Label Name: MLH1METH
Lab Discipline: Molecular Diagnostics
Institution:  Duke University Health System 
EAP ID:  LAB7925 
Last Review:  3/17/2017 10:10:12 AM
Specimen Type
Container & Volume
  Age Group   Container   Volume  
  0  - 18 Years Sterile Specimen Cup 3  ML
Collection Notes
  • This assay uses genomic DNA isolated from paraffin-embedded tumor tissue. Nine freshly cut (within one week) unstained slides should be submitted containing 5 mm2 to 20 mm2 of tissue. The slides should be prepared in the Molecular Diagnostics Lab (Histology will perform for especially small biopsies) following procedure MD-082 (Paraffin Microtomy)-external clients may send unstained slides in lieu of a block to be cut in-house. An H&E stained slide should be prepared and submitted for reference purposes (unstained slide should be taken to Histology for staining). The pathologist on service will circle the tumor-containing area on the H&E slide and indicate approximate percentage of tumor cells present.

Turn Around Time -  Routine: 10 days   Stat: N/A
Reference Values
Methylated promoter sequences NOT detected
  This assay uses methylation-specific PCR to determine the presence or absence of DNA methylation in the promoter of the MLH1 gene. An H&E stained slide for each case is first evaluated to identify the regions of greatest tumor content. These regions are then macro-dissected from adjacent unstained formalin-fixed paraffin-embedded sections and used to prepare genomic DNA. This genomic DNA is treated with sodium bisulfate to convert unmethylated cytosines to uracil. Bisulfite-modified DNA is added to a multiplex PCR amplification reaction with fluorescently-tagged (FAM) primers that specifically amplify either the methylated cytosine-containing allele (89 bp amplification product) or the unmethylated uracil-containing allele (97 bp amplification product). Amplification products are resolved and analyzed on an ABI 3130xl or 3500 genetic analyzer.

Clinical Significance and Interpretive Data
    HNPCC is an inherited tumor syndrome associated with an increased risk of developing a variety of different cancers, including colon, endometrial and gastric adenocarcinoma. Most HNPCC patients have an inherited mutation in one of four genes (MLH1, MSH2, MSH6 and PMS2). These genes are involved in DNA mismatch repair (MMR). Tumors that form in patients with these inherited mutations have a defect in MMR that manifests as a characteristic cellular phenotype termed ‘microsatellite instability (MSI)’. MSI is present in the great majority of tumors from patients with HNPCC. Thus, the absence of MSI makes a diagnosis of HNPCC very unlikely. However, MSI can also occur in patients who do not have HNPCC, but rather, sporadic, non-familial, cancer. MLH1 methylation analysis can be used to help distinguish between these two possibilities. MLH1 promoter methylation is very rare in carcinomas arising in patients with HNPCC. Thus, the presence of MLH1 promoter methylation suggests a sporadic origin of disease, making HNPCC unlikely. The absence of the MLH1 promoter methylation is not diagnostic of HNPCC, but rather suggests that additional studies are necessary to either further rule out HNPCC or establish this diagnosis. These results should be considered in the context of other clinical and laboratory findings, including a detailed family history and immunohistochemical (IHC) testing for a loss of expression of the MMR genes to determine the need for further evaluation or molecular genetic testing. Due to the unique nature of genetic screening, patients offered this test should receive pre-test and post-test genetic counseling. Counseling should help the patient understand the strengths and limitations of DNA testing and the medical implications for the patient, as well as for other family members.


Bettstetter M, 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.

Capel E et al. Assessment of MLH1 promoter methylation in relation to gene expression requires specific analysis. Oncogene 2007;26(54):7596-600.

Hampel H, et al. Screening for the Lynch syndrome (hereditary nonpolyposis colorectal cancer). N Engl J Med. 2005;352(18):1851-60.

Hampel H, et al. Feasibility of screening for Lynch syndrome among patients with colorectal cancer. J Clin Oncol. 2008;26(35):5783-8.

    Colorectal or endometrial cancer cases showing microsatellite instability and loss of MLH1 expression by immunohistochemsitry. Testing for BRAF p.V600E mutation prior to and/or along with the MLH1 promoter methylation test is the recommended best approach to identify/confirm the sporadic versus HNPCC origin of the cancer.
    This test detects only the MLH1 promoter methylation. Other genetic events that may lead to MSI would not be detected using this assay. Mutations in MLH1 associated HNPCC are not detected by this assay. The analytical sensitivity of this assay is 5%. If cells with MLH1 promoter methylation are fewer than 10% in this specimen, methylation may not be detected by this assay. Poor DNA quality, insufficient DNA quantity or the presence of PCR inhibitors can result in uninterpretable or (rarely) inaccurate results. For additional information or for help interpreting the results of this test, clinicians should contact the DUHS Clinical Molecular Diagnostics Laboratory. Patients should contact their healthcare provider with any questions related to this report.

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Molecular Diagnostics Laboratory

Medical Director:
 Michael Datto, M.D., Ph.D.
 Phone: 919-684-6965
Lab Director:
 Catherine Rehder Ph.D, FACMG
 Phone: 919-613-8434
Lab Director:
 Siby Sebastian Ph.D., DABMG
 Phone: 919-613-8432

 Wadsworth Bldg, Cytogenetics, Rm 0220
 2351 Erwin Rd
 Durham,  NC  27705
 Phone: 919-684-2698
 FAX: 919-668-5424

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