Quantitative relationship between the magnitude of variation in minor histocompatibility antigens (mHA) and graft versus host disease (GVHD) pathophysiology in stem cell transplant (SCT) donor-recipient pairs (DRP) is not established. In order to elucidate this relationship, whole exome sequencing (WES) was performed on 27 HLA matched related (MRD), & 50 unrelated donors (URD), to identify nonsynonymous single nucleotide polymorphisms (SNPs). An average 2,463 SNPs were identified in MRD, and 4,287 in URD DRP (p<0.01); resulting peptide antigens that may be presented on HLA class I molecules in each DRP were derived in silico (NetMHCpan ver2.0) and the tissue expression of proteins these were derived from determined (GTex). MRD DRP had an average 3,670 HLA-binding-alloreactive peptides, putative mHA (pmHA) with an IC50 of <500 nM, and URD, had 5,386 (p<0.01). To simulate an alloreactive donor cytotoxic T cell response, the array of pmHA in each patient was considered as an operator matrix modifying a hypothetical cytotoxic T cell clonal vector matrix; each responding T cell clone’s proliferation was determined by the logistic equation of growth, accounting for HLA binding affinity and tissue expression of each alloreactive peptide. The resulting simulated organ-specific alloreactive T cell clonal growth revealed marked variability, with the T cell count differences spanning orders of magnitude between different DRP. Despite an estimated, uniform set of constants used in the model for all DRP, and a heterogeneously treated group of patients, higher total and organ-specific T cell counts were associated with cumulative incidence of moderate to severe GVHD in recipients. In conclusion, exome wide sequence differences and the variable alloreactive peptide binding to HLA in each DRP yields a large range of possible alloreactive donor T cell responses. Our findings also help understand the apparent randomness observed in the development of alloimmune responses.
Next generation sequencing (NGS)-based comprehensive genomic profiling has emerged as a powerful tool for the large-scale detection of genomic changes. Illumina's TruSight Oncology 500 (TSO500) Comprehensive Assay employs a hybrid-capture approach for target enrichment of 523 clinically relevant cancer genes to enable detection of small variants (SNVs, insertions, and deletions), CNV (copy number variations), TMB (tumor mutational burden), and MSI (microsatellite instability) in DNA and detection of fusions and splice variants in RNA. Briefly, purified DNA and RNA were sheared to the desired length by sonication or heat mediated fragmentation. Fragmented DNA and cDNA then underwent end repair and adapter incorporation, followed by target sequence capture, amplification, and purification using a bead-based approach. The enriched libraries were then pooled and sequenced on the Illumina NovaSeq 6000. Sequencing data (≥ 80M paired-end reads for DNA and ≥ 16 M paired-end reads for RNA) was analyzed using Illumina Primary Analysis pipeline and custom scripts developed for validation data analysis.To characterize the analytical performance of variant detection using the TSO500 Comprehensive Assay following guidelines established by Clinical Laboratory Improvement Amendments of 1988 (CLIA) and College of American Pathologists (CAP), we utilized various commercially available reference standards and over 59 formalin-fixed, paraffin-embedded (FFPE) samples, characterized by selected comparator testing methods. The comparator methods were as follows: for DNA small variants, the QCCP (Q2 Solutions Comprehensive Cancer Panel) NGS assay; for CNV, the NanoString nCounter Cancer Copy Number Assay; and for RNA fusions and splice variants, the TruSeq RNA Exome assay. Variant calls for orthogonal assays were manually curated during review by the Medical Director at Q2 Solutions. Analytical sensitivity, accuracy, precision, specificity, and positive predictive value (PPV) were evaluated using both reference standards and clinical FFPE samples. We observed 95% sensitivity, > 99.99% accuracy and specificity as well as > 99.5% PPV for detecting small DNA variants with variant allele frequency (VAF) above 4.5%. The intra-run and inter-run precision for SNV, insertions/deletions were 99.54% and 99.52%, respectively. Additionally, we observed > 98% accuracy for CNV. The sensitivity, accuracy, specificity, and PPV for fusion and splice variant detection were 94.64%, 99.93%, 100%, and 100%, respectively. Robustness of input studies revealed comparable coverage depth and uniformity can be achieved with varying DNA and RNA input (30, 40, and 60 ng). In conclusion, the TSO500 Comprehensive Assay enables robust detection of variants and other clinically relevant biomarkers in FFPE samples. Citation Format: Xinyan Li, Lauren Frady, Juliana Feltz, Hiba Shaban, Jennifer Sims. Evaluation of a comprehensive genomic profiling assay for characterization of biomarkers in FFPE tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 246.
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