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The apoptotic and proliferation rate of tumour budding cells in colorectal cancer outlines a heterogeneous population of cells with various impacts on clinical outcome


Dawson, Heather; Koelzer, Viktor H; Karamitopoulou, Eva; Economou, Mary; Hammer, Caroline; Muller, Dominique-Elisabeth; Lugli, Alessandro; Zlobec, Inti (2013). The apoptotic and proliferation rate of tumour budding cells in colorectal cancer outlines a heterogeneous population of cells with various impacts on clinical outcome. Histopathology, 64(4):577-584.

Abstract

Aims In colorectal cancer (CRC), tumour buds represent an aggressive cell type at the invasive front with apparently low proliferation. The aim of this study was to determine proliferation and apoptotic rates of buds in comparison to tumour centre, front and mucosa.
Methods and results Whole tissue sections from 188 CRC patients underwent immunohistochemistry for Ki67. Ten high‐power fields (HPFs) were evaluated in mucosa, tumour centre, tumour front and tumour buds (total = 40 HPFs/case). Caspase‐3 and M30 immunohistochemistry were performed on a multipunch tissue microarray from the same cohort. Ki67, caspase‐3 and M30 immunoreactivity were correlated with outcome. The average percentage of cells showing Ki67 positivity was 5.2% in mucosa, and was not significantly different between the centre and front of the tumour (38.2% and 34.9%; P < 0.0001); 0.3% of buds showed Ki67 positivity (P < 0.0001). Caspase‐3 expression was similar in mucosa, tumour centre and tumour front, but lower in tumour buds (<0.1%; P < 0.0001). M30 staining in buds was decreased (0.01%; P < 0.0001) in comparison to other areas. Ki67 positivity in buds was detrimental to survival in univariate (P = 0.0352) and multivariate (P = 0.0355) analysis. Caspase‐3‐positive tumours showed better outcome than negative tumours (P = 0.0262); but tumours with caspase‐3‐positive buds showed a worse outcome than those with caspase‐3‐negative buds (P = 0.0235).
Conclusions Ki67, caspase‐3 and M30 staining is absent in most tumour buds, suggesting decreased proliferation and apoptosis. However, the fact that Ki67 and caspase‐3 immunoreactivity was associated with unfavourable prognosis points to a heterogeneous population of tumour buds.
Introduction
In colorectal cancer, tumour budding is defined as the presence of single cells or small clusters of up to five tumour cells at the invasive front.1 This feature has been described extensively in the literature during the past two decades, and has been found repeatedly to be an independent predictor of adverse clinicopathological features such as higher tumour stage, lymph node metastases and poor patient outcome.2-5
It is widely hypothesized that tumour buds in colorectal cancer are cells in the process of epithelial–mesenchymal transition (EMT).6 These cells are no longer regulated by cellular control mechanisms and acquire migratory abilities not otherwise seen in epithelial cells, thus undergoing the initial steps toward vascular invasion and ultimately metastases. These properties are linked closely to consequences of activation of the Wnt pathway, namely the accumulation of nuclear β‐catenin, which acts as a transcription factor for many target genes, encoding proteins involved in migration, such as laminin‐5 and matrix metalloproteinases, and cell cycle regulation, such as p16 and cyclin D1.7 Nuclear β‐catenin also leads to loss of membranous E‐cadherin, disrupting cellular adhesion. Loss of membranous E‐cadherin is one of the hallmarks of EMT.8 As expected, tumour budding is most prevalent by far in colorectal cancers harbouring mutation of the APC gene, which results in deregulation of Wnt signalling.9 Previous studies have demonstrated up‐regulation of migratory proteins and chemokines in tumour buds in comparison to the main tumour body.6
It has also been hypothesized that EMT‐derived cells are generally hypoproliferative.10 Additionally, tumour buds must develop ways to stay alive for long enough to invade lymphatic and blood vessels. To date, although the rate of proliferation at the invasive front of colorectal cancer has been studied,11 investigation of the apoptotic rate, particularly using immunohistochemical markers of early and late apoptosis, has not yet been performed. We hypothesized that tumour buds, in addition to being hypoproliferative, are also hypoapoptotic and that apoptosis in buds would correlate inversely with patient outcome. Hence, the aim of the study was to analyse the rates of proliferation and apoptosis in colorectal cancer tumour buds and their impact upon clinicopathological features and patient survival.

Abstract

Aims In colorectal cancer (CRC), tumour buds represent an aggressive cell type at the invasive front with apparently low proliferation. The aim of this study was to determine proliferation and apoptotic rates of buds in comparison to tumour centre, front and mucosa.
Methods and results Whole tissue sections from 188 CRC patients underwent immunohistochemistry for Ki67. Ten high‐power fields (HPFs) were evaluated in mucosa, tumour centre, tumour front and tumour buds (total = 40 HPFs/case). Caspase‐3 and M30 immunohistochemistry were performed on a multipunch tissue microarray from the same cohort. Ki67, caspase‐3 and M30 immunoreactivity were correlated with outcome. The average percentage of cells showing Ki67 positivity was 5.2% in mucosa, and was not significantly different between the centre and front of the tumour (38.2% and 34.9%; P < 0.0001); 0.3% of buds showed Ki67 positivity (P < 0.0001). Caspase‐3 expression was similar in mucosa, tumour centre and tumour front, but lower in tumour buds (<0.1%; P < 0.0001). M30 staining in buds was decreased (0.01%; P < 0.0001) in comparison to other areas. Ki67 positivity in buds was detrimental to survival in univariate (P = 0.0352) and multivariate (P = 0.0355) analysis. Caspase‐3‐positive tumours showed better outcome than negative tumours (P = 0.0262); but tumours with caspase‐3‐positive buds showed a worse outcome than those with caspase‐3‐negative buds (P = 0.0235).
Conclusions Ki67, caspase‐3 and M30 staining is absent in most tumour buds, suggesting decreased proliferation and apoptosis. However, the fact that Ki67 and caspase‐3 immunoreactivity was associated with unfavourable prognosis points to a heterogeneous population of tumour buds.
Introduction
In colorectal cancer, tumour budding is defined as the presence of single cells or small clusters of up to five tumour cells at the invasive front.1 This feature has been described extensively in the literature during the past two decades, and has been found repeatedly to be an independent predictor of adverse clinicopathological features such as higher tumour stage, lymph node metastases and poor patient outcome.2-5
It is widely hypothesized that tumour buds in colorectal cancer are cells in the process of epithelial–mesenchymal transition (EMT).6 These cells are no longer regulated by cellular control mechanisms and acquire migratory abilities not otherwise seen in epithelial cells, thus undergoing the initial steps toward vascular invasion and ultimately metastases. These properties are linked closely to consequences of activation of the Wnt pathway, namely the accumulation of nuclear β‐catenin, which acts as a transcription factor for many target genes, encoding proteins involved in migration, such as laminin‐5 and matrix metalloproteinases, and cell cycle regulation, such as p16 and cyclin D1.7 Nuclear β‐catenin also leads to loss of membranous E‐cadherin, disrupting cellular adhesion. Loss of membranous E‐cadherin is one of the hallmarks of EMT.8 As expected, tumour budding is most prevalent by far in colorectal cancers harbouring mutation of the APC gene, which results in deregulation of Wnt signalling.9 Previous studies have demonstrated up‐regulation of migratory proteins and chemokines in tumour buds in comparison to the main tumour body.6
It has also been hypothesized that EMT‐derived cells are generally hypoproliferative.10 Additionally, tumour buds must develop ways to stay alive for long enough to invade lymphatic and blood vessels. To date, although the rate of proliferation at the invasive front of colorectal cancer has been studied,11 investigation of the apoptotic rate, particularly using immunohistochemical markers of early and late apoptosis, has not yet been performed. We hypothesized that tumour buds, in addition to being hypoproliferative, are also hypoapoptotic and that apoptosis in buds would correlate inversely with patient outcome. Hence, the aim of the study was to analyse the rates of proliferation and apoptosis in colorectal cancer tumour buds and their impact upon clinicopathological features and patient survival.

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Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > University Hospital Zurich > Institute of Pathology and Molecular Pathology
Dewey Decimal Classification:610 Medicine & health
Scopus Subject Areas:Health Sciences > Pathology and Forensic Medicine
Health Sciences > Histology
Language:English
Date:2013
Deposited On:26 Sep 2019 12:46
Last Modified:31 Jul 2020 03:39
Publisher:Wiley-Blackwell Publishing, Inc.
ISSN:0309-0167
OA Status:Closed
Publisher DOI:https://doi.org/10.1111/his.12294
PubMed ID:24111856

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