Case Summary: Colorectal Cancer
1) Overview a. Duke’s tumor stage correlates prognosis (Late stage worse prognosis) b. Often leads to hepatic metastasis
2) Adenoma-Carcinoma sequence a. Majority of colon cancer arise from polyps (adenomas) b. Accumulation of genetic mutations / events resulting in uncontrolled growth (dysplasia) c. Over time results in carcinoma 1. APC mutation on chromosome 5 (Early adenoma) 2. K-ras mutation on chromosome 12 3. TGF β, SMAD4 mutation (Late adenoma) 4. P53 mutation on chromosome 17 (Carcinoma) 5. Metastasis
d. Evidences 1. Foci of cancer are frequently seen within large polyps 2. Adenomatous (dysplastic) tissue is frequently seen adjacent to large cancers 3. Similar distribution of polyps and cancers within the colon 4. 10-15 year progression of polyp to cancer 5. Reduction of polyps causes reduction in subsequent incidence of cancer
3) Tumorgenesis a. Chromosomal instability
1. Gain of function – oncogenes
2. Loss of function – tumor suppressor genes (APC genes in FAP) b. Epigenetic alteration (microsatellite instability)
i. Altered methylation or defective mismatch repair genes resulting in erroneous DNA synthesis Increased mutation rate and microsatellite instability ii. Exemplified by germline mutations causing HNPCC /Lynch syndrome which is characterized by premature and proximal location of colon cancer c. Common pathways
1. Mutations in Ras pathway, Wnt pathway, loss of p53 tumor suppressor, and SMAD2/4 mutations i. Decreased exit or increased entry into cell cycle, and anti-apoptotic signaling Increased cell growth, decreased cell death: Cancer
4) HNPCC (Hereditary Non-Polyposis Colorectal Cancer): 2-3% of colorectal cancer a. Most common genetic cause of colorectal cancer (Right-sided) b. Clustering in relatives without established inheritance pattern c. Early onset: ~40yos d. Association with non-colon cancer (endometrial)
e. Caused by germline mutation in one allele of MMR (mismatch repair genes) resulting microsatellite instability and unregulated cell growth 1. 60% have defective hMSH2, hMSH1, or hMSH6
2. Disease requires inactivation of second allele by mutation, loss of heterozygosity, hypermethylation
3. When both alleles are mutated, there is a high rate of mutation (regulatory genes for apoptosis, cell cycle control) f. Microsatellite instability 1. Microsatellite DNA is short repeat sequence DNA found through the genome
2. High frequency of error of replication of microsatellite DNA in cells that are defect in mismatch repair
3. BAT 26 is a microsatellite region that is unstable in MMR cells. It is 26 bp A repeat, and it is NOT the cause of HNPCC 4. Can be amplified by PCR and analyzed by gel electrophoresis i. Isolate DNA from polyps ii. PCR amplify BAT 26 regions iii. Analyze PCR products (<26 observed in mutations)
g. Screening 1. Genetic counseling, genetic testing of suspected individuals i. MSH2, MLH1, MSH6 testing: 95% sensitivity
2. Polyp and tumor tissue in suspected individual can be tested directly for microsatellite instability
5) FAP (Familial Adenomatous Polyposis): <1% of colorectal cancer a. Autosomal dominant mutation in APC gene (chromosome 5) 1. Truncated APC gene product
Increased accumulation of nuclear protein β-catenin and increased oncogene expression via Wnt pathway b. Affect both sexes equally c. Clinical features 1. Appearance of polyps in childhood High incidence of cancer in early adulthood 2. Treated with total mucosal proctocolectomy 3. APC mutations are often associated with extra-colonic diseases /tumors
6) Colorectal cancer screening a. Most cancers originate from adenomatous polyps b. Cancer formation follows orderly “Adenoma-carcinoma” sequence c. Polyps can be easily detected and removed by colonoscopy / polypectomy d. Prevalence of colonic polyps increases with age (prevalence of colon cancer follows the same) e. Utilized methods 1. Colonoscopy with polpectomy 2. Virtual colonoscopy 3. Air contrast barium enema f. Genetic based screening for sporadic colorectal cancer
1. Stool DNA testing a. Stool Isolate DNA Analyze PCR products b. Problem i. Most fecal DNA is bacterial DNA ii. Most cells are healthy
2. PCR kit (k-ras, APC) a. Use primer that anneals to common variation of k-ras, APC mutaitons
3. Epigenetics (Vimentin gene) a. Detects aberrant DNA methylatoin b. Methylation often silences the genes i. Methylation of tumor suppressor genes silences gene expression ii. Methylation Vimentin gene Vimentin gene expressed c. Vimentin does not contribute to colon cancer, it is only used as a marker d. Rationale i. Cytosine is converted to uracil by bisulfate (HSO2) ii. However, 5 methyl-cytosine is resistant to HSO2 iii. Specific primer can be designed to detect 5 methyl-cytosine region in mutated Vimentin gene
4. Increasing sensitivity for APC mutation screening a. Done by increasing bad DNA: good DNA ratio i. Obtain stool sample from patient, and isolate DNA ii. Amplify exon 15 of APC genes using only a few DNA molecules as template iii. However, perform many separate reactions (144) for each patient iv. Carry out protein truncation test for each sample
7) Treatment of colon cancer a. CT to eliminate metastatic possibility b. Removal of tumor and pathologic assessment of associated lymph nodes for staging c. Advanced cancer: 5-fluorouracil / leukovorin based chemotherapy i. 5% survival at 5 years at each stage