Molecular Classification of Colorectal Cancer

...................................................................................................................... 9 REVIEW OF THE LITERATURE .............................................................................. 11 1. GENES AND CANCER...........................................................................................................................11 1.1 Oncogenes............................................................................................................................................12 1.2 Tumor suppressor genes ......................................................................................................................13 1.2.1 Gatekeepers.................................................................................................................................13 1.2.2 Caretakers....................................................................................................................................13 1.2.3 Landscapers..................................................................................................................................13 1.3 Epigenetic mechanisms .......................................................................................................................14 1.3.1 Methylation ..................................................................................................................................14 1.3.2 Loss of imprinting (LOI) .............................................................................................................14 2. COLORECTAL CANCER......................................................................................................................15 2.1 Non-polyposis syndromes ...................................................................................................................15 2.2 Polyposis syndromes ...........................................................................................................................17 2.2.1 Adenomatous polyposis syndromes.............................................................................................17 2.2.2 Hamartomatous polyposis syndromes .........................................................................................18 2.3 Low-penetrance alleles predisposing to colorectal cancer ..................................................................19 3. PATHWAYS LEADING TO COLORECTAL CANCER...................................................................19 3.1 Suppressor pathway (The adenoma-carcinoma sequence)..................................................................19 3.2 Mutator pathway ..................................................................................................................................22 3.3 Serrated neoplasia pathway .................................................................................................................23 4. MICROSATELLITE INSTABILITY....................................................................................................24 4.1 Mismatch repair system.......................................................................................................................26 4.1.1 Defects in mismatch repair system ..............................................................................................28 AIMS OF THE STUDY ................................................................................................. 30 MATERIALS AND METHODS ................................................................................... 31 1. PATIENT SAMPLES (I, II, III, IV) .......................................................................................................31 1.1 DNA (I, II, III, IV) and RNA (IV) extraction......................................................................................31 2. MSI ANALYSIS (I, II) .............................................................................................................................31 3. IMMUNOHISTOCHEMISTRY AND MUTATION ANALYSIS OF MLH1, MSH2 AND MSH6 (I) ........................................................................................................................................................................32 4. LOCUS SPECIFIC LOH ANALYSIS (II).............................................................................................32 5. MUTATION ANALYSIS OF KRAS (II)................................................................................................34 6. MGMT, MLH1, AND EPHB2 PROMOTER HYPERMETHYLATION ANALYSIS (II, IV).........34 6.1 Bisulfite modification ..........................................................................................................................34 6.2 Methylation specific PCR....................................................................................................................34 7. TOPOGRAPHIC MUTATION HETEROGENEITY ANALYSIS (II)..............................................35 8. ALLELIC IMBALANCE (AI) ANALYSIS (III) ..................................................................................35 9. COMPARATIVE GENOMIC HYBRIDIZATION (CGH) (III).........................................................36 10. REAL–TIME QUANTITATIVE PCR.................................................................................................36 11. STATISTICAL ANALYSES (II, III) ...................................................................................................36