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.
Highlights
Lung cancer is the leading cause of cancer death, comprising 13% (1.6 million) of the total cancer cases and 18% (1.4 million) of the cancer deaths in the world in 2008 [1,2].Tumor markers have been detected in sera, urine, and tissues from patients with malignant tumors, and can be used for an exact diagnosis, discrimination of benign or malignant tumors, follow-up after therapies, and prediction of the patient’s outcome
Some sero-diagnostic markers are used for lung cancer, such as carcinoembryonic antigen (CEA) and sialyl Lewis X antigen (SLX) for adenocarcinoma (AD), and cytokeratin 19 fragment (CYFRA) and squamous cell carcinoma antigen (SCCa) for squamous cell carcinoma (SCC) [3]
Using AMeX-fixed A549 cell preparations for the immunohistochemical screening of hybridomas, we established 188 monoclonal antibodies (MoAbs) in total and a further study was performed with the KULu-5 clone, which showed intense staining in A549 cells (Fig. 1 A)
Summary
Lung cancer is the leading cause of cancer death, comprising 13% (1.6 million) of the total cancer cases and 18% (1.4 million) of the cancer deaths in the world in 2008 [1,2].Tumor markers have been detected in sera, urine, and tissues from patients with malignant tumors, and can be used for an exact diagnosis, discrimination of benign or malignant tumors, follow-up after therapies, and prediction of the patient’s outcome. Some sero-diagnostic markers are used for lung cancer, such as carcinoembryonic antigen (CEA) and sialyl Lewis X antigen (SLX) for adenocarcinoma (AD), and cytokeratin 19 fragment (CYFRA) and squamous cell carcinoma antigen (SCCa) for squamous cell carcinoma (SCC) [3]. We have exhaustively generated monoclonal antibodies (MoAbs) against various tumor-associated proteins using the pulmonary AD-derived A549 cell as an antigen with the random immunization method [4], and over 1,000 MoAbs have been obtained [5]. This method is expected to generate antibodies against proteins with tumor-specific post-translational modifications, which are difficult to obtain by conventional immunization methods
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