Kupert et al. BMC Cancer 2011, 11:513 http://www.biomedcentral.com/1471-2407/11/513 nodules. In North America, surgical resection of a lung nodule alone costs more than $20,000 [8]. Identification of malignant nodules is crucial because they represent a localized and potentially curable form of lung cancer. A few plasma biomarkers have been used to screen and diagnose cancers [9-11]. Currently, there is no single inexpensive, convenient, and reliable biomarker proven effective for the diagnosis of lung cancer [12]. Secretory phospholipase A2-IIa (sPLA2-IIa) is a phos- pholipid hydrolase enzyme that mediates the release of arachidonic acid (AA) and lysophosphatidylcholine, which are the precursors of eicosanoids and platelet- activating factor, respectively [13,14]. Eicosanoids are products of both sPLA2-IIa and cyclooxygenase-2 (Cox- 2) and exert control over many physiologic processes, such as inflammation, immunity, and tumorigenesis. It was reported that elevated eicosanoids, such as prosta- glandins, are involved in the pathogenesis of lung cancer [15]. sPLA2-IIa is a downstream effector of the NF-kB gene [16,17]. A few early studies have demonstrated that sPLA2-IIa is overexpressed in almost all human prostate cancer specimens and elevated levels of sPLA2-IIa are associated with tumor grade [18-20]. sPLA2-IIa remains elevated in androgen-independent prostate cancers fail- ing hormonal therapy and functions as a growth factor to stimulate growth in prostate cancer cells [21]. We recently reported that elevated signaling of the HER/HER2-PI3K-Akt-NF-kB pathway contributes to sPLA2-IIa overexpression and secretion in prostate can- cer cells [17,22,23]. We confirmed that sPLA2-IIa secreted by cancer cells into the circulation to a detecT- able level in mice carrying xenograft human tumor. We are the first to demonstrate that there are high levels of plasma sPLA2-IIa in prostate cancer patients, which are associated significantly with high Gleason score and advanced disease stage. In the present study, we expanded our observation to lung cancer. We found that lung cancer tissues overexpress sPLA2-IIa and high levels of plasma sPLA2-IIa may serve as a plasma bio- marker for lung cancer. Methods Patient population The study was conducted in accordance with the Declaration of Helsinki and the local Institutional Review Board approved the study protocol. All the specimens for this study were archived biospecimens with de-identified patient information. As part of the University of Cincin- nati Thoracic Tumor Registry, plasma samples were col- lected pre-operatively from patients with pulmonary nodules known or suspected to be lung cancer under- going resection (Principal investigator: Dr. Sandra Starnes) [5]. Data on final pathology of the resected nodules was collected and defined as the benign nodule- Page 2 of 10 lung cancer cohort (BNLCC). Plasma samples were also collected from lung cancer patients from the “Genetic Epidemiology of Lung Cancer” cohort (GELCC; PI: Dr. Marshall Anderson), a familial lung cancer cohort. Plasma samples from healthy donors were obtained from the Cincinnati Hoxworth Blood Center. Reagent The mouse sPLA2-IIa antibody is a custom antibody developed in our lab. Human sPLA2-IIa antibody for Immunohistochemical (IHC) was from LifeSpan BioS- ciences (Seattle, WA). Enzyme-linked immunosorbent assay (ELISA) sPLA2-IIa levels in plasma samples were determined by ELISA kit (Catalog No. 585000, Cayman Chemical Com- pany). All plasma samples were diluted ten times for ELISA as described before [17]. The concentration of sPLA2-IIa in plasma was tested in duplicate and deter- mined against a standard curve for each ELISA assay. ELISA kits for Carcinoembryonic Antigen (CEA) (EIA- 1871) and Cytokeratin-19 fragment (CYFRA 21.1) (EIA- 3943) were purchased from DRG International Inc., USA. Plasma samples were subjected to CEA and Cyfra21.1 ELISA and quantitated against a standard curve of each ELISA assay. Immunohistochemical (IHC) staining Lung disease spectrum tissue array was obtained from Biomatrix.US (Rockville, MD). Immunohistochemistry (IHC) for the sPLA-IIa protein was performed as detailed in our previous studies [17,24]. Briefly, paraffin- embedded tissue sections were deparaffinized in xylene, rehydrated in graded alcohol, and transferred to PBS. The slides were treated with a citric acid-based antigen- retrieval buffer (DAKO Co., Carpinteria, CA), followed by 3% H2O2 in methanol, incubated in blocking buffer (5% BSA and 5% horse serum in PBS) and then in the blocking buffer containing the primary antibody. After washing, the slides were incubated with a biotinylated secondary antibody (BioGenex Laboratories, San Ramon, CA), followed by washing and incubation with the strep- tavidin-conjugated peroxidase (BioGenex). A positive reaction was visualized by incubating the slides with sTable diaminobenzidine and counterstaining with Gill’s hematoxylin (BioGenex) and mounted with Universal Mount mounting medium (Fisher Scientific, Pittsburgh, PA). The intensity and extent of positive labeling for sPLA2-IIa in tissue arrays were assessed semiquantita- tively and scored as 0 (no staining), 1+ (weak and focal staining in < 25% of tissue), 2+ (moderate intensity in 25-50% of tissue), 3+ (moderate intensity in > 50% of tissue), and 4+ (strong and diffused staining in > 50% of tissue).