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

Mutational inhibition of ligation in the hairpin ribozyme: Substitutions of conserved nucleobases A9 and A10 destabilize tertiary structure and selectively promote cleavage

The hairpin ribozyme acts as a reversible, site-specific endoribonuclease that ligates much more rapidly than it cleaves cognate substrate. While the reaction pathway for ligation is the reversal of cleavage, little is known about the atomic and electrostatic details of the two processes. Here, we report the functional consequences of molecular substitutions of A9 and A10, two highly conserved nucleobases located adjacent to the hairpin ribozyme active site, using G, C, U, 2-aminopurine, 2,6-diaminopurine, purine, and inosine. Cleavage and ligation kinetics were analyzed, tertiary folding was monitored by hydroxyl radical footprinting, and interdomain docking was studied by native gel electrophoresis. We determined that nucleobase substitutions that exhibit significant levels of interference with tertiary folding and interdomain docking have relatively large inhibitory effects on ligation rates while showing little inhibition of cleavage. Indeed, one variant, A10G, showed a fivefold enhancement of cleavage rate and no detectable ligation, and we suggest that this property may be uniquely well suited to intracellular targeted RNA cleavage applications. Results support a model in which formation of a kinetically stable tertiary structure is essential for ligation of the hairpin ribozyme, but is not necessary for cleavage

<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

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

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

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

Proceedings of International Conference on Women Researchers in Electronics and Computing

This proceeding contains articles on the various research ideas of the academic community and practitioners presented at the international conference, “Women Researchers in Electronics and Computing” (WREC’2021). WREC'21 was organized in online mode by Dr. B R Ambedkar National Institute of Technology, Jalandhar (Punjab), INDIA during 22 – 24 April 2021. This conference was conceptualized with an objective to encourage and motivate women engineers and scientists to excel in science and technology and to be the role models for young girls to follow in their footsteps. With a view to inspire women engineers, pioneer and successful women achievers in the domains of VLSI design, wireless sensor networks, communication, image/ signal processing, machine learning, and emerging technologies were identified from across the globe and invited to present their work and address the participants in this women oriented conference. Conference Title: International Conference on Women Researchers in Electronics and ComputingConference Acronym: WREC'21Conference Date: 22–24 April 2021Conference Location: Online (Virtual Mode)Conference Organizers: Department of Electronics and Communication Engineering, Dr. B. R. Ambedkar National Institute of Technology, Jalandhar, Punjab, INDI