INTRODUCTION

Approximately 10% of adults diagnosed with myelodysplastic neoplasms (MDS) or acute myeloid leukemia (AML) harbor germline variants in genes associated with predisposition to myeloid neoplasms. Clonal hematopoiesis (CH) has emerged as an area of interest in the context of germline predisposition (GP) in adults. However, the lack of longitudinal studies limits our understanding of CH as either a pre-malignant condition or a compensatory mechanism in a background of GP.

This study aimed to investigate the prevalence, evolution, and clinical implications of CH in relatives of adult patients with hematologic neoplasms of germline predisposition (HNGP) carrying familial pathogenic germline variants.

METHODS

Between 2019 and 2025, suspected cases were evaluated using a germline predisposition–focused virtual panel based on exome sequencing performed on DNA extracted from hair roots. In cases with clinical features suggestive of telomere biology disorders (TBD), telomere length was assessed by flow-FISH. First- and second-degree relatives were offered diagnostic testing using Sanger sequencing on peripheral blood (PB) and/or flow-FISH, as appropriate. Relatives who carried the familial germline variant underwent evaluation with a targeted next-generation sequencing (NGS) panel of genes recurrently mutated in myeloid malignancies, using PB or bone marrow (BM) samples at baseline and after a 2- to 3-year follow-up. Relatives who tested negative for the familial germline variant were evaluated at a single time-point using the same NGS panel and served as healthy controls. In families affected by TBD, an additional NGS panel targeting genes involved in telomere maintenance was performed. CH risk was classified according to the scoring system proposed by Weeks et al. (N Engl J Med Evid 2023), and CH evolution was defined as either an increase in variant allele frequency (VAF) of at least 5% or the acquisition of new mutations.

RESULTS

Among the 201 patients with suspected HNGP, germline variants were identified in 30 index cases in the following genes: ETV6 (n=3), GATA2 (n=2), DDX41 (n=9), ELANE (n=1), CHEK2 (n=1), RUNX1 (n=1), TP53 (n=1), and 12 patients were diagnosed with TBD. All TBD cases had very short telomere length (<1%), and two of them also carried germline variants in RTEL1 and TINF2, respectively. The median age at diagnosis was 47 years (IQR: 40–55)

Out of the 30 index cases, 133 family members were studied. Of these, 50 (37.5%) were confirmed carriers of the familial variant (median age 50 years, IQR 38.5-68.5). Among them, 41 (82%) underwent NGS analysis, and 12 (24%) were found to harbor likely pathogenic/pathogenic (LP/P) somatic variants. The median age at CH detection was 62.5 years (IQR 47.5-76.5). Eight carriers were re-evaluated after 2 to 3 years, with 5 (62%) showing clonal evolution, either through an increase in VAF or the acquisition of new mutations.

A notable case involved a TP53 pathogenic variant detected at a VAF of 4%, which remained stable over the follow-up period, with no progression to myeloid malignancy. Additionally, in a family with germline ETV6 mutation, five carriers developed CH; 3 of them with somatic mutations in myeloproliferative driver genes (JAK2, KRAS, NFE2, PTPN11, and CBL), and 1 individual progressed to MDS with low blasts counts during the follow-up. DDX41 carriers presented typical somatic alterations (DNMT3A) without age-anticipation compared to the general population.

Based on Weeks' risk score, 55% of CH in family carriers was classified as intermediate- or high-risk.

Preliminary results from the telomere maintenance gene panel identified two somatic TERT variants in 2 TBD carriers (full data will be presented at the 2025 ASH conference).

Between non-carrier relatives, somatic variants were detected in 13.5% (median age 63 years, IQR 54-80.5), restricted to DNMT3A and TET2, all of them classified as low-risk CH. The difference in the frequency of CH between carriers (24%) and non-carriers (13.5%) was not statistically significant (p = 0.19), while the difference in CH risk-score showed a trend toward statistical significance (p = 0.05).

CONCLUSIONS

We observed a high-risk profile CH among family carriers compared to non-carriers, most cases showing evidence of clonal evolution over time. Notably, we report for the first time a characteristic mutational landscape in asymptomatic individuals carrying germline ETV6 variants.

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2025
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