Label Name: MSI
Lab Discipline: Molecular Diagnostics
Institution:  Duke University Health System 
EAP ID:  LAB6664 
Last Review:  3/17/2017 10:04:43 AM
Container & Volume
  Age Group   Container   Volume  
Collection Notes
  • Formalin Fixed Paraffin Embedded Tissue: The laboratory can receive either a paraffin embedded tissue block or four freshly cut (within one week)5uM thick unstained slides containing at least 3 - 20 square mm of tissue. A minimum neoplastic cell content of 15% is required for testing. Unstained slides should be accompanied by an H&E stained slide for histologic evaluation. A block (or slides) containing both tumor and normal tissue is required. Separate blocks (or slides) for tumor and normal is acceptable.
  Formalin fixed paraffin embedded tissue blocks and slides can be sent to the lab at ambient temperature.
Turn Around Time -  Routine: 14 days   Stat: N/A
Reference Values
  This assay compares short tandem repeat (STR) lengths from normal and tumor tissues to detect novel tumor STR alleles that arise from microsatellite instability (MSI). H&E stained slides for each case are first evaluated to identify both normal tissue and an area of at least 20% neoplastic cell content. These regions are then macro-dissected from adjacent unstained formalin-fixed paraffin-embedded sections and used to prepare genomic DNA. A single tube, multiplex PCR targeting five mononucleotide STRs (NR-21, BAT-26, BAT-25, NR-24 and MONO-27) and two pentanucleotide STRs (Penta C and Penta D) is performed separately on tumor and normal DNA. The resulting PCR amplification products are resolved using capillary electrophoresis on the 3130xl Genetic Analyzer (ABI). The size of each STR is determined using GeneMapper (ABI). The mononucleotide STRs are used to determine microsatellite status while the pentanucleotide STRs give patient identity assurance among sample sets. A sample is interpreted as microsatellite stable (MSS) if tumor and normal tissue from the same patient contain identical STR allele lengths. A sample is interpreted as MSI-high if two or more STR alleles contain novel repeat lengths in the tumor tissue when compared to normal. If only a single STR allele contains a novel repeat length, the sample is interpreted as MSI-low.

This test was developed and its performance characteristics determined by the DUHS Clinical Molecular Diagnostics Laboratory. It has not been cleared or approved by the U.S. Food and Drug Administration. This test is used for clinical purposes. It should not be regarded as investigational or for research. This laboratory is certified under the Clinical Laboratory Improvement Amendments of 1988 ("CLIA") as qualified to perform high complexity clinical testing.
Clinical Significance and Interpretive Data
    Approximately 2-3% of all colon cancer occurs in patients with Hereditary Nonpolyposis Colon Cancer (also called HNPCC or Lynch Syndrome). HPNCC 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 which 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. MSI can also occur in patients who do not have HNPCC, but instead have sporadic, non-familial cancer. Approximately 15% of sporadic colon cancer cases have MSI. Thus, the presence of MSI does not distinguish familial (HNPCC) from sporadic cancer.
MSI testing may also be useful in determining disease prognosis and selecting the best chemotherapeutic treatment protocol for patients with primary or metastatic colon cancer (see references below).

Hampel H et al. "Screening for the Lynch syndrome (hereditary nonpolyposis colorectal cancer)." NEJM. 2005; 352(18):1851-1860

Hampel H et al. "Feasibility of screening for Lynch syndrome among patients with colorectal cancer." J Clin Oncol. 2008; 26(35):5783-5788

Bertagnolli MM et al. "Microsatellite instability predicts improved response to adjuvant therapy with irinotecan, fluorouracil, and leucovorin in stage III colon cancer: Cancer and Leukemia Group B Protocol 89803." J Clin Oncol. 2009; 27(11):1814-1821

Kim GP et al. "Prognostic and predictive roles of high-degree microsatellite instability in colon cancer: a National Cancer Institute-National Surgical Adjuvant Breast and Bowel Project Collaborative Study." J Clin Oncol. 2007; 25(7):767-772

Des Guetz G et al. "Does microsatellite instability predict the efficacy of adjuvant chemotherapy in colorectal cancer? A systematic review with meta-analysis." Eur J Cancer. 2009; 25(10):1890-1896

Benatti P et al. "Microsatellite instability and colorectal cancer prognosis." Clin Cancer Res. 2005; 11:8332-8340

Boland CR. "Clinical uses of microsatellite instability testing in colorectal cancer: an ongoing challenge." J Clin Oncol. 2007; 25(7):754-756

Test Interpretation:

• Normal and tumor sample alleles are compared for each marker. Only shifts greater than or equal to 3 base pair difference between the normal allele and instability allele should be considered mutated.
• Shifts less than 3 bp are within range to be a natural low-level polymorphism.
• Shifts present in both normal and tumor samples represent a heterozygous allele.
• Tumor samples demonstrating two or more instability markers are MSI-high.
• Tumor samples demonstrating one instability marker are MSI-low.
• Tumor samples demonstrating no instability markers are MS-stable.

    1. Patients meeting the clinical criteria for HNPCC

Amsterdam II Criteria:

• Three or more family members, one of whom is a first-degree relative of the other two, with HNPCC-related cancers
• Two successive affected generations
• One or more of the HNPCC-related cancers diagnosed before age 50 years
• Exclusion of familial adenomatous polyposis (FAP)

Revised Bethesda Criteria:

• Colorectal cancer diagnosed in an individual younger than than age 50 years
• Presence of synchronous or metachronous colorectal, or other HNPCC-associated tumors
• Colorectal cancer with the MSI-H histology diagnosed in an individual younger than age 60 years
• Colorectal cancer diagnosed in one or more first degree relatives with an HNPCC-related tumor, with one cancer diagnosed before age 50 years
• Colorectal cancer diagnosed in two or more first- or second-degree relatives of any age

2. Patients with histology suggestive of MSI including right sided poorly differentiated colon adenocarcinomas with a mucinous histology or prominent lymphocytic infiltrate.

3. Given the relatively high incidence (3%) of HNPCC in unselected colorectal carcinoma cases and the low sensitivity of the above clinical and histologic criteria, MSI testing could be used as a general screening tool in all patients with colorectal adenocarcinoma.
    MSI testing is considered a screening tool rather than a genetic test for disease diagnosis. Patients with tumors with MSI may have HNPCC. However, most will not. Additional testing for inherited mutations in the mismatch repair genes in non-tumor tissue is required to make this diagnosis. 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. Rarely tumors with microsatellite instability will test negative (Microsatellite Stable, MSS) using this assay. This may be caused by low tumor cellularity (less than 15%) or by mutations that lead to a partial or difficult to detect defects in MMR. If a strong clinical suspicion of HNPCC remains, additional testing including immunohistochemical analysis of MLH1, MSH2, MSH6 and PMS2 may reveal defects in mismatch repair which are not detected by PCR based testing.

• This assay only detects the presence/absence of MSI at the BAT-25, BAT-26, MONO-27, NR-21 and NR-24 loci.

• This assay can detect MSI present in at least 15% of the total isolated cell population from a 100% tumor cellularity sample; or MSI present in at least 50% of isolated cells from a 30% tumor cellularity sample. MSI present in tumor cellular levels lower than specified here may not be detected.

• A MSI-high result does not confirm a HNPCC diagnosis nor does a MS-stable result rule out HNPCC. Results should be considered in the context of other clinical findings and IHC testing to determine the appropriate course of further testing.

Test Synonyms
Synonym(s): Hereditary non-polyposis colon cancer
Synonym(s): HNPCC
Synonym(s): HNPCC BY IHC
Synonym(s): IMSI
Synonym(s): instability
Synonym(s): microsatellite
Synonym(s): MSI
Molecular Diagnostics Laboratory

Medical Director:
 Michael Datto, M.D., Ph.D.
 Phone: 919-684-6965
 Email: michael.datto@duke.edu
Lab Director:
 Catherine Rehder Ph.D, FACMG
 Phone: 919-613-8434
 Email: catherine.rehder@duke.edu
Lab Director:
 Siby Sebastian Ph.D., DABMG
 Phone: 919-613-8432
 Email: siby.s@duke.edu

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

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