CMGS LS FAP MAP Training Day, 30/11/2009 Colorectal Cancer BP Training Day Ian M Frayling

1. CMGS LS FAP MAP Training Day, 30/11/2009 Colorectal Cancer BP Training Day Ian M Frayling Consultant Genetic Pathologist Director, Laboratory Genetics Service for Wales Institute of Medical Genetics, Cardiff

2. InSiGHT workshop, Palma, Majorca, April 28-29th2006 Identification of Lynch Syndrome (MSI/IHC) Ian M Frayling Consultant Genetic Pathologist & Cancer Geneticist Director, Laboratory Genetics Service for Wales Institute of Medical Genetics, Cardiff

3. Biomarkers in familial colorectal cancer screening workshop, London, Feb 2006 Microsatellite Instability Ian M Frayling Consultant Genetic Pathologist & Cancer Geneticist Director, Laboratory Genetics Service for Wales Institute of Medical Genetics, Cardiff

4. Sporadic or LS?

5. Lynch Syndrome • History: • Warthin: Family ‘G’

6. Lynch Syndrome • History: • Lynch Syndrome I: site-specific colon cancer • Lynch Syndrome II: cancer family syndrome Oncology55:103-108. (1998) Molecular genetics and clinical-pathology features of HNPCC (Lynch Syndrome): Historical Journey from Pedigree Anecdote to Molecular Genetic Confirmation. Lynch HT, Smyrk T, Lynch JF. Picture courtesy of Dr Patrick Lynch, MD Anderson Cancer Centre, Houston, TX

7. Lynch Syndrome: 2007 • Various names for Lynch syndrome have been used in the past century. A workshop in Amsterdam in 1989 agreed upon the name "HNPCC", because at that time the syndrome was unknown to most doctors. This name clarified that the syndrome described an inherited form of CRC. The appropriateness of the name was discussed again at an international meeting in Bethesda in 2004. Most participants considered the term HNPCC to be inappropriate, since the syndrome is also associated with many other tumours. It was proposed that the name "Lynch syndrome" should be reintroduced, and that this name should be reserved for families with strong evidence of MMR deficiency—for example, by the presence of an MMR defect or by the presence of MSI in tumours. The European group agreed that this name is the best available name for the syndrome. The group suggests that families that meet the original Amsterdam criteria, but do not have evidence for MMR deficiency, are referred to as having familial CRC. • Vasen HFA et al. (2007) J Med Genet44:353-362 • http://jmg.bmj.com/cgi/content/abstract/44/6/353

8. Lynch Syndrome • History: • Lynch I: hereditary site-specific colon cancer • Lynch II: cancer family syndrome • Tumours • principal: colon & rectum • major: endometrium • minor: ovary, stomach, small intestine, pancreas, hepato-biliary tract, pelvi-ureter, skin (sebaceous & keratoacanthoma - Muir-Torre), CNS glioblastoma • Germline mutation in a DNA mismatch repair gene

9. ICG Clinical Definition of LS • Familial clustering of colorectal and/or endometrial cancer • Associated cancers: gastric, ovary, ureter/renal pelvis, brain, small bowel, hepatobiliary tract and skin (sebaceous tumours) • Development of cancer at an early age • Development of multiple cancers • Features of colorectal cancer: (a) proximal colon (right-sided), (b) improved survival, (c) multiple, (d) mucinous, (e) lymphocytic infiltration and lymphoid aggregates • Features of colorectal adenoma: (a) one to a few; (b) villous histology, (c) high grade dysplasia, (d) rapid adenoma to carcinoma progression (probably) • High frequency of Microsatellite Instability (MSI) • Immunohistochemistry: loss of MLH1, MSH2, or MSH6 protein expression in tumours • Germline mutation in MMR gene: MSH2, MLH1, MSH6, PMS2 • Any combination of 1) to 9) may be present, but 9) alone defines LS Vasen HFA et al. New clinical criteria for Hereditary Non-Polyposis Colorectal Cancer (HNPCC, Lynch Syndrome) proposed by the ICG-HNPCC. Gastroenterology 1999;116:1453-1456.

10. ICG Clinical Definition of LS • Familial clustering of colorectal and/or endometrial cancer • Associated cancers: gastric, ovary, ureter/renal pelvis, brain, small bowel, hepatobiliary tract and skin (sebaceous tumours) • Development of cancer at an early age • Development of multiple cancers • Features of colorectal cancer: (a) proximal colon (right-sided), (b) improved survival, (c) multiple, (d) mucinous, (e) lymphocytic infiltration and lymphoid aggregates • Features of colorectal adenoma: (a) one to a few; (b) villous histology, (c) high grade dysplasia, (d) rapid adenoma to carcinoma progression (probably) • High frequency of Microsatellite Instability (MSI) • Immunohistochemistry: loss of MLH1, MSH2, or MSH6 protein expression in tumours • Germline mutation in MMR gene: MSH2, MLH1, MSH6, PMS2 • Any combination of 1) to 9) may be present, but 9) alone defines LS Vasen HFA et al. New clinical criteria for Hereditary Non-Polyposis Colorectal Cancer (HNPCC, Lynch Syndrome) proposed by the ICG-HNPCC. Gastroenterology 1999;116:1453-1456.

11. CMGS BPGuidelines • 1996? • Workshop March 2002 • Within Uk Guidelines (PHGU) • Within international guidelines

12. LS: Diagnosis • Problem: LS is not the only form of FCRC • Mutation detection is possible, but LS is: • genetically heterogeneous: MSH2, MLH1, MSH6 ... • allelically heterogeneous - private mutations • frequently associated with missense mutations • phenocopied • Mutation detection is a finite resource • expensive, time-consuming • not 100% efficient • cannot, in itself, exclude LS

13. LS Family History Bowel “young” Womb 40’s Bowel 72y F+W 85y ? Endometrial 38y Caecum 45y TCC Ureter 35y ?

14. LS: FH as a Diagnostic Test • Amsterdam Criteria • Designed to select families for research • Highly stringent • Inherently specific at the cost of sensitivity • Designed before LS was • A) Elucidated • B) Understood

15. Modified Amsterdam Criteria • Three or more cases of HRC* in >1 generation • One affected individual must be a first degree relative of the other two (or more) • One HRC diagnosed < 50 yrs • Familial Adenomatous Polyposis excluded • Cancers histologically confirmed • * HRC = HNPCC Related Cancers, i.e. • Colorectal, endometrial, gastric, small bowel or pelvi-ureter

16. LS: FH as a Diagnostic Test • Modified Amsterdam Criteria • # CRCa cases • CRCa average age • Endometrial and/or small bowel Ca cases • Endometrial + CRCa case • Multiple CRCa case •  Probability of MSH2 or MLH1 mutation Wijnen JT et al. Clinical findings with implications for genetic testing in families with clustering of colorectal cancer. N Engl J Med 1998; 339: 511-8.

17. Colorectal Cancer: ?LS CRCa 63y CRCa 72y CRCa 45y

18. Colorectal Cancer: ?LS CRCa 63y CRCa 72y CRCa 45y 10%

19. Colorectal Cancer: ?LS CRCa 63y CRCa 72y CRCa 45y CRCa 29y 20%

20. Colorectal Cancer: ?LS CRCa 63y ENCa 44y CRCa 72y CRCa 45y CRCa 29y 60%

21. Colorectal Cancer: ?LS CRCa 63y CRCa 42y ENCa 54y CRCa 45y CRCa 29y 80%

22. LS: FH as a Diagnostic TestWijnen Model

23. Molecular Tests for Lynch Syndrome • Microsatellite instability (MSI) • Immunohistochemistry (IHC) • BRAF V600E • Germline mutation testing

24. Molecular Tests for Lynch Syndrome • When should testing be done?

25. Molecular Tests for Lynch Syndrome • When should testing be done? • WHEN IT WILL MAKE A DIFFERENCE

26. Molecular Tests for Lynch Syndrome • MSI or IHC? • So which is better? • What is meant by better? • Sensitivity; Specificity; Predictive values; Cost • Often assumed sporadic = hereditary • Many, many variables • Very difficult to compare studies

27. Microsatellite Instability • Different laboratories use: • Different markers, PCR conditions, gels, machines, DNA extraction • Different definitions of “Extra alleles” • % of affected markers; which markers • Different tumours; patient control DNA • Different prior odds of HNPCC, etc. • No EQAS

28. Microsatellite Instability • What is MSI? • The presence of extra alleles at a microsatellite • A number of microsatellites must exhibit instability to diagnose MSI

29. Microsatellite Instability • What does it signify? • MSI-H signifies loss of DNA Mismatch Repair

30. Microsatellite Instability: 1998 - 2007 • 1998: Microsatellite Markers: NCI set • Mononucleotides • Dinucleotides • NB markers optimal in colorectal cancer • 2007: Mononucleotides alone • 5 monos, Promega • 99% concordance; 100% accuracy • Meets 2002 Bethesda NCI workshop recommendations Boland CR et al. A NCI workshop on microsatellite instability for cancer detection and familial predisposition: Development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Research 1998; 58: 5248-5257. Bacher JW et al. Development of a fluorescent multiplex assay for detection of MSI-High tumours. Disease Markers 2004; 20: 237-250.

31. Microsatellite Instability • How does it perform?

32. Microsatellite Instability: 1993 • Patterns / definitions • MSI-H • MSI-L • MSS

33. Microsatellite Instability: 2003 • Patterns / definitions • MSI • >29% markers showing instability usually incl.  1 mono • <29% markers showing instability but incl.  1 mono. • Not (HNPCC-associated) MSI • <29% markers showing instability  Halford S et al. (2002) Low-level microsatellite instability occurs in most colorectal cancers and is a nonrandomly distributed quantitative trait. Cancer Res.62: 53-7. Laiho P et al. (2002) Low-level microsatellite instability in most colorectal carcinomas. Cancer Res.62: 1166-70.

34. MSI as a Predictive Test of HNPCC Theory • 1~2% CRCa = HNPCC • MSI-H in >95% HNPCC tumours • Sporadic tumours • 20% of colon Ca have MSI (1 in 20 = HNPCC)

35. MSI as a Predictive Test of HNPCC Theory • 1~2% CRCa = HNPCC • MSI-H in >95% HNPCC tumours • Sporadic tumours • 20% of colon Ca have MSI (1 in 20 = HNPCC)

36. MSI as a Predictive Test of HNPCC Theory • NB Colorectal tumours Frayling IM.Microsatellite Instability. Gut 1999; 45: 1-4.

37. MSI as a Predictive Test of HNPCC Theory • Sporadics – ?effect of prior odds Frayling IM.Microsatellite Instability. Gut 1999; 45: 1-4.

38. MSI as a Predictive Test of HNPCC Theory

39. Microsatellite Instability • How does that test perform? • OK as an includer, but better as an excluder of HNPCC

40. Microsatellite Instability • How is the test best used?

41. Microsatellite Instability • How is the test best used? See later!

42. Immunohistochemistry • What is IHC? • Detection of protein expression in tissues by antibodies

43. Immunohistochemistry • In an ideal world MLH1 MSH2 Dr Mark Arends, University of Cambridge Department of Pathology

44. Immunohistochemistry • Different laboratories use: • Different fixation, antibodies, antigen retrieval conditions, machines (manual), detection • Different pathologists • Different definitions of “abnormal” • No EQAS • Different tumours, case mixes • Different prior odds of HNPCC, etc.

45. Immunohistochemistry • What does loss of reactivity signify? • Loss of antigenic sites of DNA mismatch repair protein • Abnormal expression = MSI • Retained expression may = MSI or MSS • NB dysfunctional protein may still react • All* tumours with abnormal IHC have MSI • Not all tumours with MSI have abnormal IHC

46. Immunohistochemistry • How does it perform?

47. Immunohistochemistry • How does it perform? • All* tumours with abnormal IHC have MSI • Not all tumours with MSI have abnormal IHC • MSH2: Sens <= MSI Spec > MSI • MLH1: Sens <= MSI Spec = MSI • On average: Sens <= MSI Spec >= MSI

48. Immunohistochemistry

49. Immunohistochemistry • How does it perform? • Mismatch repair protein immunohistochemistry within a diagnostic setting – An inter-laboratory study • Mullarkey M, Grady AO, Jasani B, Stephens M, Dodson A, Munson P, Haperfield L, Arends M, Frayling IM, Kay EW • 13 tumours in a TMA x 6 laboratories …