Lynch Syndrome - Hereditary Nonpolyposis Colorectal Cancer (HNPCC)

Dx
Screen
Monitor
Background
Lab Tests
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

Amsterdam criteria were formerly used to assess familial risk for Lynch syndrome but are now considered obsolete due to poor sensitivity and specificity
Revised Bethesda guidelines are now considered to be more relevant in identifying colon cancer patients whose tumor should be tested for MSI
- 94% sensitivity, 51% specificity for Lynch syndrome and colorectal cancer
- Up to 30% of patients with Lynch syndrome fail to meet even the revised Bethesda guidelines

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: Colorectal cancer^a diagnosed in a patient who is <50 years Presence of synchronous, metachronous colorectal, or other HNPCC-associated tumors^b, regardless of age Colorectal cancer with MSI-H^c histology^d diagnosed in a patient who is <60 years^e Colorectal cancer diagnosed in one or more first-degree relatives with an HNPCC-related tumor, with one of the cancers being diagnosed at<50 years Colorectal cancer diagnosed in two or more 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 (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 two or more 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

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

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

Colorectal cancer^a diagnosed in a patient who is <50 years
Presence of synchronous, metachronous colorectal, or other HNPCC-associated tumors^b, regardless of age
Colorectal cancer with MSI-H^c histology^d diagnosed in a patient who is <60 years^e
Colorectal cancer diagnosed in one or more first-degree relatives with an HNPCC-related tumor, with one of the cancers being diagnosed at<50 years
Colorectal cancer diagnosed in two or more 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 (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 two or more 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

Immunohistochemistry

MSI can be inferred by immunohistochemistry but is not diagnostic for Lynch syndrome
- 10-15% false-negative rate
MSI by IHC or PCR (IHC preferred) – assess MSI in the tumor by evaluation of MMR protein (MLH1, MSH2, MSH6 and PMS2) expression
- Lynch-associated tumors will show abnormal MLH1/PMS2 immunostaining due to a germline mutation in MLH1
- PMS2 testing is performed only if no mutation is found in other genes
Majority of sporadic colorectal cancers with MSI show reduced IHC stainings for MLH1 protein due to silencing by methylation
- 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
Refer to NCCN guidelines, Colorectal Cancer Screening, page LS-A, for table listing “Tumor Testing Results and Additional Testing Strategies”

Molecular Testing

IHC guides subsequent germline MMR gene evaluation
If IHC shows abnormal MLH1/PMS2, follow up with BRAF testing to distinguish between sporadic and Lynch-associated tumors
If IHC stable but clinical suspicion of Lynch syndrome exists, follow with MSI testing by PCR
- If high or indeterminate instability in at least 2 of 5 microsatellite markers – consider germline testing of mismatch repair genes
- If no instability present – Lynch syndrome unlikely

Prognosis

Potential prognostic stains – p16, p21, p27 (Kip1), p53, circulating tumor cell count
Patients with MSI colorectal cancer have longer survival time than patients without MSI

Differential Diagnosis

Sporadic colon cancer
Familial adenomatous polyposis

Screening

Family members of an identified patient should have genetic testing for the previously identified mutation
Recently, IHC and MSI screening of all colorectal and endometrial cancer patients regardless of age or family history has been implemented at some centers to identify individuals at risk for Lynch syndrome
- Endorsed by CDC’s Evaluation of Genomic Applications in Prevention and Practice Group
For patients with positive genetic test results, the following is recommended by the International Collaborative Group-HNPCC and the American Medical Association
- 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 necessarily decrease mortality, patient 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 screening; however, clinicians may find screening useful under some circumstances
      - If opting to screen, transvaginal ultrasound and potential of bilateral removal of ovaries after childbearing is finished if clustering of ovarian cancers is noted (3-14% lifetime cancer risk)
    - Gastric – endoscopy if clustering of gastric cancers is noted (10-30% lifetime cancer risk)
  - Recommended screening interval is 1-2 years (10-20% lifetime cancer risk)
If testing is negative – recommend colonoscopy every 5 years commencing 10 years before the youngest involved family member presented with a tumor

Monitoring

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

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 or hereditary nonpolyposis colorectal cancer (HNPCC). Lynch syndrome is caused by germline mutations in the genes that encode mismatch repair (MMR) factors. It is the most common cause of hereditary colorectal cancer and accounts for 2-3% of colorectal cancer.

Epidemiology

Incidence – 1/100,000-1/300,000 in the U.S.
Age – mean onset is 45-50 years
- 80% risk of developing colorectal cancer by age 70 in men
- 20-40% risk of developing colorectal cancer by age 70 in women
Sex – M:F, equal

Inheritance

Autosomal dominant with incomplete penetrance
Mutations of one of four DNA MMR genes: MLH1, MSH2, MSH6, PMS2
- 6% of colorectal cancers are due to MMR gene mutations
- MLH1 and MSH2 mutations responsible for ~90% of Lynch syndrome
- MSH6 mutations responsible for ~10% of Lynch syndrome
- PMS2 mutations are rarely responsible

Pathophysiology

Lynch syndrome 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 Lynch syndrome but is also present in ~15% of sporadic colorectal cancers
- MSI is a hyper mutable phenotype caused by the loss of DNA MMR activity; also known as the mutation phenotype
BRAF and MLH1 methylation (sporadic colorectal cancer with MSI)
- BRAF gene encodes a serine-threonine kinase and plays a role in the mitogen-activated protein kinase signaling pathway
- BRAF V600E mutation is commonly seen in sporadic colorectal cancers with MSI
- MLH1 methylation is commonly seen in sporadic microsatellite-unstable colorectal cancer
- Assessment of the unstable tumor for BRAF mutations and MLH1 methylation separates sporadic unstable tumors from those associated with Lynch syndrome

Clinical Presentation

Early onset of proximal colorectal cancer (often at 20-30 years)
- Multiple metachronous and synchronous tumors are common
Syndrome is also associated with other 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 0049302 Method: Qualitative Immunohistochemistry	Preferred test to discriminate between Lynch syndrome and sporadic mutations in colorectal cancers Surrogate test for MSI by PCR May help guide subsequent mutation analysis	83% clinical sensitivity; 90% clinical specificity	If negative, but strong suspicion for Lynch syndrome exists, follow up testing with PCR
HNPCC/Lynch Syndrome, Microsatellite Instability by PCR 0051740 Method: Polymerase Chain Reaction/Fragment Analysis	Discriminate between MSI and microsatellite-stable cancer	90% sensitivity for Lynch syndrome
HNPCC/Lynch Syndrome Deletion/Duplication 2001728 Method: Polymerase Chain Reaction/Multiplex Ligation-dependent Probe Amplification	Detect mutations and large deletions in MLH1, MSH2, MSH6 or PMS2 genes	For PMS2 testing, suspected deletions or duplication in exons 12-15 require sequencing to exclude pseudogene copy number variants
HNPCC/Lynch Syndrome (MLH1) Sequencing and Deletion/Duplication 0051650 Method: Polymerase Chain Reaction/Sequencing/Multiplex Ligation-dependent Probe Amplification	Identify mutations and large deletions in MLH1gene Present in ~45% of Lynch syndrome	Rare diagnostic errors can occur due to primer and probe site mutations Breakpoints of large deletions/duplications will not be determined Regulatory region mutations, deep intronic mutations and mutations in genes other than MLH1 will not be detected
HNPCC/Lynch Syndrome (MSH2) Sequencing and Deletion/Duplication 0051654 Method: Polymerase Chain Reaction/Sequencing/Multiplex Ligation-dependent Probe Amplification	Detect mutations and large deletions in MSH2 gene Present in ~45% of Lynch syndrome	Rare diagnostic errors can occur due to primer and probe site mutations Breakpoints of large deletions/duplications will not be determined Regulatory region mutations, deep intronic mutations and mutations in genes other than MSH2 will not be detected
HNPCC/Lynch Syndrome (MSH6) Sequencing and Deletion/Duplication 0051656 Method: Polymerase Chain Reaction/Sequencing/Multiplex Ligation-dependent Probe Amplification	Detect mutations and large deletions in MSH6 gene Present in ~10% of Lynch syndrome	Rare diagnostic errors can occur due to primer and probe site mutations Breakpoints of large deletions/duplications will not be determined Regulatory region mutations, deep intronic mutations and mutations in genes other than MSH6 will not be detected
HNPCC/Lynch Syndrome (PMS2) Sequencing and Deletion/Duplication 0051737 Method: Polymerase Chain Reaction/Sequencing/Multiplex Ligation-dependent Probe Amplification	Detect mutations and large deletions in PMS2 gene Present in a small fraction of Lynch syndrome Suspected deletions or duplications in exons 12-15 require additional sequencing to exclude pseudogene copy number variants	Rare diagnostic errors can occur due to primer and probe site mutations Breakpoints of large deletions/duplications will not be determined Regulatory region mutations, deep intronic mutations and mutations in genes other than PMS2 will not be detected PMS2 exons 3, 4, 12, 13, 14 or 15 are not evaluated for deletions/duplications
Mismatch Repair by Immunohistochemistry with Reflex to MLH1 Promoter Methylation 2005270 Method: Qualitative Immunohistochemistry/Qualitative Real-time Polymerase Chain Reaction	Distinguish between sporadic, microsatellite unstable non-colorectal cancer and Lynch-associated non-colorectal cancer	Rare diagnostic errors can occur due to primer and probe site mutations Breakpoints of large deletions/duplications will not be determined Regulatory region mutations, deep intronic mutations and mutations in genes other than MLH1 will not be detected
CDX2 by Immunohistochemistry 2003821 Method: Immunohistochemistry	Aid in histologic diagnosis of colorectal cancer Stained and returned to client pathologist; consultation available if needed
Carcinoembryonic Antigen, Monoclonal (CEA M) by Immunohistochemistry 2003824 Method: Immunohistochemistry	Aid in histologic diagnosis of colorectal cancer Stained and returned to client pathologist; consultation available if needed
Cytokeratin 20 (CK 20) by Immunohistochemistry 2003848 Method: Immunohistochemistry	Aid in histologic diagnosis of colorectal cancer Stained and returned to client pathologist; consultation available if needed
Muc-2 by Immunohistochemistry 2004005 Method: Immunohistochemistry	Aid in histologic diagnosis of colorectal cancer Stained and returned to client pathologist; consultation available if needed
p16 by Immunohistochemistry 2004064 Method: Immunohistochemistry	Prognostic stain Stained and returned to client pathologist; consultation available if needed
p21 (Waf1/Cip 1) by Immunohistochemistry 2004067 Method: Immunohistochemistry	Prognostic stain Stained and returned to client pathologist; consultation available if needed
p27 (Kip1) by Immunohistochemistry 2004070 Method: Immunohistochemistry	Prognostic stain Stained and returned to client pathologist; consultation available if needed
p53 Tissue Assay, Paraffin 0049250 Method: Immunohistochemistry	Prognostic stain Stained and returned to client pathologist; if consultation required, contact anatomic pathology, surgical consult or hematopathology
BRAF Codon 600 Mutation Detection with Reflex to MLH1 Promoter Methylation 0051750 Method: Polymerase Chain Reaction/Pyrosequencing	Detect the presence of the V600E mutation in colorectal cancers to differentiate Lynch syndrome from sporadic colorectal cancer V600E mutation accounts for ~90% of all BRAF mutations If no BRAF mutation is detected, MLH1 promoter methylation is evaluated; evaluation can also help determine whether further workup for Lynch syndrome is indicated Presence indicates colorectal cancer is sporadic and Lynch syndrome is not present	Mutations other than BRAF V600E will not be detected Rare diagnostic errors may occur due to primer- or probe-site mutations Methylation at locations other than those covered by the primers and probes will not be detected <10% presence of a mutant allele may not be detected
BRAF codon 600 Mutation Detection by Pyrosequencing 2002498 Method: Polymerase Chain Reaction/Pyrosequencing	Detect the presence of the V600E mutation in colorectal cancers Presence indicates colorectal cancer is sporadic and Lynch syndrome is not present
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 Number	Comments
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
Carcinoembryonic Antigen 0080080 Method: Quantitative Electrochemiluminescent Immunoassay
Circulating Tumor Cell Count (Cell Search™) 0093399 Method: Immunomagnetic Separation/Immunofluorescence Stain/Computer Assisted Analysis