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Original article
Relationship between expression of IGFBP7 and clinicopathological variables in gastric cancer
  1. Yuya Sato1,
  2. Mikito Inokuchi1,
  3. Yoko Takagi2,
  4. Sho Otsuki1,
  5. Yoshitaka Fujimori1,
  6. Yoshimitsu Yanaka1,
  7. Kenta Kobayashi1,
  8. Kyoko Higuchi1,
  9. Kazuyuki Kojima3,
  10. Tatsuyuki Kawano4
  1. 1Department of Gastric Surgery, Tokyo Medical and Dental University, Tokyo, Japan
  2. 2Department of Translational Oncology, Tokyo Medical and Dental University, Tokyo, Japan
  3. 3Center for Minimally Invasive Surgery, Tokyo Medical and Dental University, Tokyo, Japan
  4. 4Department of Esophageal Surgery, Tokyo Medical and Dental University, Tokyo, Japan
  1. Correspondence to Dr Mikito Inokuchi, Department of Gastric Surgery, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan; m-inokuchi.srg2{at}tmd.ac.jp

Abstract

Aims Insulin-like growth factor binding protein 7 (IGFBP7) is reported to have tumour suppressor function through an IGF-dependent pathway in various malignant tumours. However, the expression of IGFBP7 in adenocarcinoma and its relationship with tumour progression and survival differs among studies. Our aims were to investigate the relationship between the expression of IGFBP7 and clinicopathological variables and outcomes of patients with gastric cancer.

Methods Tumour samples were obtained from 219 patients with gastric cancer who underwent gastrectomy. The expression of IGFBP7 protein was examined by immunohistochemical staining. IGFBP7 mRNA levels were analysed using real-time quantitative reverse-transcriptase PCR in 24 of the gastric cancer tumours and in adjacent non-tumour tissues. Correlation of IGFBP7 expression with clinicopathological features was analysed.

Results The protein expression of IGFBP7 was positively correlated with depth of invasion, lymph node metastasis, distant metastasis or recurrence and pathological stage. High expression of IGFBP7 protein was associated with a significantly worse disease-specific survival (p<0.001) and was an independent prognostic factor in multivariable analysis (HR, 4.8; 95% CI 2.1 to 10.6; p<0.001). The IGFBP7 mRNA level was significantly higher in advanced gastric cancer than in early gastric cancer, in tumours with lymph node metastasis than in tumours without lymph node metastasis, and in tumours with distant metastasis or recurrence than in tumours without distant metastasis or recurrence.

Conclusions Overexpression of IGFBP7 was associated with tumour progression and poor survival in gastric cancer. IGFBP7 may play a role in tumour progression in gastric cancer.

  • GASTRIC CANCER
  • IMMUNOHISTOCHEMISTRY
  • IGF & IGT BINDING PROTEINS

https://doi.org/10.1136/jclinpath-2015-202987

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    Introduction

    Gastric cancer is the second leading cause of cancer-related deaths worldwide.1 ,2 Conventional therapy options for gastric cancer include surgery, chemotherapy, radiation therapy and combination treatments. In the early stages, the disease can often be cured through complete surgical removal of the tumour.3 However, a poor outcome is observed in patients with advanced gastric cancer. Many patients with advanced gastric cancer experience recurrent disease, even after a radical resection, which results in short survival times. The high mortality rate underscores the need for effective chemotherapy for patients with advanced stages of gastric cancer.3 The survival time of patients with unresectable gastric cancers has been prolonged by chemotherapy including taxans, irinotecan and fluoropyrimidines.4 ,5 However, the median survival time is less than 1 year even in patients receiving those chemotherapies.5

    Receptor tyrosine kinases (RTKs) consist of ligand-binding extracellular domains that identify the subfamilies of RTKs, a transmembrane domain and a tyrosine kinase motif, and the activation of these kinases has been shown to play an important role in the control of many fundamental processes, such as growth, differentiation, adhesion, migration and apoptosis.6–10 In a recent ToGA trial,10 trastuzumab, a monoclonal antibody against the p185HER2 protein, improved the overall survival of patients with HER2-positive tumours when combined with chemotherapy. However, only 12% of patients with far advanced gastric cancer responded to trastuzumab, and the median survival time was only 16 months in patients with HER2-positive tumours who received chemotherapy with trastuzumab.10 Further investigation of ligands, receptors and signalling pathways of other RTKs is required to increase the number of patients with gastric cancer for whom targeted treatments may be a viable clinical option.

    The insulin-like growth factor (IGF) axis plays a key role in the growth, differentiation and proliferation of mammalian cells and consists of two growth factors (IGF-I and IGF-II), their receptors (IGF-IR and IGF-IIR) and a group of insulin-like growth factor binding proteins (IGFBPs) (IGFBP1-7).11 Matsubara et al12 reported that IGF-IR expression in gastric cancer specimens was a significant predictor of poor survival in patients with advanced gastric cancer. It was suggested that anti-IGF-IR strategies may prove valuable in such patients.12 The actions of IGFs may be modulated by the IGFBPs in a positive or negative way, depending on the tissue type and the physiological/pathological status.13 Insulin-like growth factor binding protein 7 (IGFBP7) is one of 16 members of the IGFBP superfamily of proteins. IGFBP7 inhibits IGF-1 binding to the IGF1R.14 Therefore, IGFBP7 is reported to have a tumour suppressor-like function in a variety of cancers.14–16 In addition to IGF-dependent actions, IGFBP7 was found to exert IGF-independent properties. Because of its IGF-independent functions, high expression of IGFBP7 is reported to be associated with tumour progression and poor survival in some types of adenocarcinoma.17–19

    In this study, we investigated the relationship between the expression of IGFBP7 and clinicopathological variables in gastric cancer.

    Materials and methods

    Patients

    The study group comprised 219 patients with primary gastric adenocarcinomas who underwent surgery from January 2003 to December 2007 at the Department of Gastric Surgery, Tokyo Medical and Dental University. This study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board of Tokyo Medical and Dental University. Each tumour was classified according to the tumour-node-metastasis system recommended by the International Union against Cancer. The characteristics of the patients are shown in table 1. Of the 219 patients, 166 were men and 53 were women. The median age was 66 years (range: 21–92 years). All patients were evaluated for recurrent disease by diagnostic imaging (CT, ultrasonography, MRI and endoscopy) every 3–6 months. Patients with distant metastatic or recurrent disease received chemotherapy with S-1 alone or combined chemotherapy. Nineteen patients (9%) received adjuvant chemotherapy with S-1 after radical resection. All patients were followed up until January 2013. The median follow-up was 60 months (3–111). A total of 76 (35%) patients died, 65 (30%) had recurrent disease and 10 (5%) died of other causes.

    View this table:
    Table 1

    Characteristics of patients

    Immunohistochemical analysis of IGFBP7

    For immunohistochemical analysis, immunostaining was carried out with the use of a peroxidase-labelled polymer conjugated to secondary antibodies (Histofine Simple Stain MAX PO (MULTI), Nichirei Co., Tokyo, Japan). Monoclonal mouse antibodies against IGFBP7 (H-102, sc-13095) were purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA).

    All available H&E-stained slides of the surgical specimens were reviewed. For each case, representative paraffin blocks were selected for immunohistochemical studies. Four-micrometre-thick sections were cut from each formalin-fixed, paraffin-embedded tissue block. After deparaffinisation and rehydration, antigen retrieval treatment was carried out at 105°C (autoclave) for 10 min in Tris-EDTA (100 mM Tris base, 10 mM EDTA, pH 8.0), followed by treatment with 3% hydrogen peroxide for 15 min to quench endogenous peroxidase activity. The slides were incubated with the primary anti-IGFBP7 antibody (1:50) overnight at 4°C. Immunodetection was performed using the conventional streptavidin–biotin method with peroxidase-labelled SAB-PO kits (Nichirei). Diaminobenzidine substrate was used for colour development. The slides were counterstained with 1% Mayer's haematoxylin.

    Interpretation of the immunostaining results

    IGFBP7 expression in the cytoplasm was evaluated by a scoring method based on the staining extent and the staining intensity.20 The staining extent (positive frequency) was scored into four grades according to the percentage of stained tumour cells: 1 for <25%, 2 for 25% to <50%, 3 for 50% to <75%, and 4 for ≥75% stained cells. Staining intensity was scored into three grades: 0 (no staining or weakly positive), 1 (moderately positive) and 2 (strongly positive) (figure 1). Composite scores were multiplied to produce a weighted score for each case (varying from 1 to 6). For statistical analysis, composite scores of ≥4 were defined as high expression, and scores of <4 were considered low expression. Staining was assessed by two separate observers (YS and YT), who had no knowledge of patient data. Any disagreements between the two investigators were resolved by reassessment and consensus.

    Figure 1
    Figure 1

    Representative immunostaining of insulin-like growth factor binding protein 7 (IGFBP7) in gastric cancer. (A) no staining; (B) weakly positive; (C) moderately positive; and (D) strongly positive. (magnification 200×).

    RNA extraction and cDNA synthesis

    Immediately after surgery, a small piece of gastric cancer tissue and one of matched adjacent normal mucosa (taken from the borders of the surgical specimen) were separately placed directly in RNA stabilisation reagent (RNAlater, Qiagen, Valencia, California, USA) and were stored at −80°C until further analysis. Total RNA of each sample was extracted using an RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. The concentration of total RNA was determined by measuring absorption at 260 and 280 nm with an ultraviolet spectrophotometer (Beckman Coulter, Fullerton, California, USA). For cDNA synthesis, 10 μg of total RNA was reverse-transcribed into cDNA using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, California, USA) according to the manufacturer’s instructions.

    Real-time quantitative reverse transcription PCR (qRT-PCR)

    Expression levels of IGFBP7 as well as of ß-actin, used as an endogenous control, were determined by real-time quantitative PCR using a 7300 Real-Time PCR system (Applied Biosystems). TaqMan gene expression assays were purchased from Applied Biosystems (IGFBP7, Hs00266026_m1; ß-actin, Hs99999903_m1). The PCR reaction was carried out using a TaqMan Universal PCR Master mix (Applied Biosystems) with 1 μL of cDNA in a 24-μL final reaction volume. Thermal cycling conditions were: 50°C for 2 min, 95°C for 10 min, 40 cycles of 15 s denaturation at 95°C, and 1 min of annealing at 60°C. cDNA synthesised by the gastric cancer cell line MKN-45 was used for calibration. Each sample was run in duplicate for both the target and endogenous genes. The amounts of IGFBP7 normalised to the endogenous control and relative to the calibrator were determined by the comparative Ct method for relative quantification (ΔΔCt method) using Relative Quantification Study software (7300 Sequence Detection system, V.1.4, Applied Biosystems).

    Statistical analysis

    The χ2 test was used to test possible associations of IGFBP7 expression with clinicopathological variables. The Wilcoxon–Mann–Whitney test was used to compare medians of IGFBP7 mRNA expression with various clinicopathological variables. Kaplan–Meier curves were plotted to assess the effect of IGFBP7 expression on disease-specific survival (DSS). Different DSS curves were compared using the log-rank test. Multivariable proportional hazards Cox regression models were used to assess the prognostic significance of IGFBP7 and factors associated with DSS. Any association with a p value less than 0.05 by univariable analysis was included in multivariable analysis. No variables with strong linear correlations were confirmed by the coefficient of association before multivariable analysis. p Values of less than 0.05 were considered to indicate statistical significance. Statistical analysis was performed using IBM SPSS Statistics V.22 software (IBM, Armonk, New York, USA).

    Results

    Correlation between IGFBP7 protein expression and clinicopathological variables

    Expression of IGFBP7 protein, which was assessed by immunohistochemistry, was mainly observed in the cytoplasm of cancer cells (figure 1), in fibroblasts and in lymphocytes in cancer tissue. Weak expression was observed in the nuclei in some of the cancer cells. Expression of IGFBP7 was high in 131 (59.8%) of the 219 tumours. The expression of IGFBP7 was positively correlated with the depth of tumour invasion (T1 vs T2–4; p<0.001), lymph node metastasis (N0 vs N1–3; p=0.002), distant metastasis or recurrence ­(p<0.001), and tumour stage (I vs II–IV; p<0.001) (table 2). Since high expression of IGFBP7 was significantly associated with lymph node metastasis, we further immunohistochemically evaluated the expression of IGFBP7 in lymph node metastases from 88 patients. High IGFBP7 expression was seen in metastatic lymph nodes in 35 of these patients (41%).

    View this table:
    Table 2

    Differences in clinicopathological variables by IGFBP7 expression

    Correlation between IGFBP7 protein expression and DSS

    Patients with high IGFBP7 protein expression had significantly poorer DSS than those with low IGFBP7 expression (p<0.001) (figure 2). The 5-year DSS was 58% in patients with high expression of IGFBP7 and 92% in patients with low expression of IGFBP7.

    Figure 2
    Figure 2

    Kaplan–Meier curves of the disease-specific survival (DSS) of patients with expression of insulin-like growth factor binding protein-7 (IGFBP7). Patients with high IGFBP7 protein expression (131 patients) had significantly poorer DSS than those with low IGFBP7 expression (88 patients) (p<0.001).

    The prognostic relevance of IGFBP7 protein expression was assessed using a multivariable proportional-hazards model adjusted for significant prognostic factors in univariable analysis (Lauren's classification, depth of tumour invasion, lymph node metastasis and expression of IGFBP7) (table 3). The depth of tumour invasion, lymph node metastasis, and IGFBP7 expression were independent prognostic factors in multivariable analysis (HR=7.8, 95% CI 1.8–33.7, p=0.006; HR=5.8, 95% CI 2.6 to 13.1, p<0.001; HR=4.8, 95% CI 2.1 to 10.6, p<0.001, respectively).

    View this table:
    Table 3

    Prognostic factors for disease-specific survival (DSS) in univariable and multivariable Cox proportional-hazards regression models

    Comparison of IGFBP7 mRNA expression between cancerous and adjacent tissues

    The median of IGFBP7 mRNA level was significantly higher in gastric cancer than in the adjacent normal tissue (19.1 (5.6–62.9) vs 11.9 (3.4–44.5) (p=0.034)) (figure 3A).

    Figure 3
    Figure 3

    Insulin-like growth factor binding protein-7 (IGFBP7) mRNA expression in gastric cancer and correlation with clinicopathological variables. (A) IGFBP7 mRNA expression in gastric cancerous and adjacent tissues. The median of IGFBP7 mRNA level was significantly higher in gastric cancer than in the adjacent normal tissue in 24 patients (p=0.034). (B–D) Correlation between IGFBP7 mRNA expression and the clinicopathological variables of T stage (B), N stage (C), and distant metastasis and recurrence (D). The median of IGFBP7 mRNA level was significantly higher in advanced gastric cancer (n=19) than in early gastric cancer (n=5) (B), in tumours with lymph node metastasis (n=14) than in tumours without lymph node metastasis (n=10) (C), and in tumours with distant metastasis or recurrence (n=10) than in tumours without distant metastasis or recurrence (n=14) (D).

    Relationship of IGFBP7 mRNA expression with various clinicopathological variables

    The median of IGFBP7 mRNA level was significantly higher in advanced gastric cancer than in early gastric cancer (21.0 (5.6–62.9) vs 7.0 (7.0–10.2) (p=0.002)), in tumours with lymph node metastasis than in tumours without lymph node metastasis (23.0 (7.0–62.9) vs 9.3 (5.6–28.8) (p=0.002)) and in tumours with distant metastasis or recurrence than in tumours without distant metastasis or recurrence (23.0 (12.4–62.9) vs 11.1 (5.6–33.7) (p=0.019)) (figure 3B–D).

    Discussion

    Our results showed that high IGFBP7 protein and mRNA expression significantly correlated with tumour progression and poor survival independent of other clinicopathological features in gastric cancer. IGFBP7 has been reported to play a role in tumour progression in some types of adenocarcinomas. Thus, expression of IGFBP7 is an independent prognostic indicator for decreased patient survival in oesophageal adenocarcinoma19 and colorectal carcinoma.17 Smith et al19 reported DNA methylation within the IGFBP7 promoter was associated with transcriptional silencing in oesophageal adenocarcinoma and that such silencing was frequently observed in oesophageal adenocarcinoma tissues. The survival rate tended to be better in patients with methylated IGFBP7. This result suggests that IGFBP7 has a role in tumour progression in adenocarcinoma. Georges et al18 reported that the expression of IGFBP7 in colorectal cancer was dramatically increased in response to early liver metastatic growth in vivo and that IGFBP7 expression gradually returned to normal thereafter. It was assumed that this upregulation of IGFBP7 in colorectal cancer played a vital role during the process of homing into the liver and in early metastatic dissemination. In contrast, some studies have reported that IGFBP7 is a tumour suppressor. An et al20 reported that low expression of IGFBP7 was correlated with increased proliferation and poor postoperative survival in pancreatic ductal carcinoma. Ruan et al21 reported that high expression of IGFBP7 was associated with favourable prognosis in colorectal cancer. In that study, IGFBP7 could inhibit cell growth, decrease soft agar colony forming activity and induce apoptosis in two colorectal cell lines. These controversial results may be caused by the fact that IGFBP7 is not homogeneously expressed within the cancer. Thus, the expression of IGFBP7 varies with the tumour site and even within a single tumour nest. Adachi et al17 reported that IGFBP7-positive cells were frequently located at the invasive front of the tumour nest, which was linked to a poor survival rate in patients with colorectal cancer. In the present study, IGFBP7 expression was examined in the invasive front of the tumour and in the whole tumour nest including the surface of the tumour.

    For other reasons, IGFBP7 has been said to be involved in signal transduction in both IGF-dependent and IGF-independent pathways.22–24 In addition to a tumour suppressor function by inhibition of IGF-1 binding to the IGF1R,14 IGFBP7 is considered to have a role in tumour progression through IGF-independent pathways. Adachi et al17 reported that the anti-IGFBP7 antibody stained small blood vessels near to and within tumour nests more densely than normal part of the tissue in colorectal cancer. Thus, IGFBP7 was believed to be involved in angiogenesis and in modulation of the vascular functions necessary for tumour development. However, there have been few reports on the contribution of IGF-independent pathways to tumour progression. Liu et al reported low expression of IGFBP7 was associated with tumour progression and poor survival in gastric cancer.25 The results obtained in the present study were opposite to those of this report. There may be other reasons for this discrepancy apart from non-homogeneous expression of IGFBP7 or multifunctional effects of IGFBP7. First, gastric cancer may be considered a heterogeneous disease, which has potential implications for its disease biology.26–28 The expression of RTKs are different even in the same tumour nest, which is characteristic of gastric cancer.29 ,30 Thus, some studies that have analysed the expression of various RTKs in gastric cancer have yielded conflicting results with respect to correlation with clinicopathological variables.30–32 In the present study, the expression of IGFBP7 was evaluated in the whole tumour nest by immunohistochemistry and in the surface part of the tumour by qRT-PCR. Differences in the tumour sites that were evaluated in the two studies may be a cause of the controversial results. In the present study, the staining of IGFBP7 was heterogeneous and varied with the tumour site (figure 4). Second, there are differences in the IGFBP7 primer used for real-time qRT-PCR, in the IGFBP7 antibody and the staining procedure used for immunohistochemistry, and in the method of evaluation of the immunostaining results. Third, early gastric cancer was much more frequent in the present study than the former report. As a result, the survival rate of IGFBP7 high expression group in the former report is much more worth compared with that in the present study. The different distribution of tumour staging between studies may affect the discrepancy between the two studies.

    Figure 4
    Figure 4

    Representative gastric cancer case with intratumorous insulin-like growth factor binding protein-7 (IGFBP7) heterogeneity. IGFBP7 immunohistochemistry shows heterogeneous staining with variable (strong (A) and weak (B)) staining intensities in the same tumour nest.

    A limitation of our study is that the data do not allow any conclusions to be reached regarding the possible biological role of IGFBP7 in gastric cancer. To explain the heterogeneity of IGFBP7 expression in gastric cancer, IGF-independent pathways and the function of IGFBP7 should be analysed by methods such as functional analyses using cell lines.

    In conclusion, increased IGFBP7 expression was related to tumour progression and poor survival of gastric cancer in this study. IGFBP7 may play a role in tumour progression in gastric cancer.

    Take home messages

    • High expression of insulin-like growth factor binding protein 7 (IGFBP7) was associated with a significantly worse disease-specific survival and was an independent significant prognostic factor in patients with gastric cancer.

    • The IGFBP7 mRNA level tended to increase along with a progression of gastric cancer.

    • These results suggest that IGFBP7 may play a role in tumour progression in gastric cancer.

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    Supplementary materials

    • Abstract in Japanese

      This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.

    Footnotes

    • Handling editor Cheok Soon Lee

    • Contributors YS, KK, MI and TK were responsible for drafting the manuscript. YS and YT contributed to assessing immunohistochemical staining. YS and MI contributed to analysis and interpretation data. SO, YF, YY, KK and KH contributed in data collection.

    • Competing interests None declared.

    • Provenance and peer review Not commissioned; externally peer reviewed.