Germline predisposition in hematologic malignancies: A study of 22 cases with germline DNA Free

Introduction: About 7–15% of patients with leukemia or myelodysplastic syndromes (MDS) may harbor germline mutations, even in the absence of a clear family history. According to current guidelines, germline testing is recommended in specific clinical scenarios, including early-onset MDS (before age 40), family history of hematologic or solid tumors, syndromic features (e.g., lymphedema, organ dysfunction), personal history of bone marrow failure (BMF) or inherited thrombocytopenia, suggestive cytogenetic findings (Gachard et al., 2023), or high variant allele frequency mutations (VAF >30%) (Kraft & Godley, 2020). Well-established predisposition syndromes include mutations in genes such as DDX41, RUNX1, ETV6, ANKRD26, GATA2, and inherited BMF syndromes. Identifying germline mutations is essential, as this can affect treatment decisions, stem cell donor selection, and genetic counseling. Skin fibroblast culture from biopsy is a reliable source of uncontaminated DNA for germline analysis (Cazzola, 2023), though the process is time-consuming, adding approximately 28 days to testing (Lia DeRoin et al., 2022), and is not widely available. The aim of this study is to evaluate the clinical indications for germline testing and to analyze the molecular findings in a tertiary oncology and hematology laboratory in Brazil. Methods: A retrospective chart review was conducted. Since the implementation of skin fibroblast culture in 2021, 29 samples were obtained; 22 met inclusion criteria (patients >18 years with DNA for germline testing). Clinical indications were reviewed by ELN 2022 criteria for suspected predisposition. DNA from cultured fibroblasts was analyzed by hereditary myeloid panel (HMP), targeted variant testing (TVT), or whole exome sequencing (WES). Results: Among the 22 patients, 4 (18%) had a personal history of ≥2 malignancies, including at least one hematologic cancer; 3 (13.6%) had both personal and family history of hematologic malignancies; 6 (27.2%) had deleterious variants on somatic panels suggestive of germline origin; and 6 (27.2%) had hematologic malignancies diagnosed at a younger-than-expected age. Two patients met 2 of these criteria, and 1 patient formally met none but had 1q gain and borderline age, prompting testing. All fibroblast cultures yielded sufficient DNA for molecular analysis. Of the 22 cases, 11 underwent testing with a 29-gene HMP, 5 with targeted testing (MLH1, DDX41, TP53, or ETV6), 3 with WES, and 3 with an expanded 136-gene panel. Germline predisposition was confirmed in 3 patients (13%). One MDS case diagnosed before age 40 showed a pathogenic GATA2 variant [c.1017+572C>T:p(?)] identified on the HMP; one AML case with a somatic DDX41 variant (c.346G>T) had germline confirmation by TVT, classified as likely pathogenic by ACMG criteria; and one T-prolymphocytic leukemia (T-PLL) case with family history of breast and colorectal cancer showed a pathogenic MLH1 variant (c.1731G>A) on TVT. Other tests were either negative or identified variants of uncertain significance or unknown relevance to hematologic disease. Discussion: Twenty-one of 22 patients were classified into at least one of the ELN 2022 germline suspicion criteria. While indications for germline testing are better defined in myeloid neoplasms, four patients had lymphoid malignancies (2 B-ALL, 1 mantle cell lymphoma, 1 T-PLL). Two of the 3 positive cases underwent targeted testing based on strong suspicion of germline origin from somatic findings, demonstrating the utility and lower cost of focused testing when appropriate. The third had a somatic RUNX1 mutation but a germline GATA2 variant, highlighting the importance of careful test selection. In the absence of specific guidelines for which genes to test in each case, broader panels may increase diagnostic yield. Among 8 patients with potentially germline variants identified in tumor sequencing, 2 were confirmed germline and 6 were tested for the relevant gene and effectively excluded. Conclusion: Germline testing in hematologic malignancies remains complex, due to evolving knowledge of clinical indicators and implicated genes. Increased case reporting and systematic screening may improve recognition of clinical and molecular clues. Establishing fibroblast culture as a standard method for germline testing and expanding understanding of gene-specific indications are essential steps toward accurate diagnosis, tailored treatment, and appropriate family counseling.