Clonal interactions as mechanisms of polyclonality in colorectal adenomas


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Viola Walther1,Dr. Hayley Davis2,Christian Kwasi Owusu3,Ashwin Shivaharan4,Yara Fadaili5,Dr. Marnix Jansen1,Dr. Manuel Rodriguez-Justo6,Dr. Simon Leedham2,Dr. Trevor A. Graham1,Dr. Stuart McDonald1
1Barts Cancer Institute, Queen Mary University London,2Wellcome Trust Centre for Human Genetics, University of Oxford,3Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, CB10 1SA, UK,4Imperial College London,5Queen Mary University London,6Department of Histopathology, University College London

Abstract

Background

According to the somatic mutation theory, tumours are derived from a single mutated cell that clonally expands into a neoplasm. In colorectal tumourigenesis however, studies on familial adenomatous polyposis (FAP) and some sporadic microadenomas have revealed tumours that are polyclonal in origin – they are derived from more than one crypt. The mechanisms and the implications of this are unknown.

Method

We investigated the effect of dysplastic crypts on non-dysplastic crypts to determine the mechanisms of polyclonality. To characterize the field effect of adenomas on adjacent normal crypts and surrounding stroma, immunohistochemistry was performed on FAP specimens and sporadic adenomas for Ki67, γH2AX, nuclear β- catenin, intraepithelial CD8, stromal CD4/8, CD68 and α-SMA expression. The percentage of stained cells were analysed as a function of proximity of normal to dysplastic crypts. Deficiency in the mitochondrial enzyme cytochrome c oxidase was also quantified as readout of mutation burden.

Results

We found higher expression of γH2AX, Ki67 and nuclear β-catenin in non-dysplastic crypts surrounding adenomas compared with distant normal crypts; an effect decreasing over distance away from the adenoma. Cytochrome c oxidase deficiency was also increased in crypts surrounding adenomas. For all stromal markers, the percentage of positive cells decreased the further away from the adenoma. This strongly suggests that a field effect results from an inflammatory response initiated by the adenoma generating mutant adjacent crypts.

We have also determined gene expression effects of an adenoma on normal colonic epithelium using Wild type and Apc1322/+ organoids that were subjected to RNA sequencing. Target pathways were identified using Gene Set Enrichment Analysis (GSEA). Strikingly, WT organoids exposed to Apc1322/+ organoids showed an upregulation in genes involved in proliferation, cell cycle and replication after 72hrs, whilst apoptosis genes are downregulated. 

Conclusion

This study characterises polyclonality and suggests how this develops in the early events in colorectal tumorigenesis.