Bastiaan P. van Rees

Cyclooxygenase‐2 expression during carcinogenesis in the human stomach

The prolonged use of non‐steroidal anti‐inflammatory drugs (NSAIDs) has been associated with a reduced risk of gastric cancer. The best‐known target of these drugs is cyclooxygenase (COX); the COX‐2 isoform is frequently up‐regulated in gastric adenocarcinomas. Using the post‐gastrectomy stomach as a model, the expression of COX‐2 mRNA and protein has been investigated during tumour progression in the human stomach. COX‐2 expression was comparable in gastric stump carcinomas and conventional gastric carcinomas and localized primarily to the cytoplasm of the neoplastic cells. COX‐2 mRNA was elevated in biopsies containing intestinal metaplasia, as determined by reverse transcriptase polymerase chain reaction (RT‐PCR). COX‐2 immunopositivity became more frequent during progression from reactive epithelium to high‐grade dysplasia, both in the epithelial and in the stromal cell compartment. Co‐localization of COX‐2‐positive stromal cells was seen with CD68, α‐smooth muscle actin (α‐SMA), vimentin, and HLA‐DR, but an as yet unidentified subpopulation of stromal cells remained. Co‐localization with the macrophage marker CD68 was only observed in a minority of COX‐2‐positive cells. These data show that COX‐2 expression is a relatively early event during carcinogenesis in the stomach. COX‐2 expression increases during tumour progression in the stomach, suggesting a role for COX‐2 expression in gastric tumourigenesis. Copyright

Cyclooxygenase-2 and gastric carcinogenesis

Epidemiological studies have shown that the use of nonsteroid anti‐inflammatory drugs (NSAIDs) is associated with reduced risk of gastric cancer. The best‐known target of NSAIDs is the cyclooxygenase (Cox) enzyme. Two Cox genes have been cloned, of which Cox‐2 has been connected with gastric carcinogenesis. Expression of Cox‐2 is elevated in gastric adenocarcinomas, which correlates with several clinicopathological parameters, including depth of invasion and lymph node metastasis. This suggests that Cox‐2–derived prostanoids promote aggressive behavior of adenocarcinomas of the stomach. Cox‐2 expression is especially prominent in intestinal‐type gastric carcinoma and it is already present in dysplastic precursor lesions of this disease, which suggests that Cox‐2 contributes to gastric carcinogenesis already at the preinvasive stage. Our most recent data show that Cox‐2 is expressed in gastric adenomas of trefoil factor 1 deficient mice. Treatment of these mice with a Cox‐2 selective inhibitor, celecoxib, reduced the size of the adenomas. Taken together these data support efforts to initiate clinical studies to investigate the effect of Cox‐2 inhibitors as chemotherapeutic agents and as adjuvant treatment modalities against gastric neoplasias.

Expression of microsomal prostaglandin E synthase-1 in intestinal type gastric adenocarcinoma and in gastric cancer cell lines

Gastrointestinal carcinomas synthesize elevated levels of prostaglandin E2 (PGE2), which has been mechanistically linked to carcinogenesis. Recently, microsomal prostaglandin E synthase‐1 (mPGES‐1) was cloned, which seems to be inducible and linked to cyclooxygenase‐2 (Cox‐2) in the biosynthesis of PGE2. We examined expression of mPGES‐1 in intestinal type gastric adenocarcinomas and in gastric cancer cell lines. The transcript for mPGES‐1 was elevated in 57% (4/7) of gastric carcinomas as detected by Northern blot analysis. Moderate to strong mPGES‐1 immunoreactivity was observed in 56% (5/9) of the carcinomas as detected by immunohistochemistry. Furthermore, mPGES‐1 mRNA, protein and microsomal PGES activity were detected in gastric adenocarcinoma cell lines that originated from intestinal type tumors (MKN‐7 and MKN‐28). In contrast to Cox‐2, however, expression of mPGES‐1 mRNA or protein were not induced by phorbol 12‐myristate 13‐acetate (PMA) or interleukin‐1β (IL‐1β) in any of the gastric cancer cell lines tested (MKN‐1, ‐7, ‐28, ‐45 and ‐74). Two gastric cancer cell lines (MKN‐45 and MKN‐74) did not express mPGES‐1 and lacked microsomal PGES activity, but were still able to synthesize PGE2. Because all gastric cell lines expressed cPGES as detected by immunoblotting, it is possible that Cox‐2 can interact with cPGES or with some other yet unidentified PGES in gastric cancer cells. Furthermore, our data show that regulatory mechanisms that drive expression of mPGES‐1 and Cox‐2 dissociate in gastric cancer cell lines.

Elevated Cyclooxygenase-2 Expression Is Associated with Altered Expression of p53 and SMAD4, Amplification of HER-2/neu, and Poor Outcome in Serous Ovarian Carcinoma

Purpose and Experimental Design: Cyclooxygenase-2 (COX-2) is frequently expressed in human adenocarcinomas and inhibition of COX-2 suppresses tumor formation in various animal models of carcinogenesis. We analyzed expression of COX-2 protein in human serous ovarian carcinomas by immunohistochemistry (n = 442) and by Western blotting (n = 12) and COX-2 mRNA by reverse transcriptase PCR (n = 12). COX-2 immunoreactivity was correlated to clinicopathological variables and to expression of p53 and SMAD4 as detected by immunohistochemistry and to amplification of HER-2/neu as detected by in situ hybridization. Results: COX-2 mRNA expression was detected in 75% (9 of 12) and COX-2 protein in 42% (5 of 12) of the serous ovarian adenocarcinoma specimens as detected by reverse transcriptase-PCR and Western blot analysis, respectively. Moderate to strong (elevated) immunoreactivity for COX-2 was detected in 70% (310 of 442) of the tumors. Elevated COX-2 expression associated with reduced disease-specific survival (P = 0.0011), high histological grade (P < 0.0001), residual tumor size > 1 cm (P = 0.0111), and age > 57 years (P = 0.0099). Tumors with altered immunostaining pattern for p53 or SMAD4 expressed more frequently elevated levels of COX-2 when compared with the tumors with normal staining pattern of these tumor suppressor genes (P < 0.0001 and P = 0.0004, respectively). In addition, elevated COX-2 expression associated with amplification of HER-2/neu oncogene (P = 0.0479). Conclusions: Our results suggest that elevated expression of COX-2 associates with reduced survival in serous ovarian carcinomas and that expression of COX-2 may be induced in these tumors by loss of tumor suppressor genes such as p53and SMAD4and by amplification of HER-2/neuoncogene.

Early-onset gastric carcinomas display molecular characteristics distinct from gastric carcinomas occurring at a later age.

Gastric cancer is thought to result from a combination of environmental factors and accumulation of specific genetic alterations, and consequently mainly affects older patients (>50 years of age). Fewer than 10% of patients present with the disease before 45 years of age and these young patients are thought to develop carcinomas with a different molecular genetic profile from that of sporadic carcinomas occurring at a later age. Forty early‐onset gastric carcinoma resection specimens were characterized for microsatellite instability (MSI) and loss of heterozygosity status using 22 polymorphic microsatellite markers. Twenty‐four biopsies were additionally evaluated for the presence of MSI. No MSI was observed in any of the cases analysed. Losses were infrequent, but were most common for the D1S234 (26.1%) and D1S1676 (17.4%) markers, flanking the RUNX3 gene; for the p53ALU (23.1%) and TP53 (15.4%) markers, near the TP53 gene; and for the D16S2624 (17.2%) marker, near the E‐cadherin (CDH1) gene. All cases with loss of CDH1, as well as 6/7 cases with loss of TP53, displayed aberrant staining of the corresponding proteins, pointing to a functional role for these proteins in early‐onset gastric carcinogenesis. No germline CDH1, TP53 or RUNX3 mutations were detected in any of the cases analysed. No correlation was observed between non‐functional E‐cadherin and the histological type of the tumours analysed. Finally, Epstein–Barr virus was not detected in any of the cases analysed. On the basis of these results, early‐onset gastric carcinomas appear to have characteristics distinct from gastric carcinomas occurring at a later age. Copyright

Prognostic role of cyclooxygenase-2 in neoadjuvant-treated patients with squamous cell carcinoma of the esophagus

Based on our previous demonstration that elevated cyclooxygenase‐2 (COX‐2) expression is a prognostic factor for reduced survival in patients with adenocarcinoma of the esophagus, the aim of our study was to analyze the role of COX‐2 expression in esophageal squamous cell carcinoma. We analyzed COX‐2 protein expression from 117 consecutive patients by immunohistochemistry using a COX‐2 specific monoclonal antibody. Eighty‐one patients had not received any therapy before surgery whereas 36 patients received neoadjuvant chemotherapy as part of a randomized controlled trial. In the patients who received no chemotherapy, COX‐2 expression was low in 75% and high in 25% of the specimens. In this patient group, high COX‐2 expression associated with distal location of the tumor (p = 0.02), but did not correlate with any other clinicopathological parameter tested, including overall survival. In the patient group who received neoadjuvant chemotherapy, postoperative COX‐2 expression was low in 69% and high in 31%. Interestingly, in this patient group low COX‐2 expression correlated with development of distant metastases (p = 0.03) and to reduced overall survival (p = 0.02). Our results show that the prognostic significance of COX‐2 depends on the histological type of esophageal carcinoma and preoperative treatment of the patient. In conclusion, COX‐2 is not a prognostic marker in squamous cell carcinoma of the esophagus, but low COX‐2 expression is associated with poor prognosis in the neoadjuvant‐treated patients.

Loss of Heterozygosity and Immunohistochemistry of Adenocarcinomas of the Esophagus and Gastric Cardia

Purpose: Adenocarcinomas of the distal esophagus and gastric cardia are two tumors that have many features in common. They have similar prognoses, treatment modalities, and patterns of dissemination. The etiology is different, with gastroesophageal reflux disease playing a major role for esophageal adenocarcinoma, in contrast to adenocarcinoma of the gastric cardia. In the present study, we investigated several genetic and immunohistochemical features of adenocarcinomas of the distal esophagus and gastric cardia. Experimental Design: Sixty-two resection specimens of either adenocarcinoma of the esophagus or adenocarcinoma of the gastric cardia were carefully selected. The genetic analysis included loss of heterozygosity of several tumor suppressor genes known to be involved in esophagogastric carcinogenesis. Immunohistochemical studies included the analysis of p53, c-Met, c-erbB-2, β-catenin, and cyclooxygenase-2. In addition, a mutation analysis of the Tcf1 gene was done by direct sequencing. Results: Patients with cardiac carcinoma had a significantly worse tumor stage and poorer differentiation on histology. Loss of heterozygosity analysis did not reveal significant differences between esophageal adenocarcinoma and cardiac adenocarcinoma. Immunohistochemical analysis revealed significantly more nuclear accumulation of β-catenin and overexpression of cyclooxygenase-2 in patients with esophageal adenocarcinoma, compared with patients with cardiac carcinoma. No mutation was found in the Tcf1 gene in either tumor type. Conclusions: Although adenocarcinomas of the distal esophagus and gastric cardia have many features in common, we have found some evidence that they might form two different entities.

COX-2 in cancer

Multiple epidemiological studies indicate that the use of aspirin and other NSAIDs are associated with reduced risk of malignancies especially in the digestive tract. In addition, another NSAID sulindac causes regression of colorectal adenomatous polyps in patients with familial adenomatous polyposis (FAP) [1, 2]. Recent studies suggest that COX-2 is a rational target of NSAIDs in prevention of colorectal cancer. First, elevated levels of COX-2 mRNA and protein, but not those of COX-1, were found in colorectal adenocarcinomas and in their adenomatous precursors [3–5]. Second, selective COX-2 inhibitors suppress neoplasia formation in rodent models of colorectal cancer [6–10]. Importantly, genetic disruption of COX-2 suppresses the polyp formation in ApcΔ716-knockout and in Min mice, which are models for FAP [6, 11]. However, it should be noted that disruption of COX-1 gene also reduced the polyp burden in the Min mouse model [11]. Finally, elevated COX-2 expression was shown to associate with poor prognosis in colorectal carcinoma [12].