Gene amplifications associated with primary colorectal cancer tumours and matched lymph nodes

Abstract

Colorectal cancer is among the most commonly diagnosed malignancies worldwide, with metastasis being the leading cause of mortality. CRC is a genetically heterogenous disease driven by a multitude of genomic alterations. Gene amplifications contribute to tumour progression, metastasis and therapy resistance by increasing the copy number of specific DNA sequences, thus leading to overexpression of genes that can alter signalling pathways, cell proliferation and survival. This study aimed to investigate gene amplifications in primary CRC tumours and matched metastatic lymph nodes to better understand their role in metastatic progression. While many studies focus on gene mutations in CRC, there is a relative lack of research on gene amplifications and their correlations with gene expression in matched tissue samples. Gene expression analysis of both the primary tumours and the matched metastatic lymph nodes was performed using the QuantiGene™ Plex assay, revealing high expression of several genes in the primary tumours, namely MET, GNAS, IGF2, TOP1, MTIF3, CDX2, MYC, MCL1 and KLF5. Four genes – MET, GNAS, IGF2 and TOP1 – were selected for analysis on matched lymph nodes with ddPCR using primer/probe sets designed in-house. IHC staining with FN1 was also done on a select number of cases to assess protein expression levels. Of interest, comparing the presence of amplifications with the gene expression in matched primary tumours, high IGF2 expression in the primary tumour was observed in the amplified samples. ddPCR analysis of CNVs in lymph nodes, resulted in amplification frequency of MET (58.3%), GNAS (45.8%), IGF2 (6.25%) and TOP1 (95.8%) in lymph nodes (n=48). Overall, correlation of gene expression and CNV was low and hence, these findings suggest that measuring both will give a more comprehensive understanding of metastatic progression. Study limitations included degradation over the years in the FFPE material used and a limited sample size, restricting the statistical power and limiting data analysis to visual interpretation via box plots. Despite these consstraints, this study establishes the basis for further studies, primarily the comparison of matched primary tumours and lymph nodes, including comparisons with matched invasion fronts and tumour buds, an emerging histological characteristic that is currently being investigated.