Table of Contents
Clinical features of Lynch syndrome | Colorectal cancers with microsatellite instability (MSI) | Diagrams / tables | Testing algorithm | Microscopic (histologic) images | Immunohistochemistry (IHC) | MSI testing by PCR | Additional referencesCite this page: Chung, B. MSI testing Lynch syndrome / colorectal cancer. PathologyOutlines.com website. https://www.pathologyoutlines.com/topic/stainslynchcolorectal.html. Accessed January 19th, 2021.
Clinical features of Lynch syndrome
- Lynch syndrome is also known as hereditary nonpolyposis colorectal cancer (HNPCC), see Colon tumor topic
- 3% of all colorectal cancers (early onset, Gastroenterology 2010;138:2073)
- Associated with extracolonic tumors:
- Endometrium, hepatobiliary system, ovary, pancreas, renal pelvis or ureter, small intestine, stomach
- Also brain (usually glioblastomas), skin (keratoacanthomas, sebaceous adenomas)
- Autosomal dominant inheritance
- Possess germline mutation of at least one of the four following mismatch repair (MMR) genes
- MLH1, PMS2: together form the MutSα heterodimer
- MSH2, MSH6: together form the MutLα heterodimer
- Second hit to wild type copy from unaffected parent results in inactivation of that specific MMR gene through the following mechanisms:
- Loss of heterozygosity, methylation, point mutation
- Possess germline mutation of at least one of the four following mismatch repair (MMR) genes
- Majority of HNPCC (90%) and subset of non-HNPCC patients possess germline mutations in DNA mismatch repair (MMR) genes
Colorectal cancers with microsatellite instability (MSI)
- Hypermutable phenotype caused by loss of DNA mismatch repair activity
- Defects in MMR proteins
- Rapid accumulation of somatic mutations
- Detected in about 15% of all colorectal cancers
- 3% associated with Lynch syndrome
- 12% sporadic, acquired hypermethylation of MLH1 gene promoter
- Tumors with CpG island methylator (MSI-H) phenotype
- Rarely detected in familial cases with germline mutations
- Phenotype of colorectal cancers with MSI
- Tendency to arise in proximal colon
- Tumor infiltrating lymphocytes that are usually activated and cytotoxic
- Lymphocytic reaction associated with better prognosis
- Poorly differentiated mucinous or signet ring appearance
- Clinical and family history not as obvious as for familial adenomatous polyposis (FAP) syndrome
- MSI-H colonic and gastric tumors are associated with more favorable prognosis than those lacking MSI
- Often resistant to treatment with 5-fluorouracil (5-FU)
- Sensitive to irinotecan (topoisomerase I inhibitor)
Diagrams / tables
Testing algorithm
- Amsterdam criteria II (all must be present); see Gastroenterology 1999;116:1453
- At least 3 relatives with Lynch syndrome associated cancer
- 1 should be a 1st degree relative of the other 2
- At least 2 successive generations should be affected
- At least 1 should be diagnosed < 50 years of age
- FAP should be excluded in any CRC cases
- Tumors verified by pathology
- At least 3 relatives with Lynch syndrome associated cancer
- Bethesda guidelines (any can be present); see J Natl Cancer Inst 2004;96:261
- Colorectal carcinoma (CRC) diagnosis in patient < 50 years of age
- Presence of synchronous, metachronous colorectal or other Lynch syndrome related tumors regardless of age
- CRC with MSI-H histology in a patient < 60 years of age
- CRC diagnosis in 1 or more 1st degree relatives with a Lynch syndrome related tumor, with 1 of the cancers being diagnosed before age 50
- CRC diagnosis in 2 or more 1st or 2nd degree relatives with Lynch syndrome related tumors, regardless of age
- Recommended that patients with increased risk for Lynch syndrome undergo prescreening prior to referral for germline mutation testing
- Microsatellite instability (MSI) analysis
- Immunohistochemistry (IHC)
- Subsequent referral for germline mutation testing in the following cases
- High levels of MSI
- Loss of expression of MMR proteins by IHC
- Mutation testing
- Sequencing
- Southern blot
- Multiplex ligation probe amplification (MLPA)
- Deletions larger than an exon, especially with respect to MSH2 and PMS2
Immunohistochemistry (IHC)
- Complementary with MSI testing by PCR
- MSH6- tumors may be stable by PCR
- Identify tumors with deficiencies in any of the 4 MMR proteins of interest
- Nuclear expression when present
- May be difficult to interpret
- 5% of CRCs with defective MMR proteins (MSI-H tumors) show normal IHC expression
- Gene mutation and loss of MLH1 and MSH2 result in degradation of PMS2 and MSH6 but not vice versa
- MLH1 and MSH2 can bind to other proteins and are therefore, more stable
- Sensitivity and specificity of a 2 panel test of IHC for PMS2 and MSH6 is 100% to detect MMR protein deficiency so not necessary to do 4 panel testing (Pathology 2010;42:409)
MSI testing by PCR
- Currently, no FDA approved test, only laboratory developed tests (LDTs) that are validated under CLIA 88 regulations
- Complementary with IHC (IHC may not pick up missense mutations)
- Microsatellites are short (1 - 6 nt), highly polymorphic, repetitive nucleotide sequences throughout the genome, often in noncoding regions
- Microsatellite instability (MSI) often show frequent loss of MMR protein expression
- Slippage during DNA replication may result in a change in the number of repeats if not repaired by MMR proteins
- Panel of five mono / dinucleotide markers or quasimonomorphic mononucleotide markers for MSI determination via multiplex PCR (Gastroenterology 2002;123:1804, J Natl Cancer Inst 2007;99:244)
- NCI panel: BAT25, BAT26 (mononucleotide markers); D2S123, D5S346, D17S250 (dinucleotide markers)
- Promega kit: BAT25, BAT26, NR21, NR24 and MON027
- Can be performed on frozen or formalin fixed paraffin embedded (FFPE) tissue
- Generally want tumor sections to contain at least > 50% tumor
- Also must send section with normal colonic mucosa free of tumor for comparison
- Amplified products separated by capillary gel electrophoresis
- Comparison of peak patterns with a shift in PCR product size of the tumor when compared to normal represents instability
- Diagnostic results reporting
- "Microsatellite stable" (MSS) if no MSI shown in the tumor
- Test for KRAS mutation (present in 35 - 45% of colorectal cancers), most often point mutation in exon 12 or 13 (World J Gastroenterol 2012;18:5171)
- If positive, resistant to anti-EGFR treatment
- If negative, test for BRAF V600E mutation
- Located in MAP kinase pathway downstream of KRAS
- Constitutive activation of MAPK pathway
- Anti-EGFR (e.g. cetuximab) therapy is ineffective if this mutation is present in the tumor
- Associated with unfavorable prognosis
- Test for KRAS mutation (present in 35 - 45% of colorectal cancers), most often point mutation in exon 12 or 13 (World J Gastroenterol 2012;18:5171)
- "Indeterminate" or "low" (MSI-L) if MSI in only 1 repeat; may or may not indicate an MMR deficiency
- "High instability" (MSI-H) if MSI in 2 or more repeats (> 30% of microsatellite marker panel is mutated)
- "Microsatellite stable" (MSS) if no MSI shown in the tumor
- Limit of detection (LOD): ~10% but can vary between labs
- Tumor macrodissection prior to PCR can increase analytic sensitivity and results in a lower LOD
- Reflex testing if isolated loss of MLH1 protein while other 3 MMR proteins are expressed
- MLH1 promoter hypermethylation detection
- CpG island methylator phenotype (CIMP) has the poorest prognosis
- MLH1 promoter hypermethylation detection
Additional references
- NewsPath: Screening for Lynch Syndrome [Accessed 5 June 2018], POET Report: Prognostic Uses of MSI Testing [Accessed 5 June 2018]
- University of Illinois Hospital and Health Sciences System Pathology Residency Training Program Core Curriculum: Molecular Testing - Colon Cancer - April 2012 (link unavailable)