Abstract
Background: Lung cancer remains a major health problem due to its incidence and mortality. Receptor-binding cancer antigen expressed on SiSo cells (RCAS1) is a protein that can be expressed in cancer cells and is involved in tumor cell escape from immune system surveillance. Aim: The aim of this study was to evaluate the clinical significance of immunohistochemical staining for RCAS1 in non-small cell lung cancer (NSCLC). Materials and Methods: Tissue microarrays of tumor specimens from 112 consecutive patients with newly diagnosed primary NSCLC were constructed. RCAS1 and Ki-67 immunohistochemistry were studied through computerized image analysis. Associations between RCAS1 and Ki-67 expression and clinico-pathological variables and survival were analyzed. Results: RCAS1 expression was higher in grade III tumors (p=0.009), regardless of the histological type, and in adenocarcinomas with lymphovascular invasion (p=0.014). A positive correlation between RCAS1 and Ki-67 levels was observed (p=0.002). Moreover, there was an inverse correlation of overall survival with RCAS1 (hazard ratio=0.99, p<0.001) and Ki-67 (hazard ratio=1.05, p=0.003) levels. Particularly, patients with higher expression of RCAS1 or Ki-67 had a significantly shorter survival than those with lower expression. Conclusion: RCAS1 could be a useful immunohistochemical biomarker, indicating not only tumor aggressiveness but also a poorer prognosis for patients with NSCLC.
Lung cancer is an important health problem worldwide, with approximately 1.2 million new cases every year (900,000 men and 330,000 women). It is the most common cancer type in men and the third in women. It is also the first cause of death from cancer and the ninth cause of death in general worldwide, with more than one million deaths every year (1, 2). Prognosis of lung cancer depends on many parameters, such as the disease stage, patient's age at diagnosis, gender and tumor histological type (3). Two main lung cancer types exist based on the histological type: small cell and non-small cell (NSCLC) lung cancer, with an overall 5-year survival rate 4.5% and 15%, respectively (3-5).
Malignant transformation of cells due to mutation is a frequent event. Nevertheless, in most cases, this does not result in tumor development because the immune system has mechanisms for detecting and destroying malignant cells (6-8). Thus, a tumor is actually developed when such malignant cells manage to escape immunosurveillance through activation of various mechanisms and pathways (7, 8). Some types of malignant cells, for example, express factors that inhibit the production of activated T-lymphocytes, or they express ligands on their surface that induce apoptosis of cytotoxic T-lymphocytes, natural killer cells, neutrophils or macrophages (6-10). As a result, these malignant cells gain the ability to escape immunosurveillance and develop into a tumor.
Receptor-binding cancer antigen expressed on SiSo cells (RCAS1) is a membranous protein consisting of 213 amino acids that form oligomers through coiled-coil domains. It is expressed in many malignant cells and acts as ligand of a putative receptor in various cells, such as T- and B-lymphocytes, and natural killer cells (11). Binding of RCAS1 inhibits cell cycle and growth and induces apoptosis of cells expressing this receptor (12). These findings suggest that malignant cells expressing RCAS1 in their cell membrane evade immunosurveillance by inducing apoptosis of immune cells. Furthermore, RCAS1 seems to be implicated in the differentiation of malignant cells, their proliferation, tumor invasiveness and angiogenesis (13).
The current data regarding the role and clinical importance of RCAS1 in NSCLC indicated the potential implication of this molecule in the pathogenesis and prognosis of this disease but they are limited and conflicting (14-17). The aim of our study was to evaluate the clinical significance of RCAS1 expression by immunohistochemistry in primary NSCLC and to correlate the results with the patients' clinical and pathological features, especially with overall survival.
Materials and Methods
Patients. Consecutive patients with newly diagnosed primary NSCLC were included in this study from the 417 NIMTS and Navy Hospitals of Athens. The diagnosis of their disease was based on histology and tissue examination. Their medical records were reviewed in order to gather information concerning the following parameters: age, gender, performance status, smoking habit, histological data (histological type, grade, lymphovascular invasion, inflammation, necrosis and fibrosis) stage (TNM according to 7th edition of AJCC) and overall survival. The mean monitoring duration was 27.5 months, while the median monitoring duration was 17.5 months. All the participants gave their written informed consent and this study conforms to the Declaration of Helsinki.
Tissue microarrays and immunohistochemistry. Tissue microarrays were constructed by sampling one representative tissue core, 1 mm in diameter, carefully chosen from each formalin-fixed, paraffin-embedded tumor specimen (18-21). The sections of tissue microarrays were dewaxed using xylene and rehydrated through graded alcohols. The sections were treated with 0.3% hydrogen peroxide in methanol for 30 min in the dark and at room temperature in order to remove the endogenous peroxidase activity. Afterwards, a blocking reagent (Snipper, Biocare Medical, Walnut Creek, CA, USA) was applied for 5 min to block non-specific antibody binding. We used two mouse monoclonal antibodies, one against RCAS1 (MBL Int, Nagoya, Japan) and another against Ki-67 (Dako, Glostrup, Denmark), to detect RCAS1 and Ki-67 expression, respectively. Antigen retrieval was performed by microwaving slides for 15 min at 720 W in 10 mM citrate buffer (pH 6.0) and then incubating them for 1 h at room temperature with the primary antibodies. The dilutions for RCAS1 and Ki-67 were 1:750 and 1:100 in phosphate-buffered saline (PBS), respectively. The sections were then washed three times with PBS and incubated for 10 min at room temperature with biotinylated linking reagent (Biocare Medical) and for another 10 min with peroxidase-conjugated streptavidin label (Biocare Medical). The peroxidase activity was developed in 0.5% 3,3’-diaminobenzidine hydrochloride (DAB; Sigma, Saint Louis, MO, USA) in PBS with 0.03% hydrogen peroxide for 3 min. Harris' hematoxylin was used for counterstaining, and Entellan (Merck, Darmstadt, Germany) was used as a mounting medium. Furthermore, the primary antibody was omitted and substituted by an irrelevant anti-serum antibody in negative controls. Examples of these immunohistochemical stains (RCAS1 and Ki-67) in adenocarcinomas and squamous carcinomas are illustrated in Figures 1 and 2. Tumor cell proliferative capacity was estimated as the Ki-67 immunolabeling index.
Computerized image analysis. In order to evaluate the results of immunohistochemistry, we performed computerized image analysis using a semi-automated system as previously described (18,19). This semi-automated system for computerized image analysis has the following hardware features: Intel Dual-Core Pentium processor, Sony Cyber-shot digital camera (5 Mp digital analysis), Olympus CX-31 microscope (Olympus, Center Valley, Pennsylvania, USA) and the following software: Windows XP/NIS–elements image analysis software, Nikon 2009 (Nikon, Tokyo, Japan). Measurements of RCAS1 staining intensity were performed in five optical fields per case and at a magnification of ×400. Interpretation of staining intensity was assessed in a range of 256 continuous values including the RGB protocol in grey scale (0: absolute black, 255: absolute white), meaning that higher values corresponded to less expression and vice versa. Additionally, Ki-67 evaluation was based on the number of immunostained nuclei at the same magnification in the corresponding optical fields.
Statistical analysis. Chi-square test and Pearson's correlation coefficient were used to assess the associations of RCAS1 and Ki-67 expression in the neoplastic tissue with patients' clinicopathological variables, while t-test was used for comparisons between two groups and one-way ANOVA for comparisons among three or more groups. Survival curves were constructed using the Kaplan–Meier method and the differences between the curves were compared with the log-rank test. A Cox proportional-hazard regression model was developed to evaluate the association between the potential prognostic marker and overall survival. Cox regression analysis was conducted at both univariate and multivariate levels. In all cases, an alpha level of <0.05 was considered significant. Statistical analysis was performed using SPSS 17 (SPSS Inc, Chicago, IL, USA) and Stata 11 (StataCorp LP, College Station, TX, USA) programs for Windows.
Results
One hundred and twelve patients (93 men and 19 women; mean age=63.6 years, median age=64 years) with newly diagnosed primary NSCLC were included in this study. The majority of cases were adenocarcinomas 52.7% (59/112). The patients' clinicopathological features are listed in Table I.
In general, after computerized image analysis, 112 values were valid for RCAS1 and Ki-67. Regarding RCAS1 expression, the minimum value (indicating the highest RCAS1 expression) was 79.09, the maximum value (indicating the lowest expression) was 166.98, and the mean (standard deviation) was 120.864 (24, 132), while the median value was 126.380. Taking into consideration the fact that based on the literature normal values of RCAS1 expression are unknown, we divided our patients into two groups according to whether the RCAS1 expression was above (low expression) or below (high expression) the median value.
Demographic clinical and pathological features of patients (n=112).
Overall, RCAS1 expression was high in 56 patients (56/112, 50%) and low in 56 patients (56/112, 50%) (Table II). Analytically, RCAS1 expression was higher in grade III tumors in comparison with grade I (p=0.004) and grade II tumors (p=0.005), regardless of the histological type of cancer. However, there was no significant difference between cancer of grade I and grade II (Figure 3). Moreover, adenocarcinomas with lymphovascular invasion expressed more RCAS1 than those without lymphovascular invasion (p=0.014) (Figure 4). On the contrary, RCAS1 expression was not significantly associated with age, gender, smoking, alcohol use, performance status (PS), histological type, stage of disease, primary tumor (T), lymph nodes (N), metastasis (M) and inflammation, necrosis or fibrosis. Concerning Ki-67, there was a positive correlation between its expression and age (p=0.04) and males had more stained nuclei than did females (p=0.011). Nevertheless, there were no significant associations with other parameters. There was also a positive correlation between RCAS1 and Ki-67 expression (p=0.002, Figure 5).
Additionally, the survival analysis showed an inverse correlation of overall survival with RCAS1 (hazard ratio=0.99, p<0.001) and Ki-67 expression (hazard ratio=1.05, p=0.003). Particularly, when we divided our patients into RCAS1 high or low groups, we found that patients with higher expression of RCAS1 had a significantly shorter overall survival (log-rank test for RCAS1: p=0.006, Figure 6). Finally, when a multivariate survival analysis was performed using the Cox proportional hazards model, a significant negative correlation (p=0.032) between higher expression of RCAS1 and overall survival was maintained (Table III).
Correlation between RCAS1 expression and patients' clinicopathological data (n=112).
RCAS1 (left) and Ki-67 (right) expression in lung adenocarcinoma (original magnification, ×400).
RCAS1 (left) and Ki-67 (right) expression in squamous lung cancer (original magnification, ×400).
Discussion
The RCAS1 is a membranous protein that is expressed in a variety of neoplasms and acts as a ligand with a specific activity. Particularly, it induces the apoptosis of the immune system cells within the tumor microenvironment and it helps the immunoescape of cancer cells (13, 22-23). Moreover, there are data indicating the role of RCAS1 in differentiation and proliferative capacity of tumor cells, in angiogenesis and in remodelling of tumor microenvironment (13, 22). Furthermore, there are data regarding RCAS1 expression and its clinical importance in different tumor types (13).
Several studies have shown through immunohistochemistry that RCAS1 is expressed in various types of cancer, such as endometrial, skin, breast, hepatocellular, cholangiocellular, pancreatic, gastric, colorectal, gallbladder and lung cancer, with higher expression being associated with advanced stages and poorer prognosis (13, 24-35). RCAS1 has also been detected in serum and other biological fluids, such as pleural fluid, in various types of cancer, making it a potential biomarker for many malignancies (36-40).
Regarding the role of RCAS1 in NSCLC there are some data that illustrate the potential prognostic value of this molecule (14-17). In particular, in the study of Oizumi et al., the prognostic value of RCAS1 in lung adenocarcinomas was shown, but it was a small study of 70 patients (16). In another study of Izumi et al., the prognostic value of this molecule has been shown in NSCLCs and not only in adenocarcinomas (15). Besides that, in the study of Aoe et al. the informative role of RCAS1 measurement in pleural effusion for both diagnosis and prognosis in patients with lung cancer has been illustrated (17).
Multivariate survival analysis. This table represents the variables that were included in the Cox proportional hazards model (stepwise approach, entry criterion: p≤0.1) that were used for performing multivariate survival analysis on our data. For the variables T, N, PS the referent levels are T0, N0, PS=0. All the HR expressed indicate the HR for an increase in the corresponding variable of 1 unit.
High tumor grade is associated with higher expression of RCAS1. It should be noted that the measured values of RCAS1 are inversely correlated to its expression. Accordingly in Grade III the lowest measured values of RCAS1 are observed (mean±SD: 111,3±23,4) compared to Grade I (132,6±17,3, p=0.004) and Grade II (126,3±23,7, p = 0.005). Line x is the Grade of the tumors and bar y is the expression of RCAS1. Horizontal bars represent mean values and 95% confidence intervals.
Higher expression of RCAS1 is associated with lymphovascular invasion (LVI) in the adenocarcinoma subgroup of patients. It should be noted that the measured values of RCAS1 are inversely correlated to its expression. Thus, in the presence of LVI in adenocarcinomas lower measured values of RCAS1 are observed (mean±SD: 109,3±19,7 vs. 125,4±25,6, p=0.014). Line x is the presence or no of LVI in adenocarcinomas and bar y is the expression of RCAS1. Horizontal bars represent mean values and 95% confidence intervals.
Positive correlation between RCAS1 and Ki-67 expression (p=0.002). It should be noted that the measured values of RCAS1 are inversely correlated to its expression.
Kaplan-Meier graph for survival analysis of patients classified according to RCAS1 expression (RCAS1 high with measured values ≤ median value, RCAS1 low with measured values > median value). Patients in the RCAS1 low group have a higher survival likelihood (log-rank test, p= 0.006).
Moreover, there are data showing a correlation between RCAS1 and the volume of the tumor or the stage of disease (14, 15). Additionally, a previous report shows a higher RCAS1 expression in grade III lung adenocarcinomas (14). However, in the study of Oizumi et al., no correlation was found between RCAS1 and the tumor volume or the stage of disease (16). On the other hand, in the study of Iwasaki et al., the expression of RCAS1 was higher in squamous carcinomas (14) but in the study of Izumi et al., there was no correlation between RCAS1 and the histological type of NSCLCs (15). Consequently, the existing data regarding the clinical significance of RCAS1 in NSCLCs are limited and conflicting and the need for more studies is obvious.
The clinical significance of RCAS1 in prognosis and pathological evaluation of tumor aggressiveness of NSCLC is enhanced by the results of our study, which are in accordance with the majority of existing data. More specifically, RCAS1 is overexpressed in grade III NSCLC, regardless of the histological type, and in adenocarcinomas with lymphovascular invasion. Moreover, in this study, tumor cell proliferative capacity was also estimated as Ki-67 immunolabeling index, which is considered as a potential biomarker for NSCLC (41). A positive correlation between RCAS1 and Ki-67 expression was found (p=0.002), meaning that neoplasms with higher proliferative rate express more RCAS1. Thus, NSCLCs that are more aggressive tend to escape immunosurveillance by expressing RCAS1. Furthermore, survival analysis showed that there is an inverse correlation between RCAS1 and Ki-67 expression and overall survival. In particular, patients with higher levels of RCAS1 or Ki-67 have shorter overall survival than those with lower levels. In addition to this, a multivariate analysis for overall survival revealed that RCAS1 expression was a significant and independent prognostic factor in NSCLC patients (p=0.032).
The most interesting and unique features of our study were: a) the method of computerized image analysis which was used for the evaluation of RCAS1 expression, b) the correlation of grade, Ki-67 immunolabeling index and lymphovascular invasion with the expression of RCAS1, and c) the multivariate analysis for overall survival which showed RCAS1 to be an independent prognostic factor in NSCLC. On the contrary, there are some limitations in our study such as: a) the small number of patients, b) the use of one tissue core from each paraffin-embedded tumor specimen and c) the fact that normal values of RCAS1 expression are not available.
In conclusion, according to the results of our study, RCAS1 is overexpessed in more aggressive NSCLCs with a higher proliferative rate and is associated with shorter overall survival. This biomarker was proven to be a significant and independent prognostic factor in NSCLC as a result of a multivariate analysis. Consequently, RCAS1 could be considered as a useful immunohistochemical marker indicating tumor aggressiveness and predicting poor prognosis for patients with NSCLC. Moreover, RCAS1 could be considered as a possible therapeutic target in the future taking into account the recent advances in immunotherapy for NSCLC. Obviously, a new drug that could stop or block the function of RCAS1 would be helpful since it would restore the immunosurveillance of the tumor. However, more and larger studies are needed to elucidate the clinical significance of RCAS1 not only as a biomarker but also as a potential therapeutic target in lung cancer and other neoplasms.
Acknowledgements
Hellenic Society of Medical Oncology (HeSMO) for the general support in this study. Also Mrs Vaiou Christa for her valuable support and help.
Footnotes
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This work was presented at the 48th Annual Meeting of the American Society of Clinical Oncology (ASCO), 2012.
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Conflicts of Interest
All Authors declare that they have no conflict of interest.
- Received April 7, 2014.
- Revision received April 30, 2014.
- Accepted April 30, 2014.
- Copyright © 2014 The Author(s). Published by the International Institute of Anticancer Research.
