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Lynch Syndrome: Laboratory Support of Diagnosis and Management
|Laboratory Support of Diagnosis and Management|
Colorectal Cancer Overview
Colorectal cancer (CRC) is the second leading cause of cancer death in the United States, with projections of 49,000 deaths and 134,000 new cases diagnosed in 2016.1 About three-quarters of CRC cases are sporadic.2 The remaining are familial (20%) or inherited (5% to 10%).2,3 Familial CRC is not well understood and is characterized by an increased risk of CRC and an unclear pattern of inheritance. Inherited CRC, on the other hand, is caused by a germline mutation and exhibits a clear pattern of inheritance. Inherited CRC includes Lynch syndrome (formerly known as hereditary nonpolyposis colorectal cancer [HNPCC]) as well as other less common syndromes. Whether colorectal cancer is sporadic, familial, or inherited has important implications for screening and follow-up of patients and their family members.
Lynch syndrome is caused by a pathogenic germline variant in a mismatch repair (MMR) gene (MLH1, MSH2, MSH6, and PMS2) or in the EPCAM gene. These variants result in a defective MMR process in which nucleotide mismatches that occur during DNA replication are not repaired. Another consequence is insertions or deletions of nucleotides within repeated DNA nucleotide sequences known as microsatellites, ie, microsatellite instability (MSI). In Lynch syndrome, 1 copy of a defective MMR allele is inherited. Subsequent somatic alteration of the normal allele leads to the progression of a benign adenoma to CRC.4
Lynch syndrome is characterized by a higher lifetime cancer risk and, typically, an earlier age of onset than in the general population (Table 1).5 Patients with Lynch syndrome have an increased risk of cancer in the colon, rectum, stomach, small intestine, hepatobiliary tract, urinary tract, brain, skin, and pancreas.5,6 Women with the syndrome also have an increased risk for endometrial and ovarian cancers.
Table 1. Lifetime Cancer Risk in Individuals with Lynch Syndrome5-15
Among patients with Lynch syndrome, early detection and removal of colorectal adenomas and carcinomas is critical. Surveillance colonoscopy enables their detection and removal at an earlier stage and is associated with decreased CRC-specific mortality.16,17 Furthermore, a Lynch syndrome diagnosis in the family may foster testing of blood relatives and early initiation of surveillance if an MMR gene mutation is found.
The National Comprehensive Cancer Network® (NCCN) recommends that testing for Lynch syndrome be considered for all previously untested individuals with CRC.6 This approach has been incorporated into guidelines approved by the American College of Gastroenterology, the American Gastroenterological Association, the American Society for Gastrointestinal Endoscopy, and the American Society of Colon and Rectal Surgeons.18 The approach identifies more patients with Lynch syndrome than selecting patients based on the personal and family history factors outlined in the Bethesda guidelines (Table 2).19
NCCN also provides an alternative recommendation for testing those with a CRC diagnosis: test all individuals diagnosed with CRC at <70 years of age and those ≥70 years of age if they meet the Bethesda guidelines.
NCCN further recommends testing individuals if they meet Bethesda guidelines or Amsterdam criteria (Table 3), have been diagnosed with endometrial cancer before age 50, have a blood relative with Lynch syndrome, or have a ≥5% risk for Lynch syndrome based on computer models (MMRPro, PREMM[1,2,6], or MMRpredict). These models use personal and family history to calculate the likelihood of an individual carrying a mutation in an MMR gene.6
Table 3. Amsterdam II Criteria for Identifying Families with Lynch Syndrome21
FAP, familial adenomatous polyposis.
The tests recommended by the NCCN and others include screening tests such as MSI and IHC of the 4 MMR proteins (supplemented by BRAF mutation testing) as well as germline mutation testing of the MMR genes. These tests are offered by Quest Diagnostics and can be ordered using the test codes listed in Table 4. The table is provided for informational purposes only and is not intended as medical advice. A physician's test selection and interpretation, diagnosis, and patient management decisions should be based on his/her education, clinical expertise, and assessment of the patient.
Test Selection and Interpretation [return to contents]
Individuals with Lynch Syndrome-related Cancer
MSI and/or IHC testing of tumor tissue is used to identify patients who may benefit from MMR mutation testing, which is required for a Lynch syndrome diagnosis.6,24 In patients fulfilling the Bethesda guidelines (Table 2), MSI and IHC screening tests were both found to be effective in selecting patients for MMR mutation testing.4,6,25 However, using either MSI or IHC testing alone resulted in false-negative results.26 Thus, sensitivity can be maximized by using both MSI and IHC testing.6,27
Microsatellite Instability (MSI) Testing
MSI testing compares the number of nucleotide repeats in microsatellite markers with the number in normal tissue (or whole blood) from the same individual. If the number differs, MSI is present. Results are reported as MSI-high (MSI-H) if ≥2 of the 5 National Cancer Institute-recommended markers show instability. 20 MSI-low (MSI-L) results are reported if 1 of 5 is positive; negative results are reported as microsatellite stable (MSS). Although an MSI-H result is the hallmark of Lynch syndrome, it is also found in 10% to 15% of sporadic CRC cases. 28 As such, an MSI-H result requires follow-up with MMR gene mutation testing. In families with a strong suspicion of Lynch syndrome, MMR gene mutation testing should be considered regardless of MSI status, since MSI results do not rule out Lynch syndrome.20
Immunohistochemical (IHC) Testing for MMR Proteins
A pathogenic variant in an MMR gene(s) usually leads to a loss of corresponding protein expression, which can be detected using IHC testing. The absence of IHC staining thus serves as a surrogate marker of an MMR pathogenic variant. IHC testing is usually followed with genetic testing of selected MMR genes to determine whether a deleterious mutation is present. For example, loss of MSH2 expression is confirmed with MSH2/EPCAM mutation testing. The overall clinical sensitivity (Lynch syndrome detection rate) of IHC MMR protein testing of colorectal cancers is 77% (95% CI, 69% to 84%).29
MMR Gene Mutation Testing
Germline mutation testing should include analysis for various types of DNA alterations. DNA sequence changes, such as point mutations and alterations involving just a few nucleotides, are most frequent. However, roughly 17% of individuals with suspected Lynch syndrome have large rearrangements, which may include deletions and duplications involving partial, whole, or multiple gene exons.30 Approximately 32% of the mutations occur in MLH1, 39% in MSH2, 14% in MSH6, and 15% in PMS2.29
Selection of the MMR gene(s) to be analyzed can be based on the pattern of IHC staining, ie, which proteins stain negative. Four patterns constitute 98% of the IHC tumor-screening results31 and each is followed with a different mutation-testing approach (Figure 2).
Loss of Both MLH1 and PMS2 Staining
This is the most common IHC pattern.31 In most cases, it is associated with non-Lynch syndrome CRC and is primarily caused by MLH1 promoter methylation and silencing of the MLH1 gene. MLH1 loss usually results in degradation and loss of PMS2, which is unstable outside of an MLH1/PMS2 heterodimeric complex.
Since BRAF V600E mutation is the primary cause of MLH1 promoter methylation,32 loss of MLH1 and PMS2 staining should be followed with BRAF mutation testing. A positive BRAF V600E result essentially rules out Lynch syndrome.19 Note that BRAF mutation testing is appropriate only for patients with CRC since the mutation is uncommon in other Lynch syndrome-associated cancers.5,6 For the other types of cancers, proceed directly to MLH1 gene testing.
When a BRAF V600E test result is negative, Lynch syndrome is likely. MLH1 and PMS2 gene mutation testing is suggested. Testing may be performed concurrently; however, since a pathogenic variant in MLH1 is much more likely than in PMS2, PMS2 mutation testing could be performed sequentially (ie, after a negative MLH1 test).
Loss of Both MSH2 and MSH6 Staining
This is the second most common IHC pattern.31 MSH2 and MSH6 also form a heterodimeric complex. MSH6 is unstable outside this complex, so loss of MSH2 usually results in degradation and loss of MSH6. Loss of these 2 proteins should be followed with MSH2 mutation testing. MSH2 gene testing offered by Quest Diagnostics includes testing for a 3′-deletion in the EPCAM gene, which occurs in about 1% to 3% of Lynch syndrome cases.5,33 The EPCAM gene is adjacent to the MSH2 gene in the genome. Mutation of the 3' end of EPCAM can influence MSH2 gene expression. When present, a 3′-deletion in EPCAM inactivates the MSH2 gene.34 Thus, loss of MSH2 staining can be explained either by MSH2 mutation or by 3′-deletion in EPCAM. The presence of either is diagnostic of Lynch syndrome. Testing for an MSH6 mutation should be considered as well. It can be performed along with the MSH2/EPCAM mutation test or after a negative MSH2/EPCAM test.
Loss of MSH6 Only
This is the third most common pattern,31 most likely due to a mutation in the MSH6 gene.35 A deleterious mutation in either the MSH6 or the MSH2 gene, however, is a plausible etiology. Sequential or concurrent MSH6 and MSH2 mutation testing can thus be considered.6 Loss of MSH6 staining should be interpreted with caution in rectal cancers, as it may be caused by neoadjuvant chemoradiotherapy.6
Loss of PMS2 Only
This is the fourth most common pattern,31 most likely due to a mutation in the PMS2 gene.36 A deleterious mutation in either the PMS2 or the MLH1 gene is a plausible etiology. Sequential or concurrent testing for both PMS2 and MLH1 mutation may be considered for this pattern.6
These are rarely observed, and the mechanisms responsible are not always understood.31,37 Loss of staining for a particular MMR protein, however, should generally be followed by mutation testing of the corresponding gene and possibly its heterodimeric partner.6
Detection of a deleterious MMR gene mutation is diagnostic for Lynch syndrome, but failure to identify a mutation does not rule out the diagnosis.5
MMR gene mutation testing is recommended for at-risk individuals. These include blood relatives of Lynch syndrome patients and individuals who meet the Amsterdam criteria (Table 3) or who have a ≥5% risk for Lynch syndrome based on computer models (MMRPro, PREMM[1,2,6], or MMRpredict). When the family pathogenic variant is known, a targeted, gene-specific test that can detect the specific alteration can be used (eg, MyVantage™, Single Site, test code 93945). When the family mutation is not known, the Lynch Syndrome Panel (test code 91461) can be used; this panel offers comprehensive MMR gene testing. When using this panel, relatives with a Lynch syndrome-related cancer should be tested before relatives without cancer in order for test results to be most informative.6 Because not all Lynch syndrome families meet the Amsterdam criteria, the panel should also be considered when there is a strong suspicion of Lynch syndrome based on family history.6 Figure 3 demonstrates these options.
Identification of a deleterious mutation in an MMR gene confirms the clinical diagnosis of Lynch syndrome and thereby identifies individuals who may benefit from risk reduction or routine surveillance procedures. According to NCCN guidelines, CRC surveillance should be initiated at 20 to 25 years of age or 2 to 5 years earlier than the youngest age at which CRC was diagnosed in the family (whichever is earlier).6 Surveillance for other Lynch syndrome-related cancers varies; more information can be found in the NCCN guidelines.
Gene testing should precede surveillance to ensure proper patient management. If genetic testing is negative for the known pathogenic variant, cancer screening can likely be performed as for the average-risk population.
Additional assistance in test selection and interpretation of results is available from Quest Diagnostics Genomics Client Services by calling 866.GENE.INFO (866.436.3463).
Content reviewed 11/2016
* The tests listed by specialist are a select group of tests offered. For a complete list of Quest Diagnostics tests, please refer to our Directory of Services.