Microsatellite Instability Testing Research Articles

Validation of MSIntuit as an AI-based pre-screening tool for MSI detection from colorectal cancer histology slides

Mismatch Repair Deficiency (dMMR)/Microsatellite Instability (MSI) is a key biomarker in colorectal cancer (CRC). Universal screening of CRC patients for MSI status is now recommended, but contributes to increased workload for pathologists and delayed therapeutic decisions. Deep learning has the potential to ease dMMR/MSI testing and accelerate oncologist decision making in clinical practice, yet no comprehensive validation of a clinically approved tool has been conducted. We developed MSIntuit, a clinically approved artificial intelligence (AI) based pre-screening tool for MSI detection from haematoxylin-eosin (H&E) stained slides. After training on samples from The Cancer Genome Atlas (TCGA), a blind validation is performed on an independent dataset of 600 consecutive CRC patients. Inter-scanner reliability is studied by digitising each slide using two different scanners. MSIntuit yields a sensitivity of 0.96–0.98, a specificity of 0.47-0.46, and an excellent inter-scanner agreement (Cohen’s κ: 0.82). By reaching high sensitivity comparable to gold standard methods while ruling out almost half of the non-MSI population, we show that MSIntuit can effectively serve as a pre-screening tool to alleviate MSI testing burden in clinical practice.

GLIOMA-01 GENETIC INVESTIGATIONS AND COUNSELING IN FAMILIAL MSS TURCOT SYNDROME ASSOCIATED TO INFERTILITY AND CHILD MORTALITY

Abstract The aim of this study was to report MSI genetic findings in a three-generation Tunisian pedigree of Turcot syndrome 1 (TS1), characterized by an uncommon association with infertility and child mortality. Microsatellite instability testing and genetic counseling were offered to affected members and their young relatives, to provide optimal clinical and preventive care. The index case of our Tunisian pedigree was a 38 year-old female, in whom histologic assessment of endoscopic specimens through a colorectal adenoma resection during screening colonoscopy, showed a tubular adenoma of the lower rectum with low-grade intraepithelial neoplasia. The screening procedure was conducted because her two brothers, 58 and 60 years old, harbored respectively glioblastoma multiforme and colon adenocarcinoma. The first brother exhibited an intracranial hypertension syndrome and a left hemiplegia leading to the diagnosis of a brain tumor. Whereas the second brother manifested diarrhea and intestinal colic that were investigated by colonoscopy. Histologic assessment of endoscopic specimens through a right preangular budding formation showed a well-differentiated liberkhunian adenocarcinoma that was treated by a right hemicolectomy. Genetic assessment at the germinal and somatic levels was conducted for the three affected patients using conventional microsatellite instability (MSI) testing methods. bMolecular analysis determined MSI status of the tumors and classified them into stable types (MSS) without any mutation or aberrant expression of MLH1, MSH2, MSH6, and PMS2 genes. Familial history revealed, at the first generation, a consanguineous couple who lost several children at a young age. This couple gave birth to eight children, three of whom were infertile, three affected by MMS TS1 and two healthy. The third generation consists of 15 children, so far, without any tumors. Genetic counselling for TS remains difficult because of the rarity of the condition, its shifting definition over the years, and its genetic and phenotypic heterogeneity.

Testing for deficient mismatch repair and microsatellite instability : Afocused update.

Testing to detect mismatch repair deficiency (dMMR) and high-grade microsatellite instability (MSI-H) has become an integral part of the routine diagnostic workup for colorectal cancer (CRC). While MSI was initially considered to be apossible indicator of ahereditary disposition to cancer (Lynch syndrome, LS), today the prediction of the therapy response to immune checkpoint inhibitors (ICI) is in the foreground. Corresponding recommendations and testing algorithms are available for use in primary diagnosis (reviewed in: Rüschoff etal. 2021).Given the increasing importance for routine use and the expanding indication spectrum of ICI therapies for non-CRCs, such as endometrial, small intestinal, gastric, and biliary tract cancers, an updated review of dMMR/MSI testing is presented. The focus is on the challenges in the assessment of immunohistochemical stains and the value of PCR-based procedures, considering the expanded ICI indication spectrum. Apractice-oriented flowchart for everyday diagnostic decision-making is provided that considers new data on the frequency and type of discordances between MMR-IHC and MSI-PCR findings, and the possible role of Next Generation Sequencing in clarifying them. Reference is made to the significance of systematic quality assurance measures (e.g., QuIP MSI portal and multicenter proficiency testing), including regular continued training and education.

Concordance between microsatellite instability testing and immunohistochemistry for mismatch repair proteins and efficient screening of mismatch repair deficient gastric cancer.

Microsatellite instability (MSI) testing, an established technique that has gained prominence in recent years for its predictive potential regarding the efficacy of immune checkpoint inhibitors, is used to evaluate DNA mismatch repair (MMR) deficiency (dMMR). As with other methods, the immunohistochemistry (IHC) of MMR proteins is also widely adopted. Although both techniques have been validated, their concordance rate remains unknown, particularly regarding non-colorectal cancer. Therefore, the aim of the present study was to explore and elucidate their concordance in the context of gastric cancer (GC). A total of 489 surgically resected primary GC tissues were analyzed to compare the results yielded by the MSI test and those from IHC. Of 488 GC cases, 56 (11.5%) exhibited a loss of MMR proteins, whereas 52 (10.7%) were classified as high-frequency MSI (MSI-H). The concordance rate between these two categories was 99.2%. The microsatellite markers BAT26 and MONO27 demonstrated 100% sensitivity and 99.5% specificity in detecting dMMR GC. In addition, histopathological analysis revealed that MSI-H was more prevalent in GCs exhibiting coexisting Tub2 and Por1 subtypes. However, four discordant cases were observed. All four cases were microsatellite-stable cases but exhibited loss of MLH1 protein expression with hypermethylation of the MLH1 promoter. The results of the present study highlight that while there is a strong concordance between MSI and IHC testing results for determining dMMR status, IHC testing may offer superior efficacy in detecting dMMR.

Creation of a Quality Payment Program Measure for Mismatch Repair or Microsatellite Instability Biomarker Testing Status in Colorectal, Endometrial, Gastroesophageal, or Small Bowel Carcinoma.

Quality measures that are supported by evidence-based clinical practice guidelines are preferred for assessing the quality of pathologists' practices. Careful testing of a measure ensures that scores obtained by that measure reflect the quality of a pathologist's practice. To specify a new quality measure and to demonstrate through testing that it is suitable for measuring pathologists' appropriate incorporation of information regarding microsatellite instability (MSI) and/or mismatch repair (MMR) status in pathology reports for colorectal, endometrial, gastroesophageal, and small bowel carcinoma. The College of American Pathologists collaborated with the American Gastroenterological Association to specify and test the new measure. Face validity testing was used to investigate the validity of the measure. Feasibility testing was conducted to understand if data elements required by the measure specification were readily accessible. Signal-to-noise analysis was used to characterize the measure's reliability. Guideline recommendations for MSI and/or MMR testing supported specifications for the measure. Face validity testing indicated that the measure could distinguish the quality of care provided. Data elements required by the measure specification were found to be accessible, which supported the measure's feasibility. Reliability testing showed that differences in measure score were attributable to real differences in performance rather than random variation in scoring. The Mismatch Repair or Microsatellite Instability Biomarker Testing Status in Colorectal Carcinoma, Endometrial, Gastroesophageal, or Small Bowel Carcinoma measure was appropriately specified, and testing demonstrated that it is well suited for characterizing the quality of pathologists' communication of MMR and/or MSI status.

Oncologic relevance of genetic alterations in sporadic synchronous and solitary colorectal cancer: a retrospective multicenter study

BackgroundOncologic impact of genetic alteration across synchronous colorectal cancer (CRC) still remains unclear. This study aimed to compare the oncologic relevance according to genetic alteration between synchronous and solitary CRC with performing systematic review.MethodsMulticenter retrospective analysis was performed for CRC patients with curative resection. Genetic profiling was consisted of microsatellite instability (MSI) testing, RAS (K-ras, and N-ras), and BRAF (v-Raf murine sarcoma viral oncogene homolog B1) V600E mutation. Multivariate analyses were conducted using logistic regression for synchronicity, and Cox proportional hazard model with stage-adjusting for overall survival (OS) and disease-free survival (DFS).ResultsIt was identified synchronous (n = 36) and solitary (n = 579) CRC with similar base line characteristics. RAS mutation was associated to synchronous CRC with no relations of MSI and BRAF. During median follow up of 77.8 month, Kaplan–meier curves showed significant differences according to MSI-high for OS, and in RAS, and BRAF mutation for DFS, respectively. In multivariable analyses, RAS and BRAF mutation were independent factors (RAS, HR = 1.808, 95% CI = 1.18–2.77, p = 0.007; BRAF, HR = 2.417, 95% CI = 1.32–4.41, p = 0.004). Old age was independent factor for OS (HR = 3.626, 95% CI = 1.09–12.00, p = 0.035).ConclusionThis study showed that oncologic outcomes might differ according to mutation burden characterized by RAS, BRAF, and MSI between synchronous CRC and solitary CRC. In addition, our systematic review highlighted a lack of data and much heterogeneity in genetic characteristics and survival outcomes of synchronous CRC relative to that of solitary CRC.

Real-world study on microsatellite instability and mismatch repair deficiency testing patterns among patients with metastatic colorectal cancer in Spain

PurposeClinical practice guidelines recommend that all patients with metastatic colorectal cancer (mCRC) should be tested for mismatch repair deficiency (dMMR) or microsatellite instability-high (MSI-H). We aimed to describe the dMMR/MSI-H testing practice in patients with mCRC in Spanish centers.MethodsMulticenter, observational retrospective study that included patients newly diagnosed with mCRC or who progressed to a metastatic stage from early/localized stages.ResultsThree hundred patients were included in the study from May 2020 through May 2021, with a median age of 68 years, and two hundred twenty-five (75%) had stage IV disease at initial diagnosis; two hundred eighty-four patients received first-line treatment, and dMMR/MSI-H testing was performed in two hundred fifty-one (84%) patients. The results of the dMMR/MSI-H tests were available in 61 (24%) of 251 patients before the diagnosis of metastatic disease and in 191 (81%) of 236 evaluable patients for this outcome before the initiation of first-line treatment. Among the 244 patients who were tested for dMMR/MSI-H with IHC or PCR, 14 (6%) were MMR deficient. The most frequent type of first-line treatment was the combination of chemotherapy and biological agent, that was received by 71% and 50% of patients with MMR proficient and deficient tumors, respectively, followed by chemotherapy alone, received in over 20% of patients in each subgroup. Only 29% of dMMR/MSI-H tumors received first-line immunotherapy.ConclusionOur study suggests that a high proportion of patients with mCRC are currently tested for dMMR/MSI-H in tertiary hospitals across Spain. However, there is still room for improvement until universal testing is achieved.Trial registration: Not applicable.

Tumor mutation burden in gastro-entero-pancreatic-neuroendocrine neoplasms.

As rare tumors, there are limited treatment options for neuroendocrine neoplasms (NENs). Recently, microsatellite instability (MSI) and tumor mutation burden (TMB) have been emerging as potential biomarkers in various tumors. However, there is a lack of research on the use of these biomarkers in gastro-entero-pancreatic (GEP)-NENs. We analyzed 31 patients diagnosed with GEP-NEN between 2013 to 2022. The TMB and MSI analyses using next-generation sequencing (NGS) were performed for all patients. The TruSightTM Oncology 500 assay from Illumina was used as the NGS panel. Out of the 31 patients analyzed, the most frequent primary origin was the pancreas (12 patients, 38.7%), followed by the stomach (4 patients, 12.9%), gallbladder (4 patients, 12.9%), rectum (7 patients, 22.6%), small bowel (2 patients, 6.5%), and bile duct (1 patient, 3.2%). Among these patients, 19 (61.3%) were diagnosed with well-differentiated neuroendocrine tumors, with grade 2 being the most common (15 patients, 48.4%), followed by grade 3 (3 patients, 9.7%) and grade 1 (1 patient, 3.2%). Neuroendocrine carcinoma was confirmed in 12 patients (38.7%). The median number of metastases was 2.0 [interquartile range (IQR), 1.0-3.0], and the liver was the most common site of metastasis (23 patients, 74.2%). The median TMB was 4.7 (IQR, 3.1-6.3) mutations/Mb, and all tumors were classified as microsatellite stability (MSS). Only one patient had a high TMB (266.4 mutations/Mb), which was a grade 3 neuroendocrine tumor originating from the pancreas. The TMB value did not vary depending on the primary tumor site or World Health Organization (WHO) grade. This analysis showed that, despite very low incidence, there are GEP-NENs with high TMB. For precision medicine, testing for MSI and TMB is needed for this tumor type.

Organization clinical decision-making programs in the local practice of the region: problematic issues that determine the choice of therapy in the progression of endometrial cancer

Introduction. For the treatment of uterine body cancer (UBC), modern oncologists have at their disposal clinical recommendations of scientific communities, the set of drug options in which is represented by a limited range of drugs that have not been studied enough (phase II studies, no more than 52 patients in each). There is an obvious lack of information about clinical routine data systematization algorithms that describe the molecular biological features of the tumor, its prevalence, patient characteristics, existing treatment models, and form a decision-making program when planning the treatment of UBC patients.Aim. Тo develop an algorithm for routing patients with UBC on the territory of the Krasnodar Region in the existing conditions for organizing medical care and human resources.Materials and methods. Local observational retrospective study. Sources – medical records. The data were collected by the co-authors of the article in the course of daily clinical activities. Information from the medical records of 72 patients with endometrial cancer who were referred for microsatellite instability (MSI) testing at the Oncological Dispensary of Krasnodar from 01/01/2021 to 11/16/2022 was analyzed to determine the possibility of receiving immuno/immuno-targeted therapy in the second and subsequent lines medicinal treatment.Results. In the Krasnodar Oncological Dispensary all the therapy options specified in the clinical guidelines were used. One line of systemic therapy for a common disease was received by 61 patients out of 72, only 41 patients received the second line, and only 19 patients received the third line. Five patients received the immunotargeted combination in the second line and 3 in the third. One patient received immunotherapy with pembrolizumab in the third line. One patient received immunotherapy with pembrolizumab in the third line. An analysis of the factors influencing the possibility of receiving this therapy showed the importance of timely genetic testing of MSI, as well as related organizational issues, such as the ability to quickly receive treatment and ensure appropriate follow-up of patients.Conclusions. Based on the study, low awareness of clinicians about the possibility and necessity of early testing for the presence of MSI in an endometrial tumor was revealed. The organization of delivery of material from clinics that have a territorial remoteness, insufficient access to patient consultations by related specialists, and low patient compliance limit the introduction of modern methods of therapy for UBC patients. Establishing a system of close professional interaction between physicians will contribute to a wider introduction of new methods of diagnosis and treatment of UBC patients.

Investigation of Clinically Significant Molecular Aberrations in Patients with Prostate Cancer: Implications for Personalized Treatment, Prognosis and Genetic Testing.

The data on tumor molecular profiling of European patients with prostate cancer is limited. Our aim was to evaluate the prevalence and prognostic and predictive values of gene alterations in unselected patients with prostate cancer. The presence of gene alterations was assessed in patients with histologically confirmed prostate cancer using the ForeSENTIA® Prostate panel (Medicover Genetics), targeting 36 clinically relevant genes and microsatellite instability testing. The primary endpoint was the prevalence of gene alterations in homologous recombination repair (HRR) genes. Overall, 196 patients with prostate cancer were evaluated (median age 72.2 years, metastatic disease in 141 (71.9%) patients). Gene alterations were identified in 120 (61%) patients, while alteration in HRR genes were identified in 34 (17.3%) patients. The most commonly mutated HRR genes were ATM (17, 8.7%), BRCA2 (9, 4.6%) and BRCA1 (4, 2%). The presence of HRR gene alterations was not associated with advanced stage (p = 0.21), age at diagnosis (p = 0.28), Gleason score (p = 0.17) or overall survival (HR 0.72; 95% CI: 0.41-1.26; p = 0.251). We identified clinically relevant somatic gene alterations in European patients with prostate cancer. These molecular alterations have prognostic significance and therapeutic implications and/or may trigger genetic testing in selected patients. In the era of precision medicine, prospective research on the predictive role of these alterations for innovative treatments or their combinations is warranted.

The effect of immunotherapy in a young patient with mismatch repair-deficient rectal cancer - a case report.

The incidence of colorectal cancer (CRC) in the age group of young patients has been increasing. Furthermore, in these patients, CRC is more frequently an aggressive histological type of tumor, diagnosed in the late clinical stages of the disease. Another characteristic feature is a higher frequency of mismatch repair deficient (dMMR) tumors, in the treatment of which immunotherapy can be an effective treatment modality, used to prolong overall survival and improve quality of life. Conversely, the effect of chemotherapy may be lower. We present the case of a 35year-old patient treated with primary therapy for locally advanced dMMR rectal cancer. Neoadjuvant chemoradiotherapy was followed by rectal resection, which was accompanied by serious postoperative complications. In addition, there was an early local relapse of the disease, resistant to systemic chemotherapy treatment. After the progression of the disease, second line of the treatment with pembrolizumab was initiated. The treatment with pembrolizumab led to a partial regression of the disease and subsequently its stabilization, which has been lasting for 15 months. The improvement of the patient's quality of life, e. g. stabilization of the blood count and regression of diarrhea, is a significant benefit. In addition, the treatment has been given without the development of serious toxicity, so far. Microsatellite instability testing in the management of locally advanced and metastatic colorectal cancer is of fundamental importance in setting the appropriate treatment procedures. In the future, we can expect results of several studies that will try to prove the effect of immunotherapy not only in metastatic disease, but also in the neoadjuvant regimen in resectable and potentially resectable dMMR rectal and colon cancers.

Beyond germline genetic testing - heterozygous pathogenic variants in PMS2 in two children with Osteosarcoma and Ependymoma

BackgroundLynch syndrome (LS) is not considered part of childhood cancer predisposition syndromes.Case presentationAnalysis of a pediatric osteosarcoma (OS) displayed hypermutation (16.8), alternative lengthening of telomeres (ALT), loss of PMS2 expression in tumor tissue (retained in non-neoplastic cells), PMS2 loss of heterozygosity (LOH), and high-degree of microsatellite instability (MSI) tested by PCR. A heterozygous duplication c.1076dup p.(Leu359Phefs*6) in exon 10 of NM_000535.6:PMS2 was detected by SNV analysis in peripheral blood, confirming diagnosis of LS in the patient. The tumor molecular features suggest LS-associated development of OS. In a second case, whole-genome sequencing identified a heterozygous SNV c.1 A > T p.? in exon 1 of PMS2 in tumor and germline material of a girl with ependymoma. Tumor analysis displayed evidence for ALT and low mutational burden (0.6), PMS2 expression was retained, MSI was low. Multiplex ligation-dependent probe amplification identified no additional PMS2 variant and germline MSI testing did not reveal increased gMSI ratios in the patient´s lymphocytes. Thus, CMMRD was most closely excluded and our data do not suggest that ependymoma was related to LS in the child.ConclusionsOur data suggest that the LS cancer spectrum may include childhood cancer. The importance of LS in pediatric cancers necessitates prospective data collection. Comprehensive molecular workup of tumor samples is necessary to explore the causal role of germline genetic variants.

Microsatellite Instability Detection: The Current Standards, Limitations, and Misinterpretations.

Article Tools REVIEW ARTICLES Diagnostics Article Tools OPTIONS & TOOLS Export Citation Track Citation Add To Favorites Rights & Permissions COMPANION ARTICLES No companion articles ARTICLE CITATION DOI: 10.1200/PO.23.00010 JCO Precision Oncology no. 7 (2023) e2300010. Published online June 14, 2023. PMID: 37315263 Microsatellite Instability Detection: The Current Standards, Limitations, and Misinterpretations Valentina Yakushina , PhD1,2xValentina YakushinaSearch for articles by this author; Alexandra Kavun , MSc1xAlexandra KavunSearch for articles by this author; Egor Veselovsky, MSc1,3xEgor VeselovskySearch for articles by this author; Tatiana Grigoreva , MSc1,4xTatiana GrigorevaSearch for articles by this author; Ekaterina Belova , PhD1,5xEkaterina BelovaSearch for articles by this author; Alexandra Lebedeva , MSc1xAlexandra LebedevaSearch for articles by this author; Vladislav Mileyko , MSc1xVladislav MileykoSearch for articles by this author; and Maxim Ivanov , PhD1,6xMaxim IvanovSearch for articles by this author Show More 1OncoAtlas LLC, Moscow, Russian Federation2Laboratory of Epigenetics, Research Centre for Medical Genetics, Moscow, Russian Federation3Department of Evolutionary Genetics of Development, Koltzov Institute of Developmental Biology of the Russian Academy of Sciences, Moscow, Russian Federation4Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation5Lomonosov Moscow State University, Moscow, Russian Federation6Moscow Institute of Physics and Technology, Moscow, Russian Federation https://doi.org/10.1200/PO.23.00010 First Page Full Text PDF Figures and Tables © 2023 by American Society of Clinical OncologyCONTEXTKey ObjectiveAccurate microsatellite instability (MSI) assessment is critical for Lynch syndrome diagnostics, prognosis of disease progression, and success of chemotherapy and immunotherapy. In immunotherapy, false-positive MSI testing prevents putative responding patients from receiving effective treatment, and false-negative MSI testing leads to the prescription of ineffective therapy with its adverse effects.Knowledge GeneratedThis article provides the main advantages and limitations of the standard MSI testing methods and novel approaches in clinical practice, assesses possible sources of errors, discusses best practices and guidelines, and highlights the unsolved questions that remain sources of incorrect interpretation.RelevanceAmong novel approaches, next-generation sequencing (NGS) is considered one of the most promising; its advantages include high accuracy, an expanded spectrum of microsatellites analyzed, and simultaneous assessment of a comprehensive spectrum of clinically significant markers. In liquid biopsy, NGS-based approaches demonstrate outstanding results.SUPPORTSupported by the Russian Science Foundation (project No. 22-75-10154; https://rscf.ru/project/22-75-10154/).AUTHOR CONTRIBUTIONSConception and design: Valentina Yakushina, Vladislav Mileyko, Maxim IvanovData analysis and interpretation: Alexandra Kavun, Egor Veselovsky, Tatiana Grigoreva, Ekaterina Belova, Alexandra LebedevaManuscript writing: All authorsFinal approval of manuscript: All authorsAccountable for all aspects of the work: All authorsAUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTERESTThe following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated unless otherwise noted. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO's conflict of interest policy, please refer to www.asco.org/rwc or ascopubs.org/po/author-center.Open Payments is a public database containing information reported by companies about payments made to US-licensed physicians (Open Payments).Valentina YakushinaEmployment: Atlas OncodiagnosticsAlexandra KavunEmployment: OncoAtlas LLCEgor VeselovskyEmployment: OncoAtlas LLCTatiana GrigorevaEmployment: OncoAtlas LLCEkaterina BelovaEmployment: OncoAtlas LLCAlexandra LebedevaEmployment: OncoAtlas LLCVladislav MileykoEmployment: OncoAtlas LLCLeadership: OncoAtlas LLCStock and Other Ownership Interests: OncoAtlasResearch Funding: I-pharmMaxim IvanovEmployment: OncoAtlas LLCNo other potential conflicts of interest were reported.

MSI analysis in patients with GIST with lynch syndrome.

e23510 Background: Lynch Syndrome due to mutations in one of DNA mismatch repair (MMR) genes, is found in patients with colon cancer (3%).The mutations in these DNA repair genes induce genetic instability and development of ovarian, endometrial, urothelial, gastrointestinal and biliary tract carcinoma. Methods: In our center, 31 patients with Lynch syndrome are followed in Oncology Department in multidisciplinary team according to regional diagnostic therapeutic assistance paths and international guidelines. Immunohistochemical staining (IC) for mismatch repair proteins (MLH1, MSH2, MSH6, PMS2) and microsatellite instability testing (MSI) by PCR real time easyPGX were performed in colon cancer, genetic testing has been preceded by genetic counseling. When GIST diagnosis occurred in patients with colon cancers and Lynch syndrome, IC and MSI were performed in GIST too. Results: MSI-H has been detected in synchronous colo-rectal cancer in 2 patients with GIST. Pathogenetic germline mutations in MSH2 gene were observed in both patients. In 1 female patient with gist of duodenum (very low risk according to Miettinen classification) with previous endometrial cancer and sincronous PT3N0 rectal cancer, we observed MSI-stable in IC and PCR, KIT/PDGFRα wild type in Next GenSequencing analysis, BRAF wild type (PCR-real time), SDH-B + (IC), NTRK 1 (exon 9-10, exon 1-12 del, exon 12), NTRK 2 (exon 12-15.exon 16-17), exon 14, NTRK 3 (exon 14, exon 15) no gene fusion by PCR-real time (CE.IVD kit easy NTRK). In 1 male patient with ileal GIST (intermediate risk according to Miettinen classification ) with ileal adenocarcinoma pT3N0, we observed KIT exon 11 mutated, MSI-stable in IC, when we repeated the analysis using PCR real time EasyPGX, we observed high instability of microsatellites. Conclusions: For the first time we observed in Lynch Syndrome MSH2 mutated, simolutaneous GIST and colon cancer in the same site of ileum and PCR analisys detected a rare exemple of microsatellite instability in GIST.

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.

Somatic Sequencing and Microsatellite Instability Results From Mismatch Repair-deficient Endometrial Carcinoma Patients Without Lynch Syndrome ("Lynch-like" tumors): Implications for Heritable Cancer Screening, Molecular Prognostication, and Immunotherapeutic Vulnerability.

Immunostaining of endometrial carcinomas for mismatch repair (MMR) protein loss is standard-of-care for Lynch syndrome screening, but also identifies MMR-deficient tumors without germline pathogenic variants. While the majority show MLH1 hypermethylation (MLH1hm), somatic MMR pathogenic variants are increasingly recognized drivers of immunohistochemistry-germline discordance. Because MMR abnormalities with both germline and somatic origins have prognostic significance and impart susceptibility to immune checkpoint inhibitors, it is important to understand how frequently tumors with MMR immunohistochemical loss and normal germline testing ("Lynch-like" tumors) have underlying somatic MMR pathogenic variants. Somatic tumor sequencing±microsatellite instability (MSI) testing was performed on 18 endometrial cancers with MMR immunohistochemical loss but negative MMR germline results and negative MLH1hm where relevant. Tumor sequencing and MSI testing were successful in 94%. Where successful, 80% were MSI-high and 94% had a molecular correlate for the initial immunohistochemical interpretation. The single case without an identified somatic pathogenic variant was MSI-low and initially showed loss of MSH6 by immunohistochemistry but with extremely limited internal control staining. On review, MSH6 immunohistochemistry was reclassified as equivocal, and repeat staining revealed improved control expression with intact MSH6. Following reclassification of this case, 100% tumors with MMR deficiency by immunohistochemistry had at least 1 confirmed somatic MMR pathogenic variant, and 86% were MSI-high. These results demonstrate that when correctly interpreted immunohistochemistry is a strong surrogate for somatic MMR pathogenic variants and support its use as the frontline MMR biomarker in endometrial cancer for heritable screening, molecular prognostic classification, and immunotherapeutic biomarker testing purposes.

Microsatellite instability in non-endometrioid ovarian epithelial tumors: a study of 400 cases comparing immunohistochemistry, PCR, and NGS based testing with mutation status of MMR genes

Testing of microsatellite instability is not only used as a triage for possible Lynch syndrome, but also to predict immunotherapy treatment response. The aim of this study was to assess the frequency of mismatch repair deficiency (MMR-D) / microsatellite instability (MSI) in 400 cases of non-endometrioid ovarian tumors (high-grade serous, low-grade serous, mucinous and clear cell), to compare different methodological approaches of testing, and to assess the optimal approach for next generation sequencing (NGS) MSI testing. For all tumors, we evaluated immunohistochemical (IHC) expression of MMR proteins and assessed microsatellite markers by PCR-based method. Except for high-grade serous carcinoma, we correlated the findings of IHC and PCR with NGS-based MSI testing. We compared the results with somatic and germline mutation in MMR genes. Among the whole cohort, seven MMR-D cases, all clear cell carcinomas (CCC), were found. On PCR analysis, six cases were MSI-high and one was MSS. In all cases, mutation of an MMR gene was found; in two cases, the mutation was germline (Lynch syndrome). An additional five cases with a mutation in MMR gene(s) with MSS status and without MMR-D were identified. We further utilized sequence capture NGS for MSI testing. Employing 53 microsatellite loci provided high sensitivity and specificity. Our study shows that MSI occurs in 7% of CCC while it is rare or absent in other non-endometrioid ovarian neoplasms. Lynch syndrome was present in 2% of patients with CCC. However, some cases with MSH6 mutation can evade all testing methods, including IHC, PCR, and NGS-MSI.

A robust microsatellite instability detection model for unpaired colorectal cancer tissue samples.

Microsatellite instability (MSI) is a key biomarker for cancer immunotherapy and prognosis. Integration of MSI testing into a next-generation-sequencing (NGS) panel could save tissue sample, reduce turn-around time and cost, and provide MSI status and comprehensive genomic profiling in single test. We aimed to develop an MSI calling model to detect MSI status along with the NGS panel-based profiling test using tumor-only samples. From January 2019 to December 2020, a total of 174 colorectal cancer (CRC) patients were enrolled, including 31 MSI-high (MSI-H) and 143 microsatellite stability (MSS) cases. Among them, 56 paired tumor and normal samples (10 MSI-H and 46 MSS) were used for modeling, and another 118 tumor-only samples were used for validation. MSI polymerase chain reaction (MSI-PCR) was performed as the gold standard. A baseline was built for the selected microsatellite loci using the NGS data of 56 normal blood samples. An MSI detection model was constructed by analyzing the NGS data of tissue samples. The performance of the model was compared with the results of MSI-PCR. We first intersected the target genomic regions of the NGS panels used in this study to select common microsatellite loci. A total of 42 loci including 23 mononucleotide repeat sites and 19 longer repeat sites were candidates for modeling. As mononucleotide repeat sites are more sensitive and specific for detecting MSI status than sites with longer length motif and the mononucleotide repeat sites performed even better than the total sites, a model containing 23 mononucleotide repeat sites was constructed and named Colorectal Cancer Microsatellite Instability test (CRC-MSI). The model achieved 100% sensitivity and 100% specificity when compared with MSI-PCR in both training and validation sets. Furthermore, the CRC-MSI model was robust with the tumor content as low as 6%. In addition, 8 out of 10 MSI-H samples showed alternations in the four mismatch repair genes (MLH1, MSH2, MSH6, and PMS2). MSI status can be accurately determined along the targeted NGS panels using only tumor samples. The performance of mononucleotide repeat sites surpasses loci with longer repeat motif in MSI calling.

MLH1 Loss in Primary Prostate Cancer.

Among mismatch repair-deficient (MMRd) prostate cancers, loss of MLH1 is relatively uncommon and few cases have been reported in detail. Here, we describe the molecular features of two cases of primary prostate cancer with MLH1 loss detected by immunohistochemistry, and in one case, confirmed via transcriptomic profiling. Both cases were microsatellite stable on standard polymerase chain reaction (PCR)-based microsatellite instability (MSI) testing, but showed evidence of MSI on a newer PCR-based long mononucleotide repeat (LMR) assay and by next-generation sequencing. Germline testing was negative for Lynch syndrome-associated mutations in both cases. Targeted or whole-exome tumor sequencing using multiple commercial/academic platforms (Foundation, Tempus, JHU, and UW-OncoPlex) showed modestly elevated, though variable, tumor mutation burden estimates (2.3-10 mutations/Mb) consistent with MMRd, but without identifiable pathogenic single-nucleotide or indel mutations in MLH1. Copy-number analysis confirmed biallelic MLH1 loss in one case and monoallelic MLH1 loss in the second case, without evidence of MLH1 promoter hypermethylation in either. The second patient was treated with single-agent pembrolizumab and demonstrated a short-lived prostate-specific antigen response. These cases highlight the challenges in identifying MLH1-deficient prostate cancers using standard MSI testing and commercial sequencing panels, and support the utility of immunohistochemical assays and LMR- or sequencing-based MSI testing for detection of MMRd prostate cancers.

Abstract 5550: Sociodemographic disparities in access to biomarker testing in patients with advanced colorectal cancer

Abstract Introduction All patients with newly diagnosed colorectal cancer are recommended to undergo microsatellite instability (MSI) testing. In addition, mutational testing using biomarkers such as KRAS, NRAS, and BRAF is recommended for patients with metastatic cancers. Testing for these markers is vital as they guide therapeutic decision-making and serve as prognostic indicators. Our study aims to analyze disparities in testing for MSI and KRAS biomarkers based on sociodemographic factors in patients with metastatic colorectal cancer. Furthermore, we explored the survival characteristics in these patients based on sociodemographic factors and on access to biomarker testing. Methods The National Cancer Database (NCDB) was queried for patients diagnosed with metastatic colorectal cancer (MCC). At the bivariate level, we performed chi-squared statistics and multivariate logistic regression modeling to explore variables associated with patients undergoing MSI and KRAS testing. In addition, Multivariate Cox regression and Kaplan-Meier analyses were performed for survival analysis. Results N = 51,913 patients with MCC diagnosed between 2010 to 2017 were included. The median age for Whites was 68.0 years versus 64.0 years for Blacks. Blacks had a lower probability of undergoing MSI testing (OR 0.90, [0.84-0.96] p< 0.0009). Factors associated with a lower likelihood for MSI testing included treatment at a Community Cancer Program (OR 0.61, [0.55-0.66] p<0.0001), residing in areas with people having lower education (OR 0.68, [0.62-0.74] p<0.0001) and in rural areas (OR 0.74, [0.61-0.90] p-value 0024), and a median household income of < $38,000 (OR 0.88, [0.80-0.96] p 0.0040). Patients with no insurance and Medicaid/governmental insurance were also less likely to undergo both MSI and KRAS testing. Blacks had a decreased likelihood of having high MSI levels among patients who underwent testing compared to Whites (OR 0.67, [0.50-0.91] p-value 0.0107). After controlling for confounding variables, survival analysis showed poor survival in patients who did not undergo testing for MSI and KRAS (HR 1.20, [1.17-1.23] p<0.0001 and HR 1.04, [1.01-1.06] p-value 0.0016 respectively). Conclusion Our analysis reveals that sociodemographic factors such as being from a minority race, having no insurance, residing in areas with lower education and rural settings were associated with a lower probability of undergoing MSI testing in patients with metastatic colorectal cancer. This is of concern as our study also reveals that not undergoing biomarker testing is associated with poorer survival. Addressing such sociodemographic discrepancies among racial groups is essential in achieving equitable care and narrowing gaps in outcomes. Citation Format: Saad Sabbagh, Iktej Jabbal, Barbara Dominguez, Mira Itani, Mohamed Mohanna, Arun Nagarajan. Sociodemographic disparities in access to biomarker testing in patients with advanced colorectal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5550.