Microsatellite Unstable Colorectal Cancer Research Articles

Metastasis and the Microbiome: The Impact of Bacteria in Disseminated Colorectal Cancer.

Metastasis remains a leading cause of mortality for patients with solid tumors. An expanding body of literature suggests interplay between the host, gut, and tumoral microbiomes may play a role in cancer initiation and distant dissemination. These associations have been particularly well-studied in colorectal cancer, where gut dysbiosis and an endotoxin-induced inflammatory milieu foster premalignant polyp formation, setting the stage for carcinogenesis. Subsequent violation of the gut vascular barrier enables dissemination of bacterial agents to sites such as the liver, where they contribute to establishment of pre-metastatic niches, which promote tumor cell extravasation and metastatic outgrowth. Intriguingly, breakdown of this vascular barrier has been shown to be aided by the presence of tumoral bacteria. The presence of similar species, including Fusobacterium nucleatum and Escherichia Coli, in both primary and metastatic colorectal tumors, supports this hypothesis and their presence is associated with chemotherapy resistance and an overall poor prognosis. Specific gut microbial populations are also associated with differential response to immunotherapy, which has a growing role in microsatellite unstable colorectal cancers. Recent work suggests that modulation of gut microbiome using dietary modification, targeted antibiotics, or fecal microbiota transplantation may improve response to immunotherapy and oncologic outcomes. Elucidation of the precise mechanistic links between the microbiome and cancer dissemination will open the doors to additional therapeutic possibilities.

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.

N-Glycomic Profiling of Microsatellite Unstable Colorectal Cancer.

Aberrant glycosylation affects cancer progression and immune evasion. Approximately 15% of colorectal cancers (CRCs) demonstrate microsatellite instability (MSI) and display major differences in outcomes and therapeutic responses, as compared to corresponding microsatellite stable (MSS) tumors. We compared the N-glycan profiles of stage II and IV MSI CRC tumors, further subdivided into BRAFV600E wild-type and mutated subgroups (n = 10 in each subgroup), with each other and with those of paired non-neoplastic mucosal samples using mass spectrometry. Further, the N-glycans of BRAFV600E wild-type stage II MSI tumors were compared to corresponding MSS tumors (n = 9). Multiple differences in N-glycan profiles were identified between the MSI CRCs and control tissues, as well as between the stage II MSI and MSS samples. The MSI CRC tumors showed a lower relative abundance of high-mannose N-glycans than did the control tissues or the MSS CRCs. Among MSI CRC subgroups, acidic N-glycans showed tumor stage and BRAF mutation status-dependent variation. Specifically, the large, sulfated/phosphorylated, and putative terminal N-acetylhexosamine-containing acidic N-glycans differed between the MSI CRC subgroups, showing opposite changes in stages II and IV, when comparing BRAF mutated and wild-type tumors. Our results show that molecular subgroups of CRC exhibit characteristic glycan profiles that may explain certain carcinogenic properties of MSI tumors.

Neoadjuvant immunotherapy in microsatellite unstable colorectal cancer: Are we in the era of nonoperative management?

Microsatellite unstable (MSI-H) colorectal cancers (CRC) are the hallmark of Lynch Syndrome. Advances in immunotherapy have yielded a change in the treatment of those cancers. Recent publications about neoadjuvant immunotherapy in CRC are triggering a high interest to use under the umbrella of achieving a complete clinical response. Although we do not know the extent of this response over time, avoiding surgical morbidity seems to be on the horizon for this subset of CRC.

A simple approach for detecting HLA-A*02 alleles in archival formalin-fixed paraffin-embedded tissue samples and an application example for studying cancer immunoediting.

The HLA system represents a central component of the antigen presentation machinery. As every patient possesses a defined set of HLA molecules, only certain antigens can be presented on the cell surface. Thus, studying HLA type-dependent antigen presentation can improve the understanding of variation in susceptibility to various diseases, including infectious diseases and cancer. In archival formalin-fixed paraffin-embedded (FFPE) tissue, the HLA type is difficult to analyze because of fragmentation of DNA, hindering the application of commonly used assays that rely on long DNA stretches. Addressing these difficulties, we present a refined approach for characterizing presence or absence of HLA-A*02, the most common HLA-A allele in the Caucasian population, in archival samples. We validated our genotyping strategy in a cohort of 90 samples with HLA status obtained by an NGS-based method. 90% (n=81) of the samples could be analyzed with the approach. For all of them, the presence or absence of HLA-A*02 alleles was correctly determined with the method, demonstrating 100% sensitivity and specificity (95% CI: 91.40%-100% and 91.19%-100%). Furthermore, we provide an example of application in an independent cohort of 73 FFPE microsatellite-unstable (MSI) colorectal cancer samples. As MSI cancer cells encompass a high number of mutations in coding microsatellites, leading to the generation of highly immunogenic frameshift peptide antigens, they are ideally suited for studying relations between the mutational landscape of tumor cells and interindividual differences in the immune system, including the HLA genotype. Overall, our method can help to promote studying HLA type-dependency during the pathogenesis of a wide range of diseases, making archival and historic tissue samples accessible for identifying HLA-A*02 alleles.

P-280 Can immune checkpoint inhibitors plus surgery cure advanced mismatch repair-deficient cancer?

There is a strong body of evidence supporting use of immune checkpoint inhibitors (ICI) in unresectable mismatch repair-deficient (MMRd) / microsatellite unstable (MSI) high colorectal cancer (mCRC); anti-PD-1 therapy is the standard of care for first-line treatment of mCRC (Andre, NEJM 2021). Emerging evidence also demonstrates high rates of complete pathological response (pCR) following neoadjuvant ICI in patients with stage I-III MSI colon cancer (Chalabi, Nature 2020) and in MSI gastric cancer (Andre, ASCO GI 2022). This leads to the question of whether ICI with or without surgery could lead to cures in advanced MSI cancers. Herein we describe our single institutional experience of immunotherapy followed by surgery in MSI intestinal cancers. In this retrospective series the institutional electronic health record was reviewed for patients with ICI treated MSI intestinal cancers with subsequent surgical resection. Demographics and clinicopathological characteristics were collected, with ICI therapy details and toxicity, radiological response and operative details. Post-operative histology was reviewed. Overall survival was calculated using the Kaplan-Meier method. Five patients were identified; 5 (100%) female. Mean/median age was 37/46 years (range 22-78). Four (80%) had MLH1/PMS2 deficient cancers, 1 was MSH2/MSH6 deficient. One had germline MLH1 mutation, one germline pending. Two (40%) had concurrent tumour BRAF V600E mutation, two (40%) had KRAS G12D mutation. Four (80%) had colon cancer (1 ascending colon, 1 synchronous ascending colon and hepatic flexure, 1 transverse colon, 1 rectosigmoid); 1 patient jejunal adenocarcinoma. Three colon cancer patients had lymph node metastases distant from the primary, one had local pelvic side wall recurrence, one had direct invasion of liver and pancreas. All patients received ICI with initial palliative intent (1 ascending colon 2nd line nivolumab, 1 synchronous tumours 1st line nivolumab/ipilimumab, 1 rectosigmoid 2nd line nivolumab/ipilimumab, 1 transverse 1st line pembrolizumab, jejunal 1st line nivolumab). Two patients had admissions with ICI related fevers, 1 experienced ICI G3 pneumonitis and 1 experienced G3 hepatitis. All patients had radiological response to ICI. Two patients (transverse colon / jejunal cancer) experienced stricture in responding primary tumour and required surgery. One ascending colon cancer had surgery a symptomatic PET-avid mass, 1 synchronous cancer patient had right total mesocolic excision, partial hepatectomy and Whipples. One recurrent rectosigmoid colon cancer had total pelvic exenteration following ICI and radiotherapy. All patients had R0 resection. Two patients had pCR (ypT0N0) (ascending colon, rectosigmoid colon); both complete responders had experienced ICI related toxicity. Other patients’ stage were transverse colon (ypT3N0), jejunal cancer (ypT0N1) and synchronous tumours (ypT4N0 and ypT2N0). After median follow up of 15 months, no patient has recurrent disease. Median overall survival is not reached. In this series we demonstrate that a combination of ICI and surgery can render patients with advanced MSI cancers disease free by downstaging previously unresectable cancers and eliminating distant metastases. As complete responses were demonstrated in two patients with suspicious pre-operative radiological findings on resection, improved imaging techniques or ctDNA may be helpful in guiding which patients require surgery in future.

Clustered 8-Oxo-Guanine Mutations and Oncogenic Gene Fusions in Microsatellite-Unstable Colorectal Cancer.

PURPOSEColorectal carcinomas (CRCs) with microsatellite-instability (MSI) are enriched for oncogenic kinase fusions (KFs), including NTRK1, RET, and BRAF, but the mechanism underlying this finding is unclear.METHODSThe genomic profiles of 32,218 advanced CRC tumor specimens were analyzed to assess the fusion breakpoints of oncogenic alterations including KFs in microsatellite-stable and microsatellite-unstable CRC. Genomic contexts of such alterations were analyzed to obtain mechanistic insights.RESULTSGenomic analysis demonstrated that oncogenic fusion breakpoints in MSI tumors do not preferentially involve repetitive or low-complexity sequences. Instead, their junction regions showed pronounced guanine and cytosine bias and elevated mutation frequency at G:C contexts. Elevated mutation frequency at G:C bases in relevant introns predicted prevalence of associated oncogenic fusions in MSI CRCs. CRCs harboring mismatch repair signatures had enrichment of butyrate-producing microbial species, reported to be associated with induction of 8-oxoguanine lesions in the intestine.CONCLUSIONDetailed analysis of breakpoints in MSI-associated KFs support a model in which inefficient repair and/or processing of microbiome-induced clustered 8-oxoguanine damage in MSI CRC contributes to the increased incidence of specific oncogenic fusions.

Microsatellite Instability and Colorectal Cancer, Immunohistochemical and Molecular Evaluation by Using DNA Sequencing: A Single Center Experience

Background & Objective:Microsatellite instability is common in familial colorectal cancers. It can be tested by the molecular and immunohistochemical methods. There are very few studies which address comparing the clinicopathological characteristics of microsatellite stable (MSS) and microsatellite unstable (MSI) colorectal cancers from Iran. In this study, we aimed to evaluate the clinicopathological and immuno-histochemical findings of MSS and MSI colorectal cancers in our Center as the largest Center of gastrointestinal surgery and oncology in the South of Iran. We also compared the immunohistochemical method vs. molecular study using DNA sequencing.Methods:For 5 years (2015-2019), 34 patients who underwent operation in the affiliated Hospitals of Shiraz University of Medical Sciences were clinically suspected to microsatellite instability (MSI). The molecular diagnostic tests with DNA sequencing were performed. Clinicopathological and immunohistochemical findings of MSI colorectal cancers were compared with those who were stable. Results:In the South of Iran, MSI colorectal cancers were more common in males. These tumors were more common in the right side with more tendencies to produce mucin with lymphocytic infiltration. Conclusion:Immunohistochemistry would be a specific method for diagnosis of MSI colorectal cancers but may be associated with high rate of false negative results and of low sensitivity. Therefore, we recommend performing molecular studies by DNA sequencing in those colon cancers with clinical suspicion for MSI and negative immunohistochemical findings.

Abstract 1606: Potentiating effect of olaparib on anti-PD-1 treatment in colorectal cancer

Abstract Immune checkpoint inhibitors (ICIs) are approved for microsatellite unstable colorectal cancer (CRC), however, microsatellite stable (MSS) CRC that make up the majority of tumors in clinical practice have not seen any benefit with ICI. This is in part due to the lack of sufficient tumor infiltrating immune cells, particularly CD8+ T cells, in the milieu. Immunogenic cell death (ICD) is a particular form of cell death that elicits robust cross-priming of CD8 T cells against antigens expressed by dying cells in the absence of exogenous adjuvants. ICD-inducing drugs such as poly (ADP-ribose) polymerase inhibitors (PARPis) are poised to enhance cancer cell killing, boost tumor immunogenicity, increase in vivo immune infiltration, and thereby accelerate a tumor response to ICI treatment. Here, we tested the combinatorial therapeutic efficacy of a PARPi, olaparib (OLA), and an ICI, anti-PD-1, in in vitro and in vivo conditions. Combined treatment of OLA and anti-mouse PD-1 reduced tumor growth and increased survival in an immunocompetent, syngeneic animal model of BALB/c carrying allograft CT26 tumor that is MSS. Flow cytometry analysis revealed that the combo treatment significantly reduced percentages of anti-inflammatory macrophages and increased inflammatory monocytes. There was no significant difference in dendritic cell populations. We observed increased anti-tumor lymphocyte response via activation of CD4 and CD8 T cell populations upon combo treatment with OLA and anti-mouse PD-1. In addition, we noticed an increased association of expression of PD-L1 and its receptor PD-1 with OLA treatment in CRC cell lines and CT26 tumors. Our preliminary data suggests altered expressions of the CD8+ T cell exhaustion marker TIM-3 and its ligands galectin-9 and high mobility group protein 1 (HMGB1), an indicator of ICD. The pilot studies also revealed increased levels of STING-dependent type I interferon signaling initiator cGAS upon treatment with OLA in vivo. Mechanistically, OLA might induce DNA damage among tumor cells and trigger innate immune signaling including NF-κB activation and STING-dependent type I interferon signaling, both of which contribute to the maturation of myeloid antigen-presenting cells and subsequent activation of T cells. In conclusion, OLA potentiated the antitumor efficacy of anti-PD-1 in CT26 induced syngeneic CRC mouse model. Our findings indicate that OLA might trigger tumor immunogenicity, and enable a potential targeted-immunotherapeutic angle for exercising ICD and activating cytotoxic T lymphocytes in CT26 syngeneic CRC mouse model; however, further studies are needed to elucidate the mechanism. Despite ICD markers' identification as immuno-stimulating characteristics of dead cells, whether ICD-associated molecules can be used as a prognostic biomarker or therapeutic target, and the rational for such markers on tumor cells in eliciting antitumor immunity are still under investigation. Citation Format: Chintal Bhavsar, Peter John, Magnolia Abreu, Laura Bencosme, Radhashree Maitra, Sanjay Goel, Titto Augustine. Potentiating effect of olaparib on anti-PD-1 treatment in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1606.

NMD inhibition by 5-azacytidine augments presentation of immunogenic frameshift-derived neoepitopes

SummaryFrameshifted protein sequences elicit tumor-specific T cell-mediated immune responses in microsatellite-unstable (MSI) cancers if presented by HLA class I molecules. However, their expression and presentation are limited by nonsense-mediated RNA decay (NMD). We employed an unbiased immunopeptidomics workflow to analyze MSI HCT-116 cells and identified >10,000 HLA class I-presented peptides including five frameshift-derived InDel neoepitopes. Notably, pharmacological NMD inhibition with 5-azacytidine stabilizes frameshift-bearing transcripts and increases the HLA class I-mediated presentation of InDel neoepitopes. The frameshift mutation underlying one of the identified InDel neoepitopes is highly recurrent in MSI colorectal cancer cell lines and primary patient samples, and immunization with the corresponding neoepitope induces strong CD8+ T cell responses in an HLA-A∗02:01 transgenic mouse model. Our data show directly that pharmacological NMD inhibition augments HLA class I-mediated presentation of immunogenic frameshift-derived InDel neoepitopes thus highlighting the clinical potential of NMD inhibition in anti-cancer immunotherapy strategies.

CD4 T Cell-Dependent Rejection of Beta-2 Microglobulin Null Mismatch Repair-Deficient Tumors.

Inactivation of beta-2 microglobulin (B2M) is considered a determinant of resistance to immune checkpoint inhibitors (ICPi) in melanoma and lung cancers. In contrast, B2M loss does not appear to affect response to ICPis in mismatch repair-deficient (MMRd) colorectal tumors where biallelic inactivation of B2M is frequently observed. We inactivated B2m in multiple murine MMRd cancer models. Although MMRd cells would not readily grow in immunocompetent mice, MMRd B2m null cells were tumorigenic and regressed when treated with anti-PD-1 and anti-CTLA4. The efficacy of ICPis against MMRd B2m null tumors did not require CD8+ T cells but relied on the presence of CD4+ T cells. Human tumors expressing low levels of B2M display increased intratumoral CD4+ T cells. We conclude that B2M inactivation does not blunt the efficacy of ICPi in MMRd tumors, and we identify a unique role for CD4+ T cells in tumor rejection. SIGNIFICANCE: B2M alterations, which impair antigen presentation, occur frequently in microsatellite-unstable colorectal cancers. Although in melanoma and lung cancers B2M loss is a mechanism of resistance to immune checkpoint blockade, we show that MMRd tumors respond to ICPis through CD4+ T-cell activation.This article is highlighted in the In This Issue feature, p. 1601.

CTLA-4 blockade in the treatment of colorectal cancer with microsatellite instability

Context: Colorectal cancer is one of the most common tumours worldwide, with around 10-15% of these related to microsatellite instability which is, in turn, responsible for a high neoantigen load and subsequent high tumour mutational burden. These characteristics are responsible for the poor response of these tumours to chemotherapy, highlighting the need for a different approach in the treatment of patients with microsatellite unstable colorectal cancer. Immunotherapy was proven important in the treatment of these patients, with immune checkpoint inhibition such as CTLA-4 blockade being one of the most promising targets so far. Evidence Acquisition: A PubMed search was done on February 2021 where the used query obtained a total of 33 articles. After the inclusion and exclusion criteria, a total of 21 articles were obtained and used in this narrative review. Results: Several studies with microsatellite unstable colorectal tumours have been done in order to evaluate the advantages and adverse events of CTLA-4 blockade in these patients. Studies show a benefit regarding the progression-free survival, overall survival and overall response rates in patients receiving ipilimumab (anti-CTLA-4) when compared to those who weren’t. Besides, the main adverse events are manageable and are more tolerable than those observed with chemotherapy. Nonetheless, unlike PD-1 blockade, anti-CTLA-4 drugs are currently only approved for the use as a combination therapy in microsatellite unstable colorectal cancer, still awaiting approval as a monotherapy. Conclusion: Microsatellite unstable colorectal tumours deserve a different treatment path, as their characteristics make these tumours poor responders to chemotherapy while at the same time great candidates for immunotherapy, namely with CTLA-4 inhibitors. KEY-WORDS Colorectal cancer; CTLA-4; Immune checkpoint; Immunotherapy; Ipilimumab; Microsatellite instability

Pembrolizumab in the Treatment of Patients with Microsatellite Unstable Colorectal Cancer

Colorectal cancer is one of the most common tumours in the world, with microsatellite instability having an important role in the development of these tumours. Microsatellite instability is responsible for the worse response of tumours to chemotherapy, while also having a role in the better prognosis associated with these tumours. This relates to the higher immune infiltration that results from the high neoantigen load of microsatellite unstable tumours. This highly responsive immune microenvironment seen in microsatellite unstable colorectal cancer makes these tumours great candidates for therapies targeting the immune system, such as immune checkpoint inhibitors. The PD-1/PD-L1 axis is upregulated in colorectal cancer with microsatellite instability, making the blockade of this axis a good therapy alternative for these patients.

Comparison of Clinical and Histopathological Parameters amongst Microsatellite Unstable and Microsatellite Stable Cases of Colorectal Carcinomas in an Indian Setting

Introduction:Inthiseraofprognosisbasedmedicine,itisimportant to identify microsatellite unstable Colorectal Cancers (CRCs) as they offer good prospects to the patient and they respond poorly to 5-fluorouracil and platinum based chemotherapeutic regime. Aim: To find out the prevalence of Microsatellite Instability-High (MSI-H) in CRC, to identify clinicopathological features associated with Microsatellite Instability (MSI) and assess the value of surgical pathology in predicting MSI-H. Materials and Methods: The present study was a case-control study conducted in a tertiary care centre of Pune in Western India from January 2013 to December 2020. Thirty-five CRCs deficient in Mismatch Repair (MMR) proteins contrasted with 206 Microsatellite Stable (MSS) CRCs were studied and analysed for a given set of clinical and histopathological parameters to find out any correlation between the occurrence of microsatellite unstable tumours and these variables were presented as percentages. Results: In the present study, the prevalence rate of MSI-H was found to be 14.5% and the statistical analysis was carried out using the software Statistical Package for the Social Sciences (SPSS) version 27.0. Univariate analysis revealed that right-sided/proximal location of tumours, age at diagnosis less than 50 years, no lymph node deposits (N0 disease), presence of Tumour Infiltrating Lymphocytes (TILs), peri-tumoural reaction, mucinous component, increased stromal plasma cells, histological heterogeneity, signet ring/medullary component and Crohn-like reaction were all statistically significant predictors of microsatellite instability (p-value &lt;0.05). Multivariate analysis of these significant parameters revealed right-sided location of tumours, age at diagnosis less than 50 years, N0 disease, and presence of TILs, increased stromal plasma cells, histological heterogeneity and Crohn-like reaction to be independent predictors. Conclusion: Clinical parameters and histological evaluation is handy in screening for the MSI-H colorectal carcinomas. This would go a long way in selecting the patients who will require confirmatory molecular testing and thus precluding the need of Immunohistochemistry (IHC), which will be helpful in day-to-day practice as it is uncomplicated, cost-effective and easy to replicate.

Angiogenesis-Related Functions of Wnt Signaling in Colorectal Carcinogenesis.

Simple SummaryAngiogenesis belongs to the most clinical characteristics of colorectal cancer (CRC) and is strongly linked to the activation of Wnt/β-catenin signaling. The most prominent factors stimulating constitutive activation of this pathway, and in consequence angiogenesis, are genetic alterations (mainly mutations) concerning APC and the β-catenin encoding gene (CTNNB1), detected in a large majority of CRC patients. Wnt/β-catenin signaling is involved in the basic types of vascularization (sprouting and nonsprouting angiogenesis), vasculogenic mimicry as well as the formation of mosaic vessels. The number of known Wnt/β-catenin signaling components and other pathways interacting with Wnt signaling, regulating angiogenesis, and enabling CRC progression continuously increases. This review summarizes the current knowledge about the role of the Wnt/Fzd/β-catenin signaling pathway in the process of CRC angiogenesis, aiming to improve the understanding of the mechanisms of metastasis as well as improvements in the management of this cancer.Aberrant activation of the Wnt/Fzd/β-catenin signaling pathway is one of the major molecular mechanisms of colorectal cancer (CRC) development and progression. On the other hand, one of the most common clinical CRC characteristics include high levels of angiogenesis, which is a key event in cancer cell dissemination and distant metastasis. The canonical Wnt/β-catenin downstream signaling regulates the most important pro-angiogenic molecules including vascular endothelial growth factor (VEGF) family members, matrix metalloproteinases (MMPs), and chemokines. Furthermore, mutations of the β-catenin gene associated with nuclear localization of the protein have been mainly detected in microsatellite unstable CRC. Elevated nuclear β-catenin increases the expression of many genes involved in tumor angiogenesis. Factors regulating angiogenesis with the participation of Wnt/β-catenin signaling include different groups of biologically active molecules including Wnt pathway components (e.g., Wnt2, DKK, BCL9 proteins), and non-Wnt pathway factors (e.g., chemoattractant cytokines, enzymatic proteins, and bioactive compounds of plants). Several lines of evidence argue for the use of angiogenesis inhibition in the treatment of CRC. In the context of this paper, components of the Wnt pathway are among the most promising targets for CRC therapy. This review summarizes the current knowledge about the role of the Wnt/Fzd/β-catenin signaling pathway in the process of CRC angiogenesis, aiming to improve the understanding of the mechanisms of metastasis as well as improvements in the management of this cancer.

Clinicopathological and Molecular Profiles of Sporadic Microsatellite Unstable Colorectal Cancer with or without the CpG Island Methylator Phenotype (CIMP).

Simple SummaryThe 5’-C-phosphate-G-3’ island methylator phenotype (CIMP) is a phenotype of colorectal cancer associated with microsatellite instability (MSI). The aim of the present study was to analyze the CIMP phenotype and genetic mutations in MSI-high colorectal cancer. Our results demonstrated that among MSI-high colorectal cancer patients, CIMP-high tumors were associated with specific mutation profiles and clinicopahtological features compared with CIMP-low or CIMP-0 tumors, however, the CIMP status was not an independent prognostic factor.Background: The 5’-C-phosphate-G-3’ island methylator phenotype (CIMP) is a specific phenotype of colorectal cancer (CRC) associated with microsatellite instability-high (MSI-high) tumors. Methods: In this study, we determined the CIMP status using eight methylation markers in 92 MSI-high CRC patients after excluding five germline mismatch repair (MMR) gene mutations analyzed by next-generation sequencing (NGS) and confirmed by Sanger sequencing. The mutation spectra of 22 common CRC-associated genes were analyzed by NGS. Results: Of the 92 sporadic MSI-high tumors, 23 (25%) were considered CIMP-high (expressed more than 5 of 8 markers). CIMP-high tumors showed proximal colon preponderance and female predominance. The mutation profiles of CIMP-high tumors were significantly different from those of CIMP-low or CIMP-0 tumors (i.e., higher frequencies of BRAF, POLD1, MSH3, and SMAD4 mutations but lower frequencies of APC, TP53, and KRAS mutations). Multivariate analysis demonstrated that tumor, node, metastasis (TNM) stage was the independent prognostic factor affecting overall survival (OS). Among the MSI-high cases, the CIMP status did not impact the outcome of patients with MSI-high tumors. Conclusions: Only TNM stage was a statistically significant predictor of outcomes independent of CIMP profiles in MSI-high CRC patients. Sporadic MSI-high CRCs with different mechanisms of carcinogenesis have specific mutation profiles and clinicopathological features.

The shared frameshift mutation landscape of microsatellite-unstable cancers suggests immunoediting during tumor evolution

The immune system can recognize and attack cancer cells, especially those with a high load of mutation-induced neoantigens. Such neoantigens are abundant in DNA mismatch repair (MMR)-deficient, microsatellite-unstable (MSI) cancers. MMR deficiency leads to insertion/deletion (indel) mutations at coding microsatellites (cMS) and to neoantigen-inducing translational frameshifts. Here, we develop a tool to quantify frameshift mutations in MSI colorectal and endometrial cancer. Our results show that frameshift mutation frequency is negatively correlated to the predicted immunogenicity of the resulting peptides, suggesting counterselection of cell clones with highly immunogenic frameshift peptides. This correlation is absent in tumors with Beta-2-microglobulin mutations, and HLA-A*02:01 status is related to cMS mutation patterns. Importantly, certain outlier mutations are common in MSI cancers despite being related to frameshift peptides with functionally confirmed immunogenicity, suggesting a possible driver role during MSI tumor evolution. Neoantigens resulting from shared mutations represent promising vaccine candidates for prevention of MSI cancers.

(Phospho)proteomic Profiling of Microsatellite Unstable CRC Cells Reveals Alterations in Nuclear Signaling and Cholesterol Metabolism Caused by Frameshift Mutation of NMD Regulator UPF3A.

DNA mismatch repair-deficient colorectal cancers (CRCs) accumulate numerous frameshift mutations at repetitive sequences recognized as microsatellite instability (MSI). When coding mononucleotide repeats (cMNRs) are affected, tumors accumulate frameshift mutations and premature termination codons (PTC) potentially leading to truncated proteins. Nonsense-mediated RNA decay (NMD) can degrade PTC-containing transcripts and protect from such faulty proteins. As it also regulates normal transcripts and cellular physiology, we tested whether NMD genes themselves are targets of MSI frameshift mutations. A high frequency of cMNR frameshift mutations in the UPF3A gene was found in MSI CRC cell lines (67.7%), MSI colorectal adenomas (55%) and carcinomas (63%). In normal colonic crypts, UPF3A expression was restricted to single chromogranin A-positive cells. SILAC-based proteomic analysis of KM12 CRC cells revealed UPF3A-dependent down-regulation of several enzymes involved in cholesterol biosynthesis. Furthermore, reconstituted UPF3A expression caused alterations of 85 phosphosites in 52 phosphoproteins. Most of them (38/52, 73%) reside in nuclear phosphoproteins involved in regulation of gene expression and RNA splicing. Since UPF3A mutations can modulate the (phospho)proteomic signature and expression of enzymes involved in cholesterol metabolism in CRC cells, UPF3A may influence other processes than NMD and loss of UPF3A expression might provide a growth advantage to MSI CRC cells.

Differential immune microenvironmental features of microsatellite-unstable colorectal cancers according to Fusobacterium nucleatum status.

It has been suggested that Fusobacterium nucleatum (Fn) may differentially impact tumor immune responses according to microsatellite instability (MSI) status in colorectal cancers (CRCs). We aimed to reveal the detailed relationship between intratumoral Fn and immune microenvironmental features in MSI-high CRCs. A total of 126 MSI-high CRCs were subjected to analyses for intratumoral Fn DNA load using quantitative PCR and for densities of tumor-infiltrating immune cells, including CD3+ T cells, CD4+ T cells, CD8+ T cells, FoxP3+ T cells, CD68+ macrophages, CD163+ macrophages, and CD177+ neutrophils, at invasive margin (IM) and center of tumor (CT) areas using computational image analysis of immunohistochemistry. Based on the Fn load, the 126 MSI-high CRCs were classified into Fn-high, -low, and -negative subgroups. The Fn-high subset of MSI-high CRCs was significantly correlated with larger tumor size and advanced invasion depth (p = 0.017 and p = 0.034, respectively). Compared with the Fn-low/negative subgroup, Fn-high tumors demonstrated significantly lower density of FoxP3+ cells in both IM and CT areas (p = 0.002 and p = 0.003, respectively). Additionally, Fn-high was significantly associated with elevated CD163+ cell to CD68+ cell ratio in only CT areas of MSI-high CRCs (p = 0.028). In conclusion, the Fn-enriched subset of MSI-high CRCs is characterized by increased tumor growth and invasion and distinct immune microenvironmental features, including decreased FoxP3+ T cells throughout the tumor and increased proportion of M2-polarized macrophages in the tumor center. These findings collectively support that Fn may be linked to pro-tumoral immune responses in MSI-high CRCs.

PD-L1 Associates with Poorly Differentiated, Microsatellite-Unstable Colorectal Cancers in a Norwegian Population-Based Cohort

Background: In recent years, a range of cancers have been demonstrated to interfere and effectively evade the adaptive immune response by hijacking immune checkpoints and expressing proteins able to activate them. PD-L1, a ligand protein that binds programmed death receptor 1 (PD-1) in cells of the adaptive immune system and the discovery that a range of tumours exploit this pathway to avoid immune detection has led to an effort in its targeting by immunotherapy. Recently it has also been described how microsatellite instability (MSI) in tumors correlates with PD-L1 expression and how these tumors have an increased response to anti-PD-L1 therapy. In the present study, we describe PD-L1 expression and its associated clinicopathological features in a population-based cohort of colorectal cancers (CRC).