Drye, ER; (2007) Sporadic colorectal cancers with microsatellite instability: A study of vascular endothelial growth factor. Doctoral thesis , UCL (University College London).

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Abstract

Microsatellite high (MSI-H) tumours carry a better prognosis than microsatellite low (MSI-L) or stable (MSS) tumours for the reasons that have yet to be fully determined. Prognosis in CRC is associated with the degree of invasion of the primary tumour through the bowel wall and its spread there after. Such processes are governed by the Vascular endothelial growth factor (VEGF) family of cytokines, and through angiogenesis (via VEGF and lymphangiogenesis (via VEGFc). Little is known however, of the relationships of VEGF and VEGFC in CRC with MSI, which is the aim of this thesis. DNA and total cellular RNA were extracted from wax-embedded samples of CRC. MSI status was determined from extracted DNA by SSCP, examining loci BAT25, BAT26, BAT40, D5S346, D2S123, and D17S250 unstable loci, >30-40% (MSI-H), <30-40% (MSI-L), 0% (MSS). From extracted RNA transcripts of VEGFA isoforms VEGFm, VEGF 165, and VEGFC were amplified by RT-PCR. The results were analysed by scanning densitometry, measured as total integrated optical density (IOD), and the levels of gene transcription determined by ratio to housekeeping gene, GAPDH-3. Angiogenesis was determined by examining blood vessel density using immunohistochemistry and antibodies to CD34. Lymphangiogenesis was determined immunohistochemically by VEGFc protein staining. Sixty seven sporadic CRC were analysed 35 males (median 70 (range, 38-100) years), 32 females (77(53-91)) years , and 8 normal colon samples 5 males (66 (57-82) years), 3 females (71 (45-87) years . MSI-H CRC were more in females (83%, p<0.02) and in the right colon (75%, p<0.04). VEGF 121 transcription was elevated in all cancers compared to normal colon and was highest in MSI-L tumours (MSI-L 0.679, MSS 0.538, MSI-H 0.409, Normal 0.331, p<0.01). VEGF,65 transcription was reduced in all cancers compared to normal colon (MSS 0.491, MSI-L 0.428, MSI-H 0.413, Normal 0.592, p>0.05) and there was no correlation with MSI (p>0.05). There was a weak inverse correlation between VEGFm and Duke's stage (A 0.713, B 0.570, C 0.510, D 0.451, p<0.09) but there was no correlation with either VEGF165 or VEGFc gene transcription. However, there was elevated levels of VEGFc protein expression in Duke's C and D tumours (A 59.6%, B 78.4%, C 80.0%, D 81.0%, p<0.07). VEGFm and VEGFc were elevated in MSI-L tumours (with a single dinucleotide marker mutated) compared to MSI-H tumours (VEGF!2i - MSI-L 0.679, MSI-H 0.409, VEGFC - MSI-L 0.542, MSI-H 0.471, p<0.05) and there was an inverse correlation of blood vessel density to MSI status (MSS 12.5, MSI-L 11.1, MSI-H 10.1, p<0.09). Overall, this thesis demonstrates that wax embedded samples are a useful resource of CRC to investigate both gene transcription and protein expression and helps to conclude that MSI-H tumours have reduced angiogenic/lymphogenic potential whilst transcription of VEGF121 may be important in the early growth and spread of CRC. Elevated VEGF121 and VEGFC transcription with a singular di-nucleotide mutation supports the theory of a distinct MSI-L subgroup.

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