Abstract
Targeted sequencing offers an opportunity to select specific drugs for cancer patients based on alterations in their genome. However, accurate sequencing cannot be performed in cancers harboring diffuse tumor cells because of low tumor content. We performed tumor cell enrichment using tissue suspension of formalin-fixed, paraffin-embedded (FFPE) tissue sections with low tumor cell content. The enriched fractions were used to efficiently identify mutations by sequencing a target panel of cancer-related genes. Tumor-enriched and residual fractions were isolated from FFPE tissue sections of intestinal and diffuse gastric cancers harboring diffuse tumor cells and DNA of suitable quality was isolated for next-generation sequencing. Sequencing of a target panel of cancer-related genes using the tumor-enriched fraction increased the number of detectable mutations and variant allele frequency. Furthermore, mutation analysis of DNA isolated from tumor-enriched and residual fractions allowed us to estimate germline mutations without a blood reference. This approach of tumor cell enrichment will not only enhance the success rate of target panel sequencing, but can also improve the accuracy of detection of somatic mutations in archived specimens.
Highlights
Targeted sequencing offers an opportunity to select specific drugs for cancer patients based on alterations in their genome
To increase the proportion of tumor cells from which DNA could be extracted in the FFPE tissue sections, tumor cell enrichment was performed using tissue suspension
The populations considered to be of tumor cells were enriched in the tumor fractions compared to the unseparated samples, whereas in the residual fraction, these populations were decreased in both diffuse-type and intestinal gastric cancers (Fig. 2)
Summary
Targeted sequencing offers an opportunity to select specific drugs for cancer patients based on alterations in their genome. Mutation analysis of DNA isolated from tumor-enriched and residual fractions allowed us to estimate germline mutations without a blood reference This approach of tumor cell enrichment will enhance the success rate of target panel sequencing, but can improve the accuracy of detection of somatic mutations in archived specimens. Recent large-scale analyses using nextgeneration sequencing (NGS) have revealed the relationship between gene alterations and various c ancers[1,2,3] Based on these findings, sequencing of multiple target gene panels using NGS provides an opportunity for further drug selection in clinical practice. Sequencing of a target panel is performed using formalin-fixed, paraffin-embedded (FFPE) tissue sections[5] Such samples with low tumor content can be subjected to tumor cell enrichment by macrodissection. Alternative tumor cell enrichment methods besides macrodissection are required for accurate detection of mutations in the sequencing of target panel of genes for various cancer types
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