Case Presentation Diana Lim, MBBS, FRCPA, FRCPath Senior Consultant Department of Pathology, National University Health System, Singapore Assistant Professor Yong Loo Lin School of Medicine, National University of Singapore Diana_gz_lim@nuhs.edu.sg
Clinical History 55 y/o Chinese female History of CA colon (T3N0M0) s/p right hemicolectomy in 2009. No adjuvant therapy given. Last colonoscopy in 2014 was unremarkable. CEA not raised Presents with menorrhagia x 4 months, not controlled with norethisterone Ultrasound: Multiple uterine fibroids Endometrial pipelle was performed: Scant inactive endometrium Hysteroscopy: Submucosal fibroids Clinical impression: Dysfunctional uterine bleeding with failure of medical treatment Patient underwent a laparoscopic total hysterectomy with bilateral salpingo-oophorectomy
H&E AE1/3 Vimentin Desmin
Dedifferentiated Carcinoma Dedifferentiated ca: Undiff ca + FIGO grade 1 or 2 EC Morphology Solid proliferation of tumour cells without gland formation Monotonous cytology; small to intermediate sized dyshesive cells of relatively uniform cells Focal pleomorphism; focal keratinization Rhabdoid cells may be present Focal myxoid stroma
What underlying condition is the patient likely to have?
Lynch Syndrome
Lynch Syndrome Autosomal dominant disorder caused by a defect in a DNA mismatch repair (MMR) gene Characterized by development of various tumours including CRC, endometrial and other cancers Cancer Lifetime risk Colon Up to 80% Endometrium Up to 60% Ovary Up to 24% Stomach Up to 13% Sebaceous neoplasms Up to 9% Pancreas Up to 6% Small bowel Up to 6% CNS Up to 3% Adapted from NCCN guidelines version 1.2015
Why diagnose Lynch Syndrome? Allows for screening and prevention of other cancers in patient and relatives
Clinical criteria for Lynch Syndrome Amsterdam criteria II 3 relatives with a Lynch syndrome associated cancer (CRC, Ca endometrium, small bowel, ureter or renal pelvis); all of the following criteria should be present: One should be a 1st degree relative of the other two 2 successive generations should be affected 1 should be diagnosed before the age of 50 years FAP should be excluded in the CRC case(s) if any Tumours show be verified by pathological examination Revised Bethesda criteria 1. CRC diagnosed in a patient < 50 years of age 2. Presence of synchronous, metachronous CRC or other Lynch syndrome-related tumours, regardless of age 3. CRC with MSI-H phenotype diagnosed at less than 60 years of age 4. Patients with CRC and a first degree relative with a Lynch syndrome-related tumours, with one of the cancers diagnosed before age of 50 years 5. Patient with CRC with 2 first or second-degree relatives with a Lynch syndrome-related tumour, regardless of age
Clinical criteria for Lynch Syndrome 1. Age 2. Personal/family cancer pedigree (Bethesda guidelines, Amsterdam criteria) 3. Tumour morphological features
Morphological Features CRC Proximal location Mucinous differentiation Poorly differentiated or medullary-like features Tumour infiltrating lymphocytes Peritumoral lymphoid follicles ( Crohn s-like lymphocytic reaction) Endometrial Carcinoma Tumor-infiltrating lymphocytes ( 40/10 high-power fields) Dense peritumoral lymphocytes Undifferentiated tumor histology Ambiguous morphology Tumour heterogeneity Lower uterine segment origin of tumor Synchronous ovarian and endometrial tumors
What additional tests may be performed to help exclude Lynch syndrome? Microsatellite instability testing Mismatch repair IHC
Microsatellite repeats Microsatellites are tandem repeats of DNA sequences (1-6 bps in length) randomly present throughout the genome Prone to replication errors The MMR system is responsible for the identification and correction of these errors Defects in the MMR system leads to microsatellite instability (MSI)- expansions or contractions in the number of tandem repeats at microsatellite foci in the DNA
MMR is dependent on concerted actions of several proteins which function as heterodimers In their functional state within a cell, MLH1 dimerizes with PMS2 and MSH2 dimerizes with MSH6 MLH1 and MSH2 are the obligatory partners for their respective heterodimers. Germline mutations in MLH1 and MSH2 heterodimer (including PMS2 and MSH6) degradation of the In contrast, germline mutations in MSH6 and PMS2 may not result in degradation of MLH1 and MSH2, as the function of the secondary protein may be compensated by other proteins
Mismatch repair defects Defects in the system can be secondary to genetic or epigenetic mechanisms, through germline or somatic mutations in one of the MMR genes or methylation of a promoter region (most commonly MLH1) MSH2/EPCAM MSH6 MLH1 PMS2 Chr 2 Chr 3 Chr 7 Patients with Lynch syndrome have germline defects in the genes encoding for MMR proteins (most commonly MLH1, MSH2, MSH6 and PMS2), resulting in MSI
Testing for MMR deficiency IHC MSI testing
MMR IHC
Immunohistochemistry Stain for MLH1,MSH2, MSH6, PMS2 High concordance with MSI assay (>90%) Advantages: Easy availability Efficient testing at the time of histo-reporting Ability to identify which gene is mutated Ability to detect MMR-deficient cases that can potentially be missed by MSI testing, specifically MSH6 mutations (tend to have MSS or MSI-low tumors and are more common in EM cancer than CRC) Disadvantages: Subject to interpretative errors MSH6 staining is frequently patchy and weak in gynecologic tract carcinomas making confident interpretation difficult Only testing for 4 proteins
MMR-IHC staining patterns Protein Immunoexpression MLH1 - MSH2 - PMS2 - - MSH6 - - MLH1 promoter methylation or MLH1 mutation MSH2 or EPCAM mutation Likely MSH6 mutation Likely PMS2 mutation Normal expression = nuclear staining within tumor cells, using infiltrating lymphocytes as positive internal control Negative protein expression = complete absence of nuclear staining within tumour cells with positive internal control
MMR IHC Lynch Syndrome Screening Network MLH1 promoter methylation or MLH1 mutation
MSI testing
MSI Testing ProMega MSI analysis system: 5 mononucleotide microsatellite markers (BAT-25, BAT-26, NR-21, NR-24, and MONO-27) and 2 pentanucleotide repeats (Penta C and Penta D) Paired DNA samples from tumour and normal tissue samples are genotyped and analyzed by capillary electrophoresis Interpretation MSI in 2 markers : MSI-High MSI in 1 marker: MSI-Low No shifts in any marker: Microsatellite stable (MSS)
MSI testing Advantage: Can potentially identify patients with defective DNA mismatch repair but intact immuno staining as a result of non-truncating missense alterations or defects in genes other than the 4 MMR proteins routinely tested for by immunohistochemistry. Disadvantage: Requires normal tissue (may not be able to be performed in biopsy specimens) Higher cost More labour intensive Longer turnaround time
Endometrial Cancer and LS Selective testing of high risk groups May be more cost effective Universal Screening >50% of female Lynch syndrome patients present with a gynecologic cancer as their sentinel malignancy and up to 70% may not meet either Amsterdam criteria or Bethesda guidelines for Lynch syndrome screening Clinical criteria not perfectly sensitive The mean age of MSH6 endometrial cancer patients is between 50 and 61 years Some patients do not have a personal/family cancer pedigree suggestive of LS Not all tumours exhibit MMR morphology MMR morphology criteria is subject to interobserver variation
Clinical Criteria MSI testing MMR IHC Genetic Testing
Universal Screening Model Tumour tissue available +ve IHC for MMR -ve No further work-up unless other compelling reason to refer MSI and/or genetic analysis MLH1 methylation studies (MLH1 loss) Genetic counseling Identify other at-risk relatives