Analyse van morfologische, immonohistochemische en moleculaire patronen in colorectale kanker

Colorectal cancer (CRC) is worldwide the third most diagnosed cancer and is in the top four of cancer related mortality. The risk of developing colon cancer is affected by age, family history of CRC, a personal history of cancer (e.g. colon, rectum, ovary, and endometrium), chronic inflammatory bowel diseases or a history of polyps in the colon. Epidemiological studies also indicated that meat consumption, smoking and alcohol are risk factors for CRC development. Three major molecular pathways are associated with CRC development: chromosomal instability (CIN), microsatellite instability (MSI) and CpG island methylation (CIMP). Each group is characterized by specific biologic, histologic and clinical features without being mutually exclusive resulting in tumours with differences in prognosis and response to therapy. TNM staging is generally assessed in routine diagnostic practice. TNM-staging is nowadays the best prognostic indicator for CRC and indicates depth of tumour invasion (T), extent of nodal involvement (N) and presence of distant metastasis (M). Nonetheless, disease outcome and response to therapy varies significantly for patients with the same genetic background and within the same stage of disease, implying the need for new biomarkers to stratify patients more adequately. These days, most biomarker-related research in CRC focuses on the cancer’s molecular characteristics, mostly without linking it to the tumours morphology. However, human tumour tissues contain valuable information that can only be read via histology. Our approach for improving tumour classification involves the interpretation of morphologic and immunohistochemical features in CRC tissues together with a critical assessment of the molecular background. In the first part of this work, an in depth morphological characterization of MSI and microsatellite stable (MSS) primary tumours and their lymph node and haematogenous metastasis was performed together with a detailed analysis of the accompanying inflammatory infiltrate by means of a traditional microscope-based and a digital analysis, and with an assessment of the microvessel density. This work demonstrated that MSI tumours are more heterogeneous in terms of morphology. MSS tumours have in general a more glandular differentiation pattern while MSI tumours are more poorly differentiated with often glandular, solid and mucinous growth patterns that frequently present in combinations within one tumour. Characterization of the inflammatory infiltrate revealed high levels of tumour infiltrating cytotoxic T-lymphocytes in MSI tumours which are believed to have an anti-tumoural mechanism of action. Additionally, for all immune markers tested (T-lymphocytes, T-helper lymphocytes, cytotoxic T-lymphocytes, B-lymphocytes and macrophages) the number of cells that infiltrate into the neoplastic epithelium as compared to cells only infiltrating the tumoural stroma surrounding the epithelium, were higher in MSI compared to MSS tumours. Investigation of vi the spread of the inflammatory infiltrate for all morphologic components demonstrated a highly heterogeneous spread of the inflammatory infiltrate within MSI tumours which is closely linked to the morphologic differentiation pattern. Finally, it was also found that microvessel density was significantly higher in MSI compared to MSS tumours, which may hint to an impact of anti-VEGF therapy. In the second part of this work, the consequence of these findings of morphologic and inflammatory intratumoural heterogeneity for diagnostic tissue sampling and therapeutic decision making were studied. Presence of intratumoural heterogeneity is considered to have a dramatic negative impact on cancer management and we wondered to which extent morphologic heterogeneity can be used as a reference for underlying molecular heterogeneity in CRC. Including only MSI tumours characterized by mixed morphology, we were able to demonstrate that MSI status varies between MSI-high and MSI-low in the different morphologic components of the primary tumour and between MSI-high, MSI-low and MSS in the associated lymph node and liver metastasis. Presence of BRAF V600E mutations was for all tumours the same in the different primary tumour components, however in 12% of the cases BRAF mutation status was not concordant between primary tumour and metastasis. Additionally, the gene expression profiles of all clones present in two tumour cases were assessed, demonstrating significant differences between the different subclones. The more the samples resembles to one another on the level of histology, the closer they were related in terms of gene expression. In the third part of this work, we studied the importance of tumour budding (single cells or small groups up to five tumour cells that detach from the main tumour) in CRC stratification and its molecular mechanism for disease progression. Laser microdissection of tumour budding cells and tumour cells present in the tumour centre followed by gene expression profile was the basis of this work package. Analysis of the differential expression profiles demonstrated that tumour budding cells or migrate and invade or proliferate. Immunohistochemical validation for Ki-67 (a cell proliferation marker) on a series of CRC cases with variable degrees of tumour budding confirmed that there is loss of proliferation towards the invasion border and the budding cells. Furthermore, tumours with high budding and consequently a poor prognosis, were less proliferative than tumour without or with mild budding, that generally have a better prognosis. This reinforces the hypothesis that tumours with a poor prognosis are less proliferative since they present with a phenotype of migration instead of proliferation. Furthermore, stratification of tumour bulk and budding signature according to the consensus molecular subtypes demonstrated that bulk cells were generally categorized as CMS2 (canonical) and budding cells as CMS4 (mesenchymal). Therefore, the region vii within the tumour where the biopsy is taken can highly influence the result of molecular stratification. In the fourth part of this work, we investigated the expression pattern of FOXP1 in a diverse series of CRC specimen with immunohistochemistry. Over the last years, multiple markers have been tested to help further disease stratification of CRC patients and immunohistochemistry is a globally applied technique in pathology labs. Up till now, no immunohistochemical markers are integrated in routine diagnostic testing for CRC. Our work demonstrated that FOXP1 might be a potential marker for integration in clinical practice. Loss of FOXP1 is associated with a more aggressive tumour behaviour and shorter survival wile high expression of FOXP1 is linked to better survival rates. In vitro analysis on colorectal cell lines combined with gene expression profiling, demonstrated that loss of FOXP1 is associated with low proliferation rates and changes in the inflammatory infiltrate. In conclusion, this PhD work focused on disease stratification and the importance of tumour heterogeneity. First, we found that MSI tumours are more heterogeneous in terms of morphology which is reflected on the level of MSI status, BRAF mutation status and gene expression profiles. We also demonstrated that gene expression signatures of tumour budding and tumour bulk cells are significantly different and are categorized as different molecular subtypes. Therefore, based on these findings of molecular heterogeneity being reflected by morphological patterns (e.g. intratumoural clones & budding), we have strong indications that tumour histology should be considered during tissue sampling for diagnostic testing and that there might be a need for multiregion biopsies to fully characterize the tumour. Second, we demonstrated that a tumour may choose to either divide or to migrate and invade. Tumour budding cells are almost not proliferative as indicated with gene expression studies and immunohistochemical validation. Additionally, tumours with high grades of tumour budding (poor prognosis) generally proliferate less as compared to tumours without budding (good prognosis). The same findings were observed in the FOXP1 study were loss of FOXP1 was demonstrated to be a poor prognostic marker and gene expression profiling studies together with immunohistochemical analysis validated that loss of FOXP1 is associated with slower proliferation rates.status: publishe