Plasma secretory phospholipase A2-IIa as a potential biomarker for lung cancer in patients with solitary pulmonary nodules

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 Cincinnati Thoracic Tumor Registry, plasma samples were collected pre-operatively from patients with pulmonary nodules known or suspected to be lung cancer undergoing resection (Principal investigator: Dr. Sandra Starnes) [5]. Data on final pathology of the resected nodules was collected and defined as the benign nodule-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.

The mouse sPLA2-IIa antibody is a custom antibody developed in our lab. Human sPLA2-IIa antibody for Immunohistochemical (IHC) was from LifeSpan BioSciences (Seattle, WA).

sPLA2-IIa levels in plasma samples were determined by ELISA kit (Catalog No. 585000, Cayman Chemical Company). 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 determined 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.

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 streptavidin-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 semiquantitatively 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).

The means and standard deviations were calculated with confirmed significant difference in plasma sPLA2-IIa level between lung cancer specimens and benign nodule specimens. Geometric means and standard deviations were also calculated with confirmed significant difference in plasma sPLA2-IIa level between lung cancer specimens and clinical control specimens. Unpaired t test with Welch correction was performed to evaluate the difference between sPLA2-IIa means of (i) 96 lung cancer specimens versus 29 benign SPN specimens from the BNLCC; (ii) 44 T1 stage lung cancer specimens versus 18 T2 stage lung cancer specimens from the BNLCC; (iii) 44 lung cancer specimens from the GELCC versus 29 benign SPN specimens from the BNLCC. Unpaired t test with Welch correction was also used to analyze overall cancer survival year associated with plasma sPLA2-IIa level from the BNLCC.

A parametric Receiver Operating Characteristic (ROC) analysis of plasma sPLA2-IIa values was performed to evaluate the ability of the levels of plasma sPLA2-IIa to distinguish 96 patients with lung cancer from 29 patients with benign SPNs from the BNLCC. 44 lung cancer specimens from the GELCC relative to 29 benign SPNs specimens from the BNLCC were also subjected to the ROC analysis. The optimum cutoff value of plasma sPLA2-IIa was determined by calculating the Youden Index [25], which separated the combined set of sPLA2-IIa values into two groups, such that the number of correctly classified specimens was maximized, and the associated sensitivity and specificity for predicting lung cancer versus non-malignant nodule were determined.

The means and standard deviations of plasma sPLA2-IIa levels from 96 patients with lung cancer and 29 patients with benign lung nodules (SPNs) from the BNLCC were 3646 ± 407.3 and 1772 ± 306.8 pg/ml, respectively (Table 1 and 2, and see Additional file 1). Based on an unpaired t-test, the average plasma sPLA2-IIa level in lung cancer patients was significantly higher than that in the non-malignancy SPN controls (P = 0.004). The optimum cutoff value of plasma sPLA2-IIa by ROC analysis was 2.4 ng/ml, which resulted in 48% sensitivity and 86% specificity for predicting the presence of lung cancer (Figure 1a and Table 3). The area under the curve (AUC) was 0.68 (95% CI: 0.58 ~ 0.79) (Figure 2b). We also analyzed 44 lung cancer samples from the GELCC in which mean and standard deviation of plasma sPLA2-IIa were 3718 ± 407.8 (see Additional file 2). The average plasma sPLA2-IIa level in these lung cancer patients was also significantly higher than that in patients with benign SPNs from the BNLCC (P = 0.003). The optimum cutoff value of plasma sPLA2-IIa by ROC analysis was 2.4 ng/ml, which resulted in 64% sensitivity and 86% specificity for predicting lung cancer (AUC: 0.79; 95% CI: 0.69 ~ 0.90) (Table 3a). On the other hand, among 20 healthy donors, the levels of plasma sPLA2-IIa were undetecTable in 15 healthy donors, while those of the remaining 5 donors ranged up to 275 pg/ml (see Additional file 3). Age was not significantly associated with the level of plasma sPLA2-IIa in this healthy donor cohort (Table 1). ROC analysis of the plasma sPLA2-IIa levels in 96 lung cancer samples from the BNLCC and 20 healthy donor samples revealed an AUC as 1.0 (95% CI: 1.0 ~ 1.0) (Figure 2a).

Further analysis revealed that plasma sPLA2-IIa was significantly higher in T2 stage lung cancer (n = 18) relative to T1 stage lung cancer (n = 44) from the BNLCC (Two tailed t test: P = 0.005) (Figure 1b and see Additional file 1). The optimum cutoff value of plasma sPLA2-IIa by ROC analysis was 2.4 ng/ml, which resulted in 67% sensitivity for predicting T2 stage lung cancer (AUC: 0.86; 95% CI: 0.65 ~ 0.96), as compared with 45% sensitivity for predicting T1 stage lung cancer (Table 3a and Figure 2c). High levels of plasma sPLA2-IIa were also associated with a decreased overall survival (Unpaired t test with Welch correction: P = 0.0457) (see Additional file 1). The mean overall survival year for high sPLA2-IIa (> 2.4 ng/ml, n = 6) was 1.8 ± 1.3 years, while the mean overall survival year for low sPLA2-IIa (< 2.4 ng/ml, n = 11) was 3.3 ± 1.9 years.

Given the heterogeneous nature of cancer, the combined blood tests including a panel of biomarkers will increase the sensitivity for cancer prediction. Carcinoembryonic Antigen (CEA) and Cytokeratin-19 fragment (Cyfra 21.1) with the optimum cutoff value 6 ng/ml and 3.3 ng/ml, respectively, are two best biomarkers for predicting NSCLC currently under investigation [12, 26, 27]. Positive prediction for the presence of lung cancer by the combined blood tests was defined as one or more biomarkers higher than their cutoff values. We found that a combination of sPLA2-IIa (2.4 ng/ml cutoff value), Cyfra 21.1 (3.3 ng/ml cutoff value), and CEA (6 ng/ml cutoff value) tests increased the sensitivity for lung cancer prediction up to 62% from 48% by sPLA2-IIa test alone for 96 cancers relative to 29 patients with benign SPNs from the BNLCC (Table 3b and see Additional file 1 and 4). Furthermore, the combined tests increased the sensitivity for lung cancer prediction up to 59% and 72% from 45% and 67% by sPLA2-IIa test alone for 44 T1 stage cancers and 18 T2 stage cancers relative to 29 patients with benign SPNs, respectively. CEA and Cyfra 21.1 test alone had only 20% and 14% sensitivity, respectively (Table 3b).

Among the 5 patients with benign lung nodules and higher level of plasma sPLA2-IIa than the cutoff value 2.4 ng/ml, two patient suffered from localized pneumonia, one patient suffered from myolipomatous polyp, and the other two patients were diagnosed with necrotizing granulomas (Table 2). This finding indicated that active localized inflammation can occasionally lead to a moderate increased plasma sPLA2-IIa. Furthermore, our data demonstrated that there was an increased basal level of plasma sPLA2-IIa in patients with SPNs relative to those in healthy donors, which may result from SPNs and chronic inflammation.

Our data strongly suggest that plasma sPLA2-IIa can serve as a biomarker to predict more than 48% of T1 stage lung cancers and up to 67% of T2 stage lung cancer relative to patients with benign SPNs, although the plasma sPLA2-IIa is elevated occasionally in patients of benign SPNs. sPLA2-IIa is the best biomarker relative to Cyfra 21.1 and CEA, and the combined sPLA2-IIa, Cyfra 21.1 and CEA tests increase the sensitivity for lung cancer prediction relative to sPLA2-IIa test alone. In addition, plasma sPLA2-IIa may potentially serve as a poor prognosis biomarker for lung cancer.

Among 100 core lung biopsies examined by IHC, sPLA2-IIa was overexpressed in 100% of squamous cell carcinoma (20 cores), adenocarcinoma (20 cores), and bronchioalveolar carcinoma (10 cores), in 70% of small cell carcinoma (10 cores), and in 90% of metastatic squamous cell carcinoma (10 cores) (Figure 3a-e), but not detected in atypical carcinoid (5 cores) (Figure 3f). sPLA2-IIa was not detected in inflammatory pseudo tumor (5 cores), tuberculosis (5 cores), and normal lung tissue (15 cores) (Figure 3g-h). Expression of sPLA2-IIa in infiltrating macrophages and endothelial cells of new blood vessels in inflammatory pseudo tumors (Figure 3g) implicated an underlying mechanism of localized inflammation as the cause of occasional elevated levels of plasma sPLA2-IIa in patients with benign lesions (Table 2). We also found that sPLA2-IIa was overexpressed in lung cancer tissue, but not the adjacent normal type I and II epithelial cells in the spontaneous mouse lung cancer from SP-C/TAg transgenic mice, in which the transgene SV40 early region (TAg) gene was driven by a 3.7 kb promoter of human surfactant protein C (SP-C) gene (Figure 3i) [28, 29]. These data confirmed that sPLA1-IIa was overexpressed in lung cancer cells, but not in normal alveolar epithelial cells.