Somatic Mutations and Loss of Heterozygosity of HLA Genes Are Frequently Occurred and Tightly Associated with Poor Prognosis in Adult T Cell Leukemia-Lymphoma
Authors: Tamaki Keita , Morishima Satoko , Suzuki Shingo , Shigenari Atsuko , Nomura Ikumi , Yokota Yutaro , Morichika Kazuho , Nishi Yukiko , Nakachi Sawako , Okamoto Shiki , Karube Kennosuke , Fukushima Takuya , Shiina Takashi , Masuzaki Hiroaki 
Published: November 2019

Introduction: Adult T cell leukemia-lymphoma (ATL) is clinically divided into aggressive (acute and lymphoma) and indolent (chronic and smoldering) subtypes. Recent studies revealed that ATL has a wide variety of gene alternations (Nat Genet. 2015; 47:1304, Nature. 2016; 534:402). Consistent with the notion that human leukocyte antigen (HLA) play a critical role for immune response, reduced expression of HLA has been shown to associate with poor prognosis in various types of malignancies (Oncogene. 2008; 27:5869). However, it is extremely difficult to assess the impact of somatic mutations of HLA genes in cancer cells because of its highly polymorphic nature. Taking advantage of computational tools using whole-exome sequencing data, somatic mutations of HLA class I genes were previously reported in about 3% of various types of cancers (Nat Biotechnol. 2015; 33:1152). To date, HLA typing has been performed through partial sequencing data of polymorphic exons, resulting in the loss of critical information on the entire region of HLA genes. In this context, using a next generation sequence (NGS)-based HLA DNA typing method, we analyzed the entire region of HLA genes in ATL cells and its impact on clinical outcomes.
Methods: The present study was approved by the Investigational Review Board of University of the Ryukyus and Tokai University. Peripheral blood mononuclear cells collected from 25 ATL patients (Pts) (20 with acute type and 5 with chronic type) were analyzed. Cell adhesion molecular 1 (CADM1)-positive ATL cells and CADM1-negative non-ATL cells were separated by a magnetic-activated cell-sorting protocol, and genomic DNAs in each fraction were distinctively extracted. The PCR products of the eight HLA loci (HLA-A, -B, -C, -DRB1, -DQA1, -DQB1, -DPA1 and -DPB1) were obtained using the previously designed long ranged PCR primers (Tissue Antigens. 2012; 80:305) that cover the entire HLA gene sequences from the enhancer-promoter region to the 3'UTR region. The full-length level HLA genotypes were determined by mapping of sequence reads obtained by the Ion Torrent system to HLA allele reference sequences in the international ImMunoGeneTics project (IMGT)-HLA database. Somatic mutations and loss of heterozygosity (LOH) generated in ATL cells were identified by detail comparison of sequence data between the ATL and non-ATL cells. Survival curves were estimated using the Kaplan-Meier method and compared by the log-rank test.
Results: We found a total of 20 somatic mutations in ATL cells among 9 Pts, including 15 single nucleotide variations (SNVs) and 5 insertions/deletions (indels) (Table 1). Of the 15 SNVs, 3 were nonsense mutations in exon 2/3 of HLA-A/B, 7 were nonsynonymous mutations in exon 1/4/5 of HLA-A/B/C, and 2 were mutations at splice sites in intron 2 of HLA-B/C. The mutations of splice sites in intron 2 and all the 5 indels caused frameshift generating premature stop codons. In contrast to the exons, there were only 3 mutations found in introns. Of 8 Pts harboring LOH, two Pts showed LOH in HLA-A gene, 4 Pts in HLA-A, -C, and -B genes, one Pt in HLA-DRB1, -DQA1, -DQB1, -DPA1 and -DPB1 genes, and one Pt in all the HLA-genes from HLA-A to HLA-DPB1. All the somatic mutations altering the structure of the HLA proteins (St-mt) and/or LOH were found in Pts with acute type ATL, while no St-mt or LOH were detected in Pts with chronic type ATL. Pts with St-mt/LOH (n = 9) significantly worsened in 3 years overall survival rate as compared to that in Pts without St-mt/LOH (n = 16) (11 % vs. 32 %, P = 0.016) (Fig. 1A). Among Pts with acute type ATL, Pts with St-mutations/LOH (n = 9) also showed a trend to worsen in the survival rate as compared to those in Pts without St-mutation /LOH (n = 11) (11 % vs. 30 %, P=0.064) (Fig. 1B).
Conclusion: NGS-based HLA gene analyses at full-length level revealed that St-mt/LOH were frequently occurred predominantly in HLA class I loci in tumor cells from Pts with aggressive ATL. Importantly, St-mt/LOH in HLA genes were associated with aggressive phenotypes and poor prognosis, suggesting the possibility that ATL cells would develop immune escape mechanisms through a variety of acquired abnormalities in HLA gene. Since allogeneic stem cell transplantation is an essential therapeutic option for aggressive ATL, comprehensive knowledges on HLA gene abnormalities provide considerable advantage to optimize therapeutic modalities.

Disclosures
Fukushima: Daiichi-Sankyo: Research Funding.