Feasibility and limitations of cultured skin fibroblasts for germline genetic testing in hematologic disorders

Abstract

To avoid acquired variants found in the blood, cultured skin fibroblasts are a recommended DNA source for germline genetic testing in patients with hematologic disorders, but data are lacking regarding practicality and limitations. We conducted a retrospective cohort study of 350 subjects with hematologic disorders who underwent skin fibroblast culture for germline genetic testing. We analyzed nextgeneration sequencing data from the targeted capture of 144 inherited cancer and bonemarrow failure genes to identify variants at heterozygous and subclonal variant allele frequencies. Sixteen (5%) biopsies failed to culture. Culture failure was more likely in samples with delays in culture initiation (OR = 4.3; p < 0.01) or a pathogenic variant in a telomere gene (OR = 42.6; p < 0.01). Median culture time was 28 days (IQR 22−29 days). Culture time was longer for subjects with prior allogeneic stem cell transplantation (+10.7%; p = 0.02) and shorter in subjects with a heterozygous pathogenic variant (−11.9%; p < 0.01), larger biopsy size (−10.6%; p < 0.01), or lymphoid malignancy (−8.4%; p < 0.01). Subclonal variants were identified in 10 (4%) and confirmed in five (56%) of eight with alternate samples available. Subclonal and discordant variants illustrate that germline testing from cultured skin fibroblasts requires phenotypic correlation and, in rare cases, follow-up studies for optimal interpretation.

Germ line variants in patients with acute myeloid leukemia without a suspicion of hereditary hematologic malignancy syndrome

“…BMSC cultures are suitable irrespective of the tumor burden at sample collection and even in the post-alloHCT setting but take ∼3 weeks to grow, similar to skin fibroblast cultures. 27 However, BMSC cultures do not need trained personnel performing a skin biopsy and can be retrospectively obtained from cryopreserved BM. PRBM requires disease full response, but even in this situation interpretation of variants is handicapped by measurable residual disease and chemotherapy-resistant CH.…”

Germline Predisposition to Myelodysplastic Syndromes

“…The recommended source of germline DNA testing in individuals with an active hematologic malignancy is cultured skin fibroblasts obtained via punch biopsy because the peripheral blood may harbor somatic genetic changes, which can confound detection and interpretation of germline mutations. 114,115 Clinical criteria and algorithms have already been published to enhance consideration for genetic testing for germline predisposition (Fig. 1).…”

“…The main goals are to educate about disease risk and to contribute to more informed treatment decisions, especially with regard to related donor selection in HSCT. The recommended source of germline DNA testing in individuals with an active hematologic malignancy is cultured skin fibroblasts obtained via punch biopsy because the peripheral blood may harbor somatic genetic changes, which can confound detection and interpretation of germline mutations 114,115 …”

Beyond Pathogenic RUNX1 Germline Variants: The Spectrum of Somatic Alterations in RUNX1-Familial Platelet Disorder with Predisposition to Hematologic Malignancies

“…When molecular profiling reveals a pathogenic variant in hematologic tissues with a variant allele fraction of at least 30% in a gene known to confer inherited cancer risk, a germline origin should be suspected and subsequently verified [ 55 ]. This and germline testing, in general, can be carried out by analysis of DNA extracted from cultured fibroblasts [ 56 ] or by segregation analyses. For the latter, however, the germline origin of the respective variant cannot be excluded if the variant is not detected in family members.…”