FAQ - MSI ANALYSIS
MSI status is a measure of mismatch-repair deficiency commonly found in solid tumors. When the mismatch repair (MMR) system is not functioning properly, it can cause microsatellite instability (MSI) in regions of the genome. Microsatellite instability is demonstrated by insertions or deletions due to uncorrected errors created during DNA replication. This assay detects those insertions or deletions, using five different markers at five different microsatellite regions of the genome.
The platform is a fluorescent PCR-based assay developed by Promega (v1.2). Capillary electrophoresis is used to separate the PCR products and determine the instability status of five different microsatellite regions of the genome.
The Promega assay for MSI detection is a patent-protected Laboratory Developed Test. The assay was validated in a CAP/CLIA certified laboratory, using the Promega protocol and kits.
A single DNA preparation is performed using a curl taken from a tumor block. The curl will have pathology confirmation of containing both normal DNA and tumor DNA. MSI status for five different regions is determined, indicated by an increase or decrease in the length of the microsatellite region. The vast majority of individuals are monomorphic at the tested microsatellite regions, which allows for any marker with multiple allele lengths to be considered shifted. A shift in >40% (2 markers) indicates a MSI High (MSI-H) sample. A shift from a single marker indicates MSI Low (MSI-L). No shift indicates MSS (Microsatellite Stable).
For ASTERAND™ Human Tissue, MSI results are based on the assay results of a section from a single tumor block. These results are represented as a direct result for the remaining sections from which the curl was removed. In addition, the presence or absence of the specific mutation is extrapolated across any additional primary tumor specimens from the same case and designated as inferred. The mutation status will be reported with all samples within a case, however, any samples that are not tumor, will not carry the genetic result of a tumor sample.
BioIVT performs mutational analysis on sections with an estimated tumor volume (STMR) > 40 with appropriate tissue size and no known quality issues.
Due to the heterogeneity in tumor volume across a tissue block and the heterogeneous nature of tumor biospecimens, the actual detection of MSI throughout a specific biospecimen is unable to be predetermined.
BioIVT will report positive samples as MSI-H and negative samples as MSI-L or MSS. MSI-L is not currently clinically actionable, nor has it been shown to be phenotypically distinct from MSS, and is therefore included as a negative result.
Umar, Asad et al. “Revised Bethesda Guidelines for Hereditary Nonpolyposis Colorectal Cancer (Lynch Syndrome) and Microsatellite Instability.” Journal of the National Cancer Institute 96.4 (2004): 261–268. Print.
Kim, Tae-Min, Peter W. Laird, and Peter J. Park. “The Landscape of Microsatellite Instability in Colorectal and Endometrial Cancer Genomes.” Cell 155.4 (2013): 10.1016/j.cell.2013.10.015. PMC. Web. 3 Jan. 2018.
A high molecular weight DNA is used as a positive amplification control. Nuclease-free water is used as a negative amplification control. Two pentanucleotide repeats are included in the assay as a contamination control.
|Marker Name||GenBank® Number||Major Repeat Sequence||Size Range (bp)1||K562 Alleles (bp)||Primer Dye2|
|Penta C||ALI38752||(AAAAG)3-15||143-194||164, 174||TMR|
|Penta D||AC000014||(AAAAG)2-17||135-201||168, 187||FL|
1Allelle sizes were determined using the ABI PRISM® 3100 Genetic Analyzer with POP-4® polymer and a 36cm capillary. Rare alleles outside of these size ranges may exist. Allele sizes may vary when using different polymers or instrument configurations.
2TMR = carboxy-tetramethylrhodamine; FL = fluorescein; JOE = 6-carboxy-4’,5’-dichloro-2’.7’-dimethoxyfluorescein