Discovery and validation of molecular biomarkers for colorectal adenomas and cancer with application to blood testing

Results: Genome-wide analysis uncovered reproducible gene expression signatures for both adenomas and cancers compared to controls. 386/489 (79%) of the adenoma and 439/529 (83%) of the adenocarcinoma biomarkers were validated in independent tissues. We also identified genes differentially expressed in adenomas compared to cancer. KIAA1199 was selected for further analysis based on consistent up-regulation in neoplasia, previous studies and its interest as an uncharacterized gene. Plasma KIAA1199 RNA levels were significantly higher in patients with either cancer or adenoma (31/ 40) compared to neoplasia-free controls (6/20).This work was co-funded by Flinders University of South Australia and the Commonwealth Scientific and Industrial Research Organisation (CSIRO) of Australia. Drs. Dunne, Molloy and Brown are employed by CSIRO. These funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Funding was also provided by Clinical Genomics Pty Ltd., a company involved in the discovery and commercialization of biomarkers for colorectal cancer. Drs. LaPointe, Pedersen, Gaur, McEvoy and Thomas are employed by Clinical Genomics Pty Ltd and Prof. Young is a paid consultant of Clinical Genomics Pty Ltd. The funder thus played roles in study design, data collection and analysis, decision to publish, and preparation of the manuscript. Mrs. Pimlott and Dr. Wattchow have nothing to disclose. This work was co-funded by Clinical Genomics Pty Ltd, a company involved in the discovery and commercialization of biomarkers for colorectal cancer. Drs. LaPointe, Pedersen, Gaur, McEvoy and Thomas are employed by Clinical Genomics Pty Ltd. Prof. Young is a paid consultant of Clinical Genomics Pty Ltd. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials

Discovery and Validation of Molecular Biomarkers for Colorectal Adenomas and Cancer with Application to Blood Testing

BACKGROUND & AIMS: Colorectal cancer incidence and deaths are reduced by the detection and removal of early-stage, treatable neoplasia but we lack proven biomarkers sensitive for both cancer and pre-invasive adenomas. The aims of this study were to determine if adenomas and cancers exhibit characteristic patterns of biomarker expression and to explore whether a tissue-discovered (and validated) biomarker is differentially expressed in the plasma of patients with colorectal adenomas or cancer. METHODS: Candidate RNA biomarkers were identified by oligonucleotide microarray analysis of colorectal specimens (222 normal, 29 adenoma, 161 adenocarcinoma and 50 colitis) and validated in a previously untested cohort of 68 colorectal specimens using a custom-designed oligonucleotide microarray. One validated biomarker, KIAA1199, was assayed using qRT-PCR on plasma extracted RNA from 20 colonoscopy-confirmed healthy controls, 20 patients with adenoma, and 20 with cancer. RESULTS: Genome-wide analysis uncovered reproducible gene expression signatures for both adenomas and cancers compared to controls. 386/489 (79%) of the adenoma and 439/529 (83%) of the adenocarcinoma biomarkers were validated in independent tissues. We also identified genes differentially expressed in adenomas compared to cancer. KIAA1199 was selected for further analysis based on consistent up-regulation in neoplasia, previous studies and its interest as an uncharacterized gene. Plasma KIAA1199 RNA levels were significantly higher in patients with either cancer or adenoma (31/40) compared to neoplasia-free controls (6/20). CONCLUSIONS: Colorectal neoplasia exhibits characteristic patterns of gene expression. KIAA1199 is differentially expressed in neoplastic tissues and KIAA1199 transcripts are more abundant in the plasma of patients with either cancer or adenoma compared to controls

Colorectal Neoplasia Differentially Expressed (CRNDE), a Novel Gene with Elevated Expression in Colorectal Adenomas and Adenocarcinomas

An uncharacterized gene locus (Chr16:hCG_1815491), now named colorectal neoplasia differentially expressed (gene symbol CRNDE), is activated early in colorectal neoplasia. The locus is unrelated to any known protein-coding gene. Microarray analysis of 454 tissue specimens (discovery) and 68 previously untested specimens (validation) showed elevated expression of CRNDE in >90% of colorectal adenomas and adenocarcinomas. These findings were confirmed and extended by exon microarray studies and RT-PCR assays. CRNDE transcription start sites were identified in CaCo2 and HCT116 cells by 5′-RACE. The major transcript isoforms in colorectal cancer (CRC) cell lines and colorectal tissue are CRNDE-a, -b, -d, -e, -f, -h, and -j. Except for CRNDE-d, the known CRNDE splice variants are upregulated in neoplastic colorectal tissue; expression levels for CRNDE-h alone demonstrate a sensitivity of 95% and specificity of 96% for adenoma versus normal tissue. A quantitative RT-PCR assay measuring CRNDE-h RNA levels in plasma was (with a threshold of 2–ΔCt = 2.8) positive for 13 of 15 CRC patients (87%) but only 1 of 15 healthy individuals (7%). We conclude that individual CRNDE transcripts show promise as tissue and plasma biomarkers, potentially exhibiting high sensitivity and specificity for colorectal adenomas and cancers

<i>KIAA1199</i> expression in colon tissue specimens.

<p>(<b>A</b>) <i>KIAA1199</i> expression measured via probeset 212942_s_at in the discovery dataset of 454 colorectal tissue specimens (x-axis indexed by phenotype); Norm: 222 normal specimens, black; IBD: 42 ‘colitis’ specimens, green; ADE: 29 adenomas, blue; CA: 161 cancer specimens, red. Y-axis: normalized probeset intensity (log2). (<b>B</b>) <i>KIAA1199</i> expression measured via probeset 1008852-HuGene_st in the validation dataset in 68 colorectal tissue specimens. X-axis; Norm: 30 normal colon tissue specimens, black; ADE: 19 adenomas, blue; CA: 19 colorectal cancer specimens, red. Y-axis: normalized probeset intensity (log2). (<b>C</b>) A quantitative real-time SYBR-green <i>KIAA1199</i> PCR assay applied to RNA extracts used for the validation microarray data: 30 normal, black; 21 adenomas, blue; 20 colorectal cancer, red specimens. Data are mean values of duplicates, normalized against HPRT1 and depicted as delta-delta-Ct values. Note that three additional neoplastic specimens were available for the PCR experiments which were not tested by custom microarray.</p

Summary of microarray discovery and validation studies.

<p>Review of probeset numbers for hypothesis discovery and hypothesis validation data sets. Note that an ‘up’ probeset means a probeset response differentially higher in the Class B phenotype relative to the Class A phenotype.</p

Measurement of RNA levels in plasma specimens.

<p>(<b>A</b>) <i>GAPDH</i> and (<b>B</b>) <i>KIAA1199</i> RNA levels in plasma from 20 healthy subjects (black) and from 20 patients with colon adenomas (blue) and 20 CRC patients (red). Data are mean Ct values (triplicates) normalized for extraction yield differences and depicted as fold-change differences relative to the median expression measured in the 20 normal subjects. P values were calculated using two-tailed Mann-Whitney t-test.</p

Phenotypic breakdown of clinical specimens used in this study.

1<p>42 Colitis/IBD: 2 Colitis; 13 Crohn's disease; 5 Diverticulitis of colon; 2 Proctitis; 20 Ulcerative colitis.</p>2<p>19 adenomas: 1 tubular adenomas, 8 tubulovillous adenomas, 2 villous adenomas, 2 familial adenomatous polyps, 6 unknown; 19 adenocarcinomas (17 Dukes' A and 2 Dukes' B).</p>3<p>20 adenomas: 11 tubular adenomas, 5 villous adenomas, 4 unknown; 20 adenocarcinomas (1 Dukes' A, 6 Dukes' B, 4 Dukes' C, 1 Dukes' D, 8 unknown). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029059#pone-0029059-t004" target="_blank">Table 4</a> for a further breakdown of the cancer staging into T scores (a component of the TNM score).</p

Principal component analysis of microarray gene expression profiles.

<p>(A) Discovery microarray dataset: 222 normal, black; 42 colitis/IBD, green; 29 adenomas, blue; and 161 adenocarcinomas, red. (B) Validation microarray dataset: 30 normal, black; 19 adenomas, blue; and 19 adenocarcinomas, red.</p