Determining Microsatellite Instability Status Using Multiplex PCR-Based Methods

insights from industryShaun PetersonGlobal Strategic Manager, Clinical Diagnostics Business Promega Corporation

An interview with Shaun Peterson, Global Strategic Manager, Clinical Diagnostics Business, Promega Corporation

Please give an overview of microsatellite instability (MSI) status and its applications within oncology.

Historically Microsatellite Instability (MSI) has been used to screen for Lynch Syndrome, a dominant hereditary cancer propensity. Recently, MSI status has been rediscovered as a biomarker for prolonged and durable response to immune checkpoint inhibitors, making MSI status an increasingly relevant tool in genetic- and immuno-oncology.

MSI testing functionally measures the genomic accumulation of insertion or deletion (INDEL) errors caused by cells deficient mismatch-repair system (dMMR) that occurs in certain types of solid tumors, and this screening may be used to better characterize tumors and guide therapeutic choices for MSI-High cancer types.

To learn more about both the classical and new utility of microsatellite instability please visit our educational resource.

Also, to learn more about this history of MSI please read this article from our lead R&D scientist:

What recent developments have been made that make the MSI status of tumor tissue important?

May 23, 2017, the U.S. Food and Drug Administration granted accelerated approval to pembrolizumab for adult and pediatric patients with unresectable or metastatic, microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) solid tumors. This is the first time the agency has granted approval for a cancer therapeutic agnostic to tissue/site of origin indication.

Additionally, on July 31, 2017, the U.S. FDA granted accelerated approval to nivolumab for the treatment of patients 12 years and older with mismatch repair deficient (dMMR) and microsatellite instability high (MSI-H) metastatic colorectal cancer.  Since then the agency has made additional approvals for MSI-H tumors.

Tumors with MSI-High status have been shown to respond to immune checkpoint inhibitor (ICI) therapies. This outcome may be explained by MSI-driven tumor expression of mutation-associated neoantigens (MANA) that are believed to cause immune cell infiltration into the tumor microenvironment. Tumor induced inhibition of immune cell activity can be overcome with ICI therapies, allowing for tumor cell destruction by the immune cells.

What techniques can be used to establish MSI status? What are the advantages and limitations of these methods?

The current Promega Research Use Only MSI assay, is a PCR and Capillary Electrophoresis-based test that is designed to match the robustness needed when working with fragmented DNA extracted from FFPE samples.

Image credit: Promega

The assay is easy to use and has been available and used in the market as part of Lab-Developed Tests since 2004. This patent-protected technology is considered the gold standard molecular assay for detecting DNA mismatch-repair deficiency. Promega intends to seek US FDA approval and CE-IVD marking for its MSI assay to help oncologists and pathologists make treatment decisions for colorectal cancer patients. The MSI Assay is reimbursable, has a fast turnaround time and most importantly there is a large body of evidence supporting use of MSI in colorectal cancer decisions.

Immunohistochemistry (IHC) analysis of the presence or absence of mismatch repair proteins (MMR) is often considered a surrogate and equal analysis method, but peer reviewed literature demonstrates IHC-MMR is not an equal comparison to MSI by PCR.  The presence of MMR protein expression is not necessarily a conclusive measure of MMR function. There can be a loss in the function of these proteins without a corresponding loss of the protein in the cell. It has been estimated that 5-12% of MSI-H tumors are not recognized by an IHC test in part because of the expression of non-functional MMR proteins. MSI by PCR directly measures changes in DNA caused by loss of MMR protein function as opposed to measuring the proteins themselves as in IHC. This makes MSI by PCR a functional measure of mismatch repair deficiency that detects loss in MMR repair function, regardless of the origin.

While there are large NGS cancer gene panels used by specialty service providers that will provide an estimate of the MSI status of a tumor and other signatures of genomic instability, such as tumor mutation burden. However, each laboratory attempts to analyze different loci for MSI and the MSI determination is made using different bioinformatic algorithms. Thus, one should be careful in selecting the laboratory. These types of assays are often quite costly and the long turnaround time argues against using them for MSI determination unless there are other reasons to wait for the results with regard to other cancer markers covered by the panel.

What does Promega offer to determine the MSI status of tissues? What is the workflow of this technique?

The MSI Analysis System, Version 1.2 (RUO), is a fluorescent multiplex PCR-based method for detecting microsatellite instability (MSI). Microsatellites are loci or regions of DNA where one or a few bases are tandemly repeated many times. MSI is a form of genomic instability caused by the insertion or deletion of repeat elements at these loci during DNA replication and the failure of the mismatch repair system (MMR) to correct these errors.  This system has been the gold standard test available for clinical research since 2004.

Following DNA isolation, specific mononucleotide sequences within the samples are amplified using polymerase chain reaction performed on a thermocycler. Microsatellite markers can be amplified in multiple, parallel reactions or multiplexed in a single reaction. When multiplexed primers for individual markers are fluorescently labeled, many different fragments of similar sizes can be detected in the same reaction.

Following amplification, the amplified fragments are resolved by size on a capillary electrophoresis instrument. Data analysis is then performed using specialized software developed for fragment analysis.

In a routine research laboratory, the time from sample input to MSI status determination can range from overnight to two days. Most labs use our MSI Analysis System, with data ready for analysis overnight.

What are the advantages and limitations of the Promega MSI assay? How does this compare with other methods to determine MSI status such as IHC or NGS?

Promega MSI Analysis System offers greater analytical accuracy than immunohistochemistry (IHC) for identifying MSI-high samples.  The fluorescent multiplex PCR-based method utilizes Promega industry leading forensic STR technology and applies that expertise to the clinical research market. This differentiates the Promega MSI assay from other “homebrew” MSI techniques that are often not well balanced or multiplexed, which is critical when using DNA extracted from precious tumor samples.  Our system has been on the market for over 15 years, with over 140 peer-reviewed publications.

It is easier to use, less expensive and has a faster turnaround than NGS. NGS assays vary from laboratory to laboratory. Most do not use the well-established STR loci in the Promega assay and the loci used vary from test to test. It is difficult to accurately sequence the longer homopolymeric STR regions that are more sensitive indicators of MSI.

Can the Promega MSI assay be used with other techniques? How does this affect the results?

Many guideline committees in the US and EU recommend the utility of co-testing with both MSI by PCR and IHC MMR analysis.

How does this technique affect the detection of different cancers?

Promega MSI Analysis System has been utilized in many pan-tumor clinical trials being conducted around the world, supporting the approvals of several immunotherapeutics in over 16 tumor types.

What is the future of MSI assays from Promega?

In December of 2018, FALCO biosystems of Kyoto, Japan, working in collaboration with Promega and utilizing Promega's microsatellite instability (MSI) chemistry and assay design, obtained companion diagnostic approval for FALCO’s MSI-IVD from Japan’s regulatory agency, the Ministry of Health, Labour and Welfare (MHLW), which detects MSI-High within tumor tissues as a biomarker of DNA repair dysfunction.  This is the first pan-tumor companion diagnostic test in Japan. Its intended use is to identify patients suitable for treatment with the anti-PD-1 antibody. The clinical performance study conducted for the kit confirmed the detection capability of MSI-High in 16 different cancer types for stomach, uterine, breast, pancreas, etc. other than colorectal cancer.

Additionally, in January of 2019, Promega Corporation’s MSI technology was granted the innovation designation by the Chinese National Medical Products Administration (NMPA). Promega MSI technology has been validated in labs around the world. With innovation status, the path to become classified as an in vitro diagnostic (IVD) will gain elevated efficiency by having a program coordinator assigned from NMPA and priority status for multiple processes.  We are on track to launch an MSI IVD in China during the second half of 2020.

In our most recent news, Promega is excited to announce a global collaboration with Merck, known as MSD outside the United States and Canada, to develop Promega’s microsatellite instability (MSI) technology as an on-label, solid tumor companion diagnostic (CDx) for use with Merck’s anti-PD-1 therapy, pembrolizumab. The global collaboration will initially seek regulatory approval for the Promega MSI CDx in the United States and China. Plans to seek approvals in additional territories may follow.

Where can readers find more information?

Please visit the following resources:

About Shaun Peterson

Shaun Peterson is a Global Strategic Manager in the Clinical Diagnostics Business Unit at Promega and is based at Promega’s world headquarters in the United States.  Mr. Peterson has worked in Healthcare for 20 years and has been with Promega Corporation for over 5 years.  He began his career as a US Army Combat Medic and received further advanced training as an Army Nurse from both the US Army’s premier hospital Walter Reed Army Medical Center, located in Washington D.C. and the US Navy’s National Naval Medical Center, located in Bethesda, Maryland. Following his service in the US Army, Mr. Peterson returned to college studying molecular biology.  

Since the completion of his training, he has worked for clinical molecular diagnostics companies as a technical services scientist and also in both global marketing and sales management roles representing molecular products for the genotyping of infectious disease, diseases of heredity and pharmacogenomics for Third Wave Technologies, Hologic, Thermo Fisher Scientific and AssureX Health.  Prior to joining Promega Mr. Peterson was the National Sales Director of a neuropsychiatric pharmacogenomics company (AssureX Health) that out-licensed a proprietary technology from the Mayo Clinic and Cincinnati Children’s Hospital.

At Promega Corporation, his focus is in advancing biomarker diagnostics, specifically microsatellite instability (MSI) to support oncology, immuno-oncology and clinical laboratories efforts in patient selection and research.

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