To discuss. Predictive Value of BRCA Histology. Tumour morphology: BRCA-1. Hereditary breast-ovarian cancer (HBOC)

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1 The role of histopathology in iden2fying gene2c condi2ons associated with gynaecological malignancies, with special a7en2on to pathological manifesta2ons of Lynch syndrome Dr Raji Ganesan Consultant Gynaecological Pathologist Birmingham To discuss List of hereditary cancers in the female genital tract Pathology of high grade serous carcinoma with reference to BRCA Lynch syndrome pathological immunohistochemistry and screening strategies Overview of pathology in other hereditary syndromes (no guarantees for answers) Hereditary neoplastic syndromes of the female genital tract BRCA1/2 syndromes Lynch syndrome Peutz-Jeghers syndrome Dicer syndrome Cowden syndrome Gorlin syndrome Li-Fraumeni syndrome Hereditary leiomyomatosis Tuberous sclerosis complex Holman et al. Hematol Oncol Clin North Am 2012;26:13-29 Hereditary breast-ovarian cancer (HBOC) BRCA1 Dominant inheritance pattern of susceptibility Mutation in 17q21 (>100 mutations) 55-65% lifetime risk of breast cancer 11-17% lifetime risk of ovarian cancer Possible gastric & pancreas cancer risk BRCA2 Dominant inheritance pattern of susceptibility Mutation in 13q % lifetime risk of breast cancer including male 11-17% lifetime risk of ovarian cancer Possible prostate, pancreas, gastric cancer risk Am J Human Genetics 2003;72(5): J Natl Cancer Inst 2002; 94: Tumour morphology: BRCA-1 High-grade serous, undifferentiated or pseudo-endometrioid ( SET ) SET features: Predict BRCA 1 mutation Tumor intraepithelal lymphocytes (TILs) Predictive Value of BRCA Histology Negative predictive value (>95%) Positive predictive value (26%) But high likelihood that tumor with BRCA histology is associated with BRCA germline mutation if fallopian tube is also involved (43%) Fujiwara et al. Am J Surg Pathol 2012;36: Soslow et al. Mod Pathol 2012;25: Fujiwara et al. Am J Surg Pathol 2012; 36:

2 Risk reducing surgery BRCA Lessons High incidence of serous tubal intraepithelial serous carcinoma (STIC) in BRCA1/2 STIC also seen in tubal mucosa from patients with ovarian & peritoneal high grade serous carcinoma STIC associated with p53 mutations Am J Surg Pathol 2001;25: Am J Surg Pathol 2006;30: Am J Surg Pathol 2007;31: Fallopian Tube Surgical Pathology: Risk Reducing Salpingo-oophorectomy Serial sections of entire fallopian tube (longitudinal sections of fimbria) at 2-3 mm SEE-FIM protocol p53 mutation/expression is not a criterion for diagnosis of STIC Diagnosis should be made using standard morphologic criteria Virchows Arch 2007;450:25-29 Serous Tubal Intraepithelial Carcinoma STIC Increased nuclear/cytoplasmic ratio Increased mitotic activity Disorganized growth Nucleoli often present Typically seen in fimbria or distal tube 10% seen in ampulla/isthmus, 10-20% bilateral and 20% multifocal (approximates) Incidence of non invasive lesions of the tube in BRCA carriers Age: 5% <40 yrs vs 56% >60 yrs BMI: 18% <25 kg/m 2 vs 31% >25 kg/mm 2 Oral contraceptive use: 6 yrs OCP: Normal tubal mucosa 4 yrs OCP: p53 signature 2.7 yrs OCP: STIC Am J Surg Pathol 2010;34: Vicus et al. Gynecol Oncol 2010;118:295-8 The p53 Signature: Possible Precursor Lesion Histologically normal tubal epithelium At least 12 consecutive p53 mutant epithelial cell nuclei Normal proliferative index (Ki-67) Repor@ng p53 Posi@ve/overexpression/significant if diffuse, strong, nuclear reac@vity (70 80% of cells suggested) indica@ve of missense muta@on Nega@ve/null/significant if complete absence of staining indica@ve of nonsense muta@on or dele@on resul@ng in truncated protein not detected by immunohistochemistry Most stain with p53 weak, heterogenous staining of 50% or less of nuclei Report as wild type or mutant/aberrant NOT nega2ve/posi2ve 2

3 Primary site assignment in all cases SEE FIM protocol sampling Fallopian tube if: there is STIC with ovarian, peritoneal or tubal carcinoma of ovarian disease volume Ovary if: dominant ovarian mass and no STIC despite SEE FIM sampling Primary peritoneal if: no STIC + ovaries normal sized, ovarian tumour confined to surface or cortex (less than 5 x5mm), involvement in extra ovarian sites less than ovarian cor@cal involvement Tubo ovarian if: undesignated Singh N, Gilks CB, Wilkinson N, McCluggage WG. Assignment of primary site in high-grade serous tubal, ovarian and peritoneal carcinoma: a proposal. Histopathology Aug;65(2): LYNCH SYNDROME (HNPCC) Autosomal dominant mode of inheritance Predisposes to numerous malignancies not just colon Often early age of onset One defective allele is inherited; 2 nd hit happens during patient s lifetime LYNCH SYNDROME (HNPCC) Lifetime risk of cancer in women Endometrium 25-70% Colorectum 25-50% Ovary 10% Breast 11% Ureter and renal pelvis 10% Stomach 10% Pancreas 2% Small bowel 5% Biliary tract 2% Brain (usually glioblastoma as seen in Turcot syndrome) 4% Sebaceous gland adenomas and keratoacanthomas in Muir-Torre syndrome Lynch Syndrome (HNPCC) Due to germline mutations in mismatch repair (MMR) genes* 4 genes have been identified: MSH2, MSH6, MLH1, and PMS2 Epigenetic methylation of MLH1 can also lead to dysfunction - not part of Lynch Syndrome (HNPCC)*** Background: The MMR system van Lier et al. J. Cell. Mol. Med During DNA replication, insertions or deletions of one or more nucleotides and single nucleotide mismatches may occur MSH2 and MSH6 form a heterodimer and recognize the mismatch; MLH1 and PMS2 dimerize and bind to the MSH2-MSH6 complex The complex of four proteins activates an exonuclease to perform the DNA repair 3

4 Microsatellite Instability (MSI) Morphological features Endometrioid carcinoma 80% * Clear cell carcinoma <5% Undifferentiated <5% Carcinosarcoma (MMMT) <5% Serous <5% Mucinous <5% Broaddus RR, Lynch HT, Chen LM, et al. Pathologic features of endometrial carcinoma associated with HNPCC. Cancer 2006;106: Tumour topography 30% of lower uterine segment tumors are Lynch syndrome-associated LS-associated LUS tumors may show overlapping morphologic and immunophenotypic features of endocervical and endometrial carcinoma Tumour Morphology: Endometrioid Peritumoral lymphocytes Tumor infiltrating lymphocytes (TILs) Dedifferentiated endometrial carcinomas Mixed patterns e.g., endometrioid & mucinous Westin SN, et al. J Clin Oncol 2008;26: Shia et al. Hum Pathol 2008;39: Screening For Lynch Syndrome Clinical Pathological Clinical & pathological All colorectal, endometrial, ovarian cancers WHY SCREEN ENDOMETRIAL CARCINOMA FOR LYNCH SYNDROME? 20-year risk following diagnosis of endometrial cancer: Colorectal cancer 48% Kidney, renal pelvis or ureter cancer 11% Breast cancer 11% Bladder cancer 9% J Natl Cancer Inst 2013;105:

5 Risk of Carcinoma in Lynch Syndrome is MMR-Dependent Endometrial Ovarian Colorectal MLH1 21% 4% 41% MSH2 54% 29% 48% MSH6 16% 1% 12% Screening for LS Genealogy based criteria Amsterdam, Bethesda, SGO Histology based criteria Lower uterine segment Specific tumour components/subtypes IHC results Bonadona et al. JAMA 2011;305: Amsterdam I Criteria Three or more family members with confirmed diagnosis of colorectal cancer, one of whom is a first degree rela@ve of the other two. Two successive affected genera@ons. One or more colorectal cancers diagnosed under age 50 years Familial adenomatous polyposis (FAP) has been excluded. Amsterdam II Criteria Three or more family members with LS/HNPCC-related cancers, one of whom is a first degree rela@ve of the other two. Two successive affected genera@ons. One or more of the LS/HNPCC-related cancers diagnosed under age 50 years Familial adenomatous polyposis (FAP) has been excluded. Vasen et al, Gastroenterology 1999 Vasen et al, Gastroenterology 1999 Revised Bethesda Criteria Colorectal cancer diagnosed in a pa@ent who is less than 50 years of age. Presence of synchronous, metachronous colorectal, or other LS/ HNPCC-associated tumors, regardless of age. Colorectal cancer with the MSI-H histology diagnosed in a pa@ent who is less than 60 years of age. Colorectal cancer diagnosed in one or more first-degree rela@ves with an LS/HNPCC-related tumor, with one of the cancers being diagnosed under age of 50 years. Colorectal cancer diagnosed in two or more first- or seconddegree rela@ves with HNPCC-related tumors, regardless of age SGO Criteria (20-25% Risk) Pa@ents with endometrial or colorectal cancer who meet the revised Amsterdam criteria. Pa@ents with synchronous or metachronous endometrial and colorectal cancer with the first cancer diagnosed prior to age 50. Pa@ents with synchronous or metachronous ovarian and colorectal cancer with the first cancer diagnosed prior to age 50. Pa@ents with colorectal or endometrial cancer with evidence of a mismatch repair defect (i.e. microsatellite instability or immunohistochemical loss of expression of MLH1, MSH2, MSH6 or PMS2). Pa@ents with first or second degree rela@ve with a known mismatch repair gene muta@on. Umar et al. J Natl Cancer Inst 2004 Lancaster, et al. Gynecol Oncol

6 SGO Criteria (5-10% Risk) with endometrial or colorectal cancer diagnosed prior to age 50. with endometrial or ovarian cancer with a synchronous or metachronous colon or other LS/HNPCC associated tumor at any age. Pa@ents with endometrial or colorectal cancer and a first degree rela@ve with LS/HNPCC associated tumor diagnosed prior to age 50. Pa@ents with colorectal or endometrial carcinoma diagnosed at any age with two or more first or second degree rela@ves with LS/HNPCC associated tumors, regardless of age. Mismatch Repair Protein (IHC) Mismatch repair protein expression is lost in nonfunc@oning MMR IHC can detect specific MMR protein deficiency Pa@ents with a first or second degree rela@ve that meets the above criteria. Lancaster et al. Gynecol Oncol 2007 Mismatch Repair Protein (IHC) MSH2 MLH1 MLH1, PMS2, MSH2, MSH6 MLH1 and PMS2 dimer: MLH1 is dominant MSH2 and MSH6 dimer: MSH2 is dominant All 4 intact = MMR proficient Loss of 1 or 2 = MMR deficient Rarely, loss of >2 MSH2 MLH1 MSH6 PMS2 MSH6 MLH1 PMS2 PMS2 MSH2 MSH6 6

7 IHC for DNA Mismatch Repair Proteins: Patterns of Loss If IHC is normal, no loss of expression If MLH1, MSH2, MSH6 and PMS2 are present, the is unlikely to have LS. If there is a high index of suspicion, consider MSI tes@ng because IHC will miss about 5 15 percent of LS cases. If MSI is also nega@ve, no further gene@c tes@ng for LS is indicated. IHC interpreta@on These findings indicate that it is unlikely that this carcinoma associated with Lynch Syndrome (LS) since tumours in pa@ents with LS typically show abnormal mismatch repair protein expression. These immunohistochemical findings cannot en@rely exclude the possibility of LS and family/personal history of cancers is s@ll important. IHC for DNA Mismatch Repair Proteins: Patterns of Loss If MLH1 and PMS2 are absent If MLH1 and PMS2 are absent, the pa@ent likely has acquired methyla@on of the MLH1 gene promoter, a situa@on that causes lack of MLH1 protein expression (but does not cause LS). However, LS is s@ll possible. IHC for DNA Mismatch Repair Proteins: Patterns of Loss If MSH2 and MSH6 are absent If MSH2 and MSH6 are absent, the pa@ent likely has LS. Order MSH2 gene@c tes@ng using a blood sample from the pa@ent as the first step. If no muta@on is found, consider EPCAM gene@c tes@ng and consulta@on with a gene@cs specialist. If no muta@on is found, follow up with MSH6 gene@c tes@ng. IHC for DNA Mismatch Repair Proteins: Patterns of Loss If only MSH6 is absent If only MSH6 is absent, the pa@ent may have LS. Order MSH6 gene@c tes@ng using a blood sample from the pa@ent. If only PMS2 is absent If only PMS2 is absent, the pa@ent may have LS. Order PMS2 gene@c tes@ng using a blood sample from the pa@ent. IHC for DNA Mismatch Repair Proteins: Advantages Familiar methodology Quick turn-around Relatively inexpensive Can pinpoint genes of interest for sequencing Cost effective Shia et al. J Mol Diagn 2008;10: Shia et al. Am J Surg Pathol 2009;33: Resnick et al. Obstet Gynecol 2009;114:

8 IHC for DNA Mismatch Repair Proteins: Disadvantages Numerous TILs may create false impression of intact MMR expression in tumor nuclei MSH6 may be heterogeneous need to evaluate entire tumor Absence of internal positive control if tumor nuclei negative, test can only be interpreted as equivocal Fixation dependent Epigenetic Methylation in Colorectal Cancer: MLH1 Common in colorectal carcinomas Occurs in left- and right-sided tumors Trend for older individuals Trend for females May show differential response to standard (5-FU) chemotherapy Can be detected by BRAF mutation Epigenetic Methylation in Endometrial Cancer: MLH1 Common in endometrial cancer Endometrioid & mixed endometrioid/ mucinous histology; undifferentiated Average age: 65 years (range: 42-88) Majority (86%, 44/51) located in the uterine fundus Cannot be detected by BRAF mutation: must do promoter methylation analysis Lynch Syndrome: MSI Testing Microsatellites are repetitive sequences that are particularly vulnerable to error without functioning MMR system Microsatellite instability (MSI) can serve as a proxy for impaired MMR Mills et al Mod Pathol 2011;24:260A [Abstract] MSI PCR Disadvantages Requires molecular laboratory set up Insufficient tumor cell nuclei may hinder test esp problematic with colloid colorectal carcinomas May not identify MSH6 MMR protein deficient cases (MSS or MSI-L) Does not distinguish between genetic and epigenetic causes of MSI Does not identify specific MMR protein Reflex Testing Is consent needed to test with IHC? Short answer no Assure all targeted patient samples are tested Assure all relevant patients are referred for counseling 8

9 The debate When to test Whom to test Mills et al Am J Surg Pathol 2014;38: ) 605 consecu@ve endometrial emca evaluated regardless of age, family history or histology IHC for MMR proteins followed by DNA methyla@on analysis 40 cases iden@fied 37 MSH6/MSH2 deficiency and 3 MLH1/PMS2 25% below 50 years of age 15% had family or prior history of carcinoma 13% arose in LUS 23% had TILs 80% were pure endometrioid Dudley et al Am J Surg Pathol Aug;39(8): CRC and 215 endometrial carcinomas 32 tumours demonstrated isolated loss of PMS2 16 CRC and 16 EMCa Germline muta@on analysis 24% MLH1 muta@ons, 35% with PMS2 muta@ons, 12% PMS2 varia@ons of uncertain significance, 29% no muta@ons So pa@ents with isolated PMS 2 lack of expression must have germline muta@onal analysis of MLH1 and PMS2 Rabban et al Am J Surg Pathol 2014;38: consecu@ve EMCa in pa@ents older than 50 years of age 181 lacked criteria for screening 98% intact MMR, 2% (1 case) loss of MSH6 92 fulfilling 1 or more criteria - had abnormal MMR mostly MLH1/PMS2 Mills et al Am J Surg Pathol Oct 29 Although universal MMR-IHC iden@fies endometrial carcinoma pa@ents with LS who would have been missed using targeted screening algorithms, it also iden@fies cancers with discordant IHC and germline results for which the soma@c versus germline origin of the MMR defect is unclear. Further study of this LL group is required before drawing defini@ve conclusions about their familial cancer risk. Ovarian carcinoma in LS Endometrioid and clear cell carcinomas Vierkoetter KR et al Gynecol Oncol ;135(1):81-4 Chui MH Am J Surg Pathol ;38(9): Kobayashi Y et al Int J Gynecol Cancer ;25(3):440-6 Karamurzin Y and Soslow RA Am J Surg Pathol. 2013;37(4):

10 IHC for loss of MMR protein expression for all endometrial carcinomas Selective testing MMR intact per IHC but clinical suspicion of LS Order LS microsatellite instability by PCR Abnormal staining for MLH1 & PMS2 Loss of MMR per IHC Abnormal staining for MSH2 and MSH6 Abnormal staining for MSH6 Abnormal staining for PMS2 Instability at Instability 1 2/5 of microsatellite microsatellite marker markers High Indeterminate Consider germline tes@ng of mismatch repair genes No instability present Low Test for MLH1 promoter methyla@on Methyla@on Methyla@on present absent Gene@c muta@on tes@ng for LS: recommend LS MSH2 sequencing and dele@on/duplica@on as first test Gene@c muta@on tes@ng for LS: recommend LS MSH6 sequencing and dele@on/duplica@on as first test Gene@c muta@on tes@ng for LS: recommend LS PMS2 (and MLH1 if PMS2 nega@ve) sequencing and dele@on/ duplica@on as first test K Garg, and R A Soslow J Clin Pathol 2009;62: Copyright by the BMJ Publishing Group Ltd & Association of Clinical Pathologists. All rights reserved. Likely Gene@c muta@on tes@ng for sporadic LS: recommend LS MSH1 sequencing and dele@on/ endometrial duplica@on as first test carcinoma* Universal testing *If strong clinical suspicion for LS, consider MLH1 promoter methyla@on analysis of blood to evaluated for germline epigene@c MLH1 promoter methyla@on MMR Gene Mutation Testing Requires consent and counseling Expensive Labor intensive Specialized centers Only defini@ve test to establish a diagnosis of Lynch syndrome Not a screening test Surveillance for Gynaecologic Tumours in Women with Lynch Syndrome Age years Annual pelvic exam with pap smear Transvaginal and/or pelvic ultrasound Endometrial biopsy Not shown to be effective Risk Reducing Surgery in Lynch Syndrome Women who undergo prophylactic surgery hysterectomy and BSO - do not develop cancer Consider risk reducing surgery in women with Lynch syndrome after the age of 35 years or once childbirth has been completed More effective and less expensive compared to surveillance Disadvantages: Surgical complications and surgical menopause Am J Surg Pathol 2013;37: Thank you 10