Spectrum Of Genetic Alterations In Acquired Aplastic Anemia
Authors: Yoshizato Tetsuichi , Dumitriu Bogdan , Hosokawa Kohei , Makishima Hideki , Yoshida Kenichi , Okuno Yusuke , Chiba Kenichi , Tanaka Hiroko , Shiraishi Yuichi , Nagata Yasunobu , Katagiri Takamasa , Kon Ayana , Clemente Michael J. , Sanada Masashi , Miyano Satoru , Maciejewski Jaroslaw P. , Nakao Shinji , Young Neal S. , Ogawa Seishi 
Published: October 2019

Abstract
Acquired aplastic anemia (AA) is a prototype of idiopathic bone marrow failure, which is caused by immune-mediated destruction of hematopoietic progenitors. However, its natural course could be more complicated than expected for a simple immune-mediated disorder, as in the development of apparently acquired (somatic) clonal disorders such as paroxysmal nocturnal hemoglobinuria (PNH) and myelodysplastic syndrome (MDS) or acute myelogenous leukemia (AML), during its course. Although these evidences suggest a pathogeneic link between these disorders, the clonal architecture in AA has not been fully explored. In order to genetically define the origin of clonal hematopoiesis in patients with acquired AA, we sought gene mutations, by targeted deep sequencing of peripheral blood DNA from 192 Japanese patients with AA for mutations, using a panel of 51 genes including common mutational targets in myeloid malignancies using a SureSelect custom kit. An extended cohort of 293 American AA patients was further analyzed for targeted sequencing of granulocyte-derived DNA; for these cases, multiple sequential specimens with germline controls and complete clinical follow-up data were available. Exome sequencing was also performed for selected cases.
In the Japanese cohort, about 40% were severe or very severe diseases with an excellent response to immunosuppressive therapies (IST). In total, 43 somatic mutations were detected in 18% of the cases with the mean allelic burden of 18%. Mutations were most frequent in DNMT3A (3.6%), followed by ZRSR2 (3.1%), ASXL1 (2.6%), BCOR (2.0%) and more biased to nonsense (25.6%), frameshift (14.0%), splice site changes (7.0%) and non-frameshift indel (11.6%), indicating driver roles of these mutations in many cases. Mutations were associated with older age (p=0.014) and a better response to IST (p=0.040). We next examined an extended cohort of 293 AA cases from the United States, for which samples were collected at 6 months after treatment in the patients of severe or very severe disease. Except for 12 cases, CD3(+) cells were available and used to confirm the somatic origin of mutations by comparison with CD3 cells representing germline sequence. All patients had received IST, with overall response about 65%. As of the date of submission, data analysis was completed for 86 of the 293 cases, in whom we confirmed somatic mutations detected in BM samples from 45 cases (53%) 6 months after IST, with the mean number of mutations and the mean allelic burden were 1.08 and 15.3%, respectively. Similar to the finding in the Japanese cohort, BCOR (13.8%), DNMT3A (11.5%), and ASXL1 (10.3%) were most frequently mutated. PIGA (6.9%) and CSMD1 (4.6%) were also mutated in 6.9% and 4.6%, respectively. Again, mutations were associated with older age. Although there was no significant difference in response to IST (p=0.133) between mutation (+) and (-) cases, responders showed significantly higher numbers of mutations compared with non-responders (p=0.033). Evolution to MDS/AML occurred in 12 out of the 45 cases with mutations, while 13 out of the 62 cases without mutations developed MDS/AML. Therefore, candidate genes associated with some but not most evolution events. We further performed whole exome sequencing in 6 cases, for whom sequential samples were available: in 5 of 6 cases, somatic mutations were detected and the mean number of mutations was 9. There was evidence over time of clonal selection with or without progression to MDS or AML. Small clones of cells containing RUNX1 and U2AF1-mutated clones present in the initial specimen showed expansion in size with progression to MDS (0.003 to 0.46 and 0.013 to 0.097, respectively).
In conclusion, mutations in common target genes in myeloid malignancies can drive clonal evolution during the course of AA. However, overall there was no correlation between the presence of mutations and clinical evolution to MDS/AML, as many patients with evidence of clones containing mutations remained stable. Clonal expansion and the appearance and disappearance of clones occurred in some cases without clinical changes. The marrow failure environment may favor selection of mutant clones. In addition, other genetic/epigenetic alterations, including chromosomal instability induced by telomere shortening (accompanying abstract by Dumitriu and Feng) provide a mechanism of oncogenesis.

Disclosures:
Makishima: AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding. Maciejewski:Aplastic anemia&MDS International Foundation: Research Funding; NIH: Research Funding.