Methylation status of cyclooxygenase-2 in primary gastric tumors as a prognostic biomarker for disease outcome in a multicenter trial
Authors: Dave Hoon, Michiel de Maat, Pieter de Heer, Hein Putter, Anneke van Hoesel, Gerrit Meijer, Nicole van Grieken, Peter Kuppen, Anton Bilchik, Rob Tollenaar, Cornelis van de Velde
Journal: Clinical Cancer Research (2007)

A62  Gastric cancer is one of the leading causes of cancer death worldwide. Surgery still remains the current major intervention approach. However, prognosis is poor and management of this cancer is difficult due to the limited prognostic factors available to stratify patients. There are no prognostic tumor biomarkers to predict disease outcome. Our strategy was to develop prognostic biomarker(s) of primary gastric cancers to improve prediction of disease outcome. Overexpression of cyclooxygenase-2 (COX-2) in gastrointestinal cancers has been shown to influence disease progression. COX-2 variable expression assessed in tumors by immunohistochemistry (IHC) has been shown to be related to tumor progression in limited patient subset analysis. Our hypothesis was that COX-2 expression in primary gastric tumors assessed by quantitative methylation analysis would be of prognostic utility in determining disease outcome. To address this hypothesis we retrospectively assessed gastric cancer patients having primary tumor resection from a multicenter trial with long-term follow up. Specifically we investigated whether silencing by the promoter region by hypermethylation of the COX-2 gene contributes to disease outcome in gastric cancer. COX-2 methylation status was initially assessed by quantitative methylation-specific PCR followed by capillary array electrophoresis (MSP-CAE) of COX-2 using histopathology microdissected paraffin-embedded (PE) primary tumor tissues. In addition for comparison IHC staining of COX-2 protein expression was assessed in PE tissue sections.  In a pilot study 40 primary gastric cancers (phase II clinical trial) with clinical follow-up and 18 normal gastric tissues were assessed initially to optimize the assay and determine the potential assay utility. Prognostic endpoints of the analysis of COX-2 methylation status were time to recurrence, overall survival, and standard clinicopathology. MSP-CAE analysis was then validated in an independent gastric cancer population (n=137). As a validation set, patients were used from the D1D2 randomized multicenter clinical trial by the Dutch Gastric Cancer Group. The trial evaluated (sub)total gastrectomy for gastric cancer with D1/D2 lymph node dissection, of which the latter included partial removal of spleen and pancreas. COX-2 hypermethylation was detected in 23% and 28% of the pilot and validation patient groups, respectively. COX-2 expression (IHC) in gastric tumors inversely correlated with COX-2 hypermethylation status(P=0.02). COX-2 hypermethylation in primary tumors was significantly associated with lower T-, N-, and TNM-stage in the validation patient group (P=0.02, P=0.006, P=0.008, respectively). Patients with COX-2 methylated tumors had significantly longer time to recurrence and improved overall survival (OS) in a multivariate analysis in the validation patient group (HR=0.49; 95%CI, 0.24-0.99 and HR=0.62; 95%CI, 0.38-0.99, respectively). Hypermethylation of COX-2 promoter region was an independent prognostic factor and a significant regulatory mechanism of COX-2 protein expression. Adjuvant therapy targeting COX-2 inhibition in primary tumors prescreened for COX-2 expression may be of benefit to improve disease management in certain patient populations.