the NK cell-mediated surveillance of SARS-CoV-2. Both deleterious HLA alleles and inhibitory KIR appear prominently in the severe COVID-19 group focusing on the importance of NK cells in the convalescence of COVID-19.

Evaluation of 19years of international external proficiency testing for high-resolution HLA typing.

The international high-resolution external proficiency testing (EPT) started in 2004 with high-resolution typing of human leucocyte antigen (HLA) class I (HLA-A,B,C) and HLA class II (HLA-DRB1, DRB345, DQB1, and DPB1) alleles, since possibilities for such an EPT within Europe were limited and all existing EPTs at that time made use of the comparison of HLA typing results without a reference. This EPT was set up as a collaboration between the HLA laboratory of Leiden, providing DNA samples to the participants, and the laboratory of Maastricht, performing the high-resolution typing as the reference result and evaluating the results of all participants according to the prevailing European Federation for Immunogenetics (EFI) standards. Once a year, 12 samples were sent to the participating laboratories, and evaluation and certificates were provided at the end of that same year. During the years, the EPT was extended to low-resolution HLA class I and II typing, high-resolution typing including DQA1 and DPA1, and allelic resolution typing for HLA class I, the latter one being unique in this field. Evaluation of the high-resolution typing results of the last 19years showed a clear increase in the number of loci tested by the participating laboratories and a clear change of method from Sanger sequencing with additional other techniques (SSO/SSP) to the nowadays widely used next-generation sequencing method. By strictly using the EFI rules for high-resolution HLA typing, the participants were made aware of the ambiguities within exons 2 and 3 for class I and exon 2 for class II and the presence of null alleles even in a two-field HLA typing. There was an impressive learning curve, resulting in >98% correctly typed samples since 2017 and a 100% fulfillment of EFI rules for all laboratories for all loci submitted in the last 2years. Overall, this EPT meets the need of an EPT for high-resolution typing for EFI accreditation.

Impact of Allele-Level HLA-Matching on Outcomes after Double Unrelated Cord Blood Transplantation in Adults with Malignant Diseases

Introduction: In single unrelated cord blood transplantation (sCBT), an increasing number of HLA-mismatches (MM) has been associated with inferior overall survival (OS) attributed to higher transplant-related mortality (TRM). These findings have prompted current guidelines to endorse high resolution HLA typing in the selection of CB (cord blood) units, including double CBT (dCBT). However, a comprehensive appraisal of the effect of allele-level HLA matching following dCBT was likely hindered by the relatively small sample sizes in previous studies. Herein we aimed to analyze the impact of allele-level HLA-matching on outcomes of a large cohort of dCBT recipients to improve CB unit selection and further explore the unique dCBT biology. Methods: We included patients ≥18 years-old receiving a dCBT between 2006-2019 with the availability of HLA typing minimally at HLA-A, -B, -C at the antigen-level and HLA-DRB1 at the allele-level for the recipient and both CB units. A validated high-resolution imputation tool, HaploStats, was used to adjudicate allele-level match status for a subset of cord and recipient pairs (n = 173) having only antigen-level typing at HLA-A, -B or -C. A sensitivity analysis for OS was performed excluding imputed cases, and results were unchanged. HLA matching was classified according to the CB unit with the greatest degree of HLA mismatch with the patient, meaning that only the most mismatched unit was considered and that the interunit matching was not considered. Continuous covariates were dichotomized to find an optimal cutoff value by using the Contal-O'Quigley method, except for cryopreserved CD34 and total nucleated cell (TNC) count, whose cutoffs were based on previous findings reported by our group. Pairwise comparisons of the numbers of allele MM showed that receiving units with ≥4 MM had the highest negative impact on OS. Cox and Fine-Gray regression models were built for the different outcomes using the backward elimination method (p<.05 to stay). Results: 963 patients met the eligibility criteria, of which 392 received at least one CB unit with 0-3 allele MM while 571 with ≥4 allele MM. Of those receiving at least one unit with ≥4 allele MM, 18% had a maximum HLA disparity of 5/6 at lower HLA-typing resolution, 69% of 4/6 and 13% of ≤3/6. Median age at dCBT was 49 years old. Acute leukemia and myelodysplastic syndrome (MDS) accounted for 70% of the cases. There was a larger proportion of patients receiving CB units with fewer allele MM after 2012. Other patient-, disease- and CB unit-related characteristics are summarized in Table 1. The day-42 cumulative incidence of neutrophil recovery was 83% (95%CI 81-85). The number of allele MM had no significant impact on neutrophil recovery (≥ 4 MM vs 0-3 MM, multivariate HR 0.94 [0.80-1.11], P=.46). The cumulative incidence of TRM at 4 years was 36% (95% CI 32-40) for patients receiving CB units with ≥4 MM vs. 23% (95% CI 19-28) for those with 0-3 MM (multivariate HR 1.58 [95%CI 1.18-2.11], P=.002] - Fig. 1A). Infectious complications were the most frequent cause of TRM in the group receiving dCBT with ≥ 4 allele MM (40%). Relapse, grade II-IV/III-IV acute and chronic GVHD were not significantly different between the two groups. Patients receiving CB units with 0-3 allele MM had a 4-year survival of 54% (95%CI 49-59) compared to 43% (95%CI 39-47) for those receiving units with ≥4 allele MM (multivariate HR 1.40 [1.11-1.78], P=.005 - Fig. 1B). The inferior OS associated with higher allele HLA disparity was only partially mitigated by increasing the TNC count in multivariate analysis (0-3 MM + TNC ≤ 3.9x10ˆ7/kg vs.0-3 MM + TNC > 3.9x10ˆ7/kg, HR 1.50 [0.97-2.32], P=.07; ≥4 MM + TNC > 4.9x10ˆ7/kg vs. 0-3 MM + TNC > 3.9x10ˆ7/kg, HR 1.42 [1.06-1.89], P=.02; ≥4 MM + TNC ≤ 4.9x10ˆ7/kg vs. 0-3 MM + TNC > 3.9x10ˆ7/kg, HR 1.66 [1.24-2.22], P=<.001 - Fig. 1C). Discussion: Our results confirm that allele-level HLA typing is a significant factor for OS following dCBT. The inferior OS associated with the increasing number of allele-level MM was driven by higher TRM. Selection of CB units with 0-3 allele-level MM is recommended. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Estimation of Antibody-Verified Eplet Mismatch Load, 2-Field HLA Resolution vs Imputation in a Large Cohort of European Donors

The assessment of class II eplet mismatch load is useful to determine the risk of chronic rejection in solid organ transplantation. However, high-resolution (2-field) HLA typing is mandatory to accurately define eplet mismatches. The imputation of the most frequent allele has been used in retrospective studies. Here, we sought to investigate the concordant of antibody-verified (AbV) eplet load in different class II alelles between real 2-field HLA typing and HLA imputed by most frequent allele in a large White cohort. The allelic frequency of the different HLA class II loci was calculated using a database of high-resolution typing of 23,628 voluntary Spanish bone marrow donors obtained from the Spanish Registry of Bone Marrow Donors, managed by the Josep Carreras Foundation. The AbV eplet count in the different class II alleles was performed using the HLA-Matchmaker v3.1 algorithm. The probability of imputing the correct allele compared to the most frequent for DRB1 and DQB1 loci was 69.3% and 53.0%, respectively. However, studying the less frequent alleles, the same AbV eplet load was observed in 82.22% and 76.15%, respectively. Our data show that despite the potential error in the imputation of class II HLA typing, the number of AbV eplets is not significantly over- or underestimated in our population. Until high-resolution typing is widely established for solid organ transplantation, retrospective studies of AbV eplet mismatch load with imputed high-resolution types provide an acceptable outcome in White people.

Virtual crossmatching and epitope analysis in kidney transplantation: What the physician involved in kidney transplantation should know?

Solid-phase single antigen bead (SAB) assay for detection of anti-human leukocyte antigen (HLA) antibodies and high-resolution HLA typing have enabled tremendous progress in virtual crossmatch (VXM) technology in recent years. However, misinterpretation of the SAB assay may result in detrimental consequences after kidney transplantation. Meanwhile, epitope analysis could be an effective method to estimate immunizing eplets, which may provide ancillary information for better understanding of the SAB assay. To perform epitope analysis appropriately, it is necessary to understand the basic principles related to histocompatibility testing and the characteristics of the SAB assay. Therefore, knowledge of the properties and limitations of the SAB assay is critical. In this review, we aim to describe the fundamental concepts regarding immunobiological assessment, including HLA, anti-HLA antibodies, and SAB assay, and explain epitope analysis using examples.

On the clinical relevance of using complete high-resolution HLA typing for an accurate interpretation of posttransplant immune-mediated graft outcomes.

Complete and high-resolution (HR) HLA typing improves the accurate assessment of donor–recipient compatibility and pre-transplant donor-specific antibodies (DSA). However, the value of this information to identify de novo immune-mediated graft events and its impact on outcomes has not been assessed. In 241 donor/recipient kidney transplant pairs, DNA samples were re-evaluated for six-locus (A/B/C/DRB1/DQB1+A1/DPB1) HR HLA typing. De novo anti-HLA antibodies were assessed using solid-phase assays, and dnDSA were classified either (1) as per current clinical practice according to three-locus (A/B/DRB1) low-resolution (LR) typing, estimating donor HLA-C/DQ typing with frequency tables, or (2) according to complete six-locus HR typing. The impact on graft outcomes was compared between groups. According to LR HLA typing, 36 (15%) patients developed dnDSA (LR_dnDSA+). Twenty-nine out of 36 (80%) were confirmed to have dnDSA by HR typing (LR_dnDSA+/HR_dnDSA+), whereas 7 (20%) did not (LR_dnDSA+/HR_dnDSA−). Out of 49 LR_dnDSA specificities, 34 (69%) were confirmed by HR typing whereas 15 (31%) LR specificities were not confirmed. LR_dnDSA+/HR_dnDSA+ patients were at higher risk of ABMR as compared to dnDSA− and LR_dnDSA+/HR_dnDSA− (logRank < 0.001), and higher risk of death-censored graft loss (logRank = 0.001). Both LR_dnDSA+ (HR: 3.51, 95% CI = 1.25–9.85) and LR_dnDSA+/HR_dnDSA+ (HR: 4.09, 95% CI = 1.45–11.54), but not LR_dnDSA+/HR_dnDSA− independently predicted graft loss. The implementation of HR HLA typing improves the characterization of biologically relevant de novo anti-HLA DSA and discriminates patients with poorer graft outcomes.

A low-cost, sensitive and specific PCR-based tool for rapid clinical detection of HLA-B*35 alleles associated with delayed drug hypersensitivity reactions.

HLA (HLA) alleles are risk factors for CD8+ T-cell-mediated drug hypersensitivity reactions. However, as most HLA associations are incompletely predictive and/or involve risk alleles at low frequency, costly sequence-based typing can elude an economically productive cost: benefit ratio for clinical validation studies and diagnostic and/or preventative screening. Hence rapid and low-cost detection assays are now required, both for single alleles but also across risk loci associated with broader multi-disease risk; exemplified by associations with diverse alleles in HLA-B*35, including HLA-B*35:01 and green tea- or co-trimoxazole-induced liver injury. Here, we developed a cost-effective (<$10USD) qPCR assay for rapid (<2.5h) clinical detection of HLA-B*35 alleles. The assay was validated using 430 DNA samples with previous American society for histocompatibility and immunogenetics-accredited sequence-based high-resolution HLA typing, positively detecting all HLA-B*35 allelic variants in our cohort, and as expected by primer design, the six samples that expressed low-frequency B*78:01. The assay did not result in positive detection for any negative control allele. With expected detection of B*35 and B*78, our assay sensitivity (95% CI, 95.07%-100.00%) and specificity (95% CI, 98.97%-100.00%) of 100% using as low as 10ng of DNA provides a reliable HLA-B*35 screening tool for clinical validation and HLA-risk-based prevention and diagnostics.

Exclusion of HLA-C Genotype with Zero Mismatched PCR-SBT Results by Next Generation Sequencing

Three cases of rare alleles of HLA-C with zero mismatched PCR-SBT results were analyzed by full-length sequencing to determine the true genotypes. Three rare HLA-C alleles with zero mismatched PCR-SBT results were screened from clinical transplant matching samples, and the full-length sequence was detected by next-generation sequencing technology. The results of PCR-SBT typing of 3 samples were: HLA-C*03:04, 12:167; HLA-C*07:291, 15:02; HLA-C*01:43, 08:16. Other alleles were not in the CWD table of common and confirmed HLA alleles in China (version 2.3) except common allele HLA-C*03:04, HLA-C*15:02. NGS full-length sequencing revealed that the HLA-C genotypes of the three samples were a combination of common alleles and novel alleles, and the three novel alleles had a base mutation in exons 6, 2, and 4, respectively. The novel allele sequences have been submitted to the Genbank database (MK629722, MK335474, MK641803), which were officially named HLA-C*03:04:74, HLA-C*15:192, HLA-C*08:01:25 by the WHO HLA Nomenclature Committee. The HLA high-resolution typing results of 3 samples were: HLA-C*03:04:74, HLA-C*12:03; HLA-C*07:02, HLA-C*15:192; HLA-C*01:02, HLA-C*08:01:25. HLA typing results containing rare alleles should be treated cautiously, and the full-length sequence should be verified by NGS or cloning. The laboratory finally confirmed that the 3 cases of PCR-SBT zero mismatch HLA-C genotypes are the combination of common alleles and novel alleles by NGS sequencing, which provides an accurate basis for clinical transplantation matching and enriches the human HLA genetic database.

Abstract 5132: HLA-I homozygosity as a protective biomarker for developing immune related adverse events (irAE) among non-small cell lung cancer (NSCLC) patients treated with single agent immunotherapy in the first- or second-line setting

Abstract Background: Biomarkers that predict risk of immune-mediated adverse events (irAEs) among non-small cell lung cancer (NSCLC) patients may reduce morbidity and mortality associated with these treatments. While it has been suggested that human leukocyte antigen (HLA-I) homozygosity is associated with short survival among these patients, its association with toxicity has not been examined. Method: We carried out high resolution HLA typing on 193 NSCLC patients treated with anti-PD1/PDL1. Toxicity data was collected and graded as per common terminology criteria for adverse event (CTCAE) v5.0. We investigated the correlation between HLA-I/II homozygosity, different supertypes and alleles with irAE. In addition, we investigated the relationship between irAE, clinical benefit rate (CBR), progression free survival (PFS) and overall survival (OS). Results: We recorded 103 irAE among 179 (58%) patients suitable for analysis. Homozygosity at one or more HLA-I loci, but not HLA-II was associated with increased risk of irAE with relative risk RR=0.57 (95%CI 0.31-0.96, P=0.025). None of the patients with homozygosity at one or more HLA-I loci developed pneumonitis of any grade (P=0.037) or grade 3 toxicity (P=0.023). The presence of HLA-A03 (N=49) supertype or HLA-DRB1*0401 (N=22) genotype was correlated with increased risk of developing irAE, (RR=1.40, 95%CI 0.99-2.01, P=0.039) and (RR=1.51, 95%CI 1.05-2.06, P=0.023) respectively. In contrast, none of the patients with HLA-DQB1*0301 (N=5) experienced gastrointestinal toxicity (P=0.048) and HLA-DRB1*15:01 (N=16) seems to be protective against developing arthralgia (RR=0.19, 95%CI 0.03-1.0, P=0.041). The occurrence of any irAE was associated with improved ORR, PFS and OS (RR=1.94, 95%CI 1.57-2.47, P less than 0.0001), (HR=0.37, 95%CI 0.25-0.54, P less than 0.0001) and (HR=0.26, 95%CI 0.16-0.41, P less than 0.0001) respectively. The usage of steroid to treat irAE did not diminish the positive clinical outcome. Conclusions: Homozygosity at one or more HLA-I loci can be a useful biomarker to predict irAE among NSCLC patients treated with anti-PD1/PD-L1 in the first- or second-line setting. This is more important among patients who developed pneumonitis or Grade 3 toxicities. However, the occurrence of any irAE is correlated with favourable clinical outcome. Citation Format: Afaf Abed, Ngie Law, Leslie Calapre, Johnny Lo, Vikas Bhat, Samantha Bowyer, Michael Millward, Elin Gray. HLA-I homozygosity as a protective biomarker for developing immune related adverse events (irAE) among non-small cell lung cancer (NSCLC) patients treated with single agent immunotherapy in the first- or second-line setting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5132.

Unique Molecular Identifier-Based High-Resolution HLA Typing and Transcript Quantitation Using Long-Read Sequencing.

HLA typing provides essential results for stem cell and solid organ transplants, as well as providing diagnostic benefits for various rheumatology, gastroenterology, neurology, and infectious diseases. It is becoming increasingly clear that understanding the expression of patient HLA transcripts can provide additional benefits for many of these same patient groups. Our study cohort was evaluated using a long-read RNA sequencing methodology to provide rapid HLA genotyping results and normalized HLA transcript expression. Our assay used NGSEngine to determine the HLA genotyping result and normalized mRNA transcript expression using Athlon2. The assay demonstrated an excellent concordance rate of 99.7%. Similar to previous studies, for the class I loci, patients demonstrated significantly lower expression of HLA-C than HLA-A and -B (Mann–Whitney U, p value = 0.0065 and p value = 0.0154, respectively). In general, the expression of class II transcripts was lower than that of class I transcripts. This study demonstrates a rapid high-resolution HLA typing assay using RNA-Seq that can provide accurate HLA genotyping and HLA allele-specific transcript expression in 7–8 h, a timeline short enough to perform the assay for deceased donors.

HlaR: A rapid and reproducible tool to identify eplet mismatches between transplant donors and recipients

Eplet mismatch load, both overall and at the single molecule level, correlates with transplant recipient outcomes. However, precise eplet assessment requires high-resolution HLA typing of both the donor and recipient. Anything less than high-resolution typing requires imputation of HLA types. The currently available methods to identify eplet mismatch are both tedious and demanding. Therefore, we developed a software package and user-friendly web application (hlaR), that simplifies the workflow of eplet analysis, provides functions to impute high-resolution from low-resolution data and calculates both overall and single molecule eplet mismatch for single or multiple donor recipient pairs. Compared to manual assessments using currently available tools (namely, HLAMatchMaker), hlaR resulted in only minimal discrepancy in eplet mismatches (mean absolute difference of 0.56 for class I and 0.86 for class II for unique sum across loci). Additionally, output of the single molecule eplet function compared well to manual calculation, with an average single antigen count increase of 0.19. Importantly, the hlaR tool permits rapid and reproducible imputation and eplet mismatch including comparison between eplet reference tables (e.g. HLAMatchMaker version 2 or 3). Users can import data from a spreadsheet rather than relying on keystroke entry of individual donor and recipient data, thus reducing the risk of data entry errors. The resulting improved scalability of the hlaR tool is highlighted by plotting analysis time against the size of the input dataset. The new hlaR tool can provide eplet mismatch data with a streamlined workflow. With decreased effort from the end user, eplet matching and mismatch load data can be further incorporated into both research and clinical use.

P696 Genetic predisposition to infliximab immunogenicity in patients with immune-mediated inflammatory diseases – secondary analyses from a randomised clinical trial

Abstract Background Immunogenicity is a leading cause of treatment failure to TNF inhibitors. Genetic variations in HLA class II genes have been suggested to predispose to anti-drug antibody formation, but studies using high-resolution HLA typing as well as characterisation of biologically relevant haplotypes are currently lacking. The aim of this study was to assess associations between HLA loci and immunogenicity to infliximab. Methods Patients with immune-mediated inflammatory diseases (IMID) treated with infliximab (N=612) (120 rheumatoid arthritis, 181 spondyloarthritis, 72 psoriatic arthritis, 114 ulcerative colitis, 80 Crohn disease, and 45 psoriasis) were included in the randomised, Norwegian Drug Monitoring (NOR-DRUM) trial. Neutralising anti-drug antibodies were detected with an automated fluorescence assay. Next generation HLA sequencing at G group resolution were performed and trimmed to 2nd field. Haplotype analyses including the most significant HLA loci were performed, and epitope predictions for the infliximab light- and heavy-chain were assessed. Results Anti-drug antibodies to infliximab were detected in 147 (24 %) of patients. The most significant associations with immunogenicity were with the genes HLA-DQB1 (P=1.4x10-6), -DRB1 (P= 2.7x10-5), -DQA1 (P=0.00017) and -B (P=0.0009). Conditional analyses indicated that HLA-DQB1 represents the primary association(Table 1). Haplotype analyses showed that there was significantly increased risk of immnogenicity in patients carrying the HLA-DQ2 haplotypes DQB1*02:01~DQA1*05:01 (P=1.1x10-6) and DQB1*02:02~DQA1*02:01 (P=0.008), while one protective haplotype DQB1*05:01~DQA1*01:01 (P=0.0005) was identified (Figure 1a-c). Presence of HLA-DQ2 more than doubled the risk of anti-drug antibody formation (HR 2.54, 95%CI 1.82–3.56) (Figure 1d), in a model including the covariates age, sex, diagnosis, and concomitant immunosuppressive therapy. Association tests of each level of HLA analyses showed that HLA-DQ2 represents the strongest association(Figure 2). Epitope predictions revealed that the HLA-DQ2 haplotypes DQB1*02:01-DQA1*05:01 and DQB1*02:02-DQA1*02:01 share nine strong binder peptides, originating from the infliximab light chain, encompassing the amino acid core-peptide sequences INTVESEDI and VYACEVTHQ. Conclusion Using high-resolution HLA sequencing, we here demonstrate a genetic association between HLA-DQ2 and infliximab immunogenicity in IMID patients. The presence of either HLA-DQ2 risk haplotypes more than doubled the risk of anti-drug antibody formation across all diagnoses. Testing for HLA-DQ2 could be of clinical value in identifying patients at risk for immunogenicity and facilitate treatment decisions. Whether these findings apply to other TNF inhibitors should be further explored.

Unique Molecular Identifier based High-resolution HLA Typing and Transcript Quantitation using Long-Read Sequencing

Unique Molecular Identifier based High-resolution HLA Typing and Transcript Quantitation using Long-Read Sequencing

Single nucleotide polymorphisms within HLA region are associated with the outcomes of unrelated cord blood transplantation

Cord blood transplantation (CBT) provides a treatment scheme for hematologic diseases and leukemia in both children and adults. However, adverse reactions and transplantation-related death may still occur in patients receiving CBT even when donor and recipient have fully matched HLA in high-resolution HLA typing analysis. Single nucleotide polymorphisms (SNPs) of HLA-related and unrelated genes are known to associate with disease status of patients with unrelated stem cell transplantation. In this study, the genomic regions ranging from 500 base pairs upstream to 500 base pairs downstream of the eight SNPs that were reported as transplantation determinants by Petersdorf et al. were analyzed to evaluate whether genetic variants were associated with the survival status of patients, and the risk for severe (grades 3–4) graft-versus-host disease (GVHD) or cytomegalovirus (CMV) infection/reactivation. The analyses were performed in the mode of recipient genotype, donor genotype, and recipient-donor mismatching, respectively. By analysis of sixty-five patients and their HLA-matched unrelated donors, we found that five SNPs were associated with patient survival which included the recipient genotype with SNPs of rs107822 in the RING1 gene, and rs2070120, rs17220087 and rs17213693 in the HLA-DOB gene; and the recipient-donor mismatching with SNPs of rs9282369 in HLA-DOA gene, and rs2070120, rs17220087 and rs17213693 in the HLA-DOB gene. Five SNPs were associated with the risk for severe GVHD which included the donor genotype with SNPs of rs213210 and rs2523675; the recipient genotype with SNPs of rs9281491 in the HCP5 gene; and the recipient-donor mismatching with SNPs of rs209130 in the TRIM27 gene, and rs986522 in the COL11A2 gene. Six SNPs were related to the risk for CMV infection/reactivation which included the donor genotype with SNPs of rs435766, rs380924, and rs2523957; and the recipient-donor mismatching with SNPs of rs2070120, rs17220087, and rs17213693 in the HLA-DOB gene; and rs435766 and rs380924 in the MICD gene. This study provides the basis for larger analyses and if the results are confirmed, a way of selecting better unrelated CBT candidate donors.

The Copy Number of Disease-Associated HLA Alleles Predicts the Response to Immunosuppressive Therapy in Acquired Aplastic Anemia

In immune-mediated acquired aplastic anemia (AA), the presence of an HLA allele, which is highly overrepresented or lost due to somatic mutations, may represent a specific immune pathophysiology and a clinical manifestation. HLA-B*14:02 is one of the most overrepresented class I alleles in AA and is also frequently affected by a somatic loss of expression; the inherited B*14:02 genotype was correlated with high-risk clonal evolution in two independent cohorts in the U.S. (Babushok DV et al. Blood Adv 2017; Zaimoku Y et al. manuscript in preparation). In contrast, HLA-B*14:02 is virtually absent in Japanese, in whom somatic mutations of AA have frequently been detected in HLA-B*40:02, B*54:01, and A*02:06, and occasionally in A*02:01, A*02:07, A*31:01, B*13:01, B*40:01, B*40:03, B*44:03, B*55:02, and B*56:01 (Mizumaki H et al. Haematologica 2021). A class II allele HLA-DRB1*15 is highly overrepresented in AA across various ethnic groups, including those in the U.S. and Japanese. This retrospective study in the Japanese population aimed to explore the clinical significance of disease-associated non-B*14:02 HLA class I and II alleles in AA.A total of 423 enrolled patients with AA (very severe [n = 81], severe [n = 266], transfusion dependent non-severe [n = 76]; median age 60 [range, 1-86] years) had undergone genotyping for HLA-A, HLA-B, HLA-C, and HLA-DRB1 at 2-field resolution. The HLA allele frequencies in these patients were compared to those in a Japanese HLA haplotype dataset (n = 19183; Ikeda N et al. Tissue Antigens 2016).The most overrepresented allele in AA was HLA-DRB1*15:02, followed by DRB1*15:01, B*40:02, and A*02:06 (Table); DRB1*13:02 and B*44:03, which are in linkage disequilibrium, were markedly underrepresented, consistent with a well-known protective role of DRB1*13 against autoimmune diseases. HLA-DRB1*15:02 was also significantly correlated with age and its frequency among patients aged <50 years was below the level of the control group (Figure A). Frequencies of HLA-DRB1*15:01, B*40:02, and A*02:06 were increased in both older and younger groups. HLA-A*31:01 and A*02:01 found to be enriched in young patients instead of DRB1*15:02 (Figure B); B*13:01, B*55:02, and B*56:01 showed a similar tendency for an early onset. HLA-B*40:02 was correlated with disease severity and was especially overrepresented in very severe AA (Figure C).The overall response rate to anti-thymocyte globulin-based immunosuppressive therapy at 6 months was 63% (139 of 220 treated and evaluable patients). A trend for a higher response was observed in patients harboring mutation-related HLA-B alleles (except for minor alleles B*13:01, B*40:03, and B*55:02) and the highly overrepresented or protective HLA-DRB1 alleles, but not in the HLA-A alleles (Figure D). A multivariate logistic regression revealed that the combination of the presence of any favorable alleles in HLA-B (odds ratio 3.6, P < 0.0001) or in HLA-DRB1 (odds ratio 2.3, P = 0.00085) was significantly and independently associated with a hematologic response; the tendencies for a lower or higher response in very severe disease and the presence of paroxysmal nocturnal hemoglobinuria clone did not reach statistical significance. Further, there was likely an additive effect when two favorable alleles coexisted in HLA-B or HLA-DRB1 (Figure E); the copy number of the favorable HLA-B and HLA-DRB1 alleles stratified the response rate to four groups: three or four copies, 95% (19 of 20); two copies, 72% (61 of 85); one copy, 59% (50 of 85); and zero copy, 30% (9 of 30). Only eight patients displayed clonal evolution to monosomy 7, myelodysplastic syndrome, or acute myeloid leukemia after immunosuppression without significant overrepresentation or underrepresentation of the pathogenic HLA alleles.Using a large dataset of homogeneous Japanese population with high-resolution HLA typing, we revealed, for the first time, a strong relationship between disease-associated (overrepresented, inactivated, or protecting) HLA alleles and the responsiveness to immunosuppressive therapy. [Display omitted] DisclosuresTakamatsu: Bristol-Myers Squibb: Honoraria, Research Funding; SRL: Consultancy; Adaptive Biotechnologies, Eisai: Honoraria; Janssen: Consultancy, Honoraria, Research Funding. Yamazaki: Novartis Pharma: Honoraria; Kyowa Kirin: Honoraria; Kyowa Kirin: Research Funding. Nakao: Symbio: Consultancy; Kyowa Kirin: Honoraria; Novartis Pharma: Honoraria; Alexion Pharma: Research Funding.

Barriers to and Results of Allogeneic Hematopoietic Transplantation for Adult Patients with Sickle Cell Disease: An Analysis of Referrals and Transplants Performed at a Single Center in the Era of Haploidentical Donor Use

Allogeneic hematopoietic cell transplantation (alloHCT) is the only proven curative treatment for homozygous sickle cell disease (SCD) with debilitating symptoms. alloHCT may ideally be performed in children with severe manifestations of SCD before they develop irreversible organ dysfunction. However, many adult patients may also be appropriate for alloHCT. The historic lack of HLA-matched donors for this population has recently been ameliorated by the use of HLA-haploidentical donors with a T-replete graft and post-transplant cyclophosphamide (HAPLO). However, patients with SCD belong to ethnically and socio-economically underserved populations and may face several additional barriers to accessing complex and expensive curative strategies such as alloHCT. We analyzed SCD patients referred between January 2012 and April 2021 for alloHCT to our center- an adult only center with special expertise in HAPLO transplantation, located in an area with a large population of black patients. Success and barriers to progression from referral to alloHCT were assessed using our institutional database and coordinator records where they had been prospectively entered. Numbers of patients advancing from referral through stages of pre-transplant workup to alloHCT are shown in Table 1. Fifty-nine patients referred to our program for consideration of alloHCT for SCD during the period. The number of annual referrals increased during the studied period from 7 in 2012-2014 to 35 in 2018-2021. Of referred patients, 78% had an initial consult, 41% underwent high resolution HLA typing, 34% also had HLA typing of family members, 31% had an acceptable donor (HLA-identical, 8/8 matched MUD or HAPLO without donor-specific antibodies) and 14% underwent alloHCT. No significant differences were seen among the above categories with respect to age, gender, self-assigned race, year of referral and insurance status. Referrals came from a limited subset of community hematologists- with one physician each referring 9, 6, and 4 patients. Three physicians referred 3 patients, six referred 2 patients and nineteen referred 1 each. Reasons for referred patients not proceeding to alloHCT were: psychosocial issues including lack of caregiver support and history of non-compliance with treatments (n=15), insufficient disease severity by institutional criteria (n=14), failure of referred patient to attend initial consult (n=13), medical comorbidities precluding transplant (n=9), refusal of alloHCT by clinically and psycho-socially suitable patient (n=7), lack of acceptable donor despite suitable patient and search (n=4), refusal of relatives to undergo HLA-typing for an otherwise suitable patient (n=2). Patients (n=8) who underwent alloHCT a median of 253d post referral and 232d post initial consult. Transplant characteristics were: donor- HAPLO (6), MRD (1), MUD (1); graft source BM (8); intensity - NMA (4), RIC (4); median CD34 dose- 3.6x 10e6/kg (1.5-4.9); median CD3 dose 3.3x 10e7/kg (2.4-4.5); HCT-CI - <3 (2), >4(6); CMV pos patient with neg donor (3); ABO major incompatible (3). Engraftment was assessed using T-cell (CD3) and myeloid (CD33) donor chimerism. Median CD33 chimerisms were 100% on each assessment (d30, 60, 100, 180 and 360). Median CD3 chimerisms were 52%, 100%, 100%, 100%, 93% respectively. All evaluable patients achieved > 95% CD33 chimerism by d 30. Two patients (one MRD, one HAPLO) transplanted on an earlier protocol without ATG and thiotepa subsequently rejected their graft. All five HAPLO patients transplanted using the ATG/thiotepa based regimen used in BMTCTN 1507 had sustained high level engraftment (>95% CD33 chimerism) with freedom from SCD symptoms post-transplant. With a median follow-up of 39 months, three-year cumulative incidences of grade 2-4 acute GVHD, and moderate to severe NIH grade chronic GVHD were 12.5% and 0% respectively. Estimated 3 year overall survival was 85%. These data show that while most referred patients lack HLA matched donors, allo-HCT performed using HAPLO donors and RIC conditioning incorporating ATG and thiotepa with a BM graft is highly effective at eradicating SCD. However, many barriers are faced by referred patients resulting in only a small percentage of referred patients proceeding to transplant. Some of these barriers may be overcome by education of referring physicians, patients and their families [Display omitted] DisclosuresSolh: Jazz Pharmaceuticals: Consultancy; Partner Therapeutics: Research Funding; BMS: Consultancy; ADCT Therapeutics: Consultancy, Research Funding. OffLabel Disclosure:Preparative regimens for allogeneic transplantation typically involve off-label use of chemotherapy drugs