Microsatellite Instability Testing Research Articles

Microsatellite Instability: From the Implementation of the Detection to a Prognostic and Predictive Role in Cancers.

Microsatellite instability (MSI) has been identified in several tumors arising from either germline or somatic aberration. The presence of MSI in cancer predicts the sensitivity to immune checkpoint inhibitors (ICIs), particularly PD1/PD-L1 inhibitors. To date, the predictive role of MSI is currently used in the selection of colorectal cancer patients for immunotherapy; moreover, the expansion of clinical trials into other cancer types may elucidate the predictive value of MSI for non-colorectal tumors. In clinical practice, several assays are used for MSI testing, including immunohistochemistry (IHC), polymerase chain reaction (PCR) and next-generation sequencing (NGS). In this review, we provide an overview of MSI in various cancer types, highlighting its potential predictive/prognostic role and the clinical trials performed. Finally, we focus on the comparison data between the different assays used to detect MSI in clinical practice.

Mismatch Repair and Microsatellite Instability Testing for Immune Checkpoint Inhibitor Therapy: Guideline From the College of American Pathologists in Collaboration With the Association for Molecular Pathology and Fight Colorectal Cancer.

The US Food and Drug Administration (FDA) approved immune checkpoint inhibitor therapy for patients with advanced solid tumors that have DNA mismatch repair defects or high levels of microsatellite instability; however, the FDA provided no guidance on which specific clinical assays should be used to determine mismatch repair status. To develop an evidence-based guideline to identify the optimal clinical laboratory test to identify defects in DNA mismatch repair in patients with solid tumor malignancies who are being considered for immune checkpoint inhibitor therapy. The College of American Pathologists convened an expert panel to perform a systematic review of the literature and develop recommendations. Using the National Academy of Medicine-endorsed Grading of Recommendations Assessment, Development and Evaluation approach, the recommendations were derived from available evidence, strength of that evidence, open comment feedback, and expert panel consensus. Mismatch repair immunohistochemistry, microsatellite instability derived from both polymerase chain reaction and next-generation sequencing, and tumor mutation burden derived from large panel next-generation sequencing were within scope. Six recommendations and 3 good practice statements were developed. More evidence and evidence of higher quality were identified for colorectal cancer and other cancers of the gastrointestinal (GI) tract than for cancers arising outside the GI tract. An optimal assay depends on cancer type. For most cancer types outside of the GI tract and the endometrium, there was insufficient published evidence to recommend a specific clinical assay. Absent published evidence, immunohistochemistry is an acceptable approach readily available in most clinical laboratories.

Mismatch repair immunohistochemistry in endometrial cancer: Intact vs. deficient is insufficient (235)

<b>Objectives:</b> Mismatch repair (MMR) and microsatellite instability (MSI) status in endometrial cancer (EC) have been used for Lynch syndrome screening. Recently the utility has expanded to molecular/risk stratification, therapeutic decision making, and clinical trial enrollment. We sought to examine discordance between clinical MMR testing with immunohistochemistry (IHC) and MSI status in a single institution cohort. <b>Methods:</b> A single-institution cohort of EC patients with IHC for <i>MLH1, PMS2, MSH2,</i> and <i>MSH6</i> was performed and scored (intact/deficient) as part of routine clinical care. Tissues appropriate for MSI typing were available for 666 cases. MSI testing using the NCI consensus MSI panel was performed for all 666 cases. Promega V1.2 MSI analysis was performed for cases with discordant IHC and MSI findings. IHC results were re-reviewed using existing archived slides for the discordant cases to evaluate for possible explanations of discordance. We defined a subclonal staining pattern as a strong normal IHC expression seen only in a portion of the tumor whereas the remaining tumor, showing abrupt complete loss of expression, and we defined a heterogeneous staining pattern where there is some patchy staining throughout the tumor rather than large areas of the tumor showing intact or loss of expression. <b>Results:</b> MMR and MSI status was determined for 666 EC cases; 165 (24.8%) had IHC defects, and 183 (27.5%) had MSI. Discordant findings occurred in 24 (3.6%) cases, with the most common discordant result (21 of 24 discordant cases) being intact MMR IHC and the presence of MSI. Upon re-review of IHC, two cases were attributed to inter-observer variability. Of the remaining 19 cases, ten demonstrated subclonal IHC staining (all <i>MLH1/PMS2</i>), seven had heterogeneous staining (six <i>MSH6</i> and one <i>MLH1/PMS2</i>), while only two had no specific IHC abnormality (Table 1). One case of a known Lynch syndrome patient had an <i>MSH6</i> frameshift mutation and heterogeneous IHC staining. Of the 21 tumors with clinically determined intact MMR status and MSI, there were four recurrences leading to EC death. In total, 11.4% of tumors with MSI were not identified with IHC interpretation alone. All three MMR IHC deficient cases without MSI demonstrated protein loss of <i>MLH1/PMS2</i> with evidence of <i>MLH1</i> promoter methylation. <b>Conclusions:</b> Clinical MMR IHC and MSI findings were discordant in 3.6% of tumors in our cohort, consistent with previous reports; however, 11.4% of tumors with MSI were not identified with clinical IHC alone in our cohort. The majority of tumors with normal IHC interpretation and MSI were explained by subclonal or heterogeneous staining patterns of <i>MLH1/PMS2</i>. Given the importance of MMR status in Lynch syndrome screening, clinical trial inclusion, and therapeutic decision making, heterogeneous and subclonal staining patterns should prompt additional molecular testing, and MSI testing should be considered for recurrent EC patients.

An Unexpected Esophageal Lesion in a Patient With Muir-Torre Syndrome

Question: In a 52-year-old female patient with a history of sebaceous gland neoplasms of the skin (carcinoma resected in 2017 and several subsequent adenomas) and positive family history for early-onset colorectal cancer (father at the age of 39 years), Muir-Torre syndrome was suspected. This is a rare variant of Lynch syndrome (LS; approximately 10% of individuals with LS) clinically defined by the typical LS tumor spectrum (colorectal cancer as well as extracolonic tumors, eg, endomtetrial/ovarian cancer, urinary tract cancer) and additional increased risk for characteristic skin neoplasms (especially sebaceous gland neoplasms and keratoacanthoma). DNA mismatch-repair (MMR) deficiency resulting in an accumulation of somatic mutations throughout the genome, also affecting genes involved in carcinogenesis, causes LS. Genetic testing identified a pathogenic germline mutation in MSH2 (mutS homolog 2) on chromosome 2p21 and confirmed the diagnosis in 2018. Compliant with current international guidelines, the patient was included in a specific tumor-screening program. In dermatologic follow-up examinations, basal cell carcinoma and squamous cell carcinoma of the skin were detected. By annual surveillance colonoscopies up to the present, 4 colorectal adenomas with sizes from 2 to 15 mm were resected. On an individual basis, and not covered by guidelines, we performed yearly surveillance esophagogastroduodenoscopy (EGD). Interestingly, at the age of 57 years, a small irregular and slightly elevated lesion (8 mm) with small central erosion (Figure A, arrow) was observed (Paris 0-IIa) in the mid-esophagus (25 cm from incisors). The central part of the lesion stained negative during chromoendoscopy with Lugol’s iodine (1%) (Figure B, arrow) and showed an irregular surface pattern with virtual chromoendoscopy (NBI mode; narrow band imaging) (Figure C). Therefore, malignancy was suspected, and the lesion was removed en bloc by means of rubber-band ligation assisted endoscopic mucosal resection (Figures D and E). Of note, the patient was never a smoker and only occasionally consumed alcohol. Endoscopies were performed with Olympus GIF-H190 endoscopes (Olympus, Hamburg, Germany). Which diagnosis do you suspect and is this lesion LS associated? Look on page 189 for the answer and see the Gastroenterology website (www.gastrojournal.org) for more information on submitting to Gastro Curbside Consult. Histopathologic and immunohistochemical examination of the resected tumor revealed poorly differentiated invasive squamous cell carcinoma (pT1a, Pn0, V0, R0, G3) (Figure F, hematoxylin and eosin [HE] stain, ×40) with focal invasion of the lamina muscularis mucosae (Figure G, HE, ×200, arrow). Molecular microsatellite instability testing discovered instability in 4/5 markers (BAT-26, NR-21, BAT-25, and NR-24; NR-27 was stable). When an LS-associated tumor was suspected, MMR protein immunohistochemistry showed a loss of MSH2 expression within the neoplastic cells (Figure H, ×200, red arrow) while surrounding tissue stained positive for MSH2 (Figure H, black arrow) and MLH1 expression was unaffected in neoplastic (Figure I, ×200, red arrow) and nonneoplastic (Figure I, black arrow) cells. Computed tomography and endoscopic ultrasound did not find any distant or lymph-node metastases, and there was no residual tumor in a follow-up endoscopy. Because of poor tumor differentiation and invasion of the lamina muscularis mucosae (m3), additive radiotherapy (50.4 Gy/59.92 Gy boost in 28 fractions) was recommended after discussion by the tumor board. This is the first description of an LS/Muir-Torre syndrome–associated squamous cell carcinoma of the esophagus in a patient without typical risk factors for this tumor entity. Moreover, we show in-depth molecular characterization to prove LS association. Only rarely have adenocarcinomas of the esophagus been reported1Sweetser S. Chandan V.S. Baron T.H. Dysphagia in Lynch syndrome.Gastroenterology. 2013; 145: 1167-1168Abstract Full Text Full Text PDF Scopus (6) Google Scholar in LS. Until recently, squamous cell carcinoma of the skin was not counted as typical skin manifestation of Muir-Torre syndrome, but an association has been demonstrated.2Ykema B.L.M. Adan F. Crijns M.B. et al.Cutaneous squamous cell carcinoma is associated with Lynch syndrome: widening the spectrum of Lynch syndrome-associated tumours.Br J Dermatol. 2021; 185: 462-463Crossref PubMed Scopus (7) Google Scholar Of note, our patient also suffered from squamous cell carcinoma of the skin. In conclusion, clinicians should carefully consider esophageal neoplasms as a rare manifestation of LS. Despite limited data supporting EGD surveillance in LS and in contrast to current guidelines, upper endoscopy could be easily performed along with routine colonoscopy in an effort to prevent morbidity and mortality from upper GI manifestations of LS (early-stage cancer observed in about 1.5% of asymptomatic patients3Farha N. Hrabe J. Sleiman J. Beard J. et al.Clinically actionable findings on surveillance EGD in asymptomatic patients with Lynch syndrome.Gastrointest Endosc. 2022; 95: 105-114Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar).

Critical aspects of microsatellite instability testing in endometrial cancer: a comparison study

The identification of mismatch repair deficient (dMMR) and microsatellite unstable (MSI) endometrial cancers (ECs) is important in screening, diagnosis, and therapeutic stratification of patients. We compared the diagnostic performance of 4 MSI molecular tests based on fragment length assay in capillary electrophoresis (OncoMate™ MSI assay, Promega) and in microcapillary electrophoresis (TapeStation 4200, Agilent); with high-resolution melting (HRM) analysis approaches (Idylla™ MSI Test, Biocartis; EasyPGX® ready MSI, Diatech Pharmacogenetics) on a series of 56ECs, which was well characterized for MMR status with immunohistochemical approach (IHC, nonmolecular reference test). The concordance of fluorescence capillary electrophoresis with IHC (AUC 0.98) was higher respect to the other molecular methodologies. Otherwise, HRM approaches and microcapillary electrophoresis platform failed to detect MSI-ECs showing minimal microsatellite shifts. In conclusion, in colorectal site, several technologies are eligible for MSI test, whereas in ECs, MSI test should be based on fluorescent capillary electrophoresis as it identifies a higher proportion of cases that could be misdiagnosed with other strategies.

Lynch syndrome pre-screening and comprehensive characterization in a multi-center large cohort of Chinese patients with colorectal cancer.

Objective:Lynch syndrome (LS) pre-screening methods remain under-investigated in colorectal cancers (CRCs) in Asia. Here, we aimed to systematically investigate LS pre-screening and comprehensively characterize LS CRCs.Methods:Microsatellite instability (MSI) and germline variants of DNA mismatch repair (MMR) genes were examined in 406 deficient MMR (dMMR) and 250 proficient MMR CRCs. The genetic differences between LS and sporadic CRCs were studied with whole exome sequencing analysis.Results:The incidence of dMMR in Chinese patients with CRCs was 13.8%. Consistency analysis between MMR immunohistochemistry (IHC) and MSI testing showed the kappa value was 0.758. With next-generation sequencing (NGS), germline variants were detected in 154 CRCs. Finally, 88 patients with CRC were identified as having LS by Sanger sequencing. Among them, we discovered 21 previously unreported pathogenic germline variants of MMR genes. Chinese patients with LS, compared with sporadic CRCs, tended to be early-onset, right-sided, early-stage and mucinous. Overall, the performance of MMR IHC and MSI testing for LS pre-screening was comparable: the area under the ROC curve for dMMR, MSI-H, and MSI-H/L was 0.725, 0.750, and 0.745, respectively. dMMR_MSI-H LS and sporadic CRCs showed substantial differences in somatic genetic characteristics, including different variant frequencies of APC, CREBBP, and KRAS, as well as different enriched pathways of VEGF, Notch, TGFβR, mTOR, ErbB, and Rac protein signal transduction.Conclusions:MMR IHC and MSI testing were effective methods for LS pre-screening. The revealed clinical and somatic genetic characteristics in LS CRCs may have the potential to improve the performance of LS pre-screening in combination with dMMR/MSI.

MO38-6 A retrospective study on the success rate of microsatellite instability tests

Currently, there are two methods clinically available for evaluating MSI. One is MSI-IVD Kit (Promega panel) by FALCO, and the other is FoundationOne CDx (F1CDx) by Foundation Medicine (FMI). However, some cases fail in gaining valid results, mainly because of small amounts or poor quality of sample DNA.

Microsatellite instability in patients with hepato-biliary-pancreatic malignancies in clinical practice (KHBO 1903).

This study aimed to determine the prevalence of microsatellite instability (MSI)-high status in hepato-biliary-pancreatic malignancies in clinical practice and the clinical characteristics of and therapeutic results of pembrolizumab on patients with MSI-high cancers. The subjects were 283 patients who had undergone MSI tests for unresectable, metastatic hepato-biliary-pancreatic malignancies at seven hospitals. The tests were polymerase chain reaction fragment analyses using the microsatellite markers consisting of BAT25, BAT26, NR21, NR24, and MONO27. Formalin-fixed, paraffin-embedded blocks, prepared according to the guidelines of the Japan Society of Pathology were used within 4years after sampling. There were 13 patients with cancers high in MSI, including eight patients receiving pembrolizumab treatment. The clinical characteristics of these patients and therapeutic outcomes of their pembrolizumab treatment were investigated. MSI-high was detected in 13 (4.6%) of the 283 patients with hepato-biliary-pancreatic malignancies. None of these 13 patients had been diagnosed with Lynch syndrome. Of the Eight patients receiving pembrolizumab, a complete response was observed in three patients, a partial response in one patient, and stable disease in three patients. The objective response rate was 50% and the disease control rate was 87.5%. MSI-high was detected in 4.6% of patients with hepato-biliary-pancreatic malignancies. There was a 50% objective response rate to pembrolizumab treatment for MSI-high cancers. The evaluation of MSI status by the current method using appropriately prepared tissue samples was to be a reliable and accurate approach to both the determination of MSI status and estimation of the effectiveness of pembrolizumab.

A case of strongly suspected Lynch syndrome with colorectal neuroendocrine carcinoma

BackgroundNeuroendocrine cell carcinomas (NEC) of the colon and rectum are uncommon, representing ~ 0.1% of all colorectal carcinomas. They are associated with a much worse prognosis compared to adenocarcinoma of the colon and rectum, as death occurs in approximately half of all patients within 1 year. Lynch syndrome (LS) is the most common cause of inherited colorectal cancer, accounting for 2–4% of newly diagnosed colorectal cancer cases. This case is extremely rare which was strongly suspected LS as the background, and NEC as the histological type of colorectal cancer.Case presentationThe patient was a 44-year-old man presenting with vomiting as the main complaint. He had undergone ileocecal resection for cecal cancer at age 29. The diagnosis was obstructive descending colorectal cancer, and colonoscopy revealed tumors in the rectum and sigmoid colon in addition. Due to multiple occurrences of colorectal cancer and its prevalence in the patient’s family, LS was suspected. The operation which was a subtotal proctocolectomy was performed. Pathological analysis revealed complete curative resection and the descending colon cancer of the obstructed portion was at the most advanced pathological Stage IIIC in UICC TNM classification, and the tissue type was a NEC. The Ki-67 index was 70%. The results of the microsatellite instability (MSI) test showed high-frequency MSI. The BRAF V600E variant was negative. The immunoexpression of MLH1 was positive, MSH2 was negative, PMS2 was positive, and MSH6 was negative.ConclusionsExtended surgery is recommended for incipient colorectal cancer in LS cases in order to reliably reduce the risk of developing metachronous colorectal cancer. The survival outcome of surgery alone on digestive tract NECs, even locoregional lesions that are completely resection, is extremely poor. It is currently unclear if digestive tract NECs develop more readily in patients with LS. The accumulation of additional cases is necessary.

Abstract 1230: Increasing equity in MSI and TMB biomarker testing with diverse datasets and personalized informatics

Abstract Introduction: NGS-based tests that include biomarkers such as microsatellite instability (MSI) and tumor mutational burden (TMB) increase patient access to immune checkpoint inhibitors and improve the personalization of cancer care. Tumor-only NGS tests of biomarkers typically rely on germline reference sets in order to subtract germline variants from biomarker scores. Many germline reference sets lack sufficient population diversity to ensure consistent test quality for all patients. Informatic strategies such as somatic/germline zygosity prediction are often necessary to reduce bias introduced from germline variation and population-specific artefacts and mitigate the risk of inflating TMB and MSI scores. Methods: We sequenced germline samples from a curated set of research-consented patients representing diverse ancestries on a hybrid capture panel (N=1047 patients, from 10 populations). The tested groups included: African-American=416, Hispanic=162, Ashkenazi=130, Asian=127, Caucasian=85, Middle Eastern=44, Native American=27, Pacific Islander=25, Indian=25, and African=6. We confirmed that both rare population-specific alleles and population-specific technical artefacts contributed to spurious results from biomarker score inflation in underrepresented populations. To mitigate bias and universally improve scores in both MSI and TMB, we combined 1) an ancestry-diverse panel of germline samples assembled from the targeted panel, 2) population-specific public database filtering, 3) informatic germline variant subtraction, and 4) a heuristic for population-neutral target space selection. Results: Inter-population comparisons of MSI scores (Kruskal-Wallis p of 4.6*10^-69) and TMB scores (Kruskal-Wallis p of 3.7*10^-6) differed significantly in mutation signature errors prior to the mitigation work described in the methods. Application of the germline knowledge base filters and population-neutral target selection reduced the overall error rate (51% for TMB and 16% for MSI), and reduced inter-population score differences. TMB error rates reduced most for African, Pacific Islander, and African-American patients (100%, 71%, and 58%, respectively). Conversely, MSI error rates reduced most for Caucasian, Hispanic, and Asian patients (24%, 21%, and 20%, respectively). Conclusion: The incorporation of a diverse population set in this study empowered development of a more equitable bioinformatic approach, and significantly improved TMB and MSI determination across all populations. Qualitative differences in the sources of error per population and per biomarker type were associated with differing responses to each mitigation strategy. ​​These findings underscore the technical and clinical value of incorporating diverse ancestry reference sets and identifying non-biased assay targets to optimize personalized therapy selection. Citation Format: Elizabeth R. Starks, Victoria Carlton, Veena Rajaraman, Katya Kosheleva, Tamsen Dunn, Nick Kamps-Hughes, Laurie Gay, Meaghan Russell, Sarah Albritton, John J. Vincent, Nhu Ngo. Increasing equity in MSI and TMB biomarker testing with diverse datasets and personalized informatics [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 1230.

Abstract 5012: Deep Gaussian process with uncertainty estimation Improves microsatellite instability prediction based on whole slide image: A retrospective multicenter and multiethnic cohort study

Abstract Background: Microsatellite Instability (MSI) is a prognostic and predictive biomarker which can guide treatments including immunotherapy for patients with gastrointestinal cancer. Universal MSI testing would benefit to patients to receive a better therapy, but many patients remain untested. To deliver broadly accessible MSI testing, deep learning models have demonstrated their feasibility for MSI prediction using H&amp;E-stained whole-slide images (WSIs). However, to be deployed into clinical routine care, prediction models should be validated in datasets from multicenter and multiethnic groups. In addition, these models should provide uncertainty of prediction to help clinicians to make informed decisions. Method: We develop a MSI prediction model using WSIs based on Deep Gaussian process (DGP), a Bayesian model being able to model the uncertainty in prediction. We implement a DGP model in the transfer learning, where a WPI is decomposed into multiple non-overlap image patches which are converted to feature vectors by a pretrained convolutional neural network (CNN). Then, the DGP model makes a prediction for an image by averaging score function values at all the feature vectors in the image in weakly supervised learning (WSL). To test our method, we collected H&amp;E stained colorectal/stomach cancer slides (n=1,619) from multi-institutions and multiethnic groups, including TCGA Colorectal (CRC; n=351) and Stomach Adenocarcinoma (STAD; n=174) and six datasets from tertiary hospitals in Korea (i.e., Yonsei-1 (n=174), Yonsei-1-remade (n=146), Yonsei-2 (n=95), St. Mary-1 (n=48), St. Mary-2 (n=50), and Yonsei-CLASSIC trial (n=581)). We also compare our method to multiple deep learning methods, including Densenet, Resnet, Shufflenet, Googlenet, Squeezenet and two WSL models, MIL (Campanell et al., 2019) and CLAM (Lu et al., 2021). Result: In each cancer type, we train each model using TCGA dataset as discovery cohort and tested on the rest of datasets as validation cohort. We use the area under of roc curve (AUC) for the evaluation metric. For the CRC cohorts, our DGP method achieves overall best performance with 0.81 (Yonsei-1), 0.82 (Yonsei-1-remade), 0.89 (Yonsei-2), 0.85 (St. Mary-1) 0.75 (St. Mary-2) AUCs compared to the state-of-the art methods. For the STAD cohorts, our method also achieves overall best performance with 0.74 AUC (Yonsei-classic). In addition, incorporating the uncertainty in prediction measured by our method improves the model’s performance to predict MSI. Specifically, removing cases with prediction results with high uncertainty, which could lead false positive prediction, significantly improves MSI prediction performance. Taken together, these results demonstrate the robustness and generalisability of the DGP model for MSI prediction across multicenter and multiethnic datasets. Citation Format: Sunho Park, Hongming Xu, Sung Hak Lee, Jeonghyun Kang, Tae Hyun Hwang. Deep Gaussian process with uncertainty estimation Improves microsatellite instability prediction based on whole slide image: A retrospective multicenter and multiethnic cohort study [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 5012.

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.

Intratumoural expression of IL-6/STAT3, IL-17 and FOXP3 immune cells in the immunosuppressive tumour microenvironment of colorectal cancer Immune cells-positive for IL-6, STAT3, IL-17 and FOXP3 and colorectal cancer development

Immune cells in the tumour microenvironment (TME) interact with each other and with tumour cells to promote tumour development. IL-6/STAT3 pathway induces and maintains mainly pro-tumour TME. Macrophages and lymphocytes are positive for IL-6, STAT3 and IL-17, while FoxP3 cells are mainly regulatory cells. IL-17+ and FoxP3+ immune cells have impact on gut inflammation and tumourigenesis. The aim of this study was to determine IL-6+, STAT3+, IL-17+ and FoxP3+ immune cells in colorectal cancer (CRC) TME and explore their association with clinicopathological parameters and mismatch repair (MMR) status. The investigated samples were collected from 104 CRC patients. Immunohistochemistry for the aforementioned markers and microsatellite instability (MSI) markers was performed. MSI testing was used. The investigated immune cells were significantly more in the invasive front (IF) as compared to tumour stroma (TS). IL-6+ and STAT3+ immune cells were more in the early tumour stages as compared to advanced stages. IL-17+ and IL-6+ immune cells were more in well and moderately differentiated cancers. IL-6+, STAT3+ and IL-17+ immune cells prevailed in the TME in microsatellite stable patients and only FoxP3+ cells were fewer there. Higher numbers of STAT3+ cells correlated with longer survival. These results support the suggestion that the transition of normal colonic mucosa to CRC is marked by a shift of Th programme, leading to accumulation of Th17 cells and Tregs that sustain tumour cell growth through the IL-6/STAT3 signalling pathway.

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.

Microsatellite Status Detection in Gastrointestinal Cancers: PCR/NGS Is Mandatory in Negative/Patchy MMR Immunohistochemistry.

Simple SummaryMicrosatellite instability (MSI) detection has a high impact on eligibility for immune checkpoint inhibitors in gastrointestinal cancers. The appropriate detection of MSI represents the major critical issue in clinical practice, thus a better understanding of the limits related to MSI testing is needed to avoid misinterpretations. This study addresses the discordance between IHC and PCR/NGS testing in a large retrospective series of colorectal and gastric cancers in order to improve diagnosis. Our findings show a disagreement between negative/patchy expression IHC and PCR/NGS results, suggesting that molecular testing is mandatory in this subset of tumors.Background: Microsatellite instability (MSI) is a predictive biomarker for immune checkpoint inhibitors. The main goal was to investigate the discordance between IHC and PCR/NGS for MSI testing in gastrointestinal cancers. Methods: Two series were analyzed through IHC for mismatch-repair-system proteins (MMRP) and PCR, with one series of 444 colorectal cancers (CRC) and the other of 176 gastric cancers (GC). All cases with discordant results between IHC and PCR were analyzed by NGS. IHC staining was evaluated as follows: proficient MMR (pMMR), with all MMR positive; deficient MMR (dMMR), with the loss of one heterodimer; and cases with the loss/patchy expression of one MMR (lo-paMMR). Cases with instability in at least two markers by PCR were MSI-high (MSI-H) and with instability in one marker, MSI-low (MSI-L). Cases without instability were evaluated as microsatellite-stable (MSS). Results: In the CRC cohort, 15 out of 444 cases were dMMR and 46 lo-paMMR. Among the 15 dMMR, 13 were MSI-H and 2 MSS. Among the 46 lo-paMMR, 13 were MSI-H and 33 were MSS. In the GC cohort, 13 out of 176 cases were dMMR and 6 cases lo-paMMR. Among the 13 dMMR, 12 were MSI-H and only 1 was MSS. All six lo-paMMR cases were MSS. All NGS results were in agreement with PCR. Conclusions: In clinical practice, MMR–IHC could be used as a screening test and additional molecular analysis is mandatory exclusively in cases carrying loss/patchy MMR-IHC.

The clinical features and management of Lynch syndrome-associated ovarian cancer.

Lynch syndrome (LS) is one of the most common hereditary cancer syndromes, characterized by mutations in mismatch repair genes and autosomal dominant inheritance. Women with LS have an additional increased risk of gynecologic malignancies, including endometrial cancer (EC) and ovarian cancer (OC). Compared with EC, OC is relatively under investigation. This review thoroughly summarizes the current clinical evidence of surveillance, screening, and prevention strategies, and describes the molecular and clinical characteristics of LS-associated OC. An electronic search from databases of PubMed and Google Scholar was carried out using key words pertaining to Lynch syndrome and ovarian cancer. A review of the literatures including review articles, experimental, and observational studies published between 2000 and 2021 was conducted. The lifetime risk of OC in women with LS of MLH1, MSH2, and MSH6 mutations is approximately 7%, with the median age at onset being 46 years, 10-15 years earlier than that in sporadic cases. Histologically, LS-associated OCs are primarily endometrioid (40%), high-grade (25%), and low-grade (11%) serous, or clear cell (6%) in nature. Eighty-five percent of patients are diagnosed at an early stage, presenting with a good prognosis at 84% 5-year survival. Optimal screening strategies for OC in LS are controversial; combined screening of patients' clinical and family history, immunohistochemical analysis, and microsatellite instability testing for mismatch repair deficiency have been proven efficient. The clinical features were different between ovarian cancer in Lynch syndrome and sporadic cases. More research are needed for a greater understanding of the prevention and medical treatment of LS-associated OC.

Comparative molecular genomic analyses of a spontaneous rhesus macaque model of mismatch repair-deficient colorectal cancer.

Colorectal cancer (CRC) remains the third most common cancer in the US with 15% of cases displaying Microsatellite Instability (MSI) secondary to Lynch Syndrome (LS) or somatic hypermethylation of the MLH1 promoter. A cohort of rhesus macaques from our institution developed spontaneous mismatch repair deficient (MMRd) CRC with a notable fraction harboring a pathogenic germline mutation in MLH1 (c.1029C<G, p.Tyr343Ter). Our study aimed to provide a detailed molecular characterization of rhesus CRC for cross-comparison with human MMRd CRC. We performed PCR-based MSI testing (n = 41), transcriptomics analysis (n = 35), reduced-representation bisulfite sequencing (RRBS) (n = 28), and MLH1 DNA methylation (n = 10) using next-generation sequencing (NGS) of rhesus CRC. Systems biology tools were used to perform gene set enrichment analysis (GSEA) for pathway discovery, consensus molecular subtyping (CMS), and somatic mutation profiling. Overall, the majority of rhesus tumors displayed high levels of MSI (MSI-H) and differential gene expression profiles that were consistent with known deregulated pathways in human CRC. DNA methylation analysis exposed differentially methylated patterns among MSI-H, MSI-L (MSI-low)/MSS (MS-stable) and LS tumors with MLH1 predominantly inactivated among sporadic MSI-H CRCs. The findings from this study support the use of rhesus macaques as an alternative animal model to mice to study carcinogenesis, develop immunotherapies and vaccines, and implement chemoprevention approaches relevant to sporadic MSI-H and LS CRC in humans.

Initial screening by immunohistochemistry is effective in universal screening for Lynch syndrome in endometrial cancer patients: a prospective observational study.

Few prospective reports of universal screening for Lynch syndrome exist for patients with endometrial cancer. In this study, we performed immunohistochemical staining for DNA mismatch repair-related genes (MLH1, MSH2, MSH6 and PMS2), to determine the extent to which Lynch syndrome can be diagnosed in endometrial cancer patients through universal screening. We recruited 116 consecutive patients assumed to have uterine corpus malignancy from October 2019 to February 2021 in a prospective observational study. We performed immunohistochemical for mismatch repair-related proteins on samples from 100 patients who had surgicopathologically confirmed diagnoses of endometrial cancer. Samples with missing immunohistochemical results for any of the proteins had subsequent universal screening tests for microsatellite instability, DNA methylation of the MLH1 promoter region and mismatch repair genetics. We identified 19 (19.0%) patients with lost results for any of the proteins. All 19 patient samples had subsequent screening tests. We identified the microsatellite instability-high phenotype in 84.2% (16/19) of these patients and MLH1 methylation in 57.9% (11/19). Mismatch repair genetic testing detected two pathological variants, in MSH2 and MSH6, which indicated that the prevalence of Lynch syndrome was 2.0% in our cohort. Two cases of unclassified variant (MSH6) and one case of benign variant (PMS2) were also detected. Initial screening by immunohistochemical is an effective method in universal screening for Lynch syndrome in endometrial cancer patients.

Microsatellite instability in colon cancer: A single center experience from North India.

Due to the defects of mismatch repair (MMR) genes MLH1, PMS2, MSH2, and MSH6, the mutations which occur in microsatellite region are not repaired during deoxyribonucleic acid synthesis, leading to microsatellite instability (MSI). MSI is one of the major molecular changes that occur in colorectal carcinoma (CRC). Studies have shown that MMR deficient CRC has different clinicopathological characteristics and a better stage adjusted survival when compared to microsatellite stable tumors. We have retrospectively analyzed the cases of colon cancers treated in our institute for 3 years from 2017 to 2019. Most of the patients underwent surgery and received adjuvant chemotherapy. MSI testing was done in surgical specimen with immunohistochemistry. The clinical details of the patients were tabulated in Microsoft Excel, and statistical analysis was done using IBM SPSS Statistics for Windows, version 21 (IBM Corp., Armonk, NY, USA). A total of 52 patients who were treated in our institution from 2017 to 2019 were analyzed. The mean age was 46.8 ± 13.5 (19-72) years. The male-to-female ratio was 8:5. No significant association in patient demographics and clinicopathological parameters was observed between MSI stable and unstable disease. However, lymphovascular invasion showed a significantly higher trend in MSI unstable patients (P = 0.052). The median progression-free survival (PFS) of the entire cohort was 27.8 months (95% confidence interval = 22.7-32.9) and the median overall survival (OS) is not reached. The median PFS is 21.3 months in MSI stable patients whereas it is not reached in MSI unstable patients (P = 0.049). The median OS is 27.1 months in MSI stable patients, but it is not reached in MSI unstable patients and the difference shows a trend towards statistical significance (P = 0.061). MSI unstable tumors were found to have higher PFS and higher OS in our study. It needs prospective validation in larger studies in Indian scenario.