Circulating hypoxia marker carbonic anhydrase IX (CA9) in patients with hepatocellular carcinoma and patients with cirrhosis

Abstract

Background and aims: Expression of carbonic anhydrase IX (CA9), an enzyme expressed in response to hypoxia, acidosis and oncogenic alterations, is reported to be a prognostic factor in HCC patients. Here we evaluated serum CA9 levels in HCC and cirrhosis patients.

Methods: HCC and cirrhosis patients were prospectively recruited and CA9 levels were determined. CA9 levels were compared to stages of cirrhosis and HCC stages. The association of the CA9 levels and overall survival (OS) was assessed. Furthermore, immunohistochemical CA9 expression in HCC and cirrhosis was evaluated.

Results: 215 patients with HCC were included. The median serum CA9 concentration in patients with HCC was 370 pg/ml and significantly higher than in a healthy cohort. Patients with advanced cancer stages (BCLC and ALBI score) had hid significant higher levels of CA9 in the serum. HCC patients with high serum CA9 concentrations (>400 pg/ml) had an increased mortality risk (hazard ratio (HR) 1.690, 95% confidence interval (CI) 1.017-2.809, P = 0.043). Serum CA9 concentration in cirrhotic patients did not differ significantly from HCC patients. Higher CA9 levels in cirrhotic patients correlated with portal hypertension and esophageal varices. Patients with ethanol induced cirrhosis had the highest CA9 levels in both cohorts. Levels of CA9 did not correlate with immunohistochemical expression.

Conclusions: We conclude that a high CA9 level is a possible prognostic indicator for a poor outcome in HCC patients. The high CA9 levels are probably mainly associated with portal hypertension. Ductular reactions might be a possible source of serum CA9.

Fig 1. CA9 levels in HCC patients.

A CA9 levels in healthy patients, patients with cirrhosis only and patients with hepatocellular carcinoma. B CA9 levels in different stages of Child Pugh in HCC patients. C CA9 levels in different stages of HCC according to BCLC. D CA9 levels in different stages of HCC according to ALBI score. (*, P<0.01). Vertical lines indicate the range, the horizontal boundaries of the boxes represent the first and third quartile.

Fig 2. CA9 levels in patients with cirrhosis only and etiology of HCC.

A Median CA9 levels according to etiology of liver disease in patients with HCC compared to patients with cirrhosis only. (*, P<0.01). A CA9 levels in different stages of Child Pugh in cirrhotic patients. Vertical lines indicate the range, the horizontal boundaries of the boxes represent the first and third quartile.

Fig 3. Survival in patients stratified for CA9 levels.

A Mortality according to CA9 levels in patients with HCC. Patients with high serum CA9 concentrations had an increased mortality risk (hazard ratio (HR) 1.690, 95% confidence interval (CI) 1.017–2.809, p = 0.043). B Mortality according to CA9 levels in patients with cirrhosis. Patients with high serum CA9 concentrations had an increased mortality risk in this cohort (hazard ratio (HR) 2.458, 95% confidence interval (CI) 1.010–6.114, p = 0.048).

Fig 4. CA9 immunohistochemistry of HCC and cirrhosis samples.

Representative pictures (magnification 10x) of CA9 staining. White bar represents 100μm. A HCC of a patient with cryptic cirrhosis, CA9 score 4. B HCC of a patient with HCV cirrhosis, CA9 score 3. C HCC of a patient with ethanol induced cirrhosis and necrotic area, CA9 score 3. D Non-tumorous area in cryptic cirrhosis with ductular reactions. E Non-tumorous area in NASH cirrhosis with ductular reactions. F Non-tumorous area in ethanol induced cirrhosis with ductular reactions. The evaluation of the immunostainings was done according to the publication of Kang et al.[26], score 0: no expression, score 1: <5% weakly membranous staining, score 2: 5-<25%, score 3: 25-<50%, score 4: 50-<75%, score 5: >75% moderately to strongly membranous staining.