Integrating Formalin-Fixed, Paraffin-Embedded–Derived Whole-Genome Sequencing into Routine Molecular Pathology

Formalin-fixed, paraffin-embedded (FFPE) tumor tissue is the standard in pathology due to logistical and quality constraints of fresh-frozen samples. Although whole-genome sequencing (WGS) offers diagnostic promise, its validity and utility in FFPE samples remain underexplored. This study bridges the gap by comparing FFPE-derived tumor WGS with next-generation sequencing results from FoundationOneCDx (F1CDx) and a melanoma-specific panel (MelArray) in 78 metastatic melanoma samples from the Swiss Tumor Profiler Study. A diagnostic pipeline was developed for quality control, variant annotation, and clinical actionability using public and commercial databases. FFPE-derived WGS displayed robust analytical validity, detecting 95% of somatic single nucleotide variants, 98% of multi-nucleotide variants, 90% of insertions/deletions, 76% of amplifications, and 96% of homozygous deletions identified by F1CDx. Tumor mutational burden strongly correlated with F1CDx (R Z 0.98), particularly at the clinical threshold of >= 10 mutations per megabase, crucial for treatment decisions. WGS detected complex biomarkers such as UV-associated mutational signatures and genome-wide copy number alterations, aiding melanoma subtype distinction. Clinically, WGS suggested treatments or trials for all cases, identifying additional markers in 38% and 71% compared with F1CDx and MelArray, respectively. Novel therapeutic opportunities were found in 18% and 56% of cases. FFPE-derived WGS closely matches targeted panels in performance while providing comprehensive insights, enhancing therapeutic options. With decreasing costs, WGS could become a powerful routine diagnostic tool. (J Mol Diagn 2025, 27: 722-735; https://doi.org/10.1016/j.jmoldx.2025.04.011)