Background

Myeloid malignancies are clonal disorders of hematopoietic progenitor cells characterized by impaired differentiation and deregulated proliferation. These include acute myeloid leukemia (AML), myelodysplastic neoplasms (MDS), myeloproliferative neoplasms (MPN), and overlapping entities such as MDS/MPN [1]. Traditionally, their pathogenesis has been attributed to somatic mutations that perturb essential processes like self-renewal, proliferation, and differentiation [2]. These mutations can be broadly classified into five categories based on function: signal transduction proteins (e.g., JAK2, MPL, CALR, FLT3, and KIT), transcription factors (e.g., CEBPA, RUNX1, and ETV6), epigenetic modifiers (e.g., TET2, DNMT3A, ASXL1, and IDH1/2), tumor suppressors (e.g., TP53, WT1, and PHF6), and splicing factors (e.g., SF3B1, SRSF2, and U2AF1) [3,4]. In recent years, however, there has been increasing recognition that a subset of myeloid neoplasms may arise in the setting of germline predisposition [5–7]. The advent of next-generation sequencing (NGS) has uncovered a growing number of germline variants associated with inherited leukemia predisposition syndromes [8]. The fifth edition of the World Health Organization (WHO) classification of hematolymphoid tumors categorizes such myeloid neoplasms into three groups: (i) those without a pre-existing disorder, including germline mutations in CEBPA and DDX41; (ii) those with pre-existing thrombocytopenia, involving RUNX1, ANKRD26, and ETV6 mutations; and (iii) those with additional organ dysfunction, such as GATA2 deficiency or syndromic disorders like Fanconi anemia, telomere biology disorders, Noonan syndrome, and Down syndrome [1].

Compared to the well-established role of germline variants in solid tumors like breast cancer, our understanding of inherited predisposition in hematologic malignancies remains limited [9]. A unique challenge arises from the fact that several genes implicated in myeloid neoplasms, such as RUNX1, CEBPA, and TP53, can harbor both somatic and germline variants [10,11]. As a result, determining the origin of a detected variant is not always straightforward. In solid tumors, this issue is commonly addressed by paired sampling—comparing the mutation in tumor tissue to that in a non-malignant reference tissue like peripheral blood [12,13]. However, in leukemia and related disorders, both peripheral blood and bone marrow are often involved in the disease process or affected by clonal hematopoiesis, making them unreliable for germline analysis. Consequently, accurate classification of such variants in hematologic malignancies necessitates using alternate, non-hematopoietic tissues.

Among the available non-hematopoietic tissues, cultured skin fibroblasts from punch biopsy specimens have emerged as the most reliable source for germline DNA in patients with hematologic malignancies [14,15]. They offer several advantages, including elimination of contaminating hematopoietic cells, high DNA yield, and reproducibility, making them superior to alternatives such as hair follicles, nail clippings, or buccal mucosa, which often yield inadequate concentration of DNA for clinical-grade assays [14].

While several protocols for culturing fibroblasts from skin biopsies have been published, the majority remain research-oriented and lack direct clinical applicability [16–18]. These protocols often focus on enzymatic digestion techniques or specialized culture conditions, with limited guidance on how they can be effectively integrated into patient care. They also seldom address practical aspects such as bedside sample collection or the need for quick turnaround time in real-world diagnostic settings. To bridge this translational gap, we present a streamlined, enzyme-free protocol specifically optimized for clinical laboratories involved in diagnosing myeloid malignancies. Our method incorporates practical modifications, such as using mechanical separation instead of enzyme-based digestion to separate the epidermis from the dermis, eliminating the need to adhere skin pieces with gelatin, and other simple adjustments that promote faster fibroblast outgrowth. These refinements reduce culture time and contamination risk and are critical when timely and accurate germline results impact patient management. This comprehensive protocol details every step, from bedside collection of the skin punch biopsy alongside routine bone marrow sampling, to standardized fibroblast culture, confluency monitoring, and high-quality DNA extraction for germline variant analysis. The protocol was validated using multiple complementary approaches, including DNA yield assessment, flow cytometry, and immunocytochemistry (ICC). Additionally, the commonly studied JAK2V617F single-nucleotide polymorphism—frequently observed in myeloproliferative neoplasms like polycythemia vera, essential thrombocythemia, and primary myelofibrosis—was specifically used as a prototype during validation. Its detection in the patient’s bone marrow DNA and absence in fibroblast-derived DNA confirmed the lack of hematopoietic contamination and reinforced the reliability of the cultured fibroblasts as a source of pure germline DNA.

By emphasizing feasibility, reproducibility, and clinical relevance, our approach provides a practical framework for integrating fibroblast culture into routine diagnostic workflows for assessing hereditary predisposition in myeloid neoplasms.