Jeremiah McDole and Embriette Hyde
As the third most prevalent cancer worldwide, colorectal cancer (CRC) is a significant global health burden. Biomarker identification and detection has proven a powerful strategy for improving CRC diagnosis and informing targeted treatment. Microsatellite instability (MSI) is a key biomarker in CRC, and with Droplet Digital PCR (ddPCR), MSI can be accurately detected using a wider range of sample types.
CRC accounted for roughly 1.9 million new cancer cases and nearly 1 million deaths in 2020 alone (Morgan et al. 2023). Biomarker identification and detection has proven a powerful strategy for improving CRC diagnosis and enabling targeted treatment selection.
A critical CRC biomarker is microsatellite instability (MSI), the presence of alternate-sized short tandem repeat DNA sequences resulting from defective DNA mismatch repair (MMR). Defects in MMR-associated proteins, including MSH2, MSH6, PMS2, and MLH1, prevent the correction of small errors introduced during DNA replication, and these errors compound as replication progresses (Li et al. 2020). A high-MSI phenotype, characterized by the accumulation of numerous microsatellite mutations across the genome, is thus an indicator of deficient MMR.
MSI is observed in approximately 15% of all CRC cases and has important implications for cancer prognosis and treatment response (Boland & Goel 2011). Research suggests that MSI is a positive prognostic marker, particularly for early-stage cases (Kang et al. 2018). Additionally, the presence of MSI in CRC at any stage indicates a high likelihood of response to immunotherapies, while MSI in stage II and III CRC is predictive of a poor response to adjuvant chemotherapy (Motta et al. 2021). Therefore, MSI detection is a powerful tool for understanding a patient’s unique disease presentation and for selecting the treatment with the highest likelihood of success.
Assessing Microsatellite Instability: Where Traditional Approaches Fall Short
The two most common approaches used to determine MSI status in CRC cases are immunohistochemical (IHC) analysis of protein expression and PCR assessment of MMR-associated mutations. IHC is used to detect MMR-associated proteins in tissue samples, where the loss of major MMR proteins implies a deficiency in mismatch repair and, subsequently, a higher likelihood of MSI. However, IHC is not a conclusive measure of MMR function, as 5-10% of nonfunctional proteins retain antigenicity (Funkhouser et al. 2012).
Researchers and clinicians can use PCR assays to profile repeat microsatellite sequences, assessing fragment size variation to detect changes in sizing indicative of MSI. This approach provides a functional measure of MMR deficiency, regardless of the cause. PCR, used in concert with capillary electrophoresis fragment analysis, has been the gold standard method for MSI detection for over 20 years. Denaturing high-performance liquid chromatography (DHPLC) and high-resolution melting (HRM) analysis can also be used for MSI detection following PCR amplification of microsatellite markers (Baudrin et al. 2018).
In recent years, mutation analysis by liquid biopsy has become increasingly common due to a reduced risk profile compared to solid tissue collected by invasive biopsies. A significant drawback of both IHC- and most PCR-based methods is their limit of detection, which generally precludes their use to detect MSI in liquid samples like plasma. More sensitive detection methods will be vital for safer, less invasive MSI testing, enabling investigators to determine mutation status without a surgically collected biopsy.
Opening New Doors in MSI Detection with ddPCR-Based Assays
Droplet Digital PCR (ddPCR) technology presents a promising approach for sensitive, minimally invasive MSI detection from plasma samples. Data presented in a poster at the 2023 International Symposium on Minimal Residual Cancer outlined the validation of the ddPCR Microsatellite Instability (MSI) Kit for profiling MSI in circulating free DNA (cfDNA) in plasma. The assay, intended for use with FFPE tissue, is designed to detect five MMR mutations (BAT25, BAT26, NR21, NR24 and Mono27). Researchers validated the assay using both tumor and plasma samples from three independent CRC cohorts, comparing the performance of the ddPCR assay with the gold standard PCR-based method of MSI detection.
The ddPCR MSI Detection Kit was 100% concordant with the Promega MSI Analysis System, the gold standard MSI assay in clinical research for detecting MSI in CRC tumor samples. The ddPCR MSI Detection kit demonstrated 98.3% specificity, 77.8% sensitivity, and an overall concordance of 96.8% with known tumor status when tested on tumor and plasma cfDNA. Additionally, the kit facilitated accurate determination of MSI status with as little as 1 ng of input DNA, with the majority of discordant results due to cfDNA levels below this concentration. The ddPCR MSI detection kit was also more sensitive than the Promega MSI Analysis System for samples with low tumor content. Taken together, these findings indicate that ddPCR-based MSI detection could support efficient, highly sensitive assessment of MSI in cfDNA from both tissue and plasma.
Conclusions
Developing accurate, accessible, and minimally invasive methods for detecting biomarkers is a foundational goal among clinical researchers. While MSI testing is well established for use with tissue samples, the development and application of more sensitive detection methods can enable less invasive approaches for determining MSI status, such as liquid biopsy cfDNA analysis. Researchers have already achieved promising results using ddPCR-based cfDNA analysis for other oncology biomarkers, highlighting the method’s potential for application in plasma MSI testing. While continued work is necessary to further refine and validate the use of ddPCR-based MSI testing in plasma cfDNA, studies of the ddPCR MSI Detection Kit thus far support its value in clinical research.
Visit our website to learn more about how our ddPCR Microsatellite Instability (MSI) Kit* can enhance your colorectal cancer research.
* For research use only. Not for use in diagnostic procedures.
References
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Boland CR and Goel A (2010). Microsatellite instability in colorectal cancer. Gastroenterology 138, 2073–2087.
Funkhouser WK et al. (2012). Relevance, pathogenesis, and testing algorithm for mismatch repair–defective carcinomas: A report of the Association for Molecular Pathology. J Mol Diagn 14, 2, 91–103.
Kang S et al. (2018). The significance of microsatellite instability in colorectal cancer after controlling for clinicopathological factors. Medicine (Baltimore) 97, 9, e0019.
Li K et al. (2020). Microsatellite instability: a review of what the oncologist should know. Cancer Cell Int 13, 20, 16.
Morgan E et al. (2023). Global burden of colorectal cancer in 2020 and 2040: Incidence and mortality estimates from GLOBOCAN. Gut 72, 2, 338–344.
Motta R et al. (2021). Immunotherapy in microsatellite instability metastatic colorectal cancer: Current status and future perspectives. J Clin Transl Res 7, 4511–4522.