LYNCH SYNDROME SCREENING: HIGHLIGHTS OF THE NORTHERN

ALBERTA EXPERIENCE 2009-2013

Jean Deschênes, CCI,

Dept. Of Lab. Medicine and Pathology

University of Alberta

Provincial AP-Cancer Lead

AHS Laboratories

Collaborators:

Dawna Gilchrist, U of A Hospital, Medical Genetics

Iyare Izevbaye, Edmonton Zone Molecular Pathology

Neil Chua, CCI, Medical Oncology

Ross McLean, Royal Alexandra Hospital, Pathology

Neelam Sandhu, Resident, AnatomicPathology

U of Alberta, Edmonton.

CONFLICTS

• Very conflicted with my 15 y.o. little angel

• BUT

• NOTHING RELEVANT TO THIS TOPIC

OBJECTIVES

• EXPLAIN HOW PATHOLOGISTS CAN CONTRIBUTE TO LYNCH SYNDROME SCREENING

• LIST FACTORS THAT CAN BE USED TO SELECT PATIENTS FOR LYNCH SYNDROME SCREENING

• EXPLAIN THE DIAGNOSTIC ALGORITHM INDICATED FOR PATIENTS WITH ABNORMAL MMR TEST RESULTS

• INTERPRET THE SIGNIFICANCE OF THE VARIOUS MMR STAINING PATTERNS THAT CAN BE OBSERVED

The problem in 2008

• Testing since 2006-7

Originally dependent on clinicians and MSI PCR testing

MSI BASED

IHC ADJUNCT (NOT SET UP)

CLINICIAN BASED—GI GROUPS,SURGEONS,ETC…

CONSENT REQUIRED FOR TESTING

The problem in 2008

LS is massively under-recognized

Amsterdam criteria too restrictive ~25 - 50% of individuals with LS do not meet

Amsterdam –Bethesda and revised B. criteria are more sensitive but much less specific….

Accuracy of family history is poor in the small family era

Clinical interest is unreliable Estimated 1.2% of all individuals with LS are

aware of their LS diagnosis (Hampel et al 2011)

Solution – Tumor-based Screening for LS?

The Team,2008

Cross Cancer IHC lab personnel Sarah Mitchell Rick Linford Clinical and Laboratory Colleagues: Ross McLean, GI pathology Dawna Gilchrist, medical genetics Eoin Lalor, gastro-enterology Imran Mirza, molecular pathology

Edmonton Testing

• Team meetings in 2008

• 2009: MMR IHC as prime mover, new emphasis on pathology involvement

• 2012: GI biomarker group starts using MMR status to select therapy in aggressive stage II CRC

• Late 2013: surge in testing of GYN tumors

OLD VS NEW, 2008 STEPS OLD

ALGORYTHM NEW ALGORYTHM

DECIDE WHO NEEDS TO BE TESTED+ REQUEST TESTING

CLINICIAN PATH+CLIN

PRIMARY TESTING DEMANDS PATIENT CONSENT

YES NO

NOTES THE POSSILIBTY OF A MMR DEFICIENT TUMOR

PATH+CLIN PATH+CLIN

TRACKING CASES TO ENSURE GENETIC REFERRAL IF NEEDED

CLINICIAN CLIN+PATH

RESPONSIBLE FOR GENETIC FU CLINICIAN CLINICIAN PRIMARY TESTING

MSI----MMR MMR----MSI

Paper wht proof of concet de MS path

Joost et al. BMC Clinical Pathology 2013, 13:33

MS Path score

Loss MLH2/MLH6, (PMS2)

Initiation: pathologist

+-clinician interaction

IHC

1) BRAF v600 Mutation +

2) MLH1 PROMOTER

Hypermethylation

Negative results

MSI

PCR

Positive results

Normal IHC

lossMLH1

Both PRESENT

MSI-H

MSI-L or MSS

GENETICS

ONE ABSENT

1 sporadic MSI-High, CIMP-high BRAF mutation Serrated----- (8-10%) 2 CIMP-H with partial methylation BRAF mut. Serrated--------------- (8%) 3 CIMP low, KRAS mutation CIN, Adenoma or serrated------ (20%) 4 CIMP negative, mostly adenomas FAP, MUTYH, CIN ------------------(57 %) 5 LS: BRAF negative, adenomas, KRAS Mutation--------------------------- (3-5%)

Adapted from Jass 2007. CIMP: CPG ISLAND METHYLATOR PHENOPTYPE.

BRAF MUTATIONS & MLH1 PROMOTER METHYLATION STATUS in MLH1-DEFICIENCY

MLH1 promoter

hypermethylation

LYNCH

-0-2% OF LYNCH=BRAF MUTATION

-5-6% of LYNCH= MLH1 HPERMETHYLATION

-2/3 OF NON-LYNCH HAVE BRAF MUTATION

LACK OF BRAF MUTATION DOES NOT MEAN LYNCH

BRAF mutation

Initiation: pathologist

+-clinician interaction

IHC

BRAF v600 Mutation +

MLH1 PROMOTER

Hypermethylation

Negative results

MSI

PCR

Positive results

Normal IHC

lossMLH1

Loss MLH2/MLH6, (PMS2)

Both PRESENT

MSI-H

MSI-L or MSS

GENETICS

ONE ABSENT

Diagnostic testing

• Immunohistochemistry (IHC):MLH1, MSH2, MSH6 and PMS2

• Parallel MSI testing was performed: mononucleotide BAT-25, BAT-26, MON0-27, NR-21, NR-24 using PCR.

• Qiagen, MLH1 promoter hypermethylation, area C (see Deng et al 1999 and Grady et al. 2001)

• Snapshot platform for BRAF v600E and v600Ec mutations

• All ancillary tests were done in the EZ molecular and IHC labs

• Gene sequencing done in the Edmonton Genetics Laboratory (GLS);EpCam sequencing done on all MSH2 abnormal cases with no gene abnormality

SITES REQUESTING TEST

EDMONTON AND NORTH ZONE

RED DEER LETHBRIDGE CALGARY OOP TOTAL

NUMBER OF TUMORS SENT FOR TESTING

628 124 14 42 3 812

NUMBER OF PATIENTS

603 (610 profiles) 121 14 39 3 781

Comments/N (PTS,%) TUMORS W. all NORMAL PROFILE

462 (441, 73,1 %) 94 (92 ,76,0%)

14/14 PATHS. 25 (22, 68,8 %)

N (%) TUMORS W. ABNORMAL PROFILE

166 (162, 26,9 %). 30 (29, 24,0 %) 3 cases were Abnormal (0)

10 ( 10, 31,2 %) * 7 pts with incomplete profile

TESTING BY LOCATION OF PATIENTS

TUMOURS SENT FOR TESTING BY SITE

site Number of tumors

COLON 732

OTHER GI TRACT

16

NON LUMINAL GI

6

GYN 63

GU 5

SOFT TISSUE 4

OTHER,NOS 6

TOTAL 812

PATHOLOGISTS MEDICAL ONCOLOGISTS

CLINICAL STAFF, NOS

GENETICS TOT.

NUMBER OF CASES TESTED

413 (396)—65,7% 85 (85)—14,1% 24 (23)—3,8% 106 (99)--16,4% 628(603)

CASES WITH NORMAL PROFILE

281 (269) 67.9 %

68 (68) 80.0 %

20(19) 82,6 %

93(86) 86,7 %

462 (442) 73,3 %

CASES ABNORMAL PROFILE

132 (127) 32,1 %

17 (17) 20.0 %

4(4) 17,4 %

13(13) 13,1 %

166 (161) 26,7 %

CASES W RECOMMENDED REFERRAL TO MED. GENETICS

54 13,6 %

9 10,6 %

4 17,4 %

7 7,1 %

74 12,3%

CASES ACTUALLY REFERRED TO GENETICS

30 7 3 7 47

CASES WHO OBTAINED GC

20 5 3 7 35

ABNORMAL GERMLINE TESTING

15 (3,7%) (4 WITH NO GERMLINE MUTATIONS)

5 (5,9%) 1 (4,3%) 2 (2,0%) (3 WITH NO GERMLINE MUTATIONS)

23 (3,7%)

EDMONTON AND NORTH ZONE REFERRAL PATTERNS BY CARE PROVIDER

TUMORS PATIENTS PROFILES MEDICAL GENETICS MUTATIONS

MLH1 PMS2 104 101 9/ 27 cases 7=MLH1, 2= NO MUT (1 hm case), 1=N/A

MSH6 MLH1 PMS2 1 1 0/1 N/A

PMS2 8 8 3/7 2=PMS2, 1=moved

MSH2 1 1 0/1 N/A

MSH6 4 4 1 out of 4 1=NO MUTATION

MSH2 MSH6 10 10 7/10 5= MSH2, 1 PT = NO MUTATION. 1= MSH6 VUS, likely LS

All NORMAL 0/2 N/A

MSH6-U 2 1 0/1 N/A

(MLH1 PMS2)U 2 2 0/1 N/A

TOTAL 132 128*** 20/54 14 (+1 VUS)

PATTERNS OF MMR ABNORMALITIES OBSERVED IN PATHOLOGY REFERRED CASES

***ONE PATIENT HAD 2 ABNORMAL PROFILES

Sourrouille et al. Familial Cancer 2013;12:27-33

4 of 18 MMR deficient tumours have somatic mutations explaining the silencing.

From Mesenkamp et al,

Of 26 unexplained MLH1 deficient tumors,18

could be studied for SOMATIC mutations.

-- 17/18 contained mutations, with 8/18 likely

of sufficient quality/quantity to expain the

deficit.

Of 15 unexplained MSH2 deficient tumors,7

could be studied for SOMATIC mutations.

-- 6/7 contained mutations, with 5/7 likely of

sufficient quality/quantity to explain the

deficit.

Gastroenterology 2014 , DECEMBER,147: 1308

Colon and Endometrial Cancers with Mismatch Repair Deficiency can Arise from Somatic, Rather Than Germline, Mutations.

Haraldsdottir S , Hampel H , Tomsic J , Frankel WL , Pearlman R , de la Chapelle A , Pritchard C

70% OF TESTED TUMORS WITH SIGNIFICANT SOMATIC GENETIC CHANGES WHICH EXPLAIN THE MMR-D STATUS. (N=32)

ALL GENES INVOLVED.

BOTH CRC AND EM CANCERS.

MMR ABNORMALITIES correlated with MSI STATUS

MSI-H MSI non high

total

MMR deficient 45 2 47 PPV

95,6 %

Non

consecutive

MMR

Proficient

4 477 481 NPV

99.2 %

total 528

MMR ABNORMALITIES correlated with MSI STATUS

MSI-H MSI non high

total

MMR deficient

159 7 166 (26,9%)

Projected EZ and NZ

MMR

Proficient

4 458 462 (73,1 %)

total 163 465 628 (100%)

SE:97,5 % SP:98,5% CONCORDANCE: 98,2 %

HYPER-METHYLATION NO DETECTED HYPERMETHYLATION

TOTAL

BRAF +

93 4 97

BRAF -

26 23 49

TOTAL 119 27 146

BRAF AND MLH1 PROMOTER HYPERMETHYLATION STATUS

N=146 WITH NO OR VARIABLE MLH1-PMS2 EXPRESSION

Screening after 70 years of age (n=241 t.)

• Pathologists-referred cases dominate in this age group (81 % of cases, vs 62 % under 71)

• Non-specificity of pathology referral criteria (MS Path less than 50%)

• For all groups (oncologists, geneticist, etc), more abnormal MLH1 results are noted in proportion (90,2 % of results in 71+ vs 70,8% under 71)

• The patients under 71 have more meaningful MLH1 abnormalities after BRAF and HM studies: 32/80 (40%) suspicious for LS vs 11/83 (13,2%) after 70

Gyn tumor results

• 63 tumors from 58 patients ( path=26, gen=34 (29))

• 10 tumors (9 pts) from Calgary

• 53 tumors (49 pts) from the North

• 3 MSH2-MSh6, 42 normal (38 pts), 7 MLH1-PMS2, 1PMS2

• 8 patients recommended and referred to genetics: • 3 cases MLH1-PMS2 with HM, 1 NOMUT, 1 not pursued, 1 declined

• 3 cases MSH2-MSH6, 2 – NOMUT, 1-DECLINED

• 1 case MLH1-PMS2 without HM, --MLH1 MUTATION found

• 1 case PMS2, lost to follow up (moved out of town)

• Much increased in 2013-2014, issues created by surge in cases.

Lynch Syndrome Screening Algorithm,2015

Entry IHC for MMR

proteins All 4 normal

Positive

MSH2 or

PMS2, or

MSH6

abnormalities

MLH1

abnormal

BRAF mut analysis

and MLH1

hypermethylation

studies Genetic

counseling

Negative BRAF

mutated

MLH1

hypermethylation

studies

STOP

STOP

BRAF

normal

Pt ≥ 60 y.o.

Pt < 60 y.o.

+FH

Pathology & Clinical Criteria**

Patient less than 45

Special request

YES------------NO

MSI testing

HIGH---Low-stable

Key findings

• MMR IHC : reliable,sensitive and specific

• Pathologists can function as initiators

• Screening criteria are evolving for CRC

• Pathologist’s involvement within a multidisciplinary framework is productive

• MLH1 hypermethylation and BRAF mutation testing results are easily and reliably performed;

• Either can be used to rule out LS in the appropriate setting ( age and family history) and reduce costs ,especially when integrated in the screening pipeline to help assess the need for Genetics referrals

Key findings

• Cost-effectiveness and yield are markedly hampered by poor follow-up patterns in the clinical community or from patients lack of interest

• Pathology activity should not occur in silos.

• The status of GYN tumour-driven LS screening is still evolving in our province

• An official request for Provincial Health Technology Assessment is in progress to help

• clarify the best approach to screening , • secure appropriate province-wide resource allocation • determine strategic steps necessary to optimize uptake and yield.

Acknowledments

IHC lab personnel

Sarah Mitchell

Rick Linford

Robin Stock

Clinical and Laboratory Colleagues:

Ross McLean, GI pathology

Dawna Gilchrist, medical genetics

Eoin Lalor, gastro-enterology

Imran Mirza, molecular pathology

Iyare Izevbaye, molecular pathology

?

THANK YOU!