1

CANCER GENETICS: IDENTIFICATION AND

MANAGEMENT OF INDIVIDUALS WITH LYNCH SYNDROME

HENRY T. LYNCH, MDCreighton UniversitySchool of MedicineOmaha, Nebraska

2Hereditary 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 CRC 37,011-61,686FAP <1% of all CRC <12,337Familial 20% of all CRC 246,742

*GLOBOCAN. The International Agency for Research on Cancer web site. URL: http://www.iarc.fr/

8

8

Familial/Hereditary CRC in US

Annual CRC incidence in US: 142,570

Lynch syndrome 3-5% of all CRC 4,277 - 7,129

FAP <1% of all CRC <1,426

Familial 20% of all CRC 28,514

Jemal et al. CA Cancer J Clin 60:277-300,2010.

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.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 26:5783-5788, 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 26:5783-5788, 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 293:1979-1985, 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.e1- 2197.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 HNPCC is associated with germline mutations in any one of at least five genesmutations in any one of at least five genes

Chr 2Chr 2Chr 3Chr 3

Chr 7Chr 7

MSH2MSH2

PMS1PMS1

MLH1MLH1PMS2PMS2

MSH6MSH6

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 willhave a second primary CRC within 10 years, if surgery was < asubtotal 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, 2009.

23

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 20:583-587, 2010.

24

25

N Engl J Med 354: 261-269, 2006.

26

27

28

Could this behereditary

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 16:14-22,2009.

32

33

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

34

J Clin Pathol 62:679-684, 2009.

35

J Clin Pathol 62:679-684, 2009.

36

J Clin Pathol 62:679-684, 2009.

37

Sporadic

Lynch Syndrome

Familial Hereditary

FAP; AFAPMixed Polyposis SyndromeAshkenazi I1307KCHEK2 (HBCC)MUTYH (MAP)TGFBR1

PJSFJPCDBRRS

= as yet undiscovered hereditary cancer variants

HamartomatousPolyposisSyndromes

AC-1 without MMR(Familial CRC of syndrome “X”)

TACSTD1 (EPCAM)

38

Hereditary Polyposis Syndromes

39

40

Attenuated FAP

Later onset (CRC ~age 50)Later onset (CRC ~age 50) Few colonic adenomasFew colonic adenomas Not associated with Not associated with

CHRPECHRPE UGI lesions UGI lesions Associated with Associated with

mutations at extreme 5mutations at extreme 5’’, 3, 3'' ends of ends of APCAPC gene, & exon gene, & exon 9A9A

41

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 118:115-122, 2006.

**Lynch et al. J Natl Cancer Inst 99:261-263, 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 6:206-214, 2008.

44

MORPHOLOGYSUSPICIOUS

FOR MSI-H

Run PCR testfor MSI status

Is thereMSI-H?

Run mutation analysisfor BRAF V600E

Is thereBRAF V600E

mutation?

SPORADIC CRCWITH MSI-H

NO EVIDENCE OFLYNCH

SYNDROME

Is there lossof stainingwith any ofthe Abs?

IHC for MLH1,MSH2, MSH6, PMS2

PUTATIVELYNCH

SYNDROME

MMR GENES MUTATIONANALYSIS

Is therea mutation in MMR

gene?

LYNCHSYNDROME

YES

YES

NO

NO

NO

YES

YES

NO

Gatalica Z, Torlakovic E. Fam Cancer 2008;7:15-26

FAMILIAL CRCTYPE “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 99:291-299, 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 30:197-203, 2009.**Lindor et al. JAMA 293:1979-1985, 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 2009.

Transcriptional read throughHypermethylation 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.859-1462_*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 56:934-938, 1985.

Lynch et al. Cancer 56:939-951, 1985.

52First 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 Oncol25:3534-3542, 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.

57

58

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 26:5783-5788, 2008.

60

Physicians

Payers

Patients

Personalized Medicine

Need Individual managementbased upon Genetic diagnosis(deleterious mutation helpful)

Need more clinical genetic education. Example: multiplepolyps = FAP; multiple primary cancer pattern = syndrome identification

What is advantage of extensive family history; genetic counseling; genetic testing (MMR, MSI, IHC, BRCA1/2); screening?

61

62

J Clin Pathol 62:679-684, 2009.