High-level Microsatellite Instability Research Articles

Immune-related dermatological adverse events of antitumor immunotherapy with PD-1, PD-L1, CTLA-4 inhibitors

The discovery of a new group of modern anticancer drugs was a breakthrough in the treatment of cancer. Immune checkpoint inhibitors that block cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein-1 (PD-1), and programmed cell death receptor ligand (PD-L1) may improve prognosis for patients with malignant neoplasms with a high level of microsatellite instability. Despite the high effectiveness of these drugs, immune checkpoint inhibitors can lead to dysregulation of immune responses and the occurrence of adverse reactions associated with an increase in the activity of immunocompetent cells in the body. The aim of this review is to analyze the available data on the immune-related dermatological adverse events during treatment with immune checkpoint inhibitors.

Establishment and molecular characterization of HCB-541, a novel and aggressive human cutaneous squamous cell carcinoma cell line

Cutaneous squamous cell carcinoma (cSCC) is a common type of skin cancer that can result in significant morbidity, although it is usually well-managed and rarely metastasizes. However, the lack of commercially available cSCC cell lines hinders our understanding of this disease. This study aims to establish and characterize a new metastatic cSCC cell line derived from a Brazilian patient. A tumor biopsy was taken from a metastatic cSCC patient, immortalized, and named HCB-541 after several passages. The cytokeratin expression profile, karyotypic alterations, mutational analysis, mRNA and protein differential expression, tumorigenic capacity in xenograft models, and drug sensitivity were analyzed. The HCB-541 cell line showed a doubling time between 20 and 30 h and high tumorigenic capacity in the xenograft mouse model. The HCB-541 cell line showed hypodiploid and hypotetraploidy populations. We found pathogenic mutations in TP53 p.(Arg248Leu), HRAS (Gln61His) and TERT promoter (C228T) and high-level microsatellite instability (MSI-H) in both tumor and cell line. We observed 37 cancer-related genes differentially expressed when compared with HACAT control cells. The HCB-541 cells exhibited high phosphorylated levels of EGFR, AXL, Tie, FGFR, and ROR2, and high sensitivity to cisplatin, carboplatin, and EGFR inhibitors. Our study successfully established HCB-541, a new cSCC cell line that could be useful as a valuable biological model for understanding the biology and therapy of metastatic skin cancer.

Abstract 5942: Discovery of WRN inhibitors as targeted therapy in the treatment of microsatellite unstable (MSI-H) tumors

Abstract The synthetic lethality resulting from the inhibition of the WRN helicase protein has been observed in tumors characterized by high levels of microsatellite instability (MSI-H). This instability stems from a deficiency in the DNA mismatch repair (MMR) mechanisms, leading to the accumulation of DNA damage. This phenomenon is notably prevalent in 10-30% of colorectal, gastric, endometrial, and ovarian cancers. Specifically, the inhibition of WRN helicase activity induces DNA damage leading to cell cycle arrest and apoptosis exclusively in MSI-H cell lines. This selectivity underscores the therapeutic potential of WRN inhibitors, as they demonstrate efficacy against MSI-H cells while remaining non-toxic to microsatellite stable (MSS) cell lines. Ryvu is developing a chemical series of potent, selective WRN helicase inhibitors. Structure-based optimization facilitated the rapid expansion and delivery of a compound library with novel intellectual property (IP), demonstrating target engagement in cells and selective potency over other RecQ family members. The correlation between the cellular effect and selectivity upon treatment with these ligands was confirmed in the clonogenic assay, specifically in responder, microsatellite instable cell lines only, thereby leaving a substantial safety margin (>100x) relative to non-responder, microsatellite stable lines. Target engagement has been confirmed by dose dependent DNA damage pathway activation concomitant with WRN degradation in in vitro assays. The pharmacokinetic properties of these compounds are favorable for progressing to in vivo studies. The correlation between compound exposure and on-target effect was confirmed in PK/PD and efficacy studies in xenograft MSI-H cancer models. These data provide pharmacological proof-of-concept for the synthetic lethal effect of our inhibitors and support WRN inhibition as a new oncological therapy. Citation Format: Wojciech Schönemann, Agnieszka Ludwig-Słomczyńska, Łukasz Dudek, Marcin Król, Marco Farinone, Urszula Kulesza, Wojciech Jasnosz, Izabela Mac, Karol Zuchowicz, Katarzyna Wierzbicka, Aleksandra Buchcic-Szychowska, Andrzej Gondela, Anna Zagórska, Oliwia Matuszewska, Marta Sowińska, Marcin Nowogródzki, Marianna Girardi, Szymon Paluch, Svitlana Sukhomlinova, Agata Chłopek, Iana Levenets, Kamila Kozłowska-Tomczyk, Katarzyna Łagosz-Ćwik, Mateusz Stoszko, Szymon Woroszyło, Maciej Mikulski, Justyna Jabłońska, Magdalena Wiśniewska, Katarzyna Palus-Chramiec, Paulina Podkalicka-Gołda, Anita Janiga, Sylwia Sudoł-Tałaj, Magdalena Sieprawska-Lupa, Magdalena Miodek, Jacek Faber, Róża Starczak, Bozena Winnik, Agnieszka Świrska, Anna Kowal-Chwast, Dawid Gogola, Sanja Novak Ratajczak, Agata Dudek, Nilesh Gaud, Grzegorz Satała, Agata Stachowicz, Martin Swarbrick, Krzysztof Brzózka, Magdalena I. Zawadzka. Discovery of WRN inhibitors as targeted therapy in the treatment of microsatellite unstable (MSI-H) tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5942.

Interferon gamma induces higher neutrophil extracellular traps leading to tumor-killing activity in microsatellite stable colorectal cancer.

Many colorectal cancer (CRC) patients do not respond to immune checkpoint blockade (ICB) therapy, highlighting the urgent need to understand tumor resistance mechanisms. Recently, the link between the IFNγ signaling pathway integrity and ICB resistance in the CRC tumor microenvironment has been revealed. The immunosuppressive microenvironment poses a significant challenge to antitumor immunity in CRC development. Tumor-associated neutrophils (TANs) found in tumor tissues exhibit an immunosuppressive phenotype and are associated with CRC patient prognosis. Neutrophil extracellular traps (NETs), DNA meshes containing cytotoxic enzymes released into the extracellular space, may be promising therapeutic targets in cancer. This study showed increased NETs in tumor tissues and peripheral neutrophils of high levels of microsatellite instability (MSI-H) CRC patients compared to microsatellite stable (MSS) CRC patients. IFNγ response genes were enriched in MSI-H CRC patients compared to MSS CRC patients. Co-culturing neutrophils with MSI-H CRC cell lines induced more NET formation and higher cellular apoptosis than MSS CRC cell lines. IFNγ treatment induced more NET formation and apoptosis in MSS CRC cell lines. Using subcutaneous or orthotopic CT-26 (MSS)-tumor-bearing mice models, IFNγ reduced tumor size and enhanced PD-1 antibody-induced tumor-killing activity, accompanied by upregulated NETs and cellular apoptosis. These findings suggest IFNγ could be a therapeutic strategy for MSS CRC.

Enrichment of Bacteroides fragilis and enterotoxigenic Bacteroides fragilis in CpG island methylator phenotype-high colorectal carcinoma

ObjectivesData support that enterotoxigenic Bacteroides fragilis (ETBF) harbouring the Bacteroides fragilis toxin (bft) gene may promote colorectal tumourigenesis through the serrated neoplasia pathway. We hypothesized that ETBF may be enriched in colorectal carcinoma subtypes with high-level CpG island methylator phenotype (CIMP-high), BRAF mutation, and high-level microsatellite instability (MSI-high). MethodsQuantitative PCR assays were designed to quantify DNA amounts of Bacteroides fragilis, ETBF, and each bft gene isotype (bft-1, bft-2, or bft-3) in colorectal carcinomas in the Health Professionals Follow-up Study and Nurses' Health Study. We used multivariable-adjusted logistic regression models with the inverse probability weighting method. ResultsWe documented 4476 colorectal cancer cases, including 1232 cases with available bacterial data. High DNA amounts of Bacteroides fragilis and ETBF were positively associated with BRAF mutation (p ≤ 0.0003), CIMP-high (p ≤ 0.0002), and MSI-high (p < 0.0001 and p = 0.01, respectively). Multivariable-adjusted odds ratios (with 95% confidence interval) for high Bacteroides fragilis were 1.40 (1.06–1.85) for CIMP-high and 2.14 (1.65–2.77) for MSI-high, but 1.02 (0.78–1.35) for BRAF mutation. Multivariable-adjusted odds ratios for high ETBF were 2.00 (1.16–3.45) for CIMP-high and 2.86 (1.64–5.00) for BRAF mutation, but 1.09 (0.67–1.76) for MSI-high. Neither Bacteroides fragilis nor ETBF was associated with colorectal cancer-specific or overall survival. DiscussionThe tissue abundance of Bacteroides fragilis is associated with CIMP-high and MSI-high, whereas ETBF abundance is associated with CIMP-high and BRAF mutation in colorectal carcinoma. Our findings support the aetiological relevance of Bacteroides fragilis and ETBF in the serrated neoplasia pathway.

Combination of pembrolizumab and lenvatinib in second-line therapy for MSS/pMMR advanced endometrial cancer: literature review and a case report

Despite the existing standard treatments for endometrial cancer, the prognosis for these patients remained poor until recently. None of currently available cytostatics ensured long-term disease control and long-term survival of patients receiving standard platinum-based therapy. Poor treatment outcomes in patients with advanced endometrial cancer necessitated changes in therapeutic approaches and development of more effective treatment regimens. Better understanding of carcinogenesis mechanisms, emergence of a new molecular classification of endometrial cancer, and implementation of tailored treatment approaches based on tumor types led to a significant breakthrough in the treatment of advanced endometrial cancer. Currently, the most effective second-line therapy for endometrial cancer with high level microsatellite instability (MSI-h) or deficient mismatch repair system (dMMR) (25 % of cases), and no indications for surgery is pembrolizumab monotherapy. Most of cancers (about 75 %) are represented by microsatellite-stable and mismatch repair proficient tumors (MSS/рMMR). Patients with such tumors demonstrating disease progression after systemic therapy should receive a combinations of the immune checkpoint inhibitor pembrolizumab and the multi-targeted tyrosine kinase inhibitor lenvatinib. In this article, we review relevant literature and report a case of successful treatment of MSS/pMMR advanced endometrial cancer with a combination of pembrolizumab and lenvatinib as a second-line therapy (after disease progression in response to platinum-containing first-line chemotherapy), which ensured long-term disease control.

Stromal localization of inactive CD8+ T cells in metastatic mismatch repair deficient colorectal cancer.

The determinants of metastasis in mismatch repair deficiency with high levels of microsatellite instability (MSI-H) in colorectal cancer (CRC) are poorly understood. Here, we hypothesized that distinct immune and stromal microenvironments in primary tumors may discriminate between non-metastatic MSI-H CRC and metastatic MSI-H CRC. We profiled 46,727 single cells using high-plex imaging mass cytometry and analyzed both differential cell type abundance, and spatial distribution of fibroblasts and immune cells in primary CRC tumors with or without metastatic capacity. We validated our findings in a second independent cohort using immunohistochemistry. High-plex imaging mass cytometry and hierarchical clustering based on microenvironmental markers separated primary MSI-H CRC tumors with and without metastatic capacity. Primary tumors with metastatic capacity displayed a high stromal content and low influx of CD8+ T cells, which expressed significantly lower levels of markers reflecting proliferation (Ki67) and antigen-experience (CD45RO) compared to CD8+ T cells in non-metastatic tumors. CD8+ T cells showed intra-epithelial localization in non-metastatic tumors, but stromal localization in metastatic tumors, which was validated in a second cohort. We conclude that localization of phenotypically distinct CD8+ T cells within stroma may predict metastasis formation in MSI-H CRC.

Explore the alterations of downstream molecular pathways caused by ARID1A mutation/knockout in human endometrial cancer cells.

As one of the most common gynecologic malignancies, endometrial cancer (EC) is driven by multiple genetic alterations that may be targeted for treatments. AT-rich interaction domain 1A (ARID1A) gene mutations were reported as early events in endometrial carcinogenesis. To explore the alterations of downstream molecular pathways caused by ARID1A mutations and the associated therapeutic implications, we edited ARID1A gene in human endometrial cancer cell line Ishikawa using the Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-Associated Proteins (CRISPR/Cas9) technology. We successfully constructed a stable Ishikawa cell line with a confirmed 10bp deletion on the ARID1A gene, which resulted in a code-shift mutation and gene knockout. Compared with unedited wild-type cells, ARID1A knockout (KO) led to reduced apoptosis, accelerated transformation from G0/G1 to S phase, and enhanced cell proliferation. ARID1A deficiency would reduce the protein levels of p21, caspase 7, and caspase 9 in Ishikawa endometrial cancer cells compared with the wild-type cells. In addition, ARID1A KO resulted in high levels of microsatellite instability (MSI-H). Moreover, transcriptomic analyses showed that ARID1A KO can lead to activated phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling. Furthermore, experimental analyses demonstrated that ARID1A KO cells had reduced expression of genetic instability-associated markers mutL homologue 1 (MLH1) and progesterone receptor B (PR) and increased p-Akt expression. These findings support further exploration of ARID1A as a therapeutic target for EC and provide insight into developing more effective treatments in EC, such as the combinatory use of immune checkpoint inhibitors.

Detection of a status of microsatellite instability in tumors of patients with endometrioid adenocarcinoma of the ovaries and/or of uterine corpus

Introduction. Multiple primary malignant neoplasms of female reproductive organs are a rare pathology. However, over the past decades, there has been an upsurge of interest in the study of this phenomenon in oncology. This is particularly the case for the diagnosis of synchronous endometrioid adenocarcinoma of the ovaries and uterine corpus, which histogenetically belong to the same germ layer and have similar histological structure. Until recently, clinicians relied only on morphological examination in these cases, but with the development of molecular genetic technologies, new diagnostic possibilities have emerged.Aim. Is the detection of the status of microsatellite instability in tumors of patients with endometrioid adenocarcinoma of the ovaries and/or uterine corpus.Materials and Methods. A pilot retrospective molecular genetic study (n = 48) was conducted to determine the status of microsatellite instability (MSI) in the tumors of the ovaries and/or uterine corpus: it involved 33 patients with solitary endometrioid ovarian cancer and 15 patients with synchronous endometrioid adenocarcinoma of the ovaries and uterine corpus. Microsatellite instability status was detected using PCR method with subsequent fragment analysis performed on ABI PRISM 3500 genetic analyzer (8 capillaries, Applied Biosystems). DNA was isolated from paraffin blocks of surgical specimens using DNAsorb B extraction kit (AmpliSens, Russia), according to the manufacturer’s manual. DNA concentration was estimated fluorometrically using Qubit 2.0 (Life Technologies, USA). The obtained data were analyzed using GeneMapper program (Thermo Fisher, USA). In case of polymorphism of two and more markers high-level microsatellite instability (MSI-H) was observed.Results. The incidence of MSI-H in solitary endometrioid ovarian cancer (n = 33) was 12,1 % (4 cases), while in synchronous ovarian and uterine corpus tumors (n = 15) MSI-H incidence made up 20 % (n = 3). Herewith, there have been only cases of a combination of endometrioid histotypes of ovarian and endometrial cancer with identical status of microsatellite instability. Thus, the incidence of MSI-H in synchronous endometrioid adenocarcinoma of the ovaries and uterine corpus (20 %) is comparable to that in solitary endometrial cancer.Conclusion. Our pilot study became a significant complement to the previously published materials, as it allowed to confirm the clonal origin of tumors in patients with endometrioid adenocarcinoma of the ovaries and uterine corpus, that can affect the stratification of treatment strategy for this category of patients.

Comprehensive mapping of cell fates in microsatellite unstable cancer cells supports dual targeting of WRN and ATR.

Addiction to the WRN helicase is a unique vulnerability of human cancers with high levels of microsatellite instability (MSI-H). However, while prolonged loss of WRN ultimately leads to cell death, little is known about how MSI-H cancers initially respond to acute loss of WRN-knowledge that would be helpful for informing clinical development of WRN targeting therapy, predicting possible resistance mechanisms, and identifying useful biomarkers of successful WRN inhibition. Here, we report the construction of an inducible ligand-mediated degradation system in which the stability of endogenous WRN protein can be rapidly and specifically tuned, enabling us to track the complete sequence of cellular events elicited by acute loss of WRN function. We found that WRN degradation leads to immediate accrual of DNA damage in a replication-dependent manner that curiously did not robustly engage checkpoint mechanisms to halt DNA synthesis. As a result, WRN-degraded MSI-H cancer cells accumulate DNA damage across multiple replicative cycles and undergo successive rounds of increasingly aberrant mitoses, ultimately triggering cell death. Of potential therapeutic importance, we found no evidence of any generalized mechanism by which MSI-H cancers could adapt to near-complete loss of WRN. However, under conditions of partial WRN degradation, addition of low-dose ATR inhibitor significantly increased their combined efficacy to levels approaching full inactivation of WRN. Overall, our results provide the first comprehensive view of molecular events linking upstream inhibition of WRN to subsequent cell death and suggest that dual targeting of WRN and ATR might be a useful strategy for treating MSI-H cancers.

Does high [18F]FDG uptake always mean poor prognosis? Colon cancer with high-level microsatellite instability is associated with high [18F]FDG uptake on PET/CT.

High-level microsatellite instability (MSI-high) is generally associated with higher F-18 fluorodeoxyglucose ([18F]FDG) uptake than stable microsatellite (MSI-stable) tumors. However, MSI-high tumors have better prognosis, which is in contrast with general understanding that high [18F]FDG uptake correlates with poor prognosis. This study evaluated metastasis incidence with MSI status and [18F]FDG uptake. We retrospectively reviewed 108 right-side colon cancer patients who underwent preoperative [18F]FDG PET/CT and postoperative MSI evaluations using a standard polymerase chain reaction at five Bethesda guidelines panel loci. The maximum standard uptake value (SUVmax), SUVmax tumor-to-liver ratio (TLR), metabolic tumor volume (MTV), and total lesion glycolysis (TLG) of the primary tumor were measured using SUV 2.5 cut-off threshold. Student's t-test or Mann-Whitney U test was performed for continuous variables, and χ2 test or Fisher's exact test was performed for categorical variables (p value of < 0.05 for statistical significance). Medical records were reviewed for metastasis incidence. Our study population had 66 MSI-stable and 42 MSI-high tumors. [18F]FDG uptake was higher in MSI-high tumors than MSI-stable tumors (TLR, median (Q1, Q3): 7.95 (6.06, 10.54) vs. 6.08 (4.09, 8.82), p = 0.021). Multivariable subgroup analysis demonstrated that higher [18F]FDG uptake was associated with higher risks of distant metastasis in MSI-stable tumors (SUVmax: p = 0.025, MTV: p = 0.008, TLG: p = 0.019) but not in MSI-high tumors. MSI-high colon cancer is associated with high [18F]FDG uptake, but unlike MSI-stable tumors, the degree of [18F]FDG uptake does not correlate with the rate of distant metastasis. MSI status should be considered during PET/CT assessment of colon cancer patients, as the degree of [18F]FDG uptake might not reflect metastatic potential in MSI-high tumors. • High-level microsatellite instability (MSI-high) tumor is a prognostic factor for distant metastasis. • MSI-high colon cancers had a tendency of demonstrating higher [18F]FDG uptake compared to MSI-stable tumors. • Although higher [18F]FDG uptake is known to represent higher risks of distant metastasis, the degree of [18F]FDG uptake in MSI-high tumors did not correlate with the rate at which distant metastasis occurred.

PACE: A phase III trial of CAPOX versus anti-PD-1 inhibitor as adjuvant therapy for patients with dMMR/MSI-H stage III colon cancer.

TPS3637 Background: Approximately 12% of stage III colon cancer are deficient mismatch repair (dMMR), leading to high levels of microsatellite instability (MSI-H). In the KEYNOTE-177 study, the anti–PD-1 inhibitor Pembrolizumab alone significantly improved progression free survival (PFS) versus chemotherapy as first-line therapy for metastatic colorectal cancer (mCRC) patients with MSI-H/dMMR. We are conducting a phase III randomised trial to determine whether anti-PD-1 inhibitor Sintilimab monotherapy can improve disease free survival (DFS) compared with standard adjuvant chemotherapy in stage III colon cancer patients with dMMR. Methods: This is an open-label, multicentre, randomised phase III study. Patients aged ≥ 18 years with completely resected, stage III colon cancer confirmed to have dMMR/MSI-H and ECOG performance status 0-1 are eligible. Approximately 323 patients will be randomly assigned 1:1 to either Sintilimab monotherapy (200 mg IV every 3 weeks for 8 cycles) or CAPOX regimen (capecitabine, oxaliplatin for 4 or 8 cycles according to current standard practice). Adverse events (AEs) will be assessed throughout treatment and for 30 days thereafter (90 days for serious AEs) and graded per NCI CTCAE v4.0. Primary endpoint is 3-year DFS. The study was estimated to provide approximately 80% power to detect a hazard ratio of 0.56 for DFS at a two-sided alpha of 0.05. Secondary endpoints include OS, safety profile, toxicity, and quality of life. Archived tumor tissue and blood samples will be collected for correlative studies. Study enrollment is ongoing. Clinical trial information: NCT05236972 .

Microsatellite instability in sarcomas: Which tests are ideal for diagnosis, and a systematic review.

e23519 Background: There is a lack of clinically relevant biomarkers to inform immunotherapy for patients with advanced sarcoma. High levels of microsatellite instability (MSI-H) is a histology-agnostic biomarker of response to pembrolizumab. However, the frequency of MSI-H in sarcoma is low - ranging from &lt; 0.5% to 4%. MSI status can be assessed through Next Generation Sequencing (NGS), immunohistochemistry (IHC), or PCR followed by fragment analysis with capillary electrophoresis (PCR). To date, both the College of American Pathologists and the European Society of Medical Oncology have not determined the best method to evaluate MSI in sarcomas. We performed a systematic review to evaluate the methods to diagnose MSI-H in sarcomas and to evaluate the impact of this biomarker in treatment with pembrolizumab. Methods: We performed a literature search using the PubMed database to identify all English full-text available articles between January 2020 and December 2022, with the terms “sarcoma and microsatellite instability” and “sarcoma and pembrolizumab”. One independent reviewer screened the studies, two independent reviewers analyzed the selected studies, and disparities were resolved with a fourth reviewer. Results: Concerning the diagnosis of MSI-H in sarcomas, 187 abstracts were found, and 31 articles were identified for full analysis. MSI status was reported for 7958 patients - 7762 with a single test and 196 with more than one test. NGS was used for 6649, IHC for 1222, and PCR for 285. MSI-H tumors were 25 (0.4%), 38 (3.1%), and 21 (7.33%) for each technique, respectively. Two investigators performed the three available tests to evaluate concordance, with one concordant case and one discordant case. 194 cases were evaluated for both IHC and PCR, with 4 discordant results. No article that described NGS results performed a second test. To analyze the results of pembrolizumab therapy in MSI-H sarcomas, we analyzed 199 abstracts of which we selected 13 prospective and 31 retrospective papers for full analysis. The K cohort of Keynote-158 is the only prospective trial concerning therapies guided by MSI-H in sarcoma (n = 14 patients, ORR = 33%). Regarding retrospective data, we identified 2 case reports of the use of pembrolizumab in patients with advanced sarcoma – both achieved a response to therapy. Conclusions: There is limited data to establish the best diagnostic test for MSI-H in patients with sarcoma. The concordance between NGS, IHC, and PCR seems to be high, but discrepancies have been described and need to be further evaluated. There is only one prospective trial that enrolled patients with sarcomas utilizing MSI-H as a biomarker. Albeit only a small number of patients were enrolled (n = 14), efficacy is consistent with that seen in other histologies, and scarce retrospective data corroborate the potential role of MSI-H as a predictive biomarker of pembrolizumab efficacy in sarcoma.

Are High Levels of Microsatellite Instability and Microsatellite Stability Identical in DNA Mismatch Repair-Deficient Colorectal Cancer Patients?

The purpose of the current study was to determine whether there is a difference between high levels of microsatellite instability (MSI-H) and microsatellite stability (MSS) in DNA mismatch repair-deficient (DMMR) colorectal cancer (CRC) patients. A total of 452 CRC patients with DMMR from December, 2014, to April, 2021, in our hospital were selected retrospectively. However, only 105 patients underwent Sanger or next-generation-sequencing (NGS) to confirm their microsatellite status. Ultimately, 55 MSI-H patients and 20 MSS patients with intact medical record information were included in this study. The MSS group was associated with a higher mutation rate in the KRAS gene (P=0.011). Meanwhile, MSI-H was related to colon cancer (P < 0.01). However, no significant differences in other clinical characteristics were observed between the two groups of patients. There was no significant difference between the MSI-H and MSS groups in terms of overall survival (OS) (P=0.398) and disease-free survival (DFS) (P=0.307). The MSI-H status was associated with colon cancer and a lower mutation rate of the KRAS gene in DMMR patients. In CRC-DMMR patients, the MSS group exhibited better OS and DFS than the MSI-H group, although these differences were not statistically significant. Accordingly, in clinical practice, we should not confuse these two types of patients.

Discordance of microsatellite instability and mismatch repair immunochemistry occurs depending on the cancer type

Microsatellite instability (MSI) and deficiency of mismatch repair (dMMR) are key markers for predicting the response of immune checkpoint inhibitors (ICIs) and screening for Lynch syndrome (LS). This study examined the incidences of and factors associated with the concordance of MSI and MMR in human cancers. A total of 518 formalin-fixed cancer tissues were analyzed for MSI and MMR immunohistochemistry (IHC). MSI was analyzed by a PCR-based method using Promega markers. Concordance with MMR expression and factors associated with concordance were analyzed. In 2 colorectal cancer samples, MMR IHC failed due to inadequate staining conditions. In the remaining 516 cancers, a high level of MSI (MSI-H) was identified in 113 cases, and dMMR was identified in 112. The concordance of MSI and MMR IHC was 98.3%. Only 9 cases (4 pancreatobiliary, 3 colorectal, and 2 endometrial cancers) were discordant. Of the 113 MSI-H cases, 4 (3.5%) were proficient MMR (pMMR); of the 403 microsatellite stability (MSS) cases, 5 (1.2%) were dMMR. The independent factors associated with MSI-H/dMMR included meeting Amsterdam II criteria, assay purpose, and sampling method. Multivariate analysis revealed that cancer type (gastrointestinal cancers or others) was associated with concordance of MSI and MMR IHC. Three LS cases with pancreatic or endometrial cancer demonstrated MSS and dMMR, and one biliary cancer showed MSI-H and pMMR. Discordance between MSI and MMR IHC occasionally occurs in pancreaticobiliary and endometrial cancers. When suspected, both MSI and MMR IHC should be done to judge the ICI indication and screen for LS.

Case report: Undifferentiated sarcoma with multiple tumors involved in Lynch syndrome: Unexpected favorable outcome to sintilimab combined with chemotherapy

Patients with Lynch syndrome are at an increased risk of developing simultaneous or metachronous tumors, while sarcomas have been occasionally reported. Sarcomas are generally not considered part of the common Lynch syndrome tumor spectrum. However, more and more studies and case reports suggested that sarcoma could be a rare clinical manifestation of Lynch syndrome, leading to new treatment strategies for sarcoma. We report the case of a 74-year-old male patient with Lynch syndrome who had rectal mucinous adenocarcinoma and prostate adenocarcinoma and then developed undifferentiated sarcoma of the left neck two years later. Mismatch repair deficiency (dMMR) was confirmed by immunohistochemical staining for the mismatch repair proteins MSH2, MSH6, MLH1 and PMS2. The result of polymerase chain reaction (PCR) microsatellite instability (MSI) testing of sarcoma showed high-level microsatellite instability (MSI-H). Additionally, a pathogenic germline mutation in MSH2 (c.2459-12A>G) was detected by next-generation sequencing (NGS). Taking into account HE morphology, immunohistochemical phenotype, MSI status, NGS result, medical history and germline MSH2 gene mutation, the pathological diagnosis of left neck biopsy tissue was Lynch syndrome related undifferentiated sarcoma with epithelioid morphology. The patient has been receiving immunotherapy (sintilimab) combined with chemotherapy (tegafur, gimeracil and oteracil potassium capsules) and currently has stable disease. We also reviewed the literature to understand the association between sarcoma and Lynch syndrome. Sarcoma may now be considered a rare clinical manifestation of Lynch syndrome. Attention and awareness about the association between Lynch syndrome and sarcoma need to be increased. Therefore, timely detection of MMR proteins and validation at the gene level for suspicious patients are the keys to avoiding missed or delayed diagnosis and to identifying patients suited for immunotherapy, which may also help to provide appropriate genetic counseling and follow-up management for patients.

Unifocal Peritoneal Malignant Mesothelioma as Initial Presentation of Lynch Syndrome: A Case Report and Brief Review

Abstract Introduction/Objective Peritoneal malignant mesothelioma (pMM) has scarcely been reported in the context of Lynch Syndrome (LS). No cases have directly led to a diagnosis of LS. We report an unusual case of pMM presenting as a localized mass, leading to the diagnosis of LS. Methods/Case Report A 55 year old female with no prior oncologic history presented with a solitary epigastric mass. Her mother and maternal relatives had colonic, gastric and ovarian cancers. Imaging studies revealed a perigastric mass abutting the left liver. The resected 9.8 cm mass showed a variegated histology with tubulopapillary formations and solid sheets. The neoplastic cells also showed variable cytologic features including anisocytosis, pleomorphic nuclei, multinucleation, prominent nucleoli, intranuclear pseudoinclusions, and rhabdoid features. A dense intratumoral and peritumoral lymphoplasmacytic infiltrate was present. Epithelioid pMM was confirmed with expression of cytokeratin 5/6, calretinin, WT-1, and thrombomodulin and loss of MOC31. Abnormal loss of MSH2 and MSH6 was seen (retained expression MLH1/PMS2 expression), suggesting high-level microsatellite instability. A germline pathogenic variant of MSH2 was identified. Results (if a Case Study enter NA) NA. Conclusion We report the first case of LS presenting as an atypical (unifocal) pMM showing variegated histology and lymphocytic infiltrate. Microsatellite instability in pMM is rare and has only been reported in known LS patients. Awareness of the pMM within LS is evolving and atypical features may prompt evaluation of DNA microsatellite repair system, with implications for the patient (treatment/screening) and their family.

S2204 A Rare Case of Medullary Carcinoma of the Colon With Lymphatic Metastasis

Introduction: Medullary carcinoma of the colon (MAC) is a rare cancer encompassing only .03% of all colorectal carcinomas. We present a rare case of MAC occurring in an older adult with a strong tobacco use history. Case Description/Methods: A 76-year-old female with PMHx of tobacco use disorder, COPD, and known pulmonary nodules presented to the ED for surveillance of her pulmonary nodules. A review of systems was positive for chronic shortness of breath and cough. A CT scan of the chest revealed enlarging lung nodules and mediastinal adenopathy. A PET scan revealed a hypermetabolic 2.5 x 2.5 cm soft mass in the distal ascending colon and hypermetabolic lymph nodes in the right upper quadrant (Figure). Colonoscopy revealed an infiltrative 2 cm mass at the hepatic flexure, and pathology revealed an invasive, poorly differentiated adenocarcinoma with normal expression of MSH2 and MSH6 and absence of MLH1 and PMS2. These findings were consistent with medullary carcinoma of the colon. Endoscopic ultrasound with biopsy of peripancreatic and porta-hepatis lymph nodes favored metastatic medullary carcinoma. The patient underwent a right hemicolectomy and follows closely outpatient. Discussion: MAC is a rare cancer associated with a high level of microsatellite instability and deficient mismatch repair proteins. It is a non-gland forming cancer composed of large polygonal eosinophilic cells growing in solid sheets and infiltrated with small lymphocytes. The majority of reported cases revealed a lack of MLH-1 and PMS2, just as our patient did. They instead express markers that are not commonly associated with colorectal cancers, such as calretinin, CK7, SATB2, and CDH17. MACs were previously difficult to differentiate from poorly differentiated adenocarcinomas due to their unique tumor marker expression and histopathology. The current standard for diagnosis of MAC therefore requires distinct microscopic morphology and molecular staining patterns. There are no current treatment guidelines due to the rarity of MACs, although MACs do have a favorable prognosis when compared to other adenocarcinomas and surgical resection and FOLFOX chemotherapy have been used successfully in some studies. Although MACs have been sporadically reported in case reports, further studies are required to determine guidelines for both diagnosis and treatment of this rare disease.Figure 1.: Endoscopic Visualization with Final Needle Aspiration of Abnormal Lymph Node.

Current progress and future perspectives of neoadjuvant anti-PD-1/PD-L1 therapy for colorectal cancer.

Immunotherapies, especially the programmed cell death 1/programmed cell death ligand 1 (PD-1/PD-L1) inhibitors, have revolutionized the therapeutic strategies of various cancers. As for colorectal cancer (CRC), the current clinical application of PD-1/PD-L1 inhibitors are mainly used according to the mutation pattern, which is categorized into deficient mismatch repair (dMMR)/high levels of microsatellite instability (MSI-H) and proficient mismatch repair (pMMR), or non-high levels of microsatellite instability (non-MSI-H). PD-1/PD-L1 inhibitors have been proven to have favorable outcomes against dMMR/MSI-H CRC because of more T-cell infiltration into tumor tissues. Nevertheless, the effectiveness of PD-1/PD-L1 inhibitors in pMMR/non-MSI-H CRC is still uncertain. Because of the quite-lower proportion of dMMR/MSI-H in CRC, PD-1/PD-L1 inhibitors have been reported to combine with other antitumor treatments including chemotherapy, radiotherapy, and targeted therapy for better therapeutic effect in recent clinical trials. Neoadjuvant therapy, mainly including chemotherapy and radiotherapy, not only can reduce clinical stage but also benefit from local control, which can improve clinical symptoms and the quality of life. Adding immunotherapy into neoadjuvant therapy may change the treatment strategy of primary resectable or some metastatic CRC. In this review, we focus on the development of neoadjuvant anti-PD-1/PD-L1 therapy and discuss the future perspectives in CRC.

121P Spectrum of druggable gene fusions in microsatellite-unstable colorectal tumors

Colorectal carcinomas (CRCs) with high-level microsatellite instability (MSI-H) often contain gene fusions, which involve receptor tyrosine kinases. This study included 105 MSI-H CRCs. NTRK1/2/3, ALK, ROS1 and RET gene fusions were analyzed by PCR test for 5’/3’-end unbalanced expression, which is capable of detecting all translocation variants. The type of rearrangements was subsequently determined by variant-specific PCR for common fusions, and, wherever necessary, by targeted RNA next-generation sequencing (NGS). NTRK1/2/3 translocations were the most common, being detected in 8/105 (8%) CRCs (TPM3-NTRK1 (T8;N10): n = 2; ETV6-NTRK2 (E5;N15): n = 2; ETV6-NTRK3 (E5;N15): n = 4). There were 2 tumors with RET rearrangements (CCDC6-RET (C1;R12) and NCOA4-RET (N9del501;R12), respectively) and 1 instance of SPTBN1-ALK (S7;A20) chimera. 76/105 (72%) CRCs were negative for KRAS, NRAS or BRAF mutations; gene rearrangements were detected in 10 (13%) of these tumors. 29/105 (28%) carcinomas carried activating lesions in RAS/RAF oncogenes; one of these CRCs had both ETV6-NTRK3 (E5;N15) translocation and BRAF V600E substitution. Microsatellite-unstable CRCs have high frequency of druggable gene rearrangements, with NTRK1/2/3 deserving particular attention.