Molecular markers
Inherited disease molecular testing
MSI testing Lynch syndrome / colorectal cancer

Author: Betty Chung, D.O., M.P.H., M.A. (see Authors page)

Revised: 5 June 2018, last major update January 2014

Copyright: (c) 2003-2018, PathologyOutlines.com, Inc.

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

Cite this page: Chung, B. MSI testing Lynch syndrome / colorectal cancer. PathologyOutlines.com website. http://www.pathologyoutlines.com/topic/molecularpathlynchcolorectal.html. Accessed August 16th, 2018.
Clinical features of Lynch syndrome
  • 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

Images hosted on other servers:

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