Qualitative Immunohistochemistry
- First-line screening test for newly diagnosed CRC, endometrial carcinoma, and LS
- Highly recommended prior to ordering germline MMR gene testing
- Directs subsequent genetic diagnostic testing
- Testing for CRC and other solid tumors to qualify patients for certain immune checkpoint inhibitor treatment
Capillary Electrophoresis
- First-line screening test for newly diagnosed CRC, endometrial carcinoma, and LS
- Directs subsequent genetic diagnostic testing for LS
- Testing for CRC and other solid tumors to qualify patients for certain immune checkpoint inhibitor treatment
Qualitative Immunohistochemistry/Qualitative Real-time Polymerase Chain Reaction
Polymerase Chain Reaction/Pyrosequencing
- Recommended reflex test to differentiate between LS and sporadic CRC in tumors showing loss of MLH1
- If no BRAF variant is detected, MLH1 promoter methylation is evaluated
Qualitative Immunohistochemistry/Qualitative Real-time Polymerase Chain Reaction
- Reflex screening test for LS in non-CRC tumors (eg, endometrial carcinoma)
- If MLH1 expression is lost, MLH1 methylation is performed
Real-Time Polymerase Chain Reaction/Fluorescence Resonance Energy Transfer
Recommended test to distinguish between LS and sporadic non-CRC tumors with loss of MLH1
Diagnostic Germline Genetic Studies
- Specimen: peripheral blood
- Germline genetic testing is available for all 4 MMR genes known to cause LS, either separately or as part of the hereditary gastrointestinal (GI) cancer panel (see Related Tests)
Polymerase Chain Reaction/Sequencing/Multiplex Ligation-dependent Probe Amplification
- Detect germline MLH1 variants
- Use in MMR-deficient carcinoma with suggestive IHC results (loss of MLH1 and PMS2 proteins), negative for the BRAF codon 600 pathogenic variant, and with normal MLH1 promoter methylation studies
Polymerase Chain Reaction/Sequencing/Multiplex Ligation-dependent Probe Amplification
- Detect germline MSH2 variants
- Use in MMR-deficient carcinoma with suggestive IHC results (loss of MSH2 and MSH6 proteins)
- Includes evaluation of EPCAM exon 9 deletions and 10 Mb inversion of MSH2 exons 1-7
Polymerase Chain Reaction/Sequencing/Multiplex Ligation-dependent Probe Amplification
- Detect germline MSH6 variants
- Use in MMR-deficient carcinoma with suggestive IHC results (isolated loss of MSH6 protein)
Polymerase Chain Reaction/Sequencing/Multiplex Ligation-dependent Probe Amplification
- Detect germline PMS2 variants
- Use in MMR-deficient carcinoma with suggestive IHC results (isolated loss of PMS2 protein)
Polymerase Chain Reaction/Sequencing
- Useful when a pathogenic familial variant identifiable by sequencing is known
- A copy of a relative’s lab report is REQUIRED
Multiplex Ligation-dependent Probe Amplification
- Use to assess for large deletion/duplication previously identified in a family member
- A copy of a relative’s lab report is REQUIRED
See Related Tests
Colorectal cancer (CRC) exhibits the characteristics of familial clustering in ~10-15% of cases. The most common cause of hereditary CRC is Lynch syndrome (LS), also known as hereditary nonpolyposis colorectal cancer (HNPCC). LS is caused by a germline mutation in one of the genes within the DNA mismatch repair (MMR) system.
Indications for Ordering
Microsatellite instability (MSI) and/or immunohistochemistry (IHC) tumor analyses
- Universal screening for LS in all individuals with newly diagnosed CRC
- Universal screening for LS in individuals with endometrial cancer
Germline MMR gene testing
- Order following abnormal screening test result
- Confirms diagnosis of LS
Disease Overview
Incidence
- Accounts for 2-4% of CRC and ~1-2% of endometrial cancers
- Most common hereditary CRC syndrome
- Most common cause of hereditary endometrial cancer
Risk Estimates
The following lifetime risk estimates apply to individuals with MLH1 and MSH2 pathogenic variants; risks for variants in MSH6 or PMS2 may be lower :
- CRC: 52-82%
- Endometrial: 25-60%
- Prostate: ~30%
- Ovarian: 11-24%
- Gastric: 6-13%
- Hepatobiliary tract: 1-4%
- Urinary tract: 1-7%
- Small bowel: 3-6%
- Brain/central nervous system: 1-3%
- Sebaceous neoplasms: 1-9%
- Pancreatic: 1-6% (MLH1 and MSH2 only)
Genetics
Genes Tested
MLH1, MSH2, MSH6, PMS2, and EPCAM
Inheritance
Autosomal dominant
Test Interpretation
Microsatellite Instability (MSI), HNPCC/Lynch Syndrome, by PCR
Sensitivity/Specificity
Results
- High MSI (MSI-H)
- MMR deficiency, either sporadic or LS
- Indeterminate MSI (MSI-I)
- Instability in even one mononucleotide repeat can be associated with LS.
- Follow-up IHC studies are recommended.
- Microsatellite stable (MSS)
- LS unlikely
Limitations
- 10-15% of sporadic CRCs are also MSI-H.
- Preoperative chemoradiation of rectal cancer:
- May complicate IHC interpretation and/or decrease tumor mass
- May make MSI testing difficult
- Evaluation of pretreatment biopsies will avoid this limitation
- Screens for LS only and does not evaluate other hereditary causes of CRC or endometrial cancer.
Mismatch Repair by IHC
Sensitivity/Specificity
Results
- Normal: MMR proteins are normally expressed.
- MMR deficiency is unlikely.
- LS unlikely
- Abnormal: MMR protein expression is abnormal.
- Loss of expression of one or more proteins is highly predictive of MMR deficiency.
- Absence of both MLH1 and PMS2:
- MLH1 germline pathogenic variant is possible.
- Consider MLH1 methylation ± BRAF V600E studies.
- If methylation and BRAF studies are negative, follow with MLH1 germline genetic testing.
- Absence of PMS2 only:
- PMS2 germline pathogenic variant likely
- Consider PMS2 germline testing.
- If PMS2 testing does not identify a germline pathogenic variant, consider MLH1 germline testing.
- Absence of both MSH2 and MSH6:
- MSH2 germline pathogenic variant likely
- Consider MSH2 germline testing.
- Absence of MSH6 only:
- MSH6 germline pathogenic variant likely
- Consider MSH6 germline testing.
- If MSH6 testing does not identify a germline pathogenic variant, consider MSH2 germline testing.
Limitations
- ~10% of individuals with LS will have IHC tests that show normal staining of the MMR proteins.
- Because the correlation of MSI with IHC is not 100%, direct testing of MSI by PCR may be helpful.
- Screens for LS only and does not evaluate other hereditary causes of CRC or endometrial cancer.
BRAF Codon 600 Mutation Detection with Reflex to MLH1 Promoter Methylation
Sensitivity/Specificity
- Analytical sensitivity: Methylation levels >10% are reported as positive.
Results
- BRAF V600E detected
- Presence in MMR-deficient CRC indicates the tumor is probably sporadic and not associated with LS.
- Further germline testing is not typically indicated.
- MLH1 promoter methylation detected
- Presence in an MSI CRC indicates the tumor is probably sporadic and not associated with LS.
- Further germline testing is not typically indicated.
- No variants detected
- In MSI-H tumors with loss of MLH1 protein by IHC, MLH1 germline testing is indicated.
Germline Genetic Studies (MLH1, MSH2, MSH6, PMS2, or EPCAM)
Sensitivity/Specificity
- Proportion of LS attributed to pathogenic variants in specific MMR gene:
- Analytical sensitivity/specificity: 99%
Results
- Positive: one pathogenic variant detected
- Predicted to be causative for LS
- Negative: no pathogenic variants detected
- Diagnosis of LS unlikely, but not excluded
- Inconclusive: variant detected, but whether it is benign or pathogenic is unknown
Limitations
- Not evaluated:
- Regulatory region and deep intronic variants
- Sequence variants and large deletion/duplications in genes other than MLH1, MSH2, MSH6, and PMS2
- Sequence variants in EPCAM
- Large deletions/duplications in EPCAM, other than exon 9
- Large gene inversions, other than the MSH2 10 Mb exons 1-7 inversion
- Causes of hereditary CRC or endometrial cancer other than LS
- Diagnostic errors can occur due to rare sequence variations.
References
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NCCN - Genetic/Familial High-Risk Assessment: Colorectal v1.2018
NCCN clinical practice guidelines in oncology, genetic/familial high-risk assessment: colorectal, Version 1.2018. National Comprehensive Cancer Network. [Updated: Jul 2018; Accessed: Jan 2019]
Online -
NCCN - Uterine Neoplasms - Lynch Syndrome
NCCN clinical practice guidelines in oncology, uterine neoplasms, Version 2.2019. National Comprehensive Cancer Network. [Updated: Dec 2018; Accessed: Dec 2019]
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GeneReviews - Lynch Syndrome
Kohlmann W, Gruber SB. Lynch syndrome. In: Adam MP, Ardinger HH, Pagon RA, et al, editors. GeneReviews, University of Washington; 1993-2021. [Last revision: Apr 2018; Accessed: Feb 2020]
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Screening Studies (Requires Pathological Tissue)