CLINICAL SIGNIFICANCE OF CANCER STEM CELL MARKERS AND OTHER RELATED PROTEINS IN COLORECTAL CARCINOMAS – BIOLOGICAL AND METHODOLOGICAL CONSIDERATIONS ONG CHEE WEE B Sc (Hons), NUS A THESIS SUBMITTED FOR THE DEGREE OF MASTERS OF SCIENCE DEPARTMENT OF PATHOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2010 ACKNOWLEDGEMENTS The work of this thesis was carried out at the Cancer Science Institute of Singapore, with support from the Department of Pathology, National University of Singapore I am grateful to the following people who, in different ways, have contributed to this work: Associate Prof Manuel Salto-Tellez, my supervisor, for his patience, guidance and constant encouragement Professor Barry Iacopetta and Associate Professor Richie Soong, for their helpful critiques on my manuscripts and collaboration in this work Professor Chia Kee Seng, for giving me the opportunity to embark on this graduate study Ms Sandy Kim and Ms Maggie Cheung, for their invaluable help in microarrays construction and collation of clinical data Friends and colleagues at the Cancer Science Institute of Singapore and the Centre for Molecular Epidemiology Lastly, this thesis would not be possible without my wife, Lyn i TABLE OF CONTENTS ACKNOWLEDGMENTS i SUMMARY v LIST OF PUBLICATIONS viii LIST OF TABLES vi LIST OF FIGURES x CHAPTER 1: INTRODUCTION 1.1 Incidence and Survival Rates of Colorectal Cancer 1.2 Hallmarks of Cancer Development 1.3 Molecular Mechanisms of Colorectal Cancer Development 1.3.1 The Chromosomal Instability (CIN) Pathway 1.3.2 The CpG Island Methylator Phenotype (CIMP) Pathway 1.3.3 The Microsatellites Instability (MSI) Pathway 5 10 1.4 Implications of Cancer Stem Cells 11 1.5 The Putative Prognostic and Predictive Markers Examined in this Study 1.5.1 p53 1.5.2 Cyclooxygenase (COX-2) 1.5.3 p27 1.5.4 CD133 1.5.5 SOX-2 1.5.6 OCT-4 14 17 18 19 21 22 23 1.6 Advanced Histopathological Techniques Employed in this Study 1.6.1 Archival Pathological Specimens and Tissue Microarray Techniques 1.6.2 Automated Imaging and Pathological Scoring Platform 24 25 26 Aims of Study 28 1.7 ii CHAPTER 2: MATERIALS AND METHODS 2.1 2.2 Materials 2.1.1 Samples for Examining Availability of DNA and Immunohistochemical Antigenic Sites from Archival Formalin-Fixed Paraffin-Embedded Tissues 2.1.2 Samples for Studying Prognostic and Predictive Significance of Cancer Stem-Cell Markers 2.1.3 Samples for Studying the Implications of Cancer Stem-Cell Markers with KRAS, BRAF and Microsatellite Instability 2.1.4 Approval from Ethic’s Committee Methods 2.2.1 Tissue Microarray Construction 2.2.2 DNA Extraction 2.2.3 Assessment for Availability of DNA for Polymerase Chain Reactions 2.2.4 KRAS Mutational Analysis 2.2.5 BRAF Mutational Analysis 2.2.6 Microsatellite Instability Analysis 2.2.7 Immunohistochemistry 2.2.8 Pathological Scoring 2.2.8.1 Human Observer Scoring Criterion 2.2.8.2 Automated Computer Scoring Criterion 2.2.9 Statistical analysis 31 31 32 34 34 35 35 36 38 40 41 41 42 44 44 45 46 CHAPTER 3: RESULTS 3.1 3.2 Methodological Considerations 3.1.1 Comparisons between Different Methods of DNA Extraction 3.1.2 Availability of DNA and Antigenic Sites from Formalin-Fixed Paraffin-Embedded Tissues 3.1.3 Concordance between Observer and Automated Scoring 3.1.4 Time Required for Generating Pathological Scoring and Reproducibility of Results Biological Considerations 3.2.1 Protein Expression of Cancer Stem-Cell Markers 3.2.2 Selection of Protein Markers with Discriminatory Power for Prognostic and Predictive Significance 3.2.3 Associations between Markers’ Expression and Clinicopathological Features 3.2.4 Prognostic Significance of Protein Expression 3.2.5 Predictive Significance of Protein Expression 3.2.6 KRAS Mutational Analysis 3.2.7 BRAF Mutational Analysis 3.2.8 Microsatellite Instability Analysis 48 48 50 54 57 60 60 62 62 64 67 70 71 73 iii CHAPTER 4: DISCUSSION 4.1 4.2 4.3 4.4 4.5 REFERENCES Availability of DNA and Antigenic Sites from Formalin-Fixed Paraffin-Embedded Tissues Concordance between Observer and Automated Scoring Prognostic and Predictive Significance of Cancer Stem Cells in Colorectal Cancer Associations of Cancer Stem Cell Markers with KRAS, BRAF and Microsatellite Instability Conclusion 74 76 79 82 85 87 iv SUMMARY 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 stemcell markers and its related proteins in colorectal cancers were investigated Tissue microarray analysis were combined with an automated image scanning v 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 vi 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 highthroughput 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 vii LIST OF PUBLICATIONS The thesis is based on the following publications, which will be referred to in the text by their Roman numerals: I Das K, Mohd Omar MF, Ong CW, Abdul Rashid SB, Peh BK, Putti TC, Tan PH, Chia KS, Teh M, Shan N, Soong R, Salto-Tellez M TRARESA: a tissue microarray-based hospital system for biomarker validation and discovery Pathology 2008; 40(5): 441-9 II Ong CW, Kim LG, Kong HH, Low LY, Wang TT, Supriya S, Kathiresan M, Soong R, Salto-Tellez M Computer-assisted pathological immunohistochemistry scoring is more time-effective than conventional scoring, but provides no analytical advantage Histopathology 2010; 56(4):523-9 (Impact Factor: 4.13) III Ong CW, Kim LG, Kong HH, Low LY, Iacopetta B, Soong R, SaltoTellez M CD133 expression predicts for non-response to chemotherapy in colorectal cancer Mod Pathol 2010; 23(3): 450-7 (Impact Factor: 4.41) viii LIST OF TABLES Table TNM classification according to the 7th edition of the American Journal of Colorectal Cancer (AJCC) Staging Manual Table Five-year overall survival rate according to AJCC staging Table Clinicopathological features of the patient cohort 33 Table Primer sequences for producing different sized PCR products of β-actin 39 Table Optimized concentrations and manufacturers for antibodies used 39 Table Comparison of the three protocols for quantity and quality of extracted DNA 49 Level of agreement between human visual and computerassisted machine scoring methods 55 Nature of disagreement between human visual and computerassisted scoring methods for non-matching cases 55 Comparison of immunohistochemical markers expression in relation to clinical pathological features 56 Univariate analysis for survival in relation to expression of immunohistochemistry markers 58 Extent of disagreement in reproducibility of results between human visual and computer-assisted scoring methods 58 Frequency of positive expression of protein markers using cut-off scores derived from the area under the receiver operating characteristic curve 63 Correlation matrix showing relationships between the expressions of each protein marker 63 Associations between expression of cancer stem cell markers and clinicopathological features 65 Univariate disease-specific survival analysis for clinicopathological features and protein markers 66 Table Table Table Table 10 Table 11 Table 12 Table 13 Table 14 Table 15 Table 16 Multivariate (adjusted) analysis for disease-specific survival according to clinicopathological features and protein expression 68 Table 17 Associations between cancer stem cells marker expression and clinicopathological features 72 ix 4.5 Conclusion and Future Directions In conclusion, the findings from this work indicated that amplifiable DNA was extractable from formalin-fixed paraffin-embedded tissue blocks as old as 50 years of age These DNA need not be fully intact to be amplification as contaminations during the extraction process are minimized A high level of agreement between automated pathological scoring and manual observer scoring were demonstrated in this thesis A fully automated computer-assisted scoring system, although did not provide any analytical advantage, is more time-effective and is suited for high-throughput biomarker analyses The main finding of this study is that CD133 expression was found to possess predictive significance of chemoresistance in colorectal cancer patients In addition, independent prognostic significance of p27 expression as well as CD133 and OCT-4 were observed with the latter also unreported to date In our study, KRAS mutations did not yield any predictive advantage to treatment response for 5-FU treatment, suggesting that different markers are required for predicting response to different therapeutic agents CD133 is indeed a better marker suited for predicting response for 5-FU treated cases 91 The poor prognosis associated with BRAF mutated tumours in this study indicated that current therapies might not be specifically targeting mutated BRAF Cancer stem cell markers are not limited to those that are outlined in this thesis, future studies can work on various to other cancer stem cell markers, such as Lgr5 or CD44, to further improve the 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112(3): 495-502 105 ... both biological and methodological aspects of our hypothesis in determining clinical significance of cancer stem- cell marks in colorectal cancer Our main objective is to understand the clinical. .. predictive effect of cancer stem cell markers in colorectal cancers (ii) examine the associations of cancer stem cell markers with KRAS and BRAF mutations as well as microsatellite instability In order... haemotoxylin and eosin stained slide 2.1.2 Samples for Studying Prognostic and Predictive Significance of Cancer Stem- Cell Markers For the study of the prognostic and predictive significance of the cancer