There is accumulating evidence that clonal hematopoiesis is prevalent in acquired AA, a condition where the immune-mediated destruction of hematopoietic stem/progenitor cells leads to pancytopenia. While the prevalence and impact of somatic mutations in AA have been described in patients who received immunosuppressive therapy (IST), little is known about somatic mutations' impact in patients undergoing allogeneic HCT.

We retrospectively evaluated a consecutive case series of 146 patients with AA who underwent HCT at our institution from 2005-2023; and 104 had pre-HCT DNA samples. The study was approved by our IRB. NGS libraries were prepared from genomic DNA (40 ng) using the SureSelect target enrichment system (Agilent Technologies Inc.) after transposase-based fragmentation and adapter ligation. Target regions were captured using a customized SureSelect library for all coding exons plus ten flanking bases of 230 genes curated to include genes involved in myeloid neoplasms, CHIP and bone marrow failure for NGS.

The median age (n=146) was 26 years (range 3-74) at HCT, 42,5% were female, KPS was 90-100 in 60.3%. HCT CI was 0-2 in 64.3%, ≥3 in 21.2%, and unknown in 14.4%. Most patients received fludarabine (Flu)/cyclophosphamide (Cy)/ATG+/-TBI as conditioning (58.2%) and 35.5% received Cy/ATG-based regimen without Flu followed by bone marrow (92.5%) or PBSC graft (6.8%) from a matched related/unrelated (42.5%/21.1%), mismatched unrelated (9.6%), haploidentical (10.3%), or cord blood donor (0.7%). Graft-versus-host disease (GVHD) prophylaxis was calcineurin inhibitor-based (87.3%) or post-transplant Cy-based (12.3%).

After a median follow up of 6.1 years (range 1.0-18.1) for surviving patients, 5-year overall survival (OS) was 81.8% (95%CI 74.1-87.3%) for the entire cohort. The cumulative incidence of non-relapse mortality (NRM) was 11% (95%CI 6.5-16.7%) at 1 year and 18.2% (95%CI 12.2-25.3%)at 5 years. Cumulative incidence of day-100 grade 2-4, grade 3-4 acute GVHD were 28.1%, 6.8%, respectively. Cumulative incidence of all and extensive chronic GVHD at 1 year were 42.5% and 28.6%, respectively. We observed significant difference in 5-year OS by the recipient age; 90.1% if younger than 40 years, 70.2% if 40-60 years (HR 2.4, 95%CI 1.02-5.81), and 55.5% if older than 60 years (HR 7.7, 95%CI 3.05-19.33; p<0.001).

A total of 58 pathogenic mutations were detected in 36/104 patients (35%) tested for NGS. The number of mutations per patient ranged from 1 (n=27), 2 (n=7), and 4 (n=1) and 13 (n=1). The most frequently mutated gene was previously undescribed KMT2C (16/104), followed by previously reported ASXL1 (6/104), DNMT3A (3/104), GNAS (3/104) and U2AF1 (3/104). Most mutations had a VAF below 5% (35/58) and the median VAF of KMT2C was 4.25% (range: 2.3-5.6%), while 8 cases may represent germline alterations with VAF of 40-50%, including BRCA2 (n=2), FANCI, CHEK2, TERT, PMS2, NSH6, and CDKN2A. There was no significant association between age and prevalence of these mutations: 32% in age <40, -40% in age 40-60, and 50% in age>60 (p=0.48), suggesting that evolution of clonal hematopoiesis is more likely driven by autoimmune hematopoietic environment rather than aging of hematopoietic progenitor cells. Time from diagnosis to HCT was significantly longer in patients with somatic mutations (11.8 months, interquartile range: 6.8-25.8) vs those without mutations (7.5 months, interquartile range 2.8-15.2). All 14 cases with KMT2C mutations whose old record was available had IST before their HCT.

The 5-year OS in patents with high-risk mutations (ASXL1, DNMT3A, TP53, RUNX1, CSMD1) was 50% (n=8) compared with 83.1% in others (n=96 p=0.03). The 5-year OS in patents with any mutations (n=36) was 85.0% vs 72.2% in others (n=68, p=0.059). When adjusted for age, the impact of high risk or any mutations was not statistically significant (HR: 2.42 [0.78-7.49], p=0.16, and HR 1.82 [0.81-4.11], p=0.15, respectively).

Our study represents the first report of systematically evaluating somatic mutations in patients with AA undergoing HCT, showing that somatic mutations are common, associated with time from diagnosis to HCT, and that KMT2C is a newly identified frequent somatic mutation in this population. Our results also justify further studies to define the prognostic impact of mutations on transplant outcomes in a larger cohort.

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2025
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