Project description:BACKGROUND & AIM: Sessile serrated adenoma/polyps (SSA/Ps) are the likely culprit of ~20% colon cancers but they are molecularly poorly understood. We investigated their epigenetic phenotype using high-throughput analysis of DNA methylation and gene expression. METHODS: 17 SSA/Ps and, as a comparative group, 15 conventional adenomas (all with matched samples of normal mucosa) were prospectively collected during colonoscopy. DNA from the 64 tissues was analyzed, via bisulfite next generation sequencing, for methylation at ~2.7 million CpG sites located prevalently in gene regulatory regions spanning 80.5Mb (~2.5% of the genome). The transcriptome of these samples was also investigated using RNA sequencing. An independent series of 61 archival lesions was used for targeted verification of DNA methylation. RESULTS: Both SSA/Ps and conventional adenomas showed a profound remodelling of their methylome. Cytosine hypermethylation was more pervasive in SSA/Ps than adenomas, in terms of number of hypermethylated regions (22,147 vs 15,965, respectively) and genes (4938 vs 3443, respectively). In addition, the extent of hypermethylation in a given region was higher in SSA/Ps than adenomas. This methylation pattern of SSA/Ps was reminiscent of the CpG island methylator phenotype (CIMP) of their descendant cancers. We have called this phenotype proto-CIMP since most of the hypermethylation occurred in CpG islands and shores. SSA/Ps were also protected from a wave of demethylation that instead occurred in adenomas outside of CpG islands/shores (4288 vs 18,417 hypomethylated regions in SSAPs vs adenomas). Verification studies of six hypermethylated regions demonstrated the high potential of DNA methylation markers for predicting the diagnosis of SSA/Ps and/or adenomas. Proto-CIMP of SSA/Ps was surprisingly associated with a higher number of up- (618) than downregulated (349) genes, while adenomas showed the opposite trend (516 up- vs 712 downregulated genes). CONCLUSIONS: The epigenetic landscape of SSA/Ps differs substantially from that of conventional adenomas. This huge amount of epigenetic variation represents a rich source of promising diagnostic tools, such as novel DNA markers or histologic stainings, for the tailored management of the two most frequent colon cancer precursors.

Project description:Sessile serrated adenomas are now recognized as precursor lesions of a substantial subset of colorectal cancers arising via a so-called “serrated pathway”. However, their biological markers remain to be defined. The aim of our study was to identify differentially expressed genes in sessile serrated adenomas, hyperplastic polyps and tubular adenomas. Gene expression analysis demonstrated molecular differences between polyp types. Further studies using QRT-PCR on Cathepsin E demonstrated a significantly (p< 0.05) higher expression in sessile serrated adenomas as compared to both other polyp types. Trefoil Factor 1, showed the same trend of expression for sessile serrated adenomas as compared to hyperplastic polyps, and was significantly higher in both polyps compared to tubular adenomas. Immunohistochemistry for both proteins demonstrated strong cytoplasmic staining of abnormal crypts in all sessile serrated adenomas while staining in tubular adenomas and hyperplastic polyps was weak and focal. BRAF and KRAS mutation analysis were employed to further validate polyp discrimination. The findings demonstrated the positive association of the BRAF mutation, V600E, with sessile serrated adenomas and KRAS mutations with tubular adenomas (P<0.05). This study demonstrates CTSE and TFF1 over-expression in sessile serrated adenomas compared to both hyperplastic polyps and tubular adenomas. Keywords: colonic polyp tissue comparison, linear modelling, SSA Microarray analysis was performed on 13 SSAs and 11 TAs. SSAs were from 4 males and 9 females (mean age of 75) and TAs were from 5 males and 6 females (mean age of 72). Samples were directly hybridised to each other (SSA versus TA) or to a pooled normal control (SSA versus control). A linear model (Smyth 2004) was fitted to the data bringing together the three contrasts: SSA versus control, TA versus control and SSA versus TA. Human OligoLibrary (Compugen Human Oligo Library (v1) containing 18861 60-mer oligonucleotides, representing approximately 16,000 unique genes) by the Adelaide Microarray Centre (Australia) was used. Slides were scanned using a GenePix 3000B scanner (Axon Instruments, Sunnydale, CA), and the Spot package (CSIRO, Australia) was used to identify spots and estimate fore- and background intensities (using a morphological opening background estimator) (Yang, Buckley et al. 2001; Ritchie 2004). Data analysis was performed in R (www.r-project.org) using the Limma package of Bioconductor (Gentleman, Carey et al. 2004; Smyth 2004). Loess print tip method was used to correct for dye-bias and intensity within each group of adjacent spots printed by one pin (Yang, Dudoit et al. 2002). Linear modelling was performed with the Limma package of Bioconductor (Smyth 2004).

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Gene expression analysis of microvesicular hyperplastic polyps and sessile serrated polyps/adenomas

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