MLH1 Hypermethylation and BRAF Mutation Analyses, Tumor
NY State Approved Indicates the status of NY State approval and if the test is orderable for NY State clients.
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
Genetics Test Information Provides information that may help with selection of the correct test or proper submission of the test request
If this test is ordered in conjunction with the HNPCC screen (HNPCC/17073) or MLH1 immunostain (IHCO/29004) and MSI (MSIO/88566), this test (MLBRF/87931) will only be performed when clinically indicated.
Profile Information A profile is a group of laboratory tests that are ordered and performed together under a single Mayo Test ID. Profile information lists the test performed, inclusive of the test fee, when a profile is ordered and includes reporting names and individual availability.
|Test ID||Reporting Name||Available Separately||Always Performed|
|MLHHM||MLH1 Hypermethylation||Yes, (order MLH1H)||Yes|
|BRAFP||BRAF Mutation Analysis (V600E)||Yes, (order BRAF)||Yes|
|MBPIN||MLH1 Hyp/BRAF (V600E) Mut Interp||No||Yes|
Additional Tests Lists test(s) that are always performed, at an additional charge, with the initial test(s)
|Test ID||Reporting Name||Available Separately||Always Performed|
Testing Algorithm Delineates situation(s) 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 Hereditary Nonpolyposis Colorectal Cancer Testing Algorithm in Special Instructions.
Special Instructions and Forms Describes specimen collection and preparation information, test algorithms, and other information pertinent to test. Also includes pertinent information and consent forms to be used when requesting a particular test
Polymerase Chain Reaction (PCR) Analysis
(PCR is utilized pursuant to a license agreement with Roche Molecular Systems, Inc.)
Reporting Name A shorter/abbreviated version of the Published Name for a test; an abbreviated test name
MLH1 Hypermethylation/BRAF Mutation
Specimen Type Describes the specimen type needed for testing
Specimen Required Defines the optimal specimen. This field describes the type of specimen required to perform the test and the preferred volume to complete testing. The volume allows automated processing, fastest throughput and, when indicated, repeat or reflex testing.
This test is only performed on colon tumors with confirmed loss of MLH1 protein expression.
Pathology report must accompany specimen in order for testing to be performed.
Specimen Type: Tissue block or slide
1. Submit formalin-fixed, paraffin-embedded tissue block (preferred) or 1 slide stained with hematoxylin and eosin and 10 unstained, nonbaked slides (5 micron-thick sections) of the tumor tissue.
2. Sections should contain both tumor and normal tissue.
1. Molecular Genetics-Inherited Cancer Syndromes Patient Information Sheet (Supply T519) in Special Instructions
2. If not ordering electronically, submit a Molecular Genetics Request Form (Supply T245) with the specimen.
Specimen Stability Information Provides a description of the temperatures required to transport a specimen to the laboratory. Alternate acceptable temperature(s) are also included.
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.
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.
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.
Clinical Reference 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
Method Description Describes how the test is performed and provides a method-specific reference
A PCR-based assay is used to test tumor DNA for the presence of hypermethylation of the MLH1 promoter and for the presence of the V600E mutation within the BRAF gene. The MLH1 hypermethylation method is a modification of the method described by Grady et al.(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; Grady WM, Rajput A, Lutterbaugh JD, Markowitz S: Detection of aberrantly methylated hMLH1 promoter DNA in the serum of patients with microsatellite unstable colon cancer. Cancer Res 2001;61:900)
Day(s) and Time(s) Test Performed Outlines the days and times the test is performed. This field reflects the day and time the sample must be in the testing laboratory to begin the testing process and includes any specimen preparation and processing time required before the test is performed. Some tests are listed as continuously performed, which means assays are performed several times during the day.
Monday; 8 a.m.
Analytic Time Defines the amount of time it takes the laboratory to setup and perform the test. This is defined in number of days. The shortest interval of time expressed is "same day/1 day," which means the results may be available the same day that the sample is received in the testing laboratory. One day means results are available 1 day after the sample is received in the laboratory.
Maximum Laboratory Time Defines the maximum time from specimen receipt at Mayo Medical Laboratories until the release of the test result
Specimen Retention Time Outlines the length of time after testing that a specimen is kept in the laboratory before it is discarded
Extracted DNA: 3 months
Performing Laboratory Location The location of the laboratory that performs the test
CPT Code Information Provides guidance in determining the appropriate Current Procedural Terminology (CPT) code(s) information for each test or profile. The listed CPT codes reflect Mayo Medical Laboratories interpretation of CPT coding requirements. It is the responsibility of each laboratory to determine correct CPT codes to use for billing.
MLH1 Hypermethylation and BRAF Mutation Analyses, Tumor
81210-BRAF (v-raf murine sarcoma viral oncogene homolog B1) (eg, colon cancer), gene analysis, V600E variant
81479-Unlisted molecular pathology procedure
LOINC® Code Information Provides guidance in determining the Logical Observation Identifiers Names and Codes (LOINC) values for the result codes returned for this test or profile.
|Result ID||Reporting Name||LOINC Code|
|26930||Specimen ID||In Process|
|26918||Specimen ID||In Process|
|26922||Reason For Referral||42349-1|