Serial VAF Kinetics Reveal Dynamic Clonal Evolution Associated With Progression in Clonal Cytopenia of Undetermined Significance

ABSTRACT Background Clonal cytopenia of undetermined significance (CCUS) is a premalignant condition characterized by persistent cytopenias with somatic mutations but no morphologic evidence of overt myeloid neoplasia. While CCUS confers an elevated risk of progression to myeloid malignancies, the timing and biology of this transformation remain unpredictable. We hypothesized that the type of mutated gene and tracking clonal expansion over time via serial variant allele frequency (VAF) measurements may uncover early evolutionary signatures of malignant progression. Design We retrospectively analyzed 55 CCUS patients who underwent serial next‐generation sequencing (NGS) testing. Patients were stratified by their final clinical course at the time of this study as non‐progressor ( n = 26) or progressor ( n = 29). Baseline clinicopathologic variables, mutation burden, and the presence of ≥ 1 high‐risk mutation, which was defined in prior studies as SF3B1 , SRSF2 , ZRSR2 , JAK2 , TP53 , RUNX1 , FLT3 , IDH1 , and IDH2 , were compared between groups. The maximum annualized VAF slope across all tracked mutations was used as the primary dynamic clonal expansion metric. Baseline risk was assessed using the Clonal Hematopoiesis Risk Score (CHRS), and maximum VAF slope was evaluated across CHRS categories to determine whether serial clonal kinetics provided additional prognostic value beyond baseline risk stratification. Results Compared with non‐progressors, progressors had a higher baseline mutation burden ( p < 0.01), more frequently harbored ≥ 1 high‐risk mutation at baseline ( p < 0.01), and also more acquired new mutations during follow‐up ( p < 0.01). Across longitudinally tracked mutations, progressor‐associated clones more frequently demonstrated positive annualized VAF slopes, particularly involving RUNX1 (12.70%/year), STAG2 (13.09%/year), ASXL1 (6.44%/year), SRSF2 (4.29%/year), and U2AF1 (8.21%/year). Maximum annualized VAF slope correlated with CHRS risk category but also identified increased clonal expansion among progressors within the non‐high CHRS subgroup ( p = 0.006), suggesting added dynamic prognostic value beyond static baseline risk classification. Conclusion Serial VAF kinetics are helpful in predicting progression and complement baseline clinicopathologic features and CHRS risk stratification in CCUS. Expanding VAF trajectories, particularly involving RUNX1 , spliceosome genes, and cohesin‐related genes, may reflect biologically active clonal evolution toward progression to myeloid neoplasm, whereas stable or declining VAFs were more often observed in stable CCUS. These findings support that longitudinal VAF monitoring could be a practical dynamic approach to identify patients who require closer surveillance or repeat marrow evaluation.