Abstract
Background/Aim: Endothelial cell-specific molecule-1 (ESM-1) is a soluble proteoglycan which has important role in various biological events. We investigated the impact of the ESM-1 expression in cancer tissues on outcomes in stage II/III gastric cancer patients who received adjuvant S-1 chemotherapy. Patients and Methods: The ESM-1 mRNA expression in cancerous tissues and adjacent normal mucosa from 253 patients was measured. The associations between the ESM-1 gene expression and the survival and clinicopathological features were investigated. Results: A significant association was observed between high ESM-1 expression and undifferentiated adenocarcinoma. The overall survival curve was significantly lower in patients with high ESM-1 expression than in those with low expression (p=0.005). High ESM-1 expression was a significant independent prognosticator (HR=2.291, p=0.007). Conclusion: ESM-1 gene expression in cancerous tissues is an important prognosticator in stage II/III gastric cancer patients who received adjuvant S-1 chemotherapy.
Gastric cancer is the fifth most major cancer among new cases and the third most common cause of cancer-associated mortality worldwide, numbering 1,033,701 and 781,631 in 2018, respectively (1). The Adjuvant Chemotherapy Trial of S-1 for Gastric Cancer (ACTS-GC) demonstrated that adjuvant S-1 chemotherapy significantly improved the survival of patients who received D2 gastrectomy for pathological stage (pStage) II/III gastric cancer (2, 3). Furthermore, in the CLASSIC trial and JACCRO-07 trial, the effectiveness of capecitabine plus oxaliplatin therapy for pStage II/III gastric cancer and S-1 plus docetaxel therapy for pStage III was confirmed (4-6). Thus, fluoropyrimidine remains the key drug for adjuvant chemotherapy in gastric cancer patients (7).
Endothelial cell-specific molecule-1 (ESM-1) was originally cloned from a human endothelial cell cDNA library in 1996 (8). ESM1 is a soluble dermatan proteoglycan that can circulate in the bloodstream (9). The structure of ESM-1 is composed of a mature polypeptide of 165 amino acids, of which approximately 30 kDa corresponds to a single dermatan sulfate chain (8). Recently, several authors have shown that ESM-1 is overexpressed at the mRNA and protein levels in various cancers (10-15). It was reported that the overexpression of ESM-1 plays an important role in development, angiogenesis, tumor growth, and so on (16). Furthermore, previous studies have reported that ESM-1 overexpression in tumor tissue was related to poor outcomes in patients with various malignancies (17-20). However, there is no available information concerning the correlation between the ESM-1 expression and long-term outcome in gastric cancer patients who receive adjuvant S-1 chemotherapy.
The PCR primer sequences of ESM-1 and β-actin.
Thus, we examined the impact of ESM-1 mRNA expression in cancer tissues regarding outcomes in patients treated with adjuvant S-1 chemotherapy for pStage II/III gastric cancer.
Patients and Methods
Patients and samples. We retrospectively analyzed the clinical data from 146 consecutive patients who underwent curative resection followed by adjuvant S-1 chemotherapy for the treatment of pStage II/III gastric cancer at Kanagawa Cancer Center and Yokohama City University between 2002 and 2010. As a comparison group, we concurrently studied the ESM-1 expression and long-term outcome in 107 patients who did not receive adjuvant chemotherapy with S-1.
Each tissue sample was engrafted in optimum cutting temperature compound (Sakura Finetechnical Co., Ltd., Tokyo, Japan) and instantly stored at −80 °C. We stained tissue specimens with eosin and hematoxylin and examined histopathologically. Sections were consisted of >80% tumor cells and were used to extract RNA.
RNA extraction and complementary DNA (cDNA) synthesis. Total RNA was isolated from cancerous tissue and adjacent normal mucosa and was prepared with the use of Trizol (Gibco, Life Tech, Gaithersburg, MD, USA). Complementary DNA (cDNA) was synthesized from total RNA with an iScript cDNA synthesis kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Comparison of ESM-1 gene expression between gastric cancerous tissues and adjacent normal mucosa.
Quantitative reverse transcription polymerase chain reaction (qRT-PCR). qRT-PCR was performed with iQ SYBR Green Supermix (Bio-Rad Laboratories). PCR reactions were performed in a total volume of 15 μl, which included 0.2 μg of cDNA, 0.4 μM of each primer, 7.5 μl of iQ SYBR Green Supermix containing dATP, dCTP, dGTP and dTTP at concentrations of 400 μM each, and 50 units/ml of iTag DNA polymerase. After the PCR consisted of 3 min at 95 °C, the cycling conditions were as follows: 40 cycles of denaturation of the cDNA at 95°C for 10 s, annealing for 10 s at 60 °C for ESM-1 and β-actin, and a primer extension at 72 °C for 20 s, finally hold on 72 °C for 10 min. To evaluate the specific mRNA expression in samples, a standard curve was produced for each run, measuring three points of the human control cDNA (Clontech Laboratories, Inc., CA, USA). The concentration of each sample was calculated by relating its crossing point to a standard curve. The PCR primer sequences of ESM-1 and β-actin, as an internal control, are shown in Table I.
Statistical analyses. Gene expression levels were compared between gastric cancer and adjacent normal mucosa by the Wilcoxon's test. The expression of ESM-1 mRNA was categorized as low or high based on a cut-off value calculated using the maximum chi-square test (χ2). The optimal cut-off point was selected by the minimum p-value method, whereas the internal validity of the cut-off point was evaluated with a two-fold cross-validation approach (21). The relationship between the ESM-1 mRNA expression and clinicopathological factors were evaluated with the χ2 test. The survival curves were calculated using the Kaplan-Meier method and compared by the log-rank test. The Cox proportional hazards model was used for the univariate and multivariate survival analyses to determine the risk factors. p-Values<0.05 were considered to indicate statistical significance. All statistical analyses were performed using the EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for the R software program (The R Foundation for Statistical Computing, Vienna, Austria) (22).
Comparison of the clinicopathological factors between high and low ESM1 expression.
Ethics. The present study was conducted in compliance with the ‘ethical guidelines for clinical research’ and with the Helsinki Declaration of 1975, as revised in 1983. This study was approved by the Institutional Review Board (IRB) of Yokohama City University (approval number: 18-7A-4) and Kanagawa Cancer Center (approval number: epidemiological study-29). Written informed consent for using clinical data without identifying personal information was obtained from all patients the initiation of the study.
Results
ESM-1 mRNA expression. The results revealed that the ESM-1 mRNA expression was significantly higher in cancer tissue [1.062 (0.000-15.349)] than in normal gastric mucosa [0.426 (0.000-119.618)] (p<0.001) (Figure 1).
Patient characteristics. Using the optimal cut-off point of the expression of the ESM-1 mRNA, patients were classified into those with low expression of ESM-1 mRNA and high expression of ESM-1 mRNA. The patients' demographic and clinical characteristics are summarized in Table II. Tumors with an undifferentiated type had a significantly higher ESM-1 expression than those with a differentiated type (p=0.016).
Survival analysis. Patients with a high expression of ESM-1 mRNA have a significantly worse OS than those with a low expression (p=0.005; Figure 2). The 5-year OS rate was 57.5% in the patients with a high expression of ESM-1 mRNA and 77.8% in those with a low expression of ESM-1 mRNA. The OS curve in the reference group of pStage II/III gastric cancer patients who did not receive adjuvant S-1 chemotherapy are shown in Figure 3. There was no significant difference in the survival between the patients with a high expression of ESM-1 mRNA and those with a low expression (p=0.141).
The clinicopathological factors were analyzed to determine their prognostic significance for OS (Table III). The univariate analyses demonstrated that the TNM stage and ESM-1 mRNA expression were significant prognostic factors for OS. The lymph node metastasis was marginally significant prognostic factor. The multivariate analyses demonstrated that the TNM stage and ESM-1 mRNA expression were significant independent prognostic factors for OS.
Comparison of overall survival between high and low ESM-1 gene expression in pathological stage II/III gastric cancer patients who received curative gastrectomy followed by adjuvant S-1 chemotherapy.
Discussion
The present study evaluated the impact of the ESM-1 mRNA expression on long-term outcome in pStage II/III gastric cancer patients who received curative resection followed by adjuvant S-1 chemotherapy. The major finding of this study was that the patients with high expression of ESM-1 mRNA had significantly worse survival than those with low expression of ESM-1 mRNA. Our results suggested that the ESM-1 gene expression in cancerous tissue is an important prognosticator in these patients.
We first examined the expression of ESM-1 mRNA in cancerous tissues and adjacent normal mucosa. Several previous studies have compared the relative mRNA expression of the ESM-1 gene between various types of cancer tissue and adjacent normal mucosa (10, 20, 23). It was reported that the ESM-1 expression was higher in gastric cancer tissue than in non-cancerous tissue in 159 samples (20). Our results are consistent with those previous findings, as the expression of ESM-1 mRNA was significantly higher in the gastric cancer tissue than in the paired adjacent normal mucosa.
We, next, examined the relationship between the ESM-1 mRNA expression and the clinicopathological factors in gastric cancer. Liu et al. reported that the overexpression of ESM-1 mRNA was significantly correlated with distant metastasis, vascular invasion, and Borrmann type IV after radical resection for gastric cancer (20). Furthermore, they reported that the local expression of ESM-1 was correlated with vascularity and tumor aggressiveness (20). In the present study, tumors with an undifferentiated type had a high ESM-1 expression.
Comparison of overall survival between high and low ESM-1 gene expression in pathological stage II/III gastric cancer patients who received curative gastrectomy, but did not receive adjuvant S-1 chemotherapy.
We then assessed the relationship between ESM-1 gene expression and long-term outcome in pStage II/III gastric cancer patients who received curative resection followed by adjuvant S-1 chemotherapy. Previous studies reported that a high ESM-1 mRNA expression was associated with a poor outcome in patients with various cancers, including gastric cancer (17-20). In the present study, a high ESM-1 mRNA expression was associated with significantly poorer outcomes than a low expression in locally advanced gastric cancer patients who received adjuvant chemotherapy with S-1. Furthermore, a multivariate analysis revealed that a high expression of ESM-1 mRNA was an independent risk factor for poor outcomes. In contrast, in the patients who did not receive adjuvant S-1 chemotherapy, the overall survival did not significantly differ between the patients with a high expression of ESM-1 mRNA and those with a low expression of ESM-1 mRNA. These results suggest that a high expression of ESM-1 mRNA in gastric cancer tissue indicates a high risk of recurrence in pStage II/III gastric cancer patients who received curative resection followed by adjuvant S-1 chemotherapy. Although further studies are necessary, such patients may be better treated with only close follow-up at an outpatient or with the combination of S-1 plus other anticancer agents.
The mechanism underlying the association of a high expression of ESM-1 mRNA in cancerous tissue with a poor prognosis in pStage II/III gastric cancer patients who received curative resection followed by adjuvant S-1 chemotherapy is poorly understood at present. However, previous reports have hypothesized several mechanisms: ESM-1 is expressed in the tumor endothelium and is upregulated by angiogenic growth factors, such as vascular endothelial growth factor (VEGF) (24). The expression of ESM-1 is also correlated with the balance of positive PKC/NFKB and negative PI3K/AKT/FKHRL1 signaling pathways (25). In addition, it has been recently shown that hypoxia-inducible factor-1a (HIF-1a) was regulated by ESM-1 (26). HIF-1a expression reduced apoptosis in cancer cells through modulation of the expression of apoptotic proteins, such as Bcl-2, Bid, leading to drug resistance against chemotherapeutic agents like 5-fluorouracil (27, 28).
Uni- and multivariate Cox proportional hazards analyses of clinicopathological factors for the overall survival.
Caution is required when interpreting the current results, since the present study has several potential limitations. First, this study examined the ESM-1 mRNA expression in cancerous tissues. It will be necessary to examine both the mRNA expression and protein expression using the same specimen to determine the clinical utility of a protein as a biomarker. Second, there is the issue of heterogeneity in cancerous tissue. The sample from which the mRNA was extracted was 5 mm×5 mm×10 μm (for 3 sheets) of cancer tissue. Although that tissue included the deepest part of the tumor, they did not accurately represent the entire tumor.
From the results of this study, it would be suggested to administrate the appropriate regimen based on the ESM-1 mRNA expression level for pStage II/III gastric cancer in clinical practice. For example, a more efficient adjuvant chemotherapy, such as S-1 plus docetaxel (6), should be administrated if the ESM-1 mRNA expression level is high in gastric cancer tissue samples after surgery in patients with pStage II/III gastric cancer. On the other hand, no adjuvant chemotherapy and careful monitoring may administrate if the ESM-1 mRNA expression level is low in patients with stage II gastric cancer who have organ dysfunction, such as in elderly patients. However, validations and prospective studies in are necessary.
In conclusion, a high expression of ESM-1 mRNA in cancer tissue is an important prognosticator in stage II/III gastric cancer patients who received adjuvant S-1 chemotherapy.
Acknowledgements
The Authors thank Kazue Yoshihara for her technical support.
Footnotes
Authors' Contributions
KK and TO contributed to the conception and design of the study. KK, YK, IH, KH, YM, TA, HF, TY, TO, HC, TY, YM, and TO contributed to the data collection and assembly. All of the authors contributed to the data acquisition and interpretation of the analyzed data. KK wrote the manuscript. All of the Authors contributed to the critical revision of the paper. All of the Authors read and approved the final manuscript.
This article is freely accessible online.
Conflicts of Interest
The Authors have no conflicts of interest to declare regarding this study.
- Received October 19, 2019.
- Revision received November 4, 2019.
- Accepted November 6, 2019.
- Copyright © 2020 The Author(s). Published by the International Institute of Anticancer Research.
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