Test Catalog

Test ID: ML1HM    
MLH1 Hypermethylation Analysis, Tumor

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

An adjunct to MSI / Microsatellite Instability (MSI), Tumor and Mismatch Repair (MMR) Protein Immunohistochemistry Only, Tumor when colon or endometrial 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 or endometrial tumor demonstrates MSI-H and loss of MLH1 protein expression

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

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

Hereditary nonpolyposis colon cancer (HNPCC), also known as Lynch syndrome, is an inherited cancer syndrome caused by a germline mutation 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 HNPCC/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) 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 MSI / Microsatellite Instability (MSI), Tumor and Mismatch Repair (MMR) Protein Immunohistochemistry Only, Tumor 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 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 interpretative report will be provided.

Interpretation Provides information to assist in interpretation of the test results

An interpretive report will be provided. The likelihood of a germline (inherited) mutation is very low in those cases where the tumor demonstrates MLH1 promoter hypermethylation and the normal tissue is unmethylated. The likelihood of a germline mutation is high in those cases where the tumor and normal tissue lack MLH1 promoter hypermethylation. In cases where the tumor and normal tissue demonstrate MLH1 promoter hypermethylation, this result will be interpreted as equivocal and a blood sample 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

Testing tumors other than colon or endometrial for MLH1 hypermethylation has not been fully evaluated, and these specimens are not accepted for testing.


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 if there is high clinical suspicion of HNPCC.

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

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