Clinical Science (2010) 118, 717–725 (Printed in Great Britain) doi:10.1042/CS20090537 717 Increased plasma concentrations of anterior gradient 2 protein are positively associated with ovarian cancer Tracey A. EDGELL ∗ , Dong L. BARRACLOUGH†‡, Antonio RAJIC ∗ , Janu DHULIA ∗ , Kate J. LEWIS ∗ , Jane E. ARMES§, Roger BARRACLOUGH§, Philip S. RUDLAND§, Gregory E. RICE ∗ ‖ and Dominic J. AUTELITANO ∗ ∗ HealthLinx Ltd, 576 Swan Street, Richmond, Victoria 3121, Australia, †School of Clinical Sciences, University of Liverpool, Daulby Street, Liverpool L69 3GA, U.K., ‡School of Biological Sciences, University of Liverpool, Daulby Street, Liverpool L69 3GA, U.K., §Mater Pathology, Mater Health Services, Brisbane, Queensland 4101, Australia, and ‖Baker IDI Heart and Diabetes Institute, 75 Commercial Road, Melbourne, Victoria 3134, Australia A B S T R A C T Ovarian cancer is often asymptomatic and is diagnosed at an advanced stage with poor survival rates, thus there is an urgent need to develop biomarkers for earlier detection of ovarian cancer. In the present study, we demonstrate for the first time that the previously reported metastasis- inducing protein AGR2 (anterior gradient protein 2) can be detected in the blood of ovarian cancer patients. Using a newly developed ELISA, we show significantly increased concentrations of AGR2 protein in plasma from cancer patients relative to normal controls. Plasma AGR2 concentrations were highest in stages II and III ovarian cancer patients and were similarly elevated in patients with both serous and non-serous tumours. The identification of elevated plasma concentrations of AGR2 may provide a useful biomarker to aid in the discrimination of normal and ovarian cancer patients particularly when used in combination with CA125. INTRODUCTION Ovarian cancer is the most lethal of gynaecological cancers and represents one of the most frequent causes of cancer-related deaths in women in Europe and the United States. Since early stage ovarian cancer is often asymptomatic, the disease is usually diagnosed at an advanced stage, with 5-year survival rates of less than 30 % arguing that improved early detection is paramount for enhanced survival [1]. At present, CA125 represents the best characterized and most widely used ovarian cancer biomarker, being elevated in more than 80% of patients with epithelial ovarian cancer, but only in approximately 50 % of patients with stage I disease [1]. Therefore, there is an urgent need to develop novel diagnostic tests to be able to detect ovarian cancers as early as possible, when survival and treatment opportunities are greatest. AGR2 (anterior gradient 2) protein is the human homologue of the cement-gland XAG-2 protein that was previously described in Xenopus laevis, where this protein has been shown to be a crucial factor involved in cellular differentiation and development [2]. In normal mammalian tissue, AGR2 is found in mucin-containing cells and may play a role in the production of intestinal MUC2 [3]. AGR2 mRNA has been shown to be co-expressed with ER (oestrogen receptor) in human breast cancer cell lines and in human breast cancer tissue [2,4]. AGR2 was also shown to be markedly elevated in the majority of prostate Key words: anterior gradient protein 2 (AGR2), biomarker, CA125, ELISA, ovarian cancer. Abbreviations: AGR2, anterior gradient protein 2; AUC, area under the curve; ESI, electrospray ionization; HRP, horseradish peroxidase; MAb, monoclonal antibody; MALDI, matrix-assisted laser-desorption ionization; PAb, polyclonal antibody; rhAGR2, recombinant human AGR2; MS/MS, tandem MS; ROC, receiver operator characteristic; TBS, Tris-buffered saline; TFA, trifluoroacetic acid; TOF, time-of-flight. Correspondence: Dr Dominic J. Autelitano (email d.autelitano@healthlinx.com.au). C  The Authors Journal compilation C  2010 Biochemical Society