Sustained clonal hematopoiesis by HLA-lacking hematopoietic stem cells without driver mutations in aplastic anemia

Clonal hematopoiesis by hematopoietic stem progenitor cells (HSPCs) that lack an HLA class I allele (HLA^- HSPCs) is common in patients with acquired aplastic anemia (AA); however, it remains unknown whether the cytotoxic T lymphocyte (CTL) attack that allows for survival of HLA^- HSPCs is directed at nonmutated HSPCs or HSPCs with somatic mutations or how escaped HLA^- HSPC clones support sustained hematopoiesis. We investigated the presence of somatic mutations in HLA^- granulocytes obtained from 15 AA patients in long-term remission (median, 13 years; range, 2-30 years). Targeted sequencing of HLA^- granulocytes revealed somatic mutations (DNMT3A, n = 2; TET2, ZRSR2, and CBL, n = 1) in 3 elderly patients between 79 and 92 years of age, but not in 12 other patients aged 27 to 74 years (median, 51.5 years). The chronological and clonogenic analyses of the 3 cases revealed that ZRSR2 mutation in 1 case, which occurred in an HLA^- HSPC with a DNMT3A mutation, was the only mutation associated with expansion of the HSPC clone. Whole-exome sequencing of the sorted HLA^- granulocytes confirmed the absence of any driver mutations in 5 patients who had a particularly large loss of heterozygosity in chromosome 6p (6pLOH) clone size. Flow-fluorescence in situ hybridization analyses of sorted HLA⁺ and HLA^- granulocytes showed no telomere attrition in HLA^- granulocytes. The findings suggest that HLA^- HSPC clones that escape CTL attack are essentially free from somatic mutations related to myeloid malignancies and are able to support long-term clonal hematopoiesis without developing driver mutations in AA patients unless HLA loss occurs in HSPCs with somatic mutations.