ALBERT

Description: [Background] Acquired aplastic anemia (AA) is thought to be caused by cytotoxic T lymphocyte (CTL) attack specific to antigens presented by class I HLA alleles on hematopoietic stem cells (HSCs) although the target antigens on HSCs are still unknown. HLA alleles that are responsible for the auto-antigen presentation, which can be inferred from the presence of leukocytes that lack particular HLA class I alleles (HLA-lacking leukocytes [HLA-LLs]), may provide useful information on the identification of autoantigens in AA. We previously reported that deep sequencing of HLA class I genes of HLA-LLs revealed various loss-of-function mutations in alleles, including HLA-A*02:06 and B*40:02, suggesting that limited HLA-class I alleles are involved in the autoantigen presentation of AA (ASH 2018). Intriguingly, 60% of patients possessing HLA-LLs due to 6pLOH or several inactivating mutations in different HLA-A or HLA-B genes shared a nonsense mutation in exon 1 (Exon1mut) of which allelic frequency was very low (range 1.0-37.3%, median 4.5%). We hypothesized that the nonsense mutation, which efficiently lacks the corresponding HLA-allelic expression, might have been overlooked due to its low VAFs, and if we could establish a highly sensitive assay for detecting Exon1mut and determine the HLA alleles that undergo the mutation for a large number of AA patients, we might be able to define all HLA alleles that are involved in autoantigen presentation of AA. [Objectives/Methods] To test this hypothesis, we developed a highly sensitive droplet digital PCR (ddPCR) assay for precisely detecting Exon1mut in the peripheral blood (PB) of AA patient. In brief, the exon 1 regions of HLA-A and HLA-B alleles were amplified using two different sets of primer pairs that are complementary to the consensus sequences of the HLA-A and HLA-B alleles. The amplicons were subjected to a ddPCR assay using TaqMan probes complementary to wild-type (WT) and mutant-specific (MT) sequences, which were labeled with different fluorochromes (6-FAM for MT and HEX for WT). Peripheral blood leukocytes from 363 patients with AA (mean 64 [range, 11-93]) years of age, 134 with severe AA and 229 with non-severe AA; 173 males and 190 females; 84 6pLOH[+] and 279 6pLOH[-]) were subjected to the ddPCR assay. All blood samples were analyzed for 6pLOH by SNP array-based methods or a ddPCR assay as previously described. The HLA allele that underwent Exon1mut was determined by targeted deep sequencing using a unique molecular identifier (UMI), which enabled us to detect variant calling at a VAF as low as 0.1%, or deduced from the alleles contained in the lost haplotype, which are known to be the frequently lost alleles due to 6pLOH. [Results] Using 2 different ddPCR mixtures for HLA-A and HLA-B, the presence of Exon1mut was evaluable in all 363 AA patients. The sensitivity of the ddPCR assay for detecting Exon1mutwas 0.07%. 6pLOH was detected in 84 (23.1%) of the 363 AA patients. Ninety-nine (27.3%) of the 363 patients with (55 [65.5%] of 84) or without (44 [15.8%] of 279) 6pLOH were found to be positive for Exon1mut. The median allele frequency of Exon1mut in DNA from the Exon1mut(+) patients was 0.6% (range, 0.074% to 21.3%). In 17 patients whose blood samples were serially available, Exon1mutwas persistently detected in 13 and disappeared in 4 patients for 10-77 months (Figure 1). Among 43 different HLA-A and HLA-B alleles carried by the Exon1mut(+) patients, those with Exon1mutcould be identified by targeted deep sequencing in 54 patients. In 13 of the remaining 42 patients with Exon1mut, the Exon1mut-involved HLA alleleswere deduced from the alleles contained in the lost haplotype due to 6pLOH. These were 12 alleles and included A*02:06 (n=11), A*31:01 (n=3), B*13:01 (n=2), B*40:01 (n=3), B*40:02 (n=26), B*40:03 (n=1), B*54:01 (n=6), A*02:01 (n=2), A*02:07 (n=1), B*44:03 (n=1), B*55:02 (n=2) and B*56:01 (n=1) (Figure 2). The last five infrequent alleles were newly identified as "risk alleles" using the Exon1mutdetection. Two-hundred and twenty (92%) of 239 patients with PNH-type cells possessed at least 1 of the 12 alleles, while 103 (85%) of 121 patients without PNH-type cells did (P=0.045). [Conclusions] The Exon1mutdetection assay identified 12 HLA-alleles that are closely and exclusively involved in the autoantigen presentation of AA in Japanese patients. Similarity analyses of their antigen-presentation motifs may help to identify autoantigen peptides in AA. Disclosures Nakao: Takeda Pharmaceutical Company Limited: Honoraria; SynBio Pharmaceuticals: Consultancy; Ono Pharmaceutical: Honoraria; Novartis Pharma K.K: Honoraria; Bristol-Myers Squibb: Honoraria; Kyowa Kirin: Honoraria; Alaxion Pharmaceuticals: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Daiichi-Sankyo Company, Limited: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Celgene: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria.

Description: [Background] HLA-class I allele-lacking (HLA[-]) leukocytes are detected in approximately 30% of patients with acquired aplastic anemia (AA), and are thought to represent the involvement of cytotoxic T lymphocyte attack against hematopoietic stem cells (HSCs) in the development of AA, based on the high response rate to immunosuppressive therapy (IST) in patients with such aberrant leukocytes. Similar to glycosylphosphatidylinositol-anchored protein (GPI-AP)-deficient (GPI[-]) leukocytes in patients with paroxysmal nocturnal hemoglobinuria (PNH), HLA(-) leukocytes in AA patients are often clonal or oligoclonal and expand to account for more than 50% of the total leukocytes. Despite such overwhelming proliferation, somatic mutations in driver genes as well as telomere shortening that portend clonal evolution are rarely detected in HLA(-) granulocytes, suggesting the genetic stability of HLA(-) HSCs and the persistence of the immune pressure on HSCs that favors expansion of HLA(-) HSCs (Imi, et al. Blood Adv). However, recent studies from the United States have shown a higher incidence of clonal evolution to secondary myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) in AA patients with HLA(-) leukocytes than in those without such leukocytes, a finding inconsistent with the results of our previous study. Given the high prevalence of HLA(-) leukocytes in AA patients, it is critical to determine whether or not the presence of the aberrant leukocytes is associated with clonal evolution. We therefore addressed this issue by studying the prognosis of a large number of AA patients with or without HLA(-) leukocytes who had been followed for a long term period. We also studied the clonal composition of granulocytes in AA patients with HLA(-) cells, wherein aberrant clones other than HLA(-) cells might be responsible for clonal evolution to MDS/AML. [Methods] We retrospectively analyzed the clinical characteristics of 633 AA patients and peripheral blood samples were examined for the presence of HLA(-) leukocytes using a high-sensitivity flow cytometry (FCM) assay, droplet digital PCR, single-nucleotide polymorphism arrays, or next generation sequencing (NGS) between 2010 and 2020. GPI(-) cells were detected using a high-sensitivity FCM assay as previously described. [Results] HLA(-) granulocytes were detected in 127 (20.1%) of the 633 patients with a median clone size of 16.9% (range, 0.04%-100%); the aberrant granulocytes accounted for greater than 50% of the total granulocytes in 29 (22.8%) of 127 patients. Eighty-nine (70.0%) of the 127 patients possessed aberrant clones other than HLA(-) clones, which included 0.005% to 91.6% GPI(-) cells (n=86), del(13q) cells (n=3), t(1;10) cells (n=1), t(9;13) cells (n=1), inv12 cells (n=1), and trisomy 8 cells (n=1). The prevalence of GPI(-) cells was not significantly different between patients with and without HLA(-) cells (67.7% vs 65.4%). Eighty-five of 102 (83.3%) patients with HLA(-) cells responded to IST, whereas 231 of 318 (72.6%) without HLA(-) cells responded (p90% of granulocytes, suggesting that these few escape clones were enough to sustain the hematopoietic function of the patients. The prognosis survey revealed no clonal evolution to MDS/AML in any of the 127 AA patients with HLA(-) leukocytes after a follow-up period of the median 5 years. In contrast, 15 of 234 (6.4%) patients without HLA(-) cells who were trackable evolved to MDS/AML during a median 5 year follow-up. [ Conclusions] The presence of HLA(-) leukocytes and concomitant aberrant clones was not associated with clonal evolution to MDS/AML in Japanese AA patients, even in those possessing a large (〉50% of the total granulocyte) HLA(-) cell population. The discrepancy between our results and the data from the United States may be due to the difference in the race and mechanism underlying HLA loss. These data suggest that HSC clones that escape immune attack, such as HLA(-) and GPI(-) clones, are healthy enough to support hematopoiesis for a long term in AA patients. Disclosures Ishiyama: Novartis: Honoraria; Alexion: Research Funding. Yamazaki:Novartis: Honoraria; Kyowa Kirin: Honoraria, Research Funding. Ogawa:Eisai Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company; Otsuka Pharmaceutical Co., Ltd.: Research Funding; KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding. Nakao:Alexion: Research Funding; Kyowa Kirin: Honoraria; Novartis: Honoraria; Symbio: Consultancy.