Molecular Diagnostics

Carbonic anhydrase 9 (CA9) expression in non-small-cell lung cancer: correlation with regulatory FOXP3+T-cell tumour stroma infiltration



Low pH suppresses the proliferation and cytotoxic activity of CD8+ cytotoxic and natural killer lymphocytes. The hypoxia-regulated transmembrane protein, carbonic anhydrase CA9, converts carbon dioxide produced by the Krebs cycle to bicarbonate and protons that acidify the extracellular milieu. We examined whether CA9 is also involved in intratumoural immunosuppression pathways.


A series of 98 tissue samples of primary non-small-cell lung carcinomas (NSCLC) from patients treated with surgery were analysed for the expression of CA9 and programmed-death ligand PD-L1 by cancer cells, and of FOXP3 by tumour-infiltrating lymphocytes (TILs).


There was no direct association of CA9 with PD-L1 expression or the density of TILs in the tumour stroma, but CA9 was directly related to the extent of FOXP3+ TIL density (p = 0.008). Double-stratification survival analysis showed that patients with high CA9 expression and low TIL score had significantly poorer survival compared with all other groups (p < 0.04). In a multivariate analysis stage (p < 0.0001, HR 1.95, 95% CI: 1.3–2.7), TIL score (p = 0.05, HR 0.55, 95% CI: 0.2–1.0) was an independent prognostic variable of death events. CA9 expression by cancer cells is associated significantly with FOXP3+ regulatory T-cell abundance in the tumour stroma of NSCLC.


The study provides a basis for testing CA9 as a marker of resistance to immune-checkpoint inhibitors and as a therapeutic target to enhance the efficacy of immunotherapy.

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Fig. 1: Immunohistochemical figures and figures showin associations between CA9 expression and lymphocyte-related parameters.
Fig. 2: Kaplan–Meier overall (disease-specific) survival curves.
Fig. 3: Figures of survival analysis.


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We would like to thank Ms K. Devetzi for assisting in the immunohistochemical elaboration of the tissue material.

Author information

A.G., A.L.H. and M.I.K.: conception and design, analysis and interpretation of the data and writing of the paper. A.H.B.: analysis and interpretation of the data, writing of the paper. C.A.C.: acquisition of the data, analysis and interpretation of the data and writing of the paper.

Correspondence to Michael I. Koukourakis.

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Ethical approval was obtained from the Scientific Committee and the Ethics Research Committee of the University Hospital of Alexandroupolis (study approval number ES11-26-11-18). As the study is based on archival material, and anonymity of patients was guaranteed, patient informed consent was waved by the ethics board. The study was conducted according to the criteria set by the declaration of Helsinki.

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All data reported in the study are available in our departments.

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A.L.H. is the Editor-in-Chief of the British Journal of Cancer. The remaining authors declare no competing interests.

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The study has been financially supported by the Democritus University of Thrace Special Account, project no 81006.

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Giatromanolaki, A., Harris, A.L., Banham, A.H. et al. Carbonic anhydrase 9 (CA9) expression in non-small-cell lung cancer: correlation with regulatory FOXP3+T-cell tumour stroma infiltration. Br J Cancer 122, 1205–1210 (2020).

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