The role of perineural invasion in predicting survival in patients with primary operable colorectal cancer: a systematic review

Perineural invasion is a clear route for cancer cell spread however, the role of nerves in cancer progression is relatively unknown. Recent work would suggest that nerves can actively infiltrate the tumour microenvironment and stimulate cancer cell growth. Therefore, the aim of the present study was to systematically review the identification and associations of perineural invasion and survival in patients with primary operable colorectal cancer. From initial search results of 912 articles, 38 studies were selected. Using H&E stains; five studies including 1835 patients reported on survival stratified by perineural invasion in colon cancer with weighted average detection rates of 26%; eleven studies including 3837 patients reported on rectal cancer with weighted average detection rates of 25% and; sixteen studies including 9145 patients reported on survival stratified by perineural invasion in colorectal cancer with weighted average detection rates of 17%. Using special techniques (S100), six studies including 1458 patients reported on the identification of perineural invasion in colorectal cancer. In comparison to H&E staining alone, the use of immunohistochemistry with S100 increased the detection of perineural invasion to approximately 70%. However, those studies did not examine the relationship with outcomes, so further research is required to establish the clinical significance of perineural invasion detected by immunohistochemistry. In conclusion, perineural invasion deserves special attention for improved prognostic stratification in patients with colorectal cancer. Further work is required to standardise pathology assessment and reporting of perineural invasion, in particular its definition, use of special stains and routine inclusion in pathology practice. Reliable assessment is required for investigations into mechanisms of perineural invasion, its role tumour spread and prognostic value

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

Signal transduction and activator of transcription-3 (STAT3) in patients with colorectal cancer: associations with the phenotypic features of the tumour and host

Purpose: In patients with colorectal cancer (CRC), a high-density local inflammatory infiltrate response is associated with improved survival, whereas elevated systemic inflammatory responses are associated with poor survival. One potential unifying mechanism is the IL-6/JAK/STAT3 pathway. The present study examines the relationship between tumour total STAT3 and phosphorylated STAT3Tyr705 (pSTAT3) expression, host inflammatory responses and survival in patients undergoing resection of stage I-III CRC. Experimental Design: Immunohistochemical assessment of STAT3/pSTAT3 expression was performed using a tissue microarray and tumour cell expression divided into tertiles using the weighted histoscore. The relationship between STAT3/pSTAT3 expression and local inflammatory (CD3+, CD8+, CD45R0+, FOXP3+ T-cell density and Klintrup-Mäkinen grade) and systemic inflammatory responses and cancer-specific survival were examined. Results: 196 patients were included in the analysis. Cytoplasmic and nuclear STAT3 expression strongly correlated (r=0.363, P<0.001); nuclear STAT3 and pSTAT3 expression weakly correlated (r=0.130, P=0.068). Cytoplasmic STAT3 was inversely associated with the density of CD3+ (P=0.012), CD8+ (P=0.003) and FOXP3+ T-lymphocytes (P=0.002) within the cancer cell nests and was associated with an elevated systemic inflammatory response as measured by modified Glasgow Prognostic Score (mGPS2: 19% vs. 4%, P=0.004). The combination of nuclear STAT3/pSTAT3 stratified five-year survival from 81% to 62% (P=0.012), however was not associated with survival independent of venous invasion, tumour perforation or tumour budding. Conclusion In patients undergoing CRC resection, STAT3 expression was associated with adverse host inflammatory responses and reduced survival. Up-regulation of tumour STAT3 may be an important mechanism whereby the tumour deregulates local and systemic inflammatory responses

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

Pre-operative, biopsy-based assessment of the tumour microenvironment in patients with primary operable colorectal cancer

The tumour microenvironment (TME) is recognised as an important prognostic characteristic and therapeutic target in patients with colorectal cancer (CRC). However, assessment generally utilises surgically resected specimens, precluding neoadjuvant targeting. The present study investigated the feasibility of intra‐epithelial CD3+ T‐lymphocyte density and tumour stroma percentage (TSP) assessment using preoperative colonoscopic biopsies from 115 patients who had undergone resection of stages I–III CRC, examining the relationship between biopsy and surgically resected specimen‐based assessment, and the relationship with cancer‐specific survival (CSS). High biopsy CD3+ density was associated with high CD3+ density in the invasive margin, cancer stroma and intra‐epithelial compartments of surgically resected specimens (area under the curve > 0.62, p < 0.05 for all) and with high Immunoscore. High biopsy TSP predicted high TSP in resected specimens (p = 0.001). Intra‐class correlation coefficient for both measures was >0.7 (p < 0.001), indicating excellent concordance between individuals. Biopsy CD3+ density (hazard ratio [HR] 0.23, p = 0.002) and TSP (HR 2.23, p = 0.029) were independently associated with CSS; this was comparable to the prognostic value of full section assessment (HR 0.21, p = 0.004, and HR 2.25, p = 0.033 respectively). These results suggest that assessment of the TME is comparable in biopsy and surgically resected specimens from patients with CRC, and biopsy‐based assessment could allow for stratification prior to surgery or commencement of therapy targeting the TME

Spatially resolved transcriptomics deconvolutes prognostic histological subgroups in patients with colorectal cancer and synchronous liver metastases

Background: Patients demonstrating strong immune responses to primary colorectal cancer (CRC) have a survival benefit following surgery, while those with predominantly stromal microenvironments do poorly. Biomarkers to identify patients with colorectal cancer liver metastases (CRLM) who have 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 CRC and CRLM, and to interrogate the underlying functional biology that drives disease progression. Methods: Patients undergoing synchronous resection of primary CRC and CRLM underwent detailed histological assessment, panel genomic and bulk transcriptomic assessment, immunohistochemistry (IHC) and GeoMx Spatial Transcriptomics (ST) analysis. Integration with genomic features, pathway enrichment analysis and immune deconvolution were performed. Results: High-immune metastases were associated with improved cancer specific survival (HR, 0.36, P=0.01). 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 Interferon signalling (NES=-2.05 P.Adj<0.005) and MHC-Class II Antigen Presentation (NES=-2.09 P.Adj<0.005). In contrast, patients with poor prognosis demonstrated increased abundance of regulatory T-cells and neutrophils with enrichment of Notch (NES=2.2 P.Adj=0.022) and TGF-β (NES=2.2 P.Adj=0.02) signalling pathways at the metastatic tumor centre. Conclusions: Histological assessment stratifies outcome in patients undergoing synchronous resection of CRLM. ST analysis reveals significant intra-tumoral and inter-lesional heterogeneity with underlying transcriptomic programmes identified in driving each phenotype

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

An investigation into the relationships between tumour invasiveness, the tumour micro-environment and survival in patients with primary operable colorectal cancer

Colorectal cancer is the second commonest cause of cancer-related death after lung cancer. Prognostic features of colorectal cancer are important in determining the optimal treatment for an individual patient. The management of colorectal cancer is further complicated by the need to translate prognosis based on morphology alone as the TNM staging system describes only the anatomical extent of colorectal cancer. However, there are other prognostic factors that have impact on disease progression and can be used as adjuncts to the TNM classification. In particular, features of invasiveness of the tumour may be helpful in the identification of an aggressive phenotype of colorectal cancer and were further investigated. The hypothesis of an aggressive phenotype of colorectal cancer was explored; tumour budding reflects a detachment of tumour cells at the invasive front and presumed to be an early step in the metastatic process. As such, tumour budding has received some attention in colorectal cancer as it has been proposed as an additional factor that may stratify patients into risk categories and therefore considered to be a promising prognostic factor in colorectal cancer. Chapter 2 examined lymphatic invasion (LI) and blood vessel invasion (BVI) in context to the feasibility of methods in routine practice with and without immunohistochemistry. Results suggested the use of immunohistochemistry (IHC) for detection of lymphatic invasion as feasible; D2-40 improved the identification of lymphatic invasion and was associated with N stage. Elastica staining improved detection rates of blood vessel invasion was associated with T stage and had independent prognostic value. However, the usefulness of CD31 could not be demonstrated. 2 Thus, immunostaining with D2-40 as predictor of nodal metastasis has potential as a marker of lymph node metastasis in early stage colorectal cancer. The detection of blood vessel invasion appeared to be optimised by utilising Elastica stain, resulting in improved detection rates and improved prediction of survival. Therefore, the routine use of Elastica was recommended. These results also point to the relative roles of lymphatic and blood vessel invasion in tumour progression and dissemination in patients with colorectal cancer. In Chapter 3, Elastica staining in blood vessel invasion was further investigated. In study A, the impact of Elastica staining was examined by comparing two cohorts of patients before and after the routine implementation of the stain. Despite that Elastica staining has been shown to enhance detection rates of venous invasion with improved stratification of risk for patients with colorectal cancer, the Royal College of Pathologists advise its routine use only as a measure of quality control if venous detection rates are below 30%. Results from this study concluded that detection of venous invasion appeared to be optimised by utilising Elastica/ H&E stain that resulted in improved detection rates and improved prediction of survival. The routine use of Elastica/ H&E staining was therefore recommended. In study B, venous invasion in mouse models based on the most common mutated genes of colorectal cancer were examined. Evaluation of the depth of invasion (T stage) was used as an indicator of the extent of tumour growth and venous invasion was used as indicator of metastatic spread. The results showed that Elastica staining can be of use in the assessment of mouse models as it highlighted the elastin in veins and vascular structures were recorded in all models. However, venous invasion was only present in model 2 suggesting the 3 metastatic potential of this model. Therefore, in Model 2 the addition of activated Kras promoted formation of invasive tumours. Kras has a known role in metastatic colorectal cancer. Therefore, Elastica staining can be used to contribute to the current understanding of metastatic spread in colorectal cancer. Chapter 4 examined tumour invasion in nerves as a potential supplement to the TNM staging system. Metastatic spread can occur in nerves however, the identification of perineural invasion can be difficult with routine staining -H&E (haematoxylin and eosin) alone. Therefore, the prognostic role of perineural invasion (PNI) in Stage I colorectal cancer, using immunohistochemical staining (S100) was investigated. No associations were demonstrated between perineural invasion and clinopathological features. However, the combination of venous invasion and perineural invasion (VI&PNI) were associated with poorer overall survival on univariate analysis while age had independent prognostic value. Therefore, immunohistochemistry using S100 improved the identification of perineural invasion however, alone; the prognostic value was limited unless used in combination with venous invasion. These findings suggested that the detection of early metastatic invasion (Venous/lymphatic/perineural invasion- ―VELIPI‖) in Stage I colorectal cancer can potentially be helpful in the prediction. In Chapter 5 tumour budding were further investigated. First, the prognostic value of tumour budding using of the 10HPF (high powered field) method were examined. H&E slides were used and the number of tumour buds was counted using the 10HPF method. An optimal threshold score for the determination of high-grade budding was performed by a ROC analysis using survival as endpoint. 4 The study concluded that the presence of tumour budding was an independent adverse prognostic factor in patients with primary operable colorectal cancer. Therefore, the 10HPF method demonstrated to be a promising method for the assessment of tumour budding in H&E sections and should be considered for implementation in routine clinical practice. Next , the relationship between tumour budding and clinopathological characteristics, tumour micro-environment and survival in patients with primary operable colorectal cancer were examined. Results showed that tumour budding was associated with TNM stage, serosal involvement, venous invasion and a weaker inflammatory cell infiltrate and more stroma. The study concluded that the presence of tumour budding was associated with elements of the tumour micro-environment and was an independent adverse prognostic factor in patients with primary operable colorectal cancer. Specifically, high tumour budding was associated with and stratified effectively the prognostic value of TNM stage, venous invasion and GMS. Taken together tumour budding should be assessed routinely in patients with primary operable colorectal cancer. Further, the prognostic significance of intratumoural budding were investigated, when compared to peritumoural budding in patients with primary operable colorectal cancer. Results showed that intratumoural budding was an independent prognostic factor, as such supporting further studies to investigate intratumoural budding in a larger cohort of preoperative biopsies applicable to routine clinical use. The work presented in this thesis highlights the importance of the additional factors associated with tumour invasiveness and reports associations with the tumour micro environment and local inflammation in patients with colorectal 5 cancer. In addition, this work adds weight to the body of evidence suggesting that the recognition of an aggressive phenotype may improve stratification of treatment for patients with colorectal cancer. The thesis concluded that additional prognostic factors associated with tumour invasiveness can contribute to the current TNM staging systems and have potential to be implemented with automated assessment for future use in routine practice, the implementation of tumour budding should be further explored

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

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