1 CANCER GENETICS: IDENTIFICATION AND MANAGEMENT OF INDIVIDUALS WITH LYNCH SYNDROME HENRY T. LYNCH, MD Creighton University School of Medicine Omaha, Nebraska

2 Hereditary Cancer Syndromes: Nuts and Bolts Family history; Hereditary cancer syndrome diagnosis; Genetic counseling; DNA studies; Highly targeted surveillance/management; Extend to all at-risk relatives; Physician education; Research problem of discrimination (insurance, employment); Strategies for wide-spread interest of familial cancer approach to cancer control, malpractice, molecular genetics, other.

3 Patient’s Modified Nuclear Pedigree

6 Why Pursue Cancer of All Anatomic Sites? Pertinent for any hereditary cancer syndrome diagnosis; Most identified by pattern of cancer expression, e.g.: breast and ovary (HBOC syndrome); CRC, endometrium, ovary, others (Lynch syndrome); sarcomas, breast, brain, multiple others in SBLA (Li- Fraumeni syndrome); medullary thyroid carcinoma and pheochromocytoma (MEN-2a and MEN-2b); melanoma and pancreatic cancer with CDKN2A (p16) mutation (FAMMM syndrome); diffuse gastric cancer and lobular breast cancer with CDH1 mutation (HDGC syndrome);...and the list goes on.

7 Colorectal Cancer Worldwide estimates for colorectal cancer during 2008*: Incidence – 1,233,711 Mortality – 608,644 Worldwide estimates for familial/hereditary CRC during 2008*: Lynch syndrome 3-5% of all CRC37,011-61,686 FAP <1% of all CRC<12,337 Familial 20% of all CRC246,742 *GLOBOCAN. The International Agency for Research on Cancer web site. URL:

8 8 Familial/Hereditary CRC in US Annual CRC incidence in US: 142,570 Lynch syndrome3-5% of all CRC4, ,129 FAP<1% of all CRC<1,426 Familial 20% of all CRC28,514 Jemal et al. CA Cancer J Clin 60: ,2010.

9 Magnitude of the Problem Question: Why are these figures of such significant public health impact? Answer: Each hereditary cancer comes from a family that could benefit immensely from genetic counseling. DNA testing, surveillance, and highly-targeted management are the key!

Genetic Counseling Mandatory Centers of Cancer Genetic Expertise Physician Role, unfortunately, often insufficient 10

11 Should we test all colorectal cancer for Lynch Syndrome? YES! Test everybody.

12 Search for LS Among CRC Affecteds* Evidence: Among 500 CRC patients, 18 (3.6%) had LS. Of these 18:  18 (100%) had MSI-H CRCs;  17 (94%) were correctly predicted by IHC;  only 8 (44%) were dx < 50 years;  only 13 (72%) met the revised Bethesda guidelines;  1/35 cases of CRC show LS. *Hampel et al. J Clin Oncol 26: , 2008.

13 Molecular Genetic Screening for LS Recommendation*: All incident CRC and EC cases should be molecularly screened for LS. MSI highly sensitive (89.3%). IHC equally sensitive (91.2%), is inexpensive, is more readily available, and predicts the nonworking gene. IHC is preferred method to screen for LS*. *Hampel et al. J Clin Oncol 26: , 2008.

14 Cost-effectiveness of DNA Testing Estimate the cost-effectiveness of genetic testing strategies to identify LS among newly dx CRC patients using MSI and IHC.* Conclusion: Preliminary tests seem cost-effective from the U.S. health care system perspective. Detects nearly twice as many cases of LS as targeting younger patients. MMR testing is not cost effective. *Mvundura et al. Genet Med 12:93-104, 2010.

15 Familial CRC Type “X” Amsterdam Criteria positive but lacking MSI and MMR mutations will constitute ~ 40% of those AC-I without MMR mutations and therein referred to as familial CRC type X.* 1) CRC > left side 2)  CRC and extra colonic CRC 3) Later age CRC onset 4) Molecular genetics (MSI and IHC or MMR mutation) ABSENT! *Lindor et al. JAMA 293: , 2005.

16 Screening for Amsterdam Criteria LS* a) Screening of all CRC patients meeting Amsterdam Criteria (AC) would fail to detect half of all cases; b) Screening those aged  50 would detect only half of all cases; c) Screening of all patients using Bethesda Guidelines for MSI would fail to detect at least 1/3 of all cases. *Boland & Shike. Gastroenterology 138:2197.e e7, 2010.

17 Familial CRC Familial clustering of CRC, like that for carcinoma of the breast and stomach, has been discussed for more than 100 years. What does it mean from the standpoint of risk? Best answer – First- degree relative of CRC affected has 2-3 fold excess risk for CRC compared to population expectations. But is type X different? Answer – Risk remains elusive!

18 Genetic Heterogeneity in HNPCC HNPCC is associated with germline mutations in any one of at least five genes Chr 2 Chr 3 Chr 7 MSH2 PMS1 MLH1 PMS2 MSH6 Mismatch Repair (MMR) Mutations

19 Cardinal Features of Lynch Syndrome Family pedigree shows autosomal dominant inheritance pattern for syndrome cancers. Proximal (right-sided) CRC predilection: 70-85% of Lynch syndrome CRCs are proximal to the splenic flexure. Earlier average age of CRC onset than in the general population: - Lynch syndrome: 45 years; - general population: 63 years. Accelerated carcinogenesis, i.e., shorter time for a tiny adenoma to develop into a carcinoma: - Lynch syndrome: 2-3 years; - general population: 8-10 years. High risk of additional CRCs: 25-30% of patients who have surgery for a LS-associated CRC will have a second primary CRC within 10 years, if surgery was < a subtotal colectomy.

20 Increased risk for certain extracolonic malignancies  Endometrial  Ovary  Stomach  Small bowel  Pancreas  Liver and biliary tree  Muir-Torre cutaneous features  Brain, (glioblastoma) – Torre syndrome features  Prostate cancer  Breast  Possible Adrenal cortical carcinoma and others.

21 Cardinal Features of Lynch Syndrome Differentiating pathology features of LS CRCs: - more often poorly differentiated; - excess of mucoid and signet-cell features; - Crohn’s-like reaction; - significant excess of infiltrating lymphocytes within the tumor. Increased survival from CRC. Sine qua non for diagnosis is identification of germline mutation in MMR gene (most commonly MLH1, MSH2, MSH6) segregating in the family.

22 COLONOSCOPY  Initiate age 20 – 25  every other year to age 40; annually thereafter  must get good cleanout and visualize cecum  CRC – need subtotal colectomy

EC Screening Effectiveness of screening for EC is unproven; Consequently, prophylactic surgery is the best option for ♀ who have completed their families.* *Manchanda et al. Curr Opin Obstet Gynecol 21:31-38,

Screening for EC in LS* No screening tool has been validated. Ultrasonography (US) used to screen for atypical hyperplasia and cancer. Considered normal if no polyps or intrauterine abnormalities seen and if maximum endometrial thickness < 4mm in postmenopausal ♀ on hormonal replacement therapy or < 6mm in other ♀. *Lécuru et al. Int J Gynecol Cancer 20: ,

25 N Engl J Med 354: , 2006.

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28 Could this be hereditary Colon Cancer

29 Targeted CRC Screening Screening is melded to LS’s natural history: Proximal location  colonoscopy Early age of onset  beginning at age 25 Accelerated carcinogenesis  every 1-2 yrs < age 40, then annually Pattern of extracolonic cancers  targeted screening

30 Dis Colon Rectum 53:77-82, 2010.

31 Extended Colectomy* Continued: Times to subsequent CRC and subsequent abdominal surgery were significantly shorter in the control group (P <.006 and P <.04, respectively). No significant difference in survival time between the cases and controls. Conclusion: Even though no survival benefit the increased incidence of metachronous CRC and increased abdominal surgeries among controls warrant subtotal colectomy in patients with LS. *Dis Colon Rectum 53:77-82, 2010.

Cancer Control 16:14-22,

33 Meyer et al. Cancer Control 16:14-22,2009.

34 J Clin Pathol 62: , 2009.

35 J Clin Pathol 62: , 2009.

36 J Clin Pathol 62: , 2009.

37 Sporadic Lynch Syndrome Familial Hereditary FAP; AFAP Mixed Polyposis Syndrome Ashkenazi I1307K CHEK2 (HBCC) MUTYH (MAP) TGFBR1 PJS FJP CD BRRS = as yet undiscovered hereditary cancer variants Hamartomatous Polyposis Syndromes AC-1 without MMR (Familial CRC of syndrome “X”) TACSTD1 (EPCAM)

38 Hereditary Polyposis Syndromes

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40 Attenuated FAP l Later onset (CRC ~age 50) l Few colonic adenomas l Not associated with CHRPE l UGI lesions l Associated with mutations at extreme 5 ’, 3 ' ends of APC gene, & exon 9A

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42 Molecular Diagnosis of LS: Toward a Consensus If tumor is MSI-positive, IHC is then done to direct mutational testing to a specific MMR gene, which MSI alone cannot do.* If tumor is MSS, must weigh low probability of an informative IHC test and cost of performing it.** *Engel et al. Int J Cancer 118: , **Lynch et al. J Natl Cancer Inst 99: , 2007.

43 BRAF V600E mutation and LS BRAF V600E mutation can sort this out since when detected it excludes LS and contributes to improved cost- effectiveness of genetic testing for LS. *Clin Gastroenterol Hepatol 6: , 2008.

44 MORPHOLOGY SUSPICIOUS FOR MSI-H Run PCR test for MSI status Is there MSI-H? Run mutation analysis for BRAF V600E Is there BRAF V600E mutation? SPORADIC CRC WITH MSI-H NO EVIDENCE OF LYNCH SYNDROME Is there loss of staining with any of the Abs? IHC for MLH1, MSH2, MSH6, PMS2 PUTATIVE LYNCH SYNDROME MMR GENES MUTATION ANALYSIS Is there a mutation in MMR gene? LYNCH SYNDROME YES NO YES NO Gatalica Z, Torlakovic E. Fam Cancer 2008;7:15-26 FAMILIAL CRC TYPE “X”

45 Frequency of MMR Mutations* ~60% of Amsterdam+ families with clinically defined LS phenotype carry point mutations or large genomic deletions in the transcription of either MLH1 or MSH2 genes. Conversely, the pathogenic change inactivating the MMR system is not known or not fully understood in the remaining ~40%. *Lagerstedt-Robinson et al. J Natl Cancer Inst 99: , 2007.

46 Frequency of MMR Mutations* A portion of this ~40% lacking MMR mutations is caused by a mutation mechanism in the gene known as EPCAM. Others have been classified as familial colorectal cancer Type “X”.** *Kovacs et al. Hum Mutat 30: , **Lindor et al. JAMA 293: , 2005.

47 Epithelial Cell Adhesion Molecule (EPCAM) Gene and Its Lynch Syndrome Connection Impacts Diagnosis Genetic Counseling Phenotype site specific CRC Pathogenesis Pharmacogenetics

48 Polyadenylation Sequence 5’ EPCAM deletion Exons 8 and 9 and polyadenylation sequence Ligtenberg MJ, Nature Genetics Transcriptional read through Hypermethylation of the MSH2 promoter

49 Why LS with Site-Specific CRC? Deletion in EPCAM results in hypermethylation and incomplete silencing of MSH2. EPCAM mutation carriers may have phenotypic features that differ from carriers of MSH2 mutations – namely, an almost exclusive expression of site-specific CRC, thereby lacking extracolonic cancers.

50 c _*1999del (4.9 kb, starting in intron 7 and including exons 8 & 9) EPCAM MSH2 American and Dutch families have the same deletion in the EPCAM gene Deletion Lightenberg, Nature Genetics 2009.

51 History of Family R* Ascertained by us in 1970 and followed continuously. 700 blood line relatives 327 individuals age ≥ 18, ≥ 25% pedigree risk Phenotype strikingly similar to LS but integral extracolonic cancers absent (site-specific CRCs) *Lynch et al. Cancer 56: , Lynch et al. Cancer 56: , 1985.

52 First patient identified with EPCAM mutation CRC affecteds EPCAM results

53 American and Dutch EPCAM mutations originate from a common ancestor Deletion and Region inherited from common ancestor Family R and the Dutch families share a 6.1 MB region surrounding the same EPCAM deletion indicating a common ancestor. Based on the size of the shared region it is estimated the deletion occurred 10 generations ago. Dutch Families Chromosome 2 Family R Chromosome 2

54 J Clin Oncol25: , 2007.

55 Who Should Be Tested? 1. Pedigree consistent with hereditary colorectal cancer (CRC) syndrome; 2. Known germline mutation predisposing to cancer; 3. Patients at acceptable high cancer risk status; 4. Presence of cancer syndrome stigmata (phenotype): e.g., polyposis in FAP; 5. Genetic counseling, risks/benefits understood; 6. Consent given; 7. Results: full explanation of surveillance/management advice.

56 Family Information Service (FIS) Cost-effective and highly efficient way of educating and counseling all available family members from a geographic catchment area during a single setting. Makes best use of physician’s time and effort, has group therapy potential and patients welcome it.

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59 Conclusions for EPCAM Conclusions: 1) Cancer control compliance in Family R profound; 2) 40% of AC-I cases lack MMR mutations – how many may qualify as EPCAM? 3) Likely EPCAM phenotype site-specific CRC; 4) What can we learn from molecular features of EPCAM for pharmacologic benefit? 5) 1/35 CRC affecteds likely LS (Hampel et al.*). *J Clin Oncol 26: , 2008.

60 Physicians Payers Patients Personalized Medicine Need Individual management based upon Genetic diagnosis (deleterious mutation helpful) Need more clinical genetic education. Example: multiple polyps = FAP; multiple primary cancer pattern = syndrome identification What is advantage of extensive family history; genetic counseling; genetic testing (MMR, MSI, IHC, BRCA1/2); screening?

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62 J Clin Pathol 62: , 2009.