Incidence and prognostic impact of U2AF1 mutations and other gene alterations in myelodysplastic neoplasms with isolated 20q deletion

Abstract Background In myelodysplastic neoplasms (MDS), the 20q deletion [del(20q)] is a recurrent chromosomal abnormality that it has a high co‐occurrence with U2AF1 mutations. Nevertheless, the prognostic impact of U2AF1 in these MDS patients is uncertain and the possible clinical and/or prognostic differences between the mutation type and the mutational burden are also unknown. Methods Our study analyzes different molecular variables in 100 MDS patients with isolated del(20q). Results & Conclusions We describe the high incidence and negative prognostic impact of U2AF1 mutations and other alterations such as in ASXL1 gene to identify prognostic markers that would benefit patients to receive earlier treatment.

The 20q deletion [del(20q)] is a recurrent chromosomal aberration in myelodysplastic neoplasms (MDS), and as a single abnormality is associated with a favorable outcome according to the Revised International Prognostic Scoring System (IPSS-R). 1 U2AF1 mutations have been associated with chromosomal instability by cell replication obstruction and are frequent (15%-20%) in MDS with del(20q). 2,3Nevertheless, their prognostic impact in these MDS patients is uncertain and the possible clinical and/ or prognostic differences between mutation type and mutational burden are also unknown. 4Other alterations in genes such as ASXL1, SF3B1, or SRSF2 may also play a prognostic role in MDS with del(20q). 3,5he aim of the current study was to assess the incidence and prognostic impact of U2AF1 mutations and other gene alterations in MDS patients with isolated del(20q).
From 2001 to 2020, 100 adult MDS patients with isolated del(20q) were studied.The median follow-up of the series was 17 months (range, 1-199 months).All patient samples were acquired at diagnosis after written informed consent, in compliance with the Declaration of Helsinki and with the approval of the local ethics review committee.As in a previous analysis, ASXL1, SF3B1, SRSF2, U2AF1, DNMT3A, IDH1, IDH2, TP53, RUNX1, and SETBP1 mutations were analyzed by high-resolution melting (HRM) and ASXL1 chromosomal deletion was studied by FISH (Supplemental Methods). 5Three quantitative allelespecific PCR (ASO-qPCR) were designed to determine variant allele frequency (VAF) and to increase sensitivity in detection of U2AF1 mutations (S34F, VAF≥0.70%;Q157P, VAF ≥0.15%; Q157R, VAF ≥1.30%; Supplemental Methods).Statistical analysis was generated using the SPSS® statistical data package v.20 and p < 0.05 was considered as statistically significant.Pairwise comparisons between patients' characteristics were performed using the Mann-Whitney test for continuous variables and the χ 2 test or Fisher exact test for categorical variables.Overall survival (OS) was measured from time of diagnosis to last follow-up or death and was also censored at the time of hematopoietic stem cell transplantation.Survival curves were generated using the Kaplan-Meier method and compared by log-rank test.A Cox proportional hazard model was constructed for multivariable analysis.Transfusion dependence was defined as patients requiring two or more red blood cells (RBC) transfusions per month or at least one transfusion every 8 weeks in a 4-month period.Response to azacitidine treatment was assessed using the modified international working group criteria. 6atient characteristics are in Table S2.The median age in the overall series was 74 years and 74% were male patients.According to the IPSS-R, 74% of patients were categorized as lower-risk, 15% as intermediate-risk, and 11% as higher-risk.Twenty-four patients without response to first-line treatment or with higher-risk MDS received azacitidine.Fourteen patients progressed to acute myeloid leukemia (AML).The median OS for patients was 49 months.
Regarding other genes, SF3B1 mutations were associated with a lower hemoglobin level (median, 8.4 vs. 10.4 g/ dL, p = 0.033), treatment with erythropoiesis-stimulating agents (ESAs) (85% vs. 29%, p < 0.001) and fewer patients in higher-risk categories of the IPSS-R (IPSS-R HR ) (15% vs. 28%, p = 0.012).Patients with ASXL1 mutations (n = 20) showed a lower hemoglobin level (median, 8.2 vs. 10.6 g/ dL, p = 0.043), and patients with ASXL1 chromosome deletion (n = 21; Figure S3) had a higher progression to Results & Conclusions: We describe the high incidence and negative prognostic impact of U2AF1 mutations and other alterations such as in ASXL1 gene to identify prognostic markers that would benefit patients to receive earlier treatment.
The increase in the number of gene mutations was also associated with poor clinical features such as higher ferritin levels and lower OS (median ferritin, 967 vs. 434 vs. 221 ng/mL, p = 0.025; median OS, 13 vs. 36 vs. 65 months, for ≥2, 1, and 0 mutations, respectively, p = 0.015; Figure S2C).Additionally, response to azacitidine was analyzed according to clinical and molecular variables, and a lower age remained as the only significant predictor of better response (responders, 53% vs. 11%, for <70 and ≥70 years, respectively, p = 0.048).
As in previous analyses, we found a high incidence of U2AF1 mutations in MDS patients with 20q deletion. 3,5In a new finding, U2AF1 mutations had a negative prognostic impact on the isolated 20q condition.The significance of U2AF1 mutations in MDS cohorts without del(20q) is variable, in a few cohorts they have no prognostic impact or are confined to low-risk patients, 7,8 while in others U2AF1 mutations show a significant correlation with a worse OS. 4,9,10 Currently, U2AF1 mutations are considered detrimental prognostic factors in MDS and have been associated with adverse risk in the new Molecular International Prognostic Scoring System for MDS (IPSS-M). 10,11Furthermore, in our analysis, U2AF1 Q157 patients had worse clinical parameters and lower OS than U2AF1 S34 patients, suggesting functional differences between the two mutations based on their different effects in the pre-mRNA splicing. 4,12As in a previous study, U2AF1 mutation burden was also found to influence prognosis; however, further studies are needed to explore the relationship between high VAF and disease complexity due to an increase in other mutations. 13ther splicing genes were also found to be mutated in a significant number of patients, and SF3B1 mutations in particular were related to anemia, resulting in high dependence of ESAs.On the other hand, ASXL1 alterations were also frequently detected in MDS with del(20q) and determined a poor clinical outcome. 3,5Finally, in concurrence with previous reports, the number of driver mutations was found to provide prognostic information. 14he absence of a more comprehensive gene analysis could limit the scope of this study.So, further studies in other MDS with del(20q) cohorts with a more complete gene characterization could be necessary to confirm these novel results.
In summary, risk stratification remains an essential step before treatment decision-making, and although isolated del(20q) is associated with favorable prognosis in MDS, accompanying gene alterations may have a marked negative impact on clinical outcome.In this context, U2AF1 mutations and ASXL1 alterations are present in approximately half of MDS patients with isolated del(20q) and detection at diagnosis would help to identify different subgroups with worse prognosis that could benefit from earlier treatment.

F I G U R E 1 2
Gene mutations and ASXL1 chromosomal deletion in the series.Each column represents an individual sample.Kaplan-Meier curves.(A) Overall survival (OS) in the series according to U2AF1 mutations.(B) OS in the series according to the co-occurrence of U2AF1 and ASXL1 alterations.U2AF1 WT (n=73) U2AF1 MUT (n=27) P= 0.016 P= 0.001