Gene expression biomarkers for colorectal neoplasia

Author: Lawrence C LaPointe

LaPointe, Lawrence C, 2009 Gene expression biomarkers for colorectal neoplasia, Flinders University, School of Medicine

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The aim of this research was to assemble sufficient experimental evidence about candidate gene transcript expression changes between non-neoplastic and neo- plastic colorectal tissues to justify future assay development involving promis- ing leads. To achieve this aim, this thesis explores the hypothesis that gene expression-based biomarkers can be used to accurately discriminate colorectal neoplastic tissues from non-neoplastic controls. This hypothesis was tested by first analysing multiple, large, quality controlled data sets comprising gene expression measurements across colorectal phenotypes to discover potential biomarkers. Candidate biomarkers were then subjected to validation testing using a custom-design oligonucleotide microarray applied to independently derived clinical specimens. A number of novel conclusions are reached based on these data. The most important conclusion is that a defined subset of genes expressed in the colorectal mucosa are reliably differentially ex- pressed in neoplastic tissues. In particular, the apparently high prediction accu- racy achieved for single gene transcripts to discriminate hundreds of neoplastic and non-neoplastic tissues provides compelling evidence that the resulting can- didate genes are worthy of further biomarker research. In addition to addressing the central hypothesis, additional contributions are made to the field of colorectal neoplasia gene expression profiling. These contributions include: The first systematic analysis of gene expression in non-diseased tissues along the colorectum To better understand the range of gene expression in non-diseased tissues, RNA extracts taken from along the longitudinal axis of the large intestine were studied. The development of quality control methodologies for high dimen- sional gene expression data Complex data collection platforms such as oligonucleotide microarrays introduce the potential for unrecognized confound- ing variables. The exploration of quality control parameters across five hundred microarray experiments provided insights about quality control techniques. The design of a custom microrray comprised of oligonucleotide probe- sets hybridising to RNA transcripts differentially expressed in neo- plastic colorectal specimens A custom design oligonucleotide microarray was designed and tested combining the results of multiple biomarker discovery projects. Introduction of a method to filter differentially expressed genes dur- ing discovery that may improve validation efficiencies of biomarker discovery based on gene expression measurements Differential expression discovery research is typically focused only on quantitative changes in transcript concentration between phenotype contrasts. This work introduces a method for generating hypotheses related to transcripts which may be quali- tatively “switched-on” between phenotypes. Identification of mRNA transcripts which are differentially expressed between colorectal adenomas and colorectal cancer tissues Transcripts differentially expressed between adenomatous and cancerous RNA extracts were discovered and then tested in independent tissues. In conclusion, these results confirm the hypothesis that gene expression profiling can discriminate colorectal neoplasia (including adenomas) from non-neoplastic controls. These results also establish a foundation for an ongoing biomarker development program.

Keywords: gene expression,colorectal cancer,neoplasia,microarrays,bioinformatics,discriminant analysis,biomarkers

Subject: Medicine thesis

Thesis type: Doctor of Philosophy
Completed: 2009
School: School of Medicine
Supervisor: Prof. Graeme Young