Genetics of Colorectal Cancer Peter Lee MD Central Ohio Colon & Rectal Center

Overview Molecular biology of cancer Epidemiology of colorectal cancer Inherited colorectal cancers Screening implications of colorectal cancer Summary

Cancer is a disease of the cell cycle Cells either grow and divide with restraint ...or not! The many kinds of malignant growth that the term "cancer" represents all have one lethal attribute in common: The cells of the malignancy go through the cell cycle without restraint. These cells "defy" the control mechanisms that lie with them. What Is the Connection Among Cancer, the Cell Cycle, and Genetics? There are many protein molecules involved in the cell cycle, each is the product of a single gene. When there is a mutation in one of these genes, it can: increase the likelihood that a cell will become cancerous and eventually, through repeated, unrestrained division, overtake the normal cells, become malignant; and possibly spread, or metastasise throughout the body. Cancer can develop at almost any stage in life. There are some forms of cancer that develop very early, such as retinoblastoma (a cancer of the eye); others tend to develop in childhood, such as various forms of leukaemia, a cancer of the blood; and, of course, there are many forms that develop during adulthood. In each case, cancer is the result of a mutated gene, or a series of mutated genes, that lead to unregulated cell growth and haphazard controls over cell proliferation. “Carcinoma is a genetic disease but it is not necessarily inherited”

Knudsen’s “two hit” hypothesis

Types of genes which may mutate to cause cancer: Oncogenes Tumor suppressor genes DNA repair genes p53

Oncogenes Cellular oncogene c-onc Viral oncogene v-onc Proto-oncogene, activated by mutation to c-onc

Proto-oncogene activation

Tumour suppressor genes The gene’s normal function is to regulate cell division. Both alleles need to be mutated or removed in order to lose the gene activity. The first mutation may be inherited or somatic. The second mutation will often be a gross event leading to loss of heterozygosity in the surrounding area.

Types of proto-oncogene Growth factor e.g. SIS oncogene (PDGF) G proteins e.g. ras Nuclear transcription factors e.g. MYC

p53 Suppress progression through the cell cycle in response to DNA damage Initiate apoptosis if the damage to the cell is severe Is a transcription factor and once activated, it represses transcription of one set of genes (several of which are involved in stimulating cell growth) while stimulating expression of other genes involved in cell cycle control

Transformation is a multistep process

Transformation is a multistep process

DNA Mismatches Damage to nucleotides in ds-DNA Misincorporation of nucleotide Missed or added nucleotides

Acquired DNA Damage M -C-A- -T-A- -G-T- -G-T- Demethylation

Nucleotide Misincorporation -C-A-G-C-T- -G-T-C-C-A- CT substitution -C-A-G-C-T- -G-T-T-C-A- -G-T-C-C-A- -C-A-G-C-T- -G-T-C-C-A- correctly copied

Added Nucleotides -C-A-G-C-T- -G-T-C-C-A- -C-A-G-C-T- -G-T-C C-A- correctly copied nucleotide added -C-A-G-C-T- -G-T-C-C-A-

Human Mismatch Repair Genes MLH1 (3p21) PMS1 (2q31-33) PMS2 (7p22) MSH2 (2p16) MSH3 (5q3) MSH6 (2p16) (=GT Binding Protein)

Mismatch Repair Genes Recognition and repair of mismatches Other functions Repair of branched DNA structures Prevent recombination of divergent sequences Direct non-MMR proteins in nucleotide excision and other forms of DNA repair

Other MMR Proteins DNA ligase Replication protein A Replication factor C Proliferating Cell Nuclear Antigen Exonucleases DNA polymerase 

Defective Mismatch Repair Defects in MMR Genes and Function Microsatellite Instability Cancer development 90% of HNPCC colorectal cancers 20% of sporadic colorectal cancers 30% of sporadic uterine cancers

Cancer Development Activation of Oncogenes Inactivation of Tumour Suppressor Genes Defects in DNA mismatch repair Susceptible to mutation

Genetics of Colorectal Cancer

Colorectal Cancer 11% of cancer-related deaths Tumor progression may take 10-35 years Adenomatous polyp develops into carcinoma

Chromosome changes in colorectal cancer Cancer karyotype Stable karyotype

Worldwide Statistics for Colorectal Cancer (CRC) 875,000 cases in 1996 8.5% of all new cases of cancer 556,000 deaths Incidence rates vary (Up to 20 fold) Highest in North America, Western Europe, Australia, New Zealand, Japan Lowest in India, Northern Africa

Estimated New Cancer Cases of 10 Leading Sites by Gender for the US 2000

Colorectal Cancer Statistics in the US Second overall leading cause of cancer-related deaths in the US Estimated 130,000 new cases and 56,300 deaths in the year 2000 Declining trends between 1990 and 1996 Incidence rate: 2.1% per year Mortality rates: 1.7% per year

Average Annual Age-Specific US Incidence and Mortality Rates of CRC, 1992-1996

Prevalence of adenomas Age ≥ 50 with any adenomas: 25-40% Lifetime risk of cancer at age 50 5% for females 6% for males Advanced adenomas at highest risk

Risk Factors for Colorectal Cancer (CRC) Aging Personal history of CRC or adenomas High-fat, low-fiber diet Inflammatory bowel disease Family history of CRC Hereditary colon cancer syndromes 2

Risk of Colorectal Cancer (CRC) General population 5% Personal history of colorectal neoplasia 15%–20% Inflammatory bowel disease 15%–40% 70%–80% HNPCC mutation >95% FAP 20 40 60 80 100 Lifetime risk (%) 3

Familial Risk for CRC Lifetime CRC risk (%) 70% 17% 10% 6% 8% 2% None One 1° and two 2° One 1° age <45 Two 1° HNPCC mutation Aarnio M et al. Int J Cancer 64:430, 1995 Houlston RS et al. Br Med J 301:366, 1990 St John DJ et al. Ann Intern Med 118:785, 1993

Inherited Colorectal Cancers

Heredity of Colorectal Cancer 5-8% of all cases of CRC are hereditary 15-20% are “familial” / multifactorial 75% of cases are sporadic ~75% of cases of CRC are sporadic only 7% of sporadic cases occur <55 ~15-20% are “familial” / multifactorial – genetic testing not generally available for low penetrance genes associated with increased risk of CRC ~5-8% are hereditary (defined cancer susceptibility syndromes caused by single genes) Feuer EJ: DEVCAN: National CA Inst. 1999

Causes of Hereditary Susceptibility to CRC Sporadic (65%–85%) Familial (10%–30%) Rare CRC syndromes (<0.1%) Hereditary nonpolyposis colorectal cancer (HNPCC) (5%) Familial adenomatous polyposis (FAP) (1%) Adapted from Burt RW et al. Prevention and Early Detection of CRC, 1996

Features of Familial CRC Family history of CRC with no clear inheritance pattern Age at onset typical of sporadic CRC Multiple causes Few or no adenomas Sporadic Familial CRC FAP HNPCC Rare CRC syndromes

Progression of Colorectal Cancer Loss of APC Activation of K-ras Deletion of 18q Loss of TP53 Other alterations Normal epithelium Hyper- proliferative epithelium Early adenoma Inter- mediate adenoma Late adenoma Carcinoma Metastasis Adapted from Fearon ER. Cell 61:759, 1990

Adenomatous polyp Adenomatous polyp Can take 5-10 years for polyp to develop Up to 10% of polyps develop into cancer Size and histology are risk factors for polyp to cancer progression

Characteristics of Average Risk No well-defined threshold between sporadic and familial CRC at this time Probably safe to include individuals with: No personal risk factors or family history of CRC One 2nd or 3rd degree relative with CRC >60 years with no other family history of CRC

Characteristics of Familial CRC “Clustering” of colon cancer cases in the family (age> 50 at diagnosis) without clear dominant pattern One close relative with CRC <60 yrs & family history does not meet criteria for known hereditary CRC syndromes Likely to be multiple low penetrant genes plus environmental factors Family members warrant earlier CRC screening Starting at 40 years or 5-10 yrs earlier than age of diagnosis of the youngest affected relative Winawer et al., Gastroenterology 2003:124:544-560

Characteristics of Hereditary CRC Multiple relatives with colorectal cancer One or more diagnosed at an early age (<50) Sequential generations affected Except in autosomal recessive syndromes Other cancers in the family known to be associated with CRC (uterine, ovarian, GI) Multiple primary tumors or polyps

Hereditary CRC syndromes Hereditary Non-Polyposis Colorectal Cancer (HNPCC) Variants: Muir-Torre, Turcot Familial Adenomatous Polyposis (FAP) Variants: Gardner, Turcot Attenuated FAP APC mutation in Ashkenazi Jews Others: Multiple adenomatous polyposis syndrome/MYH gene (MAP) Peutz-Jeghers syndrome (PJS) Familial Juvenile Polyposis (FJP)

HNPCC: AKA “Lynch syndrome” 2-3% of all colorectal cancer cases Autosomal dominant; high penetrance Typical age of CA onset is 40-50 yrs Multiple affected generations 60-70% right-sided/proximal CRC tumors Polyps may be present, multiple primaries common. Can overlap with AFAP High lifetime risk of CRC and other cancers beginning age 20 Typical age of onset is 40-50, range from 14-82 yrs Preponderance of right-sided/proximal tumors – 60% Polyps may be present (usually few to < 100), multiple primaries common. Can overlap AFAP so consider this diagnosis if >20 colon polyps detected.

Cancer Risks in HNPCC 100 80 60 % with cancer 40 20 20 40 60 80 Colorectal 78% 60 % with cancer Endometrial 43% 40 Stomach 19% 20 Biliary tract 18% Urinary tract 10% Ovarian 9% 20 40 60 80 Age (years) Aarnio M et al. Int J Cancer 64:430, 1995

Lifetime cancer risks: HNPCC Colorectal 80% Endometrial 20-60% Gastric 13-19% Ovarian 9-12% Biliary tract 2% Urinary tract 4% Small bowel 1-4% Brain/CNS 1-3% Cancer Risk with HNPCC: CRC - 80% lifetime, 40% for 2nd primary

Contribution of Gene Mutations to HNPCC Families Sporadic Familial Unknown 30% MSH2 30% HNPCC Rare CRC syndromes FAP MLH1 30% MSH6 (rare) PMS1 (rare) PMS2 (rare) Liu B et al. Nat Med 2:169, 1996

Amsterdam Criteria (HNPCC) 3 or more relatives with an HNPCC-related cancer, one of whom is a 1st degree relative of the other two 2 or more successive generations affected 1 or more cancers diagnosed before age 50 Failure to meet these criteria does not exclude HNPCC

Mutations in HNPCC Caused by mutations or deletions in mismatch repair (MMR) genes MSH2, MLH1, MSH6, (PMS2) 90% of detectable mutations in MSH2 and MLH1 50% of families meeting Amsterdam II Criteria have detectable mutations Testing/screening options: Direct genetic testing of MMR genes (in select families) Initial screening of the tumor tissue by MSI/IHC Autosomal dominant condition caused by mutations in one of a number of mismatch repair genes MSH2, MLH1, MSH6, PMS1, or PMS2, others(?) Sequencing of the MSH2 and MLH1 genes can identify up to 60-70% of HNPCC Microsatellite Instability (MSI) and Immunohistochemistry (IHC) testing on tumor tissue can be used to screen for possible HNPCC Genetic testing should be done on an affected family member, only after genetic counseling and informed decision-making

When to perform genetic testing Family history fulfills Amsterdam II criteria or Patient has two HNPCC related cancers or Patient has CRC and a 1st degree relative with HNPCC-related cancer, with at least one cancer diagnosed <50 years of age Always test an affected family member first!

Cancer in MSH2 mutation family Endometrial Cancer Colorectal/Upper GI Cancer Ovarian Cancer Other Cancer

MSI/IHC screening Microsatellite Instability (MSI) on tumor tissue can be used to screen for HNPCC in select cases Immunohistochemistry (IHC) on tumor tissue can be used to detect the presence or absence of the mismatch repair proteins (MSH2, MLH1, etc.) “Screen positive” individuals can be offered cancer genetic counseling/assessment and targeted genetic testing

Microsatellite Instability (MSI) 10%–15% of sporadic tumors have MSI 95% of HNPCC tumors have MSI at multiple loci Normal MSI tumor Electrophoresis gel

Criteria for MSI/IHC screening CRC or endometrial CA <50 yrs 2 HNPCC cancers in same person CRC with “MSI-H histology” diagnosed <60 yrs Infiltrating lymphocytes, Crohn’s-like lymphocytic reaction, mucinous/signet ring differentiation, medullary growth pattern CRC and one or more 1st degree relative with an HNPCC-related cancer, one diagnosed <50 yrs CRC and two or more 1st or 2nd degree relatives with HNPCC-related cancers, any age Umar A et al: J Natl Cancer Inst, 2004; 96(4):261-268

Genetic Testing for HNPCC Susceptibility Begin genetic testing with affected family member Positive result Negative result Offer testing to at-risk family members Continued risk of unidentified familial mutation

HNPCC Surveillance Gene carriers or at-risk relatives: CRC: colonoscopy age 20-25, repeat 1-2 yrs Women: gyn exam, endometrial aspiration, TV U/S, CA-125 (?) age 25-35, repeat 1-2 yrs If one HNPCC family member affected w/the following: Stomach CA: EGD age 3-35, repeat 1-2 yrs Urinary tract CA: urine cytology age 30-35, repeat 1-2 yrs NCCN practice guidelines in oncology – v.1.2003

• Diagnostic Screening

FAP Prevalence 0.01% 100’s to 1000’s of colonic adenomas by teens Cancer risk: colon, gastric, duodenum (periampulla), small bowel, pancreas, papillary thyroid, childhood hepatoblastoma 7% risk of CRC by 21 yrs; 93% by 50 yrs Variants: Gardner, Turcot

Clinical Features of FAP Estimated penetrance for adenomas >90% CHRPE may be present congenital hypertrophy of the retinal pigment epithelium Untreated polyposis leads to 100% risk of cancer

Genetics of FAP Autosomal dominant inheritance Caused by mutations in APC tumor suppressor gene on chromosome 5q Up to 30% of patients have de novo germline mutations Most families have unique mutations Most mutations are protein truncating Genotype/phenotype relationships emerging

The APC Tumor Suppressor Gene Mutations Codon 1309 5' 3' 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

FAP – surveillance Colon Annual sigmoidoscopy, age 10-12 yrs Prophylactic colectomy following polyp detection w/continued surveillance of rectum, ileal pouch Duodenal/gastric EGD age 25, repeat 1-3 yrs Gastroenterology 2001; 121: 195. AGA Statement

Genetic Testing: FAP/AFAP Test an affected family member first! After genetic counseling and informed consent APC gene testing can confirm a suspected diagnosis Family members of a person with a known APC mutation can have mutation-specific testing Genetic testing for children at risk for FAP can be considered before beginning colon screening APC gene analysis (either by protein truncation testing [PTT] or sequencing) can confirm a suspected diagnosis PTT detects 80-90% of affected persons (FAP) Gene sequencing detects up to 95% of affecteds Family members of a person with a known APC mutation can be tested for carrier status Testing at-risk children – can be considered before beginning screening (10-12yrs) Detection rate for AFAP is not known

Attenuated FAP 20 to 100 polyps, usually more proximal Onset later than FAP, average age of onset = 50 Lifetime risk of CRC = 80% Extracolonic tumors occur at same rate as FAP Not associated with CHRPE Variant of FAP, mutations in same APC gene Surveillance: annual colonoscopy starting late teens or early 20’s Option of subtotal colectomy Option of subtotal colectomy if polyps too numerous – AFAP does not generally affect the rectum, although continued surveillance is recommended.

APC gene mutation in Ashkenazi Jews Missense mutation (I1307K) associated with increased risk of CRC and adenomas in Ashkenazi Jews (AJ) Found in 6% of the general AJ population 12% of AJs with CRC 29% of AJs with CRC and a positive family history Lifetime risk of CRC in mutation carrier is 10-20% Screening: colonscopy every 2-5 yrs starting at 35 yrs

MAP syndrome/MYH gene Multiple adenomatous polyposis (MAP) syndrome Autosomal recessive; mutations in the MYH gene Median number of polyps = 55 Mean age of polyp diagnosis = 30-50 years Small, mildly dysplastic tubular adenomas Some tubulovillous, hyperplastic, serrated adenomas, microadenomas 30% of individuals with 15-100 polyps have homozygous mutations in the MYH gene Genetic testing should be offered if >15 polyps (and APC gene testing negative)

Peutz-Jeghers syndrome <1% of all CRC cases Hamartomatous polyps of GI tract as early as 1st decade Mucocutaneous hyperpigmentation lips, mouth, buccal mucosa, fingers Usually seen in children < 5 yrs Cancer risk: colon, small intestine, stomach, pancreas, breast, ovaries, uterus, testes, lungs, kidneys Mutations in STK11 gene found in 70% of familial cases and 30-70% of sporadic cases

Familial Juvenile Polyposis <1% of all CRC cases Autosomal dominant 5 or more juvenile polyps in colon or GI tract Appear in 1st or 2nd decade 50% lifetime risk of CRC; AOO in 30’s Increased risk gastric, GI, pancreatic CA ~50% of cases have mutations in either the MADH4 or BMPR1A genes

Consider Genetics Referral for: Two or more family members with CRC* at least one <50 Three or more family members w/CRC*; any age Patient with colon cancer before 40 yrs Endometrial cancer and family history of CRC <50 Persons with more than one primary CRC <50 yrs or with both endometrial CA and CRC Family or personal history of CRC and one or more 1st degree relative with an HNPCC-related cancer, one diagnosed <50 yrs. *Same side of family Any patient with 2 or more family members (same side) with early onset CRC (<50 yrs) Any colon cancer case before 40 yrs Woman with endometrial cancer <45 and family history of early onset CRC Persons with more than one primary CRC <50 yrs or with both endometrial and CRC Autosomal dominant pattern of cancers in the same lineage Insurance coverage: Some insurers and some plans will cover cancer genetic risk assessment/cancer genetic counseling. Others will not. More difficult to obtain coverage for unaffected person with a family history than an affected person. Pre-authorization recommended. Pre-authorization for cancer genetic testing is important as tests are expensive. The genetic counselor will facilitate the pre-auth for testing if indicated and if patient decides to have testing. Insurance discrimination – needs to be discussed before insurer is contacted regarding coverage.

Consider Genetics Referral for: MSI and/or IHC tumor results suspicious for HNPCC Autosomal dominant pattern of cancers in the family Persons with 15 or more adenomatous colorectal polyps Persons with multiple hamartomatous or juvenile GI polyps Persons with a family history of a known hereditary cancer syndrome

Screening Implications of Inherited Colorectal Cancers

Risk of inherited CRC Risk for CRC based on family history increases with: Closer degree of relationship and # of affected members Younger age of onset Presence of extracolonic tumors, multiple primaries About 10% of all people have a 1st degree relative with CRC, which increases risk 2-fold Risk for CRC increases with: degree of relationship and # of affected members younger age of onset presence of extracolonic tumors, multiple primaries

Family History: Empiric Risks Lifetime Risk CRC General population in US 2 to 6% One 1st degree relative w/CRC 2-3 fold Two 1st degree relatives w/CRC 3-4 fold 1st degree relative w/CRC <50 3-4 fold One 2nd or 3rd degree relative w/CRC 1.5-fold Two 2nd degree relatives w/CRC 2-3 fold

Goal: Classification Assessment Risk Classification Intervention Standard prevention recommendations Average Moderate (“Familial”) Personalized prevention recommendations Family Hx Referral for genetic evaluation with personalized prevention recommendations High/Genetic

CRC Risk Management Age to Begin Average Risk 50 yrs No family history CRC OR One 2nd or 3rd degree relative with CRC ACBE = air contrast barium enema. FOBT = fecal occult blood test FOBT annually + Flex sig every 5 yrs; OR Colonoscopy every 10 yrs; OR DCBE every 5 yrs

Average risk screening CRC Risk Management Moderate/Family history Age to begin 1. Two 1st degree relatives with CRC any age 40 years* or one 1st degree relative with CRC < 60 2. One 1st degree relative with CRC >60 or 40 years two 2nd degree relatives with CRC any age * Or 5-10 yrs earlier than earliest case in family Colonoscopy every 5 yrs Average risk screening Gastroenterology: 2003;124:544-560

Adenoma-Carcinoma Sequence Accumulation of Mutations DCC, MCC, p53, K-ras, APC, MSH2, MLH1, etc.

CRC Risk Management Age to Begin HNPCC or suspected HNPCC 20-25 yrs FAP or suspected FAP 10-12 yrs Colonoscopy every 1-2 yrs Genetic counseling; consider genetic testing Flex sig or colonoscopy every1-2 yrs Genetic counseling; consider genetic testing

Chemoprevention Evidence that ASA, NSAIDs, Calcium, and COX-2 inhibitors may reduce incidence of CRC by reducing # of adenomas 40-50% risk reduction for developing CRC regardless of location in colon, age, gender, and race Generally performed by RCT’s in patients with prior CRC followed for recurrence of adenomas Diet, fiber, and antioxidant vitamins have not been shown by RCT’s to decrease risk of recurrent adenomas COX-2i’s and Sulindac have been shown to reduce the number of adenomas found in FAP alone Not effective for sporadic colon CA Actually can cause regression of adenomas

Summary

Summary Risk factors for colon cancer Genetic basis for colon cancer Inherited Acquired (sporadic)-adenomatous polyp, IBD Genetic basis for colon cancer Inherited (FAP, HNPCC, to be defined) Sporadic polyp-different pathways 

Summary Genetic counseling and testing HNPCC FAP Implications for screening/surveillance Family members Other malignancies

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