Lynch Syndrome - Hereditary Nonpolyposis Colorectal Cancer (HNPCC)

Primary Author Samowitz, Wade S., MD.

Key Points

Lynch syndrome (formerly referred to as hereditary nonpolyposis colorectal cancer or HNPCC)

  • Universal screening of all colorectal cancer (CRC) specimens (NCCN, 2015; ASCO, 2014) and endometrial cancer specimens (ASCO, 2014; ESMO, 2013; NCCN, 2015) is recommended
  • LS is an autosomal dominant inherited cancer syndrome that predisposes to colorectal, endometrial, gastric, ovarian, upper urinary tract, and other cancers
  • The presence of mismatch repair (MMR) deficiency helps identify patients at risk for Lynch syndrome (LS); however, it also occurs in ~15% of sporadic colorectal cancers 
    • Differentiating colorectal tumors with MMR deficiency due to a sporadic somatic event from colorectal tumors with MMR deficiency due to a LS germline mutation is important
  • Microsatellite Instability (MSI)

    Microsatellite Instability (MSI)

    Functions as a surrogate marker for MMR deficiency

    • Manifests as expansion or contraction of  microsatellite* repeats because of defects in MMR genes

    Adenomas are the precursor lesion for LS; however, not all adenomas are MMR deficient

    • Only 40-50% of adenomatous polyps in LS will be MMR deficient
      • Greater size of polyp tends to be associated with higher risk of MMR deficiency
    • Rare in non-LS polyps
    • Occurs in ~15% of sporadic colorectal cancers
    • 5-10% of LS-associated colorectal carcinomas will have normal MMR protein expression as evaluated by immunohistochemistry (IHC) staining (NCCN, 2015)

    Mutation(s)

    • LS
      • Epimutation – monoallelic
    • Sporadic colorectal cancer
      • Epimutation – biallelic
      • Acquired promoter methylation of MLH1
      • Absence of MLH1 and PMS2 proteins on IHC
      • Frequent mutations in BRAF

    * Microsatellites are simple repeat sequences of 1-6 base pairs that are repeated up to 100 times and are prone to DNA replication errors.  MMR genes function to identify and correct the replication errors.

    MMR Gene Mutations

    MMR Gene Mutations

    Mutations include point mutations and large genomic deletions or rearrangements

    Common mutations

    ~90% of mutations in LS

    • MLH1 (mutL homolog1) – 50% 
    • MSH2 (mutS homolog 2) – 40%

    Less common mutations

    • PMS2 (postmeiotic segregation increased 2)
    • MSH6 (mutS homolog 6)

    Heterodimeric complexes

    Obligatory partners

    • MLH1 and PMS2
      • MLH1 loss leads to concomitant PMS2 loss due to PMS2 degradation
    • MSH2 and MSH6
      • MSH2 loss leads to concomitant MSH6 loss due to MSH6 degradation
    • MSH6 or PMS2 losses are not associated with any other loss
    BRAF V600E Gene Mutations
    BRAF V600E Gene Mutations

    BRAF V600E (c1779T>A)

    • Not identified in patients with MLH1 germline mutations
    • Identified in ~15% of sporadic colorectal cancers
    • Highly associated with promoter methylation of MLH1
    Diagnostic Testing for Lynch Syndrome (LS)

    Initial testing*

    • Use immunohistochemistry staining (IHC) or PCR initially to eliminate expense of full gene sequencing. Both tests are sensitive and usually produce concordant results.

    Test

    Test Interpretation

    IHC  

    • Uses antibodies to MMR proteins MLH1, MSH2, MSH6, and PMS2
    • Protein loss identified on IHC directs mutation testing
    • 5-10% false-negative rate (NCCN, 2015)
    • Isolated PMS2 loss has been associated with germline MLH1

    IHC Result

    Likely Gene Mutation

    • MLH1, PMS2 loss
    • MLH1
    • MSH2, MSH6 loss
    • MSH2
    • PMS2
    • PMS2
    • MSH6
    • MSH6

    BRAF V600E or MLH1 methylation

    • When MLH1 loss is identified using IHC, perform prior to LS mutation analysis
    • Loss of MLH1 is due to acquired hypermethylation in sporadic tumors, versus a germline mutation as in LS

    If BRAF or MLH1 hypermethylation is positive

    • LS unlikely; probably sporadic colorectal cancer
    • LS gene mutation analysis not necessary unless high suspicion for LS still exists

    PCR (panel of five mononucleotide microsatellites)

    • Expansion or contraction of microsatellite repeats in the tumor
    • Does not detect specific protein
    • Patient tumor tissue is compared to patient normal tissue
    • Highest sensitivity achieved when PCR is combined with IHC

    Determination of MSI

    High – 2 or more markers with instability

    • Consider gene mutation testing
    • IHC can be used to target specific mutation testing

    Indeterminate – 1 marker with instability

    • Instability in even 1 mononucleotide repeat can be associated with LS – suggest IHC

    Stable – no markers detected with instability

    • Very low risk of LS
    • Add IHC and possibly gene mutation testing if high suspicion of LS exists

    MOLECULAR ANALYSIS

    • IHC result guides choice of gene to target
    • Mutation testing is gold standard for diagnosing LS
    *Refer to Lynch Syndrome Testing (HNPCC) algorithm

Diagnosis

Indications for Testing

  • All colorectal cancer (CRC) and/or endometrial tumors

Laboratory Testing

  • For additional information regarding testing strategies, refer to the following
    • Key Points section for this topic
    • Lynch Syndrome Testing (HNPCC) algorithm
    • NCCN guidelines, Genetic/Familial High Risk Assessment (2015), page LS-2, for algorithm

Prognosis

  • Patients with MMR-deficient colorectal cancer have improved prognosis compared to patients without MMR deficiency

Screening

  • Refer to NCCN guidelines, Genetic/Familial High Risk Assessment (2015), ASCO (2014), ESMO (2013)

Monitoring

  • Refer to NCCN guidelines, Genetic/Familial High Risk Assessment (2015)

Clinical Background

Colon cancer exhibits the characteristics of familial clustering in ~10-15% of cases. The most common cause of hereditary colon cancer is Lynch syndrome (LS – hereditary nonpolyposis colorectal cancer [HNPCC]). LS is caused by a germline mutation in one of the genes within the DNA mismatch repair system.

Epidemiology

  • Prevalence – 2-4% of colorectal cancer (CRC) (NCCN, 2015)
  • Age – mutation dependent (44-66 years mean)
    • >75% risk of developing CRC by 70 years
  • Sex – M:F, equal

Inheritance

  • Autosomal dominant with incomplete penetrance
  • Germline mutations in 1 of 4 DNA MMR genes
    • MLH1 50%
    • MSH2 40%
      • Small percentage of MSH2 inactivation is due to EPCAM deletions
        • Included with MSH2 testing
    • MSH6 – 7-10%
    • PMS2 <5%

Clinical Presentation

  • Early onset of proximal (right side) colorectal cancer – often <50 years
    • Multiple metachronous and synchronous tumors are common
  • Early-onset extra colonic tumors, including endometrial, ovarian, small bowel, brain, pancreatic, gastric, renal pelvis, ureter, hepatobiliary tract
    • Risk depends on mutation present
  • Penetrance variable; patients may present with tumors at older age
    • Especially true for MSH6 and PMS2
    • Universal screening will identify these tumors and patients will not be mistakenly classified as sporadic CRC

Indications for Laboratory Testing

  • Tests generally appear in the order most useful for common clinical situations
  • Click on number for test-specific information in the ARUP Laboratory Test Directory
Test Name and Number Recommended Use Limitations Follow Up
Mismatch Repair by Immunohistochemistry with Reflex to BRAF Codon 600 Mutation and MLH1 Promoter Methylation 2002327
Method: Qualitative Immunohistochemistry/Qualitative Real-time Polymerase Chain Reaction

Preferred screening test for Lynch Syndrome (LS) in individuals with colorectal cancer (CRC)

Reflex pattern – if MLH1 IHC is abnormal, evaluations of BRAF codon 600 and, possibly, MLH1 methylation are performed

Clinical sensitivity – 90%

~10% of individuals with LS will have IHC tests which show normal staining of the MMR proteins

Since the correlation of MSI with IHC is not 100%, direct testing of MSI by PCR may be helpful

Definitive diagnosis of LS requires additional targeted MMR germline mutation studies

Mismatch Repair by Immunohistochemistry with Reflex to MLH1 Promoter Methylation 2005270
Method: Qualitative Immunohistochemistry/Qualitative Real-time Polymerase Chain Reaction

Preferred reflex screening test for LS in non-CRC tumors (eg, endometrial carcinoma) 

Reflex pattern – if MLH1 IHC is abnormal, MLH1 methylation is performed

Clinical sensitivity – 90%

~10% of individuals with LS will have IHC tests which show normal staining of the MMR proteins

Since the correlation of MSI with IHC is not 100%, direct testing of MSI by PCR may be helpful

 
Mismatch Repair by Immunohistochemistry 0049302
Method: Qualitative Immunohistochemistry

First-line screening test for LS; directs additional molecular diagnostic testing for LS

Highly recommended prior to ordering MMR germline mutation analysis

Results guide subsequent genetic testing

Clinical sensitivity – 90%

~10% of individuals with LS will have IHC tests which show normal staining of the MMR proteins

Since the correlation of MSI with IHC is not 100%, direct testing of MSI by PCR may be helpful

 
Microsatellite Instability (MSI), HNPCC/Lynch Syndrome, by PCR 0051740
Method: Polymerase Chain Reaction/Fragment Analysis

First-line screening test for LS; directs additional molecular diagnostic testing for LS

Clinical sensitivity – 90%

Analytic sensitivity/specificity – 99%

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

 
HNPCC/Lynch Syndrome (MLH1) Sequencing and Deletion/Duplication 0051650
Method: Polymerase Chain Reaction/Sequencing/Multiplex Ligation-dependent Probe Amplification

Detect germline MLH1 mutations, diagnostic for LS

Use in MMR-deficient carcinoma with suggestive IHC (loss of MLH1 and PMS2 protein), absence of BRAF codon 600 mutation, and normal MLH1 methylation studies

Clinical sensitivity – >95% (combined sensitivity across all 4 genes [MLH1, MSH2, MSH6, and PMS2])

Analytic sensitivity/specificity – 99%

Gene sequencing may identify mutations of unknown clinical significance

Rare false negatives can occur due to primer- and probe-site mutations

Breakpoints of large deletions/duplications will not be determined

Deep intronic mutations or promoter mutations of the MLH1 gene will not be detected

 
HNPCC/Lynch Syndrome (MSH2) Sequencing and Deletion/Duplication 0051654
Method: Polymerase Chain Reaction/Sequencing/Multiplex Ligation-dependent Probe Amplification

Detect germline MSH2 mutations

Use in MMR-deficient carcinoma with suggestive IHC (loss of MSH2 and MSH6 protein)

Detects large MSH2 deletions and EPCAM 3 prime deletions

Clinical sensitivity – >95% (combined sensitivity across all 4 genes [MLH1, MSH2, MSH6, and PMS2])

Analytic sensitivity/specificity – 99%

Gene sequencing may identify mutations of unknown clinical significance

Rare false negatives can occur due to primer- and probe-site mutations

Breakpoints of large deletions/duplications will not be determined

Deep intronic mutations or promoter mutations of the MSH2 gene will not be detected

 
HNPCC/Lynch Syndrome (MSH6) Sequencing and Deletion/Duplication 0051656
Method: Polymerase Chain Reaction/Sequencing/Multiplex Ligation-dependent Probe Amplification

Detect germline MSH6 mutations

Use in MMR-deficient carcinoma with suggestive IHC (isolated loss of MSH6 protein)

Clinical sensitivity – >95% (combined sensitivity across all 4 genes [MLH1, MSH2, MSH6, and PMS2])

Analytic sensitivity/specificity – 99%

Gene sequencing may identify mutations of unknown clinical significance

Rare false negatives can occur due to primer- and probe-site mutations

Breakpoints of large deletions/duplications will not be determined

Deep intronic mutations or promoter mutations of the MSH6 gene will not be detected

 
HNPCC/Lynch Syndrome (PMS2) Sequencing and Deletion/Duplication 0051737
Method: Polymerase Chain Reaction/Sequencing/Multiplex Ligation-dependent Probe Amplification

Detect germline PMS2 mutations

Use in MMR-deficient carcinoma with suggestive IHC (isolated loss of PMS2 protein)

Clinical sensitivity – >95% (combined sensitivity across all 4 genes [MLH1, MSH2, MSH6, and PMS2])

Analytic sensitivity/specificity – 99%

Gene sequencing may identify mutations of unknown clinical significance

Rare false negatives can occur due to primer- and probe-site mutations

Breakpoints of large deletions/duplications will not be determined

Deep intronic mutations or promoter mutations of the PMS2 gene will not be detected

 
BRAF Codon 600 Mutation Detection with Reflex to MLH1 Promoter Methylation 0051750
Method: Polymerase Chain Reaction/Pyrosequencing

Recommended reflex test for differentiating between LS and sporadic CRC in tumors showing loss of MLH1

Reflex pattern – if no BRAF mutation is detected, MLH1 promoter methylation is evaluated

Analytic sensitivity – methylation levels >10% are reported as positive

Mutations other than BRAF V600E will not be detected

Rare false negatives may occur due to primer- and probe-site mutations

 
BRAF Codon 600 Mutation Detection by Pyrosequencing 2002498
Method: Polymerase Chain Reaction/Pyrosequencing

Single gene assay

Detects activating BRAF mutations (codon 600) associated with anti-EGFR therapy resistance

Use prior to BRAF V600E inhibitor initiation

Clinical sensitivity – activating BRAF mutation found in ~10% of CRCs

Analytic sensitivity/ specificity – 100%

Limit of detection

  • MassARRAY and pyrosequencing − 10% mutant alleles
  • NGS – 5% mutant alleles

MassARRAY and pyrosequencing – oncogenic mutations outside of codon 600 will not be detected

 
HNPCC/Lynch Syndrome Deletion/Duplication 2001728
Method: Polymerase Chain Reaction/Multiplex Ligation-dependent Probe Amplification

Order only if sequencing studies have been performed at another laboratory

Both sequencing and deletion/duplication testing are necessary to detect all pathogenic mutations in MMR genes

Requires permission from ARUP's Genetic Counselors (800-242-2787, x2141) before ordering

   
Additional Tests Available
 
Click the plus sign to expand the table of additional tests.
Test Name and NumberComments
Familial Mutation, Targeted Sequencing 2001961
Method: Polymerase Chain Reaction/Sequencing

Consultation with a genetics counselor is advised

Order to detect a mutation previously identified in a family member