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

van Wyk, Hester C. (2017) An investigation into the relationships between tumour invasiveness, the tumour micro-environment and survival in patients with primary operable colorectal cancer. PhD thesis, University of Glasgow.

Abstract

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.
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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
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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.
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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
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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.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Tumour invasion, colorectal cancer.
• Subjects: R Medicine > RD Surgery
• Colleges/Schools: College of Medical Veterinary and Life Sciences
• Supervisor's Name: McMillan, Professor D.C., Horgan, Professor P. and Roxburgh, Mr. C.S.D.
• Date of Award: 2017
• Embargo Date: 2017
• Depositing User: DR HC van Wyk
• Unique ID: glathesis:2017-8335
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 15 Aug 2017 07:53
• Last Modified: 06 Oct 2017 09:50
• URI: https://theses.gla.ac.uk/id/eprint/8335