Identification of osteopontin as a prognostic plasma marker for head and neck squamous cell carcinomas

Abstract

Purpose: Tumor hypoxia modifies treatment efficacy and promotes tumor progression. Here, we investigated the relationship between osteopontin (OPN), tumor pO(2), and prognosis in patients with head and neck squamous cell carcinomas (HNSCC).

Experimental design: We performed linear discriminant analysis, a machine learning algorithm, on the NCI-60 cancer cell line microarray expression database to identify a gene profile that best distinguish cell lines with high Von-Hippel Lindau (VHL) gene expression, an important regulator of hypoxia-related genes, from those with low expression. Plasma OPN levels in 15 volunteers, 31 VHL patients, and 54 HNSCC patients were quantitatively measured by ELISA. The relationships between plasma OPN levels, tumor pO(2) as measured by the Eppendorf microelectrode, freedom from relapse (FFR), and survival in HNSCC patients were evaluated.

Results: Microarray analysis indicated that OPN gene expression inversely correlated with that of VHL. These findings were confirmed by Northern blot analysis. ELISA studies and Western blot in a HNSCC cell line demonstrated that hypoxia exposure resulted in increased OPN secretion. Patients with VHL syndrome had significantly higher plasma OPN levels than healthy volunteers. Plasma OPN level inversely correlated with tumor pO(2) (P = 0.003, r = -0.42). OPN levels correlated with clinical outcomes. The 1-year FFR and survival rates were 80 and 100%, respectively, for patients with OPN levels <or=450 ng/ml and 43 and 63%, respectively, for levels >450 ng/ml (P = 0.002 and 0.0005). Multivariate analysis revealed that OPN was an independent predictor for FFR and survival.

Conclusions: Plasma OPN levels appeared to correlate with tumor hypoxia in HNSCC patients and may serve as noninvasive tests to identify patients at high risk for tumor recurrence.

Fig. 1

VHL versus OPN expression. A, identification of 11 low VHL expressers (VHL log₂ ratio < −0.5) and 7 high VHL expressers (VHL log₂ ratio > 0.5). The remaining cell lines had VHL log₂ ratios between 0.5 and −0.5 and were not considered further in this analysis. Using LDA, we were able to identify OPN as a VHL regulated gene that encoded a secreted product. B, Northern blot analysis of OPN and VHL gene expressions in seven human cancer cell lines identified from LDA analysis. These results support the gene arrays data. Methylene blue staining showed equal loading of all lanes. C, comparison of mean plasma OPN levels in 31 VHL patients and 15 healthy volunteers by ELISA assay. VHL patients had significantly higher OPN plasma levels than that of age-matched volunteers.

Fig. 2

OPN and tumor hypoxia. A, ELISA analysis of OPN expression in serum-free conditioned media and cell lysates from SCC4 cells after a time course of hypoxia exposure or 24 h of normoxia. Error bars represented replicated measurements. B, Western blot showing OPN expression in serum-free conditioned media from SCC4 cells after (a) 16 h of normoxia, (b) 20 h of normoxia, (c) 40 h of normoxia, (d) 16 h of hypoxia, (e) 16 h of hypoxia + 4 h of reoxygenation, (f) 16 h of hypoxia + 24 h of reoxygenation. Ponceau-S staining show equal loading for all lanes. Hypoxia and hypoxia and reoxygenation enhanced OPN secretion into the media. C, correlation between plasma OPN levels and median tumor pO₂ in patients with HNSCC.

Fig. 3

A, Kaplan-Meier estimates of FFR by OPN plasma levels. B, overall survival by OPN plasma levels. C, overall survival by OPN plasma levels in patients with N_0–2 neck nodes. D, overall survival by OPN plasma levels in patients with N₃ neck nodes.