Molecular Pathogenesis of Gastrointestinal Neoplasia
Moderators: Dr. Wataru Yasui and Dr. Jeremy Jass
Section 5 -
Molecular Pathology of "Budding" in Colorectal Cancer
Institute of Pathology , University of Munich
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. They can be named migrating cancer
stem cells, and they might be the subpopulation of colorectal cancer cells which drives tumour
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