Abstract
Background: The overexpression of microRNA-21 (miR-21) in pancreatic cancer has been implicated in drug resistance to gemcitabine. Thus far, miR-21 has gained wide attention as a potential biomarker to predict the clinical response in patients with pancreatic cancer receiving gemcitabine. The aim of this study was to evaluate the predictive value of miR-21 expression, determined by locked nucleic acid in situ hybridization (LNA-ISH), in patients with pancreatic cancer who underwent adjuvant gemcitabine after curative surgery. Materials and Methods: Tumor miR-21 expression was analyzed via LNA-ISH and correlated with the clinical outcomes of the patients treated with adjuvant gemcitabine. Results: The overexpression of miR-21 in pancreatic cancer, determined by LNA-ISH, was significantly and independently associated with a shorter disease-free survival in patients who received adjuvant gemcitabine after curative resection. Conclusion: The LNA-ISH analysis of miR-21 may serve as a significant predictor for gemcitabine resistance in patients with pancreatic cancer undergoing adjuvant gemcitabine after curative resection.
Pancreatic cancer remains a malignancy with one of the worst prognoses despite recent advances in cancer treatment. At present, surgery is the only curative approach, and combined treatment with adjuvant gemcitabine, which represents the current gold-standard therapy for resected pancreatic cancer, improves the outcomes, although with limited benefits (1). Therefore, predicting the outcomes of such patients is a major challenge for the identification of a subset of patients who may benefit from adjuvant therapy in this patient population.
MicroRNAs (miRNAs) are small (22 to 26 nucleotides in length) non-coding RNA gene products that negatively regulate the gene expression at the post-transcriptional level by imperfectly binding to their target mRNA, and play an important role in regulation of all major cellular processes such as proliferation, differentiation, metabolism, and cell death (2). Moreover, in addition to their role in various biological processes, the altered expression of miRNAs is involved in tumor development and progression of various cancer types (3-5).
MicroRNA-21 (miR-21) is one such oncogenic miRNA which is aberrantly expressed in pancreatic cancer and promotes tumor growth and progression (6-8), as in cancer of the breast, colon, and stomach (4). The forced overexpression of miR-21 by transfection of miR-21 precursor in pancreatic cancer cell lines increased proliferation and invasion activities in vitro (9). Clinically, overexpression of miR-21 in pancreatic cancer has been associated with a more aggressive tumor grade, stage, lymph node status, and poor clinical outcomes of patients (8, 10, 11).
Recently, pre-clinical in vitro studies have implicated miR-21 in drug resistance to gemcitabine in pancreatic cancer (9, 12, 13). The transfection of miR-21 precursor in pancreatic cancer cell lines induced chemoresistance to gemcitabine, with increased proliferation, reduced apoptosis and an increased cancer cell survival (9, 12). Conversely, inhibition of miR-21 induced increased apoptosis, a lower cancer cell survival and reduced proliferation (9, 12, 13).
For these reasons, miR-21 has attracted interest as a potential biomarker for predicting the efficacy and clinical outcomes of patients with pancreatic cancer receiving gemcitabine chemotherapy (14). A previous study which evaluated the miR-21 expression in micro-dissected pancreatic cancer tissue using quantitative polymerase chain reaction (PCR) analysis demonstrated that higher expression of miR-21 in cancer cells was significantly associated with a shorter progression-free survival (PFS) and overall survival (OS) of patients treated with gemcitabine in both metastatic and adjuvant settings (12). However, at present, only few clinical reports are available which focused on this matter. Additional confirmatory studies are needed to clarify the predictive and prognostic value of miR-21 in patients with pancreatic cancer undergoing gemcitabine chemotherapy.
The aim of this study was to clarify whether the miR-21 expression in pancreatic cancer, measured by locked nucleic acid in situ hybridization (LNA-ISH) method, that allows estimation of both the expression intensity and distribution of miR-21, can predict the clinical outcomes of the patients undergoing adjuvant gemcitabine monotherapy after curative surgery.
Materials and Methods
Patients. A total of 39 patients with pancreatic cancer (22 males and 17 females), aged 44-77 years (median=62 years), who received adjuvant gemcitabine monotherapy after curative surgical resection at Kanagawa Cancer Center between June 2006 and May 2010, were included in this study and retrospectively examined for the predictive/prognostic significance of miR-21 expression. Tumor staging was performed according to the International Union against Cancer (UICC) classification guidelines (2009) (15). Informed consent was obtained from all patients for use of their specimens for this study according to the institutional rules of the Kanagawa Cancer Center. The study protocol conformed to the ethical guidelines of the 2004 Declaration of Helsinki.
Adjuvant treatment. Each patient received adjuvant gemcitabine monotherapy using one of the following protocols: a biweekly gemcitabine protocol (1,000 mg/m2 gemcitabine, biweekly for 6 months) or the standard gemcitabine protocol (1,000 mg/m2 gemcitabine on days 1, 8 and 15; every 4 weeks for 6 months). Eleven patients received the biweekly gemcitabine protocol, and 28 patients received the standard gemcitabine protocol. The treatment was initiated within 10 weeks after surgery. Fourteen patients completed the full course of either protocol, and the median cumulative total dose of gemcitabine was 12.0 g/m2 (range=2.0 to 21.0 g).
LNA-ISH. LNA-ISH was carried out on formalin-fixed and paraffin-embedded (FFPE) tissue sections according to the kit manufacturer's instructions (miRCURY LNA™ microRNA ISH Optimization Kit; Exiqon Inc., Vedbaek, Denmark). Briefly, the sections were deparaffinized in xylene and rehydrated with graded ethanol. The sections were then incubated with Proteinase-K, and hybridized with the miR-21 double-digoxigenin (DIG)-labeled LNA™ probe. A specific anti-DIG antibody directly conjugated with alkaline phosphatase (AP) was applied, and then the sections were counterstained with Nuclear Red.
Baseline patient characteristics.
Determination of the miR-21 ISH score. The staining intensity of miR-21 ISH and the distribution of positive tumor cells were scored. The staining intensity for miR-21 ISH was assigned a score from 0 to 3 according to staining: 0: no staining; 1: weakly positive; 2: moderately positive; and 3: strongly positive. The percentage of positively stained tumor cells was scored as follows: 0, no positively stained tumor cells; 1, <50% positively stained cells; 2, 50-80% positively stained cells; and 3, >81% positively stained cells. Finally, a composite score (miR-21 score) was obtained by calculating the sum of these two scores. According to the miR-21 score, we classified tumors with scores of 0-3 as having a low miR-21 expression and tumors with scores of 4-6 as having a high miR-21 expression.
Statistical analysis. The DFS and OS rates were estimated using the Kaplan–Meier method and then ware compared by the log-rank test. The predictors of the outcome were assessed by univariate and multivariate analyses (a stepwise variable selection) using the Cox proportional hazard regression model. For all statistical analyses, the level of significance was set at 0.05, and the SPSS statistical software program (SPSS for Windows 11.0J; SPSS Inc.; Chicago, IL, USA) was used.
Results
Patients' characteristics. The patients' demographics and clinical characteristics are presented in Table I. A total of 22 patients had a primary tumor located in the head of the pancreas, and 17 in the body to tail regions. Of the 39 patients, all patients had pT3 stage and 29 patients had pN1 disease. The follow-up period ranged from 5.4 months to 114.1 months (median=16.8 months). Thirty-four patients developed recurrent disease, and 21 of these patients received palliative chemotherapies for recurrent disease. Thirty-four patients died of pancreatic cancer and one patient from cardiovascular disease.
Representative results of locked nucleic acid in-situ hybridization (LNA-ISH) for miR-21 in pancreatic adenocarcinoma. Weak to strong cytoplasmic staining was observed in cancer cells (original magnification, ×200).
LNA-ISH analyses. Representative results of LNA-ISH staining for miR-21 in pancreatic adenocarcinoma tissue sections are shown in Figure 1. Weak to strong cytoplasmic staining was observed in cancer cells. According to the miR-21 ISH score, 14 patients were assigned to the high miR-21 expression group (scores 4-6) and 27 patients to the low miR-21 expression group (score 0-3).
Survival analysis. Figure 2 shows the DFS and OS curves for patients who had undergone adjuvant gemcitabine therapy after curative surgery, stratified by the miR-21 expression of the tumor. Patients with a high miR-21 expression in the tumor had a significantly shorter DFS (p=0.041, log-rank test) than patients with a low miR-21 expression. The median DFS was 7.9 months [95% confidence interval (CI)=6.0-9.8 months) in the high mir-21 group and 9.8 months (95% CI=6.9-12.6 months) in the low-expression group. However, no significant difference was detected in the OS between patients with a high miR21 expression and those with a low miR-21 expression (p=0.129, log-rank test). The univariate and multivariate analyses using three variables, namely miR-21 expression, microscopic lymphatic invasion and microscopic perineural invasion, indicated that a high miR-21 expression was independently associated with a shorter DFS, along with microscopic lymphatic invasion (Table II).
Discussion
In this study, we evaluated the miR-21 expression in pancreatic cancer using the LNA-ISH method and correlated the mir-21 expression levels with the clinical outcomes of patients who underwent adjuvant gemcitabine monotherapy after curative surgery. The results of the present study show that overexpression of miR-21 in pancreatic cancer measured by LNA-ISH is strongly associated with poor clinical outcomes of these patients. Patients with a high miR-21 expression in the tumor had a significantly shorter DFS than those with a low miR-21 expression, and a high expression of miR-21 in the tumor was indicated to be a significant and independent predictor for DFS by multivariate analyses.
The disease-free survival (DFS) and overall survival (OS) according to tumor miR-21 expression. The DFS of patients with tumors that had a high miR-21 expression was significantly shorter (p=0.041, log-rank test) than patients whose tumors had a low miR-21 expression. No significant difference was detected in the OS between patients with a high miR-21 expression and those with a low miR-21 expression (p=0.129, log-rank test).
The result of the present study is in agreement with that of a previous study that evaluated the miR-21 expression in laser-microdissected tumors from gemcitabine-treated patients with pancreatic cancer using PCR and that demonstrated a significant association between miR-21 expression and clinical outcomes in both metastatic and adjuvant settings (12).
miR-21 has been intensely studied among potential diagnostic and prognostic biomarkers for pancreatic cancer. An expression-profiling study using real-time PCR showed significant changes in the miR-21 expression between pancreatic adenocarcinoma and normal pancreas (6), and a distinct aberrant miRNA expression pattern, including the overexpression of miR-21, was able to differentiate ductal adenocarcinoma of the pancreas from normal pancreas and chronic pancreatitis (7). Some studies using RT-PCR or ISH also showed the prognostic values of miR-21 in patients with pancreatic cancer who underwent surgical resection (8, 10, 11). The higher expression of miR-21 in pancreatic cancer, determined by RT-PCR, was significantly associated with tumor stage and a poor prognosis (10). An expression-profiling study assessed by RT-PCR in prospectively collected fresh-frozen tissue samples from patients with pancreatic cancer undergoing surgery showed that a high expression of miR-21 was significantly associated with a poor survival, along with resection margin involvement, venous invasion, and lack of adjuvant therapy (8). A strong miR-21 expression in pancreatic cancer determined by LNA-ISH was associated with a shorter OS after surgical resection in a subset of patients with node-negative disease (11).
Recently, some pre-clinical studies have implicated miR-21 in drug resistance to gemcitabine, in addition to its role in tumor development and progression (9, 12, 13). Thus far, miR-21 has gained wide attention as a potential biomarker for predicting the clinical response to chemotherapy with gemcitabine in patients with pancreatic cancer. However, only few clinical reports are available which investigated the predictive value of miR-21 in this patient population (12). To the best of our knowledge, our report is the first to show a significant association between the miR-21 expression, as evaluated by LNA-ISH, and the clinical outcomes of patients with pancreatic cancer receiving adjuvant gemcitabine monotherapy after curative surgery. Our study, in addition to a previous study investigating this matter using the PCR method (12), strongly suggests that the intratumoral miR-21 expression may represent a significant predictive/prognostic biomarker for patients with pancreatic cancer undergoing adjuvant gemcitabine therapy after surgery.
In this study, we applied the LNA-ISH method to visualize the expression intensity and distribution of miR-21 in cancer FFPE tissues. LNA is a unique nucleic-acid modification possessing very high binding affinity, increasing the specificity and sensitivity of miRNA detection (16), and the applicability of LNA-ISH for research purposes has been documented in a number of recent reports (17, 18). The LNA-ISH method allowed us to semiquantitatively evaluate the miR-21 RNA expression localized in pancreatic cancer cells and the distribution of miR-21-expressing positively stained cancer cells in the cancerous lesion on formalin-fixed tissues, leading to scoring of the expression level of miR-21. This technique is much faster and easier than PCR and could be widely applied.
Univariate and multivariate analyses for disease-free survival.
In summary, the overexpression of miR-21 in pancreatic cancer determined by LNA-ISH was significantly associated with a shorter DFS in patients who received adjuvant gemcitabine monotherapy after curative surgery. The LNA-ISH method to visualize miR-21 expression may serve as a significant predictive/prognostic marker for appropriately selecting patients for gemcitabine-based adjuvant therapy or for selecting a more suitable drug for these patients. However, the current study was small-scaled and retrospective; therefore, further investigation in a larger prospective study is required to confirm the findings of the present study.
This report was presented in part at the 2014 Gastrointestinal Cancers Symposium of the American Society of Clinical Oncology, as part of the General Poster Session (San Francisco, California, January 2014).
Acknowledgements
This work was partially supported by grants from the Kanagawa Prefectural Hospitals Cancer Fund. The Authors declare that they have no potential conflicts of interest.
Footnotes
Disclosure
The Authors declare that they have no potential conflicts of interest.
- Received December 28, 2015.
- Revision received February 12, 2016.
- Accepted February 16, 2016.
- Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
