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)

  • The presence of microsatellite instability (MSI) helps identify patients at risk for Lynch syndrome (LS); however, it also occurs in ~15% of sporadic colorectal cancers 
  • MSI is the accumulation of sequencing errors in microsatellites, or short tandem repeats, which occur during the DNA replication process
  • Differentiating colorectal tumors with MSI due to a sporadic somatic event from colorectal tumors with MSI due to a LS germline mutation is important
  • LS is an autosomal dominant inherited cancer syndrome that predisposes to colorectal, endometrial, gastric, ovarian, upper urinary tract, and other cancers
  • Microsatellite Instability (MSI)

    Microsatellite Instability (MSI)

    Functions as a surrogate marker for defects of mismatch repair (MMR) genes

    • Manifests as increases or, more frequently, decreases in the number of  microsatellite* repeats because of defects in MMR genes

    Frequently develops late in tumor development

    • Only 40-50% of adenomatous polyps in LS will have MSI
    • Rare in non-LS polyps
    • Occurs in ~15% of sporadic colorectal cancers
    • 10-15% of LS-associated colorectal carcinomas will have normal MMR protein expression as evaluated by immunohistochemistry staining (IHC)

    Mutation(s)

    • LS
      • Epimutation – monoallelic
    • Sporadic colorectal cancer
      • Epimutation – biallelic
      • Acquired promoter methylation of MLH1

    * 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

    Associated with proximal tumors

    • MLH1 (mutL homolog1) – ~30% 
    • MSH2 (mutS homolog 2) – ~60%

    Less common mutations

    • PMS2 (postmeiotic segregation increased 2)
    • MSH6 (mutS homolog 6) – often associated with distal tumors and endometrial cancer
    • 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  

    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

    Encoded protein loss associated with constitutive activation of the epidermal growth factor receptor (EGFR) signaling cascade

    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 will direct mutation testing

    IHC Result

    Likely Gene Mutation

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

    BRAF V600E with hypermethylation for MLH1

    • IF MLH1 loss is identified with IHC, perform prior to mutation analysis
    • Loss of MLH1 is due to acquired hypermethylation in sporadic tumors, not a germline mutation as in LS
    • ~15% of tumors with MLH1 MSI are sporadic colorectal cancers

    If BRAF or MLH1 hypermethylation is positive

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

    PCR (panel of five mononucleotide microsatellites)

    • Relies on detection 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

    Low (indeterminate) – 1 marker with instability

    • Low risk of LS
    • Consider IHC if high suspicion of LS exists

    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

  • Early onset of colorectal cancer or family history of colon cancer in multiple family members
  • Women <50 years of age diagnosed with endometrial cancer

Criteria for Diagnosis

  • Revised Bethesda guidelines are relevant for identifying colon cancer patients whose tumor should be tested for MSI
    • 90% sensitivity, 80% specificity for Lynch syndrome (LS) and colorectal cancer
  • Revised Bethesda guidelines

    Revised Bethesda Guidelines for Testing
    Colorectal Tumors for Microsatellite Instability (MSI)

    Tumors from individuals should be tested for MSI in the following situations:

    1. Colorectal cancera diagnosed in a patient who is <50 years
    2. Presence of synchronous, metachronous colorectal, or other HNPCC-associated tumorsb, regardless of age
    3. Colorectal cancer with MSI-Hc histologyd diagnosed in a patient who is <60 yearse
    4. Colorectal cancer diagnosed in ≥1 first-degree relatives with an HNPCC-related tumor, with one of the cancers being diagnosed at <50 years
    5. Colorectal cancer diagnosed in ≥2 first- or second-degree relatives with HNPCC-related tumors, regardless of age

    a Endometrial cancer in women <50 years of age is not included in the revised Bethesda guidelines; however, recent evidence suggests that they should be evaluated for Lynch syndrome

    b HNPCC-related tumors include colorectal, endometrial, gastric, ovarian, pancreas, ureter and renal pelvis, biliary tract, brain tumors (usually glioblastoma as seen in Turcot syndrome), tumors, sebaceous gland adenomas and keratoacanthomas (as seen in Muir-Torre syndrome), and carcinoma of the small bowel

    c MSI-H = high MSI in tumors refers to changes in ≥2 of the five National Cancer Institute-recommended panels of microsatellite markers

    d Presence of tumor infiltrating lymphocytes, Crohn-like lymphocytic reaction, mucinous/signet-ring differentiation, or medullary growth pattern

    e There was no consensus among the workshop participants on whether to include the age criteria in guideline 3 above; participants voted to keep <60 years of age in the guidelines

  • Characteristic features of sporadic colorectal with MSI include the following
    • Absence of family clustering
    • Biallelic methylation of MLH1 promoters
    • Absence of MLH1 and PMS2 proteins on immunohistochemistry
    • Frequent mutation in BRAF
  • For additional information regarding testing strategies, refer to the following
    • Key Points tab for this topic
    • Lynch Syndrome Testing (HNPCC) algorithm
    • NCCN guidelines, Colorectal Cancer Screening, page LS-A 2 of 2, for table listing “Tumor Testing Results and Additional Testing Strategies”

Prognosis

  • Patients with MSI colorectal cancer have longer survival time than patients without MSI
  • Circulating tumor cell count (CTC) – may aid in prediction of survival

Differential Diagnosis

  • Sporadic colon cancer
  • Familial adenomatous polyposis

Screening

  • Family members of an individual with confirmed Lynch syndrome (LS) tumor
    • Should undergo genetic testing for the previously identified mutation
  • All colorectal and endometrial cancer patients
    • Recently, IHC and MSI screening of these individuals, regardless of age or family history, has been implemented at some centers to identify individuals at risk for LS
    • Screening endorsed by CDC’s Evaluation of Genomic Applications in Prevention and Practice Group
  • Individuals with gene mutation for LS
    • Colonoscopy every 1-2 years starting at 20-25 years
    • Extra colonic screening should also be initiated (statistics below are based on highly penetrant kindreds)
      • While not proven to decrease mortality, individual should at least be aware of potential risks of other tumors
      • Tumors at risk include the following
        • Endometrial – data does not support routine screening for Lynch screening; however, clinicians may find screening useful under some circumstances
          • If opting to screen, annual pelvic exam and aspiration biopsy at 30-35 years (20-60% lifetime cancer risk)
        • Ovarian – data does not support routine screening for Lynch; however, clinicians may find screening useful under some circumstances
          • If opting to screen, transvaginal ultrasound and potential of bilateral removal of ovaries after childbearing if familial clustering of ovarian cancers is noted (3-14% lifetime cancer risk)
        • Gastric – endoscopy if familial clustering of gastric cancers is noted (10-30% lifetime cancer risk)
      • Recommended screening interval is 1-2 years (10-20% lifetime cancer risk)
    • Average age of colon cancer onset for MSH6 and PMS2 mutation carriers is somewhat higher than for MLH1 and MSH2 carriers
      • May delay starting colon screening for 5 years (ie, to age 30) but may need to start earlier in some families, depending on ages of family members when cancers were observed (NCCN, 2012)
  • For individuals with negative genetic test results
    • Recommend colonoscopy every 5 years beginning 10 years prior to the earliest diagnosis in the family for patients with ≥1 affected first-degree relatives

Monitoring

  • Patients with resected Lynch tumors have >30% risk for synchronous and metachronous cancers
    • Perform colonoscopy 1 year following resection; if normal, repeat in 2-3 years

Clinical Background

Colon cancer exhibits the characteristics of familial clustering in >10-15% of cases. The most well-characterized 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. It is the most common cause of hereditary colorectal cancer.

Epidemiology

  • Incidence – 1/100,000-1/300,000 in the U.S.
    • Most common hereditary colorectal cancer
  • Age – 61 years (mean onset)
    • 70% risk of developing colorectal cancer by age 70 in men
    • 50% risk of developing colorectal cancer by age 70 in women
  • Sex – M:F, equal

Inheritance

  • Autosomal dominant with incomplete penetrance
  • Mutations in 1 of 4 DNA MMR genes
    • MLH1 50%
    • MSH2 40%
    • MSH6 – 7-10%
    • PMS2 <5%

Pathophysiology

  • LS is associated with accelerated carcinogenesis, causing tiny adenomas to develop into carcinomas within 2-3 years instead of the usual 8-10 years that occurs in sporadic colorectal cancer
  • Tumors are more often poorly differentiated with excess mucinous and signet ring cell features
  • Microsatellite instability (MSI) is the hallmark of LS but is also present in ~15% of sporadic colorectal cancers
    • MSI is a hypermutable phenotype caused by the loss of DNA MMR activity; also known as the mutation phenotype
  • BRAF and MLH1 methylation (sporadic colorectal cancer with MSI)
    • Presence of BRAF V600E mutation or MLH1 promoter methylation in microsatellite unstable tumor indicates that the tumor is probably sporadic, and further workup for LS may not be warranted
    • Lack of BRAF V600E mutation indicates that the tumor may either be sporadic or Lynch-associated

Clinical Presentation

  • Early onset of proximal colorectal cancer (often <50 years)
    • Multiple metachronous and synchronous tumors are common
  • LS is also associated with early-onset tumors in other organs, including endometrial, ovarian, small bowel, brain, pancreatic, gastric, renal pelvis, ureter

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 LS in individuals with CRC

If MLH1 expression is abnormal, evaluations of BRAF codon 600 and, possibly, MLH1 methylation are performed

 

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) 

If MLH1 expression is lost, MLH1 methylation is performed

   
Mismatch Repair by Immunohistochemistry 0049302
Method: Qualitative Immunohistochemistry

First-line screening test for LS

Use to determine mismatch repair deficiency and to direct additional molecular diagnostic testing for LS

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

First-line screening test for LS

Use for tumor and normal tissue to distinguish MSI from microsatellite stable tumors

   
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 MSI carcinoma with suggestive immunohistochemistry (loss of MLH1 and PMS2 protein), absence of BRAF codon 600 mutation, and normal MLH1 methylation studies

Rare diagnostic errors 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, diagnostic for LS

Use in MSI carcinoma with suggestive immunohistochemistry (loss of MSH2 and MSH6 protein)

Rare diagnostic errors 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, diagnostic for LS

Use in MSI carcinoma with suggestive immunohistochemistry (isolated loss of MSH6 protein)

Rare diagnostic errors 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, diagnostic for LS

Use in MSI carcinoma with suggestive immunohistochemistry (isolated loss of PMS2 protein)

Rare diagnostic errors 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

If no BRAF mutation is detected, MLH1 promoter methylation is evaluated

Mutations other than BRAF V600E will not be detected

Rare diagnostic errors may occur due to primer- and probe-site mutations

Methylation at locations other than those covered by the primers and probes will not be detected

Methylation levels <10% are not reported

 
BRAF V600E by Immunohistochemistry 2008710
Method: Immunohistochemistry

Aid in histologic diagnosis of Lynch syndrome

Stained and returned to client pathologist for interpretation; consultation available if needed

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

Use to predict effectiveness of therapies targeting the EGFR pathway

For endometrial tissue evaluation, refer to MLH1 Promoter Methylation, Paraffin

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

Analytic sensitivity/specificity – 99%

   
Familial Mutation, Targeted Sequencing 2001961
Method: Polymerase Chain Reaction/Sequencing

Evaluate family members for a known family mutation in an MMR gene

   
Additional Tests Available
 
Click the plus sign to expand the table of additional tests.
Test Name and NumberComments
Carcinoembryonic Antigen 0080080
Method: Quantitative Electrochemiluminescent Immunoassay
Circulating Tumor Cell Count 0093399
Method: Immunomagnetic Separation/Immunofluorescent Stain/Computer Assisted Analysis

Use to determine prognosis, assess treatment efficacy, and aid in treatment decisions for patients with colorectal cancer

Cutoffs vary by tumor cell type

KRAS Mutation Detection with Reflex to BRAF Codon 600 Mutation Detection 2001932
Method: Polymerase Chain Reaction/Pyrosequencing

Determine eligibility for anti-EGFR (cetuximab and panitumumab) therapy in patients with metastatic colorectal cancer

Familial Adenomatous Polyposis Panel: (APC) Sequencing and Deletion/Duplication, (MUTYH) 2 Mutations 2004915
Method: Polymerase Chain Reaction/Sequencing/Multiplex Ligation-dependent Probe Amplification

Differential diagnosis of Lynch syndrome