Protein expression of S100A2 reveals it association with patient prognosis and immune infiltration profile in colorectal cancer.

PURPOSE: Colorectal cancer (CRC) is the third most diagnosed cancer worldwide. Despite a well-established knowledge of tumour development, biomarkers to predict patient outcomes are still required. S100 calcium-binding protein A2 (S100A2) has been purposed as a potential marker in many types of cancer, however, the prognostic value of S100A2 in CRC is rarely reported. MATERIAL AND METHODS: In this study, immunohistochemistry (IHC) was performed to identify the prognostic role of S100A2 protein expression in the tumour core of the tissue microarrays (TMAs) in colorectal cancer patients (n=787). Bulk RNA transcriptomic data was used to identify significant genes compared between low and high cytoplasmic S100A2 groups. Multiplex immunofluorescence (mIF) was performed to further study and confirm the immune infiltration in tumours with low and high cytoplasmic S100A2. RESULTS: Low cytoplasmic protein expression of S100A2 in the tumour core was associated with poor survival (HR 0.539, 95%CI 0.394-0.737, P<0.001) and other adverse tumour phenotypes. RNA transcriptomic analysis showed a gene significantly associated with the low cytoplasmic S100A2 group (AKT3, TAGLN, MYLK, FGD6 and ETFDH), which correlated with tumour development and progression. GSEA analysis identifies the enriched anti-tumour and immune activity group of genes in high cytoplasmic S100A2. Additionally, mIF staining showed that high CD3+FOXP3+ and CD163+ inversely associated with low cytoplasmic S100A2 (P<0.001, P=0.009 respectively). CONCLUSION: Our finding demonstrates a prognostic value of S100A2 together with the correlation with immune infiltration in CRC.

Spatially Resolved Transcriptomics Deconvolutes Prognostic Histological Subgroups in Patients with Colorectal Cancer and Synchronous Liver Metastases.

Strong immune responses in primary colorectal cancer correspond with better patient survival following surgery compared with tumors with predominantly stromal microenvironments. However, biomarkers to identify patients with colorectal cancer liver metastases (CRLM) with good prognosis following surgery for oligometastatic disease remain elusive. The aim of this study was to determine the practical application of a simple histological assessment of immune cell infiltration and stromal content in predicting outcome following synchronous resection of primary colorectal cancer and CRLM and to interrogate the underlying functional biology that drives disease progression. Samples from patients undergoing synchronous resection of primary colorectal cancer and CRLM were evaluated in detail through histological assessment, panel genomic and bulk transcriptomic assessment, IHC, and GeoMx spatial transcriptomics (ST) analysis. High immune infiltration of metastases was associated with improved cancer-specific survival. Bulk transcriptomic analysis was confounded by stromal content, but ST demonstrated that the invasive edge of the metastases of long-term survivors was characterized by adaptive immune cell populations enriched for type II IFN signaling and MHC-class II antigen presentation. In contrast, patients with poor prognosis demonstrated increased abundance of regulatory T cells and neutrophils with enrichment of Notch and TGFß signaling pathways at the metastatic tumor center. In summary, histological assessment can stratify outcomes in patients undergoing synchronous resection of CRLM, suggesting that it has potential as a prognostic biomarker. Furthermore, ST analysis has revealed significant intratumoral and interlesional heterogeneity and identified the underlying transcriptomic programs driving each phenotype. SIGNIFICANCE: Spatial transcriptomics uncovers heterogeneity between patients, between matched lesions in the same patient, and within individual lesions and identifies drivers of metastatic progression in colorectal cancer with reactive and suppressed immune microenvironments.

CXCL8 expression is associated with advanced stage, right sidedness, and distinct histological features of colorectal cancer.

CXCL8 is an inflammatory chemokine elevated in the colorectal cancer (CRC) tumour microenvironment. CXCR2, the major receptor for CXCL8, is predominantly expressed by neutrophils. In the cancer setting, CXCL8 plays important roles in neutrophil chemotaxis, facilitating angiogenesis, invasion, and metastasis. This study aimed to assess the spatial distribution of CXCL8 mRNA expression in CRC specimens, explore associations with clinical characteristics, and investigate the underlying biology of aberrant CXCL8 levels. CXCR2 expression was also assessed in a second cohort of unique CRC primary tumours and synchronously resected matched liver metastases. A previously constructed tissue microarray consisting of a cohort of stage I-IV CRC patients undergoing surgical resection with curative intent (n = 438) was probed for CXCL8 via RNAscope®. Analysis was performed using HALO® digital pathology software to quantify expression in the tumour and stromal compartments. Scores were assessed for association with clinical characteristics. Mutational analyses were performed on a subset of these patients to determine genomic differences in patients with high CXCL8 expression. A second cohort of stage IV CRC patients with primary and matched metastatic liver tumours was stained via immunohistochemistry for CXCR2, and scores were assessed for clinical significance. CXCL8 expression within the stromal compartment was associated with reduced cancer-specific survival in the first cohort (p = 0.035), and this relationship was potentiated in right-sided colon cancer cases (p = 0.009). High CXCL8 within the stroma was associated with driving a more stromal-rich phenotype and the presence of metastases. When stromal CXCL8 scores were combined with tumour-infiltrating macrophage counts or systemic neutrophil counts, patients classified as high for both markers had significantly poorer prognosis. CXCR2+ immune cell infiltration was associated with increased stromal invasion in liver metastases (p = 0.037). These data indicate a role for CXCL8 in driving unfavourable tumour histological features and promoting metastases. This study suggests that inhibiting CXCL8/CXCR2 should be investigated in patients with right-sided colonic disease and stroma-rich tumours.

The relationship between the Glasgow Microenvironment Score and markers of epithelial-mesenchymal transition in TNM II-III colorectal cancer.

Recently published work on the Glasgow Microenvironment Score (GMS) demonstrated its relevance as a biomarker in TNM II-III colorectal cancer (CRC). Epithelial-mesenchymal transition (EMT) markers in CRC have also shown promise as prognostic biomarkers. This study aimed to assess the relationship between GMS and markers of EMT in stage II-III CRC. A previously constructed tissue microarray of CRC tumors resected between 2000 and 2007 from the Western Infirmary, Stobhill, and Gartnavel General Hospitals in Glasgow was used. Immunohistochemistry was performed for 5 markers of EMT: E-cadherin, ß-catenin, Fascin, Snail, and Zeb1. Two-hundred and thirty-eight TNM II-III CRC with valid scores for all EMT markers and GMS were assessed. The prognostic significance of markers of EMT in this cohort and relationships between GMS and markers of EMT were determined. High cytoplasmic and nuclear ß-catenin and membrane Zeb-1 were significant for worse cancer-specific survival (hazard ratio [HR] 1.67, 95% confidence interval [CI] 1.01-2.76, P < .05; HR 2.22, 95% CI 1.24-3.97, P < .01; and HR 2.00, 95% CI 1.07-3.77, P = .03, respectively). GMS 0 was associated with low membrane Fascin (P = .03), whereas membrane and cytoplasmic Fascin were observed to be highest in GMS 1, but lower in GMS 2. Nuclear ß-catenin was lowest in GMS 0, but highest in GMS 2 (P = .03), in keeping with its role in facilitating EMT. Novel associations were demonstrated between GMS categories and markers of EMT, particularly ß-catenin and Fascin, which require further investigation in independent cohorts.

Systematic review of tumour budding and association with common mutations in patients with colorectal cancer.

INTRODUCTION: Despite a well-known prognostic role in colorectal cancer, the genomic profiling of tumour budding remains to be elucidated. We aim to review the association of common mutations with tumour budding. METHODS: A systematic review of studies relating to tumour budding and genetic mutation in CRC was performed. The relationship between mutational status and tumour budding was evaluated using meta-analysis. RESULTS: A total of 6153 patients from 17 articles were included. According to the meta-analysis, high-grade tumour budding was significantly associated with KRAS mutation (OR = 1.52, 95 %CI: 1.13-2.02, P = 0.005) and MSS/pMMR (OR = 2.06, 95 %CI: 1.42-2.97, P = 0.0001). CONCLUSION: The significant association between high-grade tumour budding and mutated KRAS or MSS/pMMR may suggest a role of these mutations in the development of the tumour budding phenotype and be useful for stratifying patient outcome in CRC.

The Glasgow Microenvironment Score associates with prognosis and adjuvant chemotherapy response in colorectal cancer.

BACKGROUND: The Glasgow Microenvironment Score (GMS) combines peritumoural inflammation and tumour stroma percentage to assess interactions between tumour and microenvironment. This was previously demonstrated to associate with colorectal cancer (CRC) prognosis, and now requires validation and assessment of interactions with adjuvant therapy. METHODS: Two cohorts were utilised; 862 TNM I-III CRC validation cohort, and 2912 TNM II-III CRC adjuvant chemotherapy cohort (TransSCOT). Primary endpoints were disease-free survival (DFS) and relapse-free survival (RFS). Exploratory endpoint was adjuvant chemotherapy interaction. RESULTS: GMS independently associated with DFS (p = 0.001) and RFS (p < 0.001). GMS significantly stratified RFS for both low risk (GMS 0 v GMS 2: HR 3.24 95% CI 1.85-5.68, p < 0.001) and high-risk disease (GMS 0 v GMS 2: HR 2.18 95% CI 1.39-3.41, p = 0.001). In TransSCOT, chemotherapy type (pinteraction = 0.013), but not duration (p = 0.64) was dependent on GMS. Furthermore, GMS 0 significantly associated with improved DFS in patients receiving FOLFOX compared with CAPOX (HR 2.23 95% CI 1.19-4.16, p = 0.012). CONCLUSIONS: This study validates the GMS as a prognostic tool for patients with stage I-III colorectal cancer, independent of TNM, with the ability to stratify both low- and high-risk disease. Furthermore, GMS 0 could be employed to identify a subset of patients that benefit from FOLFOX over CAPOX.

The Relationship Between Tumor Budding, Tumor Microenvironment, and Survival in Patients with Primary Operable Colorectal Cancer.

BACKGROUND: Tumor budding is an independent prognostic factor in colorectal cancer (CRC) and has recently been well-defined by the International Tumour Budding Consensus Conference (ITBCC). OBJECTIVE: The aim of the present study was to use the ITBCC budding evaluation method to examine the relationship between tumor budding, tumor factors, tumor microenvironment, and survival in patients with primary operable CRC. METHODS: Hematoxylin and eosin-stained slides of 952 CRC patients diagnosed between 1997 and 2007 were evaluated for tumor budding according to the ITBCC criteria. The tumor microenvironment was evaluated using tumor stroma percentage (TSP) and Klintrup-Makinen (KM) grade to assess the tumor inflammatory cell infiltrate. RESULTS: High budding (n = 268, 28%) was significantly associated with TNM stage (p < 0.001), competent mismatch repair (MMR; p < 0.05), venous invasion (p < 0.001), weak KM grade (p < 0.001), high TSP (p < 0.001), and reduced cancer-specific survival (CSS) (hazard ratio 8.68, 95% confidence interval 6.30-11.97; p < 0.001). Tumor budding effectively stratifies CSS stage T1 through to T4 (all p < 0.05) independent of associated factors. CONCLUSIONS: Tumor budding effectively stratifies patients' survival in primary operable CRC independent of other phenotypic features. In particular, the combination of T stage and budding should form the basis of a new staging system for primary operable CRC.

Relationship between tumour PTEN/Akt/COX-2 expression, inflammatory response and survival in patients with colorectal cancer.

In patients with colorectal cancer (CRC), local and systemic inflammatory responses have been extensively reported to associate with cancer survival. However, the specific signalling pathways responsible for inflammatory responses are not clear. The PTEN/Akt pathway is a plausible candidate as it may play a role in mediating inflammation via COX-2, and has been associated with cancer progression. This study therefore examined the relationship between tumour PTEN/Akt/COX-2 expression, inflammatory responses and survival in CRC patients using a tissue microarray.In 201 CRC patients, activation of tumour-specific PTEN/Akt significantly associated with poorer CSS (12.0yrs v 7.3yrs, P=0.032), poorer differentiation (P=0.032), venous invasion (P=0.008) and peritoneal involvement (P=0.004). Patients were stratified for peri-nuclear expression of COX-2 to examine associations with inflammatory responses. In patients with absent peri-nuclear COX-2 expression, activation of tumour-specific PTEN/Akt significantly associated with poorer CSS (11.9yrs v 5.4yrs, P=0.001), poorer differentiation (P=0.018), venous invasion (P=0.003) and peritoneal involvement (P=0.001). However, no associations were seen with either the local or systemic inflammatory responses.In CRC patients, tumour-specific PTEN/Akt pathway activation was significantly associated with poorer CSS, particularly when peri-nuclear COX-2 expression was absent. However, activation of the PTEN/Akt pathway appears not to be responsible for the regulation of inflammatory responses.

The relationship between tumour budding, the tumour microenvironment and survival in patients with primary operable colorectal cancer.

BACKGROUND: Tumour budding has been reported to reflect invasiveness, metastasis and unfavourable prognosis in colorectal cancer. The aim of the study was to examine the relationship between tumour budding and clinicopathological characteristics, tumour microenvironment and survival in patients with primary operable colorectal cancer. METHODS: A total of 303 patients from a prospective data set of patients with primary operable colorectal cancer were included in the study. The presence of budding was determined through assessment of all tumour-containing H&E slides and the number of tumour buds was counted using a 10 high-powered field method. Routine pathologic sections were used to assess: tumour necrosis, the tumour inflammatory cell infiltrate using Klintrup-Makinen (KM) grade and tumour stroma percentage (TSP) combined as the Glasgow Microenvironment Score (GMS). RESULTS: High-grade tumour budding was present in 39% of all tumours and in 28% of node-negative tumours respectively. High-grade budding was significantly associated with T stage (P<0.001), N stage (P<0.001), TNM stage (P<0.001), serosal involvement (P<0.001), venous invasion (P<0.005), KM grade (P=0.022), high tumour stroma (P<0.001) and GMS (P<0.001). Tumour budding was associated with reduced cancer-specific survival (CSS) (HR=4.03; 95% confidence interval (CI), 2.50-6.52; P<0.001), independent of age (HR=1.47; 95% CI, 1.13-1.90; P=0.004), TNM stage (HR=1.52; 95% CI, 1.02-2.25; P=0.040), venous invasion (HR=1.73; 95% CI, 1.13-2.64; P=0.012) and GMS (HR=1.54; 95% CI, 1.15-2.07; P=0.004). CONCLUSIONS: The presence of tumour budding was associated with elements of the tumour microenvironment and was an independent adverse prognostic factor in patients with primary operable colorectal cancer. Specifically high tumour budding stratifies effectively the prognostic value of tumour stage, venous invasion and GMS. Taken together, tumour budding should be assessed routinely in patients with primary operable colorectal cancer.

The detection and role of lymphatic and blood vessel invasion in predicting survival in patients with node negative operable primary colorectal cancer.

Although vascular invasion in colorectal cancer has been recognised since 1938, detection methods and results remain inconsistent. Vascular invasion is currently an independent prognostic factor in colorectal cancer influencing disease progression and survival. The vascular system consists of three components, arterial, venous and lymphatic vessels, all of which can be invaded but accurate distinction between the components remains difficult with routine staining techniques. Even though higher detection rates with elastica staining, for large vessel invasion, and recent techniques for immunohistochemistry for small vessel invasion, have been reported, a standardised method of detection has not been agreed upon which is reflected in the variability of published results. As a result of the Bowel Cancer Screening Programme in the UK it will be necessary to attempt to identify and stratify patients better, to be able to handle the stage migration to early node negative colorectal cancer. At present up to a third of patients, with node-negative colorectal cancer on conventional histopathological analysis, ultimately die of recurrent disease. It is therefore important to develop and standardised methods to identify lymphatic and blood vessel invasion which will influence ultimate survival. The present review summarises the current status of detection methods for these components of vascular invasion.