1. Colorectal cancer (CRC) Professor Yaron Niv Rabin Medical Center Tel Aviv University

2. CRC n Colon anatomy n The problem n Clinical picture n Diagnosis n Staging and prognosis n Etiology/pathogenesis n Genetic pathways n Genetic applications

4. CRC – the problem n 150,000 new cases every year in USA n lifetime probability is 6% (death = 3%) n Dukes D 5-y-survival = 6-12% n Colorectal cancer kills 55,000 Americans every year n 3000 new cases every year in Israel, 1500 deaths

5. CRC – clinical presentation n Men:women = 1.2:1 n Sporadic CRC related to age, rare < 40y n Genetic syndromes – younger n Early stages – asymptomatic n Average risk vs. high risk populations n Symptoms/signs – advanced stages: Abdominal pain, rectal bleeding, weight loss, iron deficiency anemia (>R), change in bowel habits (>L)

6. CRC – diagnosis (sensitivity/specificity) n Physical examination n Rectal examination n Iron deficiency anemia n Elevated CEA n FOBT n BAE/virtual colonoscopy n CT/US n Colonoscopy/sigmoidoscopy

7. Dukes’ Staging Dukes 5-year-survival A 95% A 95% B 80-85% B 80-85% C 50-70% C 50-70% D 6-12% D 6-12%

8. TNM System - 1958 n T = tumor, N = nodes, M = metastasis M = metastasis Stage I = T1,N0,M0 Stage IV = T1,N1,M1 n Survival n Treatment n Clinical trials n Accurate communication n Uniform reporting Grade: Grade: Well, Moderately well, Poorly differentiated, Mucinous adenocarcinoma

9. Multi-step carcinogenesis proliferation, differentiation, apoptosis, angiogenesis Series of mutational events that release the cell from some level of growth restraint, followed by waves of clonal expansion of these cells. Series of mutational events that release the cell from some level of growth restraint, followed by waves of clonal expansion of these cells.

11. Vogelgram

12. Genetic definitions - colorectal cancer n Oncogenes n Tumor suppressor genes n DNA repair genes n Epigenetic processes

13. Oncogenes m Transformed proto-oncogenes m Signal trasduction and regulation of gene expression m SRC, RAS, MYC, FOS, JUN m Mutation increases the activity of the encoded proteins - proliferation m Dominantly acting m JCV, DNA virus encodes for T Ag, transform cells of GI tract transform cells of GI tract

14. Tumor suppressor genes l Act in normal tissues to restrain growth and maintain the differentiated phenotype. l Recessively acting: one copy undergoes an inactivating mutation (heterozygosity - silent), the second copy is deleted (loss of heterozygosity or LOH - no more protection). l Tumor specific l 5q(APC), 17p(P53), 18q(DCC,SMAD)

16. Gene silencing prevents translation and expression and may start tumorigenesis n Genetic: Mutation, deletion, insertion TSG (LOH, dominant - 2 hits) TSG (LOH, dominant - 2 hits) Oncogene (recessive – 1 hit) Oncogene (recessive – 1 hit) n Epigenetic: Promoter (CpG island) methylation leads to loss of imprinting (LOI) n When both alleles (maternal and paternal) are methylated, imprinting patterns are lost – carcinogenesis

17. DNA methylation in CRC n Activated by DNA-methyltransferase (uses S- adenosyl methionine) adds CH3 to Cytosine followed by Guanine (CpG island) n MLH1 hypermethylation is responsible for up to 80% of MSI tumors n Therapeutic potential: 1. Inhibition by 5-deoxy-azacytidine 1. Inhibition by 5-deoxy-azacytidine 2. MSI-H tumors are resistant to 2. MSI-H tumors are resistant to 5FU-based chemotherapy 5FU-based chemotherapy

18. Microsatellite instability (MSI – H) n Stretches of DNA – a short motif (1-5 nucleotides) is repeated several times n Mutation (germ-line or somatic) or hypermethylation (somatic, epigenetic) of DNA repair gene (MLH1) n Diagnosis: genotyping fluorescently labeled PCR products with the use of an automated sequencer n A panel of 5 microsatellite markers – MSI-H = 2 or more of them is a positive result

19. Genomic instability in CRC n 50% - Suppressor pathway - chromosomal instability (CIN, LOH) n 15% - Mutator pathway - microsatellite instability (MSI) microsatellite instability (MSI) n 35% - CpG island methylation phenotype of TSG (CIMP)

20. Colorectal cancer *Chromosomal instability (CIN) LOH, aneuploidy Predominantly Lt. sided Highly differentiated Little lymphocytic infiltration Rarely mucinous Worse prognosis 50%15% MSI-H Diploidy Predominantly Rt. Sided Poorly differentiated Lymphocytic infiltration Often mucinous Better prognosis HNPCCEpigenetic silencing of MLH1 5% 10% *CpG island methylator phenotype (CIMP) – 35% Serrated adenoma pathway Rt, women, old, BRAF+ 35%

21. Genetic applications n Diagnosis of genetic (germ line) syndromes n Diagnosis (screening) of sporadic CRC n Prognosis of sporadic CRC

22. Distribution of CRC (%)

23. FAP n Hundreds to thousands of adenomas at the age of 16, CRC risk = 100% (39y) n Gardner, AAPC, Turcot n Annual sigmoidoscopy start at age 10-12y

24. FAP, Extra-Colonic Cancers, Lifetime risk (%)

25. Hereditary NonPolyposis Colorcetal Cancer n Amsterdam II criteria: 3 1st degree relatives with CRC (female genitalia, other GI), 2 generations, 1 case < 50 y 1 case < 50 y n LOH of DNA mismatch repair genes n Microsatellite instability n Proximal colon, fast growing adenomas, better prognosis, relatively resistant to chemotherapy n CRC risk 80% n Colonoscopy every 2y, start age 25y or 10 years younger the earliest case

26. HNPCC, Extra-Colonic Cancers - Lifetime Risk (%)

27. Stool: DNA quantitation Loktinov, Clin Caner Res 98;4:337 n Normal cells - apoptosis - short DNA n Cancer cells - necrosis - long DNA n 17 CRC - 2133  407 ng/ml 28 healthy - 469  65 ng/ml 28 healthy - 469  65 ng/ml p=0.0005 p=0.0005

29. Tumor MSI status – predictor of benefit from 5- FU-based adjuvant chemotherapy for CRC Ribic, N Engl J Med 2003;349:247 n 570 patients – CRC tissue specimens MSI-H MSS MSI-H MSS N 95 475 5-y-s 78% 72% No adjuvant 88% 68% No adjuvant 88% 68% Adjuvant 71% 76% Adjuvant 71% 76%