Integrated Whole-Genome and RNA Sequencing Analysis of FFPE Lung Cancer Samples: Feasibility and Pathological Validation

Whole-genome sequencing (WGS) and RNA sequencing (RNA-seq) can be applied to formalin-fixed paraffin-embedded (FFPE) specimens for genomic and transcriptomic analyses, including gene rearrangement detection. We tested whether PCR-free WGS and RNA-seq could detect driver alterations in FFPE lung adenocarcinoma samples prepared under standardized automated fixation and embedding conditions. Two adenocarcinoma cases were analyzed. Before this study, immunohistochemistry had suggested an EGFR alteration in one case and an ALK alteration in the other, although neither had been confirmed at the genomic or chromosomal level. In this analysis, PCR-free WGS yielded stable sequencing metrics and successfully detected small variants, including the canonical EGFR p.Leu858Arg mutation. By contrast, conventional structural variant calling failed to identify the EML4::ALK rearrangement. However, a custom algorithm based on supplementary alignment tags recovered the fusion candidate from WGS data despite support from only a single junction read, and RNA-seq independently confirmed expression of the EML4::ALK fusion transcript. Break-apart fluorescence in situ hybridization demonstrated tumor-wide clonal ALK rearrangement. These findings show that a single junction read can be biologically meaningful only when supported by orthogonal pathological validation and highlight the importance of integrating sequencing findings with pathological validation for the interpretation of gene rearrangements in FFPE specimens.