Although the worldwide incidence of gastric cancer has declined in recent years, it remains the most common cancer and the leading cause of cancer-related death in China.1 The prognosis for patients with advanced cancers is less predictable and generally poorer. At present, therapeutic decisions are based on clinicopathological parameters, including age, TNM stage and histological grade. Although useful, these factors often fail to differentiate between more and less aggressive tumor types. As a result, there is an urgent need to find special markers, which are closely related to bionomic characteristics, outcome of gastric adenocarcinoma and performance of antigen specific therapeutic targeting strategy.
Insulin-like growth factor-II mRNA-binding protein 3 (IMP3) known in previous studies as L523S or KOC (K-homologous domain containing protein overexpressed in cancer), is a newly identified oncofetal RNA-binding protein that binds to mRNA such as insulin-like growth factor-II (IGF-II) transcripts.2,3 IMP3 is a member of the human IGF-II mRNA-binding protein family (IMPs) whose other members are IMP1 snd IMP2. These proteins are capable of strong and specific binding with mRNA transcripts including those encoding IGF-II, cell adhesion molecules and others. IMPs have also been implicated in various post-transcriptional processes, such as mRNA localization,3 turnover,4 and translational control.4 IMP3 is expressed during embryogenesis but not in most adult tissues.5,6 By using cDNA library subtraction and large-scale DNA screening techniques, the IMP3 gene has been found to be expressed in pancreatic and lung carcinomas.7,8 IMP3 protein consists of 580 amino-acid residues and is encoded by a 4350-nucleotide mRNA transcript and the gene is located on chromosome 7p11.5. IMP3 is a regulatory binding protein believed to be involved in the stabilization and intracellular trafficking of IGF-II mRNA to facilitate IGF-II production.9
IMP3 is expressed in many cells of developing fetus, but not in most cells of adults except in the gonads. Antibody specific for IMP3 has been used successfully with routine immunohistochemical methods to study overexpression of IMP3 protein in a number of malignant tumors, such as malignant pancreatic lesions, renal carcinomas, uterine cervical and endometrial carcinomas, gonads and testicular cancer.10-12 IGF-II receptor gene-167 genotype increases the risk of oral squamous cell carcinoma in humans.13
However, expression of IMP3 in gastric adenocarcinoma (GAC) has rarely been studied previously. In order to further understand the role of IMP3 in neoplastic pathology, in this study, we detected the expression of IMP3 in GAC with immunohistochemical techniques, correlated IMP3 expression with clinicopathological features and the proliferation marker Ki-67. Also, we compared the expression of IMP3 between GAC and adjacent normal mucosa (ANM), between the primary and metastatic lesions.
Patients and tissue samples
Tumor specimens were obtained from 92 patients (56 males and 36 females; mean age 56.1 years, range from 24 to 82 years) who underwent surgery for GAC from November 2005 to April 2007. None had received prior chemotherapy or radiotherapy. Clinical and pathological records and slides were available for all cases. H&E-stained slides of GAC were reviewed and one block with tumor and (in 92 cases with) ANM tissue was selected for immunohistochemical staining. Informed consent was obtained from each patient before the study was conducted.
Processing of specimens and immunohistochemistry
Sections (4 µm) of tissue blocks were transferred to an adhesive-coated slide. A 3-step immunoperoxidase technique using the streptavidin-peroxidase (S-P) was employed for IMP3 detection. All the sections were routinely deparaffinized and rehydrated, then the sections were rinsed in phosphate-buffered saline (PBS, pH=7.4), subsequently were treated for antigen retrieval (10 minutes, Microwave oven, High Power, citrate buffer, pH 6.0). After cooling at room temperature for 20 minutes, the sections were rinsed in PBS, and then immersed in 3% H2O2 for 15 minutes to block the endogenous enzymes. Thereafter, the sections were incubated with normal goat serum at 37°C for 15 minutes to block nonspecific antibodies. The primary antibody was a polyclonal goat antiserum for IMP3 (N-19, Santa Cruz, USA), which was diluted at 1:50 dilution. The primary antibody was a monoclonal mouse antiserum for Ki-67 (MAB-0129, Maixin, China), which was diluted at 1:200 dilution. After being incubated with primary antibody at 37°C for 60 minutes, the sections were rinsed in PBS and incubated with biotinylated secondary antibody (SP kit, Zhongshan Co., China) and rinsed in PBS again. After interaction with streptavidin-HRP (SP kit, Zhongshana Co.) and being rinsed in PBS, the sections were visualized by reaction with 3′-diaminobenzidine and counterstained with hematoxylin. Adequate positive controls of pancreatic carcinoma were used according to the manufacturer’s recommendations, and normal goat serum and PBS substituting the primary antibody were used as negative controls.
Scoring of the results
A positive stain for IMP3 was defined as brown stain seen in the cytoplasm. Negative stain was scored as 0. The intensity of the staining was evaluated as weak (score 1), or strong (score 2).10 Immunoreactivity of Ki-67 was evaluated as follows: 0, <20% of the tumor cells showed positive immunoreactivity; 1, 20%–50% of these cells showed positive immunoreactivity; 2, >50% of these cells showed positive immunoreactivity. All the staining results were evaluated by two independent researchers.
The Mann-Whitney U test was used to compare immunohistochemical reactivity in the distribution of the categorical variables (i.e. age, sex, and different gastric mucosae). The Kruskal-Wallis H test was used to compare immunohistochemical reactivity in the distribution of the categorical variables (i.e. histologic grade, WHO type, lymph node involvement, and TNM stage). The correlation of IMP3 protein expression with Ki-67 was evaluated with Sperman correlation analysis.
Survival curves of patients were plotted using the method of Kaplan-Meier, and the log-rank test was used to determine statistical differences between life tables. The prognostic effect of various parameters (i.e. age, sex, histologic grade, WHO type, lymph node involvement, TNM stage and IMP3 expression) on clinical outcome (i.e. death) of disease was tested by the Cox-regression model. Patients dying of other causes during the follow-up period were treated as censored data.
Statistical analysis was performed using the SPSS for Windows Software, version 13.0. A P value of less than 0.05 was considered indicative of a statistically significant difference.
IMP3 protein expression in different gastric mucosae
Out of 92 cases of ANM, 10 cases with dysplasia demonstrated weak expression of IMP3 and 82 without dysplasia showed negative expression. Out of 92 cases of GAC, positive immunohistochemical stain for IMP3 was identified in 75 (82%) cases. Among which, immunohistochemical reaction intensity for IMP3 was identified to be weak in 41 (45%) cases and strong in 34 (37%) cases (Figure 1). The Mann-Whitney U test showed that expression of IMP3 in GAC was significantly stronger than that in ANM (Z=–2.917, P=0.004, Table 1).
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Figure 1. Immunohistochemical staining (Original magnification: ×200). A: No immunoreactive staining of IMP3 protein in the adjacent normal mucosa. B: Cytoplasmic expression (weak, 1+) of IMP3 in gastric adenocarcinoma tissues. C: Cytoplasmic expression (intermediate, 2+) of IMP3 in GAC. D: Nuclear expression of Ki-67 in gastric adenocarcinoma tissues.
Correlation of IMP3 protein expression to clinicopathological features
Expression of IMP3 was detected more strongly in GAC with one or more team lymphoid metastasis than with no lymphoid metastasis (P=0.005, Table 2). In 51 cases of GAC with more team lymphoid metastasis, stronger positive staining was detected than that with no lymphoid metastasis (51/92 vs. 20/92, Z=–3.042, P=0.002).
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Table 1. Expression of IMP3 protein in ANM and GAC
Table 2. Correlation of IMP3 protein to clinicopathological features and Ki-67 in GAC
There was a significant difference in TNM stage between GAC with and without IMP3 expression (P
=0.003, Table 2). Tumors at higher stage showed higher level of IMP3 expression. The positive IMP3 expression levels were 40% (2/5), 40% (4/10), 93% (39/42) and 86% (30/35) in stage I, II, III and IV, respectively (II vs. IV: P
=0.001; II vs. III: P
It was indicated that IMP3 expression was not significantly correlated with age, sex, and histological grade (P >0.05, Table 2).
Correlation of IMP3 protein expression to Ki-67 expression in GAC
As shown in Figure 1, the immunoreaction of Ki-67 was localized in the nuclei. The positive Ki-67 protein expression level was 44.6% in GAC. Additionally, it was observed that when IMP3 gene was overexpressed, there was a strong association with high Ki-67 labelling index (rs=0.247, P=0.017).
Correlation of IMP3 protein expression to prognosis
Univariate survival analysis demonstrated that patients with positive staining of IMP3 (1+ and 2+) had a significantly worse overall survival compared to patients with negative IMP3 immunoreactivity (P=0.012, Figure 2). Mean survival time for these 17 patients whose tumor showed negative IMP3 expression was 33 months, which was much longer than 23 months of patients with positive staining of IMP3 (1+ and 2+). In multivariate analysis (Cox regression model, Forward stepwise), IMP3 emerged as an independent predictor of survival (Wald x2=4.047, P=0.044).
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Figure 2. Kaplan-Meier analysis of postoperative survival in patients whose tumors showed negative IMP3 expression (n=17) vs. those positive (1+ and 2+) (n=75). Patients of positive expression of IMP3 had much shorter survival than those of negative expression (P=0.012).
The IMP3 gene was initially identified in pancreatic carcinomas in 1996,7 and subsequently cloned in 1999.5 IMP3 together with IMP1 and IMP2, members of human IGF-II mRNA binding protein family were first purified from the human rhabdomyosarcoma cell line RD in 1999.5 They have been shown to play an important role in the binding, trafficking, and stabilization of the fetal subtype of IGF-II mRNA during embryogenesis.3,11 As a result, IGF-II expression is indirectly regulated by IMP3 and other proteins in the IMP family. IGF-II overexpression has been implicated in the pathogenesis of the tumors in multiple organ systems as seen in the Beckwith-Wiedemann syndrome, and the development of breast and liver tumors in transgenic mouse models as well as hepatocellular carcinoma in viral hepatitis mouse models.14-16 Therefore, it is hypothesized that IMP family members are involved in carcinogenesis by stabilizing IGF-II mRNA. However, the IMP proteins also bind and affect other mRNAs and this may also influence the malignant potential of cells.
In the present study, we have clearly demonstrated the cytoplasm expression of IMP3 protein by immuno- histochemistry in a series of GAC. According to our results, IMP3 was expressed in carcinoma lesions, while it almost did not show up in adjacent non-tumor epithelia. Similar associations were demonstrated where IMP3 expression was correlated with older age, larger tumour size, deep tumour invasion and lymph node metastasis.17
We selected Ki-67 to represent the proliferation status of cells. Ki-67, a proliferation-associated antigen, is strictly correlated with the progression of cell cycle and expresses in G1, S, G2 and mitosis. In our study, it was observed that when IMP3 gene was overexpressed, there was an association with high Ki-67 labelling index. Recent studies have shown the importance of IMP3 in tumor cell proliferation and invasion, which indicates that IMP3 is an oncofetal protein that may play a critical role in malignant transformation and tumor progression.4,18,19 Our results also confirmed IMP3 protein as an important proliferation marker for tumor cells again.
IMP3 protein expression in tumor cells has represented itself as a robust indicator for predicting metastases in patients treated for clinically localized renal cell carcinoma.20 In this study, we showed that IMP3 was highly expressed in GAC with lymphoid metastasis as compared to non-metastatic tumors. The relationship between IMP3 expression and lymphoid metastasis suggests that IMP3 may promote the lymphoid metastasis in GAC. Further, there was a significant TNM stage difference between GAC with and without IMP3 expression. Yaniv et al21 reported that IMP3 in Xenopus laevis is required for the migration of cells forming the roof plate of the neural tube and, subsequently, for neural crest migration, which suggested that IMP3 is important for promoting cell migration. These findings could explain the association of IMP3 expression with tumor progression and metastasis. However, further study is required to investigate whether IMP3 plays a direct role in the biological behavior of GAC.
Survival analyses indicated IMP3 expression to be negatively linked to favorable prognosis of colorectal adenocarcinoma,18 renal cell carcinoma,20 and hepatocellular carcinoma.22 In the current study, we observed that patients with GAC showed positive immunoreactivity of IMP3 and had shorter survival time than those of negative staining. Mean survival time of patients with negative IMP3 staining was 33 months, which was much longer than 23 months with positive staining.
In conclusion, we have demonstrated that IMP3, a novel oncofetal mRNA-binding protein, is frequently expressed in GAC. The findings suggest that IMP3 is implicated in the carcinogenesis and cancer progression especially in lymphoid metastasis of cancers, but the exact role of this multifunctional molecule in the neoplastic process merits further investigation. Highly intense immunoreactivity of IMP3 was associated with a lymphoid metastatic tumor and a poor patient outcome. Immunohistochemistry for IMP3 could be a novel proliferation and prognostic indicator in patients with gastric adenocarcinomas.
Acknowledgments: We thank Manut Tandon Ph.D research associate from the Department of Pharmacology & Chemical Biology, University of Pittsburgh, for editorial assistance with the manuscript.
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