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 28-29 th 2006 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:History: –Warthin: Family ‘G’

6. Lynch Syndrome History:History: –Lynch Syndrome I: site-specific colon cancer –Lynch Syndrome II: cancer family syndrome Picture courtesy of Dr Patrick Lynch, MD Anderson Cancer Centre, Houston, TX Oncology 55:103-108. (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.

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.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 Genet 44:353-362Vasen HFA et al. (2007) J Med Genet 44:353-362 http://jmg.bmj.com/cgi/content/abstract/44/6/353http://jmg.bmj.com/cgi/content/abstract/44/6/353

8. Lynch Syndrome History:History: –Lynch I: hereditary site-specific colon cancer –Lynch II: cancer family syndrome TumoursTumours –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 geneGermline mutation in a DNA mismatch repair gene

9. ICG Clinical Definition of LS 1Familial clustering of colorectal and/or endometrial cancer 2Associated cancers: gastric, ovary, ureter/renal pelvis, brain, small bowel, hepatobiliary tract and skin (sebaceous tumours) 3Development of cancer at an early age 4Development of multiple cancers 5Features of colorectal cancer: (a) proximal colon (right-sided), (b) improved survival, (c) multiple, (d) mucinous, (e) lymphocytic infiltration and lymphoid aggregates 6Features of colorectal adenoma: (a) one to a few; (b) villous histology, (c) high grade dysplasia, (d) rapid adenoma to carcinoma progression (probably) 7High frequency of Microsatellite Instability (MSI) 8Immunohistochemistry: loss of MLH1, MSH2, or MSH6 protein expression in tumours 9Germline 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 1Familial clustering of colorectal and/or endometrial cancer 2Associated cancers: gastric, ovary, ureter/renal pelvis, brain, small bowel, hepatobiliary tract and skin (sebaceous tumours) 3Development of cancer at an early age 4Development of multiple cancers 5Features of colorectal cancer: (a) proximal colon (right-sided), (b) improved survival, (c) multiple, (d) mucinous, (e) lymphocytic infiltration and lymphoid aggregates 6Features of colorectal adenoma: (a) one to a few; (b) villous histology, (c) high grade dysplasia, (d) rapid adenoma to carcinoma progression (probably) Microsatellite Instability (MSI) 7High frequency of Microsatellite Instability (MSI) 8Immunohistochemistry 8Immunohistochemistry: loss of MLH1, MSH2, or MSH6 protein expression in tumours 9Germline mutation in MMR gene 9Germline mutation in MMR gene: MSH2, MLH1, MSH6, PMS2 Any combination of 1) to 9) may be present, but 9) alone defines LSAny 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 BP Guidelines 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 Caecum45y Bowel“young” TCC Ureter 35y Endometrial38y Womb 40’s Bowel 72y ? ? F+W85y

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 45y CRCa 63y CRCa 72y

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

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

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

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

22. LS: FH as a Diagnostic Test LS: FH as a Diagnostic Test Wijnen Model

23. Molecular Tests for Lynch Syndrome Microsatellite instability (MSI)Microsatellite instability (MSI) Immunohistochemistry (IHC)Immunohistochemistry (IHC) BRAF V600EBRAF V600E Germline mutation testingGermline mutation testing

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

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

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

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

28. Microsatellite Instability What is MSI?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?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?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. Theory 1~2% CRCa = HNPCC MSI-H in >95% HNPCC tumours Sporadic tumours –20% of colon Ca have MSI (1 in 20 = HNPCC) MSI as a Predictive Test of HNPCC

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

36. Frayling IM. Microsatellite Instability. Gut 1999; 45: 1-4. Theory NB Colorectal tumours

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

38. MSI as a Predictive Test of HNPCC Theory

39. Microsatellite Instability How does that test perform?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?How is the test best used?

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

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

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

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

45. Immunohistochemistry What does loss of reactivity signify?What does loss of reactivity signify? –Loss of antigenic sites of DNA mismatch repair protein Abnormal expression = MSIAbnormal expression = MSI Retained expression may = MSI or MSSRetained 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?How does it perform?

47. Immunohistochemistry –All* tumours with abnormal IHC have MSI –Not all tumours with MSI have abnormal IHC –MSH2:Sens MSI –MLH1:Sens <= MSISpec = MSI –On average:Sens = MSI

48. Immunohistochemistry

49. Immunohistochemistry How does it perform?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 EWMullarkey 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 …

50. MSH2MSH6MLH1PMS2 abcdefabcdefabcdefabcdef 1 2 3 4 5 6 7 8 9 10 11 12 13

51. Immunohistochemistry How does that test perform?How does that test perform? –Variably! –There are issues: –Biological reagents –Different antibodies, machines, methods, ag retrieval –Scoring subjective; not standardised –No EQAS (UK NEQAS IHC) –?MSI …

52. Is it better to do IHC or MSI? It can be best to do both,... but it depends on the question. Tumour testing

53. 1) Clinically HNPCC (>20%), but which gene? –IHC result: Abnormal IHC allows –Confident diagnosis of HNPCC –Targeted mutation detection –Mutation interpretation Normal IHC doesn’t change anything –MSI result: Lack of MSI excludes HNPCC - in that tumour/individual Presence of MSI confirms clinical suspicion, even if Normal IHC Tumour testing

54. 2) Hi-Mod Familial risk, <20% odds of HNPCC –MSI result: Lack of MSI excludes HNPCC - in that tumour/individual Presence of MSI suggests HNPCC, even if Normal IHC –IHC result: Abnormal IHC allows –Confident diagnosis of HNPCC –Targeted mutation detection –Mutation interpretation –BRAF testing may permit better discrimination of sporadics Normal IHC doesn’t change anything Tumour testing

55. Odds >20% Odds <20% Courtesy Dr Hans Vasen

56. Odds >20% Odds <20% Courtesy Dr Hans Vasen 47 21 20 26 22 4 3

57. Odds >20% Odds <20% Courtesy Dr Hans Vasen 47 21 20 26 22 4 3 364 56 38 48 8 3 308 304 4 1 6

58. Cost –MSI potentially semi-automatable, but needs DNA extraction –IHC needs extra pathologist time, MSI needs Clinical Scientists: £ –Both need equipment, reagents, space etc. –Can’t say if one is cheaper –Needs better data Tumour testing

59. Cost –MSI potentially semi-automatable, but needs DNA extraction –IHC needs extra pathologist time, MSI needs Clinical Scientists: £ –Both need equipment, reagents, space etc. –Can’t say if one is cheaper –Needs better data, and a health economist

60. Somatic BRAF V600E mutationSomatic BRAF V600E mutation –Associated with sporadic MSI+ MLH1- colon cancers (40%; 82/206) –Not present in HNPCC cancers (0%; 0/111) –Technically far easier to test for than MLH1 promoter methylation Domingo E et al. (2004) BRAF screening as a low-cost effective strategy for simplifying HNPCC genetic testing. J Med Genet 41:664-8. Molecular Tests for Lynch Syndrome

61. BRAF V600E Courtesy: Hood Mugalassi, All-Wales Molecular Genetics Laboratory, Institute of Medical Genetics, Cardiff V600E V600

62. Biomarkers in familial colorectal cancer screening Public Health Genetics Unit Expert workshop, 14th February 2006 www.phgu.org.uk/ www.phgfoundation.org/ (from 1/6/07)