Detection Of Microsatellite Instability Research Articles

Microsatellite instability and its associations with the clinicopathologic characteristics of diffuse large B-cell lymphoma.

Microsatellite instability (MSI) has been investigated as a prognostic and predictive factor for chemotherapy in colorectal cancer and has recently been demonstrated to be predictive of the PD‐1/PD‐L1 checkpoint blockade response in various solid tumors. However, MSI status in diffuse large B‐cell lymphomas (DLBCLs) has not been thoroughly explored. This study investigated MSI status in DLBCLs and analyzed the associations between MSI and clinicopathologic characteristics and clinical outcomes. Ninety‐two cases of primary DLBCLs treated with R‐CHOP/CHOP chemotherapy between 2009 and 2017 were collected. MSI detection was performed by the Promega MSI Analysis System. The protein expression of MLH1, MSH2, MSH6, and PMS2 was detected by immunohistochemistry. The associations of MSI‐H and MSI‐L with progression‐free survival (PFS) and overall survival (OS) were assessed by COX models and Kaplan–Meier curves. The correlations of complete response (CR) after R‐CHOP/CHOP chemotherapy with MSI‐H and MSI‐L were examined by univariate and multivariate logistic regression analyses, respectively. 3 of 92 cases (3.2%) were high MSI (MSI‐H), and 9 cases (9/92, 9.8%) exhibited low MSI (MSI‐L). One case with MSI‐H showed negative expression of MSH2 and MSH6. Univariate analysis indicated that MSI‐L was correlated with poor response to R‐CHOP/CHOP chemotherapy in DLBCLs (OR, 0.178; 95% CI, 0.041‐0.776; P = .022). Multivariate analysis showed that MSI‐L was an independent predictive factor for non‐CR to R‐CHOP/CHOP chemotherapy (OR, 0.144; 95% CI, 0.027‐0.761; P = .023). Kaplan‐Meier curves showed that there was a trend that MSI‐H patients had favorable PFS (P = .36) and OS (P = .48), which did not have statistical significance and MSI‐L was not significantly correlated with PFS (P = .24) and OS (P = .52).Our study indicated that there existed MSI‐H and MSI‐L in DLBCLs. MSI‐L could be an independent predictive factor for the chemotherapy response in DLBCLs.

MSIsensor-pro: Fast, Accurate, and Matched-normal-sample-free Detection of Microsatellite Instability

Microsatellite instability (MSI) is a key biomarker for cancer therapy and prognosis. Traditional experimental assays are laborious and time-consuming, and next-generation sequencing-based computational methods do not work on leukemia samples, paraffin-embedded samples, or patient-derived xenografts/organoids, due to the requirement of matched normal samples. Herein, we developed MSIsensor-pro, an open-source single sample MSI scoring method for research and clinical applications. MSIsensor-pro introduces a multinomial distribution model to quantify polymerase slippages for each tumor sample and a discriminative site selection method to enable MSI detection without matched normal samples. We demonstrate that MSIsensor-pro is an ultrafast, accurate, and robust MSI calling method. Using samples with various sequencing depths and tumor purities, MSIsensor-pro significantly outperformed the current leading methods in both accuracy and computational cost. MSIsensor-pro is available at https://github.com/xjtu-omics/msisensor-pro and free for non-commercial use, while a commercial license is provided upon request.

Application of msh3 immunohistochemistry and tetranucleotide microsatellite analysis to identify Elevated Microsatellite Alterations at Selected Tetranucleotide repeats (EMAST) in colorectal tumours

Background and aims: Reduced function of the DNA mismatch repair enzyme MSH3 in tumour cells results in elevated microsatellite alterations at selected tetranucleotide repeats (EMAST). In contrast to microsatellite instability (MSI) caused by MLH1/PMS2/MSH2/MSH6 defects, EMAST in CRCs is a marker of poor prognosis. This study examined the prevalence of EMAST-positive CRCs in a Western Australian population and critically evaluated immunohistochemical and molecular detection methods. Methods and results: MSH3 immunostaining of 250 archival CRCs identified aberrant MSH3 expression (focal or widespread reduction of nuclear MSH3, cytoplasmic MSH3 mis-localisation) in 66% cases. Tetranucleotide MSI (EMAST) was detected in 46% cases, a proportion consistent with limited previous reports. Discordance between MSH3 immunostaining and molecular detection of tetranucleotide MSI resulted from over-detection of abnormal MSH3 immunostaining, which is focal and confounded by common tissue damage (crush, autolysis). Summary and conclusions: Aberrant MSH3 function in CRCs results from elevated inflammatory signalling, a potentially modifiable risk factor that may be targeted to reduce or prevent disease relapse. A combination of MSH3 immunostaining and molecular methods can be used to identify EMAST-positive CRCs, however further antibody development and establishment of consistent tetranucleotide panels will improve the reliability and reproducibility of its detection in routine practice.

Detection of Mismatch Repair and Microsatellite Instability in Colorectal Cancer Patients

Introduction: Colorectal cancer is the third most common cancer worldwide. Microsatellite instability (MSI) contributes to be one of the main mechanisms in colorectal cancer. Individuals with MSI tumors have loss of expression of one or more Mismatch Repair proteins. MSI tumors have better survival rate than microsatellite stable (MSS) tumors, poor response to 5FU-based adjuvant chemotherapy and relatively successful immunotherapy in metastatic MSI tumors. Immunohistochemistry recognizes altered gene by recognizing loss of its protein product. Based on the presence or absence of Mismatch repair proteins, groups are classified into Mismatch repair proficient (MMR-p) and Mismatch repair deficient (MMR-d). Aim: To investigate the immunohistochemical profile of Mismatch repair proteins namely: hMLH1, hMSH2, hMSH6, and hPMS2 in surgically resected colorectal cancer specimens. Materials and Method: A total of 76 cases were selected from the Histopathology Department of HTAA to determine MMR protein expression status. Cases were either MMR-p or MMR-d. Results: Of the specimens which were properly immunostained, seventeen out of seventy-six cases (22.37%) showed loss of one or more MMR proteins expression and thus were MMR-d. MLH1, MSH2, MSH6 and PMS2 protein expression was detected as 85.53% (65/76), 81.6% (62/76), 88.16% (67/76), and 76.32% (58/76), respectively. Conclusion: Mismatch repair proteins profile should be done using immunohistochemistry in local laboratories on these selected cases before referring for the expensive molecular test.

Validation of Microsatellite Instability Detection Using a Comprehensive Plasma-Based Genotyping Panel.

To analytically and clinically validate microsatellite instability (MSI) detection using cell-free DNA (cfDNA) sequencing. Pan-cancer MSI detection using Guardant360 was analytically validated according to established guidelines and clinically validated using 1,145 cfDNA samples for which tissue MSI status based on standard-of-care tissue testing was available. The landscape of cfDNA-based MSI across solid tumor types was investigated in a cohort of 28,459 clinical plasma samples. Clinical outcomes for 16 patients with cfDNA MSI-H gastric cancer treated with immunotherapy were evaluated. cfDNA MSI evaluation was shown to have high specificity, precision, and sensitivity, with a limit of detection of 0.1% tumor content. In evaluable patients, cfDNA testing accurately detected 87% (71/82) of tissue MSI-H and 99.5% of tissue microsatellite stable (863/867) for an overall accuracy of 98.4% (934/949) and a positive predictive value of 95% (71/75). Concordance of cfDNA MSI with tissue PCR and next-generation sequencing was significantly higher than IHC. Prevalence of cfDNA MSI for major cancer types was consistent with those reported for tissue. Finally, robust clinical activity of immunotherapy treatment was seen in patients with advanced gastric cancer positive for MSI by cfDNA, with 63% (10/16) of patients achieving complete or partial remission with sustained clinical benefit. cfDNA-based MSI detection using Guardant360 is highly concordant with tissue-based testing, enabling highly accurate detection of MSI status concurrent with comprehensive genomic profiling and expanding access to immunotherapy for patients with advanced cancer for whom current testing practices are inadequate.See related commentary by Wang and Ajani, p. 6887.

Noninvasive Detection of Microsatellite Instability and High Tumor Mutation Burden in Cancer Patients Treated with PD-1 Blockade.

Microsatellite instability (MSI) and high tumor mutation burden (TMB-High) are promising pan-tumor biomarkers used to select patients for treatment with immune checkpoint blockade; however, real-time sequencing of unresectable or metastatic solid tumors is often challenging. We report a noninvasive approach for detection of MSI and TMB-High in the circulation of patients. We developed an approach that utilized a hybrid-capture-based 98-kb pan-cancer gene panel, including targeted microsatellite regions. A multifactorial error correction method and a novel peak-finding algorithm were established to identify rare MSI frameshift alleles in cell-free DNA (cfDNA). Through analysis of cfDNA derived from a combination of healthy donors and patients with metastatic cancer, the error correction and peak-finding approaches produced a specificity of >99% (n = 163) and sensitivities of 78% (n = 23) and 67% (n = 15), respectively, for MSI and TMB-High. For patients treated with PD-1 blockade, we demonstrated that MSI and TMB-High in pretreatment plasma predicted progression-free survival (hazard ratios: 0.21 and 0.23, P = 0.001 and 0.003, respectively). In addition, we analyzed cfDNA from longitudinally collected plasma samples obtained during therapy to identify patients who achieved durable response to PD-1 blockade. These analyses demonstrate the feasibility of noninvasive pan-cancer screening and monitoring of patients who exhibit MSI or TMB-High and have a high likelihood of responding to immune checkpoint blockade.See related commentary by Wang and Ajani, p. 6887.

Consensus on the detection of microsatellite instability in colorectal cancer and other related solid tumors in China

Microsatellite instability (MSI) which resulted from the deficiency of DNA mismatch repair (MMR), is an important clinical significance in the related solid tumors, such as colorectal cancer and endometrial cancer. There are several methods to detect MSI status, including immunohistochemistry for MMR protein, multiplex fluorescent polymerase chain reaction (PCR) for microsatellite site and MSI algorithm based on next generation sequencing (NGS). The consensus elaborates the definition and clinical significance of MSI as well as the advantages and disadvantages of the three detection methods. Through this expert consensus, we hope to promote the screening which based on MSI status in malignant tumors and improve the acknowledge of clinicians about various testing methods. Thereby, they could interpret the results more accurately and provide better clinical services to patients.

639P - Microsatellite instability detection in colorectal cancer: 44-Center comparison between the Idylla MSI assay and routine molecular and immunohistochemistry tests on formalin-fixed paraffin-embedded tissue

BackgroundMicrosatellite instability (MSI) is observed in 15% of all colorectal cancers (CRC), including Lynch syndrome caused by germline mutation of an MMR gene. Guidelines recommend assessing the MSI status of all CRC patients for screening for Lynch syndrome as well as for prognostic stratification. MSI-high tumors are more vulnerable to the immune system and therefore generally have a better prognosis. MethodsThe performance of the Idylla™ MSI Assay was compared to routine immunohistochemistry (IHC) and molecular tests (including Bethesda panel) on 1,301 archival CRC formalin-fixed, paraffin-embedded (FFPE) tissue sections, at 44 centers worldwide. ResultsThe failure rate of the Idylla™ MSI Assay was 0.23%, while the molecular methods had a higher failure rate overall of 0.86% probably due to their use of a longer amplicon. IHC had a much higher failure rate of 4.37%, possibly related to interpretation difficulties of the results. Routine method failure rates might be an underestimation as the current analysis was done retrospectively on samples with known routine results. Compared to IHC, the results of 37 of the 1,075 samples tested were discordant, resulting in an overall concordance agreement of 96.6%. When taking molecular results and further analysis of discordant samples into account, the Idylla™ MSI result was confirmed in 16 samples. Compared to molecular methods, 16 of the 812 samples tested were discordant, resulting in an overall concordance agreement of 98.0%. Taking IHC results and further analysis of discordant samples into account, the Idylla™ MSI result was confirmed in 8 samples. ConclusionsResults of the Idylla™ MSI Assay were highly concordant with results of routine tests and lower failure rates were observed. Further advantages are the lack of need for matched normal tissue, the low dependence on pre-analytical conditions, the simplified workflow, the short turnaround times, the automated result interpretation, and the very limited hands-on work. Editorial acknowledgementTeams involved in the MSI, Idylla MSI Assay, Colon Rectal Cancer, Multicenter Study. Legal entity responsible for the studyIrblleida. FundingBiocartis. DisclosureX. Matias-guiu: Non-remunerated activity/ies: Biocartis.

1041P - Clinical features and frequency of mismatch repair protein deficiency in ovarian clear cell and endometrioid carcinoma patients

BackgroundThe rates of mismatch repair (MMR) protein deficiency and microsatellite instability (MSI) in ovarian clear cell (CC) and endometrioid (EM) cancer patients were investigated. The clinical features of CC and EM were also analyzed. MethodsPatients postoperatively diagnosed as CC or EM carcinoma from January 2006 to December 2015 were eligible. Diagnosis of all cases was confirmed by the Central Pathological Review Board. MMR protein was analyzed by IHC using a monoclonal antibody and MSI was examined by a multiplex PCR assay for five microsatellite markers, BAT25, BAT26, NR-21, NR-24, and MONO-27. MLH1 IHC-negative cases were examined for DNA hypermethylation of the MLH1 promoter. ResultsWe enrolled 339 cases consisting of 219CC, 116 EM and 4 mixed type (MT) cases. One case could not be evaluated by IHC. MMR protein deficiency was confirmed in five CC (2.3%), 16 EM (13.9%) and 1MT case (25%). Deficiency was observed in 4 cases for MLH1 and PMS2, in 13 cases for MSH2 and MSH6, in 1 case for MSH2 and PMS2, in 2 cases for MSH6, and in 1 case for PMS2. DNA hypermethylation of the MLH1 promoter was detected in one of the four MLH1 and PMS2 deficiency cases. Three of the 339 cases could not be evaluated by MSI. A high rate of MSI was confirmed in 13 cases (3.9%) and MMR deficiency was detected by IHC in these cases. However, MSI was not detected in the other nine cases where MMR deficiency was detected by IHC. The median age of all 339 cases was 54 (CC 54, EM 51) and that of CC and EM cases with MMR deficiency was 39 and 48 years, respectively. ConclusionsThe rate of MMR deficiency in CC and EM was not high, whereas CC in young women was more frequent. IHC is a more successful screening method of MMR deficiency in ovarian CC and EM compared with MSI detection. The rate of MLH1 promoter hypermethylation (epigenetic MMR deficiency) in ovarian CC and EM was less than that of endometrial cancer. Legal entity responsible for the studyThe author. FundingNational Hospital Organization of Japan. DisclosureThe author has declared no conflicts of interest.

255 Yield of Non-Lynch Gene Mutations During Testing for Lynch Syndrome

INTRODUCTION: Multigene panels are commonly used for genetic testing when Lynch syndrome is suspected. However, the data about the relative yield of Lynch vs. non-Lynch mutations are sparse. METHODS: In this retrospective, single-center study, all patients with a diagnosis of colon or uterine cancer between January 1, 2016 and June 30th, 2018 were included. The patients were identified using search in the pathology database for SNOMED codes. Additionally, the search results of SNOMED codes were tallied with the cancer center database to capture all patients with colon cancer. A structured RedCap electronic database was created to systematically abstract data about demographics, location and stage of colon cancer, PREMM5 scores, genetic testing and a detailed 3-generation family pedigree. After being screened for Lynch syndrome using immunohistochemical assays or PCR-based detection of microsatellite instability, patients with abnormal results underwent genetic testing using well-established multi-gene commercial assays. The yields of Lynch and non-Lynch syndromes were compared. Statistical comparisons were performed using Chi-square test for categorical variables and Student's t-test for numerical variables. RESULTS: A total of 737 participants (whites 84.1%, mean age of 62.7) were included. A diagnosis of Lynch syndrome was made in 21 of the 737 participants (2.85%). Non-Lynch mutations were detected in 12 of the 737 participants (1.63%). When colon and uterine cancers were analyzed separately, non-Lynch mutations were detected in 2.1% and 1.5% of participants respectively (Table 1). The non-Lynch genetic mutations detected included: APC, MAP syndrome, CHEK2, BRCA1, BRCA2, MUTYH, PALB2, AXIN2, SMARC4, and NTLH1. CONCLUSION: Germline testing has a significant yield of non-Lynch mutations in patients suspected to have Lynch syndrome. Our data support the current practice of multigene panels to define syndromic cancer risk.

Quantitative next-generation sequencing-based analysis indicates progressive accumulation of microsatellite instability between atypical hyperplasia/endometrial intraepithelial neoplasia and paired endometrioid endometrial carcinoma

Atypical hyperplasia/endometrial intraepithelial neoplasia is an accepted precursor to endometrioid-type endometrial carcinoma. Mismatch repair-deficient endometrial carcinomas are also known to be a biologically and clinically distinct subset of tumors. However, the development of microsatellite instability in endometrial carcinogenesis has not yet been evaluated by novel next-generation sequencing-based methods. We examined 17 mismatch repair-deficient endometrioid endometrial carcinomas and their paired atypical hyperplasia/endometrial intraepithelial neoplasia precursors using a next-generation sequencing panel with quantitative microsatellite instability detection at 336 loci. Findings were compared to histological features, polymerase chain reaction-based microsatellite instability testing, immunohistochemical expression of mismatch repair proteins, and tumor mutational burden calculations. All 17 endometrial carcinomas and 8/17 atypical hyperplasia/endometrial intraepithelial neoplasia showed microsatellite instability by next-generation sequencing-based testing. Endometrial carcinoma specimens showed significantly more unstable microsatellite loci than paired atypical hyperplasia/endometrial intraepithelial neoplasia (mean: 40.0% vs 19.9 unstable loci, respectively). Out of nine microsatellite-stable atypical hyperplasia/endometrial intraepithelial neoplasia specimens, four showed mismatch repair loss by immunohistochemistry. All atypical hyperplasia/endometrial intraepithelial neoplasia and endometrial carcinoma specimens with microsatellite instability were also mismatch repair-deficient by immunohistochemistry. Tumor mutational burden was significantly greater in endometrial carcinoma than in paired atypical hyperplasia/endometrial intraepithelial neoplasia specimens, and tumor mutational burden was significantly correlated with percent unstable microsatellite loci. Paired atypical hyperplasia/endometrial intraepithelial neoplasia and endometrial carcinoma specimens show progressive accumulation of unstable microsatellite loci following loss of mismatch repair protein expression. Comprehensive next-generation sequencing-based testing of endometrial carcinomas offers new insights into endometrial carcinogenesis and opportunities for improved tumor surveillance, diagnosis, and management.

Low temperature isothermal amplification of microsatellites drastically reduces stutter artifact formation and improves microsatellite instability detection in cancer.

Microsatellites are polymorphic short tandem repeats of 1–6 nucleotides ubiquitously present in the genome that are extensively used in living organisms as genetic markers and in oncology to detect microsatellite instability (MSI). While the standard analysis method of microsatellites is based on PCR followed by capillary electrophoresis, it generates undesirable frameshift products known as ‘stutter peaks’ caused by the polymerase slippage that can greatly complicate the analysis and interpretation of the data. Here we present an easy multiplexable approach replacing PCR that is based on low temperature isothermal amplification using recombinase polymerase amplification (LT-RPA) that drastically reduces and sometimes completely abolishes the formation of stutter artifacts, thus greatly simplifying the calling of the alleles. Using HT17, a mononucleotide DNA repeat that was previously proposed as an optimal marker to detect MSI in tumor DNA, we showed that LT-RPA improves the limit of detection of MSI compared to PCR up to four times, notably for small deletions, and simplifies the identification of the mutant alleles. It was successfully applied to clinical colorectal cancer samples and enabled detection of MSI. This easy-to-handle, rapid and cost-effective approach may deeply improve the analysis of microsatellites in several biological and clinical applications.

Microsatellite instability detection using a large next-generation sequencing cancer panel across diverse tumour types

AimMicrosatellite instability (MSI), a hallmark of DNA mismatch repair deficiency, is a key molecular biomarker with multiple clinical implications including the selection of patients for immunotherapy, identifying patients who may...

Sensitive Detection of Microsatellite Instability (MSI) in Liquid Biopsies from Early Stage Colon Cancer Patients using Nuclease-based Enrichment and Standard-Marker or NGS based approaches

Sensitive Detection of Microsatellite Instability (MSI) in Liquid Biopsies from Early Stage Colon Cancer Patients using Nuclease-based Enrichment and Standard-Marker or NGS based approaches

Development and analytical validation of a next-generation sequencing based microsatellite instability (MSI) assay.

We have developed and analytically validated a next-generation sequencing (NGS) assay to classify microsatellite instability (MSI) in formalin-fixed paraffin-embedded (FFPE) tumor specimens. The assay relies on DNA-seq evaluation of insertion/deletion (indel) variability at 29 highly informative genomic loci to estimate MSI status without the requirement for matched-normal tissue. The assay has a clinically relevant five-day turnaround time and can be conducted on as little as 20 ng genomic DNA with a batch size of up to forty samples in a single run. The MSI detection method was developed on a training set (n = 94) consisting of 22 MSI-H, 24 MSS, and 47 matched normal samples and tested on an independent test set of 24 MSI-H and 24 MSS specimens. Assay performance with respect to accuracy, reproducibility, precision as well as control sample performance was estimated across a wide range of FFPE samples of multiple histologies to address pre-analytical variability (percent tumor nuclei), and analytical variability (batch size, run, day, operator). Analytical precision studies demonstrated that the assay is highly reproducible and accurate as compared to established gold standard PCR methodology and has been validated through NYS CLEP. This assay provides clinicians with robust and reproducible NGS-based MSI testing without the need of matched normal tissue to inform clinical decision making for patients with solid tumors.

A Novel Next-Generation Sequencing Approach to Detecting Microsatellite Instability and Pan-Tumor Characterization of 1000 Microsatellite Instability–High Cases in 67,000 Patient Samples

Microsatellite instability (MSI) is an important biomarker for predicting response to immune checkpoint inhibitor therapy, as emphasized by the recent checkpoint inhibitor approval for MSI-high (MSI-H) solid tumors. Herein, we describe and validate a novel method for determining MSI status from a next-generation sequencing comprehensive genomic profiling assay using formalin-fixed, paraffin-embedded samples. This method is 97% (65/67) concordant with current standards, PCR and immunohistochemistry. We further apply this method to >67,000 patient tumor samples to identify genes and pathways that are enriched in MSI-stable or MSI-H tumor groups. Data show that although rare in tumors other than colorectal and endometrial carcinomas, MSI-H samples are present in many tumor types. Furthermore, the large sample set revealed that MSI-H tumors selectively share alterations in genes across multiple common pathways, including WNT, phosphatidylinositol 3-kinase, and NOTCH. Last, MSI is sufficient, but not necessary, for a tumor to have elevated tumor mutation burden. Therefore, MSI can be determined from comprehensive genomic profiling with high accuracy, allowing for efficient MSI-H detection across all tumor types, especially those in which routine use of immunohistochemistry or PCR-based assays would be impractical because of a rare incidence of MSI. MSI-H tumors are enriched in alterations in specific signaling pathways, providing a rationale for investigating directed immune checkpoint inhibitor therapies in combination with pathway-targeted therapies.

Comprehensive application of various screening strategies of Lynch syndrome

Lynch syndrome (LS), which is the most common hereditary colorectal cancer, accounts for about 3% of all colorectal cancers. However, due to its various clinical manifestations, it is difficult to be diagnosed. The diagnosis of LS requires comprehensive application of various screening criteria (such as the Amsterdam criteria, Bethesda criteria), predictive models, risk factors, immunohistochemistry test of mismatch repair (MMR) protein, microsatellite instability (MSI) detection, MLH1 methylation detection, BRAF gene mutation detection, germline gene mutation detection, and so on. LS can be diagnosed only after the identification of pathogenic germline mutation of MMR gene. The first-degree and second-degree relatives of LS patients are recommended to be tested for the identified mutant gene. For LS patients and gene mutation carriers, LS associated cancer can be detected early or even prevented by monitoring and preventive surgery. Reproductive techniques can be used to prevent this disease from being passed down to the next generation.

Abstract 4252: Detecting microsatellite instability in ffpe tissue from crc subjects using next generation sequencing

Abstract Background Microsatellite instability (MSI) is a hypermutable phenotype resulting from DNA mismatch repair deficiency and is observed in up to 10-15% of early-stage colorectal cancers (CRC). It manifests as abnormal lengthening or shortening of DNA repeats at specific genomic loci. MSI-High (MSI-H) CRC patients have been shown to respond favorably towards both chemotherapy and immunotherapy and have different prognosis than MSS patients and the FDA has approved Pembrolizumab as the first cancer therapy based on a class of biomarkers rather than a cancer type. With increasing options for targeted therapy in solid tumors, being able to combine MSI testing with other cancer biomarkers for approved therapies is beneficial. We show proof of concept of a pan-cancer gene panel using an NGS-based assay and bioinformatics workflow that enables classification of MSI status along with detection of SNVs, fusions, indels and CNVs from tumor tissue samples. Unlike the gold standard PCR-based test, our method does not require a matched normal sample, thus enabling MSI detection based on tumor tissue alone. Methods Our workflow combines whole genome library preparation, hybrid capture target enrichment, high-throughput sequencing and a proprietary analysis algorithm. We expanded the number of genomic loci to improve the sensitivity of MSI detection. The analysis algorithm was trained using DNA from pure and mixed cell lines, tumor tissue and normal samples with known MSI status to classify the molecular alterations between MSI-H and MSS genotypes from sequencing reads. The algorithm leverages repeat lengths in homopolymeric MSI loci in order to predict instability of individual loci followed by aggregation using a statistical framework to make the final call on MSI status. Results The above algorithm was evaluated on a cohort of 134 stage II and stage III CRC subjects who underwent curative intent surgery. MSI status for these samples was orthogonally tested using a PCR-based MSI Analysis System (31 positives, 103 negatives) and a dMMR system. Using a pre-determined threshold, the algorithm yielded 100% sensitivity and 100% specificity on the above cohort without using matched normal tissue. A second cohort evaluated includes 47 CRC patients that are enriched for MSI positive status by virtue of their selection criteria (BRAF positive). Very high concordance on MSI status with an orthogonal method was observed. Data demonstrating high concordance with a PCR_based MSI system will be available at the presentation. Conclusions Here we present an NGS-based assay and bioinformatics workflow with robust analytical performance for MSI detection in FFPE tissue samples without a paired normal. Citation Format: Amrita Pati, Hao Wang, Hamid Mirebrahim, Seng Saelee, Joshua Lefkowitz, Sean Chien, Ashla Singh, Fergal Casey, Vera Rapoport, Xiaoju Max Ma, John Lee, Alex Lovejoy, Daniel Klass, Hans-Peter Adams. Detecting microsatellite instability in ffpe tissue from crc subjects using next generation sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4252.

Targeted Cancer Next-Generation Sequencing as a Primary Screening Tool for Microsatellite Instability and Lynch Syndrome in Upper Gastrointestinal Tract Cancers.

No consensus guideline has been established for microsatellite instability testing in upper gastrointestinal tract cancers. This study aims to determine whether targeted cancer next-generation sequencing can accurately detect microsatellite instability in upper gastrointestinal tract cancers and screen for patients with Lynch syndrome. In a cohort of 645 upper gastrointestinal tract cancers, targeted next-generation sequencing assessed microsatellite instability by identifying characteristic insertion and deletion mutations. Sequencing classification was compared with mismatch repair protein IHC. Cancers with microsatellite instability by sequencing were analyzed using a testing protocol to identify patients with Lynch syndrome. Sequencing identified microsatellite instability in 3.6% (23/645) of upper gastrointestinal tract cancers, including 28% (8/29) of small intestinal and 9% (9/97) of gastric carcinomas. In 20 cancers classified as having microsatellite instability, 19 demonstrated loss of expression of MLH1, PMS2, MSH2, or MSH6, and one cancer was indeterminate by IHC. In contrast, 52 control cancers demonstrated retained expression of all mismatch repair proteins. Using targeted sequencing as the initial screening test, 1.1% (7/645) of patients were identified to have pathogenic germline variants confirming a diagnosis of Lynch syndrome. Targeted cancer next-generation sequencing is an accurate first-line test to detect microsatellite instability in upper gastrointestinal tract cancers. This study provides a proof of concept for the use of targeted next-generation sequencing to detect microsatellite instability and screen for Lynch syndrome.

Abstract 1701: Improvement of tumor mutation burden measurement by removal of deaminated bases in FFPE DNA

Abstract Tumor mutation burden (TMB) is a positive predictive factor for response to immune-checkpoint inhibitors in certain types of cancer. The Oncomine™ Tumor Mutation Load Assay, a 1.7Mb targeted next generation sequencing (NGS) panel, measures TMB and detects mutations in 409 cancer genes. The TMB values obtained using targeted sequencing are positively correlated with TMB measured by whole exome sequencing in NSCLC, colorectal, endometrial and gastric cancers. TMB from these tumor samples are correlated with other phenotypes associated with genomic instability, specifically microsatellite instability (MSI) and mutations involved in mismatch repair and cancer related genes. Analysis of the Oncomine™ TML Assay results with Torrent Suite and Ion Reporter software uniquely measures the degree of deamination of cytosines to uracils in fixed tissues. FFPE preservation methods can lead to significant cytosine deamination of the isolated DNA, resulting in decreased sequencing quality. In these samples, uracils are propagated as thymines and result in false C>T substitutions. To minimize the influence that excess deamination has on TMB results, we have incorporated a UDG enzyme treatment to eliminate damaged targets and improve usable TMB values of DNA from damaged FFPE tumor tissue. The Oncomine™ TML assay for TMB on Ion GeneStudio™ S5 in conjunction with MSI detection is informative and potentially predictive for the use of checkpoint inhibitors in multiple cancer types. Citation Format: Warren Tom, Ruchi Chaudhary, Vinay Mittal, Dinesh Cyanam, Iris Casuga, Elaine Wong-Ho, Rob Bennett, Fiona Hyland, Seth Sadis, Janice Au-Young. Improvement of tumor mutation burden measurement by removal of deaminated bases in FFPE DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1701.