Hello, my name is Lindsay Meganheart and I'm a medical science liaison at Primeca. I'd like to welcome you to today's webinar current gold standards for MSI testing and the clinical relevance of the MSI high biomarker. Before we begin, I would like to cover a few housekeeping items. On your screen there are multiple windows, all of which are movable and resizable. So please feel free to move them around to get the most out of your experience. Today you will notice we have many ways for you to be interactive. You can submit a question at any time during the webinar. We will receive these questions and answer these live. After the presentation has concluded, we also have a survey available. Please take a moment to answer these few questions. We really appreciate and value your feedback. There is also continuing education credit available for your participation today. In order to claim your credit, please complete both the webinar survey and the post test. After the webinar has concluded, you can also download your Certificate of Attendance for your records. Lastly, please check out our Resource library. It's located on your screen and will provide some additional educational materials. Additionally, a copy of the presentation slides from today's webinar will be available tomorrow for you to download. Now, I'd like to introduce today's presenter, Annette Berghaus. Annette serves as a Senior Medical Science Liaison at Primeca. She earned her PhD in Molecular Virology from Case Western Reserve University and completed postdoctoral training at The Ohio State University, focusing on viral inhibitor resistance and host targets for novel therapeutics. She began her career creating viral diagnostic assays at Diagnostic Hybrids, now part of Codell Corporation. Since joining Permega, Annette has been instrumental in shaping the Medical Affairs Department, with a special focus on precision oncology diagnostics. She has actively participated in major medical congresses, championed updates and clinical practice guidelines and served on the ASCO CAP Task force for predictive immuno oncology biomarkers. Through these roles, she has dedicated her experience to supporting the healthcare community and advancing diagnostic innovations for improved outcomes. And with that, I will turn it over to Annette to start today's presentation. Thank you for joining me today for our webinar, where we'll be talking about the current gold standards for MSI testing and the clinical relevance of the MSI high biomarker. Let's get started for a brief introduction. My name is Annette Berkhaus and I'm a Senior Medical Science Liaison at Prabega within the Medical Affairs Department. Our primary role is to provide the most up to date information to physicians and other healthcare providers around the world. If you would like to contact us, please e-mail us at medicalaffairs@promega.com. Today we'll delve into two key areas. First, we'll explore the gold standards in microsatellite instability or MSI testing. This includes understanding MSI, its relationship with DNA mismatch repair or MMR deficiency detection methods, and a comparative analysis of these methods, advantages and limitations. Second, we'll examine the clinical significance of the MSI high biomarker. This will cover the pathological and epidemiological traits of MSI High tumors, their implication and biomarker, biomarker and Lynch syndrome screening, and their influence on patient outcomes and immunotherapy approaches. References that underpin the presented material will be provided upon request should you need them. Please just reach out to us at medicalaffairs@hermega.com. To start off, let's review what MSI is and how we detect it. Let's begin by first understanding microsatellites. These are short, repetitive DNA sequences in our genome. For instance, as shown on the top right, we have a series of Adenosines which is a mononucleotide repeat. There are also other types like dinucleotide repeat, where two nucleotides are repeated in a sequence. These microsatellite regions comprise around 3% of the human genome. The crucial aspect of microsatellites to remember is that they're high error rate during DNA replication. Due to the repetitive pattern, DNA polymerases or the enzymes that synthesize DNA can mistakenly insert or delete nucleotides in these sequences. Under typical conditions, a cellular process called the DNA mismatch repair system or MMR system detects and rectifies these errors. However, when there's about bialylic inactivation, meaning both copies of a mismatch repaired gene are turned off or an epigenetic inactivation where gene expression is suppressed without changes in the DNA sequence, this MMR system can fail to correct these mistakes. This failure leads to an accumulation of errors and a condition known as microsatellite instability or MSI. We utilize various molecular techniques to examine these regions for errors. The upcoming video will demonstrate this process and highlight its significance in the development of cancer cells due to MMR deficiency. What is microsatellite instability or MSI microsatellites? Are stretches of nucleotide repeats within the genome that can act as hotspots where errors in DNA replication occur frequently. Normally these errors are repaired by the four major mismatch repair, or MMR proteins. Sometimes in cancer cells, the ability to correct replication errors is disrupted by mutations or methylation silencing of these MMR proteins. This is called mismatch repair deficiency or DMMR, and allows replication errors to accumulate. While all microsatellites accumulate replication errors, mononucleotide sequences are most sensitive and specific as biomarkers for DMMR. This accumulation of errors due to mismatch repair deficiency in microsatellite regions is known as microsatellite instability or MSI. It's also worth mentioning that we can analyze mismatch repair deficiency, or DMMR through a method called immunohistochemistry. It's a different approach from MSI testing, but is a vital complementary method in tumor analysis. This technique specifically checks for the absence of MMR proteins in cells. Now let's turn our attention to the technologies for detecting microsatellite instability. Historically, the detection of MSI began with polyacrylamide gel electrophoresis following PCR amplification of DNA for match normal and tumor samples. The essence of this technique lies in the amplification of both wild type and mutant alleles from the tumor DNA during electrophoresis. These PCR products are size fractionated on a polyacrylamide gel. The Gel's resolving power allows us to discern size discrepancies stemming from insertion or deletions that are characteristic of MSI. Typically, a normal DNA sample will display a singular distinct band corresponding to the wild type allele. Conversely, A tumor sample harboring deletions will show an additional smaller band indicative of a mutant allele. This differential banding pattern was instrumental in the initial detection and understanding of MSI in tumor samples. Today the gold standard method for MSI detection is PCR amplification of microsatellite loci coupled with a capillary electrophoresis for fragment analysis. During PCR fluorescent markers are incorporated enabling the detection and sizing of fragments through their emitted fluorescence. In the analysis phase, we compare the pre peak profiles representing fluorescence intensity from normal and tumor samples. Shift and peak position such as the leftward shift seen here signal a deletion of nucleotides in the tumor sample. It's crucial to understand though that MSI within a single lopus doesn't automatically classify a sample as MSI high. We look for instability across multiple loci to determine the MSI status of a tumor According to current National Cancer Institute or NCI guidelines. The determination of mismatch repair deficiency and MSI status is guided by specific parameters for DMMR. Absence of one or more MMR proteins on IHC tumor staining classifieds. A tumor as mismatch repair deficient for MSIA tumor is classified as MSI high if there is a shift in two out of five specific loci compared to normal tissue, or if more than 30% of loci interrogated show instability. When using a more extensive panel. Samples with shifts in fewer than 30% of the loci are considered MSI low. However, clinical and histological distinctions between MSI low and MSI Stable or those where no instability is observed are not always clear, leading many labs to adopt A binary classification MSI High or MSI stable with streamlined classification aids in defining samples for PCR based MSI assays. While PCR amplification followed by capillary electrophoresis is the gold standard, several alternative methods for MSI testing exist. These include denaturing High Performance liquid chromatography or DHPLC, high resolution melt curve analysis and Next generation sequencing or NGS. DHPLC involves PCR amplification of microsatellite loci and then analyzes the amplified DNA based on its retention time during chromatography, which correlates with differences in the DNA sequence between normal and tumor samples. While DHPLC has been pivotal in research settings, is not commonly employed in clinical laboratories. High resolution melt analysis, on the other hand, employs fluorescent probes that bind to PCR products. As the temperature increases, these probes dissociate from the DNA at rates that are dependent on sequence composition, allowing us to differentiate between normal and tumor DNA based on their melting profile. Advanced machine learning algorithms assist in interpreting these profiles to pinpoint MSI at specific loci. Lastly, Next Generation Sequencing offers a comprehensive approach by sequencing either matched normal tumor samples or tumor only samples. Post sequencing, various MSI scoring algorithms specific to the panel and Sequencing platform used are applied to detect MSI. Each of these methods has its own set of strengths and application niches. Within the landscape of MSI detection, Next Generation Sequencing or NGS, is the most common alternative to PCR for MSI testing. Unlike PCR, NGS is not typically used in isolation or clinical MSI testing. It's more often part of a broader genomic profiling suite for patients with advanced cancers. Now let's consider the distinction between NGS based MSI testing and the traditional PCR approach. Let's distinguish between MGS for comprehensive genomic profiling and single biomarker testing. On the one hand, single biomarker tests like MSI by PCR evaluated through IHCPCR or fragment analysis focus on individual biomarkers. They are usually linked to specific clinical actions guiding immediate treatment decisions based on their well defined clinical implications. On the other hand, comprehensive genomic profiling, which is often conducted via NGS, evaluates A broader spectrum including numerous biomarkers such as fusion genes, single nucleotide variants and copy number variants. This approach extends to complex genomic signatures that assess a wide array of sequences to deduce a composite outcome and to multi gene markers that pinpoint particular phenotypes. The resultant profiling report encapsulates a vast range of data, including novel variants that may not yet be clinically actionable, leading to potential outcomes that are not as clearly defined for immediate clinical decision making. Comparing the workflows of NGS and PCR, we see a stark contrast in turn around times, NGS can take anywhere from 4 to 14 days from sample receipt to generating a result with variability depending on the lab equipment, NGS workflow, and sample volume. Starting with sample preparation for NGSDNA, extraction and quality assessment are critical to ensure samples are fit for library preparation and enrichment. The methods for pulling specific genes vary widely, which can affect the sequencing setup. During sequencing, the choice of chemistry platform and operational throughput of the lab come into play. Labs often batch run samples to maximize efficiency, influencing the overall time to result. The bioinformatic analysis encompasses the final steps the assessing the sequencing run quality, aligning reads to the genome and evaluating for unique sequences and error rates. This phase is critical to meet the MSI scoring algorithms quality control standards. These algorithms themselves are diverse. They consider different loci sets, have varying QC thresholds, and may require paired tumor normal DNA. Once a sample passes, QCMSI scoring takes place, leading to data interpretation. The results categorize samples as MSI high, MSI stable or if the data quality is insufficient, MSI indeterminate. However, NGS is typically part of a larger tumor profiling service as we just discussed. This comprehensive approach generates an extensive range of variant data from gene mutations in indulge to copy number alterations and tumor mutation burden. The interpretation of this data is multifaceted, incorporating both clinically actionable and unactionable mutations. This detailed genetic profiling plays a vital role for patients with advanced cancer, especially as it can inform clinical trial eligibility and guide personalized treatment strategies. In comparison to NDS, the PCR workflow is more streamlined, consisting of four main steps. Initially, as with NGSDNA, is isolated, quantified, and subjected to quality checks. Once it passes these checks, the sample proceeds to PCR amplification, A straightforward process in contrast to the library preparation needed for NGS. Following amplification, PCR products are size separated via capillary electrophoresis. The resulting peak profiles are then analyzed, often using automated software. Supplemented by a molecular pathologist reviewing the data when necessary. A key advantage of PCR is its rapid turn around time. Typically, the process from sample to result is completed within one to two days, reflecting its targeted approach and the efficiency of focusing on a single biomarker. There are many advantages and limitations of these methods, and here we'll review those. IHC or immunohistochemistry is valued for its high sensitivity in detecting loss of MMR proteins and for identifying which proteins are missing. It aligns well with routine histopathological work and typically has a fast turn around time of between one to three days. However, limitations include a 5 to 10% false negative rate due to non functional proteins that should still show staining and analysis can be complicated by heterogeneous staining or reduced protein expression post chemo radiation in some patient samples. Comparatively, PCR based MSI testing has the benefit of using a standard panel of MSI markers that are known for their sensitivity in detecting mismatch repair deficiencies. It requires A minimal DNA input, allowing it to be performed even when limited tumor tissue is available, and offers a straightforward result without the need for bioinformatics, making it suitable for medium to high throughput labs with basic molecular expertise. This assay does have some limitations, including that it does not have the ability to identify gene mutations and so does not indicate which mismatch repaired genes were mutated and may need to be investigated for germline mutations. False positives are also possible due to MSI polymorphisms when used without a matching normal sample. However, this is less likely in assays that use mononucleotide. Quasi monomorphic loci NGS has the ability to detect MMR gene mutations alongside other genetic alterations and to identify novel variants, making it useful for both clinical and research applications. It can process large sample batches, but requires significant DNA quantity and quality and necessitates advanced molecular and bioinformatic skills. A crucial drawback of NGS for MSI testing is the current absence of standardization across sequencing platforms, algorithms and panels, which we'll discuss further in relation to clinical practice guidelines. So these are the basic comparisons of advantages and limitations of these detection methods. Now we're going to switch gears and talk about the clinical relevance of the MSI high biomarker, starting off with the pathological and epidemiological characteristics of these tumors. Looking at the characteristics of MSI High tumors, several key features stand out. These tumors often exhibit a loss of function in a mismatch repair protein which can be detected through IHC as we've seen or by sequencing that identifies known pathogenic mutations in MMR genes. Additionally, MSI High tumors are marked by a heightened presence of tumor infiltrating lymphocytes, and they commonly show increased expression of immune checkpoint proteins. In the specific context of colorectal cancer, pathologists often note distinct histological features in MSI high tumors. Typically, they display poor differentiation, A mucinous component, and a characteristic absence of dirty necrosis. Despite their poorly differentiated state, these tumors usually maintain a preserved chromosomal architecture. MSI High has been identified across the spectrum of solid tumors, yet its prevalence does vary. For instance, in colorectal, gastric, and certain gynecologic cancers such as endometrial, uterine and ovarian cancers, MSI High is detected in over 10% of cases. These cancers are often linked to Lynch syndrome, a hereditary condition associated with a higher risk of certain types of cancer. MSI High is also observed in other solid tumors, albeit less frequently. This includes various sarcomas, gliomas, as well as pancreatic, prostate, thyroid and breast cancers, among others. However, in certain cancer types like lung cancer, the prevalence of MSI High is not as well established and continues to be an active area of research. MSI High or DMMR tumors exhibit certain characteristics in their clinical presentation. Generally, patients with these tumors are more likely to be diagnosed at an earlier stage as opposed to a later one. There's a notable prevalence of MSI High and older age groups aligning with the general trend of increased cancer incidence with age. Interestingly, A deviation from this pattern occurs in younger patients, particularly those with early onset hereditary cancers like colorectal cancer, which are often associated with genetic syndromes such as Lynch syndrome. When we consider the incidence of MSI high colorectal cancers across different races in the United States, studies suggest similar rates among various racial groups. This is despite the varying risks for development of colorectal cancer overall, where, for instance, African American individuals are known to have a higher risk compared to other populations. Now let's discuss the role of this biomarker in Lynch syndrome screening, which is the hereditary cancer type mentioned previously. There are several cancer syndromes associated with microsatellite instability. However, today we're going to be focusing on one important cancer syndrome, referred to as Lynch syndrome. Lynch syndrome is the most prevalent genetic predisposition for hereditary cancers, affecting approximately one in 279 individuals. It's a condition rooted in mismatch repair deficiency, typically resulting from inherited mutations in one of the mismatch repaired genes such as MLH 1, MSH 2, MSH 6 or PMS two, or occasionally due to alterations in the EPCAM promoter. These germline mutations are passed down in an autosomal dominant pattern for the syndrome to manifest phenotypically, a second hit typically occurs which inactivates the remaining wild type allele. This can lead to an elevated risk for certain cancer types, with colorectal cancer being among the most significant risks linked to Lynch syndrome. The second hit in the wild type copy may arise from a range of genetic events, including point mutations, loss of heterozygosity, or epigenetic changes like hypermethylation. Recognizing Lynch syndrome is crucial for public health as it directly informs screening strategies and diagnostic protocols. Knowing a cancer patient's MSI status and consequently the risk for Lynch syndrome, means that we can adjust the frequency of cancer screening appropriately. Given the substantially increased risk for various cancers compared to the general population, more frequent screenings for individuals with Lynch syndrome can lead to earlier detection. Early detection through vigilant screening can not only mitigate cancer risk, but also improve outcomes and provide options for preventive care. This heightened surveillance extends beyond individual benefit. It serves familial health. By identifying one person with Lynch syndrome, we potentially uncover a network of family members who might also carry germline mutations associated with the syndrome and help them to make informed healthcare decisions. To screen for Lynch syndrome, two primary methods are employed, microsatellite instability testing by PCR and mismatch repair protein testing by Immunohistorchemistry. Neither method diagnosis Lynch syndrome directly, but both can indicate A deficient mismatch repair system in a tumor, potentially pointing towards the presence of Lynch syndrome and necessitating further diagnostic work. MSIPCR and IHC individually show high sensitivity but are not infallible. MSI by PCR may miss approximately .3 to 10% of cases and IHC may underestimate around 5 to 11% of cases. Combining these tests, a method called Co testing has been shown to increase sensitivity, potentially reaching near 100%. The medical community increasingly acknowledges the benefits of the stool testing approach to improve the accuracy of Lynch syndrome screening. The occasional discrepancies between MSI by PCR and IHC result can be due to factors such as retained antigenicity of mismatch repair protein which can affect IHC results, and tumor heterogeneity or MSI polymorphisms that can influence PCR outcomes. Co testing mitigates these issues by providing a more comprehensive view, ensuring that tumors with MSI high are more accurately identified. Current clinical practice Guidelines_the importance of screening for MSI and MMR to assist in diagnosing Lynch syndrome. These guidelines are endorsed internationally, with organizations in Europe, the United States, and Asia recommending universal screening for colorectal and endometrial cancer patients, which are commonly associated with hereditary cancer syndrome. Beyond these, screening is also advocated for an other tumor type linked to hereditary cancers. MSI testing by PCR and mismatch repair testing by IHC are the most recommended methods for this screening. Numerous professional organizations around the world advocate for Lynch syndrome screening, reflecting its crucial role in cancer care. These guidelines encompass the range of tumor types known to be associated with Lynch syndrome. Some of the key organizations issuing these recommendations include the American Society of Clinical Oncology, the European Society for Medical Oncology, the Japanese Society of Medical Oncology, and the National Comprehensive Cancer Network. The cancer types for which screening is recommended include, but are not limited to, colorectal and demetrio, gastric, ovarian and pancreatic cancers. Each of these organizations recognizes the importance of detection through screening for Lynch syndrome. If we take a look at one of the major guidelines from the United States National Comprehensive Cancer Network or NCCN, we see that they recommend universal screening of all colorectal and endometrial cancers in order to maximize sensitivity for identifying individuals with Lynch syndrome. They also recommend screening for other types of cancers independent of HI diagnosis. Further discussion and recommendations for this universal screening approach include the use of IHC and or MSI as the primary approach for those lab based screenings. They recommend one approach initially, but if normal results are obtained but Lynch syndrome is strongly suspected, then another test should be performed together. Universal screening provides sensitivity of up to 100% and a specificity of 93% for helping identify individuals who might have Lynch syndrome. NCDN guidelines define MSI high tumors as those with significant alterations across a set panel of markers indicative of loss of mismatch repair activity. They also state that the implications of these tests are similar to that of IHC and that these tests are complementary. There are two main panels that they indicate for MSI testing. The first panel is the Promega panel which includes 5 mononucleotide loci, and the 2nd is the Bethesda panel or NCI panel which includes 2 mononucleotide loci and three dinucleotide loci. Of note, they indicate that laboratories do vary in their approach, but that panels such as the Bethesda panel, which employ dinucleotide loci may be less specific than panels like the Promega panel, which utilizes only mononucleotide loci. The NCCN acknowledges that MSI can be detected through the bioinformatic analysis of NGS data. Unlike other techniques that may focus on a limited panel of markers, NGS enables the detection of a wide array of loci, which can differ significantly between tests. The methodology for comparison also varies, with some approaches using paired tumor normal samples, others using the tumor alone or comparing tumor DNA to a baseline reference sample. However, the NCCN indicates that additional research is necessary to evaluate the sensitivity and specificity of NGS in comparison to the established gold standard methods like PCR or IHC. They emphasize the need for validation studies, particularly in the specific cancer context where the NGSMSI test is being applied. In addition to the NCCN global recommendations for MSI testing and Lynch Syndrome, associated tumors are well established. For instance, the European Society of Medical Oncology suggests using a panel of five polyadenosine mononucleotide repeats as a current standard, with the Bethesda panel as an alternative. However, if alterations are observed only in dinucleotide repeats with the Bethesda panel, these guidelines advise additional testing with a mononucleotide repeat panel for confirmation. In the United Kingdom, the National Institute for Health and Care Excellence guidelines advocate for IHC or MSI testing for all colorectal cancer patients. This initial screening is to identify tumors with deficient mismatch repair, which can then direct further sequential testing for Lynch syndrome. The Japanese Society of Medical Oncology includes a strong recommendation for MSI testing. Their guidelines encompass PCR and IHC as well as NGS based testing. Notably, they recognize the Falco Biosystem MSI test, which uses a panel of five mononucleotide markers and is approved in Japan as a companion diagnostic for identifying MSIMSI high tumors before initiating treatment with pembrolizumab. These various guidelines converge on the importance of systemic screening for MSI status with specific methodologies tailored to regional approvals and practices. Collectively, they reflect the critical role MSI testing plays in the detection and management of Lynch Syndrome associated cancers. For patients with Lynch syndrome, testing for MSI and MMR is not only diagnostic but also integral to shaping a treatment plan. Take colorectal cancer as an example. Your MSI testing is commonly paired with IHC for MMR proteins and BRAF mutational testing. This combination of biomarkers can provide insight into a patient's prognosis. In particular, patients with MSI high tumors may not benefit as much from 5 Fluorurus cell based chemotherapy, a standard treatment for colorectal cancer. MSI status therefore has therapeutic implications. It can help oncologists decide on the most appropriate treatment regimen and can be predictive of treatment response. In summary, understanding a patient's MSI status along with other biomarker data can be important to determine both prognosis and response to treatment. Next, we're going to talk about the clinical relevance of the MSI hive biomarker in immunotherapy treatment decision making. So we've reviewed previously that MSI high has been detected across solid tumors and 1 hallmark feature is the notable increase in tumor infiltrating lymphocytes, which is attributed to the process of neo antigen formation. As we have discussed, MSI high status is a result of mismatch repair deficiency. This MMR deficiency leads to widespread microsatellite instability affecting both non coding and coding DNA regions. When instability occurs in coding regions, it often results in frame shift mutations that produce truncated non functional proteins. These aberrant proteins are then presented as antigens to the immune system, typically to T cells within the tumor microenvironment. As a result, this presentation elicits the proliferation of T cell clones that recognize these neo antigens. A response is characteristic of MSI high tumors. Consequently, we observe A robust infiltration of T cells which does not occur to the same extent in microsatellite stable tumors. Tumor infiltrating lymphocytes and MSI high tumors are particularly adept at recognizing frame shifted peptides. Moreover, these tumors frequently exhibit increased expression of immune checkpoint protein such as PDL ONE. In the tumor microenvironment, the interaction between PD1 on T cells and PDL ONE on tumor cells typically results in an off signal preventing the immune system from attacking the tumor. With the administration of anti PD ONE or anti PDL 1 antibodies, this inhibitory interaction is disrupted, effectively removing the brakes on the immune system and enabling it to mount a robust anti tumor response. Consequently, MSI high tumors characterized by their high neo antigen load and immune cell infiltration are often responsive to checkpoint blockade immunotherapy. This therapeutic approach has revolutionized the treatment for patients with these types of tumors, offering improved outcomes in cases where traditional therapies might not be as effective. In 2017, the US Food and Drug Administration set a precedent in personalized cancer treatment by granting approval to pembrolizumab and anti PD1 immunotherapy for the treatment of MSI high solid tumors. This landmark approval was particularly notable for being tumor agnostic based not on the site of the tumor but rather on the biomarker presence, marking the first time a drug was approved for any cancer with a specific genetic feature. Following this, the regulatory landscape saw a series of approvals from various immunotherapies in the United States and Europe targeting different tumor types with MSI high status in 2019. Japan joined these advancements approving pembrolizumab for MSI high solid tumors alongside a companion diagnostic MSI assay that utilize the Promega panel of markers. These regulatory milestones_the importance of MSI status as a predictive biomarker for immunotherapy, immunotherapy response across cancer types in the pivotal clinical trials that paved the way for the approval of immunotherapies for MSI High or DMMR cancers. MSI by PCR and DMMR by IHC were the primary methods employed to select patients. These trials hinged on the ability of these tests to accurately identify patients with the biomarker profiles that are most likely to respond to the therapies under investigation in certain trials. A code testing approach employing both MSI by PCR and MMR by IHC was utilized to ensure comprehensive screening. This methodological rigor was instrumental in establishing A robust correlation between MSI High or DMMR status and a positive response to checkpoint inhibitors. We'll review in a moment the impact that using both methods together may have in the context of individual patient testing. The College of American Pathologists has published clinical practice guidelines to help navigate MSI and MMR testing, which are critical in deciding on immunotherapy for cancer patients. Their recommendations, based on a comprehensive review of literature through March of 2021, offer specific guidance for various tumor types. For colorectal tumors they recommend to use IHC and or MSI by PCR as the preferred testing methods. They state NGS may be used but the assay must be validated against IHC or MSI by PCR in colorectal tumors. For endometrial tumors they recommend the use of IHC over MSI by PCR or NGS. For gastroesophageal and small bowel cancer, they recommend IHC and or MSI by PCR over MSI by NGS. However, this recommendation does not include esophageal squamous cell carcinoma for other solid tumor types. They state that testing should occur, but that the optimal approach has not yet been established. They also note that assays must be adequately validated for the specific cancer type being tested. The last recommendation is a statement that tumor mutation burden or TMB, another genomic signature biomarker, should not be used as a surrogate for the detection of DNA mismatch repair defects. In addition to the recommendation statements, they also highlight a number of good practice statements which include comments that in the event of discordant results from more more than one method, that any evidence of MMR deficiency regardless of method, should be interpreted as a positive result for patients to be eligible for immune checkpoint inhibitor therapy. But the discordant results should be reviewed carefully. If one method provides an indeterminate result, an alternative method or repeat testing on a different tumor block should occur. If subclonal loss by IHC is observed, MSI by PCR should be performed using tissue dissected from the area of the tumor with MMR protein loss to confirm DNA mismatch repair status. While these guidelines offer clear direction for certain tumor types, it is clear additional clinical evidence is needed to define optimal approaches for numerous other cancer types. A key study by Cohen ET al. In 2019 brought to light the crucial role of Co testing for MMR and MSI. This study found instances of misdiagnosis and subsequent therapeutic resistance when only IHC or MSIPCR were used individually. Their work underlined the necessity of routinely testing for microsatellite instability or mismatch repair deficiency before administering immune checkpoint inhibitors. As a result of these findings, the European Society of Medical Oncology updated their recommendations to include Co testing using both IHC and PCR. This dual testing approach ensures a more accurate identification of MSI and DMMR status in patients with advanced cancer, optimizing their eligibility for immunotherapy globally. Clinical guidelines now recognize the value of MSI and MMR testing in determining patients suitability for anti PD ONE and anti PDL 1 therapies. While specifics may differ based on tumor type and geographic region, the consensus is clear these tests are integral to selecting most effective treatment and enhancing patient outcomes. To conclude from this presentation, MSI is a functional indicator of mismatch repair deficiency crucial for identifying Lindt syndrome and predicting immunotherapy response. MSI by PCR, particularly using a panel of mononucleotide loci, stands as the current gold standard for assessing MSI in solid tumors. We've observed that MSI High tumors share certain clinical and pathological characteristics regardless of the type of solid tumor. For optimal detection of Lynch syndrome, the synergy of Co testing by combining MSI by PCR and DMMR by IHC has been established as a more effective approach. And lastly, the significance of MSI high status as a predictive biomarker cannot be overstated. It is a vital determinant of the potential efficacy of immunotherapy across various solid tumors. This highlights the integral role of MSI testing in the landscape of personalized medicine and cancer care. Thank you for joining us today. If you have additional questions, please feel free to reach out to us at any time. My contact information is listed here or feel free to e-mail the Medical Affairs Department at medicalaffairspermega.com. We will now be transitioning to the live Q&A session. Thanks, Annette. We will now transition to our Q&A session. As a reminder, if you have a question, please submit that now into the Ask a Question box that's located on the dashboard. So, Annette, we have our first question which asks can MSI RISE and PMMR? In other words, are there any other known mechanisms apart from DMMR that may lead to MSI? Yeah. Thanks so much, Lindsay, for the question. Yes, so I think there's a couple of things here maybe to tease apart. You can see samples where you could have a positive MSI result, and by IHC the result may be proficient dispatch repair. I think it's important to distinguish from the way in which the assays are performed that that result could be a discordant between the two. So it's important to remember that for IHC, typically only the four major proteins of the mismatch repair system are actually interrogated, but there are other minor partners and partner proteins. The root impacts of mutations on those could in fact result in microsatellite instability. That's not as well understood, but it is part of what could contribute to seeing the discrepancy or just ordinance between results of the two assets. I think it's also important to recognize that MSI is really the hallmark of a dysfunction within the DNA mismatch repair system. But the way in which that dysfunction occurs could be through a number of different mutational pathways. As we saw, there could be sporadic mutations, inherited mutations, and even chromosomal loss of heterozygosity and certain regions that can ultimately impair that system's ability to correct errors, and we see that specifically because of how prevalent mutations are within microsatellite regions. That's great. Thanks Annette for that response. So we have another question. It looks like someone has asked what makes MSI testing by PCR the preferred or gold standard method for identifying microcelled instability and cancer diagnostics. Yeah. Thanks so much. I think what makes PCR the gold standard really relates to to a couple of different factors. The first primarily being that a very standard set of markers has been utilized in PCR analysis. You really have two major sets we talked about the mononucleotide panel and then you have the Bethesda panel and it's really over the last 20 years. Those are the marker panels that have established the relationships between the MSI hive biomarker and some of the prognostic and predictive impacts of that biomarker that we've seen. And so that's really what what's driving that. I think you also have in terms of just the ease of using the assay, the quickness of getting a result using that particular workflow for laboratories is really what's what's driven that. They're very sensitive for DNA mismatch repair deficiency and it's a very quick and easy assay for laboratories, which gets a critical result for physicians. Great. Thanks so much, Annette. So it looks like we have another question and the question asks can you explain how combining MSI by PCR and IHC and Co testing enhances the accuracy of detecting Lynch syndrome compared to using either method alone? Yeah. Thanks so much Lindsay. So I think it relates to what we were just talking about a moment ago. Each assay is querying slightly different things and as a result of that, they are not equivalent to one another. They're very complementary. So we just talked about, although IHC is very sensitive, we're looking at whether the MMR proteins that are interrogated are present or absent. There are other minor proteins within the system which could still relate to dysfunction of the overall system and lead to MSI. Similarly, MSI is very dependent on the markers that are being interrogated and how sensitive those microsatellite regions are to a deficient mismatch repair system. And so combining those two together, you're really getting a more comprehensive and complimentary view of what's happening. And so if we're one assay may a minor amount of the time miss something. The idea is that the other assay should be able to to cover that leading to 100% coverage of really what's happening and whether or not MMR deficiency is present in that particular tumor all. Right, That's great. Thank you. I think we have time for just one more question. So we have here asking what benefits does MSI testing by PCR offer compared to MSI testing by Next Generation Sequencing or NGS? Yeah. So I think as we mentioned, one of the major benefits of PCR is simply that you're using a standard set of panels. We can relate data from a variety of studies because we're using a standard definition. The markers are consistent, the definition of what is MSI is consistent. Whereas in NGS, it's really an emerging technology. And so we haven't gotten to that stage of standardization. You're often looking at microsatellite regions. That are present within a targeted gene panel and those regions can differ panel to panel, assay to assay. And so we're not getting as comprehensive of a view and as consistent of a view. Also I think it relates to turn around time. We're very targeted as we talked about the single gene or single biomarker test versus a comprehensive test. We're really looking across a variety of different mutations still very important for advanced cancer patients that to have that view. But I think it it kind of relates to when you're, when you're looking to make a clinical decision sometimes you you need an answer as soon as possible for that patient. Yeah, absolutely. Thanks so much, Annette. I think we have run out of time. I do notice we have a few other questions in the chat, so we'll make sure to reach out to you each of you individually to make sure you have answers to your questions. I do want to just provide a quick reminder that we are offering continuing education credit for the webinar. So please, if you're interested, please make sure you complete both the webinar survey and the post test, both of which are located on your dashboard. So this will conclude our webinar. I appreciate everyone who joined us today and I hope you all have a wonderful day. Thank you. Thank you. _1734116247390