Xingpei Hao

Early expression of cyclo‐oxygenase‐2 during sporadic colorectal carcinogenesis

Regular administration of non‐steroidal anti‐inflammatory drugs (NSAIDs) may reduce the incidence of colorectal cancer by targeting cyclo‐oxygenase‐2 (Cox‐2), a key enzyme in arachidonic acid metabolism. To evaluate the role of Cox‐2 in sporadic colorectal cancer development, Cox‐2 expression was investigated by immunohistochemistry in 85 adenomas, 53 carcinomas, 34 hyperplastic lesions and 104 samples of histologically normal mucosa adjacent to adenoma or carcinoma. In addition, Cox‐2 mRNA expression was assessed by reverse transcription‐polymerase chain reaction (RT‐PCR) in six adenomas and 14 carcinomas with paired grossly normal mucosa. Immunohistochemistry for the proliferation‐associated antigen Ki‐67 and in situ end labelling for demonstrating apoptotic bodies were also used to analyse the associations between Cox‐2 expression and proliferation and apoptosis. Cox‐2 protein expression was increased in 76/85 (89·4 per cent) adenomas and 44/53 (83·0 per cent) carcinomas compared with normal mucosa. Cox‐2 protein expression was unrelated either to the degree of dysplasia or to the size of the adenomas (p > 0·50, p > 0·10, respectively) or to differentiation, Dukes stage or lymph node metastasis of carcinomas (all p > 0·50). Interestingly, 20/34 (58·8 per cent) hyperplastic lesions adjacent to adenomas or carcinomas displayed expression higher than in normal mucosa (18·3 per cent) (p < 0·0001) but lower than in adenomas or carcinomas (p < 10−5, p < 0·001, respectively). There were no correlations between Cox‐2 protein expression and proliferative or apoptotic index in either adenomas or carcinomas (all p > 0·25). Cox‐2 mRNA expression was significantly increased in adenomas and carcinomas compared with normal mucosa (p < 0·005, p < 0·001, respectively). There were no differences between adenomas and carcinomas in either protein or mRNA levels (p > 0·25, p > 0·90, respectively). These data indicate that enhanced expression of Cox‐2 occurs early during colorectal carcinogenesis and may contribute to tumour formation. Copyright

Reciprocity between membranous and nuclear expression of β-catenin in colorectal tumours

Abstract β-Catenin has a central role not only in linking the cadherin-mediated cell adhesion system but also in the intercellular signalling pathway. To investigate alterations of β-catenin in the development of colorectal carcinoma, the pattern of β-catenin expression was studied using immunohistochemistry in 74 sporadic colorectal adenomas, in histologically normal mucosa adjacent to 65 of these adenomas, and in 52 carcinomas arising in adenomas. All normal epithelia displayed cell boundary staining for β-catenin. Adenomas and carcinomas showed varying degrees of membranous staining. However, some tumours also showed nuclear staining of β-catenin protein. Decreased membranous and increased nuclear β-catenin staining were associated with increasing degrees of dysplasia in adenomas (P < 0.005, P < 0.05, respectively). Carcinomas manifested significantly reduced membranous, but enhanced nuclear β-catenin expression compared with their associated adenomas (P < 0.001, P < 0.005, respectively). An inverse correlation was found between decreased membranous and increased nuclear staining of β-catenin in both adenomas and carcinomas (P < 0.025, P < 0.05, respectively). The data confirm that reduced membranous and increased nuclear expression of β-catenin is associated with the progression of colorectal adenomas to carcinomas. Our results also suggest that decreased membranous expression of β-catenin may result from aberrant localisation of the protein in the cell nucleus.

Changes in the expression of syndecan-1 in the colorectal adenoma-carcinoma sequence.

Abstract Syndecan-1, a transmembrane heparan sulphate proteoglycan (HSPG), functions as a matrix receptor on the basal surface of epithelial cells. It also co-localizes with E-cadherin at the lateral cell surface where its function is uncertain. Tumour development in the large bowel is associated with loss of normal epithelial adhesion and altered patterns of expression of cell adhesion molecules, possibly including syndecan-1. To evaluate changes in syndecan-1 expression during the development of colorectal neoplasia, 59 adenomas and 20 carcinomas arising from adenomas were investigated by immunohistochemistry. The staining intensity and distribution of syndecan-1 and E-cadherin in sequential sections was examined, semi-quantified and compared. Staining of syndecan-1 and E-cadherin was uniform in normal colorectal epithelial cells, and located at the basolateral surface. No significant change was seen in either molecule in mildly or moderately dysplastic adenomas. A significant reduction in expression of both syndecan-1 and E-cadherin was seen in severely dysplastic epithelium as compared to moderate dysplasia (P=0.001 and P=0.004 respectively). Similarly, there was a significant reduction of both molecules in carcinomas compared with associated adenomas (syndecan-1 P=0.00003; E-cadherin P=0.002). In both cases the loss of syndecan-1 expression was more striking than that of E-cadherin. Previous in vitro studies have shown that epithelial cells made deficient in syndecan-1 cease to express E-cadherin, suggesting a causal association. Our results support these findings and indicate that disruption of cell-matrix adhesion is critical in colorectal carcinogenesis, probably preceding changes in the purely homotypic cell-cell adhesion mediated by E-cadherin.

Loss of Bcl-2 expression correlates with tumour recurrence in colorectal cancer

Aims—To investigate the association between immunohistochemical expression of Bcl-2 and p53 in colorectal cancer and tumour recurrence following surgery. Methods—Sixty six cases of Dukes’ B colorectal carcinoma were studied. All tumours were moderately differentiated and were shown to be histologically clear of the resection margins. Immunohistochemistry was performed on formalin fixed paraffin wax embedded tissue using monoclonal antibodies for p53 and Bcl-2. The Bcl-2 staining was assessed separately for relative intensity of staining and percentage of positive tumour cells and given a final score which combined the two factors. The p53 staining was assessed on number of positive tumour cells only. The patterns of immunostaining of those cases in which there had been tumour recurrence were compared with those cases in which there was no tumour recurrence (controls). Results—A statistically significant inverse association was found between Bcl-2 score and tumour recurrence (median Bcl-2 score of 6 (interquartile range (IQR) 2–9) in patients with recurrent disease; median Bcl-2 score of 8 (IQR 6–10) in those without recurrence; p=0.03). When examined separately, both the intensity of expression and percentage of positive tumour cells were significantly associated with tumour recurrence (p=0.04 in each case). There was no association between p53 staining and tumour recurrence. Conclusion—Results suggest that, when controlled for differentiation, Bcl-2 expression is a prognostic marker and may be useful as an adjunctive test in clinical decision making.

β-Catenin expression and allelic loss at APC in sporadic colorectal carcinogenesis

Abstract β-catenin is involved in E-cadherin-mediated cell adhesion, intracellular signal transduction, and also interacts with adenomatous polyposis coli (APC) protein. We previously found that 31% of colorectal adenomas and 84% of carcinomas showed reduced membranous staining of β-catenin, while 46% of adenomas and 79% of carcinomas displayed β-catenin nuclear expression. Importantly, a reciprocal relationship between reduced membranous and increased nuclear β-catenin expression was demonstrated in the development from adenoma to carcinoma. To clarify whether this relates to an abnormality of the APC gene (APC), we have now studied allele loss in microdissected tissues from 74 adenomas and 21 carcinomas (sporadic cases, previously immunostained for β-catenin) by analysis of the microsatellites D5S346, D5S82 and D5S299. Fifty-five tumors (57.8%) showed allele loss at APC (no difference between adenomas and carcinomas). Thirty-one of these 55 (31/55, 56.4%) displayed both increased nuclear localization and reduced membranous staining of β-catenin, and thirteen tumors (13/55, 23.6%) manifested either nuclear expression without changes in membranous expression or reduced membranous staining without nuclear expression (9 and 4 cases, respectively), while 11 (11/55, 20.0%) preserved normal membranous expression. Adenomas and carcinomas showing both nuclear and reduced membranous expression of β-catenin, compared with those with normal membranous expression, tended to show allele loss (P<0.01). In addition, 24 (24/95, 25.6%) tumors showed a change in the pattern of β-catenin expression, but did not exhibit allele loss. These results suggest that although there may be a number of mechanisms responsible for changes in β-catenin expression in colorectal tumors, dysfunction of APC may be the major cause of this phenomenon.

Expression of Bcl-2 and p53 in the Colorectal Adenoma-Carcinoma Sequence

To evaluate the respective roles of Bcl-2 and p53 during colorectal tumour growth, 73 sporadic adenomas and 54 carcinomas arising in adenomas were studied by immunohistochemistry. Bcl-2 protein was expressed in 72 of 73 (98.6%) adenomas and in 25 of 54 (46.3%) carcinomas, whereas p53 protein expression was found in 18 of 73 (24.6%) adenomas and 23 of 54 (42.6%) carcinomas. Bcl-2 expression was related to the size and increasing severity of dysplasia (p < 0.05), while p53 expression was associated with the degree of dysplasia, but not with size (p < 0.025 and p > 0.10, respectively). Bcl-2 expression in carcinomas arising in adenomas was significantly reduced when compared with the associated adenoma (p < 0.005). However, there was no such difference in p53 expression between carcinomas and associated adenomas. Interestingly, there were inverse correlations between p53 and Bcl-2 expression in both adenomas and carcinomas (p < 0.05 and p < 0.005, respectively). We conclude that increased expression of Bcl-2 in adenomas may imply increasing protection against apoptosis during the progression from mild to severe dysplasia. Reduced expression of Bcl-2 may be due to downregulation by mutant p53 and may mark the point of transition from adenoma to carcinoma.

The spectrum of p53 mutations in colorectal adenomas differs from that in colorectal carcinomas

Background: p53 mutations are frequently observed in colorectal carcinomas but they have also been found in colorectal adenomas, although considerably less frequently. Aims: To explore p53 mutations in benign tumours, we have screened 70 colorectal adenomas for allelic loss at, and point mutations in, TP53 by analysis of selected microdissected cell populations. Results: Sixteen (22.8%) adenomas were found to have allelic loss, of which 11 (15.7%) had p53 mutations. In adenomas with mild, moderate, or severe dysplasia, mutation or allelic loss occurred in 4.8%, 16.7%, and 52.6%, respectively (p<0.001). Seven different mutations were found, all missense changes or inframe deletions: one (Thr150Arg) has not been found before while three (Gln144His, Gly245Arg, and Glu285Gln) have not been described previously in colorectal tumours. The other three mutations (Arg175Gly, ΔPro190, and Gly245Ser) have been found in colorectal carcinomas, the last commonly. Adenomas harboured a spectrum of p53 mutations which was significantly different from cancers as regards the position in the gene and a higher frequency of G→C/C→G changes. Conclusions: Combining our data on adenomas with data already published and in comparison with the spectrum of mutations in colorectal carcinomas, it is suggested that some p53 mutations have a weaker effect than others and are therefore more likely to be found in adenomas which have not progressed to carcinomas.