Areas of Crush Nuclear Streaming Should Be Included as Tumor Content in the Era of Molecular Diagnostics

SIMPLE SUMMARY: To expand the pool of samples available for genetic analysis, the quality and utility of DNA and RNA extracted from degenerated tumor tissues were examined. The DNA obtained from the nuclear streaming samples was preserved, enabling the identification of reliable variants using next-generation sequencing (NGS) analysis. The NGS metrics and variant allele frequency of the nuclear streaming samples did not differ from those of the control. This suggests that non-tumor cells exhibiting nuclear streaming could be used for genetical analysis and therefore, should be considered as tumor content to avoid misinterpreting the variant allele frequency. ABSTRACT: Degenerated tissues are frequently observed in malignant tumors, but are not analyzed. We investigated whether nuclear streaming and necrosis samples could be used for genetic analysis to expand the sample pool. A total of 81 samples were extracted from small cell carcinoma and lymphoma FFPE tissue blocks and classified into three histological cohorts: 33 materials with well-preserved tumor morphology, 31 nuclear streaming samples, and 17 necrosis samples. DNA and RNA integrity numbers, percentage of RNA fragments with >200 nucleotides, and next-generation sequencing quality metrics were compared among the cohorts. DNA quality did not significantly differ between nuclear streaming materials and materials with well-preserved morphology, whereas that of the necrosis samples was inferior. RNA quality decreased in the following order: materials with well-preserved morphology > nuclear streaming > necrosis. The sequencing metrics did not differ significantly between the nuclear streaming samples and materials with well-preserved morphology, and reliable variants were detected. The necrosis samples extracted from resections exhibited sequencing failure and showed significantly fewer on-target aligned reads and variants. However, variant allele frequency did not differ among the cohorts. We revelated that DNA in nuclear streaming samples, especially within biopsies, could be used for genetic analysis. Moreover, degenerated non-tumor cells should be counted when evaluating tumor content to avoid misinterpreting the variant allele frequency.