Multiple ASXL1 mutations and G646fs alterations confer increased risk of leukemic Transformation in patients with myelofibrosis

Abstract Introduction The epigenetic regulator additional sex combs-like 1 (ASXL1) is the most commonly mutated high molecular risk gene in myleloid neoplasms and is associated with inferior clinical outcomes. Although the adverse prognostic value of ASXL1 mutations in myelofibrosis (MF) is invariable, most previous studies primarily focused on comparisons between mutated and wild-type (wt) ASXL1 patients. Intriguingly, Giada Rotunno et al. had reported the presence of multiple ASXL1 mutations (multi-ASXL1) in MF patients, a phenomenon also observed in clinical practice. However, whether the presence of multi-ASXL1mutations represents a molecular signature with prognosis impact remains to be explored. In this study, we aimed to evaluate the clinical significance of multi-ASXL1 and specific high-risk variants in predicting leukemic transformation (LT) among MF patients. Methods Molecular characterization was performed by next-generation sequencing (NGS) using a custom panel targeting 84 myeloid-associated genes. We retrospectively enrolled 222 MF patients with NGS data from the First Affiliated Hospital, Zhejiang University School of Medicine. Multi-ASXL1 was defined as the presence of two or more distinct ASXL1 mutations identified by NGS, regardless of VAF. Patient characteristics were compared using Chi-Square test for categorical variables and Kruskal-Wallis test for continuous variables. Leukemia-free survival (LFS) was assessed using the Kaplan-Meier analysis and log-rank testing. Furthermore, a 2-year landmark analysis was performed to evaluate long-term risk of LT. Variant-level analysis was conducted to assess the enrichment and prognostic impact of ASXL1 G646fs mutation. Results ASXL1 mutations were detected in 69 patients (31.1%), with 76.8% classified as mono-ASXL1 and 23.2% as multi-ASXL1. Compared with the wt counterpart, those harboring ASXL1 mutations had significantly higher age, WBC count, LDH levels, and MF grade. When stratified into three groups (wt, mono-ASXL1, and multi-ASXL1), WBC, LDH, and MF grade remained significantly different across groups. The incidence of complex karyotypes was numerically highest in the multi-ASXL1 group (43.8% vs 24.5% vs 21.2%). No significant difference in the distribution of driver mutations among the three groups was observed. Notably, the G646fs mutation, the most common ASXL1 variant, was significantly enriched in the multi-ASXL1 group compared with the mono-ASXL1 group (31.3% vs 18.9%, p < 0.001), and all the mutation sites were displayed in a lollipop plot. The incidence of LT was significantly higher in the multi-ASXL1 group (31.3% vs 26.5% vs 13.8%, p = 0.045), indicating a potential dose-dependent prognostic effect of mutation burden. Median LFS was not reached in the wt group, 114 months in mono-ASXL1, and 54 months in multi-ASXL1 patients. Compared to wt, ASXL1-mutated patients had a significantly shorter LFS (HR=2.22, 95% CI 1.16–4.23, p =0.016). When stratified by mutation burden, only the multi-ASXL1 group retained a significant increase in LT risk versus wt (HR=4.25, 95% CI 1.18–15.3, P=0.027), while mono-ASXL1 showed no significant difference. A landmark analysis at 2 years demonstrated a significant divergence in LFS between multi- and mono-ASXL1 patients beyond the 24-month mark (log-rank p = 0.024), proving a long-term adverse impact of multi-hit status. Among ASXL1-mutant patients, those with G646fs had significantly inferior LFS compared to those without (HR=4.06, 95% CI 1.48–11.1, p = 0.006), further supporting its role as a high-risk molecular signal as previously reported. Conclusion Multi-ASXL1 mutations are associated with a significantly increased risk of LT, particularly in the long-term setting. The enrichment of high-risk variants such as G646fs within the multi-ASXL1 cohort may partially explain this adverse outcome. Further study is warranted to validate these findings. Acknowledgement This research was funded by the Zhejiang Provincial Health High-level Innovative Talent Project (2022-2026). *Correspondence to Jian Huang, M.D., Ph.D., Department of Hematology, The First Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310003, Zhejiang, China. E-mail: [email protected]