Identification Of Tumor Antigens In Human Lung Squamous Carcinoma By Serological Proteome Analysis

There is currently substantial interest in the identification of human tumor antigens for diagnosis and immunotherapy of cancer. In our previous study, we have applied a proteomic approach to lung squamous carcinoma (LSC) and identified 17 tumor proteins that elicit a humoral response in patients with LSC. In fact, autoantibodies against 2 of the 17 proteins, triosephosphate isomerase (TPI) and peroxiredoxin 6 (PRDX6), were detected in sera from 25% and 30% patients with LSC, but none of the normal controls. Western blot analysis showed secretion character and increased expression of TPI in LSC sera. PRDX6 was known to have its secretory form in the sera. All these showed that the two proteins may be two potential serum biomarkers for LSC. In this study, we further studied the association between TPI and PRDX6 with LSC.First, the serum level of TPI and PRDX6 was evaluated by sandwich ELISA in additional 61 LSC patients, 14 lung adeocarcinoma patients, 28 other types of cancer (OTC) patients and 71 non-cancer controls (NCC) including 59 normal controls (NC) and 12 patients with lung benign diseases. The mean levels of the two proteins in the normal controls group were set to be 100%, that was, the protein level was expressed to be a ratio of the OD value of each sample to the mean OD value of all the normal controls, as Rtpi and Rprdx6, respectively. Results showed that the serum level of TPI in LSC patients was significantly higher than that in lung adeocarcinoma patients and in OTC patients and in NCC (P<0.05). Likewise, the serum level of PRDX6 in LSC patients was significantly higher than that in OTC patients and in NCC. However, no significant difference of PRDX6 was found comparing to that in lung adeocarcinoma patients. Differentiating LSC patients and NCC, Receive Operating Characteristic curve (ROC curve) with TPI and PRDX6 showed AUC with 0.811(p=0.037<0.05) and 0.738(p=0.043<0.05), respectively. Based on the ROC curve and the Youden index analysis, the optimal cut-off value to distinguish the LSC from the NCC was used. As a result, with a cut-off value of 1.122, Rtpi can differentiate 62.3% of the LSC patients and 85.9% of the NCC controls. For Rprdx6, with a cut-off value of 1.010, 65.6% of the LSC patients and 67.6% of the NCC controls can be correctly classified. In addition, A discriminant analysis was applied to differentiate LSC patients and NCC using the serum levels of the two proteins as variables together, the result showed 72.1%(44/61) of the LSC and 84.5% (60/71) of NCC was correctly classified , that was 78.8% of the original grouped cases correctly classified. With the discriminant function, we found 2 of the lung adeocarcinoma but non OTC patients were wrongly classified to be LSC. In addition, the cross-validation showed a similar result with 70.5% (43/61) of the LSC patients and 84.5% (60/71) of the NCC correctly classified. All of the results indicated that not only their autoantibody, the two proteins themselves may also be two novel serum markers for LSC.In addition, within the group of LSC, statistic analysis showed that PRDX6 level in sera had statistically significant positive correlation with the differentiation of LSC and the LSC patients with distant metastasis (M1) had higher level of TPI in their sera compared with LSC patients with M0 (p<0.05), which indicates positive correlation between PRDX6and LSC differentiation and some association between TPI and LSC distant metastasis. For the research of the mechanism of the antigenic property of the two proteins, 2D western blot was applied to find if the sera which reacted with the two proteins in the tumor tissues could react with TPI and PRDX6 in the normal lung tissues. The results showed that the same positive sera could react with the two proteins in the normal tissues, indicating no structure modification happened with the two in the tumor tissues or the modification didn't account for the antigenic property for them. Immunohistochemistry analysis showed higher expression of TPI but not PRDX6 in 30 LSC tissues than that in their paired normal tumor-adjacent bronchial epithelial tissues. And as the ELISA results showed, it is possibly that overexpression of TPI in tumor tissues and increased expression of TPI and PRDX6 in LSC sera may account for their autoantibody development.