To detect the expression of Raf kinase inhibitory protein (RKIP) in gastrointestinal stromal tumors (GISTs) and to analyze its relationship with clinicopatholgical characteristics and prognosis of this disease.

Sixty-three patients with pathologically diagnosed GISTs who underwent surgical resection at the Shengjing Hospital of China Medical University from January 2011 to January 2015 and had complete clinical, pathological, and follow-up data were included. Immunohistochemical method was used to detect the expression of RKIP in GIST tissue samples from these patients. Kaplan-Meier method was used to calculate the survival rate of 60 patients with complete follow-up data, and Cox regression analysis was performed to identify factors affecting the prognosis of patients GISTs to evaluate further the diagnostic and prognostic value of RKIP in GISTs.

In GIST tissues, RKIP positive signals, manifesting as brownish yellow or brown granules, were located in the cytoplasm or on the membrane. Of 63 tissue samples included in this study, 34 (54%) were positive and 29 (46%) were negative for RKIP expression. Statistical analysis showed that RKIP expression in GISTs was significantly associated with tumor size, National Institutes of Health (NIH) risk grade, and mucosal invasion, but had no significant association with age, gender, tumor location, or the number of mitotic figures. Univariate Kaplan-Meier analysis revealed that the 1-, 3-, and 5-year survival rates were 94.4%, 89.2%, and 80.5% for patients with positive RKIP expression, and 88.6%, 68.2%, and 48.2% for patients with negative RKIP expression, suggesting that patients with high RKIP expression had significantly higher survival rates than those with low expression (Log-rank test, P = 0.0015). Cox regression analysis demonstrated that NIH risk grade was significantly associated with the prognosis of GISTs (P = 0.037), suggesting that NIH risk grade is a significant predictor of the prognosis of GISTs. RKIP expression had a tendency to predict the survival of GISTs (P = 0.122), suggesting that RKIP expression may have appreciated value to predict the prognosis of GISTs.

This study demonstrated that: (1) RKIP expression in GISTs is associated with tumor size, NIH risk grade, and mucosal invasion, and low or no expression of RKIP predicts a high malignancy potential; (2) high RKIP correlates positively with the survival of patients with GISTs; and (3) RKIP expression has appreciated value for predicting the survival of patients with GISTs, although it is not an independent prognostic factor in GISTs.

Gastrointestinal stromal tumors (GISTs) have previously been classified or pathologically diagnosed as leiomyosarcomas, leiomyomas, or leiomyoblastomas. For many years, surgical resection was the only effective treatment for GISTs. Until 1983, Mazur and Clark, two professors of pathology at the State University of New York, found that these “leiomyomas” have neither smooth muscle characteristics nor Schwann characteristics, and they for the first time proposed the concept of GISTs[1,2]. In 1998, Hirota et al[3] at the Osaka University reported that GISTs contained activated c-kit mutations. Further immunohistochemical examination showed that GIST tissue was positive for CD34 and CD117. Since then, the diagnosis of GISTs entered the standardization phase[4].

GISTs are now considered the most common gastrointestinal mesenchymal tumors, accounting for about 1%-4% of all gastrointestinal neoplasms. With the application of endoscopic ultrasound (EUS) in the gastrointestinal tract, more GISTs have been detected and distinguished form other subepithelial lesions[5-7]. The annual incidence of GISTs is about 2/100000[8,9]. Approximately 90% of GISTs are located in the stomach and small intestine, with gastric lesions being the most common (~60%). Geneticists estimate that about 10% of all individuals suffer from deleterious gene mutations[10], and current research shows that the development of GISTs is associated with multiple gene mutations, such as c-kit and PDGFRα (platelet derived growth factor receptor alpha) mutations. C-kit gene mutations generally occur in exon 11 (~60%) and include deletions, point mutations, and insertions. In some GISTs without c-kit mutations, PDGFRα mutations may occur, which account for about 8% of all GISTs. PDGFRα mutations occur mainly in exon 18 (~6% of all GISTs) and occasionally in exons 12 and 14 (~1.5% and 0.5% of all GISTs, respectively). Exons 18 and 12 encode the intracellular tyrosine kinase domain and jaxtamembrane domain, respectively. Of note, a small portion of GISTs are wild-type tumors, without c-kit or PDGFRα abnormalities.

GISTs are basically a malignancy, and there are almost no absolutely benign GISTs. Seemingly benign GISTs have the potential to be malignant. For many years, treatment of GISTs was limited to surgical resection and the application of chemotherapy regimens for sarcomas, but with poor efficacy. In 2000, the first successful case using the targeted drug “imatinib” in GISTs was reported. Imatinib is a small molecule tyrosine kinase inhibitor that targets c-kit and PDGFRα and can prevent the initiation of the downstream oncogenes by inhibiting theses tyrosine kinases.

The application of the targeted drug imatinib in GISTs was a milestone in the understanding and gene therapy of GISTs. However, with the increased application of imatinib in clinical cases, drug resistance is beginning to emerge. Thus, it is important to investigate further the pathogenesis of GISTs and discover new therapeutic targets for this malignancy.

Raf kinase inhibitory protein (RKIP), also known as phosphatidylethanolamine-binding protein 1 (PEBP1), is an important endogenous modulator of many kinases and the star protein of recent oncology research. This protein was initially identified in the brain of cattle[11]. RKIP belongs to a highly conserved family of phospholipid-binding proteins, which contains more than 400 members and is widely distributed in microbes, plants, and mammals. RKIP can regulate multiple signaling transduction pathways, including the Raf/MAP kinase (MAPK) pathway, the β-adrenergic (β-AR) pathway, and the NF-kappa B pathway[12-14]. RKIP is not only a kinase inhibitor but also a target of phosphorylation, having important and complex functions, such as regulating tumor growth and metastasis and affecting cell cycle and apoptosis.

Recently, there have been many studies on RKIP, including in gastric cancer, colon cancer, esophageal cancer, and gynecological tumors. However, there have been very few reports on RKIP in GISTs. The role of this protein in the growth and metastasis of GISTs, the relationship between RKIP expression and clinical characteristics of GISTs, and the effect of RKIP expression on the prognosis of GISTs remain unclear. To address these problems, we detected the expression of RKIP in GISTs by immunohistochemistry and analyzed the clinical and prognostic significance of RKIP expression in GISTs, with an aim to provide a basis for GIST diagnosis, prognosis evaluation, and identification of new tumor therapeutic targets.

GISTs are the most common gastrointestinal mesenchymal neoplasms. In the past 30 years, the pathogenesis of GISTs has gradually been elucidated, and their diagnosis and treatment has become standardized[15-19]. However, there is still a significant number of recurrent or metastatic GISTs. Because of their resistance to radiation and chemotherapy, recurrent or metastatic GISTs were once considered an incurable disease. GIST patients with metastasis have a median survival period as low as 20 mo, and for patients with locally recurrent GISTs, the median survival period is only 9-12 mo[20,21]. Imatinib, which is a drug that targets c-kit and PDGFRα gene mutations, is the first choice of molecular therapy for GISTs. However, there are currently no other targets for therapy and prognosis monitoring in GISTs.

RKIP is a structurally complex protein that can act as a “multidirectional switch” to regulate multiple signaling transduction pathways, including the Raf/MAPK pathway, the β-AR pathway, and the NF-kappa B pathway[12,13]. RKIP protein is not only a kinase inhibitor but also a target of phosphorylation. For example, in the MAPK pathway, RKIP binds and inhibits Raf protein, and in the G protein-coupled pathway, RKIP binds to GRK2. This functional shift is achieved through the phosphorylation of the S153 residue by protein kinase C (PKC)[22]. Recently, the regulatory function of RKIP protein in tumors has gradually become a hot research topic, but the data on its expression in GISTs are limited.

In this study, we found in GIST tissue that RKIP was mainly expressed in the cytoplasm and on the membrane, presenting as brownish yellow or brown granules. Such an expression pattern is consistent with many previous reports. RKIP is mainly expressed in the human cytoplasm or on the membrane. The RKIP gene is located on chromosome 12q24.23 and contains four exons, spanning a length of 1435 bp and encoding a 21 KDa protein containing 187 amino acid residues[23]. Immunohistochemical results showed that the expression of RKIP in GISTs tissue was correlated with tumor size; in tumors of < 2 cm, 3-5.9 cm, 6-10 cm, or > 10 cm, the positive expression rates of RKIP were 90.9%, 75%, 27.8%, and 28.6%, respectively (P < 0.01). As the tumor size increased, the degree of malignancy increased, and the expression of RKIP was gradually reduced or absent. This result suggests that the expression of RKIP may be associated with the growth of GISTs. Consistent with this finding, Eves et al[24] reported the lack of RKIP protein expression in hepatoma cells, which can lead to rapid cell division and increased cell proliferation instability[25]. As far as the mechanism is concerned, RKIP protein is an important regulatory factor in the MAPK signaling transduction pathway, and its ligand Raf-1 can regulate the cell cycle of mitotic cells[26]. In addition, the activation of downstream protein ERK1/2 in the MAPK pathway can control important structures that are related to cell division, such as centromeres, spindles, and intermediates[27,28]. Collectively, RKIP can regulate tumor cell cycle and mitosis by regulating the phosphorylation and activation of Raf-1 and the activation of downstream MEK/ERK. In GIST cells, the low or no expression of RKIP protein can reduce its inhibitory effect on the MAPK pathway, thus resulting in the proliferation of tumor cells[29]. Therefore, there is a negative correlation between tumor volume and RKIP expression.

When analyzing the relationship between RKIP protein expression and tumor invasion, we found that there was a significant difference in the positive rate of RKIP between GISTs with and without mucosal invasion (P = 0.015). Previously, many reports on RKIP in other tumor types have found that RKIP protein was related to invasion or metastasis suppression, suggesting a general role of RKIP protein in tumor metastasis and invasion. In the earliest research on RKIP in prostate cancer, Fu et al[30] performed a gene chip analysis of mRNA expression in a prostate cancer cell line with a low metastatic potential (LNCaP cells) and a prostate cancer cell line with a high metastatic potential (C42B cells) and found that the expression level of RKIP was lower in C42B cells than in LNCaP cells. The study of specimens from patients with prostate cancer also showed that the expression of RKIP was lower in metastatic prostate cancer. These results suggest that there is a correlation between RKIP expression and tumor metastasis. Furthermore, an in vitro tumor invasion assay demonstrated that down-regulation of RKIP expression in LNCaP cells can enhance their invasion ability, while restoring RKIP expression in C42B cells can weaken their ability of invasion[31]. In both in vivo and in vitro studies of prostate cancer and melanoma, high expression of RKIP can reduce vessel invasion and reduce metastasis risk[32]. Similar results have also been reported in studies on breast cancer with lymph node metastasis[33], insulinoma[34], colon cancer[35,36], liver cancer[25], ovarian cancer[37], and thyroid cancer[38].

In the present study, the positive rate of RKIP expression differed significantly among different NIH risk grades (very low risk: 80.0%; low risk: 84.2%; moderate risk: 30.0%; high risk: 29.2%; P < 0.01). Fletcher discovered that tumor size and the number of mitotic figures are the main prognostic factors to evaluate the malignant potential of GISTs; and, therefore, proposed a risk grading system (NIH grading), which is of great importance in clinical and pathological diagnosis. In this study, the expression of RKIP was correlated with the size of GISTs: the larger the tumor and the higher the malignancy, the higher the possibility of negative expression of RKIP. However, our results indicated that the expression of RKIP did not significantly correlate with the number of mitotic figures. This result is similar to the study of Miettinen, who found that small intestinal stromal tumors had higher malignant potential than gastric stromal tumors. Gastric stromal tumors had good biological behavior as long as the number of mitotic figures was no more than 5/50 HPFs, and duodenal stromal tumors had good prognosis only when the number of mitotic figures was less than 2/50 HPFs[39]. These findings suggest that other factors (such as tumor location) may also affect the weight of the number of mitotic figures as one of the only two indicators of the NIH risk grading system. To overcome this problem, Miettinen further proposed a new risk assessment system to use anatomical site as an independent assessment factor. In 2013, Sebatian et al[40] performed an immunohistochemical analysis of 161 surgical specimens and a survival analysis based on the clinical and pathological data of these surgical patients and found that the low expression of RKIP was significantly correlated with both high NIH risk grade (P = 0.033, moderate vs low risk group) and high Miettnen grade (P = 0.044, moderate vs low risk group).

In order to have a more direct understanding of the impact of RKIP expression on the prognosis of patients with GISTs, we followed 60 GISTs patients (median follow-up period, 54.96 mo) and plotted the survival curve of these patients according to RKIP expression using univariate Kaplan-Meier method. The results showed that the survival rate differed significantly between patients with positive and negative expression of RKIP (Log-rank test, P = 0.0015). The 1-, 3-, and 5-year survival rates were 94.4%, 89.2%, and 80.5% for patients with positive RKIP expression, and 88.6%, 68.2%, and 48.2% for patients with negative RKIP expression, suggesting that patients with high RKIP expression had higher survival rates than those with low expression. Thus, RKIP expression has appreciated value for evaluating the prognosis of patients with GISTs.

Further, Cox regression analysis showed that the prognosis of GISTs patients was related to NIH risk grade (P = 0.037), indicating that NIH risk grade is an important parameter to evaluate the prognosis of patients. RKIP expression had a tendency to predict the prognosis of GISTs (P = 0.122), suggesting that RKIP expression may have appreciated value to predict the survival of GISTs. RKIP as a prognostic factor has been reported in many other tumors. For example, Fu et al[41] used prostate cancer tissue chip to detect RKIP expression in non-tumor tissues, primary tumor tissues, and metastasis tumor tissues and found that RKIP was strongly expressed in the majority of non-tumor tissues. Although RKIP was weakly expressed in advanced prostate cancer (Gleason score, 6-7), it was strongly expressed in the rest prostate cancer tissues. RKIP expression, however, almost disappeared in metastasis tumor tissues. These results are consistent with the above-mentioned observation that RKIP may act as a metastasis suppressor gene, and also indicate that RKIP can be used as a prognostic factor for prostate cancer. In gliomas, the absence of RKIP correlated with a higher malignancy and a shorter survival period. Yu et al[42] performed a meta-analysis of RKIP expression in gastrointestinal tumors, in which they systematically summarized and analyzed 28 articles that met the criteria, and found that low expression of RKIP is associated with a poor prognosis and short survival time.

Although both univariate and multivariate analyses indicated that RKIP expression had appreciated significance in prognosis evaluation in patients with GISTs in this study, multivariate analysis demonstrated that RKIP expression cannot be an independent prognostic factor in GISTs (P = 0.122). Similarly, Marcus Valadao concluded that RKIP expression does not affect the overall survival (P = 0.73), progression free survival (P = 0.22), or objective response rate (P = 0.30)[43]. Sebatian et al[40] performed a multiple regression analysis of factors affecting overall survival (age, gender, imatinib chemotherapy, Flecher grade, etc.) in patients with GISTs and suggested that low expression of RKIP does not have prognostic significance. This may be because current studies are all limited to detection of RKIP expression at the protein level using immunohistochemical method. Because of the qualitative nature and limited number of experimental samples, there is a lack of quantitative research and detection of RKIP expression at the genetic level.

In conclusion, this study demonstrated that: (1) RKIP expression in GISTs is associated with tumor size, NIH risk grade, and mucosal invasion, and low or no expression of RKIP predicts a high malignancy potential; (2) high RKIP correlates positively with the survival of patients with GISTs; and (3) RKIP expression has appreciated value for predicting survival of patients with GISTs, although it is not an independent prognostic factor in GISTs. These findings suggest that RKIP expression in GISTs is closely related to the diagnosis and treatment of this disease and RKIP may be used as a novel target for therapy and prognostic evaluation in GISTs. Future studies should further investigate the role and underlying mechanism of RKIP in GISTs.