Test Catalog

Test ID: BRMLH    
MLH1 Hypermethylation and BRAF Mutation Analysis, Tumor

Useful For Suggests clinical disorders or settings where the test may be helpful

An adjunct to MSI / Microsatellite Instability (MSI), Tumor and IHC / Mismatch Repair (MMR) Protein Immunohistochemistry Only, Tumor testing, 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


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

Genetics Test Information Provides information that may help with selection of the correct genetic test or proper submission of the test request

If this test is ordered in conjunction with the MLH1 immunostain (IHC / Mismatch Repair [MMR] Protein Immunohistochemistry Only, Tumor) and MSI (MSI / Microsatellite Instability [MSI], Tumor), this test will only be performed when clinically indicated.

Testing Algorithm Delineates situations when tests are added to the initial order. This includes reflex and additional tests.

When this test is ordered, BRAF analysis and MLH1 hypermethylation analysis will always be performed. The exception would be if the tissue origin is an endometrial tumor; in those cases only the MLH1 hypermethylation analysis component will be performed.


When this test is ordered, slide review will always be performed at an additional charge.


See Lynch Syndrome Testing Algorithm in Special Instructions.

Clinical Information Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test

Lynch syndrome is an inherited cancer syndrome caused by a germline pathogenic variant in one 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 Lynch syndrome, including testing tumor tissue for the presence of microsatellite instability (MSI-H) and loss of protein expression for any one 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) etiology, 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 alteration 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 alteration is rarely identified in cases with germline MLH1 pathogenic variants. Thus, direct assessment of MLH1 promoter methylation status and testing for the BRAF V600E alteration can be used to help distinguish between germline etiologyand epigenetic/somatic inactivation of MLH1. Tumors that have the BRAF V600E alteration and demonstrate MLH1 promoter hypermethylation are almost certainly sporadic, whereas tumors that show neither are most often caused by an inherited (germline) pathogenic variant.


Although testing for the BRAF V600E alteration 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 Lynch syndrome, especially when testing is performed in conjunction with MSI / Microsatellite Instability (MSI), Tumor and IHC / Mismatch Repair (MMR) Protein Immunohistochemistry Only, Tumor. 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 Lynch Syndrome Testing Algorithm in Special Instructions.

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.

Interpretation Provides information to assist in interpretation of the test results

An interpretive report will be provided.

Cautions Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances

Testing tumors other than colon (in the evaluation of Lynch syndrome) for BRAF and MLH1 hypermethylation 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 a Lynch syndrome family. Therefore, evaluation of other family members should still be considered in cases with MLH1 promoter hypermethylation and absence of the BRAF V600E alteration if there is high clinical suspicion of Lynch syndrome.

Clinical Reference Recommendations for in-depth reading of a clinical nature

1. Cunningham JM, Kim CY, 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

5. Gupta S, Provenzale D, Llor X, et al: NCCN Guidelines Insights: Genetic/familial high-risk assessment: colorectal, version 2.2019. J Natl Compr Canc Netw. 2019;17(9):1032-1041

Special Instructions Library of PDFs including pertinent information and forms related to the test