A gene-panel blood test for the detection of colorectal adenomas and cancer.

Abstract

e15728 Background: Colorectal cancer (CRC) is among the most treatable cancers, yet it remains the second leading cause of cancer deaths in men and third in women. Early detection of CRC precursors and early-stage CRC could significantly reduce mortality rates. The existing screening tests, including stool-based tests and the invasive colonoscopy with its challenging bowel preparation, often face low compliance. Therefore, a more convenient, accurate, and cost-effective screening test for CRC could greatly improve patient quality of life and most importantly, increase survival rates. The protein, N-myristoyltransferase-2 (NMT2), was discovered to be overexpressed in the peripheral blood mononuclear cells (PBMC) of CRC patients and individuals with adenomatous polyps (AP). In a proof of concept (POC) study, we demonstrated that an NMT2-based immunohistochemical (IHC) test is highly effective in detecting precancerous colorectal polyps and CRC with high sensitivity (91%) and specificity (81%) and surpasses other FDA approved tests, including the fecal immunochemical test (FIT), that has a sensitivity of 73%. A study involving a larger cohort would validate NMT2 as an effective biomarker that can be used for a commercial blood test to screen for adenomas and CRC and triage patients for colonoscopies. We are developing a quantitative real-time polymerase chain reaction (qRT-PCR)-based blood test to analyze the expression pattern of the NMT2 gene, its paralog, NMT1, and upstream target MetAP2 in the PBMC of CRC or AP cases and subjects with no evidence of disease (NED). By comparing the results of the subjects' colonoscopy procedure to those of the qPCR and IHC tests, we aim to use NMT2 as a biomarker to discriminate between CRC, AP, and NED. Methods: qRT-PCR was used to validate endogenous control genes and analyze the expression pattern of NMT2, NMT1, and MetAP2 . DNA libraries were prepared for next-generation sequencing according to the manufacturer's protocol [Illumina] and sequencing was conducted at The Centre for Applied Genomics in Toronto, Ontario. Results: We assessed the expression stability of candidate genes in CRC, AP, and NED PBMC samples and validated two endogenous control genes, RPS17 and RPL37A , for a qRT-PCR-based screening assay, using robust statistical algorithms such as the geNorm and NormFinder tools. Upon validation, we assessed the relative expression of NMT2 , NMT1 , and MetAP2 across sample types and observed differential expression, which aligns with previous IHC results. In parallel, we used NGS to identify two novel synonymous mutations in NMT2 [ID:rs137889266] and NMT1 [ID:rs1132898] within the CRC samples, providing further insights into tumorigenic mechanisms. Conclusions: Our findings demonstrate that RPS17 and RPL37A are reliable control genes for qRT-PCR assays, the potential of NMT2 as a biomarker, and highlights the potential of NGS to uncover clinically relevant mutations.