Genes da instabilidade de microssatélites no câncer colorretal: papel na inflamação e estresse oxidativo / Microsatellite instability genes in colorectal cancer: role in inflammation and oxidative stress

A instabilidade de microssatélites é um fenômeno genético caracterizado pela alteração na repetição de sequências de nucleotídeos conhecidas como microssatélites. Esta instabilidade pode ocorrer devido a defeitos nos genes reparadores de DNA, como os genes MLH1, MSH2, MSH6 e PMS2. A inflamação crônica tem sido associada ao desenvolvimento do câncer colorretal. Os genes da instabilidade de microssatélites estão envolvidos na regulação da resposta inflamatória, podendo influenciar a progressão tumoral. Estudos demonstraram que a presença de instabilidade de microssatélites em tumores colorretais está relacionada a uma maior infiltração de células imunes, como linfócitos T, macrófagos e neutrófilos, que podem modular a resposta inflamatória no microambiente tumoral. O estresse oxidativo é caracterizado pelo desequilíbrio entre a produção de espécies reativas de oxigênio e a capacidade antioxidante do organismo e desempenha um papel importante na carcinogênese. Os genes da instabilidade de microssatélites podem influenciar a resposta ao estresse oxidativo, afetando a capacidade das células tumorais de lidar com o dano oxidativo e promovendo a sobrevivência celular. O objetivo deste trabalho consiste na compreensão dos genes envolvidos na instabilidade de microssatélites no câncer colorretal e como eles contribuem para o desenvolvimento da doença, relacionando com processos inflamatórios e estresse oxidativo nas células tumorais. Justifica-se pela necessidade de compreensão das interconexões entre a instabilidade de microssatélites, inflamação e o estresse oxidativo em pacientes com câncer colorretal.

Microsatellite instability is a genetic phenomenon characterized by changes in the repetition of nucleotide sequences known as microsatellites. This instability may occur due to defects in DNA repair genes, such as the MLH1, MSH2, MSH6 and PMS2 genes. Chronic inflammation has been linked to the development of colorectal cancer. Microsatellite instability genes are involved in regulating the inflammatory response and may influence tumor progression. Studies have shown that the presence of microsatellite instability in colorectal tumors is related to a greater infiltration of immune cells, such as T lymphocytes, macrophages and neutrophils, which can modulate the inflammatory response in the tumor microenvironment. Oxidative stress is characterized by the imbalance between the production of reactive oxygen species and the body's antioxidant capacity and plays an important role in carcinogenesis. Microsatellite instability genes can influence the response to oxidative stress, affecting the ability of tumor cells to deal with oxidative damage and promoting cell survival. The objective of this work is to understand the genes involved in microsatellite instability in colorectal cancer and how they contribute to the development of the disease, relating it to inflammatory processes and oxidative stress in tumor cells. It is justified by the need to understand the interconnections between microsatellite instability, inflammation and oxidative stress in patients with colorectal cancer.

Integrative analysis of the immunological significances of guanylate binding protein family genes in microsatellite stability colorectal cancer.

Background: Microsatellite stability (MSS) colorectal cancer (CRC) has poor sensitivity to immunotherapy and its underlying mechanisms are still unclear. Guanylate binding proteins (GBPs) are a family of GTPase involving innate immune responses by providing defense against invading microbes and pathogens. However, the immunological significances of GBPs in MSS CRC remain unknown. Methods: We utilized bioinformatic tools to comprehensively analysis the expression pattern, clinical relevance, prognostic value, biological function, and immunoregulation effect of distinct GBP members in MSS CRC. Results: The expression of all seven GBPs in MSS samples are remarkably decreased compared to microsatellite instability-high (MSI-H) samples. Among them, GBP1/2/4/5 are obviously correlated with distant metastasis status. High expression of GBP1/4/5/6 was remarkably related to favorable overall survival (OS) and progression-free survival (PFS) in CRC patients with MSS tumor. Subsequent enrichment analysis revealed that Interferon-gamma (IFN-γ) and NOD-like receptor signaling are the most relevant functions. Besides, the expression patterns of GBPs are remarkably associated with several tumor infiltrated immune cells (e.g. regulatory T cells, CD4+ T cells, and macrophages) and diverse immunoregulatory molecules (e.g. immune checkpoint biomarkers (ICBs) and major histocompatibility complex (MHC) molecules). Moreover, high GBP1/2/4/5 expression predicted better immunotherapy responsiveness in immunotherapy cohorts. Conclusion: These findings might provide novel insights for the identification of therapeutic targets and potential prognostic biomarkers of GBP family in CRC with MSS samples.

Simvastatin induces ferroptosis and activates anti-tumor immunity to sensitize anti-PD-1 immunotherapy in microsatellite stable gastric cancer.

BACKGROUND: Gastric cancer (GC), especially the case with microsatellite stability (MSS) phenotype, has limited efficacy for immune checkpoint blockade (ICB) therapy. Metabolism reprogramming is newly recognized to affect tumor immune microenvironment (TIME). However, the relationship between metabolism reprogramming and immunotherapy for MSS GC has not been reported. METHODS: A metabolic stratification for GC was developed based on the glycolysis/cholesterol synthesis axis using the R package "ConsensusClusterPlus". The T cell inflamed score was used to define "immune-hot" and "immune-cold" phenotypes in MSS GC. The anti-tumor and immunological effects of simvastatin were explored using in vitro and in vivo experiments. RESULTS: Three metabolic subtypes were identified in GC patients, including cholesterol, glycolysis and quiescent subtypes. The cholesterol subtype was associated with poorer clinical features and higher tumor purity. Correspondingly, we demonstrated that simvastatin, a specific inhibitor of cholesterol synthesis, significantly inhibited the proliferation, migration, and induced ferroptosis in GC cells. Interestingly, simvastatin markedly inhibited tumor growth in immunocompetent mice, while no significant effect in immunodeficient mice. Upregulation of chemokines and increased recruitment of CD8+ T cells were observed after simvastatin treatment. Consistently, the cholesterol subtype exhibited a less inflamed TIME and coincided significantly with the "immune-cold" phenotype of MSS GC. Finally, we confirmed simvastatin enhanced PD-1 blockade efficacy via modulating the TIME and activating anti-tumor immunity in tumor-bearing mice. CONCLUSION: Our data revealed the significance of cholesterol synthesis in GC and demonstrated simvastatin served as a promising sensitizer for ICB therapy by inducing ferroptosis and anti-tumor immunity in MSS GC patients.

The ATOM-Seq sequence capture panel can accurately predict microsatellite instability status in formalin-fixed tumour samples, alongside routine gene mutation testing.

Microsatellite instability (MSI) occurs across a number of cancers and is associated with different clinical characteristics when compared to microsatellite stable (MSS) cancers. As MSI cancers have different characteristics, routine MSI testing is now recommended for a number of cancer types including colorectal cancer (CRC). Using gene panels for sequencing of known cancer mutations is routinely performed to guide treatment decisions. By adding a number of MSI regions to a small gene panel, the efficacy of simultaneous MSI detection in a series of CRCs was tested. Tumour DNA from formalin-fixed, paraffin-embedded (FFPE) tumours was sequenced using a 23-gene panel kit (ATOM-Seq) provided by GeneFirst. The mismatch repair (MMR) status was obtained for each patient from their routine pathology reports, and compared to MSI predictions from the sequencing data. By testing 29 microsatellite regions in 335 samples the MSI status was correctly classified in 314/319 samples (98.4% concordance), with sixteen failures. By reducing the number of regions in silico, comparable performance could be reached with as few as eight MSI marker positions. This test represents a quick, and accurate means of determining MSI status in FFPE CRC samples, as part of a routine gene mutation assay, and can easily be incorporated into a research or diagnostic setting. This could replace separate mutation and MSI tests with no loss of accuracy, thus improving testing efficiency.

Cuproptosis in microsatellite stable colon cancer cells affects the cytotoxicity of CD8+T through the WNT signaling pathway.

The microsatellite stable (MSS) colon cancer (CC) has long been considered resistant to immunotherapy. Cuproptosis, as a novel form of cell death, may interact with tumor immunity. This project focused on the impact of cuproptosis on the cytotoxicity of CD8+T in MSS CC, aiming to provide effective clues for improving the treatment strategy of MSS CC. The study developed an MSS CC cuproptosis model using 50 nM elesclomol and 1 µM CuCl2. Cuproptotic SW480 cells were directly co-cultured with CD8+ T cells. Cuproptosis levels were assessed via intracellular copper ion detection, western blot, and confocal laser scanning microscopy. CCK-8, Hochest/PI staining, CFSE cell proliferation assay, LDH cytotoxicity detection, and ELISA were used to evaluate CD8+ T cell immune activity and cytotoxicity. Transcriptome sequencing and bioinformatics analysis identified regulated signals in cuproptotic SW480 cells. A rescue experiment utilized a WNT pathway activator (BML-284). PD-L1 expression in cells/membranes was analyzed using qRT-PCR, western blot, and flow cytometry. NSG mice were immunoreconstituted, and the effects of cuproptosis on immune infiltration and cancer progression in MSS CC mice were assessed using ELISA and immunohistochemistry (IHC). Treatment with 50 nM elesclomol and 1 µM CuCl2 significantly increased cuproptosis in SW480 cells. Co-culture with CD8+ T cells enhanced their cytotoxicity. Sequencing revealed cuproptosis-mediated modulation of immune and inflammatory pathways, including WNT signaling. Rescue experiments showed downregulation of WNT signaling in cuproptotic SW480 cells. Indirectly, CD8+ T cell immune function was enhanced by reducing PD-L1 expression. In mice, cuproptosis resulted in increased infiltration of CD8+ T cells in tumor tissue, leading to delayed cancer progression compared to the control group. Cuproptosis in MSS CC cells enhances the cytotoxicity of CD8+ T cells, which may be achieved through downregulation of the WNT signaling pathway and decreased expression of PD-L1. In the future, drugs that can induce cuproptosis may be a promising approach to improve MSS CC immunotherapy.

A single-institution retrospective study of comprehensive genomic profiling tests based on C-CAT findings for advanced solid cancers.

BACKGROUND: In recent years, as the availability of precision therapies expands, there is increasing reliance on genomic profiling assays to help identify the most appropriate treatment options for patients with advanced cancers. We retrospectively investigated the results of comprehensive genomic profiling tests from the time insurance coverage began until recently and examined the status of genetic analysis. METHODS: We retrospectively reviewed the analysis results of 300 patients with advanced solid tumors who consented to comprehensive genomic profiling tests from October 2019 to December 2022. RESULTS: Of the 300 patients who underwent comprehensive genomic profiling tests, analysis results for 274 patients were obtained, and were reviewed by the Clinical Genome Expert Panel. Six specimens (2%) were discontinued due to patient deaths and deteriorations in general condition. The three most frequently occurring actionable genomic alterations observed were TP53 (47.4%), KRAS (28.1%) and CDKN2A (20.4%). The most common druggable variant was CDKN2A, which was noted in 52 (19%) of 274 patients. The next most common were PIK3CA, BRAF, KRAS and PTEN. The cancer types that showed a greater median number of actionable alterations comprised thyroid cancer, pancreatic cancer and colorectal cancer. CONCLUSIONS: In conclusion, comprehensive genomic profiling tests have the potential to be valuable in identifying genomic abnormalities. Even if there is no effective treatment at present, it may lead to a treatment in the future. Comprehensive genomic profiling tests should be considered for any cancer.

Clinicopathologic and molecular characterization of stages II-IV gastric cancer with Claudin 18.2 expression.

BACKGROUND: Claudin 18.2 (CLDN18.2) is a promising target for targeted therapies in gastric cancer (GC). This study investigated the prevalence of CLDN18.2 expression in patients with stages II-IV GC or gastroesophageal junction (GEJ) adenocarcinoma and its correlation with clinicopathologic features and other crucial GC biomarkers. METHODS: We enrolled 1000 patients diagnosed with stages II-IV GC after surgical treatment. Immunohistochemistry for CLDN18 (43-14A clone), PD-L1 (22C3 pharmDx), HER2, and FGFR2 was performed. CLDN18.2 positivity was defined as moderate-to-strong (2+/3+) membranous staining in ≥75% of tumor cells. CLDN18.2 expression was compared with biomarker expression, Epstein-Barr virus (EBV) association and microsatellite instability status, and clinicopathologic features. RESULT: CLDN18.2 was positive in 34.4% of the patients. CLDN18.2 positivity was significantly higher in the middle and upper thirds than in the lower third gastric location (P < .001), but there was no correlation with age, sex, or stage (P > .05). CLDN18.2 positivity was rare (2.8%) in mucinous adenocarcinoma but frequent (90.9%) in a majority of gastric carcinomas with lymphoid stroma. CLDN18.2 positivity was higher in EBV-associated (P < .001) and PD-L1-positive (PD-L1 CPS ≥ 5) GC (P = .014) but lower in HER2 positive GC (P = .005). CLDN18.2 positivity was not significantly associated with overall survival and disease-free survival. CONCLUSION: This study provides a comprehensive evaluation of CLDN18.2 status and its correlation with the clinicopathologic characteristics of patients with stages II-IV GC in Korea and with crucial biomarkers. It may be valuable for guiding future drug development, expanding treatment options, and ultimately improving patient outcomes in GC.

Stage IV gastric cancer with microsatellite instability-high achieving long-term survival by gastrectomy after nivolumab as third-line therapy: a case report and literature review.

BACKGROUND: The prognosis for stage IV gastric cancer remains poor; however, the advent of immune checkpoint inhibitors (ICIs) such as nivolumab has increased the number of patients with long-term survival. Patients with microsatellite instability (MSI)-high gastric cancer have been recognized as a highly effective population for ICIs. Herein, we report a patient with MSI-high advanced gastric cancer treated with gastrectomy after the administration of nivolumab as third-line therapy. CASE PRESENTATION: A 73-year-old woman presented with a type 3 tumor in the lower part of the gastric body, which was diagnosed as gastric cancer through biopsy. Staging laparoscopy revealed that the tumor had invaded the pancreas and the posterior lobe of the transverse mesocolon, and disseminated nodules were found near the ligament of Treitz. After 4 courses of S-1 plus cisplatin therapy, laparoscopic gastrojejunal bypass was performed because of difficulty in oral intake. She received S-1 plus oxaliplatin therapy after a gastrojejunal bypass; however, her regional lymph nodes were enlarged. After six courses of paclitaxel plus ramucirumab as second-line chemotherapy, computed tomography (CT) showed exacerbation of peritoneal dissemination; thus, nivolumab was selected as the third-line therapy. The tumor was characterized by MSI-high. At 24 courses, CT and gastroscopy revealed a complete clinical response of the tumor; however, re-growth of the primary tumor was observed at 36 courses. The patient underwent distal gastrectomy with D1 + lymph node dissection, and received S-1 monotherapy as adjuvant therapy for 1 year. No recurrence was noted at 39 months after the surgery. CONCLUSIONS: We report a patient with highly advanced gastric cancer with peritoneal dissemination, which worsened during second-line therapy and was successfully treated with gastrectomy after nivolumab administration as a third-line therapy. MSI-high gastric cancer is a target that should be actively considered for the administration of ICIs, such as nivolumab, and multidisciplinary treatment combined with chemotherapy and gastrectomy, including conversion surgery, can lead to patients' long-term survival.

Use of the EmsB microsatellite-based next generation sequencing for genotyping of Echinococcus granulosus sensu lato in hydatid cyst tissue samples from animals and humans.

Echinococcus granulosus (Batsch, 1786), a cestode of the Teniidae family, causes human cystic echinococcosis (CE) also known as hydatid disease. Echinococcus granulosus sensu lato includes the G1, G3, G4, G5, G6/7 and G8/10 genotypes which are known to cause human CE. This study aimed to differentiate genotypes of E. granulosus s.l. complex by employing EmsB, a tandemly repeated multilocus microsatellite, using next-generation sequencing (MIC-NGS). Human and animal histopathology-confirmed hydatid cyst tissue samples and reference DNA samples of E. granulosus G1, G3, G4, G5, G6/7 and G10 underwent MIC-NGS assay with custom primers amplifying a 151 bp EmsB DNA fragment. NGS data were analysed using online Galaxy analysis pipeline, a phylogenetic tree was constructed by MEGA software, and haplotype networking was performed with PopArt 1.7. All sixty samples (49 from animals and 11 from humans) included were successfully identified and genotyped with a 100 % success rate. The study showed improved discrimination power to distinguish all study samples including closely related E. granulosus s.s. genotypes G1-G3. The maximum likelihood tree reaffirmed the monophyly of E. granulosus s.l. The median-joining haplotype networking revealed 12 distinct haplotypes. In conclusion, MIC-NGS assay was shown to be sensitive, specific and simple to apply to clinical samples offering a powerful discriminatory tool for the genotyping of E. granulosus s.l.

Mining expressed sequence tag (EST) microsatellite markers to assess the genetic differentiation of five Hynobius species endemic to Taiwan.

Taiwan harbors five endemic species of salamanders (Hynobius spp.) that inhabit distinct alpine regions, contributing to population fragmentation across isolated "sky islands". With an evolutionary history spanning multiple glacial-interglacial cycles, these species represent an exceptional paradigm for exploring biogeography and speciation. However, a lack of suitable genetic markers applicable across species has limited research efforts. Thus, developing cross-amplifying markers is imperative. Expressed sequence-tag simple-sequence repeats (EST-SSRs) that amplify across divergent lineages are ideal for species identification in instances where phenotypic differentiation is challenging. Here, we report a suite of cross-amplifying EST-SSRs from the transcriptomes of the five Hynobius species that exhibit an interspecies transferability rate of 67.67%. To identify individual markers exhibiting cross-species polymorphism and to assess interspecies genetic diversity, we assayed 140 individuals from the five species across 84 sampling sites. A set of EST-SSRs with a high interspecies polymorphic information content (PIC = 0.63) effectively classified these individuals into five distinct clusters, as supported by discriminant analysis of principal components (DAPC), STRUCTURE assignment tests, and Neighbor-joining trees. Moreover, pair-wise FST values > 0.15 indicate notable between-cluster genetic divergence. Our set of 20 polymorphic EST-SSRs is suitable for assessing population structure within and among Hynobius species, as well as for long-term monitoring of their genetic composition.

Single-cell AI-based detection and prognostic and predictive value of DNA mismatch repair deficiency in colorectal cancer.

Testing for DNA mismatch repair deficiency (MMRd) is recommended for all colorectal cancers (CRCs). Automating this would enable precision medicine, particularly if providing information on etiology not captured by deep learning (DL) methods. We present AIMMeR, an AI-based method for determination of mismatch repair (MMR) protein expression at a single-cell level in routine pathology samples. AIMMeR shows an area under the receiver-operator curve (AUROC) of 0.98, and specificity of ≥75% at 98% sensitivity against pathologist ground truth in stage II/III in two trial cohorts, with positive predictive value of ≥98% for the commonest pattern of somatic MMRd. Lower agreement with microsatellite instability (MSI) testing (AUROC 0.86) reflects discordance between MMR and MSI PCR rather than AIMMeR misclassification. Analysis of the SCOT trial confirms MMRd prognostic value in oxaliplatin-treated patients; while MMRd does not predict differential benefit of chemotherapy duration, it correlates with difference in relapse by regimen (PInteraction = 0.04). AIMMeR may help reduce pathologist workload and streamline diagnostics in CRC.

Ipilimumab with Nivolumab in molecular-Selected patients with castration-resistant PRostate cancer: primary analysis of the Phase 2 INSPIRE trial.

BACKGROUND: Metastatic castration-resistant prostate cancer (mCRPC) typically exhibits resistance to immune checkpoint inhibitors (ICIs). However, a subset of mCRPC patients displays a more immunogenic profile. This study examines efficacy and safety of dual ICI therapy in molecularly selected mCRPC. PATIENTS AND METHODS: This single-arm, phase II trial, included 69 molecularly selected mCRPC patients with mismatch repair deficiency (MMRd), non-synonymous tumour mutational burden ≥7.1 muts/Mb (hTMB), a BRCA2 mutation (BRCAm), or biallelic CDK12 inactivation (CDK12i). Efficacy was assessed in ICI-naïve patients (cohort A) with RECIST1.1 (A1) and PCWG3 (A2) measurable disease. Safety was evaluated in cohorts A and B (prior ICI monotherapy). Treatment included nivolumab 3 mg/kg and ipilimumab 1 mg/kg every 3 weeks for 4 cycles, followed by nivolumab 480 mg every 4 weeks for up to 1 year. The primary endpoint was disease control rate beyond 6 months (DCR>6), aiming to surpass a DCR>6 of 22%. RESULTS: Patients initiated treatment between January 2021 and February 2024. Cohort A included 65 patients. Of these, 21 had MMRd (32%), 8 hTMB (12%), 20 BRCAm (31%), and 16 CDK12i (25%). DCR>6 was achieved in 38% (95% CI 27-51), and was highest in MMRd (81%), followed by hTMB (25%), CDK12i (19%), and BRCAm (15%). Objective and PSA50 response rates in cohort A were 38% (95% CI 22-55) and 47% (95% CI 34-60), respectively. Median progression-free survival was 4.0 months (95% CI 3.5-12.0) in cohort A, and 32.7 months (95% CI 21.8-NR) in MMRd patients. Treatment-related adverse events (TRAEs) led to permanent discontinuation in 14 of 69 patients (20%). Grade ≥3 TRAEs occurred in 48% of patients, with diarrhoea and elevated transaminases each in 10%. There was one treatment-related death due to a bowel perforation and a second following euthanasia after grade 4 toxicity. CONCLUSIONS: This trial of dual ICIs in molecularly selected mCRPC met its primary endpoint, showing DCR>6 in 40% of patients. Dual ICIs exhibited modest responses in the hTMB, BRCAm and CDK12i subgroups, but demonstrated exceptional efficacy in MMRd.

Deep Learning Algorithm­Based MRI Radiomics and Pathomics for Predicting Microsatellite Instability Status in Rectal Cancer: A Multicenter Study.

RATIONALE AND OBJECTIVES: To develop and validate multimodal deep-learning models based on clinical variables, multiparametric MRI (mp-MRI) and hematoxylin and eosin (HE) stained pathology slides for predicting microsatellite instability (MSI) status in rectal cancer patients. MATERIALS AND METHODS: A total of 467 surgically confirmed rectal cancer patients from three centers were included in this study. Patients from center 1 were randomly divided into a training set (242 patients) and an internal validation (invad) set (105 patients) in a 7:3 ratio. Patients from centers 2 and 3 (120 patients) were included in an external validation (exvad) set. HE and immunohistochemistry (IHC) staining were analyzed, and MSI status was confirmed by IHC staining. Independent predictive factors were identified through univariate and multivariate analyses based on clinical evaluations and were used to construct a clinical model. Deep learning with ResNet-101 was applied to preoperative MRI (T2WI, DWI, and contrast-enhanced T1WI sequences) and postoperative HE-stained images to calculate deep-learning radiomics score (DLRS) and deep-learning pathomics score (DLPS), respectively, and to DLRS and DLPS models. Receiver operating characteristic (ROC) curves were plotted, and the area under the curve (AUC) was used to evaluate and compare the predictive performance of each model. RESULTS: Among all rectal cancer patients, 82 (17.6%) had MSI. Long diameter (LD) and pathological T stage (pT) were identified as independent predictors and were used to construct the clinical model. After undergoing deep learning and feature selection, a final set of 30 radiomics features and 30 pathomics features were selected to construct the DLRS and DLPS models. A nomogram combining the clinical model, DLRS, and DLPS was created through weighted linear combination. The AUC values of the clinical model for predicting MSI were 0.714, 0.639, and 0.697 in the training, invad, and exvad sets, respectively. The AUCs of DLPS and DLRS ranged from 0.896 to 0.961 across the training, invad, and exvad sets. The nomogram achieved AUC values of 0.987, 0.987, and 0.974, with sensitivities of 1.0, 0.963, and 1.0 and specificities of 0.919, 0.949, and 0.867 in the training, invad, and exvad sets, respectively. The nomogram outperformed the other three models in all sets, with DeLong test results indicating superior predictive performance in the training set. CONCLUSION: The nomogram, incorporating clinical data, mp-MRI, and HE staining, effectively reflects tumor heterogeneity by integrating multimodal data. This model demonstrates high predictive accuracy and generalizability in predicting MSI status in rectal cancer patients.

PROTAC-mediated conditional degradation of the WRN helicase as a potential strategy for selective killing of cancer cells with microsatellite instability.

Multiple studies have demonstrated that cancer cells with microsatellite instability (MSI) are intolerant to loss of the Werner syndrome helicase (WRN), whereas microsatellite-stable (MSS) cancer cells are not. Therefore, WRN represents a promising new synthetic lethal target for developing drugs to treat cancers with MSI. Given the uncertainty of how effective inhibitors of WRN activity will prove in clinical trials, and the likelihood of tumours developing resistance to WRN inhibitors, alternative strategies for impeding WRN function are needed. Proteolysis-targeting chimeras (PROTACs) are heterobifunctional small molecules that target specific proteins for degradation. Here, we engineered the WRN locus so that the gene product is fused to a bromodomain (Bd)-tag, enabling conditional WRN degradation with the AGB-1 PROTAC specific for the Bd-tag. Our data revealed that WRN degradation is highly toxic in MSI but not MSS cell lines. In MSI cells, WRN degradation caused G2/M arrest, chromosome breakage and ATM kinase activation. We also describe a multi-colour cell-based platform for facile testing of selective toxicity in MSI versus MSS cell lines. Together, our data show that a degrader approach is a potentially powerful way of targeting WRN in MSI cancers and paves the way for the development of WRN-specific PROTAC compounds.

PD-1 blockade combined with chemotherapy and bevacizumab in DNA mismatch repair-proficient/microsatellite stable colorectal liver metastases.

Background: Single-agent immunotherapy is less effective in patients with DNA mismatch repair-proficient/microsatellite stable (pMMR/MSS) metastatic colorectal cancer (mCRC). Whether pMMR/MSS mCRC patients benefit from combination immunotherapy remains unclear. This study aimed to evaluate the efficacy and safety of anti-programmed cell death protein 1 (PD-1) therapy combined with chemotherapy and bevacizumab in pMMR/MSS colorectal liver metastases (CRLM) patients. Methods: A total of 12 patients with pMMR/MSS CRLM treated at The Sixth Affiliated Hospital of Sun Yat-sen University were enrolled. All patients were treated with at least 4 doses of PD-1 monoclonal antibody combined with chemotherapy and bevacizumab as neoadjuvant/adjuvant therapy. Results: A total of 10 of the 12 patients received the combined therapies before primary tumor resection; the disease control rate (DCR) was 100% (10/10), and the objective response rate (ORR) was 70% (7/10). The ORR of liver metastases was 75% (9/12). Pathological complete response (pCR) was achieved in 1 primary tumor patient and 2 patients with hepatic lesions. A total of 5 patients underwent simultaneous resection of the primary tumor and liver metastases; 9 patients underwent microwave ablation for liver metastases. A total of 7 patients were assessed as having no evidence of disease (NED) with a median progression-free survival (PFS) interval of 9.2 (1.5-15.8) months after multimodality treatments for both primary and metastatic lesions. No severe immune-related adverse events (irAEs) and operational complications were observed. Conclusions: PD-1 blockade combined with chemotherapy and bevacizumab might be safe and effective for patients with pMMR/MSS CRLM. This treatment strategy might lead to better tumor regression and a higher chance of achieving NED.

Immune microenvironmental heterogeneity according to tumor DNA methylation phenotypes in microsatellite instability-high colorectal cancers.

The detailed association between tumor DNA methylation, including CpG island methylation, and tumor immunity is poorly understood. CpG island methylator phenotype (CIMP) is observed typically in sporadic colorectal cancers (CRCs) with microsatellite instability-high (MSI-H). Here, we investigated the differential features of the tumor immune microenvironment according to CIMP status in MSI-H CRCs. CIMP-high (CIMP-H) or CIMP-low/negative (CIMP-L/0) status was determined using MethyLight assay in 133 MSI-H CRCs. All MSI-H CRCs were subjected to digital pathology-based quantification of CD3 + /CD8 + /CD4 + /FoxP3 + /CD68 + /CD204 + /CD177 + tumor-infiltrating immune cells using whole-slide immunohistochemistry. Programmed death-ligand 1 (PD-L1) immunohistochemistry was evaluated using the tumor proportion score (TPS) and combined positive score (CPS). Representative cases were analyzed using whole-exome and RNA-sequencing. In 133 MSI-H CRCs, significantly higher densities of CD8 + tumor-infiltrating lymphocytes (TILs) were observed in CIMP-H tumors compared with CIMP-L/0 tumors. PD-L1 TPS and CPS in CIMP-H tumors were higher than in CIMP-L/0 tumors. Next-generation sequencing revealed that, compared with CIMP-L/0 tumors, CIMP-H tumors had higher fractions of CD8 + T cells/cytotoxic lymphocytes, higher cytolytic activity scores, and activated immune-mediated cell killing pathways. In contrast to CIMP-L/0 tumors, most CIMP-H tumors were identified as consensus molecular subtype 1, an immunogenic transcriptomic subtype of CRC. However, there were no differences in tumor mutational burden (TMB) between CIMP-H and CIMP-L/0 tumors in MSI-H CRCs. In conclusion, CIMP-H is associated with abundant cytotoxic CD8 + TILs and PD-L1 overexpression independent of TMB in MSI-H CRCs, suggesting that CIMP-H tumors represent a typical immune-hot subtype and are optimal candidates for immunotherapy in MSI-H tumors.

Genomic landscape of cutaneous, acral, mucosal, and uveal melanoma in Japan: analysis of clinical comprehensive genomic profiling data.

BACKGROUND: Cutaneous melanoma (CM) is the most common type in Caucasians, while acral melanoma (AM) and mucosal melanoma (MM), which are resistant to immunotherapies and BRAF/MEK-targeted therapies, are more common in East Asians. Genomic profiling is essential for treating melanomas, but such data are lacking in Japan. METHODS: Comprehensive genomic profiling data compiled in the Center for Cancer Genomics and Advanced Therapeutics (C-CAT) were analyzed. RESULTS: A total of 380 melanomas was analyzed, including 136 CM, 46 AM, 168 MM, and 30 uveal melanoma (UM). MM included conjunctival, sinonasal, oral, esophageal, anorectal, and vulvovaginal melanomas. No significant difference in the median tumor mutational burden (TMB) of CM (3.39 mutations/megabase), AM (2.76), and MM (3.78) was the key finding. Microsatellite instability-high status was found in one case. BRAF V600E/K was found in only 45 patients (12%). Key driver mutations in CM were BRAF (38%), NRAS (21%), NF1 (8%), and KIT (10%), with frequent copy number alterations (CNAs) of CDKN2A, CDKN2B, and MYC. AM was characterized by altered KIT (30%), NRAS (26%), and NF1 (11%) and CDKN2A, CDKN2B, CDK4, MDM2, and CCND1 CNAs. MM was characterized by altered NRAS (24%), KIT (21%), and NF1 (17%) and MYC, KIT, and CDKN2A CNAs, with differences based on anatomical locations. UM bore GNAQ or GNA11 driver mutations (87%) and frequent mutations in SF3B1 or BAP1. CONCLUSION: The distinct genomic profiling in Japanese patients, including lower TMB, compared to Caucasians, is associated with poorer treatment outcomes. This result underscores the need for more effective therapeutic agents.

Efficacy and safety of hepatic arterial infusion chemotherapy combined with fruquintinib and tislelizumab for patients with microsatellite stable colorectal cancer liver metastasis following failure of multiple-line therapy.

Background and aim: The prognosis of microsatellite stable (MSS)-colorectal cancer liver metastasis (CRCLM) following failure of multi-line therapy remains dismal. The aim of this study is to evaluate the efficacy and safety of hepatic arterial infusion chemotherapy (HAIC) plus fruquintinib and tislelizumab (HAIC-F-T treatment) for MSS-CRCLM which failed from multiple-line therapy. Methods: From February 2021 to June 2023, 45 patients with MSS-CRCLM after failure of multiple-line therapy who received HAIC combined with fruquintinib and tislelizumab (HAIC-F-T triple treatment) were enrolled. The combination therapy included HAIC regimens with oxaliplatin and 5-fluorouracil or irinotecan, oxaliplatin, and 5-fluorouracil on days 1-2, intravenous tislelizumab (200 mg) before HAIC on day 1, and oral fruquintinb (3 mg/d) on day 3-21, every 4 weeks. Overall survival (OS) and progression-free survival (PFS) were estimated using the Kaplan-Meier method. Results: The follow-up ended on June 22, 2024, with a median follow-up time of 17.5 months. The objective response rate was 42.2%, and the disease control rate was 82.2%. The median OS was 15.3 months (95% confidence interval [CI]:12.634-17.966), and the median PFS was 7.5 months (95% CI:5.318-9.682). The independent risk factors related to worse OS were previous PD-1 immunotherapy (P = 0.021) and the number of HAIC-F-T triple treatment cycles of ≤ 2 (P = 0.007). The incidence of grade 3 or higher adverse events (AEs) was 20%, with the most frequent grade 3 or higher AEs being abdominal pain (3/45, 6.7%). Conclusion: HAIC combined with fruquintinib and tislelizumab may be an alternative salvage treatment for patients with MSS-CRCLM following failure of multiple-line therapy.

Comprehensive Analysis of Microsatellite Instability in Canine Cancers: Implications for Comparative Oncology and Personalized Veterinary Medicine.

Microsatellite instability (MSI) is a crucial feature in cancer biology, yet its prevalence and significance in canine cancers remain largely unexplored. This study conducted a comprehensive analysis of MSI across 10 distinct canine cancer histotypes using whole-exome sequencing data from 692 tumor-normal sample pairs. MSI was detected in 64% of tumors, with prevalence varying significantly among cancer types. B-cell lymphomas exhibited the highest MSI burden, contrasting with human studies. A novel "MSI-burden" score was developed, correlating significantly with tumor mutational burden. MSI-high (MSI-H) tumors showed elevated somatic mutation counts compared to MSI-low and microsatellite stable tumors. The study identified 3632 recurrent MSI-affected genomic regions across cancer types. Notably, seven of the ten cancer types exhibited MSI-H tumors, with prevalence ranging from 1.5% in melanomas to 37% in B-cell lymphomas. These findings highlight the potential importance of MSI in canine cancer biology and suggest opportunities for targeted therapies, particularly immunotherapies. The high prevalence of MSI in canine cancers, especially in B-cell lymphomas, warrants further investigation into its mechanistic role and potential as a biomarker for prognosis and treatment response.

Exploring the value of multiple preprocessors and classifiers in constructing models for predicting microsatellite instability status in colorectal cancer.

Approximately 15% of patients with colorectal cancer (CRC) exhibit a distinct molecular phenotype known as microsatellite instability (MSI). Accurate and non-invasive prediction of MSI status is crucial for cost savings and guiding clinical treatment strategies. The retrospective study enrolled 307 CRC patients between January 2020 and October 2022. Preoperative images of computed tomography and postoperative status of MSI information were available for analysis. The stratified fivefold cross-validation was used to avoid sample bias in grouping. Feature extraction and model construction were performed as follows: first, inter-/intra-correlation coefficients and the least absolute shrinkage and selection operator algorithm were used to identify the most predictive feature subset. Subsequently, multiple discriminant models were constructed to explore and optimize the combination of six feature preprocessors (Box-Cox, Yeo-Johnson, Max-Abs, Min-Max, Z-score, and Quantile) and three classifiers (logistic regression, support vector machine, and random forest). Selecting the one with the highest average value of the area under the curve (AUC) in the test set as the radiomics model, and the clinical screening model and combined model were also established using the same processing steps as the radiomics model. Finally, the performances of the three models were evaluated and analyzed using decision and correction curves.We observed that the logistic regression model based on the quantile preprocessor had the highest average AUC value in the discriminant models. Additionally, tumor location, the clinical of N stage, and hypertension were identified as independent clinical predictors of MSI status. In the test set, the clinical screening model demonstrated good predictive performance, with the average AUC of 0.762 (95% confidence interval, 0.635-0.890). Furthermore, the combined model showed excellent predictive performance (AUC, 0.958; accuracy, 0.899; sensitivity, 0.929) and favorable clinical applicability and correction effects. The logistic regression model based on the quantile preprocessor exhibited excellent performance and repeatability, which may further reduce the variability of input data and improve the model performance for predicting MSI status in CRC.