MSI testing Lynch syndrome / colorectal cancer

Stains & molecular markers
MSI testing Lynch syndrome / colorectal cancer

Author: Betty Chung, D.O., M.P.H., M.A.

Topic Completed: 1 January 2014

Minor changes: 8 September 2020

Copyright: 2003-2020, PathologyOutlines.com, Inc.

PubMed Search: MSI testing[TIAB] Lynch syndrome colorectal cancer

Page views in 2019: 16

Page views in 2020 to date: 1,433

Table of Contents

Cite this page: Chung, B. MSI testing Lynch syndrome / colorectal cancer. PathologyOutlines.com website. https://www.pathologyoutlines.com/topic/stainslynchcolorectal.html. Accessed September 20th, 2020.

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
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

Contributed by Shrihari S. Kadkol, M.D., Ph.D.

Algorithm - colon cancer testing

MSI electropherogram

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
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

Microscopic (histologic) images

Images hosted on other servers:

MSI tumor showing loss of MLH1

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
- "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)
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

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)