An investigation of metastatic colorectal cancer

Colorectal cancer (CRC) is the third most common cancer worldwide and the fourth most common cause of death. This complex disease process starts in the colon or rectum, as a non-cancerous polyp which can become malignant over time. In the later stages of CRC, tumour cells become detached from the primary tumour, migrate and enter the blood or lymphatic vessels and ultimately form a secondary tumour at another site (distant metastasis). CRC metastasis may also arise from residual tumour cells that persist after treatment. Identifying biomarkers for potentially metastatic disease or residual disease may provide novel tools for early detection and therapy monitoring patients prior to metastasis and tumour recurrence. Genetic and epigenetic changes are required during every step in metastatic spread and these may have use as biomarkers as well as providing information about the mechanisms behind these changes. In addition, tumour cells release cellular contents into the bloodstream as a consequence of cell death during the metastatic process. These circulating free (cf) contents have potential to be cancer biomarkers for treatment monitoring and residual diseases detection. This thesis investigated the molecular events associated with metastatic CRCs in order to improve diagnosis and prognosis in this disease. DNA was extracted from 82 cases of formalin-fixed paraffin-embedded (FFPE) human CRCs. Mutation and methylation analysis was performed by QMC-PCR followed by high resolution melting (HRM) analysis. Using statistical methods, we analysed the association between the targeted mutations and lymph node involvement, local recurrence, and distant metastasis. The findings showed significant association of KRAS, PIK3CA, PTEN, SMAD4 mutations and P16 promoter methylation with lymph node involvement, advanced disease and local/metastatic recurrences. The study also confirmed that CRCs with microsatellite instability (MSI) were significantly associated with mutant BRAF. MSI occurs in sporadic tumours and tumours arising in Lynch Syndrome and BRAF is commonly found in sporadic tumours but almost never in Lynch Syndrome tumours. However not all sporadic tumours with MSI have mutation in BRAF and therefore a new assay was developed to discriminate sporadic tumours with MSI from tumours arising due to Lynch Syndrome. In order to develop tests to test for residual disease, blood samples of 25 CRC patients were collected pre-operation and daily post-operation (until discharge) and plasma was extracted for the analysis of cfDNA/ctmiRNA following operation. The matched primary tumours were also collected. A protocol for COLD-HRM (a combination of COLD-PCR and HRM designed for detection of low frequency of mutant alleles) was optimized to screen for KRAS and BRAF mutation. This protocol was subsequently used to screen cfDNA for mutations. ctmiRNA expression was quantified by Q-PCR. Findings in this study showed that patients can be divided into a group which either loses or retains mutant cfDNA/ctmiRNA following operation. Detection of mutations in cfDNA is a good means of non-invasive screening for CRC and may provide a novel method of assessing surgical clearance and testing for recurrence. The activation of GNAS1 by mutation leads to several biological possibly metastasis promoting events including cell proliferation, survival and motility. GNAS1 was found to be mutated in CRCs and therefore investigated for its activity in CRC cell lines. GNAS1 was knocked-down in two CRC cell lines (RKO, and SW620). Gene knockdown was undertaken by transfecting small interfering RNAs into the cells and this was followed by an evaluation of cell proliferation and motility. The findings of this study revealed that inhibition of GNAS1 expression does not show any effects on cell proliferation or migration in the CRC cell lines, RKO and SW620. In conclusion, this study identified specific targets, such as KRAS, PIK3CA, PTEN, SMAD4 mutations and P16 promoter methylation, in correlation with lymph node involvement, advance stage CRCs and local/distant recurrences. Further analysis and investigation for their functional role in CRC progression is required to further identify their exact impact on CRC cell proliferation and motility. This study also confirmed that cfDNA/cfmiRNA is detectable in plasma of CRC patients and may provide potential biomarker for surgical clearance and residual disease. In addition, it was shown that GNAS1 knock-down did not increase both cell proliferation and migration in the CRC cell lines, RKO and SW620. However, further validation for these findings may enhance the understanding of these molecular markers in invasion-metastatic transformation in CRC