OR6 Next generation sequencing HLA typing in patients with copy neutral loss of heterozygosity

Abstract

Aim Next Generation Sequencing (NGS) technology is well documented as a highly sensitive and specific method for HLA typing. Here we investigate the possibility that copy neutral loss of Heterozygosity (cnLOH) involving the HLA complex located at 6p21 is a potential confounder of interpreting NGS HLA typing results. Methods HLA typing for 11 cases with cnLOH of 6p21 identified by Chromosomal Genomic Array Testing (CGAT), were evaluated by NGS and rSSOP. Tumor burden (TB) estimates for CGAT are based on standard practice. The TB estimate for NGS is based on allele frequency (AF) and calculated using the formula: TB = 100–2 * AF. The baseline of AF is established by evaluating 36 controls without LOH. Results One of 11 patients was a post-transplant case with mixed chimerism. The remaining 10 patients were not transplanted and had confirmed 20%–80% cnLOH at 6p21 by CGAT and Flow Cytometry (FC). In NGS HLA typing, the average baseline AF for HLA-A, B, C, DRB1, DQA1, DQB1, DPA1 and DPB1 loci was 43% ± 5%. In patients who had less than 50% cnLOH by CGAT, the average HLA AF and estimated TB were 34.3% ± 5.2% and 29.8% ± 10.5%, respectively. However, patients who had over 51% of cnLOH, the average HLA AF and TB were 18.8% ± 6.2% and 62.6% ± 12.6%, respectively; suggesting that the higher TB results in greater allele imbalance. Although cnLOH involved the HLA region of 6p21 in all patients, both NGS and rSSOP methods accurately typed HLA alleles except in two patients. In the first case, the patient had received related haplo-identical transplant and was known to be chimeric with 65% of donor cells; this case demonstrated loss of the non-shared haplotype. In the second patient, the specimen had a TB of greater than 80% by CGAT and FC, and HLA typing was not possible for the B and C loci by both NGS and rSSOP. In this patient, the border of cnLOH detected by CGAT involved only the HLA class I genes with the proximal border resting close to the HLA-C and B loci. Conclusions Our findings suggest that HLA locus specific cnLOH could be closely tied with exact molecular location of cnLOH detected by CGAT. The calculated AF by the TruSight Assign software in NGS typing decreases proportionally to increasing TB in cnLOH at 6p21. Our findings indicate that caution is warranted in cases with low frequency alleles and in cases with multiple contiguous homozygous genes.