CMGS LS FAP MAP Training Day, 30/11/2009CMGS LS FAP MAP Training Day, 30/11/2009

Colorectal Cancer BP Training DayColorectal Cancer BP Training Day

Ian M FraylingIan M FraylingConsultant Genetic PathologistConsultant Genetic Pathologist

Director, Laboratory Genetics Service for WalesDirector, Laboratory Genetics Service for WalesInstitute of Medical Genetics, CardiffInstitute of Medical Genetics, Cardiff

InSiGHT workshop, Palma, Majorca, April 28-29InSiGHT workshop, Palma, Majorca, April 28-29 thth 20062006

Identification of Lynch Syndrome Identification of Lynch Syndrome (MSI/IHC)(MSI/IHC)

Ian M FraylingIan M FraylingConsultant Genetic Pathologist & Cancer GeneticistConsultant Genetic Pathologist & Cancer Geneticist

Director, Laboratory Genetics Service for WalesDirector, Laboratory Genetics Service for WalesInstitute of Medical Genetics, CardiffInstitute of Medical Genetics, Cardiff

Biomarkers in familial colorectal cancer screening workshop, London, Feb Biomarkers in familial colorectal cancer screening workshop, London, Feb 20062006

Microsatellite InstabilityMicrosatellite Instability

Ian M FraylingIan M FraylingConsultant Genetic Pathologist & Cancer GeneticistConsultant Genetic Pathologist & Cancer Geneticist

Director, Laboratory Genetics Service for WalesDirector, Laboratory Genetics Service for WalesInstitute of Medical Genetics, CardiffInstitute of Medical Genetics, Cardiff

Sporadic or LS?

Lynch SyndromeLynch Syndrome• History:History:

– Warthin: Family ‘G’ Warthin: Family ‘G’

Lynch SyndromeLynch Syndrome• History:History:

– Lynch Syndrome I: site-specific colon cancerLynch Syndrome I: site-specific colon cancer– Lynch Syndrome II: cancer family syndrome Lynch Syndrome II: cancer family syndrome

Picture courtesy of Dr Patrick Lynch, MD Anderson Cancer Centre, Houston, TXPicture 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.

Lynch Syndrome: 2007Lynch Syndrome: 2007• Various names for Lynch syndrome have been used in the past century. A Various names for Lynch syndrome have been used in the past century. A

workshop in Amsterdam in 1989 agreed upon the name "HNPCC", because workshop in Amsterdam in 1989 agreed upon the name "HNPCC", because at that time the syndrome was unknown to most doctors. This name clarified at that time the syndrome was unknown to most doctors. This name clarified that the syndrome described an inherited form of CRC. The appropriateness that the syndrome described an inherited form of CRC. The appropriateness of the name was discussed again at an international meeting in Bethesda in of the name was discussed again at an international meeting in Bethesda in 2004. Most participants considered the term HNPCC to be inappropriate, 2004. Most participants considered the term HNPCC to be inappropriate, since the syndrome is also associated with many other tumours. It was since the syndrome is also associated with many other tumours. It was proposed that the name "proposed that the name "Lynch syndromeLynch syndrome" should be reintroduced, and that " should be reintroduced, and that this name should be reserved for families with strong evidence of MMR 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 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 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 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 that meet the original Amsterdam criteria, but do not have evidence for MMR deficiency, are referred to as having familial CRC.deficiency, are referred to as having familial CRC.

• Vasen HFA et al. (2007) Vasen HFA et al. (2007) J Med GenetJ Med Genet 4444:353-362:353-362• http://jmg.bmj.com/cgi/content/abstract/44/6/353http://jmg.bmj.com/cgi/content/abstract/44/6/353

Lynch SyndromeLynch Syndrome• History:History:

– Lynch I: hereditary site-specific colon cancerLynch I: hereditary site-specific colon cancer– Lynch II: cancer family syndrome Lynch II: cancer family syndrome

• TumoursTumours– principalprincipal: colon & rectum: colon & rectum– majormajor: endometrium: endometrium– minorminor: : ovary, stomach, small intestine, pancreas, hepato-ovary, stomach, small intestine, pancreas, hepato-

biliary tract, pelvi-ureter, skin (sebaceous & keratoacanthoma - biliary tract, pelvi-ureter, skin (sebaceous & keratoacanthoma - Muir-TorreMuir-Torre), CNS glioblastoma), CNS glioblastoma

• Germline mutation in a DNA mismatch repair geneGermline mutation in a DNA mismatch repair gene

ICG Clinical Definition of LS1 Familial clustering of colorectal and/or endometrial cancer2 Associated cancers: gastric, ovary, ureter/renal pelvis, brain, small bowel,

hepatobiliary tract and skin (sebaceous tumours)3 Development of cancer at an early age4 Development of multiple cancers5 Features of colorectal cancer: (a) proximal colon (right-sided), (b) improved survival,

(c) multiple, (d) mucinous, (e) lymphocytic infiltration and lymphoid aggregates6 Features of colorectal adenoma: (a) one to a few; (b) villous histology, (c) high grade

dysplasia, (d) rapid adenoma to carcinoma progression (probably)7 High frequency of Microsatellite Instability (MSI)8 Immunohistochemistry: loss of MLH1, MSH2, or MSH6 protein expression in tumours9 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.

ICG Clinical Definition of LS1 Familial clustering of colorectal and/or endometrial cancer2 Associated cancers: gastric, ovary, ureter/renal pelvis, brain, small bowel, hepatobiliary

tract and skin (sebaceous tumours)3 Development of cancer at an early age4 Development of multiple cancers5 Features of colorectal cancer: (a) proximal colon (right-sided), (b) improved survival, (c)

multiple, (d) mucinous, (e) lymphocytic infiltration and lymphoid aggregates6 Features of colorectal adenoma: (a) one to a few; (b) villous histology, (c) high grade

dysplasia, (d) rapid adenoma to carcinoma progression (probably)7 High frequency of Microsatellite Instability (MSI)Microsatellite Instability (MSI)8 ImmunohistochemistryImmunohistochemistry: loss of MLH1, MSH2, or MSH6 protein expression in tumours9 Germline mutation in MMR geneGermline 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.

CMGS BP Guidelines

• 1996? • Workshop March 2002• Within Uk Guidelines (PHGU)• Within international guidelines

LS: DiagnosisLS: 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

LS Family HistoryLS Family History

CaecumCaecum45y45y

BowelBowel““young”young”

TCC UreterTCC Ureter35y35y

EndometrialEndometrial38y38y

Womb 40’sWomb 40’sBowel 72yBowel 72y

??

??

F+WF+W85y85y

LS: FH as a Diagnostic TestLS: 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

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

LS: FH as a Diagnostic TestLS: 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.

Colorectal Cancer: ?LSColorectal Cancer: ?LS

CRCa 45y

CRCa63y

CRCa 72y

Colorectal Cancer: ?LSColorectal Cancer: ?LS

CRCa 45y

CRCa63y

CRCa 72y

10%

Colorectal Cancer: ?LSColorectal Cancer: ?LS

CRCa 45y

CRCa63y

CRCa 29y

CRCa 72y

20%

Colorectal Cancer: ?LSColorectal Cancer: ?LS

CRCa 45y

CRCa63y

CRCa 29y

ENCa 44y CRCa 72y

60%

Colorectal Cancer: ?LSColorectal Cancer: ?LS

CRCa 45y

CRCa63y

CRCa 29y

CRCa 42y ENCa 54y

80%

LS: FH as a Diagnostic TestLS: FH as a Diagnostic TestWijnen Model

Mutation probability by family type (P1)

0

20

40

60

80

100

15 20 25 30 35 40 45 50 55 60 65 70 75 80

mean age of colorectal cancer

Pm

ut (

%) EnCa+ AC+

EnCa- AC+

EnCa+ AC-

EnCa- AC-

Molecular Tests for Lynch SyndromeMolecular Tests for Lynch Syndrome

• Microsatellite instability (MSI)Microsatellite instability (MSI)

• Immunohistochemistry (IHC)Immunohistochemistry (IHC)

• BRAF BRAF V600EV600E

• Germline mutation testingGermline mutation testing

Molecular Tests for Lynch SyndromeMolecular Tests for Lynch Syndrome

• When should testing be done?When should testing be done?

Molecular Tests for Lynch SyndromeMolecular 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

Molecular Tests for Lynch SyndromeMolecular 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

Microsatellite InstabilityMicrosatellite Instability• Different laboratories use:Different laboratories use:

• Different markers, PCR conditions, gels, machines, Different markers, PCR conditions, gels, machines, DNA extractionDNA 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

Microsatellite InstabilityMicrosatellite Instability

• What is MSI?What is MSI?

– The presence of extra alleles at a microsatelliteThe presence of extra alleles at a microsatellite

– A number of microsatellites must exhibit instability to A number of microsatellites must exhibit instability to diagnose MSIdiagnose MSI

Microsatellite InstabilityMicrosatellite Instability

• What does it signify?What does it signify?

– MSI-H signifies loss of DNA Mismatch RepairMSI-H signifies loss of DNA Mismatch Repair

Microsatellite Instability: 1998 - 2007Microsatellite 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.

Microsatellite InstabilityMicrosatellite Instability

• How does it perform?How does it perform?

Microsatellite Instability: 1993Microsatellite Instability: 1993

• Patterns / definitions– MSI-H

– MSI-L

– MSS

Microsatellite Instability: 2003Microsatellite 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.

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 HNPCCMSI 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)

MSI as a Predictive Test of HNPCCMSI as a Predictive Test of HNPCC

MSI as a Predictive Test of HNPCCMSI as a Predictive Test of HNPCC

MSI detected in: One Ca One Ad Two Ca Two Ad

Sensitivity 95% 95% 90% 81%

Specificity 86% 98% 98% >99%

PPV 6% 32% 29% 95%

NPV >99% >99% 98% >99%

Frayling IM. Microsatellite Instability. Gut 1999; 45: 1-4.

Theory

• NB Colorectal tumours

MSI as a Predictive Test of HNPCCMSI as a Predictive Test of HNPCC

MSI detected in: One Ca One Ad Two Ca Two Ad

Sensitivity 95% 95% 90% 81%

Specificity 86% 98% 98% >99%

PPV 6% 32% 29% 95%

NPV >99% >99% 98% >99%

Frayling IM. Microsatellite Instability. Gut 1999; 45: 1-4.

Theory

• Sporadics – ?effect of prior odds

Predictive Value of MSI testing: sens 95%, spec 85%

0%

20%

40%

60%

80%

100%

0% 20% 40% 60% 80% 100%

Odds of Lynch Syndrome

PV PPV

NPV

MSI as a Predictive Test of HNPCCMSI as a Predictive Test of HNPCC

Theory

Microsatellite InstabilityMicrosatellite Instability

• How does that test perform?How does that test perform?

– OK as an includer, but better as an excluder of OK as an includer, but better as an excluder of HNPCCHNPCC

Microsatellite InstabilityMicrosatellite Instability

• How is the test best used?How is the test best used?

Microsatellite InstabilityMicrosatellite Instability

• How is the test best used?How is the test best used? See later! See later!

ImmunohistochemistryImmunohistochemistry

• What is IHC?What is IHC?

– Detection of protein expression in tissues by antibodiesDetection of protein expression in tissues by antibodies

ImmunohistochemistryImmunohistochemistry

• In an ideal worldIn an ideal world

Dr Mark Arends, University of Cambridge Department of PathologyDr Mark Arends, University of Cambridge Department of Pathology

MSH2MSH2 MLH1MLH1

ImmunohistochemistryImmunohistochemistry• Different laboratories use:Different laboratories use:

• Different fixation, antibodies, antigen retrieval Different fixation, antibodies, antigen retrieval conditions, machines (manual), detection conditions, machines (manual), detection

• Different pathologistsDifferent pathologists

• Different definitions of “abnormal”Different definitions of “abnormal”

• No EQAS No EQAS

• Different tumours, case mixesDifferent tumours, case mixes

• Different prior odds of HNPCC, etc.Different prior odds of HNPCC, etc.

ImmunohistochemistryImmunohistochemistry

• What does loss of reactivity signify?What does loss of reactivity signify?

– Loss of antigenic sites of DNA mismatch repair proteinLoss 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 reactNB dysfunctional protein may still react

– All* tumours with abnormal IHC have MSIAll* tumours with abnormal IHC have MSI

– Not all tumours with MSI have abnormal IHCNot all tumours with MSI have abnormal IHC

ImmunohistochemistryImmunohistochemistry

• How does it perform?How does it perform?

ImmunohistochemistryImmunohistochemistry

• How does it perform?How does it perform?

– All* tumours with abnormal IHC have MSIAll* tumours with abnormal IHC have MSI

– Not all tumours with MSI have abnormal IHCNot all tumours with MSI have abnormal IHC

– MSH2:MSH2: Sens <= MSISens <= MSI Spec > MSISpec > MSI

– MLH1:MLH1: Sens <= MSISens <= MSI Spec = MSISpec = MSI

– On average:On average: Sens <= MSISens <= MSI Spec >= MSISpec >= MSI

Predictive Value of IHC testing: sens 90%, spec 85%

0%

20%

40%

60%

80%

100%

0% 20% 40% 60% 80% 100%

Odds of Lynch Syndrome

PV PPV

NPV

ImmunohistochemistryImmunohistochemistry

ImmunohistochemistryImmunohistochemistry

• How does it perform?How does it perform?

– Mismatch repair protein immunohistochemistry within Mismatch repair protein immunohistochemistry within a diagnostic setting – An inter-laboratory studya diagnostic setting – An inter-laboratory study

• Mullarkey M, Grady AO, Jasani B, Stephens M, Dodson A, Mullarkey M, Grady AO, Jasani B, Stephens M, Dodson A, Munson P, Haperfield L, Arends M, Frayling IM, Kay EW Munson P, Haperfield L, Arends M, Frayling IM, Kay EW

– 13 tumours in a TMA x 6 laboratories …13 tumours in a TMA x 6 laboratories …

MSH2 MSH6 MLH1 PMS2

a b c d e f a b c d e f a b c d e f a b c d e f

1                                                

2                                                

3                                                

4                                                

5                                                

6                                                

7                                                

8                                                

9                                                

10                                                

11                                                

12                                                

13                                                

ImmunohistochemistryImmunohistochemistry

• How does that test perform?How does that test perform?

– Variably!Variably!

– There are issues:There are issues:

– Biological reagentsBiological reagents

– Different antibodies, machines, methods, ag retrievalDifferent antibodies, machines, methods, ag retrieval

– Scoring subjective; not standardisedScoring subjective; not standardised

– No EQAS (UK NEQAS IHC)No EQAS (UK NEQAS IHC)

– ?MSI …?MSI …

• Is it better to do IHC or MSI?

It can be best to do both,

... but it depends on the question.... but it depends on the question.

Tumour testingTumour testing

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 testingTumour testing

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 testingTumour testing

Odds >20%

Odds <20%

Courtesy Dr Hans Vasen

Odds >20%

Odds <20%

Courtesy Dr Hans Vasen

47

2120

2622

4

3

Odds >20%

Odds <20%

Courtesy Dr Hans Vasen

47

2120

2622

4

3

364

563848

8 3

308304

4 1

6

• 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 testingTumour testing

Tumour testingTumour testing• 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

• Somatic Somatic BRAFBRAF V600E mutation V600E mutation

– Associated with sporadic MSI+ MLH1- colon cancers Associated with sporadic MSI+ MLH1- colon cancers (40%; 82/206)(40%; 82/206)

– NotNot present in HNPCC cancers (0%; 0/111) present in HNPCC cancers (0%; 0/111)

– Technically far easier to test for than Technically far easier to test for than MLH1MLH1 promoter promoter methylationmethylation

Domingo E Domingo E et al.et al. (2004) BRAF screening as a low-cost effective strategy for (2004) BRAF screening as a low-cost effective strategy for simplifying HNPCC genetic testing. simplifying HNPCC genetic testing. J Med GenetJ Med Genet 4141:664-8.:664-8.

Molecular Tests for Lynch SyndromeMolecular Tests for Lynch Syndrome

BRAFBRAF V600E V600E

Courtesy: Hood Mugalassi, All-Wales Molecular Genetics Laboratory, Institute of Medical Genetics, CardiffCourtesy: Hood Mugalassi, All-Wales Molecular Genetics Laboratory, Institute of Medical Genetics, Cardiff

V600E

V600

Biomarkers in familial Biomarkers in familial colorectal cancer colorectal cancer

screening screening

Public Health Genetics UnitPublic Health Genetics Unit

Expert workshop, 14th February Expert workshop, 14th February 20062006

www.phgu.org.uk/

www.phgfoundation.org/

(from 1/6/07)(from 1/6/07)