Microsatellite Instability Testing

Performing Lab:

UCSF Clinical Cancer Genomics Lab

CPT Code:

81301, 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
The Idylla MSI test is an in vitro diagnostic test intended for the qualitative detection of a novel panel of seven monomorphic biomarkers (ACVR2A, BTBD7, DIDO1, MRE11, RYR3, SEC31A, SULF2) for identification of microsatellite status. The Idylla MSI test uses FFPE tissue sections, from which nucleic acids are liberated by a combination of chemical reagents, enzymes, heat, and high-intensity focused ultrasound. Next, the seven targets are amplified with biomarker-specific primers. The specific targets are detected using fluorescent-labeled molecular beacons. These beacons differentially dissociate from the wild-type or mutated amplicons with increasing temperature. The MSI-specific software will first check the validity of the measured fluorescent profiles. Next, a pattern-recognition approach calculates a similarity score (MSI score) for any given valid biomarker-specific profile. This MSI score expresses the probability of a pattern being wild-type or mutant. Each biomarker is scored individually and reported as no mutation detected, mutation detected, or invalid. The MSI status of a sample can be determined with high confidence if at least five marker-specific fluorescence profiles can be analyzed (otherwise the MSI status of the sample is called invalid). The presence of at least two mutant markers will result in the status being MSI-H, otherwise the status is scored as microsatellite stable (MSS). MSI-low is not reported by this assay.
Turnaround Time: 7-14 days

TEST INDICATIONS:

Microsatellite Instability by PCR is designed to aid in the identification of patients who harbor germline mutations in mismatch repair (MMR) proteins that are associated with HNPCC. It is important to note that defective MMR resulting in MSI can also arise spontaneously in a tumor (i.e., without inheritance of a mutant MMR gene), and therefore the presence of MSI is not diagnostic for Lynch syndrome/Hereditary Non-Polyposis Colorectal Cancer (HNPCC).

Criteria for HNPCC testing (revised Bethesda guidelines)

Colorectal cancer diagnosed in a patient who is less than 50 years of age.
Presence of synchronous, metachronous colorectal, or other HNPCC-associated tumors, regardless of age. HNPCC-associated tumors include colorectal, endometrial, stomach, ovarian, pancreas, ureter and renal pelvis, biliary tract, and brain (usually glioblastoma as seen in Turcot syndrome) tumors; sebaceous gland adenomas and keratoacanthomas in Muir-Torre syndrome; and small bowel carcinoma.
Colorectal cancer with the MSI-H histology diagnosed in a patient who is less than 60 years of age. Note that inclusion of the age criteria is controversial.
Colorectal cancer in a patient with one or more first-degree relatives with an HNPCC-related tumor, with one of the cancers being diagnosed under age 50 years.
Colorectal cancer in a patient with two or more first- or second-degree relatives with HNPCC-related tumors, regardless of age.

UCSF Clinical criteria for HNPCC testing in gynecologic tumors
Nearly half of women with HNPCC will present with a gynecologic tumor, rather than a colon cancer.

Endometrioid histology at any GYN location in a patient under 50 in a definitive surgical procedure (i.e., not just biopsy, wait for primary surgery).
Any adenocarcinoma at any GYN location in a patient with colon cancer.
Clinician suspicion for Lynch syndrome.

HOW THE TEST WORKS:

A histologic section of formalin-fixed, paraffin-embedded tissue is examined by a pathologist to identify areas of tumor and normal tissue. DNA is extracted separately from tumor and normal areas on adjacent unstained slides and amplified in the multiplex PCR reaction. Fluorescently labeled PCR products are identified and sized by capillary electrophoresis. Markers are scored for stability or instability by comparison of marker allele sizes from tumor and normal samples. The presence of a marker allele size in the tumor sample that is not present in the normal sample is indicative of instability for that marker. The number of unstable markers is used to determine the presence or absence of instability.

Marker instability is interpreted as follows:

Microsatellite stable: No markers show instability
MSI-Low: Instability in one marker
MSI-High: Instability in two or more markers

LIMITATIONS OF THE TEST:

This test is typically run in conjunction with immunohistochemical staining for mismatch repair proteins, performed by the UCSF Pathology Laboratory. Microsatellite Instability by PCR may also be ordered as a separate test.

Results from this test should be correlated with the patient’s clinical presentation, family history and any findings from immunohistochemical staining for mismatch repair proteins in the tumor. Genetic counseling may be recommended for some cases.

MLH1 Methylation Testing or BRAF Mutation Testing (for colorectal samples only) may be performed on samples that are MSI-High and show loss of MLH1 by immunohistochemistry to distinguish the sporadic MSI-high cases (BRAF mutation positive/MLH1 methylation positive) from the MSI-high cases associated with Lynch syndrome (BRAF mutation negative/MLH1 methylation negative). Note that not all MSI-H colorectal cancers that lack detectable MLH1 methylation or BRAF mutation are due to Lynch syndrome.

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

SPECIMEN REQUIREMENTS FOR A SUCCESSFUL TEST:

Testing should be ordered only on surgical excision samples. Biopsy material is not appropriate. Blocks selected for MSI PCR testing must contain both tumor and normal tissue. If only tumor is present on the block, select an additional block with normal 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. Label slides with pathology case number and block identification.

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 or normal tissue present on slide as determined by pathologist
Outside slides not labeled or not accompanied by printed copy of test order

HOW TO ORDER THE TEST:

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.