ETV6 Mutations in Myeloid Neoplasia: Unraveling Clinical Features and Molecular Signatures

Introduction

The ETS variant transcription factor 6 (ETV6) has a crucial role in normal hematopoiesis. Autosomal dominant germline mutations (ETV6^GL) are responsible for familial thrombocytopenia and associated with increased risk of hematologic malignancies by an unclear mechanism. Although rare, somatic mutations of ETV6 (ETV6^SM) have been reported in 2-3% of patients (pts) with myeloid malignancies. In this study, we analyzed the prevalence, clinical, and molecular profile of ETV6^GL and ETV6^SM among pts with myeloid neoplasia (MN).

Methods

We reviewed all the cases with available targeted next generation sequencing (NGS) between 2016 and 2024 in the Yale New Haven Health network. All Identified variants were searched in ClinVAR and COSMIC databases and assigned as ETV6^GL or ETV6 ^SM if ever reported. ETV6^GL were further classified into benign/likely benign (B/LB), pathogenic/ likely pathogenic (P/LP) and variants of undetermined significance (VUS). According to our molecular pathology pipeline, somatic variants were classified into three levels: level-1 (pathogenic), level-2 (likely pathogenic), and level-3 (variants of undetermined significance). Clinical and molecular features for pts with ETV6^SM, ETV6^GL (P/LP and VUS variants), and ETV6 wild-type (ETV6^WT) were compared. The clonal burdens for ETV6^GL VUS variants were compared with general population allelic frequency if available.

Results

Overall, 2978 pts with available molecular data were included of whom 82 (2.8%) had ETV6 variants: 23 (27%) ETV6^SM, 52 (61%) ETV6^GL, 17 (20%) reported as both ETV6^SM/ ETV6^GL, and 27 (32%) previously unreported ETV6 variants. Of the 82 pts, 84% had MN as follows: 36% MDS, 25% AML, 19% MPN, 12% CMML, and 9% CCUS. The median age of the pts with MN was 74 (37-87) and 35% were female. The median platelet count was 123 (3-1352 x10⁹/L). Normal karyotype (45%), complex karyotype (9%), and -7/deletion 7q (9%) were the most frequent cytogenetics.

The frequency of ETV6^SM among MN pts was 30.4% (21/69). MDS (57%) and MPN (29%) were the most common diagnosis associated with ETV6^SM. The median platelet count was 155 (5-1352 x 10⁹/L). In terms of variants, 18 (86%) were missense and 3 (24%) were nonsense (all p. Arg359*). The median variable allele frequency (VAF) of ETV6^SM was 43% (range: 3-51%). Comparing ETV6^SM vs. ETV6^WT), the most frequently occurring co-mutations were ASXL1^MT (33% vs. 9%, p<0.001), KRAS^MT/NRAS^MT (24% vs. 6%, p<0.001), SETBP1^MT (19% vs. 1%) and DNMT3A^MT (19% vs. 11%, p<0.001).

ETV6^GL were classified as: B/LB (n=20, 47%), P/LP (n=10, 23%), and VUS (n=13, 30%). The frequency of ETV6^GL (P/LP and VUS) among MN pts was 10 (43%) and 13 (57%). Median platelet count of pts with P/LP and VUS ETV6^GL was 195 and 143 x10⁹/L, respectively. The type of P/LP ETV6^GL mutations (n) were nonsense (4), missense (4), frameshift (1), and splicing site (1). The median VAF of ETV6^GL was 47%. ASXL1^MT (70% vs. 9%, p<0.001), SRSF2^MT (40% vs. 9%, 0.001) and KRAS^MT/NRAS^MT (30% vs. 6%, p=0.001), were the most common co-mutations within the ETV6^GL P/LP cohort (compared to ETV6^WT and other ETV6^MT). Compared to other cases, VUS cases had high frequency of TP53^MT (23% vs. 9%, p=0.074) and KRAS^MT/NRAS^MT (15% vs. 6%, p=0.132). Allelic burden of p.Arg39Gln ETV6^GL (n=2, 0.0006) was higher than the general population (0.00002).

Of the new previously unreported variants, 9/23 were Level-2: 4 frameshift, 2 missense, 2 splicing sites, and 1 nonsense MT. Interestingly, only one developed MDS while 4 had AML (2 APML), 3 had MPN and one had CCUS. The median platelet count was 99 (13-1243 x10⁹/L). Most of the remaining variants (10/23) were Level-3 missense MT and only one was a frameshift deletion.

We then analyzed serial molecular profiles of 13 pts with P/LP ETV6^{SM & GL} to characterize clonal evolution and of these, 38% (5) pts were found to have acquired new mutations compared to the baseline molecular profile; 3 acquired RAS^MT and 2 acquired new SETBP1^MT. In one case, an MDS pt acquired new SETBP1^MT (VAF:19.6%) on top of 2 NRAS^MT (21+22%), 2 TET2^MT (24+26%), EZH2^MT (92%), and ETV6^MT (21%). Of the remaining 8 pts, 2 had RAS^MT and 3 had both RAS^MT and SETBP1^MT at time of diagnosis which did not change over time.

Conclusion

In our study, ETV6^SM occurred rarely among pts with MN. Pts with ETV6 ^{SM & GL} have a distinct molecular signature and are significantly associated with ASXL1^MT, RAS^MT, and SETBP1^MT, with the latter two being potentially acquired prospectively.