Microsatellite Instability Assay Research Articles

One-instrument, objective microsatellite instability analysis using high-resolution melt.

In recent years, immune checkpoint inhibitors have proved immense clinical progression in the treatment of certain cancers. The efficacy of immune checkpoint inhibitors is correlated with mismatch repair system deficiency and is exceptionally administered based exclusively on this biological mechanism independent of the cancer type. The promising effect of immune checkpoint inhibitors has left an increasing demand for analytical tools evaluating the mismatch repair status. The analysis of microsatellite instability (MSI), reflecting an indirect but objective manner the inactivation of the mismatch repair system, plays several roles in clinical practice and, therefore, its evaluation is of high relevance. Analysis of MSI by PCR followed by fragment analysis on capillary electrophoresis remains the gold standard method for detection of a deficient mismatch repair system and thereby treatment with immune checkpoint inhibitors. Novel technologies have been applied and concepts such as tumor mutation burden have been introduced. However, to date, most of these technologies require high costs or the need of matched non-tumor tissue as internal comparator. In this study, we present a novel, one-instrument, fast, and objective method for the detection of MSI (MicroSight® MSI 1-step HRM Analysis), which does not depend on the use of matched non-tumor tissue. The assay analyzes five well-described mononucleotide microsatellite sequences by real-time PCR followed by high-resolution melt and evaluates microsatellite length variations via PCR product melting profiles. The assay was evaluated using two different patient cohorts and evaluation included several DNA extraction methodologies, two different PCR platforms, and an inter-laboratory ring study. The MicroSight® MSI assay showed a high repeatability regardless of DNA extraction method and PCR platform, and a 100% agreement of the MSI status with PCR fragment analysis methods applied as clinical comparator.

A Validated Highly Sensitive Microsatellite Instability Assay Accurately Identifies Individuals Harboring Biallelic Germline PMS2 Pathogenic Variants in Constitutional Mismatch Repair Deficiency.

Constitutional mismatch repair deficiency (CMMRD) is a rare and extraordinarily penetrant childhood-onset cancer predisposition syndrome. Genetic diagnosis is often hampered by the identification of mismatch repair (MMR) variants of unknown significance and difficulties in PMS2 analysis, the most frequently mutated gene in CMMRD. We present the validation of a robust functional tool for CMMRD diagnosis and the characterization of microsatellite instability (MSI) patterns in blood and tumors. The highly sensitive assessment of MSI (hs-MSI) was tested on a blinded cohort of 66 blood samples and 24 CMMRD tumor samples. Hs-MSI scores were compared with low-pass genomic instability scores (LOGIC/MMRDness). The correlation of hs-MSI scores in blood with age of cancer onset and the distribution of insertion-deletion (indel) variants in microsatellites were analyzed in a series of 169 individuals (n = 68 CMMRD, n = 124 non-CMMRD). Hs-MSI achieved high accuracy in the identification of CMMRD in blood (sensitivity 98.5% and specificity 100%) and detected MSI in CMMRD-associated tumors. Hs-MSI had a strong positive correlation with whole low-pass genomic instability LOGIC scores (r = 0.89, P = 2.2e-15 in blood and r = 0.82, P = 7e-3 in tumors). Indel distribution identified PMS2 pathogenic variant (PV) carriers from other biallelic MMR gene PV carriers with an accuracy of 0.997. Higher hs-MSI scores correlated with younger age at diagnosis of the first tumor (r = -0.43, P = 0.011). Our study confirms the accuracy of the hs-MSI assay as ancillary testing for CMMRD diagnosis, which can also characterize MSI patterns in CMMRD-associated cancers. Hs-MSI is a powerful tool to pinpoint PMS2 as the affected germline gene and thus potentially personalize cancer risk.

Epigenetic MLH1 silencing concurs with mismatch repair deficiency in sporadic, naturally occurring colorectal cancer in rhesus macaques

BackgroundNaturally occurring colorectal cancers (CRC) in rhesus macaques share many features with their human counterparts and are useful models for cancer immunotherapy; but mechanistic data are lacking regarding the comparative molecular pathogenesis of these cancers.MethodsWe conducted state-of-the-art imaging including CT and PET, clinical assessments, and pathological review of 24 rhesus macaques with naturally occurring CRC. Additionally, we molecularly characterized these tumors utilizing immunohistochemistry (IHC), microsatellite instability assays, DNAseq, transcriptomics, and developed a DNA methylation-specific qPCR assay for MLH1, CACNA1G, CDKN2A, CRABP1, and NEUROG1, human markers for CpG island methylator phenotype (CIMP). We furthermore employed Monte-Carlo simulations to in-silico model alterations in DNA topology in transcription-factor binding site-rich promoter regions upon experimentally demonstrated DNA methylation.ResultsSimilar cancer histology, progression patterns, and co-morbidities could be observed in rhesus as reported for human CRC patients. IHC identified loss of MLH1 and PMS2 in all cases, with functional microsatellite instability. DNA sequencing revealed the close genetic relatedness to human CRCs, including a similar mutational signature, chromosomal instability, and functionally-relevant mutations affecting KRAS (G12D), TP53 (R175H, R273*), APC, AMER1, ALK, and ARID1A. Interestingly, MLH1 mutations were rarely identified on a somatic or germline level. Transcriptomics not only corroborated the similarities of rhesus and human CRCs, but also demonstrated the significant downregulation of MLH1 but not MSH2, MSH6, or PMS2 in rhesus CRCs. Methylation-specific qPCR suggested CIMP-positivity in 9/16 rhesus CRCs, but all 16/16 exhibited significant MLH1 promoter hypermethylation. DNA hypermethylation was modelled to affect DNA topology, particularly propeller twist and roll profiles. Modelling the DNA topology of a transcription factor binding motif (TFAP2A) in the MLH1 promoter that overlapped with a methylation-specific probe, we observed significant differences in DNA topology upon experimentally shown DNA methylation. This suggests a role of transcription factor binding interference in epigenetic silencing of MLH1 in rhesus CRCs.ConclusionsThese data indicate that epigenetic silencing suppresses MLH1 transcription, induces the loss of MLH1 protein, abrogates mismatch repair, and drives genomic instability in naturally occurring CRC in rhesus macaques. We consider this spontaneous, uninduced CRC in immunocompetent, treatment-naïve rhesus macaques to be a uniquely informative model for human CRC.Graphical abstract

Benchmarking mismatch repair testing for patients with cancer receiving immunotherapy.

179 Background: Mismatch repair deficiency (MMR-D) serves as a theranostic marker for directing immune checkpoint inhibitor (ICI) treatment across various cancer stages and tumor types. The College of American Pathologists endorses immunohistochemistry (IHC) as the primary approach for assessing mismatch repair (MMR) testing in colorectal (CRC) and endometrial cancer (EC) patients. An alternative method involves genetic evaluation of microsatellite instability (MSI). Previous studies have shown that resistance to immune checkpoint inhibitors (ICIs) may occur due to inaccurate MSI or MMR IHC status determination. Accurate assessment of MMR status is critical for treatment decision-making. Methods: We hypothesized that tumor-only next generation sequencing (NGS) for MMR mutation signature assessment for identification of MMR-D tumors is more sensitive than MMR IHC testing. Using a standardized, clinically validated, tumor-only NGS mutation signature pipeline in place in the Oncopanel assay at DFCI/BWH, we compared the concordance in MMR determination between NGS and IHC. We then evaluated a subset of ICI-treated patients to assess the clinical implications of these findings. Results: 1655 colorectal cancer (CRC) and endometrial carcinoma (EC) patients who had both NGS MSI (NGSd = deficient or NGSp = proficient) and MMR IHC (IHC- = absent or IHC+ = intact) assays performed were assessed. Discordant NGSd/IHC+ results were identified in 1% of CRC and 5% of EC cases that were IHC+. Importantly, patients with discordant NGSd/IHC+ (median OS: not reached [NR], 95% CI, 9.9 months-NR; median TTF: 44.4 months, 95% CI, 4.1-NR) and concordant NGSd/IHC- results, showed a similar overall survival and time to treatment failure with ICI treatment (p=non-significant) which contrasted with results for patients with concordant NGSp/IHC+ results (median OS: 14.9 months, 95% CI, 7.3-24.6, p-value 0.0025 and < 0.0001; median TTF: 5.0 months, 95% CI, 3.2-7.3; p-value 0.0014 and < 0.0001). Patients with EC or CRC and discordant NGSd/IHC+ results had better survival when treated with ICI-based regimens, compared to those who received non-ICI treatments (p= 0.054 and 0.017). Conclusions: In 2022, 151,030 new CRC and 65,950 new EC cases were diagnosed in the USA. Extrapolating our findings to this larger population, use of NGS mutational signature analysis instead of IHC would amount to about 1510 additional MMR-D CRC cases and 3297 additional MMR-D endometrial cases identified each year for whom first-line ICI therapy is preferred. Our findings support revisiting guideline recommendations for diagnostic testing of MMR to incorporate both IHC and targeted NGS tumor panel testing using sensitive and specific mutation signature calling algorithms. Larger prospective studies are warranted to confirm these findings in patients with mCRC and mEC treated with ICIs. Evaluation of this approach in other cancer types is also warranted.

Hereditary Colorectal Cancer in Brunei Darussalam

Introduction: Hereditary colorectal cancers (CRC) are associated with early age of disease onset and high risks of developing other cancers. In this study, we estimate the prevalence of hereditary CRC over a 6-year period, and report the socio-demographics and clinical profile of patients in Brunei Darussalam. Methods: A retrospective study of data from 146 patients diagnosed with CRC from January 2017 to December 2022 was obtained from The Brunei Cancer Centre. Suspected or confirmed hereditary CRC patients were flagged from the 146 cases based on microsatellite instability (MSI) assay testing, immunohistochemistry (IHC) four-panel staining on mismatch repair (MMR) genes MLH1, MSH2, MSH6, and PMS2, and BRAFV600E test results. Results: Among the 146 patients with CRC, 41.7% (n=61) patients underwent MSI testing where 7.5% (n=11) reported high instability in their microsatellite assay (MSI-H), of which 2 were associated with Lynch syndrome. Among the 11 patients with “suspected” or “confirmed” hereditary colorectal cancer, majority presented with abdominal pain prior to diagnosis (81.8%; n=9), Stage 2 cancer (36.4%; n=4), moderately differentiated tumour (72.7%; n=8), tumour located in caecum (36.4%; n=4) and all patients had surgery as first-line management, with more than half receiving chemotherapy (54.5%; n=6). Conclusion: 7.5% of patients of CRC cases seen at a tertiary cancer centre over a 6-year period was found to have “suspected” or “confirmed” hereditary CRC. The lack of genetic testing performed in local settings may indicate that the actual prevalence may be higher.

Performance of Immunohistochemical and Molecular Methods in Detecting Microsatellite Instability in Gastric Cancer: A Multicenter Study

Introduction: Microsatellite instability (MSI) is an important prognostic molecular biomarker for gastric cancer (GC). MSI status may be detected by immunohistochemistry (IHC) for mismatch repair (MMR) proteins and polymerase chain reaction (PCR). Idylla™ MSI assay has not been validated for GC but may prove to be a valid alternative. Methods: In a series of 140 GC cases, MSI status was evaluated by IHC for MLH1, PMS2, MSH2, and MSH6; gold-standard pentaplex PCR panel (PPP) (BAT-25, BAT-26, NR-21, NR-24, and NR-27); and Idylla. Statistical analysis was performed using SPSS 27.0. Results: PPP identified 102 microsatellite stable (MSS) cases and 38 MSI-high cases. Only 3 cases showed discordant results. Compared with PPP, the sensitivity was 100% for IHC and 94.7% for Idylla. Specificity was 99% for IHC and 100% for Idylla. MLH1 IHC alone showed sensitivity and specificity of 97.4% and 98.0%, respectively. IHC identified three indeterminate cases; all were MSS according to PPP and Idylla. Conclusion: IHC for MMR proteins represents an optimal screening tool for MSI status in GC. If resources are limited, isolated MLH1 evaluation may constitute a valuable option for preliminary screening. Idylla may help detect rare MSS cases with MMR-loss and define MSI status in indeterminate cases.

Prevalence of mismatch repair deficiency in a large prospective prostate cancer cohort.

5031 Background: Mismatch repair (MMR) deficiency and microsatellite instability (MSI) are predictive biomarkers for immunotherapy response. The best approach to identify patients with such tumors is unclear in prostate cancer. Methods: This study included men diagnosed with primary prostate cancer during prospective follow-up of the Health Professionals Follow-up Study (HPFS) and Physicians’ Health Study (PHS). The highest-grade/index lesions of tumor tissue from radical prostatectomy (95%) or transurethral resections of the prostate were mounted in triplicate on tissue microarrays. Immunohistochemistry for the MMR proteins MLH1, MSH2, MSH6, and PMS2 was performed, with scoring as MMR-deficient requiring a visible staining in a non-tumor internal positive control of the same case. For validation, a polymerase chain reaction-based MSI assay (Idylla MSI Test, Biocartis) was performed on tumor DNA of MMR-deficient cases and a selection of MMR-intact cases. Results: The study included 1016 men with prostate cancer. Tumor stage was predominantly pathologically localized (71% stage pT1/2, 18% T3a/b) with a full distribution of Gleason scores, including 20% Gleason score 6 (grade group 1), 36% 3+4 (grade group 2), 23% 4+3 (grade group 3), 8% 8 (grade group 4) and 13% 9-10 (grade group 5). MMR tumor scoring could be performed for MLH1 in 747 cases (75% of those with tumor tissue), for MSH2 in 903 cases (90%), for MSH6 in 708 cases (74%), and for PMS2 in 703 cases (72%). The remaining tumors were unevaluable due to lack of non-tumor tissue necessary as an internal positive control. Of the 903 tumors evaluable for MSH2 protein loss, 4 tumors had loss of MSH2 (prevalence 0.4%, 95% confidence interval [CI] 0.2–1.1%), and 3 of 708 evaluable tumors had concomitant loss of MSH6 (prevalence 0.4%, 95% CI 0.1–1.2%). No tumor had loss of MLH1 or PMS2. The MMR-deficient tumors had Gleason scores of 3+4, 8 (two cases), and 9–10. Tumor DNA of 1 of the 4 MMR-deficient tumors met the manufacturer’s cut-off for MSI-high; two additional MMR-deficient cases had non-zero repeats, with good reproducibility between tumor cores and technical replicates. One MSH2-deficient tumor and 6 DNA samples from 3 MMR-intact cases had repeat scores of zero. Conclusions: In this nationwide prospective study, MMR deficiency was rare in primary, surgically treated prostate cancer. This low prevalence contrasts with hospital-based studies of MMR deficiency and MSI that may have represented tumors with certain clinical features. The low prevalence and the need for an internal positive control for reliable scoring calls into question to what extent immunohistochemistry-based screening for MMR deficiency on limited tissue specimens, such as prostate biopsies, is routinely feasible.

Mismatch repair deficiency testing in Lynch syndrome-associated urothelial tumors.

Lynch syndrome-associated cancer develops due to germline pathogenic variants in one of the mismatch repair (MMR) genes, MLH1, MSH2, MSH6 or PMS2. Somatic second hits in tumors cause MMR deficiency, testing for which is used to screen for Lynch syndrome in colorectal cancer and to guide selection for immunotherapy. Both MMR protein immunohistochemistry and microsatellite instability (MSI) analysis can be used. However, concordance between methods can vary for different tumor types. Therefore, we aimed to compare methods of MMR deficiency testing in Lynch syndrome-associated urothelial cancers. Ninety-seven urothelial (61 upper tract and 28 bladder) tumors diagnosed from 1980 to 2017 in carriers of Lynch syndrome-associated pathogenic MMR variants and their first-degree relatives (FDR) were analyzed by MMR protein immunohistochemistry, the MSI Analysis System v1.2 (Promega), and an amplicon sequencing-based MSI assay. Two sets of MSI markers were used in sequencing-based MSI analysis: a panel of 24 and 54 markers developed for colorectal cancer and blood MSI analysis, respectively. Among the 97 urothelial tumors, 86 (88.7%) showed immunohistochemical MMR loss and 68 were successfully analyzed by the Promega MSI assay, of which 48 (70.6%) were MSI-high and 20 (29.4%) were MSI-low/microsatellite stable. Seventy-two samples had sufficient DNA for the sequencing-based MSI assay, of which 55 (76.4%) and 61 (84.7%) scored as MSI-high using the 24-marker and 54-marker panels, respectively. The concordance between the MSI assays and immunohistochemistry was 70.6% (p = 0.003), 87.5% (p = 0.039), and 90.3% (p = 1.00) for the Promega assay, the 24-marker assay, and the 54-marker assay, respectively. Of the 11 tumors with retained MMR protein expression, four were MSI-low/MSI-high or MSI-high by the Promega assay or one of the sequencing-based assays. Our results show that Lynch syndrome-associated urothelial cancers frequently had loss of MMR protein expression. The Promega MSI assay was significantly less sensitive, but the 54-marker sequencing-based MSI analysis showed no significant difference compared to immunohistochemistry. Data from this study alongside previous studies, suggest that universal MMR deficiency testing of newly diagnosed urothelial cancers, using immunohistochemistry and/or sequencing-based MSI analysis of sensitive markers, offer a potentially useful approach to identification of Lynch syndrome cases.

Microsatellite Instability Assay as a Potential Approach to Evaluate Genotoxicity: Lead Exposure in a Nestling Passerine Bird at the Stage of Intensive Erythropoiesis.

Although many avian studies have investigated the toxic effects of lead on important biochemical and physiological processes, organ and system function, and behavior, studies evaluating the specific genotoxic effects of exposure to lead are scarce. Nowadays, rapid technological advances can provide new molecular techniques in this regard. In this study, as a novel approach in bird studies, we used a panel of ten microsatellite loci to investigate the microsatellite instability (MSI) in response to experimental lead intoxication in a common hole-nesting species, the great tit Parus major. For this purpose, an experiment based on an intentional single supplementation of a lead (II) acetate trihydrate compound was conducted, with the use of two different doses, applied to randomly chosen great tit nestlings from randomly selected broods, being at the stage of intensive erythropoiesis. Although this preliminary study did not find any MSI in the seven microsatellite markers retained for the final comparison, it contributes to the examination of this molecular technique in field conditions as being potentially applicable in ecotoxicological bird studies. We believe that certain issues should be considered in finding an explanation for our result. First, the single doses of lead used in this study may have been too weak to induce genetic instability. Second, the panel of microsatellite markers studied may have been unsusceptible to lead genotoxicity in general. Third, the relatively short time interval (5 days) between the experimental procedure (lead exposure) and the sampling of post-exposure material (blood) for genetic analyses could have limited the effect of lead genotoxicity. Further analyzes are needed to verify these findings and to evaluate the scope of application of the MSI analysis in wild bird population studies.

Establishment of a gastric cancer cell line with high microsatellite instability, OCUM-13, derived from Borrmann type-2 primary tumor.

Gastric cancer (GC) with microsatellite instability (MSI) has been reported to be sensitive to immunotherapy, however some of GC cases with MSI remain resistant to immunotherapy. Cancer cell lines showing MSI might be useful for the analysis of mechanisms of immunotherapy, while only a few GC cell lines with MSI are available so far. In this study, we established a unique GC cell line with MSI, OCUM-13, from a primary GC with abundant tumor-infiltrating lymphocytes. MSI assay indicated that OCUM-13 cells as well as the primary tumor showed a band shift in more than 3 of 5 microsatellite loci, suggesting that OCUM-13 did have high MSI. The subcutaneous inoculation of OCUM-13 cells into mice performed tumor formation. Insulin-like growth factor 1 receptor inhibitor decreased the growth of OCUM-13 cells. The newly established cell line with MSI, OCUM-13, might be useful for the analysis of cancer therapy for GC with MSI.

Endometrial Cancer and Molecular Methods: A Case Study of MSI Heterogeneity based on Metastatic Location with IdyllaTM

Abstract Introduction/Objective MMR deficiency (dMMR) and Microsatellite Instability (MSI) are hallmarks of not only Lynch Syndrome and Colorectal Cancer (CRC), but Endometrial cancer. Endometrial cancer is the most common female gynecological cancer, yet molecular tests for MSI often focus solely on CRC validation. MSI in Endometrial cancer correlates with higher staging and more aggressive pathology, resulting in a need for reliable testing methods. Immunohistochemistry (IHC) staining is the current standard for MSI detection, due to the known association between dMMR and MSI-H. Direct detection of MSI requires molecular methods, however, there is a lack of standardization of protocol between labs. Idylla™ (Biocartis, Jersey City, NJ), an automated cartridge-based PCR assay, could help eliminate this ambiguity. Methods/Case Report To determine whether the Idylla™ MSI assay can be reliably used to detect MSI in Endometrial cancers, both primary and metastatic sites for two known MSI-H patients were examined using the Idylla ™ MSI assay and an IHC MMR panel (MLH1, MSH2, MSH6, PMS2). Results (if a Case Study enter NA) The results showed MSI-H for both patients uterine primary sites on the Idylla™. Examination of the Idylla™ results for metastatic sites found they contradicted the IHC panel results for some sites. Idylla™ produced expected results for the omentum and intestinal metastases in patient IN-20-1. The Idylla™ results for the ovarian and abdominal metastatic lesions in patient IN-15/17-2 directly contradicted the MMR findings and the MSI-H result provided for the uterine primary site. Conclusion This discrepancy between primary and metastatic site results is likely due to MSI heterogeneity in these metastatic lesions than test errors. At this time, it is not recommended to use the Idylla™ MSI test for Endometrial cancers, particularly those with abdominal or ovarian metastasis. Further testing will be needed to verify if intestinal metastatic sites are consistently MSI-H. IHC should remain the prime mode of detection for possible dMMR MSI-H Endometrial tumors until an assay catered specifically towards Endometrial cancers is made.

Comprehensive characterisation of Lynch syndrome and screening strategies: a cohort study of individuals at risk from Latin American genetic registries

Systematic screening for Lynch syndrome is non-existent in most Latin American countries. Until regional guidelines are developed and implemented, patients with colorectal cancer in Latin America are being followed up according to international recommendations. The aim of this study was to characterise the current Lynch syndrome screening method in the region, and to explore the prevalence of Lynch syndrome in a large cohort of individuals at risk. We collected clinical, molecular, and genetic data from patients with high-risk colorectal cancer from 24 existing genetic registries and centres from nine countries in Latin America (Argentina, Brazil, Chile, Colombia, Ecuador, Mexico, Peru, Puerto Rico, and Uruguay). Information on clinical history, personal or family history of cancer, screening strategy, and molecular and genetic testing was collected by the participating registries through standardised reporting tools. The compiled information was curated and imported into a PostgreSQL database at the Institute of Cancer Research (Oslo, Norway). Data from 1684 patients were included. Universal screening was the most commonly reported screening strategy involved in detection of Lynch syndrome (used to detect Lynch syndrome in 1133 [67·3%] of 1684 patients). The most common method included only immunohistochemistry (336 [29·7%] of 1133), followed by a combination of immunohistochemistry plus microsatellite instability assay (168 [14·8%] of 1133), or immunohistochemistry, BRAFV600E testing, microsatellite instability assay, and MLH1 methylation analysis (133 [11·7%] of 1133). The mutation rate detected with germline mismatch repair sequencing when applying the universal screening strategy was 60 (51%) of 117 versus 87 (47%) of 182 with the clinical criteria. The median age of patients with Lynch syndrome was 40 years (IQR 34-49) and the predominant tumour location was the right colon (109 [43%] of 254), for which the most common stage at diagnosis was stage II (92 [36%] of 254). No multiple tumours were detected in 171 (67%) of 252 patients. Amsterdam criteria were met by 159 (63%) of 254 patients and revised Bethesda guidelines were met by 136 (54%) patients. Our results have implications for the design of future Lynch syndrome screening programmes, but further studies are needed to assess the cost-effectiveness and burden on genetic counselling services with expanded universal testing for Lynch syndrome. Norwegian Cancer Society (contract 194751-2017).

Abstract 1017: Impact of microsatellite instability status and immune scores on tumor after neoadjuvant chemoradiation therapy in locally advanced rectal cancer

Abstract Purpose: Neoadjuvant chemoradiation therapy (CRT) is a widely and effectively used preoperative treatment strategy in locally advanced rectal cancers. Tumors demonstrating microsatellite instability (MSI) are known to be infiltrated by higher numbers of lymphocytes and to be characterized by a more favorable prognosis as compared with tumors with intact repair systems, and MSI is the only potential biomarker to predict the outcome of immune checkpoint inhibitors. However, the relationship between MSI and response to neoadjuvant CRT and changes in MSI status after CRT in rectal cancer are not well understood. The aim of the present study was to investigate the MSI status before and after CRT and its association with CRT response in rectal cancer. Materials and methods: Formalin-fixed paraffin-embedded tissue specimens from pre-operative biopsy via sigmoidoscopy and surgical resection of the primary tumor were obtained from 84 rectal adenocarcinoma patients who underwent neoadjuvant CRT between March 2010 and May 2018. MSI assays were performed on DNA extracted from FFPE and matched normal tissues the extracted DNA was amplified by PCR with fluorescent dye-labeled primers targeting five microsatellite loci: BAT25, BAT26, D5S346, D2S123 and D17S250. Three immune biomarker scores were calculated from the 19 patients who were able to perform RNA sequencing analysis to compare the immune activities of the specimens between responder (n = 8) and non-responder (n = 11). The gene lists included in each score calculation were as follows: CXCL9, CXCL10, IDO1, IFNG, HLA-DRA, and STAT1; GZMA and PRF1; and CD247, CD2, CD3E, GZMH, NKG7, PRF1 and GZMK for the interferon-γ (IFN-γ) signature,1 the cytolytic activity2 and the immune signature,3 respectively. Results: Patients were classified, according to Mandard tumor regression grade (TRG), as responders (grade 1 or 2; 25 patients, 29.8%) and non-responders (grade 3, 4 or 5; 59 patients, 70.2%). The TRG of 84 patients was significantly different according to the pre-CRT MSI status (P = 0.024), but was not different according to the post-CRT MSI status among 60 patients who were eligible for the paired MSI test (P = 0.788). Changes in MSI status were observed in 11 of the 60 patients; MSS to MSI-low in 9 patients, MSS to MSI-high in 1 patient, and MSI-low to MSS in 1 patient. All three immune biomarker scores (the IFN-γ signature, the cytolytic activity and the immune signature) were higher in the responder group without significant difference (p = 0.536, p = 0.152, and p = 0.272, respectively). Conclusion: Our findings demonstrated that neoadjuvant CRT modulated the MSI status of rectal cancer and pre-CRT MSI status was significantly associated with response of CRT. Future studies on various immuno-profiles that can influence the CRT response using more samples are needed. Citation Format: Sung Uk Bae, Shin Kim, Sang Jun Byun, Hye Won Lee. Impact of microsatellite instability status and immune scores on tumor after neoadjuvant chemoradiation therapy in locally advanced rectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1017.

Comparison of 3 PCR-based assays for microsatellite instability detection in formalin-fixed paraffin-embedded tissues of patients with colorectal cancer.

e15042 Background: Routine assessment of microsatellite instability (MSI) status in colorectal cancer (CRC) patients can help with initial screening for Lynch syndrome, prediction of tumor prognosis and selection of patients for immunotherapy. Immunohistochemistry (IHC) and PCR-based approaches represent the current clinical laboratory standard for the evaluation of MSI status. Both Bethesda and Promega marker panels have been used widely in the world and the Bethesda panel and the six mononucleotides panel (NR21/BAT25/NR24/NR27/MONO27/BAT26) have been approved by the National Medical Products Administration (NMPA) in China. The Idylla MSI Test, a fully automated system based on real-time PCR, which incorporates 7 novel MSI loci (ACVR2A/BTBD7/DIDO1/MRE11/RYR3/SEC31A/SULF2) can perform MSI analysis within 150min using 1-3 tissue slides. The aim of this study was to evaluate the performance of the three 3 PCR-based assays for MSI detection including Idylla MSI Test, Bethesda and that six mononucleotides panel. Methods: One hundred and fifty-three formalin-fixed paraffin-embedded (FFPE) tumor tissues from patients with CRC were collected and their MSI status were analyzed using 3 PCR-based assays, respectively, i.e. the Idylla MSI, “2B3D” NCI panel and the six mononucleotides panel. All samples were previously evaluated by IHC method. Results: In the all 153 patients, IHC identified 27 cases to be MMR-deficient (dMMR), and 126 to be MMR-proficient (pMMR). Using IHC as a reference, the Idylla MSI had a higher consistency that showed overall agreement, sensitivity and specificity as 93.46%, 92.89% and 93.65%, respectively, while the corresponding values of the six mononucleotides panel were 84.31%, 92.59% and 82.54% respectively, and the “2B3D” NCI panel showed a sensitivity of 92.16%, a specificity of 88.89% and an overall concordance of 92.86%. The PCR-based approaches identified 11 more MSI-H cases than IHC while 22 dMMR cases identified by IHC were detected by all the 3 assays, 2 dMMR by 2 assays, 1 dMMR by one assay only and 1 dMMR by neither assays above. Moreover, the Idylla MSI showed advantages in ease of use, turnaround time (TAT) and small dose of tissue input. Conclusions: Our findings support that the Idylla MSI assay provides a rapid, reliable, and ease-to-use solution to MSI detection in Chinese CRC patients with high sensitivity and specificity. The inconsistent cases between IHC and PCR-based approaches need a further analysis.

Simple IHC reveals complex MMR alternations than PCR assays: Validation by LCM and next-generation sequencing.

Evaluation of the status of mismatch repair (MMR) in tumors is crucial for determining the application of immune checkpoint inhibitors (ICIs). Conventional PCR (MSI-PCR) is the gold standard for confirming the MMR status. However, it requires visual confirmation and presents difficulties in determining MMR status. Immunohistochemistry (IHC) is a simple method and can confirming MMR protein expression in the whole tumor. We aim to investigate IHC is more suitable for evaluating MMR status in the tumor. We compared MSI-PCR and IHC by testing 319 samples from 284 patients across 14 cancer types. In discordant cases, we performed laser-capture microdissection and microsatellite instability assay by next-generation sequencing (MSI-NGS). The concordance rate between IHC and MSI-PCR testing was 98.1% (313/319). Two reasons for these discrepancies were ambiguous MSI-PCR results and heterogeneous MSI status within the tumor. Among six cases (1.9%), three were judged as MSI-H by MSI-PCR but with proficient MMR by IHC. The results of MSI-NGS revealed microsatellite stable in these three cases. The remaining three cases, two of three were MSI-H and one was MSS in whole tumor in MSI-PCR. IHC showed a "mosaic" pattern containing both proficient MMR and deficient MMR portions by IHC in all three cases. We performed microdissection and MSI-PCR and found intratumoral heterogeneity of MMR status. These results indicated the advantages of IHC and performed expanded samples (n=1082) and two additional mosaic cases were identified. Our results clearly indicated that simple IHC is the best choice for determining MMR alterations in critical cases for ICIs treatment.

Microsatellite Instability Testing for Lynch Syndrome in Colorectal Adenomas

BackgroundPatients with Lynch Syndrome (LS), a hereditary condition linked to increased risk of colorectal cancer (CRC) and a variety of other cancers, carry at least one germline variant in a DNA mismatch repair protein (MMR) gene, which results in microsatellite instability (MSI). Screening for MMR/MSI status in CRCs helps identify patients with LS, which is imperative for treatment and surveillance. MSI high instability (MSI‐H) status has been detected in some colorectal adenomatous polyps (CRAs). If MSI‐H can be detected in CRAs from LS patients, this could aid in the prevention of cancer development and facilitate early surveillance. Currently, MMR/MSI testing in CRAs is not used to identify cases of LS. Here we analyze a set of CRAs to assess the utility of using MSI testing to identify patients with LS.DesignAfter completing a retrospective review of our pathology database, we selected 54 CRAs (including tubular, villous, and tubulovillous CRAs) from 32 patients with a history of LS, 21 CRAs from 14 patients with history of sporadic CRC with BRAF mutation or MLH1 promotor methylation, and 38 CRAs from 34 patients who underwent screening colonoscopy. Results from previous molecular testing confirmed germline mutations in the LS group and BRAF mutations or MLH1 promotor methylation in the sporadic CRC group. FFPE tissue was analyzed for MSI status using the Idylla MSI assay (Biocartis, Belgium). Patient demographics and clinical characteristics, pathologic features CRAs, and MSI results were analyzed.ResultsFrequency of MSI‐H CRA was significantly higher in the LS group (18.5%, [10/54]) than in the sporadic CRC (4.8%, [1/21]) and screening colonoscopy (0/38) groups. CRAs analyzed included 106 tubular CRAs, 2 villous CRAs and 5 tubulovillous CRAs. The MSI‐H CRAs in the LS group included 9 tubular CRAs and one villous CRA. One MSI‐H tubular CRA was identified in the sporadic CRC group. All CRAs from the screening colonoscopy group were MSI stable. Mean ages in years for patients in each study group were 56.9 in the LS group, 79.2 in the sporadic CRC group, and 60.6 in the screening colonoscopy group.ConclusionMSI‐H status is detectable in CRAs from patients with LS. MSI‐H CRAs are more frequently detected in patients with LS than non‐LS patients. These findings demonstrate the utility of MSI testing in CRAs. This can help to identify LS at a pre‐malignant stage, which prevents cancer development and facilitates early surveillance.

Validation of Long Mononucleotide Repeat Markers for Detection of Microsatellite Instability

Mismatch repair deficiency (dMMR) predicts response to immune checkpoint inhibitor therapy in solid tumors. Long mononucleotide repeat (LMR) markers may improve the interpretation of microsatellite instability (MSI) assays. Our cohorts included mismatch repair (MMR) proficient and dMMR colorectal cancer (CRC) samples, MMR proficient and dMMR endometrial cancer (EC) samples, dMMR prostate cancer samples, MSI-high (MSI-H) samples of other cancer types, and MSI-low (MSI-L) samples of various cancer types. MMR status was determined by immunohistochemical staining and/or MSI Analysis System Version 1.2 (V1.2). The sensitivity and specificity of the LMR MSI panel for dMMR detection were both 100% in CRC. The sensitivity values of the MSI V1.2 and LMR MSI panels in EC were 88% and 98%, respectively, and the specificity values were both 100%. The sensitivity of the LMR panel was 75% in dMMR prostate cancer detected by immunohistochemistry. The 22 samples of other cancer types that were previously classified as MSI-H were also classified as MSI-H using the LMR MSI panel. For the 12 samples that were previously classified as MSI-L, 1 sample was classified as microsatellite stable using the LMR MSI panel, 8 as MSI-L, and 3 as MSI-H. The LMR MSI panel showed high concordance to the MSI V1.2 panel in CRC and greater sensitivity in EC. The LMR MSI panel improves dMMR detection in noncolorectal cancers.

Microsatellite Instability Analysis (MSA) for Bladder Cancer: Past History and Future Directions.

Microsatellite instability (MSI), the spontaneous loss or gain of nucleotides from repetitive DNA tracts, is a diagnostic phenotype for gastrointestinal, endometrial, colorectal, and bladder cancers; yet a landscape of instability events across a wider variety of cancer types is beginning to be discovered. The epigenetic inactivation of the MLH1 gene is often associated with sporadic MSI cancers. Recent next-generation sequencing (NGS)-based analyses have comprehensively characterized MSI-positive (MSI+) cancers, and several approaches to the detection of the MSI phenotype of tumors using NGS have been developed. Bladder cancer (here we refer to transitional carcinoma of the bladder) is a major cause of morbidity and mortality in the Western world. Cystoscopy, a gold standard for the detection of bladder cancer, is invasive and sometimes carries unwanted complications, while its cost is relatively high. Urine cytology is of limited value due to its low sensitivity, particularly to low-grade tumors. Therefore, over the last two decades, several new “molecular assays” for the diagnosis of urothelial cancer have been developed. Here, we provide an update on the development of a microsatellite instability assay (MSA) and the development of MSA associated with bladder cancers, focusing on findings obtained from urine analysis from bladder cancer patients as compared with individuals without bladder cancer. In our review, based on over 18 publications with approximately 900 sample cohorts, we provide the sensitivity (87% to 90%) and specificity (94% to 98%) of MSA. We also provide a comparative analysis between MSA and other assays, as well as discussing the details of four different FDA-approved assays. We conclude that MSA is a potentially powerful test for bladder cancer detection and may improve the quality of life of bladder cancer patients.

Oncomine™ Comprehensive Assay v3 vs. Oncomine™ Comprehensive Assay Plus.

Simple SummaryThe detection of genetic alterations in cancer is important to obtain knowledge of the underlying mutational tumor composition. Knowing the mutational profile can assist oncologists on tailoring optimal personalized treatments. Moreover, obtaining additional information from a broader cancer-related gene panel, without compromising performance, can benefit both current and future patients. In this study, we assessed the performance of gene mutations identified from sequencing using the newly Oncomine™ Comprehensive Assay Plus (OCA-Plus). The assessment was performed in comparison to gene mutations identified from sequencing using the Oncomine™ Comprehensive Assay v3 (OCAv3), currently used in our routine clinical setting. Therefore, an investigation of their performance was conducted on intersecting nucleotide positions within overlapping genes covered by both the OCA-Plus and the OCAv3. We show here that there is a 91% concordance between identified pathogenic and likely pathogenic classified variants. The usage of next generation sequencing in combination with targeted gene panels has enforced a better understanding of tumor compositions. The identification of key genomic biomarkers underlying a disease are crucial for diagnosis, prognosis, treatment and therapeutic responses. The Oncomine™ Comprehensive Assay v3 (OCAv3) covers 161 cancer-associated genes and is routinely employed to support clinical decision making for a therapeutic course. An improved version, Oncomine™ Comprehensive Assay Plus (OCA-Plus), has been recently developed, covering 501 genes (144 overlapping with OCAv3) in addition to microsatellite instability (MSI) and tumor mutational burden (TMB) assays in one workflow. The validation of MSI and TMB was not addressed in the present study. However, the implementation of new assays must be validated and confirmed across multiple samples before it can be introduced into a clinical setting. Here, we report the comparison of DNA sequencing results from 50 ovarian cancer formalin-fixed, paraffin-embedded samples subjected to OCAv3 and OCA-Plus. A validation assessment of gene mutations identified using OCA-Plus was performed on the 144 overlapping genes and 313,769 intersecting nucleotide positions of the OCAv3 and the OCA-Plus. Our results showed a 91% concordance within variants classified as likely-pathogenic or pathogenic. Moreover, results showed that a region of PTEN is poorly covered by the OCA-Plus assay, hence, we implemented rescue filters for those variants. In conclusion, the OCA-Plus can reflect the mutational profile of genomic variants compared with OCAv3 of 144 overlapping genes, without compromising performance.

Frequency of microsatellite instability (MSI) in upper tract urothelial carcinoma: comparison of the Bethesda panel and the Idylla MSI assay in a consecutively collected, multi-institutional cohort

AimsUpper tract urothelial carcinoma (UTUC) is a rare malignancy with a poor prognosis which occurs sporadically or in few cases results from a genetic disorder called Lynch syndrome. Recently, examination...