High levels of carbonic anhydrase IX in tumour tissue and plasma are biomarkers of poor prognostic in patients with non-small cell lung cancer

Abstract

Background: Carbonic anhydrase IX (CAIX) is an enzyme upregulated by hypoxia during tumour development and progression. This study was conducted to assess if the expression of CAIX in tumour tissue and/or plasma can be a prognostic factor in patients with non-small cell lung cancer (NSCLC).

Methods: Tissue microarrays containing 555 NSCLC tissue samples were generated for quantification of CAIX expression. The plasma level of CAIX was determined by ELISA in 209 of these NSCLC patients and in 58 healthy individuals. The CAIX tissue immunostaining and plasma levels were correlated with clinicopathological factors and patient outcome.

Results: CAIX tissue overexpression correlated with shorter overall survival (OS) (P=0.05) and disease-specific survival (DSS) of patients (P=0.002). The CAIX plasma level was significantly higher in patients with NSCLC than in healthy individuals (P<0.001). A high level of CAIX in the plasma of patients was associated with shorter OS (P<0.001) and DSS (P<0.001), mostly in early stage I+II NSCLC. Multivariate Cox analyses revealed that high CAIX tissue expression (P=0.002) was a factor of poor prognosis in patients with resectable NSCLC. In addition, a high CAIX plasma level was an independent variable predicting poor OS (P<0.001) in patients with NSCLC.

Conclusion: High expression of CAIX in tumour tissue is a predictor of worse survival, and a high CAIX plasma level is an independent prognostic biomarker in patients with NSCLC, in particular in early-stage I+II carcinomas.

Figure 1

CAIX expression detected by immunohistochemistry in tissue microarray cores. Staining levels for CAIX in NSCLC histological subtypes: low (A, E, I, M), intermediate (B, F, J, N) and strong (C, G, K, O). CAIX membrane staining in adenocarcinoma (ADC) (A–D), squamous cell carcinoma (SCC) (E–H), large cell carcinoma (LCC) (I–L), and sarcomatoid carcinoma (SC) (M–P). Normal bronchial epithelium (Q) and alveolar tissue (R) are devoid of staining. Strong membrane staining in head and neck squamous cell carcinoma (HNSCC) cores used as positive control of CAIX immunostaining (S–T). Panels D, H, L, P and T are higher magnifications showing details of cells within the corresponding tumour shown on panels C, G, K, O, and S, respectively.

Figure 2

Quantitative evaluation of the plasma CAIX level by ELISA in patients with NSCLC and in healthy controls. (A) Box plots showing median (central dots), 25–75% quartile ranges (boxes), and minimum/maximum levels (whiskers) of plasma CAIX levels in healthy individuals (n=58) vs patients with NSCLC (n=209). (B) ROC curve analysis of CAIX as a plasma marker for NSCLC. X axis, 1-specificity; y axis, sensitivity. (C) Plasma concentration of CAIX according to tumour size in NSCLC and in healthy individuals. NS; non significant.

Figure 3

Kaplan–Meier curves of overall survival (top panels A and C), and disease-specific survival (bottom panels B and D) duration stratified according to the tissue CAIX expression detected by immunohistochemistry (left panels A and B) and the plasma CAIX levels as determined by ELISA (right panels C and D). The cut-off value for the CAIX immunostaining score was arbitrarily defined as superior or equal to 45 grey levels. The cut-off point for the CAIX plasma levels was empirically defined as superior or equal to 100 pg ml^–1. The curves are labelled with the corresponding scores.