Osteopontin and thrombospondin-1 play opposite roles in promoting tumor aggressiveness of primary resected non-small cell lung cancer

The Gustave Roussy Cancer Center and the Marie Lannelongue Institute Institutional Review Boards gave approval for this study. Written informed consent was obtained from included patients, and patient confidentiality was protected throughout the study.

Between January and December of 2012, 171 patients with primary NSCLC who underwent curatively intended surgical resection at Marie Lannelongue Hospital, France, were included in this study. Patients were staged according to the 7th edition of the IASLC/ATS/ERS classification [27‐29] and the presence of adverse features such as visceral pleural invasion, lympho-vascular invasion and evidence of residual tumor at the resection margin were reviewed by a consultant histopathologist with expertise in lung cancer. Blood samples were obtained from each patient at baseline the day before surgery and centrifuged within 1 h of collection at 3600 × g for 10 min. Serums were stored at −20 °C until analysis was completed. Samples were then aliquoted and stored at −80 °C. Post-operative follow-ups included clinical and radiological examination (CT or conventional X-ray of the chest) at 3-month intervals for the first 2 years. Tumor recurrence and death during routine post-surgical follow-ups were recorded. First relapse was confirmed by pathologic diagnosis of the biopsy specimen.

OPN serum levels were measured using a commercially available enzyme test (Human OPN Quantikine ELISA Kit, R&D Systems, Minneapolis, MN, USA) and reported in ng/mL. All specimens were tested blinded and in duplicate. Serum samples from each patient were diluted 1:10 with calibrator Diluent RD5-24 and incubated in a micro titer plate coated with OPN antibody (Human OPN Quantikine ELISA Kit, R&D Systems, Minneapolis, MN, USA) for 2 h at room temperature (RT). After four washes, 200 μl of OPN conjugate (polyclonal antibody against OPN conjugated to horseradish peroxidase) was added to each well and incubated for 2 h at RT. Following four washes, 200 μl of substrate (hydrogen peroxide and chromogen) was added to each well and incubated for 30 min at RT. The absorbance of the samples was measured on a plate reader (Labsystems integrated EIA Management System) at 450 nm with wave length correction at 570 nm. Standard curve and sample values were calculated using Graph Pad Prism version 5.0 (GraphPad Software, Inc La Jolla, CA, USA). TSP-1 serum level was assessed using the same procedure, and each sample was assayed using Human TSP-1 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Controls were obtained from 20 healthy individuals (blood donors; 1:1 sex ratio).

For each patient, a pathologist selected one representative formalin-fixed, paraffin-embedded (FFPE) tumor block of the primary tumor. Immunohistochemistry was performed on 3 μm thick whole sections using a validated standard protocol on a Ventana Discovery Ultra autostainer (Ventana Medical Systems, Roche Tissue Diagnostics, Tucson, AZ, USA). After deparaffinization and antigen retrieval in CC1 buffer for 32 min at 98 °C, sections were incubated with either a primary goat polyclonal antibody (Santa Cruz Biothechnology; clone; concentration; dilution 1:100) against human OPN or a primary mouse monoclonal antibody (Santa Cruz Biothechnology; clone A 6.1; concentration; dilution 1:100) against TSP-1 for 1 h at room temperature. Amplification was achieved using an UltraView anti-rabbit HRP kit and with diaminobenzidine as chromogen. Slides were counterstained with hematoxylin. A pathologist who was blinded to the clinical data scored the immunohistochemical staining intensity. Two different scores were used to assess OPN and TSP-1 tissue expression because of the type of staining. OPN staining on whole slides was heterogeneous within different areas in many tumors while TSP-1 staining was much more homogeneous. H-score including both intensity of staining and percentage of stained tumor cells was used as an exploratory evaluation for OPN IHC staining. Thus, the percentage of positive tumor cells was multiplied by the staining intensity of tumor cell to obtain a final semi quantitative H score (0–300). For TSP-1, a scoring based only on staining intensity (0 to 3) was used, similarly to intensity scoring in the H-score. Then, the intensity of the staining in tumor cells was scored using a semi-quantitative scale: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). The intensity of the tumor infiltrating immune cells expressing each marker was also scored using a semi-quantitative scale from 0 (no infiltrate), 1 (mild infiltration), 2 (moderate infiltration) to 3 (dense infiltration). Image acquisition was performed with a Virtual Slides microscope VS120-SL (Olympus, Tokyo, Japan), 20X air objective (0.75 NA).

Systematic molecular analysis was performed on each tumor sample at the Genomics Platform (Gustave Roussy Institute, Villejuif, France) to identify EGFR, HER2, KRAS, BRAF, and PI3K mutation status. DNA was extracted from sections of FFPE tissues. Histological examination showed that more than 30 % of the cells were tumor cells. Genomic DNA was extracted from 4 × 10 μm thick FFPE block sections using the DNeasy Blood and tissue kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. All coding sequences of exons 18 to 21 of EGFR gene (GeneBank NM005228.3); exons 2 and 3 of Kirsten rat sarcoma viral oncogene (K-RAS) gene (NM033360-2); exon 15 of v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) gene (NM004333.4); exons 10 and 21 of phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) gene (NM006218.2); and exon 20 of human epidermal growth factor receptor-2 (HER2) gene (NM 004448.2) were analyzed. Sanger direct sequencing was performed using Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) after polymerase chain reaction amplification of targeted exons. Sequencing reactions were analyzed on 48-capillary 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Sequence reading and alignment were performed with Seq Scape software (Applied Biosystems, Forster City, CA, USA). Detected mutations were confirmed by an independent t test. ALK rearrangement status was determined by fluorescent in situ hybridization assay on tumor tissues using Dako Pre-treatment Kit (Dako) and Vysis ALK Break Apart Rearrangement Probe Kit (Abott Molecular Inc., Des Plaines, IL, USA) according to protocols described previously [30].

Statistical analysis was carried out using R [31]. Two survival end points were evaluated: (i) distant metastasis-free survival (DMFS), defined as the time interval between surgery and date of distant relapse or death, and (ii) overall survival (OS), defined as the time interval between surgery and death. Patients who were alive (OS) or without distant relapse (DMFS) were censored at the date of last contact. The prognostic values of serum OPN and TSP-1 were first tested as individual variables. Then we assessed the significance of the combination of these two variables. Hazard ratios (HR) and 95 % confidence interval (95 % CI) were calculated with the Cox proportional hazard regression model. Independent clinico-pathological variables were first analysed with univariate analysis. Variables shown in the univariate analysis that were significantly associated with DMFS and OS (with p < 0.2) were further analyzed in a multivariate cox proportional hazards regression model. Only the results of those variables that were significantly associated with the DMFS and OS (p < 0.05) using the stepwise variable selection method were reported. The required assumptions of proportionality in the multivariate survival analysis were checked graphically and by Schoenfeld’s test. DMFS and OS curves were estimated using the Kaplan–Meier method and values between groups were compared using the log-rank test. The association between OPN and TSP-1 serum levels and clinico-pathological variables were tested using Mann–Whitney U-test. Percentage, median, and range were reported to statistically describe the data. The correlation between OPN serum level and OPN tissue expression level was calculated using the Spearman method. All the statistical tests were two sided with a p value of 0.05.

Baseline characteristics of the patients are reported in Table 1. A total of 171 subjects were identified with a sex ratio of 2:1 (male: female) and a median age of 62 years (range 40–93). Non-smokers represented 16 % of the population. Primary lung adenocarcinoma was the most prevalent histotype (63 %), followed by squamous cell carcinoma (23 %). Tumor EGFR mutation, KRAS mutation and ALK rearrangement status were available for 124 (72.5 %), 126 (74 %), and 167 (98 %) patients, respectively. Pathological examination classified 92 % of the patients as having stage I to IIIA, including 47 % stage I, 21 % stage II, and 24 % stage IIIA. Thirteen patients (8 %) were classified as having stage IIIB. The majority (74 %) of resected specimen tumor sizes were lower than 5.0 cm in greatest dimension (range 0.8–11.0). All patients underwent mediastinal lymph node sampling or dissection. Lymphatic diffusion to N1, N2 and N3 nodes was present in 16 %, 22 %, and 1 % of the patients, respectively. No patient received neoadjuvant chemotherapy or radiotherapy. According to clinical criteria, a total of 48 (29 %) patients received adjuvant systemic platinum-based chemotherapy; among which combined adjuvant radio-chemotherapy was initiated in eight subjects (5 %).

Baseline serums were analyzed in 171 patients before primary tumor removal. OPN serum levels ranged from 7 to 191 ng/ml, with a median value of 27.6 ng/ml; TSP-1 serum levels ranged from 2946 to 30940 ng/ml, with a median value of 14520 ng/ml. From the control group of healthy individuals (n = 20), the median OPN and TSP-1 levels were 8.8 ng/ml [4‐45], and 31 ng/ml (0–12060), respectively. The difference in serum levels between patients and donors was statistically significant for both OPN and TSP-1 (p < 0.005) (Additional file 1). The association between clinicopathologic parameters and serum levels is presented in Table 1 and Additional file 2. Patients over 65 years old were more likely to have higher levels of OPN (32.1 vs 26.3 ng/mL, respectively, p = 0.07) and significantly lower TSP-1 serum levels compared to younger patients (13171 vs 15057 ng/mL, respectively, p = 0.01). No difference was found in serum levels with regard to smoking history. We noticed an increase of OPN serum level from stage I to IIIB. However, no significant difference was observed for either OPN or TSP-1 serum levels when the patient population was classified into pTNM stage. OPN serum level was significantly lower in patients with tumor size <5 cm than in those with size ≥5 cm (p < 0.0001). In regard to pathologic features, neither OPN nor TSP-1 serum levels was statistically associated with pleural involvement or lympho-vascular invasion. OPN serum level was lower in lung adenocarcinoma with borderline significance (p = 0.06) whereas no statistical association was found regarding pathological status and TSP-1. Small differences in serum levels among the molecular status were observed, however the difference did not exhibit statistical significance. Patients with KRAS mutation tend to have higher TSP-1 serum levels compared to those with KRAS wild-type (15323 vs13171, respectively, p = 0.08).

Median follow-up was 26 months (6–34 months). Univariate and multivariate analysis evaluating the association between survival and individual or combinatorial markers are reported in Table 2 (OS) and Table 3 (DMFS). OPN serum levels as continuous variables were significantly associated with both DMFS and OS in univariate analysis (Table 2). For each 50 units increment of serum OPN, an increased risk of metastasis by 69 % (unadjusted HR 1.69, 95 % CI 1.12–2.56, p = 0.01) and an increased risk of death by 95 % (unadjusted HR 1.95, 95 % CI 1.15–3.32, p = 0.01) were observed. Conversely, TSP-1 serum levels as continuous variables were inversely correlated with OS in univariate analysis. For each 10 units increment in TSP-1, the risk of death was decreased by 85 % (unadjusted HR 0.15, 95 % CI 0.03–0.89; p = 0.04). No statistically significant correlation was found between TSP-1 serum level and DMFS (p = 0.23). The combination of OPN and TSP-1 serum levels as continuous variable, measured as the ratio of OPN to log 10 transformation of TSP-1, was significantly correlated with both DMFS and OS in univariate analysis. For an increment of 6 ng/mL in this ratio, a 30 % increased risk of metastasis (unadjusted HR 1.3, 95 % CI 1.06–1.59, p = 0.01) and 40 % increased risk of death (unadjusted HR 1.4, 95 % CI 1.08–1.81, p = 0.01) were observed. In stage I to IIIA, absence of pleural involvement and lympho-vascular invasion were also associated with better clinical outcome in univariate analysis (Tables 2 and 3).

Three separate multivariate analyses that included prognostic clinical parameters and each of the three following variables: OPN and TSP-1 serum levels, and their combination were performed. As reported in Table 2, both OPN and TSP-1 serum levels as well as their combination maintained a strong prognostic value in the multivariate model for OS (HR 1.71, 95 % CI 1.04–2.82, p = 0.04; HR 0.18, 95 % CI 0.04–0.87, p = 0.03 and HR 1.31, 95 % CI 1.03–1.67, p = 0.03, for OPN, TSP-1 and combination, respectively). No correlation was found between these variables and DMFS (p > 0.05). The other independent prognostic factor was pathological stage. The relationship between the three markers and survival were illustrated using Kaplan-Meier survival curves represented in Fig. 1.

An overview of the immunohistochemical expression patterns of OPN and TSP-1 in tumor cells and stroma is presented in Figs. 2 and 3. The staining intensities of OPN and TSP-1 were positive in the tumor cells and the tumor stroma with heterogeneous expression. Thirty eight percent of cases showed strong cytoplasmic staining of OPN in tumor cells, while 26 % of cases were moderate, and 36 % negative (Fig. 2a,b,c). Patients with negative stroma staining (score = 0) were compared to the other cases (score = 1 or 2) (Fig. 2d). Sixty-seven percent of the samples were stained positive for TSP-1, with 37 % of cases showing strong staining and 30 % displaying moderate staining (Fig. 3a and b). In the tumor stroma, some immune cells were stained, prominently macrophages for OPN (Fig. 2d), and lymphocytes and plasmocytes for TSP-1 (Fig. 3d). There was no significant association between TSP-1 serum level and TSP-1 tissue expression level. OPN serum level and OPN tissue expression level were weakly correlated (Spearman coefficient correlation = 0.26) (Tables 2 and 3).