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

Abstract

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.

Figure 1. Principal component analysis of microarray gene expression profiles.

(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.

Figure 2. NFE2L3 – prototypical ‘switched ON’ gene in colorectal neoplastic tissue relative to non-neoplastic tissue specimens.

(A) Discovery data set, 222 normal, black; 42 IBDs, green; 29 adenomas, blue; 161 adenocarcinomas, red. (B) Validation data set: 30 normal, black; 19 adenomas, blue; 19 adenocarcinomas, red. Y-axis: Normalized probeset intensity (log2).

Figure 3. KIAA1199 expression in colon tissue specimens.

(A) KIAA1199 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) KIAA1199 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). (C) A quantitative real-time SYBR-green KIAA1199 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.

Figure 4. Measurement of RNA levels in plasma specimens.

(A) GAPDH and (B) KIAA1199 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.