Clonal Hematopoiesis By HLA Class I Allele-Lacking Hematopoietic Stem Cells and Concomitant Aberrant Stem Cells Is Rarely Associated with Clonal Evolution to Secondary Myelodysplastic Syndrome and Acute Myeloid Leukemia in Patients with Acquired Aplastic Anemia

[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 (p<0.05). In 13 patients who had been in hematological remission for more than 7 years, HLA(-) cells and other concomitant aberrant cells accounted for >90% 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.

Secondary myelodysplastic syndrome and leukemia in acquired aplastic anemia and paroxysmal nocturnal hemoglobinuria

Acquired aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH) are pathogenically related nonmalignant bone marrow failure disorders linked to T-cell–mediated autoimmunity; they are associated with an increased risk of secondary myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Approximately 15% to 20% of AA patients and 2% to 6% of PNH patients go on to develop secondary MDS/AML by 10 years of follow-up. Factors determining an individual patient’s risk of malignant transformation remain poorly defined. Recent studies identified nearly ubiquitous clonal hematopoiesis (CH) in AA patients. Similarly, CH with additional, non-PIGA, somatic alterations occurs in the majority of patients with PNH. Factors associated with progression to secondary MDS/AML include longer duration of disease, increased telomere attrition, presence of adverse prognostic mutations, and multiple mutations, particularly when occurring early in the disease course and at a high allelic burden. Here, we will review the prevalence and characteristics of somatic alterations in AA and PNH and will explore their prognostic significance and mechanisms of clonal selection. We will then discuss the available data on post-AA and post-PNH progression to secondary MDS/AML and provide practical guidance for approaching patients with PNH and AA who have CH.

Secondary myelodysplastic syndrome identified via next-generation sequencing in non-small cell lung cancer patients.

e19531 Background: Chemotherapy and radiotherapy used in cancer treatment are associated with increased risk of secondary myelodysplastic syndrome (MDS). MDS is characterized with specific chromosomal abnormalities and genomic alterations in JAK2, KRAS, CBL, DNMT3A, TET2, IDH1/2, EVI1, RUNX1, GATA2, EZH2, ASXL1, SF3B1, U2AF1, SRSF2 and ZRSR2. Since next generation sequencing (NGS) techniques have been widely used for the detection of actionable genomic variation in solid tumor patients, analysis of NGS data for chromosomal abnormalities and gene mutations may be supplementary for diagnosis of secondary MDS. Methods: We retrospectively analyzed targeted NGS data of paired tumor and blood samples from patients with non-small cell lung cancer (NSCLC) for chromosomal abnormalities and gene mutations. Sequence panel covered whole exons of 381 genes associated with cancers. Sequence data were processed using a customized analysis pipeline designed to accurately detect multiple classes of genomic alterations in routine clinical specimens. All testing was done in a CLIA-certified, CAP-accredited laboratory. Clinical information of cases with chromosomal abnormalities was reviewed, including treatment for previous cancer, blood and bone marrow tests. Results: We found 4 cases carrying MDS associated chromosome abnormalities among 3523 NSCLC patients with the prevalence of0.1%. They were old (from 66 to 71 years old), and were more likely to be male (3 males and 1 females). All of 4 cases included common abnormalities associated with MDS like 5q-, 7q-, -7, +21. 3 cases had complex (≥3) abnormalities and 1 case had double abnormalities. Point mutations were identified in blood samples of 3 cases. Mutations in TP53 occurred in 3 cases, mutations in ASXL1, DNMT3A, FLT3 and CBL occurred in one case respectively. Among the 4 cases, 2 cases were confirmed as MDS by blood and bone marrow tests. Conclusions: We identified 4 MDS associated chromosomal abnormalities from 3523 NSCLC patients through analyzing NGS data. NGS techniques could serve as supplementary for diagnosis of secondary MDS. [Table: see text]