Genetic Aberrations Associated With Metastasis in Colorectal Cancer: An Insight Into Tumour Heterogeneity

Al-Mulla, Fahd (1999) Genetic Aberrations Associated With Metastasis in Colorectal Cancer: An Insight Into Tumour Heterogeneity. PhD thesis, University of Glasgow.

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The purpose of this thesis was to obtain some insight into the role of genetic changes in progression of human colorectal cancer. Two approaches were adopted: (i), determination of frequency and nature of mutations in KA-ras and (ii), determination of the nature and extent of genetic aberrations, at chromosomal level, in advanced colorectal cancer. The main theme of the work was the use of paired tumour samples from the same patients in an attempt to measure the extent of genetic heterogeneity and clonal diversity between primary tumours and their synchronous metastases. For Ki-ras gene, I have determined the point mutations occurring in codons 12 and 13 of exon 1 of the Ki-ras gene in 78 patients with colorectal carcinoma (31 Dukes' A and B, 21 Dukes' C, 26 Dukes' D) by allele specific oligonucleotide hybridisation and sequencing. Duplicate samples of invasive primary carcinoma, adjacent normal tissue and available lymph node and liver metastases from the same patients were microdissected from paraffin sections. There were no differences in incidence of Kl-ras mutations between primary carcinomas and secondary deposits: 26 of 78 (33%) primary carcinomas, 10 of 32 (31%) lymph node metastases, and 10 of 26 (38%) liver metastases. Multiple sampling revealed frequent heterogeneity within carcinomas: 9 of 26 primaries with Ki-ras mutations also contained areas of carcinoma with only the wild-type gene, implying that Ki-ras mutation, even when present in a colonic carcinoma, may not have been necessary for establishing the malignant phenotype. Also, 2 of 26 (8%) Dukes' D patients had a mutation in their primary carcinoma but none in liver metastases and 6 of 47 (13%) Dukes' C and D patients had mutations in liver or lymph-node metastases but none in the primary carcinoma. Mutation of codon 12 from GGT (glycine) to GTT (valine) was more prevalent in primary and metastatic deposits of Dukes' C/D carcinomas (p=0.01) than in primary carcinomas from Dukes' A/B patients. Mutations of codon 12 to GAT, AGT, GCT and codon 13 GGC to GAC were also found, but no correlation with carcinoma aggressiveness was apparent. Follow-up of 71/78 patients (up to 12 years) revealed decreased overall survival (P=0.001) in patients with the GGT to GTT transversion in codon 12, even when the analysis was restricted to Dukes' D cases, supporting the suggestion that this mutation may confer a more aggressive phenotype in colorectal carcinoma. I then wanted to determine whether the correlation between valine-12 mutant Ki- RAS and tumour aggressiveness observed in vivo could be reflected in vitro. For this, Rat-1 fibroblasts transfected with valine-12 or aspartate-12 mutant or wild-type Ki-ras gene were assessed in terms of transformation, VEGF production and in vitro invasion. Both mutants demonstrated equal abilities to transform Rat-1 cells and induce VEGF production while cells expressing the wild-type Ki-Ras protein failed to do so. Clones of Rat-1 cells expressing valine-12 mutant Ki-Ras protein demonstrated increased ability to invade matrigel and to stimulate stromelysin-1 production compared to cells expressing aspartate-12 mutant Ki-Ras. A central question in the role of mutant Ras protein in tumour biology is why should one particular mutation be associated with aggressive behaviour while the other is not? To address this question, I have used molecular modelling on the wild type, valine-12 and aspartate-12 Ras molecules and shown there to be a significant conformational differences in the GTP-bound state between the two mutants involving the effector binding domain of Ras. These 'topographic' differences could be responsible for the different biological activities between the two mutants. It is arguable, based on both in vivo and in vitro work, that the biological effects of the two different Ki-ras mutations cannot be assumed to be the same and the use of valine-12 mutant Ki-ras as a clinical marker for worse prognosis should be considered.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: George D Birnie
Keywords: Oncology, Genetics
Date of Award: 1999
Depositing User: Enlighten Team
Unique ID: glathesis:1999-75493
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 19:39
Last Modified: 19 Nov 2019 19:39

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