Clinical significance of cancer stem cell markers and other related proteins in colorectal carcinomas - Biological and methodological considerations

Abstract

Colorectal cancer remains one of the leading causes of cancer-related deaths in Singapore. The colorectal cancer is a heterogeneous disease. There are at least three major molecular pathways to colorectal cancer development. These include the predominant chromosomal instability (CIN) pathway, which accounts for up to 85% of cases. Secondly, there is the CpG island methylator phenotype (CIMP) pathway that is the other major pathway to sporadic colorectal cancers. Finally, there is the pure MSI pathway which results from germline mutation of a DNA mismatch repair (MMR) gene. Hereditary nonpolyposis colorectal cancer (HNPCC) develops via the pure MSI pathway.

In the last few years, there has been a growing hypothesis that human cancer should be considered as an alternative form of stem cell disease. This concept states that tumours are not to be viewed as simple monoclonal expansions of transformed cells - but rather as complex tissues where abnormal growth is driven by a minority of cancer stem cells. These cancer stem cells possess tumour-related features such as uncontrolled growth and the ability to form metastases. They also maintain their inherent stem cell capacity to self-renew and differentiate.

In this thesis, the molecular and clinical significance of cancer stem-cell markers and its related proteins in colorectal cancers were investigated. Tissue microarray analysis were combined with an automated image scanning and analysis platform to examine the immunohistochemical expression of a panel of nine markers, of which three are cancer stem cell related proteins. In addition, BRAF and KRAS mutation analysis and microsatellite instability testing were performed to explore the possibility of any implications between cancer stem cells and the pathways responsible for colorectal cancer development. Lastly, possible relations to therapeutic responses were also examined.

In this thesis, the findings indicated that expression of CD133, a putative cancer stem cell protein marker, possesses predictive significance of chemoresistance in colorectal cancer tumours. Independent prognostic significance in p27, as well as cancer stem cell related proteins CD133 and OCT-4 were observed. In addition, no correlations between cancer stem cells proteins and BRAF or KRAS mutations. To achieve these biological observations with clinical implications, several steps were taken, namely a) a hospital-based model for translational research making use of amplifiable DNA from formalin-fixed paraffin-embedded tissue blocks archived as early as 50 years ago; and b) a reliable method for the use of computer assisted IHC scoring which achieved a high level of concordance with manual human observer scoring.

In summary, the work of this thesis presents, for the first time, the predictive significance of a cancer stem cell marker and its lack of correlation with BRAF, KRAS or microsatellite instability genotypes. By doing so, it allowed the setup and validation of a comprehensive platform to expand the possibilities for molecular pathologic studies in large cohort-centric epidemiological research. The results of this thesis provide a basis for high-throughput standardization of immunohistochemical markers, which would enable the identification, or validation of tissue-based biomarkers. This is a valuable tool in determining prognosis and prediction of treatment responses in Asian colorectal cancer patients for future studies in the local institutions.