Strong Predictors of Chromosomal Aberrations and Myeloid Neoplasia Following Immunosuppression Therapy for Severe Aplastic Anemia: A Retrospective Cohort Study
Authors: Groarke Emma M. , Patel Bhavisha A. , Shalhoub Ruba , Gutierrez-Rodrigues Fernanda , Desai Parth , Leuva Harshraj , Paton Casey , Spitofsky Nina , Lotter Jennifer , Rios Olga , Childs Richard W. , Young David J. , Dulau-Florea Alina , Dunbar Cynthia E. , Calvo Katherine R. , Wu Colin O. , Young Neal S. 
Published: November 2021

Abstract
Introduction: Immune aplastic anemia (AA) is effectively treated with either immunosuppressive treatment (IST) or allogeneic hematopoietic stem cell transplant (HSCT). Clonal evolution remains the most feared long-term complication after IST. We investigated predictor factors, genetic characteristics, and long-term outcomes of patients who developed either secondary myeloid neoplasia or isolated chromosomal abnormalities without morphologic dysplasia after immunosuppression.
Methods: All patients with severe AA treated at the NIH Clinical Center with IST from 1989-2020 who underwent clonal evolution were categorized as "high-risk" (overt myeloid neoplasia, or isolated chromosome 7 abnormality / complex cytogenetics) or "low-risk" (isolated chromosome abnormalities without overt myeloid neoplasia or dysplasia; isolated chromosome 7 abnormality or complex cytogenetics were characterized as high-risk). Univariable analysis was performed using the Fine-Gray competing risk regression model using death as a competing risk to determine predictors of clonal evolution. Classification and regression tree analysis of time to clonal evolution was performed on continuous baseline variables to partition the data based on the best categorical cutoff. Long term outcomes assessed included overall survival (OS) and HSCT. Error corrected next-generation sequencing (ECS) was used to assess for pathogenic somatic variants in known myeloid cancer genes in clonal evolvers both at time of evolution and in serial samples prior when available.
Results: Of 659 patients with severe AA included in this study, 95 developed clonal evolution: 59 high-risk and 36 low-risk. Age &gt;48 years at diagnosis and pre-treatment ANC &gt;0.87x10 9/L were strong predictors of high-risk clonal evolution. High-risk clonal evolution was increased in patients aged &gt;48 years, with cumulative incidence (CI) of 13.9% by 5 years compared to patients aged &lt;48 years of 3.8% by 5 years (p&lt;0.001). Baseline ANC &gt;0.87 x10 9/L (independent of age) predicted an even higher risk of evolution; CI for high-risk evolution was 17% by 5 years (p&lt;0.001). Combined high ANC and older age (&gt;48 years) were prognostic of the greatest risk of high-risk evolution, with a hazard ratio (HR) of 5.51; conversely, ANC &lt;0.87 x10 9/L and age &lt;48 years was protective, with HR 0.32. High-risk clonal evolution was not significantly increased by use of eltrombopag with IST versus IST only (p=0.3), but there was an increase when all clonal evolution was considered (p=0.02). Overall survival in high-risk evolution was 35% at 5 years and in low-risk evolution was 84% (p&lt;0.001). Patients with high-risk evolution who underwent HSCT (n=26) had better OS compared to those treated with chemotherapy or supportive care (p=0.005).
RUNX1 (13 variants in 8 [35%] patients) and ASXL1 (13 variants in 10 [43%] patients) were the most frequent mutated genes at time of clonal evolution in high-risk patients, and BCOR/L1 (14 variants in 8 [32%] patients) was the most frequently mutated in the low-risk group. Longitudinal data were available in five high-risk and eight low-risk patients. Three of five high-risk patients had acquisition or expansion of RUNX1 clones at evolution. Small RUNX1 variants were present in two patients as early as three years prior to high-risk evolution. Splicing factor genes and RUNX1 somatic variants were detected exclusively in the high-risk group; DNMT3A, BCOR/L1 and ASXL1 gene mutations were present in both groups.
Conclusion: Age and pre-treatment ANC strongly predict high-risk clonal evolution in AA patients after IST and may be used determine at-risk patients for long term follow-up. Outcomes in patients with low-risk evolution are favorable but poor in high-risk evolution without HSCT. The clonal landscape differs between high-risk and low-risk evolution; MDS-associated genetic mutations are enriched in high-risk evolution, in particular RUNX1. Further study of the role of RUNX1 in high-risk clonal evolvers may give insight into leukemogenesis in AA.
Figure 1: Cumulative incidence (CI) of clonal evolution since immunosuppression with death treated as competing risk. (A) CI for development of all clonal evolution in patients &gt;37 years (B) and high-risk clonal evolution in patients &gt;48 years (C) CI for development of all clonal evolution when baseline ANC &gt;0.87x10 9/L and (D) high-risk clonal evolution when baseline ANC &gt;0.87x10 9/L.
Figure 1 Figure 1.

Disclosures
Young: Novartis: Research Funding.