Molecular and cytogenetic analysis of cervical and vulvar cancer

Transcription

1 Title Molecular and cytogenetic analysis of cervical and vulvar cancer Advisor(s) Ngan, HYS Author(s) Huang, Fung-yu.; 黃鳳如 Citation Huang, F. [ 黃鳳如 ]. (2002). Molecular and cytogenetic analysis of cervical and vulvar cancer. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from Issued Date 2002 URL Rights The author retains all proprietary rights, (such as patent rights) and the right to use in future works.

2 CONTENTS Abstract....ii Declaration....v Acknowledgments vi List of Figures vii List of Tables. viii CHAPTER 1. INTRODUCTION General introduction Aims of study 3 CHAPTER 2. LITERATURE REVIEW Squamous cell carcinoma of the cervix Introduction Classification and staging of cervical squamous cell carcinoma Etiologic factors of cervical squamous cell carcinoma Association of virus Behavioral factors Environmental factors Squamous cell carcinoma of the vulva Introduction Histopathology of vulvar squamous cell carcinoma Etiologic factors of squamous cell carcinoma of vulva Viral factors Behavioral factors Environmental factors Genetic alterations in cancers..11

3 2.3.1 Importance of detecting genetic alternations in cancer Comparative genomic hybridization, CGH Cytogenetic alterations in cervical cancer and vulvar cancer LOH study on cervical and vulvar cancers Introduction LOH in squamous cell carcinoma of the cervix LOH in squamous cell carcinoma of the vulva Tumor suppressor genes in cervical and vulva cancer Oncogenes in cervical and vulvar squamous cell carcinoma Importance of analyzing candidate tumor associated gene..23 CHAPTER 3. MATERIALS AND METHODS Introduction Sample collection Collection of cervical and vulva tissue samples Collection of cell lines Collection of normal amniotic cells and lymphocytes Cell culture Extraction and Purification of high molecular weight DNA Materials Procedure of DNA extraction from cultured cells, lymphocytes and frozen tissue Comparative Genomic Hybridization Materials Introduction of comparative genomic hybridization Preparation of the probes and metaphase spread....29

4 Preparation of the probes Preparation of the metaphase spread from amniotic cells Preparation of APES-coated slides Suspension harvest for amniotic culture Pretreatment of the metaphase spreads Pre-hybridization and hybridization for CGH Post-hybridization washing Image acquisition and analysis CGH controls Polymerase Chain Reaction (PCR) Materials General description of PCR Human papillomavirus detection Study of DNA amplification on genes selected from chromosome 5p and 1q Selection of genes from chromosome 5p and 1q Fluorescent differential PCR Optimization of conditions for quantitative PCR Multiples fluorescent differential PCR amplification Electrophoresis and analysis of PCR products Large-scale study of the genes in 5p for finding potential DNA markers Extraction of total RNA from cultured cells and tissues Reverse Transcriptase PCR (RT-PCR) First-strand cdna synthesis Semi-quantitative RT-PCR Statistical analysis....52

5 CHAPTER 4. RESULTS Detection of HPV DNA in cervical and vulvar cancer Comparative genomic hybridization Fluorescent differential PCR studies Large Scale analysis of gene amplification in chromosome 5p and clinical outcomes and patient survival Analysis of gene expression by semi-quantitative RT-PCR.. 94 CHAPTER 5. DISCUSSION HPV genotyping detection in cervical and vulvar cancers Genetic imbalances detected in the tumors Genetic imbalances detected in the tumors Summary for chromosomal gains Losses of chromosomal materials Summary of losses of genetic materials Summary of CGH analysis DNA amplification status and expression of genes in chromosome DNA amplification status of genes in chromosome 1q Association of amplified genes and cervical cancer Summary of gene amplification study References

6 List of Figures Figure 2.1. Diagram showing working principle of comparative genomic hybridization (CGH) 14 Figure 3.1. Diagram showing examples of gel electrophoresis in testing the integrity of genomic DNA...37 Figure 3.2. Gel-electrophoresis pattern of nicked translated probes..37 Figure 3.3. An example of digital images acquired for each chromosome spread...38 Figure 3.4. An example of CGH ratio profile interpretation Figure 3.5. Average fluorescence ratio profile of breast cell line MPE Figure 3.6. Average fluorescence ratio profile from the same normal individual 40 Figure 3.7. Average fluorescence ratio profile from two normal individuals..40 Figure 3.8. Testing of optimum amplification cycle for the amplification of genes at an exponential phase Figure PCR amplification of HPV DNA by consensus primers Figure PCR amplification of HPV Figure PCR amplification of HPV Figure PCR amplification of beta-actin gene. 55 Figure Average CGH profile of cervical cancer cases

7 Figure Average CGH profile of 5 cervical cell lines 78 Figure Average CGH profile of vulvar cases Figure Average CGH profile of 2 vulva cell line Figure Representative CGH profiles and ideograms of chromosomes showing DNA copy number changes occurred in cervical tissues 86 Figure Electrophoretogram of fluorescent differential PCR showing the calculation on the amplification of the genes 89 List of Tables Table 2.1. Clinical staging system of carcinoma of the cervix according to FIGO system.. 5 Table 3.2. Primer sequence for PCR amplification Table 3.3. Candidate tumor associated gene for fluorescent semi-quantitative differential PCR. 46 Table CGH findings of the 5 cervical cancer cell lines Table CGH findings of 28 cervical tissue samples.. 60 Table Patient data and CGH findings of the 8 vulvar tissue samples and 2 vulvar cell lines 62

8 Table Summary of common gains and losses found in all the samples studied by comparative genomic hybridization 63 Table Gene amplification in samples with 5p amplification 90 Table Gene amplification in samples with 1q amplification 90 Table Clinical characteristics of 128 patients with cervical cancer 91 Table Spearman rho analysis of correlations of gene markers...92 Table Relationship between clinical features and gene amplification 93 Table Univariate Cox regression analysis of gene expression...93 Table Forward stepwise Cox analysis of patient survival Table RT-PCR analysis of gene expression 95