MLH1 Hypermethylation and BRAF Mutation Analyses, Tumor
Clinical Information Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test
Hereditary nonpolyposis colon cancer (HNPCC), also known as Lynch syndrome, is an inherited cancer syndrome caused by a germline mutation in 1 of several genes involved in DNA mismatch repair (MMR), including MLH1, MSH2, MSH6, and PMS2. There are several laboratory-based strategies that help establish the diagnosis of HNPCC/Lynch syndrome, including testing tumor tissue for the presence of microsatellite instability (MSI-H) and loss of protein expression for any 1 of the MMR proteins by immunohistochemistry (IHC). It is important to note, however, that the MSI-H tumor phenotype is not restricted to inherited cancer cases; approximately 20% of sporadic colon cancers are MSI-H. Thus, MSI-H does not distinguish between a somatic (sporadic) and a germline (inherited) mutation, nor does it identify which gene is involved. Although IHC analysis is helpful in identifying the responsible gene, it also does not distinguish between somatic and germline defects.
Defective MMR in sporadic colon cancer is most often due to an abnormality in MLH1, and the most common cause of gene inactivation is promoter hypermethylation (epigenetic silencing). A specific mutation in the BRAF gene (V600E) has been shown to be present in approximately 70% of tumors with hypermethylation of the MLH1 promoter. Importantly, the V600E mutation is rarely identified in cases with germline MLH1 mutations. Thus, direct assessment of MLH1 promoter methylation status and testing for the BRAF V600E mutation can be used to help distinguish between a germline mutation and epigenetic/somatic inactivation of MLH1. Tumors that have the BRAF V600E mutation and demonstrate MLH1 promoter hypermethylation are almost certainly sporadic, whereas tumors that show neither are most often caused by an inherited mutation.
Although testing for the BRAF V600E mutation and MLH1 promoter hypermethylation are best interpreted together, they are also available separately to accommodate various clinical situations and tumor types. These tests can provide helpful diagnostic information when evaluating an individual suspected of having HNPCC/Lynch syndrome, especially when testing is performed in conjunction with HNPCC/17073 Hereditary Nonpolyposis Colorectal Cancer (HNPCC) Screen, which includes MSI and IHC studies. It should be noted that these tests are not genetic tests, but rather stratify the risk of having an inherited cancer predisposition and identify patients who might benefit from subsequent genetic testing.
See Hereditary Nonpolyposis Colon Cancer Diagnostic Testing Algorithm in Special Instructions. Also, see Hereditary Colorectal Cancer: Hereditary Nonpolyposis Colon Cancer (November 2005, Communique) in Publications.
As an adjunct to HNPCC/17073 Hereditary Nonpolyposis Colorectal Cancer (HNPCC) Screen, when colon tumor demonstrates microsatellite instability (MSI-H) and loss of MLH1 protein expression, to help distinguish a somatic versus germline event prior to performing expensive germline testing
As an adjunct to negative MLH1 germline testing in cases where colon tumor from the same patient demonstrates MSI-H and loss of MLH1 protein expression
An interpretive report will be provided. The likelihood of a germline (inherited) mutation is very low in those cases where the tumor demonstrates both MLH1 promoter hypermethylation and the BRAF V600E mutation. The likelihood of a germline mutation is high in those cases where the tumor lacks both MLH1 promoter hypermethylation and the BRAF V600E mutation. In cases where the tumor and normal tissue demonstrate MLH1 promoter hypermethylation and absence of the BRAF V600E mutation, this result will be interpreted as equivocal and a blood specimen will be requested to confirm potential germline hypermethylation.
Cautions Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances
This test is not recommended as a first-tier screening measure for hereditary nonpolyposis colon cancer (HNPCC). Please refer to HNPCC/17073 Hereditary Nonpolyposis Colorectal Cancer (HNPCC) Screen. Testing will only be performed on colon tumors demonstrating microsatellite instability or immunohistochemistry results indicating loss of MLH1 protein expression.
Testing tumors other than colon or endometrial for MLH1 hypermethylation has not been fully evaluated, and these specimens are not accepted for testing.
Testing tumors other than colon (in the evaluation of HNPCC) for BRAF has not been fully evaluated; therefore other specimens are not accepted.
Colon cancer is relatively common and it is possible for a sporadic colon cancer to occur in an HNPCC family. Therefore, evaluation of other family members should still be considered in cases with MLH1 promoter hypermethylation and absence of the BRAF V600E mutation if there is high clinical suspicion of HNPCC.
Reference Values Describes reference intervals and additional information for interpretation of test results. May include intervals based on age and sex when appropriate. Intervals are Mayo-derived, unless otherwise designated. If an interpretive report is provided, the reference value field will state this.
An interpretive report will be provided.
Clinical References Provides recommendations for further in-depth reading of a clinical nature
1. Cunningham JM, Kim C-Y, Christensen ER, et al: The frequency of hereditary defective mismatch repair in a prospective series of unselected colorectal carcinomas. Am J Hum Genet 2001;69:780-790
2. Wang L, Cunningham JM, Winters JL, et al: BRAF mutations in colon cancer are not likely attributable to defective DNA mismatch repair. Cancer Res 2003;63:5209-5212
3. Domingo E, Laiho P, Ollikainen M, et al: BRAF screening as a low-cost effective strategy for simplifying HNPCC genetic testing. J Med Genet 2004;41:664-668
4. Bettstetter M, Dechant S, Ruemmele P, et al: Distinction of hereditary nonpolyposis colorectal cancer and sporadic microsatellite-unstable colorectal cancer through quantification of MLH1 methylation by real-time PCR. Clin Cancer Res 2007;13:3221-3228