OR48 Resolution of conflicting hla assignment due to loss of heterozygosity in the hla region by NGS typing

Abstract

57 year old female patient diagnosed with acute myeloid leukemia (AML) was referred to bone marrow transplant. Transplant workup and search for compatible unrelated donor was started immediately, along with induction therapy for AML. HLA typing performed from peripheral blood using high-resolution SSO method showed homozygous typing for all loci, except HLA C. However, the quality of the typing data, along with the unusual B and C allele association prompted confirmatory typing by next-generation sequencing (NGS), which revealed the presence of a previously undetected HLA B∗40:01:02 allele at 14%, and confirmed the presence of HLA C∗03:03:01 allele at 16%, consistent with a dramatic allelic imbalance. Research on the patient’s clinical history revealed that at the time of diagnosis and sampling, a significant population of atypical monocytic cells with promonocytes/monoblasts was detected by flow cytometry, accounting for at least 55% of the total leukocytes in the peripheral blood. Genomic microarray testing showed a 35.3 Mb terminal long contiguous stretch of homozygosity 6p25.3p21.31, which includes the HLA locus, resulting in copy-neutral loss of heterozygosity (LOH) acquired likely due to the malignancy. The LOH explains why one HLA haplotype was underrepresented in the initial blood sample acquired before the treatment. Buccal DNA sample was requested and tested with SSO method, which confirmed the presence of the alleles detected by NGS. In addition, confirmatory HLA typing performed 3 months later from peripheral blood post-treatment gave identical results. This supports the finding that the initial typing discrepancy was due to the overwhelming presence of malignant cells with LOH involving the HLA locus in the patient’s peripheral blood, collected before initiation of treatment. NGS typing using the Omixon Holotype platform was able to provide accurate typing of a sample with allelic disequilibrium, and allowed not only for detection but also for quantification of the minor alleles. This case highlights the importance of confirmatory typing pre-transplant for patients with hematological malignancies, since the presence of malignant cells in the peripheral blood may bias typing data if LOH involving the 6p region is present, which is a recurrent finding in patients with aplastic anemia or AML.