Key points about MSI by PCR
- Directly measures changes in DNA caused by loss of MMR protein
- Functional measure of mismatch repair deficiency that detects loss in MMR repair function, regardless of cause
- Does not identify the MMR gene to investigate
MSI by PCR uses changes in fragment size caused by unrepaired mismatched bases to detect a defect in mismatch repair function.
Key points about MMR by IHC
- Detects the presence or absence of MMR proteins
- Not a conclusive measure of MMR function
- Shows which MMR gene to investigate
dMMR by IHC detects the presence or absence of MMR proteins.
Key points about NGS
- Can be automated
- Requires highly stringent DNA quality
- Lacks standard, validated parameters (sequencing technology, MSI algorithm, Gene Panel, etc.) for detecting dMMR in tumors
Clinical Co-Testing Guidelines
“Although loss of MMR protein immunoreactivity is generally detected in dMMR colorectal cancer, normal immunoreactivity can be seen in up to 10% of dMMR cases; therefore, MSI DNA testing may be performed either stepwise or as a concurrent test”(6).
|Overview||Detects the functional failure of tumor MMR proteins, resulting in changes (instability) in microsatellite allele length||Detects the presence or absence of tumor MMR proteins using monoclonal antibodies against the major mismatch repair genes||Detects mutations within microsatellite sequences of tumor samples|
|Cost||$45/sample||$50–70/slide (x 4 slides)||$1–3K/sample (variable)|
|Throughput||Up to 96 samples (med to high)||Low to medium||Variable (more information = lower throughput)|
|Sample Input||1ng (<1 FFPE curl)||4 slides||10–20 slides|
|Turnaround Time||~10 Hours||1–3 days||2–3 weeks (2–6 weeks if send-out)|
|False Negative Rate||0.3–4%(5)||5–10%(6)||Variable (not standardized)|
|Multiplexed||Yes||No (must do separate slides)||Yes|
|Markers Analyzed||5 mononucleotide loci (NCI recommended, Revised Bethesda Panel)(8)||4–5 MMR proteins (MSH6, MSH2, PMS2, MLH1)||No standard panel or analysis method established or recommended|
|Pros||Functional test for dMMR, low false negative rate, proven technology, NCI recommended standardized loci panel available , 225+ peer-reviewed publications, minimal sample needed||Shows which genes to investigate, familiar technology||Provides more information besides just MMR (MSI), high throughput|
|Limitations||Only characterizes MSI, requires molecular training, does not indicate MMR genes to investigate||5–10% false negative rate, uses more sample(6), throughput, indirect measure of dMMR||Large sample requirements (>20ng DNA), lack of standardization, requires advanced molecular training, technical issues lead to misclassification or false results, highly stringent DNA quality requirements|
- Li, K. et al. (2020) Microsatellite instability: a review of what the oncologist should know. Cancer Cell International. 20, 16.
- Funkhouser, W.K. Jr. et al. (2012) Relevance, Pathogenesis, and Testing Algorithm for Mismatch Repair–Defective Colorectal Carcinomas: A Report of the Association for Molecular Pathology J. Mol. Diagnostics. 14, 91–103.
- Zhang, et al. (2008) Immunohistochemistry versus Microsatellite Instability Testing for Screening Colorectal Cancer Patients at Risk for Hereditary Nonpolyposis Colorectal Cancer Syndrome: Part II. The Utility of Microsatellite Instability Testing. J. Mol. Diagnostics. 10, 301–7.
- Dudley, J.C. et al. (2016) Microsatellite Instability as a Biomarker for PD-1 Blockade. Clin. Cancer Res. 22, 813–820.
- Based on an internal analysis of publications comparing MSI-PCR v. IHC-dMMR in colorectal cancer from 2004–2018. Literature bundle available from Promega Medical Affairs upon request.
- Sepulveda, A.R. et al. (2017) Molecular Biomarkers for the Evaluation of Colorectal Cancer: Guideline From the American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and American Society of Clinical Oncology. J. Mol. Diag. 19, 187–225.
- Luchini, C. et al. (2019) ESMO recommendations on microsatellite instability testing for immunotherapy in cancer, and its relationship with PD-1/PD-L1 expression and tumour mutational burden: a systematic review-based approach. Annals of Oncol. 30, 1232–1243.
- Umar et al. (2004) Revised Bethesda Guidelines for Hereditary Nonpolyposis Colorectal Cancer (Lynch Syndrome) and Microsatellite Instability. J. Natl. Cancer Inst. 96, 261–8.