CAXII Is a Sero-Diagnostic Marker for Lung Cancer

Abstract

To develop sero-diagnostic markers for lung cancer, we generated monoclonal antibodies using pulmonary adenocarcinoma (AD)-derived A549 cells as antigens by employing the random immunization method. Hybridoma supernatants were immunohistochemically screened for antibodies with AMeX-fixed and paraffin-embedded A549 cell preparations. Positive clones were monocloned twice through limiting dilutions. From the obtained monoclonal antibodies, we selected an antibody designated as KU-Lu-5 which showed intense membrane staining of A549 cells. Based on immunoprecipitation and MADLI TOF/TOF-MS analysis, this antibody was recognized as carbonic anhydrase XII (CAXII). To evaluate the utility of this antibody as a sero-diagnostic marker for lung cancer, we performed dot blot analysis with a training set consisting of sera from 70 lung cancer patients and 30 healthy controls. The CAXII expression levels were significantly higher in lung cancer patients than in healthy controls in the training set (P<0.0001), and the area under the curve of ROC was 0.794, with 70.0% specificity and 82.9% sensitivity. In lung cancers, expression levels of CAXII were significantly higher in patients with squamous cell carcinoma (SCC) than with AD (P = 0.035). Furthermore, CAXII was significantly higher in well- and moderately differentiated SCCs than in poorly differentiated ones (P = 0.027). To further confirm the utility of serum CAXII levels as a sero-diagnostic marker, an additional set consisting of sera from 26 lung cancer patients and 30 healthy controls was also investigated by dot blot analysis as a validation study. Serum CAXII levels were also significantly higher in lung cancer patients than in healthy controls in the validation set (P = 0.030). Thus, the serum CAXII levels should be applicable markers discriminating lung cancer patients from healthy controls. To our knowledge, this is the first report providing evidence that CAXII may be a novel sero-diagnostic marker for lung cancer.

High Expression Level of Preoperative Serum Uroplakin III is Associated with Biologically Aggressive Bladder Cancer

“…Serum uroplakin III levels were detected by using an automated micro-dot blot array with a 256 solid-pin system (Kakengeneqs Co., Ltd., Chiba, Japan). In brief, the removal of albumin and IgG from sera was performed using a ProteoExtract Albumin/IgG removal kit (Merck, Darmstadt, Germany) according to the manufacturer's instructions (Kobayashi et al, 2012). 1 μl each of 20-times diluted albumin-and IgG-depleted sera was spotted onto polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA, USA).…”

Clinical Impact and Reliability of Carbonic Anhydrase XII in the Differentiation of Malignant and Tuberculous Pleural Effusions

“…Furthermore, high CAXII expression is also significantly associated with better overall and disease-specific survival of patients with resectable NSCLC. Kobayashi et al (2012) demonstrated that the CAXII protein flows out into the sera and its levels in patients with lung cancer are significantly higher than in healthy controls in both the training set (P<0.0001) and validation set (P=0.03). In lung cancers, the CAXII expression levels are significantly higher in patients with SCC than with AD (P=0.035).…”

“…The possibility of CAXII overexpression has been demonstrated in resectable non-small cell lung cancers (NSCLC) (Ilie et al, 2011). Serum CAXII levels are significantly higher in lung cancer patients than healthy controls (Kobayashi et al, 2012). And there is no allegation at present to show that it is overexpression in tuberculosis patients.…”

Monoclonal antibodies raised against 167–180 aa sequence of human carbonic anhydrase XII inhibit its enzymatic activity

“…This enzyme is now being recognized as a potential biomarker for different tumours and a promising target for tumour-specific therapy 26 . Along with CA IX, an established tumour marker, the significance of CA XII in tumour growth is now under investigation.…”

“…CA XII is still less studied and understood enzyme, involved in tumour progression. Recently, two research groups established different MAbs against CA XIIfor immunohistochemistry and flow cytometry using random immunization method with A549 cell lysate or live cells, respectively 26,27 . It was demonstrated that the MAb 6A10 raised against A549 cells inhibits CA XII enzymatic activity and tumour growth in vitro 27 .…”