Section 5 -

Molecular Pathology of "Budding" in Colorectal Cancer Thomas Kirchner Institute of Pathology , University of Munich Germany

Invasion and metastasis are hallmarks of cancer. They require the loss of epithelial differentiation (dedifferentiation) and adhesion, followed by the migration and dissociation of cancer cells. In the majority of colorectal carcinomas, these processes have been observed at the invasive margin and described as tumour budding. An isolated single cancer cell and a cluster composed of fewer than five cancer cells were defined as budding foci. The grading of the budding intensity was proposed as a surrogate marker for the invasiveness of colorectal cancer. Applying a x250-magnification (x25 objective lens) and counting the budding foci in a field, the detection of <9 budding foci was considered as low grade, and the detection of >10 budding foci as high grade budding intensity. Budding foci can already be detected in hematoxilin-eosin-stains, but are highlighted by immunohistochemistry using a pan-cytokeratin antibody. The immunostaining for cytokeratin additionally shows cytoplasmic pseudo-fragments, which are dendritic cell processes in continuity with the budding cells, around the budding foci. Such cytoplasmic pseudofragments may be a marker for an activated budding phenotype that is associated with cell motility following the dedifferentiation and dissociation of cancer cells. Numerous clinico-pathological studies have established the tumour budding as a relevant index to estimate the potential aggressiveness of colorectal cancer and to predict the prognosis for colorectal cancer patients. Multivariate analysis revealed budding as a significant prognostic factor of postoperative survival in stage II and stage III colon carcinomas. High grade budding was useful to select high risk patients of stage II colon carcinoma exhibiting a similar outcome as patients of stage III, which deserve adjuvant chemotherapy. In addition, budding turned out to be a diagnostic determinant for the optimal treatment and the decision between local excision or radical surgery in pT1 colorectal carcinoma. Finally, the budding intensity was one of the best predictors of lymph node metastases and the detection of isolated cancer cells in regional lymph nodes. Based on the histological findings and clinico-pathological correlations it can be assumed that the factors, which are involved in the molecular pathology of tumour budding, are relevant for the invasiveness, metastases and progression of colorectal cancer. beta-catenin, which is deregulated by the loss of function mutation of APC, plays a major role in colorectal cancer progression. Its membranous expression is usually found in central areas of colorectal carcinomas, where cancer cells adhere to each other forming tubular structures. In contrast, a loss of the membranous expression, an intracellular accumulation and a nuclear expression of beta-catenin are found at the invasive margin of tumors, where the budding occurs. We and others have shown, that a molecular complex formed by the nuclear beta-catenin and nuclear factors as TCF 4 and LEF 1 acts as major transcription factor for invasiveness in colorectal carcinoma. Thus the TCF 4 / beta-catenin complex is able to activate a battery of genes, which are relevant for the dedifferencation of colon epithelial cells, for the loss of adhesion, for the matrix degradation and for the migration as well as for the control of the cell cycle. Genes, which are associated with dedifferencation, matrix degradation and migration are specifically upregulated and expressed at the invasive margin of colorectal cancer, where the strongest nuclear expression of beta-catenin is observed. The expression of some of these factors – e.g. MMP 7, laminin 5 gamma 2 – was shown to occur in the budding foci and had a similar prognostic impact as the budding index. Moreover, a correlation between budding and nuclear beta-catenin expression as well as between budding and the frequency of somatic APC-mutations were reported. These observations suggest that the deregulation of the WNT-pathway and the nuclear activity of beta-catenin are decisively implicated in the molecular pathology and development of tumour budding. However, nuclear beta-catenin might not be the sole cause of budding. In a recent study of 466 colorectal carcinomas nuclear beta-catenin expression increased from the central area towards invasive margin, but did not predict budding. Therefore, the nuclear activity of beta-catenin in the invasive margins of colorectal cancer could only provide the molecular background for tumour budding, but additional co-stimulatory signals or deregulations might be decisive for the induction and modulation of budding intensity. The co-stimulation could be due to the micro-environment of the cancer cells and to the cancer-cell-stroma-interaction. According to our results from gene expression profiling after microdissection of colorectal cancer, we suggest that on the background of molecular beta-catenin deregulation caused by the APC-mutation a stromal signalling related to inflammation and wound healing induces the dedifferentation, dissociation and migration of neoplastic cells at the front of invasion. Moreover, we were able to demonstrate that the dissociated (isolated) neoplastic cells in the budding foci exhibit markers of stem cells as well as factors for migration. 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