MLH1 Promoter Methylation

Performing Lab: 
UCSF Clinical Cancer Genomics Lab
CPT Code: 
81288, 88381

Specimen Type: Formalin-fixed, paraffin-embedded tissue.  Tissue block or unstained slides.  If sending unstained slides, we require five (5) 10-micron tissue sections on uncharged glass slides. One adjacent H&E stained slide should be included.
Methodology: Bisulfite treatment followed by high resolution melting curve analysis.
Turnaround Time: This assay is run once per month, 40 days


MLH1 promoter methylation is common in sporadic microsatellite unstable tumors, including colorectal cancer and endometrial cancer, and is associated with loss of MLH1 protein expression.  It is rarely detected in Lynch syndrome (hereditary nonpolyposis colorectal cancer or HNPCC).  Therefore, in tumors with high-level microsatellite instability (MSI-H) and/or loss of MLH1 staining by immunohistochemistry, the absence of MLH1 promoter methylation is associated with an increased likelihood of germline mutation in a DNA mismatch repair protein.


A histologic section of formalin-fixed, paraffin-embedded tissue is examined by a pathologist to identify areas with sufficient tumor for detection.  DNA is extracted from tumor areas on adjacent unstained slides and treated with bisulfite, which converts unmethylated cytosine residues to uracil.  Methylated cytosines are protected from conversion.  Treated DNA is then amplified by real-time PCR.  A 100 bp segment of the MLH1 promoter known to contain eight CpG dinucleotides and be subject to methylation is targeted.  The amplified product is detected with the intercalating dye EvaGreen®.  The melt characteristics of the generated amplicons are determined by loss of EvaGreen® fluorescence as the double-stranded molecule is heated and becomes single-stranded.  The temperature at which the amplicon dissociates is dependent upon the sequence, and indicates whether the sequence was methylated or unmethylated.


Lack of MLH1 methylation identification by this assay is not sufficient for a diagnosis of Lynch syndrome.  Additionally, up to 1% Lynch syndrome cases appear to be caused by constitutional MLH1 promoter methylation.  Therefore, a diagnosis of Lynch syndrome cannot be definitively confirmed or ruled out on the basis of the MLH1 promoter methylation testing alone.  Correlation with patient history and other testing (including mismatch repair immunohistochemistry, microsatellite instability, MLH1 gene sequencing, and in cases of colorectal carcinoma, BRAF mutation) is recommended.  This assay cannot rule out the presence of an epigenetic alteration outside of the promoter region analyzed.  This test only detects methylation, and will not detect point mutations, insertions, deletions, or inversions.

This test is intended for use in tumors that show high-level microsatellite instability (MSI-H) and/or loss of MLH1 staining by immunohistochemistry.

This test was validated by the UCSF Clinical Cancer Genomics Laboratory to confirm performance characteristics, in compliance with current guidelines for clinical implementation


In general, the tumor should be a minimum of 0.4 cm in size.  Blocks selected for MLH1 Promoter Methylation testing must contain tumor tissue.  Areas of tumor must contain at least 50% tumor cells. The lab can remove adjacent non-tumor tissue, so the entire slide does not need 50% tumor cells, just the area with tumor.  Contact the laboratory at 415.502.3252 or [email protected] if the specimen suitability is uncertain.  Label slides with pathology case number and block identification.

Specimen rejection criteria include: All required slides not included. Insufficient tumor tissue present on slide as determined by pathologist. Outside slides not labeled or not accompanied by printed copy of test order.


UCSF Clinicians — this test can be ordered through APeX
Outside Physicians — this test can be ordered using the CCGL Requisition Form

For all specimens, an interpretation of this test by a laboratory physician will automatically be performed and billed for separately.

CCGL Test Order Submission

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