Clonal evolution of monosomy 7 from HLA class I allele-lacking clones. (A) A 56-year-old female at the time of severe AA diagnosis, who received hATG, CsA, and mycophenolate mofetil (MMF), and multiple rounds of salvage IST, including rabbit ATG, daclizumab and danazol, and alemtuzumab, without adequate response, had clonal evolution to monosomy 7 approximately 7.5 years after her initial diagnosis of AA and died 6 months later. Her blood, sampled 4 years after the institution of initial IST, contained a PNH clone and HLA-A0201-lacking cells (A0201^-), consistent with the diagnosis of immune AA. Multiple clones with HLA-A*02:01 deactivating mutations constituted sorted A0201^- cells (multiple clones with missense mutations also did A0201⁺ cells), from which chromosome 7 deletion was not detected by fluorescent in situ hybridization (FISH). After 3.5 years, virtually all hematopoiesis was replaced by a preexisting A0201^- clone with an intron 1 mutation (intron 1^mut; c.74-19A>G) that had acquired monosomy 7. (B) A 72-year-old man at the time of severe AA diagnosis was treated with hATG, CsA, and EPAG with a partial response at 6 months but had declining cytopenia which responded to reinitiation of CsA. About 2.5 years from the initial diagnosis and IST treatment, BM cytogenetics revealed monosomy 7, and myelodysplastic syndrome was diagnosed. The patient received symptomatic treatment (infrequent blood transfusions twice a year) and died of pneumonia 3.5 years later. HLA flow cytometry of cryopreserved cells revealed a gradual expansion of a clone partially lacking HLA-B4002 (B4002^dim), which was attributed to an HLA-B*40:02 intron 1 mutation (c.74-9C>A). A small monosomy 7 clone that was not visible by conventional BM cytogenetics by Giemsa-banding was detected by FISH from the sorted B4002^dim cells at least 2 years before clinical diagnosis of monosomy 7. NA, not assessed (due to deficient cryopreserved cells for analysis). See also supplemental Tables 8 and 9.