Benchmarking NGS-Based HLA Typing Algorithms.

Knowledge of the expected accuracy of HLA typing algorithms is important when choosing between algorithms and when evaluating the HLA typing predictions of an algorithm. This chapter guides the reader through an example benchmarking study that evaluates the performances of four NGS-based HLA typing algorithms as well as outlining factors to consider, when designing and running such a benchmarking study. The code related to this benchmarking workflow can be found at https://github.com/nikolasthuesen/springers-hla-benchmark/ .

Corneal Allografts: Factors for and against Acceptance.

Cornea is one of the most commonly transplanted tissues worldwide. However, it is usually omitted in the field of transplantology. Transplantation of the cornea is performed to treat many ocular diseases. It restores eyesight significantly improving the quality of life. Advancements in banking of explanted corneas and progressive surgical techniques increased availability and outcomes of transplantation. Despite the vast growth in the field of transplantation laboratory testing, standards for corneal transplantation still do not include HLA typing or alloantibody detection. This standard practice is based on immune privilege dogma that accounts for high success rates of corneal transplantation. However, the increasing need for retransplantation in high-risk patients with markedly higher risk of rejection causes ophthalmology transplantation centers to reevaluate their standard algorithms. In this review we discuss immune privilege mechanisms influencing the allograft acceptance and factors disrupting the natural immunosuppressive environment of the eye. Current developments in testing and immunosuppressive treatments (including cell therapies), when applied in corneal transplantation, may give very good results, decrease the possibility of rejection, and reduce the need for retransplantation, which is fairly frequent nowadays.

Challenges for the standardized reporting of NGS HLA genotyping: Surveying gaps between clinical and research laboratories.

Next generation sequencing (NGS) is being applied for HLA typing in research and clinical settings. NGS HLA typing has made it feasible to sequence exons, introns and untranslated regions simultaneously, with significantly reduced labor and reagent cost per sample, rapid turnaround time, and improved HLA genotype accuracy. NGS technologies bring challenges for cost-effective computation, data processing and exchange of NGS-based HLA data. To address these challenges, guidelines and specifications such as Genotype List (GL) String, Minimum Information for Reporting Immunogenomic NGS Genotyping (MIRING), and Histoimmunogenetics Markup Language (HML) were proposed to streamline and standardize reporting of HLA genotypes. As part of the 17th International HLA and Immunogenetics Workshop (IHIW), we implemented standards and systems for HLA genotype reporting that included GL String, MIRING and HML, and found that misunderstanding or misinterpretations of these standards led to inconsistencies in the reporting of NGS HLA genotyping results. This may be due in part to a historical lack of centralized data reporting standards in the histocompatibility and immunogenetics community. We have worked with software and database developers, clinicians and scientists to address these issues in a collaborative fashion as part of the Data Standard Hackathons (DaSH) for NGS. Here we report several categories of challenges to the consistent exchange of NGS HLA genotyping data we have observed. We hope to address these challenges in future DaSH for NGS efforts.

A practical guide to chimerism analysis: Review of the literature and testing practices worldwide.

BACKGROUND AND PURPOSE: Currently there are no widely accepted guidelines for chimerism analysis testing in hematopoietic cell transplantation (HCT) patients. The objective of this review is to provide a practical guide to address key aspects of performing and utilizing chimerism testing results. In developing this guide, we conducted a survey of testing practices among laboratories that are accredited for performing engraftment monitoring/chimerism analysis by either the American Society for Histocompatibility & Immunogenetics (ASHI) and/or the European Federation of Immunogenetics (EFI). We interpreted the survey results in the light of pertinent literature as well as the experience in the laboratories of the authors. RECENT DEVELOPMENTS: In recent years there has been significant advances in high throughput molecular methods such as next generation sequencing (NGS) as well as growing access to these technologies in histocompatibility and immunogenetics laboratories. These methods have the potential to improve the performance of chimerism testing in terms of sensitivity, availability of informative genetic markers that distinguish donors from recipients as well as cost. SUMMARY: The results of the survey revealed a great deal of heterogeneity in chimerism testing practices among participating laboratories. The most consistent response indicated monitoring of engraftment within the first 30 days. These responses are reflective of published literature. Additional clinical indications included early detection of impending relapse as well as identification of cases of HLA-loss relapse.

[Definition and standardization of histocompatibility requests depending on patient course and donor type: Guidelines from the Francophone Society of Bone Marrow Transplantation and Cellular Therapy (SFGM-TC) and the Francophone Society of Histocompatibility and Immunogenetics (SFHI)]. / Définition et standardisation des bilans d'histocompatibilité en fonction du parcours du patient et du type de donneur : recommandations de la Société francophone de greffe de moelle et de thérapie cellulaire (SFGM-TC) et de la Société Francophone d'Histocompatibilité et Immunogénétique (SFHI).

Standardization of histocompatibility tests for allogeneic hematopoietic cell transplants, harmonization of information transmitted to clinicians are part of quality improvement and optimization of human and economic resources. New HLA typing technologies provide high-resolution information within a reasonable time frame. Knowledge of high-resolution HLA typing for the patient and their relatives is essential for a better interpretation of compatibilities. HLA-DPB1 typing must be considered in transplant field regardless of the donor type. The benefits of using search and match programs are considerable. It saves time and reduces additional typing costs by providing rapid information about the likelihood to identify a matched unrelated donor. A backup therapy considering alternative cell sources or treatment can therefore be quickly implemented. The importance of knowledge and consideration of patient immunization for donor choice was explored in previous workshops of the SFGM-TC (2018 and 2019). The published recommendations remain applicable. The routine follow-up protocol and in case of desensitization will be detailed here. This harmonization must be accompanied by the standardization of information to be returned to the clinician regarding the donor finding possibilities for the patient. This will guarantee a similar quality level in every center.

Concurrent typing of over 4000 samples by long-range PCR amplicon-based NGS and rSSO revealed the need to verify NGS typing for HLA allelic dropouts.

Hematopoietic stem cell transplantation (HSCT) from HLA-matched donors significantly decreases the risks of graft-rejection and graft-versus-host disease. Long-range PCR- amplicon-based next-generation sequencing (NGS) is increasingly used as a standalone method in clinical laboratories to determine HLA compatibility for HSCT and solid-organ transplantation. We hypothesized that an allelic dropout is a frequent event in the long-range PCR amplicon-based NGS HLA typing method. To test the hypothesis, we typed 4,006 samples concurrently using a commercially available long-range PCR amplicon-based NGS-typing and short exon-specific amplicon-based reverse sequence-specific oligonucleotide (rSSO) methods. The concordance between the NGS and rSSO typing results was 100% at HLA-A, -B, -C, -DRB1, -DRB3, -DRB5, -DQA1, DPA1 loci. However, 4.5% of the samples (179/4006) showed allelic-dropouts at one of the other three loci: HLA-DRB4 (3.9%), HLA-DPB1 (0.4%), and HLA-DQB1*(0.15%). The allelic-dropouts are not associated with specific haplotypes, and some dropouts can be reagent lot-specific. Although DRB1-DRB3/4/5-DQB1 linkages help to diagnose these allelic-dropouts in some cases, the rSSO typing was crucial to identify the dropouts in DQB1 and DPB1 loci. These results uncover the critical limitations of using long-range PCR amplicon-based NGS as a standalone method in clinical histocompatibility laboratories and advocate the need for strategies to diagnose and resolve allelic-dropouts.

Abrogating biologics interference in flow cytometric crossmatching.

The flow cytometric crossmatch is currently the gold standard for evaluating donor and recipient histocompatibility. The assay however does have limitations and is sensitive to false positive reactions resulting from the presence of non-HLA antibodies or therapy related immune biologics. Such false positive reactions can lead to the inappropriate decline of an acceptable donor organ or unnecessary therapeutic intervention. Here we describe the successful validation of anti-idiotype blocking antibodies in prevention of false positive flow crossmatch results caused by biologic therapy. Blocking antibodies specific for the Fab portion of Rituximab and/or Alemtuzumab were incubated with biologic containing patient serum prior to use in flow cytometric crossmatching. Biologic blocking successfully negated false positive crossmatch results with Rituximab (B cell ave. % change = -97%) or Alemtuzumab (T cell ave. % change = -99%, B cell ave. % change = -95%) infused sera respectively. Simultaneous blocking of these biologics was also successful. A complex case is presented to demonstrate the application of this procedure.

Benchmarking the Human Leukocyte Antigen Typing Performance of Three Assays and Seven Next-Generation Sequencing-Based Algorithms.

With the great progress made recently in next generation sequencing (NGS) technology, sequencing accuracy and throughput have increased, while the cost for data has decreased. Various human leukocyte antigen (HLA) typing algorithms and assays have been developed and have begun to be used in clinical practice. In this study, we compared the HLA typing performance of three HLA assays and seven NGS-based HLA algorithms and assessed the impact of sequencing depth and length on HLA typing accuracy based on 24 benchmarked samples. The algorithms HISAT-genotype and HLA-HD showed the highest accuracy at both the first field and the second field resolution, followed by HLAscan. Our internal capture-based HLA assay showed comparable performance with whole exome sequencing (WES). We found that the minimal depth was 100X for HISAT-genotype and HLA-HD to obtain more than 90% accuracy at the third field level. The top three algorithms were quite robust to the change of read length. Thus, we recommend using HISAT-genotype and HLA-HD for NGS-based HLA genotyping because of their higher accuracy and robustness to read length. We propose that a minimal sequence depth for obtaining more than 90% HLA typing accuracy at the third field level is 100X. Besides, targeting capture-based NGS HLA typing may be more suitable than WES in clinical practice due to its lower sequencing cost and higher HLA sequencing depth.

Protocol Biopsies in Patients With Subclinical De Novo Donor-specific Antibodies After Kidney Transplantation: A Multicentric Study.

BACKGROUND: De novo donor-specific antibodies (DSAs) are associated with antibody-mediated rejection (AMR) and allograft loss. Whether monitoring of de novo DSA (dnDSA) paired with systematic kidney biopsy should become routine remains to be established. METHODS: A retrospective multicentric study (9 French kidney transplant units of the Spiesser group) included patients without graft dysfunction biopsied because of the presence of dnDSA (One Lambda, mean fluorescence intensity [MFI], >1000). RESULTS: One hundred twenty-three patients (85 male/38 female; mean age, 49.5 ± 13.1 y old) were biopsied after the detection of a dnDSA, 65.3 months (median) after kidney transplantation. Graft function was stable within 3 months before biopsy (estimated glomerular filtration rate, 55.3 ± 18.9 mL/min/1.73 m). Fifty-one subclinical AMRs (sAMRs) (41.4%) were diagnosed, of which 32 (26%) active and 19 (15.5%) chronic active sAMR. Seventy-two biopsies revealed no AMR (58.5%). Predictive factors associated with the diagnosis of active sAMR were MFI of immunodominant DSA >4000, MFI of the sum of DSA >6300, age of the recipient <45 years old, and the absence of steroids at biopsy. The presence of proteinuria >200 mg/g was predictive of chronic active sAMR. The decrease of estimated glomerular filtration rate at 5 years post-biopsy was significantly higher in patients with acute sAMR (-25.2 ± 28.3 mL/min/1.73 m) and graft survival significantly lower. CONCLUSIONS: Performing a kidney graft biopsy for the occurrence of dnDSA without renal dysfunction leads to the diagnosis of a sAMR in over 40% of cases. Nevertheless, we did not observe any effect of standard treatment in acute sAMR.

Ensuring HLA-matched platelet support requires an ethnic diverse donor population.

BACKGROUND: Patients refractory for platelet transfusions benefit from human leukocyte antigen (HLA)-matched platelet transfusions. Differences in ethnic background of patients and donors could hamper the availability of sufficient numbers of HLA-matched donors for all patients. We evaluated our HLA-matched donor program and explored the role of ethnic background of patients related to the number of available donors. METHODS: We performed a cohort study among consecutive patients who received HLA-matched platelet concentrates in the Netherlands between 1994 and 2017. The number of available matched donors was determined per patient. Haplotypes were constructed from genotypes with computer software (PyPop). Based on haplotypes, HaploStats, an algorithm from the National Marrow Donor Program, was used to assess the most likely ethnic background for patients with 5 or fewer and 30 or more donors. RESULTS: HLA typing was available for 19,478 donors in September 2017. A total of 1206 patients received 12,350 HLA-matched transfusions. A median of 83 (interquartile range, 18-266) donors were available per patient. For 95 (10.3%) patients, 5 or fewer donors were available. These patients were more likely to have an African American background, whereas patients with 30 or more donors were more often from Caucasian origin, compared with Caucasian origin for patients with 30 donors. CONCLUSION: Adequate transfusion support could be guaranteed for most but not all refractory patients. More non-Caucasian donors are required to ensure the availability of HLA-matched donors for all patients in the Netherlands.

SweHLA: the high confidence HLA typing bio-resource drawn from 1000 Swedish genomes.

There is a need to accurately call human leukocyte antigen (HLA) genes from existing short-read sequencing data, however there is no single solution that matches the gold standard of Sanger sequenced lab typing. Here we aimed to combine results from available software programs, minimizing the biases of applied algorithm and HLA reference. The result is a robust HLA population resource for the published 1000 Swedish genomes, and a framework for future HLA interrogation. HLA 2nd-field alleles were called using four imputation and inference methods for the classical eight genes (class I: HLA-A, HLA-B, HLA-C; class II: HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRB1). A high confidence population set (SweHLA) was determined using an n-1 concordance rule for class I (four software) and class II (three software) alleles. Results were compared across populations and individual programs benchmarked to SweHLA. Per gene, 875 to 988 of the 1000 samples were genotyped in SweHLA; 920 samples had at least seven loci called. While a small fraction of reference alleles were common to all software (class I = 1.9% and class II = 4.1%), this did not affect the overall call rate. Gene-level concordance was high compared to European populations (>0.83%), with COX and PGF the dominant SweHLA haplotypes. We noted that 15/18 discordant alleles (delta allele frequency >2) were previously reported as disease-associated. These differences could in part explain across-study genetic replication failures, reinforcing the need to use multiple software solutions. SweHLA demonstrates a way to use existing NGS data to generate a population resource agnostic to individual HLA software biases.

Posttransplant Outcomes for cPRA-100% Recipients Under the New Kidney Allocation System.

BACKGROUND: There is concern in the transplant community that outcomes for the most highly sensitized recipients might be poor under Kidney Allocation System (KAS) high prioritization. METHODS: To study this, we compared posttransplant outcomes of 525 pre-KAS (December 4, 2009, to December 3, 2014) calculated panel-reactive antibodies (cPRA)-100% recipients to 3026 post-KAS (December 4, 2014, to December 3, 2017) cPRA-100% recipients using SRTR data. We compared mortality and death-censored graft survival using Cox regression, acute rejection, and delayed graft function (DGF) using logistic regression, and length of stay (LOS) using negative binomial regression. RESULTS: Compared with pre-KAS recipients, post-KAS recipients were allocated kidneys with lower Kidney Donor Profile Index (median 30% versus 35%, P < 0.001) but longer cold ischemic time (CIT) (median 21.0 h versus 18.6 h, P < 0.001). Compared with pre-KAS cPRA-100% recipients, those post-KAS had higher 3-year patient survival (93.6% versus 91.4%, P = 0.04) and 3-year death-censored graft survival (93.7% versus 90.6%, P = 0.005). The incidence of DGF (29.3% versus 29.2%, P = 0.9), acute rejection (11.2% versus 11.7%, P = 0.8), and median LOS (5 d versus 5d, P = 0.2) were similar between pre-KAS and post-KAS recipients. After accounting for secular trends and adjusting for recipient characteristics, post-KAS recipients had no difference in mortality (adjusted hazard ratio [aHR]: 0.861.623.06, P = 0.1), death-censored graft failure (aHR: 0.521.001.91, P > 0.9), DGF (adjusted odds ratio [aOR]: 0.580.861.27, P = 0.4), acute rejection (aOR: 0.610.941.43, P = 0.8), and LOS (adjusted LOS ratio: 0.981.161.36, P = 0.08). CONCLUSIONS: We did not find any statistically significant worsening of outcomes for cPRA-100% recipients under KAS, although longer-term monitoring of posttransplant mortality is warranted.

HLA antibodies in haematopoietic stem cell transplantation.

The negative impact of donor specific HLA alloantibodies in solid organ transplantation is well known and understood within the histocompatibility and immunogenetics community. However the influence of donor-specific antibodies in the outcome of haematopoietic stem cell transplantation is less well regarded. As donor choices have evolved from HLA matched siblings and extremely well matched unrelated donors to mismatched cord blood and haplo-identical-related donors, we are now identifying more patients with antibodies reactive against their donor mismatches. The clinical significance of the antibodies that can be detected has not yet been fully elucidated.

Selection of unrelated donors and cord blood units for hematopoietic cell transplantation: guidelines from the NMDP/CIBMTR.

Allogeneic hematopoietic cell transplantation involves consideration of both donor and recipient characteristics to guide the selection of a suitable graft. Sufficient high-resolution donor-recipient HLA match is of primary importance in transplantation with adult unrelated donors, using conventional graft-versus-host disease prophylaxis. In cord blood transplantation, optimal unit selection requires consideration of unit quality, cell dose and HLA-match. In this summary, the National Marrow Donor Program (NMDP) and the Center for International Blood and Marrow Transplant Research, jointly with the NMDP Histocompatibility Advisory Group, provide evidence-based guidelines for optimal selection of unrelated donors and cord blood units.

Acute Myeloid Leukemia, Version 3.2019, NCCN Clinical Practice Guidelines in Oncology.

Acute myeloid leukemia (AML) is the most common form of acute leukemia among adults and accounts for the largest number of annual deaths due to leukemias in the United States. Recent advances have resulted in an expansion of treatment options for AML, especially concerning targeted therapies and low-intensity regimens. This portion of the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for AML focuses on the management of AML and provides recommendations on the workup, diagnostic evaluation and treatment options for younger (age <60 years) and older (age ≥60 years) adult patients.

Recipients Receiving Better HLA-Matched Hematopoietic Cell Transplantation Grafts, Uncovered by a Novel HLA Typing Method, Have Superior Survival: A Retrospective Study.

HLA matching at an allelic-level resolution for volunteer unrelated donor (VUD) hematopoietic cell transplantation (HCT) results in improved survival and fewer post-transplant complications. Limitations in typing technologies used for the hyperpolymorphic HLA genes have meant that variations outside of the antigen recognition domain (ARD) have not been previously characterized in HCT. Our aim was to explore the extent of diversity outside of the ARD and determine the impact of this diversity on transplant outcome. Eight hundred ninety-one VUD-HCT donors and their recipients transplanted for a hematologic malignancy in the United Kingdom were retrospectively HLA typed at an ultra-high resolution (UHR) for HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 using next-generation sequencing technology. Matching was determined at full gene level for HLA class I and at a coding DNA sequence level for HLA class II genes. The HLA matching status changed in 29.1% of pairs after UHR HLA typing. The 12/12 UHR HLA matched patients had significantly improved 5-year overall survival when compared with those believed to be 12/12 HLA matches based on their original HLA typing but were found to be mismatched after UHR HLA typing (54.8% versus 30.1%, P = .022). Survival was also significantly better in 12/12 UHR HLA-matched patients when compared with those with any degree of mismatch at this level of resolution (55.1% versus 40.1%, P = .005). This study shows that better HLA matching, found when typing is done at UHR that includes exons outside of the ARD, introns, and untranslated regions, can significantly improve outcomes for recipients of a VUD-HCT for a hematologic malignancy and should be prospectively performed at donor selection.

Isolated pancreas transplantation: Is rank list position related to outcomes of imported grafts?

Transplant centers may decline an import pancreas offer based on demographics and laboratory test results, without information on actual gland quality. The relationship between position on the match run, indicative of the number of centers that chose not to use a pancreas, and patient and death-censored graft survival, is not known. We studied all 199 isolated pancreas grafts transplanted at the University of Wisconsin since July 2000 and compared overall patient and death-censored graft survival based on import vs local status. Of the 199 isolated pancreas transplants, 184 (92.5%) were imported from another donor service area with a median match rank of 49 (interquartile range 14-129). Median cold ischemia time was longer for imported pancreata (16.6 vs 13.4 hours, P = .02). In multivariate Cox modeling, there was no association with position on the rank list and patient (P = .44) or death-censored graft survival (P = .99). There was an overall rate of 6.5% of graft failure within 30 days; however, there was no association with position on the rank list and graft failure at 30 days (P = .33). Although the logistics may be challenging, sound judgment to accept offers independent of prior centers' decisions can result in quality utilization of imported pancreata.

Proposal for the International Society for Cell & Gene Therapy position statement on assays for the quality control and potency assessment of adoptive cellular immunotherapies.

Translation of cell and gene therapies from pre-clinical experiments to clinical trials and final drug licensing brings requires the development, verification and even validation of the assays essential for the definition of the drug product. The technical and scientific challenges in doing this are far greater than they seem at first and are compounded by a lack of approved standards for assays used to support (c)GMP manufacture. This paper highlights some of those challenges and proposes solutions based on the experience of our colleagues using similar assay platforms in regulated pathology laboratories.

Minimal data reporting standards for serological testing for histocompatibility.

Histocompatibility testing, and HLA antibody screening in particular, varies in practice among laboratories. Currently, standards are lacking regarding the reporting of testing methods in publications. It is essential that scientific methods are rigorously and transparently described upon publication, so that results can be accurately interpreted and independently corroborated. Additionally, this would allow work groups to compile diverse data to achieve clinically significant conclusions from meta-analyses. These efforts are hindered when there is a paucity of method descriptions and where variability in serum treatment, protocol modifications, and assay thresholds affecting assay interpretation are known to exist. Thus, the ASHI Science and Technology Initiatives Committee (ASHI STIC) undertook the task of formulating recommendations for reporting HLA antibody testing by solid phase assays, and the associated HLA typing required for interpretation, in scientific publications. Herein we put forth standards for minimum information about HLA antibody testing methods reported in histocompatibility publications.

A new hybrid approach for MHC genotyping: high-throughput NGS and long read MinION nanopore sequencing, with application to the non-model vertebrate Alpine chamois (Rupicapra rupicapra).

The major histocompatibility complex (MHC) acts as an interface between the immune system and infectious diseases. Accurate characterization and genotyping of the extremely variable MHC loci are challenging especially without a reference sequence. We designed a combination of long-range PCR, Illumina short-reads, and Oxford Nanopore MinION long-reads approaches to capture the genetic variation of the MHC II DRB locus in an Italian population of the Alpine chamois (Rupicapra rupicapra). We utilized long-range PCR to generate a 9 Kb fragment of the DRB locus. Amplicons from six different individuals were fragmented, tagged, and simultaneously sequenced with Illumina MiSeq. One of these amplicons was sequenced with the MinION device, which produced long reads covering the entire amplified fragment. A pipeline that combines short and long reads resolved several short tandem repeats and homopolymers and produced a de novo reference, which was then used to map and genotype the short reads from all individuals. The assembled DRB locus showed a high level of polymorphism and the presence of a recombination breakpoint. Our results suggest that an amplicon-based NGS approach coupled with single-molecule MinION nanopore sequencing can efficiently achieve both the assembly and the genotyping of complex genomic regions in multiple individuals in the absence of a reference sequence.