Microsatellite Instability In Cancer Research Articles

Discovery of WRN inhibitor HRO761 with synthetic lethality in MSI cancers

The Werner syndrome RecQ helicase WRN was identified as a synthetic lethal target in cancer cells with microsatellite instability (MSI) by several genetic screens1–6. Despite advances in treatment with immune checkpoint inhibitors7–10, there is an unmet need in the treatment of MSI cancers11–14. Here we report the structural, biochemical, cellular and pharmacological characterization of the clinical-stage WRN helicase inhibitor HRO761, which was identified through an innovative hit-finding and lead-optimization strategy. HRO761 is a potent, selective, allosteric WRN inhibitor that binds at the interface of the D1 and D2 helicase domains, locking WRN in an inactive conformation. Pharmacological inhibition by HRO761 recapitulated the phenotype observed by WRN genetic suppression, leading to DNA damage and inhibition of tumour cell growth selectively in MSI cells in a p53-independent manner. Moreover, HRO761 led to WRN degradation in MSI cells but not in microsatellite-stable cells. Oral treatment with HRO761 resulted in dose-dependent in vivo DNA damage induction and tumour growth inhibition in MSI cell- and patient-derived xenograft models. These findings represent preclinical pharmacological validation of WRN as a therapeutic target in MSI cancers. A clinical trial with HRO761 (NCT05838768) is ongoing to assess the safety, tolerability and preliminary anti-tumour activity in patients with MSI colorectal cancer and other MSI solid tumours.

Abstract 3364: PELO is a synthetic lethal target in chromosome 9p21-deleted or MSI cancers

Abstract Homozygous deletion of chromosomal region 9p21.3 is one of the most frequently observed somatic copy number alterations in cancers, occurring in approximately 15% of all cancers. Many of the 9p21.3-deleted cancers are further characterized by poor clinical outcomes including subsets of glioblastoma, pancreatic, esophageal, and urothelial cancers. Large deletions of chromosomal regions may also provide unique therapeutic opportunities. Specifically, cancer-specific therapeutic opportunities can arise from co-deletions of passenger genes neighboring tumor suppressors. As such, we sought to identify novel therapeutic targets associated with Ch9p21.3 loss in cancers and identified the pelota mRNA surveillance and ribosome rescue factor (PELO) as the top preferential dependency in 9p21.3-deleted cancers. Intriguingly in another analysis, we identified PELO to be amongst the top preferential dependencies in cancers with microsatellite instability (MSI), which accounts for subsets of colorectal, endometrial, gastric, and ovarian cancers. Given the strength of PELO as a candidate synthetic lethal target in two distinct cancer subsets, we performed focused validation with CRISPR interference across multiple cell lines. Knockdown of PELO preferentially impaired the viability of MSI or 9p21-deleted as compared to control (9p21-proficient and microsatellite stable) cells. Further validation with patient-derived organoid and in vivo xenograft models confirmed these findings. We next sought to refine the predictive biomarkers for PELO dependency and identified loss of FOCAD (a 9p21.3 gene and an interactor of the SKI mRNA surveillance pathway) as necessary and sufficient for PELO dependency in the 9p21.3-deleted context. Our findings implicate splicing alterations involving TTC37, a key protein in the SKI mRNA surveillance complex, as the underlying cause of PELO dependency in MSI cancers. Together, we found that loss of large deletions of 9p21.3 and MSI independently impair the SKI complex, uncovering a dependency upon PELO and highlighting a novel functional interaction between PELO and the SKI complex. Furthermore, the strength of this synthetic lethal interaction in a broad range of cancers with poor outcomes underscores the potential of PELO as a promising drug target for patients with 9p21.3-deleted or MSI cancers. Citation Format: Edmond M. Chan, Patricia Borck, Nolan Bick, Kristina Jankovic, Isabella Boyle, Lucy Parker-Burns, Kyla Foster, Anthony Lau, Ashir Borah, Joshua Dempster, Francisca Vazquez. PELO is a synthetic lethal target in chromosome 9p21-deleted or MSI cancers [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 3364.

Abstract 7602: Discovery of GH1581, a potent and selective WRN inhibitor as a cancer therapy

Abstract Defects in DNA mismatch repair (MMR) proteins could induce damage of genome, resulting microsatellite instability (MSI). MSI is highly correlated with the occurrence of several types of cancers, mainly in colon, gastric, endometrial, and ovarian cancers. Despite advances in the treatment of MSI-high cancers, tumor evolution and drug resistance remain the leading causes of treatment failure and death in cancer patients. About 50% MSI cancers have no response to immune checkpoint inhibitors. There is still unmet needs to be fulfilled for MSI-high patients. WRN is a RecQ family helicase involves in genome stability maintenance, DNA repair, replication, transcription, and telomere maintenance. Recent studies have identified WRN as a synthetic lethal target of MSI-high cancers. Employing structure-based drug design strategy, we have discovered a potent and selective WRN inhibitor, GH1581. In vitro, GH1581 displayed specific < 10 nM potency in ADP-Glo assay and < 10 nM potency in cell proliferation assay using MSI cancer cell lines. In vivo, GH1581 demonstrated favorable pharmacokinetic properties in different species. In SW48 xenograft model, it inhibited the tumor growth with 100% TGI at QD dosing of 10 mg/kg without severe adverse effect on body weight. In summary, we discovered GH1581 as a novel inhibitor of WRN with high in vitro and in vivo efficacy. Citation Format: Jiapeng Li, Guiping Zhang, Jie Jack Li. Discovery of GH1581, a potent and selective WRN inhibitor as a cancer therapy [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 7602.

Targeting ATP-binding site of WRN Helicase: Identification of novel inhibitors through pocket analysis and Molecular Dynamics-Enhanced virtual screening

WRN helicase is a critical protein involved in maintaining genomic stability, utilizing ATP hydrolysis to dissolve DNA secondary structures. It has been identified as a promising synthetic lethal target for microsatellite instable (MSI) cancers. However, few WRN helicase inhibitors have been discovered, and their potential binding sites remain unexplored. In this study, we analyzed potential binding sites for WRN inhibitors and focused on the ATP-binding site for screening new inhibitors. Through molecular dynamics-enhanced virtual screening, we identified two compounds, h6 and h15, which effectively inhibited WRN's helicase and ATPase activity in vitro. Importantly, these compounds selectively targeted WRN's ATPase activity, setting them apart from other non-homologous proteins with ATPase activity. In comparison to the homologous protein BLM, h6 exhibits some degree of selectivity towards WRN. We also investigated the binding mode of these compounds to WRN's ATP-binding sites. These findings offer a promising strategy for discovering new WRN inhibitors and present two novel scaffolds, which might be potential for the development of MSI cancer treatment.

Discovering potential WRN inhibitors from natural product database through computational methods

Microsatellite instability (MSI) is a relatively common feature associated with multiple cancers, and Werner syndrome (WRN) ATP-dependent helicase has been recognized as a novel target for treating MSI cancers, such as colorectal cancer. A small-molecule inhibitor targeting WRN would be a promising strategy for treating colorectal cancer with high MSI expression. In this study, we employed a computer-assisted drug discovery strategy to screen over 30,000 natural product molecules. By using a combination of docking, ligand efficiency, Molecular Mechanics/Generalized Born Surface Area (MM/GBSA), and thermodynamic integration (TI) calculations, we identified MOL008980, MOL010740, MOL011832, T4743, TN1166, and TNP-002173 as potential WRN inhibitors. Subsequent molecular dynamics simulation revealed that these screened natural products possessed better binding dynamic characteristics than ATP substrate and were capable of inhibiting the dynamic process of WRN, making them potential strong ATP competitive inhibitors. In conclusion, our computational approach revealed potential WRN inhibitors from a natural product database, providing a theoretical basis for future research.

Predicting mechanism of immune response in microsatellite instability colorectal cancer

Colorectal cancer (CRC), also known as colon cancer, is the third most common cancer and the fourth most cause of cancer-related death in the world. CRC can be classified into two major subtypes, including microsatellite instability (MSI) and microsatellite stability (MSS), which showed different characteristics in immunotherapy. Low sensitivity of diagnostic biomarkers and metastasis are still the principal cause of mortality, especially in MSI. Here, applying computational programs, we identified recurring expression programs based on single cell RNA sequencing (scRNA-Seq) data of CRC cell lines. Notably, three MSI specific recurring modules were identified by non-negative matrix factorization (NMF). High NMF score genes enriched in the function of metabolism and inflammatory response. Focusing on top specific active transcription factor (TF), RUNX3 (Runt-related transcription factor 3), our results suggest that T cell infiltration was increased in RUNX3 high MSI CRC samples. Unbiased Gene Set Enrichment Analysis (GSEA) showed that RUNX3 was strongly associated with immune and metastasis related functions, such as Interferon Gamma (IFN-γ) and EPITHELIAL MESENCHYMAL TRANSITION (EMT). In addition, RUNX3 shows specific highly activated at epigenetic level in MSI compared with other gastrointestinal carcinomas. Positive correlation between RUNX3 and most immune checkpoints further confirmed RUNX3 might have crucial roles in MSI cancer progression and immunotherapy. Taken together, these results indicate significant tumor heterogeneity of two CRC subtypes at single-cell level and epigenetic modification level. These results also linked transcriptional dysregulation with immune infiltration at single-cell level in MSI, which may advance the application of scRNA-Seq technology in immunotherapy and contribute to developing novel biomarkers of this malignancy.

Discovery of thiophen-2-ylmethylene bis-dimedone derivatives as novel WRN inhibitors for treating cancers with microsatellite instability

Microsatellite instability (MSI) is a hypermutable condition caused by DNA mismatch repair system defects, contributing to the development of various cancer types. Recent research has identified Werner syndrome ATP-dependent helicase (WRN) as a promising synthetic lethal target for MSI cancers. Herein, we report the first discovery of thiophen-2-ylmethylene bis-dimedone derivatives as novel WRN inhibitors for MSI cancer therapy. Initial computational analysis and biological evaluation identified a new scaffold for a WRN inhibitor. Subsequent SAR study led to the discovery of a highly potent WRN inhibitor. Furthermore, we demonstrated that the optimal compound induced DNA damage and apoptotic cell death in MSI cancer cells by inhibiting WRN. This study provides a new pharmacophore for WRN inhibitors, emphasizing their therapeutic potential for MSI cancers.

Beyond PD(L)-1 Blockade in Microsatellite-Instable Cancers: Current Landscape of Immune Co-Inhibitory Receptor Targeting.

High microsatellite instability (MSI-H) derives from genomic hypermutability due to deficient mismatch repair function. Colorectal (CRC) and endometrial cancers (EC) are the tumor types that more often present MSI-H. Anti-PD(L)-1 antibodies have been demonstrated to be agnostically effective in patients with MSI-H cancer, but 50-60% of them do not respond to single-agent treatment, highlighting the necessity of expanding their treatment opportunities. Ipilimumab (anti-CTLA4) is the only immune checkpoint inhibitor (ICI) non-targeting PD(L)-1 that has been approved so far by the FDA for MSI-H cancer, namely, CRC in combination with nivolumab. Anti-TIM3 antibody LY3321367 showed interesting clinical activity in combination with anti-PDL-1 antibody in patients with MSI-H cancer not previously treated with anti-PD(L)-1. In contrast, no clinical evidence is available for anti-LAG3, anti-TIGIT, anti-BTLA, anti-ICOS and anti-IDO1 antibodies in MSI-H cancers, but clinical trials are ongoing. Other immunotherapeutic strategies under study for MSI-H cancers include vaccines, systemic immunomodulators, STING agonists, PKM2 activators, T-cell immunotherapy, LAIR-1 immunosuppression reversal, IL5 superagonists, oncolytic viruses and IL12 partial agonists. In conclusion, several combination therapies of ICIs and novel strategies are emerging and may revolutionize the treatment paradigm of MSI-H patients in the future. A huge effort will be necessary to find reliable immune biomarkers to personalize therapeutical decisions.

Clinical features and oncological outcomes of bladder cancer microsatellite instability.

Excellent anticancer effect for solid tumors with microsatellite instability (MSI)-high by anti-PD-1 antibody has been reported. In this study, we investigated the clinical impact of MSI status in bladder cancer. This study included 205 Japanese patients who underwent transurethral resection for bladder cancer between 2005 and 2021. The prevalence rates of microsatellite stable (MSS), MSI-low (MSI-L), and MSI-high (MSI-H) were determined using molecular testing. We examined the association of MSI status (MSS versus MSI-L/H) with clinicopathological characteristics and oncological outcomes. MSI-L/H tumors were associated with higher T-category in non-muscle invasive bladder cancer (NMIBC). Additionally, MSI-L/H tumors were associated with a higher risk of intravesical recurrence in NMIBC patients treated with intravesical bacillus Calmette-Guérin (BCG) but not with non-BCG therapy. This study suggested that the MSI status might serve as a predictive marker for intravesical recurrence after BCG intravesical therapy in NMIBC and highlighted an unmet need for an alternative treatment in patients with MSI-L/H tumors.

Abstract PR007: Discovery of HRO761, a novel, first-in-class clinical stage WRN inhibitor with potent and selective anti-tumor activity in cancers with microsatellite instability

Abstract The RecQ DNA helicase WRN was identified as a synthetic lethal target in tumors with microsatellite instability (MSI) by several genetic screens. Despite recent advances in the treatment of MSI tumors by immune checkpoint inhibitors, a significant proportion of patients still fails to respond to or relapses after single agent anti-PD1 or combination of anti-PD1 plus anti-CTLA4 treatments. We present the biochemical, cellular and pharmacological characterization of the first potent and selective WRN helicase inhibitor, HRO761. We show that HRO761 is an allosteric WRN inhibitor that binds at the interface of the D1 and D2 helicase domains, locking WRN in an inactive conformation. Pharmacological inhibition of WRN by HRO761 recapitulates the phenotype observed by WRN genetic suppression, leading to activation of the DNA damage response and inhibition of tumor cell growth selectively in MSI but not in MSS cells. We further show that WRN inhibition leads to WRN protein degradation only in MSI tumor cells. Although HRO761 activates a p53-related response, anti-proliferative effects were observed in both p53 wild-type as well as p53 null or mutated tumor cells. HRO761 exposure in mice is linear with oral dose and results in dose-dependent in vivo DNA damage induction and tumor growth inhibition in MSI cell- and patient-derived xenograft models. Altogether these findings represent preclinical pharmacological validation of WRN as a selective therapeutic target in MSI cancers. A global first in human clinical trial with HRO761 (NCT05838768) is currently ongoing to assess the safety, tolerability and preliminary anti-tumor activity in patients with MSI colorectal cancer and other MSI solid tumors. Citation Format: Marta Cortes-Cros, Henrik Moebitz, Stephanie Barbe, Jutta Blank, Vincent Bordas, Giorgia Clementi, Ernesta Dammassa, Andrea Decker, Noemi Di Nanni, Ruben de Kanter, Marion Dourdoigne, Elena Gavioli, Stephane Ferretti, Marc Hattenberger, Christelle Hemmerlin, Jacques Hamon, Juergen Hinrichs, Isabel Jaco, Dragana Jankovic, Eloisa Jimenez Nunez, Yifang Li, Hans-Joerg Martus, Michele Moschetta, Cornelia Quadt, Markus Reschke, Vincent Romanet, Clemens Scheufler, Joseph Schoepfer, Ross Strang, Maxime Therier, Hans Voshol, Markus Wartmann, Sarah Welly, Monica Pham, Laurent Laborde. Discovery of HRO761, a novel, first-in-class clinical stage WRN inhibitor with potent and selective anti-tumor activity in cancers with microsatellite instability [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr PR007.

Microsatellite unstable colorectal cancers are associated with increased CD1a- and CD83-positive dendritic cell infiltration

Microsatellite instability (MSI) is characterized by a highly immunogenic tumor phenotype and abundant lymphocytic infiltrates. The aim of this study was to investigate the association between four immunohistochemically determined classes of dendritic cells (DC) with microsatellite instability status of 258 colorectal cancer (CRC) patients and to explore the possible role of those cells as prognostic factors for survival. We observed a distinct infiltration pattern of DCs both in tumor stroma (TS) and invasive front (IF), with DCs significantly prevailing in the IF (p < 0.0001). MSI cancer biopsies showed significantly higher infiltration of CD1a + and CD83+ DCs in the TS and IF compared to microsatellite stable CRCs. Survival analysis revealed that higher CD1a + and CD83+ DC numbers both in TS and IF correlated with longer survival of the patients after surgical therapy (p < 0.05, Log rank test). Cox multivariate analysis showed that lower infiltration with CD1a + DCs in TS (p = 0.039) and CD83+ DCs in IF (p = 0.022) was an independent prognostic factor for unfavorable outcome for CRC patients. The results of our study suggest that the immunohistochemically determined CD1a + and CD83+ DCs could be used as a feature of microsatellite instability and could be further explored as prognostic markers for patients’ outcome.

Identification of 2-Sulfonyl/Sulfonamide Pyrimidines as Covalent Inhibitors of WRN Using a Multiplexed High-Throughput Screening Assay.

Werner syndrome protein (WRN) is a multifunctional enzyme with helicase, ATPase, and exonuclease activities that are necessary for numerous DNA-related transactions in the human cell. Recent studies identified WRN as a synthetic lethal target in cancers characterized by genomic microsatellite instability resulting from defects in DNA mismatch repair pathways. WRN's helicase activity is essential for the viability of these high microsatellite instability (MSI-H) cancers and thus presents a therapeutic opportunity. To this end, we developed a multiplexed high-throughput screening assay that monitors exonuclease, ATPase, and helicase activities of full-length WRN. This screening campaign led to the discovery of 2-sulfonyl/sulfonamide pyrimidine derivatives as novel covalent inhibitors of WRN helicase activity. The compounds are specific for WRN versus other human RecQ family members and show competitive behavior with ATP. Examination of these novel chemical probes established the sulfonamide NH group as a key driver of compound potency. One of the leading compounds, H3B-960, showed consistent activities in a range of assays (IC50 = 22 nM, KD = 40 nM, KI = 32 nM), and the most potent compound identified, H3B-968, has inhibitory activity IC50 ∼ 10 nM. These kinetic properties trend toward other known covalent druglike molecules. Our work provides a new avenue for screening WRN for inhibitors that may be adaptable to different therapeutic modalities such as targeted protein degradation, as well as a proof of concept for the inhibition of WRN helicase activity by covalent molecules.

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.

Controlling the confounding effect of metabolic gene expression to identify actual metabolite targets in microsatellite instability cancers

BackgroundThe metabolome is the best representation of cancer phenotypes. Gene expression can be considered a confounding covariate affecting metabolite levels. Data integration across metabolomics and genomics to establish the biological relevance of cancer metabolism is challenging. This study aimed to eliminate the confounding effect of metabolic gene expression to reflect actual metabolite levels in microsatellite instability (MSI) cancers.MethodsIn this study, we propose a new strategy using covariate-adjusted tensor classification in high dimensions (CATCH) models to integrate metabolite and metabolic gene expression data to classify MSI and microsatellite stability (MSS) cancers. We used datasets from the Cancer Cell Line Encyclopedia (CCLE) phase II project and treated metabolomic data as tensor predictors and data on gene expression of metabolic enzymes as confounding covariates.ResultsThe CATCH model performed well, with high accuracy (0.82), sensitivity (0.66), specificity (0.88), precision (0.65), and F1 score (0.65). Seven metabolite features adjusted for metabolic gene expression, namely, 3-phosphoglycerate, 6-phosphogluconate, cholesterol ester, lysophosphatidylethanolamine (LPE), phosphatidylcholine, reduced glutathione, and sarcosine, were found in MSI cancers. Only one metabolite, Hippurate, was present in MSS cancers. The gene expression of phosphofructokinase 1 (PFKP), which is involved in the glycolytic pathway, was related to 3-phosphoglycerate. ALDH4A1 and GPT2 were associated with sarcosine. LPE was associated with the expression of CHPT1, which is involved in lipid metabolism. The glycolysis, nucleotide, glutamate, and lipid metabolic pathways were enriched in MSI cancers.ConclusionsWe propose an effective CATCH model for predicting MSI cancer status. By controlling the confounding effect of metabolic gene expression, we identified cancer metabolic biomarkers and therapeutic targets. In addition, we provided the possible biology and genetics of MSI cancer metabolism.

Predicting colorectal cancer microsatellite instability with a self-attention-enabled convolutional neural network

This study develops a method combining a convolutional neural network model, INSIGHT, with a self-attention model, WiseMSI, to predict microsatellite instability (MSI) based on the tiles in colorectal cancer patients from a multicenter Chinese cohort. After INSIGHT differentiates tumor tiles from normal tissue tiles in a whole slide image, features of tumor tiles are extracted with a ResNet model pre-trained on ImageNet. Attention-based pooling is adopted to aggregate tile-level features into slide-level representation. INSIGHT has an area under the curve (AUC) of 0.985 for tumor patch classification. The Spearman correlation coefficient of tumor cell fraction given by expert pathologist and INSIGHT is 0.7909. WiseMSI achieves a specificity of 94.7% (95% confidence interval [CI] 93.7%-95.7%), a sensitivity of 84.7% (95% CI 82.6%-86.9%), and an AUC of 0.954 (95% CI 0.948-0.960). Comparative analysis shows that this method has better performance than the other five classic deep learning methods.

Microsatellite Instability and Aberrant Pre-mRNA Splicing: How Intimate Is It?

Cancers that belong to the microsatellite instability (MSI) class can account for up to 15% of all cancers of the digestive tract. These cancers are characterized by inactivation, through the mutation or epigenetic silencing of one or several genes from the DNA MisMatch Repair (MMR) machinery, including MLH1, MLH3, MSH2, MSH3, MSH6, PMS1, PMS2 and Exo1. The unrepaired DNA replication errors turn into mutations at several thousand sites that contain repetitive sequences, mainly mono- or dinucleotides, and some of them are related to Lynch syndrome, a predisposition condition linked to a germline mutation in one of these genes. In addition, some mutations shortening the microsatellite (MS) stretch could occur in the 3'-intronic regions, i.e., in the ATM (ATM serine/threonine kinase), MRE11 (MRE11 homolog) or the HSP110 (Heat shock protein family H) genes. In these three cases, aberrant pre-mRNA splicing was observed, and it was characterized by the occurrence of selective exon skipping in mature mRNAs. Because both the ATM and MRE11 genes, which as act as players in the MNR (MRE11/NBS1 (Nibrin)/RAD50 (RAD50 double strand break repair protein) DNA damage repair system, participate in double strand breaks (DSB) repair, their frequent splicing alterations in MSI cancers lead to impaired activity. This reveals the existence of a functional link between the MMR/DSB repair systems and the pre-mRNA splicing machinery, the diverted function of which is the consequence of mutations in the MS sequences.

LT-RPA: An Isothermal DNA Amplification Approach for Improved Microsatellite Genotyping and Microsatellite Instability Detection.

Microsatellites are short tandem repeats of one to six nucleotides that are highly polymorphic and extensively used as genetic markers in numerous biomedical applications, including the detection of microsatellite instability (MSI) in cancer. The standard analytical method for microsatellite analysis relies on PCR amplification followed by capillary electrophoresis or, more recently, next-generation sequencing (NGS). However, their amplification during PCR generates undesirable frameshift products known as stutter peaks caused by polymerase slippage, complicating data analysis and interpretation, while very few alternative methods for microsatellite amplification have been developed to reduce the formation of these artifacts. In this context, the recently developed low-temperature recombinase polymerase amplification (LT-RPA) is an isothermal DNA amplification method at low temperature (32°C) that drastically reduces and sometimes completely abolishes the formation of stutter peaks. LT-RPA greatly simplifies the genotyping of microsatellites and improves the detection of MSI in cancer. In this chapter, we describe in detail all the experimental steps necessary for the development of LT-RPA simplex and multiplex assays for microsatellite genotyping and MSI detection, including the design, optimization, and validation of the assays combined with capillary electrophoresis or NGS.

A systematic pan-cancer analysis identifies TRIM28 as an immunological and prognostic predictor and involved in immunotherapy resistance.

Tripartite motif-containing protein 28 (TRIM28), as a transcriptional cofactor, has pleiotropic biological effects, such as silencing genes, promoting cellular proliferation and differentiation, and facilitating DNA repair. It is reported that TRIM28 is also correlated with immune infiltration in liver cancer that highlights an unnoticed function of TRIM28 in immune system. However, the prognostic and immunotherapeutic role of TRIM28 in human cancer has not been elucidated. In this study, we conducted a systematic pan-cancer analysis and partial experimental validation of TRIM28 as an immunological and prognostic predictor and its involvement in immunotherapy resistance. We found that TRIM28 expression was higher in various tumor tissues than in normal tissues. Higher TRIM28 expression was associated with poorer prognosis in multiple cancers. The expression of TRIM28 was positively correlated with the presence of T cells, macrophages and neutrophils, and TRIM28 also promoted the infiltration of a series of immune cell. Moreover, TRIM28 affected a wide range of cancer-related scores, and the abnormal expression of TRIM28 was also involved in tumor mutational burden, drug sensitivity, and microsatellite instability in cancer. The results suggest that TRIM28 is a potentially valuable immune response indicator and a molecular biomarker for predicting the prognosis of cancer patients.

184P A phase I study of the cancer-specific vaccine FMPV-1 in healthy male subjects to assess safety and immune response

FMPV-1 is a novel injectable vaccine to generate anti-cancer T cell responses. The vaccine is an immunogenic frameshift mutated transforming growth factor β receptor 2 (TGFβR2) peptide commonly occurring in microsatellite instability cancers (MSI-H). FMPV-1 is administered in combination with the adjuvant granulocyte macrophage colony stimulating factor (GM-CSF). This is the first clinical study to assess the safety and immunogenicity of FMPV-1/GM-CSF in man.

Canonical and uncanonical pathogenic germline variants in colorectal cancer patients by next-generation sequencing in a European referral center

Despite increasing use of next-generation sequencing (NGS), data concerning the gain in germline pathogenic variants (PVs) remain scanty, especially with respect to uncanonical ones. We aimed to verify the impact of different cancer predisposition genes (CPGs) on colorectal cancer (CRC) in patients referred for genetic evaluation. We enrolled for NGS, by Illumina TruSight Cancer panel comprising 94 CPGs, 190 consecutive subjects referred for microsatellite instability (MSI) CRC, polyposis, and/or family history. Overall, 51 (26.8%) subjects carried 64 PVs; PVs coexisted in 4 (7.8%) carriers. PVs in mismatch repair (MMR) genes accounted for one-third of variant burden (31.3%). Four Lynch syndrome patients (20%) harbored additional PVs (HOXB13, CHEK2, BRCA1, NF1 plus BRIP1); such multiple PVs occurred only in subjects with PVs in mismatch syndrome genes (4/20 versus 0/31; P= 0.02). Five of 22 (22.7%) patients with MSI cancers but wild-type MMR genes harbored PVs in unconventional genes (FANCL, FANCA, ATM, PTCH1, BAP1). In 10/63 patients (15.9%) with microsatellite stable CRC, 6 had MUTYH PVs (2 being homozygous) and 4 exhibited uncanonical PVs (BRCA2, BRIP1, MC1R, ATM). In polyposis, we detected PVs in 13 (25.5%) cases: 5 (9.8%) in APC, 6 (11.8%) with biallelic PVs in MUTYH, and 2 (3.9%) in uncanonical genes (FANCM, XPC). In subjects tested for family history only, we detected two carriers (18.2%) with PVs (ATM, MUTYH). Uncanonical variants may account for up to one-third of PVs, underlining the urgent need of consensus on clinical advice for incidental findings in cancer-predisposing genes not related to patient phenotype.