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Performance of 16S Metagenomic Profiling in Formalin-Fixed Paraffin-Embedded versus Fresh-Frozen Colorectal Cancer Tissues.

ABSTRACT: Formalin-fixed, paraffin-embedded (FFPE) tissues represent the most widely available clinical material to study colorectal cancer (CRC). However, the accuracy and clinical validity of FFPE microbiome profiling in CRC is uncertain. Here, we compared the microbial composition of 10 paired fresh-frozen (FF) and FFPE CRC tissues using 16S rRNA sequencing and RNA-ISH. Both sample types showed different microbial diversity and composition. FF samples were enriched in archaea and representative CRC-associated bacteria, such as Firmicutes, Bacteroidetes and Fusobacteria. Conversely, FFPE samples were mainly enriched in typical contaminants, such as Sphingomonadales and Rhodobacterales. RNA-ISH in FFPE tissues confirmed the presence of CRC-associated bacteria, such as Fusobacterium and Bacteroides, as well as Propionibacterium allowing discrimination between tumor-associated and contaminant taxa. An internal quality index showed that the degree of similarity within sample pairs inversely correlated with the dominance of contaminant taxa. Given the importance of FFPE specimens for larger studies in human cancer genomics, our findings may provide useful indications on potential confounding factors to consider for accurate and reproducible metagenomics analyses.

SUBMITTER: Borgognone A

PROVIDER: S-EPMC8582506 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: Gene expression profiling of formalin-fixed, paraffin-embedded (FFPE) samples represents a valuable approach for advancing oncology diagnostics and enhancing retrospective clinical studies; however, at present, this methodology still requires optimization and thus has not been extensively used. Here, we utilized thorough quality control methods to assess RNA extracted from FFPE samples and then compared it to RNA extracted from matched fresh-frozen (FF) counterparts. We preformed genome-wide expression profiling of FF and FFPE ovarian serous adenocarcinoma sample pairs and compared their gene signatures to normal ovary samples. METHODS: RNA from FFPE samples was extracted using two different methods, Ambion and Agencourt, and its quality was determined by profiling starting total RNA on Bioanalyzer and by amplifying increasing size fragments of beta actin (ACTB) and claudin 3 (CLDN3) by reverse-transcriptase polymerase chain reaction. Five matched FF and FFPE ovarian serous adenocarcinoma samples, as well as a set of normal ovary samples, were profiled using whole genome Agilent microarrays. Reproducibility of the FF and FFPE replicates was measured using Pearson correlation, whereas comparison between the FF and FFPE samples was done using a Z-score analysis. RESULTS: Data analysis showed high reproducibility of expression within each FF and FFPE method, whereas matched FF and FFPE pairs demonstrated lower similarity, emphasizing an inherent difference between the two sample types. Z-score analysis of matched FF and FFPE samples revealed good concordance of top 100 differentially expressed genes with the highest correlation of 0.84. Genes characteristic of ovarian serous adenocarcinoma, including a well known marker CLDN3, as well as potentially some novel markers, were identified by comparing gene expression profiles of ovarian adenocarcinoma to those of normal ovary. CONCLUSION: Conclusively, we showed that systematic assessment of FFPE samples at the RNA level is essential for obtaining good quality gene expression microarray data. We also demonstrated that profiling of not only FF but also of FFPE samples can be successfully used to identify differentially expressed genes characteristic of ovarian carcinoma.

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Performance of 16S Metagenomic Profiling in Formalin-Fixed Paraffin-Embedded versus Fresh-Frozen Colorectal Cancer Tissues.

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