Many studies confirmed that the expression abnormalities presented by cell adhesion molecules were closely correlated with the tumor invasion and metastasis, and might be one of the determinants of tumor cells to gain the ability of invasion and distant metastasis. Cell adhesion molecules CD44v6 and integrin-β1 show high expression levels in many tumor cell lines and solid human tumor tissues. In clinical studies, a number of studies addressed the correlation between the two markers (CD44v6 and integrin-β1) and the prognosis of many cancers. However, the roles of CD44v6 and integrin-β1 in predicting the prognosis for advanced PC, especially in predicting prognosis for cryosurgery against PC, were seldom reported. Several studies have reported that blood mRNA, in addition to DNA, are practical markers for clinical use for healthy individuals and for patients suffering from cancer [23, 24]. Since tissue specimens must be removed by tumor resection or biopsy, we often can not prepare enough specimens for the detection of biomarker expression, which is essential for the metastasis and prognosis evaluation of many tumors. Therefore, it is important to investigate gene expression in blood samples, instead of gene expression in tumor tissue, and determine the potential roles of related genes as biological markers for the prognosis assessment of PC. Besides, in most of previous studies, animal models and cell lines were adopted as experimental objects to evaluate the role of biomarkers in the prognosis of cancer. However, tumor progression process in animal models and cell lines differed greatly from that of human. Besides, the conventional detection methods used in these studies, such as RT-PCR and immunohistochemistry, may lead to a high false-positive rate in the investigation of clinical samples. Thus, the CD44v6 and integrin-β1 expression levels can not be precisely quantified, resulting in the errors of data analysis in these studies. Hence, the controversy on the roles of CD44v6 and integrin-β1 in tumor metastasis and differentiation are aroused. Currently, the determination of relative gene expression levels through quantitative real-time PCR is considered to be a more sensitive and more quantitative methodology than immunohistochemistry. In this method, an internal reference gene is adopted as a control for DNA quality for each sample assayed. It also acts as internal baseline measurement of the amount of DNA and allows to determining the smaller differences between samples. Thus these results are more precise than immunohistochemistry-measured protein expression. In this study, the mRNA and protein expression levels of CD44v6 and integrin-β1 in 54 PC patients and 20 healthy individuals were determined by the triplex quantitative real-time RT-PCR and quantitative ELISA assay. By constructing standard curves, CD44v6 and integrin-β1 expression levels of samples were exactly tested.
Our study confirmed that both mRNA and protein expression levels of CD44v6 and integrin-β1 in PC patients were significantly up-regulated compared with those in healthy individuals (P<0.05), proving the correlation between the two markers (CD44v6 and integrin-β1) and the development of PC, which was consistent with previous results . We also found that the CD44v6 mRNA and protein up-regulated expression was correlated with tumor differentiation, clinical stage, LNM and liver metastasis, implying that CD44v6 could facilitate the proliferation, invasion and metastasis of PC. The rising CD44v6 expression in blood may become one of the important new indicators in predicting early development, metastasis and recurrence of PC. In addition, both integrin-β1 mRNA and protein expression levels were correlated with clinical stage, LNM and liver metastasis (P<0.05), indicating that integrin-β1 could increase distant metastasis. The results show that CD44v6 and integrin-β1 may play a more important role in predicting the development and metastasis of PC. Some previous studies have revealed that CD44v6 and integrin-β1 can enhance the adhesion of tumor cells to mesothelial cells and extracellular matrix, promoting the release and activation of proteolytic enzyme in tumor cells, as well as the proliferation of tumor vessels . Tumor cells with abnormal CD44v6 and integrin-β1 expression may be possibly assisted by the “camouflage” from lymphocytes and evade the identification and elimination from the human immune system, thus gaining easier access to lymph nodes and forming the metastasis [27, 28].
Previous studies have tested CD44v6 and integrin-β1 expression levels in paraffin-embedded tumor samples in patients with gastric carcinoma and lung cancer and have found a significant correlation between decreasing survival rate and increasing expression levels of CD44v6 and integrin-β1. Our study is the first time to analyze the correlation between CD44v6 or integrin-β1 expression in blood and survival in patients with PC. Our data firstly demonstrated that PC patients with high mRNA and protein expression levels of CD44v6 or integrin-β1 had a significantly reduced survival time than patients with low CD44v6 or integrin-β1 expression (P<0.05). Univariate and multivariate analyses indicate that CD44v6 mRNA and protein expression can serve as independent prognostic factors for OS and DFS in patients with PC, while integrin-β1 mRNA and protein expression level is prognostic factors for OS, showing that the prediction value of CD44v6 expression on DFS is better than integrin-β1 expression.
Chemotherapy and radiotherapy are the main treatment selection of advanced PC. However, most studies have indicated that median survival time is less than 10 months [29, 30]. Therefore, it is necessary to explore a new treatment method of PC. In recent years, argon-helium cryosurgery, as a new tumor ablation technology, has been widely used in the treatment of various solid tumors. However, only a few studies showed the outcome of PC patients after the treatment of cryosurgery [9, 31]. In order to analyze the outcome of PC patients after the treatment of cryosurgery, the trends of CD44v6 and integrin-β1 expression after cryosurgery were firstly analyzed. Our results found that mRNA and protein expression levels of CD44v6 or integrin-β1 increased in patients in 10 days after cryosurgery. In cryosurgery, freezing temperature exerts a largely different damaging effect on tumor cells and releases large amounts of intact tumor antigens in the form of necrotic tumor cells, leading to the up-regulated expression of CD44v6 and integrin-β1 in the initial stage after cryosurgery. After then, CD44v6 or integrin-β1 expression levels decreased dramatically in the later stage. Moreover, CD44v6 or integrin-β1 mRNA and protein expression levels in patients in 3 months after treatment were significantly decreased compared with patients before treatment (P<0.05). Besides, no obvious difference was found in CD44v6 or integrin-β1 mRNA expression levels between patients in 3 months after treatment and control group (P>0.05). In addition, the other parts of PBMC and plasma in 54 PC patients were measured in Stem Cell Research Center, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences according to the protocol mentioned in the subsection of materials and methods, presenting the same results with our study. The reason is that the initial necrotic tumor cells released into blood are gradually removed by the process of metabolism. Besides, cryosurgery can induce the formation of extracellular and intracellular ice crystals, producing vascular capillary changes and circulatory stagnation. Thus, tumor cells have anti-angiogenesis characteristics and inhibit the differentiation and mature of cell adhesion molecules . At last, CD44v6 and integrin-β1 expression levels in patients in 6 months after treatment showed a moderate increase. This may be related to the recurrence of PC. Since abnormal expression of CD44v6 and integrin-β1 is always related to the poor differentiation, rapid progress, easy metastasis and poor prognosis of PC. Our results demonstrate that cryosurgery may be able to inhibit the development and metastasis of PC. CD44v6 and integrin-β1 expression in blood may become effective biomarkers for evaluating the outcome of cryosurgery in PC patients.