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An abnormally high expression of ISL-1 represents a potential prognostic factor in gastric cancer
Human Pathology (2015) xx, xxx–xxx
www.elsevier.com/locate/humpath
Original contribution
An abnormally high expression of ISL-1 represents a potential prognostic factor in gastric cancer☆,☆☆ Chen Guo MS, Weiping Wang PhD, Qiong Shi BS, Ping Chen BS, Chunyan Zhou MD, PhD ⁎ Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Key Laboratory of Molecular Cardiovascular Sciences (Ministry of Education), Key Laboratory of Protein Posttranslational Modifications and Cell Function (Beijing), Peking University,38 Xue Yuan Rd, Beijing 100191, China Received 14 February 2015; revised 6 May 2015; accepted 8 May 2015
Keywords: ISL-1; Prognostic biomarker; Gastric cancer; Immunohistochemistry; Overall survival
Summary Insulin gene enhancer binding protein–1 (ISL-1) is a transcription factor involved in development of the heart, motor neurons, and pancreas. Our previous study indicated that ISL-1 was overexpressed in gastric cancer but not in other gastrointestinal tumors. However, no immunohistochemical or clinicopathological studies of ISL-1 in gastric carcinoma have been performed. The aim of this study was to determine the expression and prognostic value of ISL-1 in gastric carcinoma. A nude mouse xenograft model was established to study the role of ISL-1 on cancer genesis and development in vivo. Overexpression of ISL-1 significantly enhanced the tumorigenicity of NIH3T3 cells in vivo. ISL-1 expression was evaluated using immunohistochemistry in 456 human gastric carcinoma and normal tissues. ISL-1 was significantly overexpressed in gastric adenocarcinoma compared with normal gastric tissues. ISL-1 expression was significantly associated with depth of invasion, lymph node metastasis, TNM stage, and histological grade (P b .05, χ2 test). Positive ISL-1 expression was associated with poorer 5-year overall survival in gastric cancer (P = .001, log-rank test). Multivariate Cox regression analysis demonstrated that ISL-1 expression (P = .047) could be an independent prognostic factor for overall survival in gastric carcinoma. This study suggests that ISL-1 may be a useful prognostic biomarker and may represent a novel therapeutic target for gastric adenocarcinoma. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Although its incidence has been declining for several decades, gastric cancer was the leading cause of cancer☆
Conflict of interest: The authors declare no conflict of interest. This work was supported by the National Natural Science Foundation of China, Beijing (81071675, 81170713, 8137023, 81472022), the Natural Science Foundation of Beijing (5122021), the Leading Academic Discipline Project of Beijing Education Bureau, and the 111 Project of China (B07001). ⁎ Corresponding author. E-mail addresses: [email protected] (C. Guo), [email protected] (W. Wang), [email protected] (Q. Shi), [email protected] (P. Chen), [email protected] (C. Zhou). ☆☆
http://dx.doi.org/10.1016/j.humpath.2015.05.006 0046-8177/© 2015 Elsevier Inc. All rights reserved.
related deaths worldwide until the mid-1990s. A total of 989 600 new cases and 738 000 deaths due to gastric cancer were estimated to have occurred in 2008, accounting for 8% and 10% of all cases and cancer-related deaths, respectively, with two-thirds of these cases in developing countries [1]. Gastric cancer is now relatively rare in North America and most of Northern and Western Europe; however, the incidence is higher in Eastern Europe, Russia, and selected areas of Central and South America and East Asia [2,3]. In China, gastric cancer is the most common type of cancer and the second leading cause of cancer deaths after lung cancer [4]. The overall morbidity and mortality of gastric cancer are doubled in male patients than female patients in China [1,4].
2 Disease stage is the most important factor for predicting the treatment outcome of patients [5,6]. Despite the fact that most cases of early stage gastric carcinoma can be cured by surgical resection, metastasis contributes to the high rate of mortality in gastric carcinoma [7]. Although numerous immunohistochemical markers such as human epidermal growth factor receptor 2, matrix metallopeptidase–2, and epithelium cadherin have been recognized as biomarkers in other types of cancer, these markers are not commonly used to assess biological behavior and prognosis in gastric cancer because of low levels of sensitivity and specificity [8–13]. Therefore, there is an urgent need to identify novel prognostic and predictive biomarkers to improve the diagnosis and clinical management of patients with gastric cancer, which in turn may help to develop more effective treatment strategies. Insulin enhancer binding protein–1 (ISL-1), a LIMhomeodomain transcription factor, was originally isolated as an insulin-responsive protein that binds to the islet β-cell–specific enhancer element [14]. The rat, hamster, and human ISL-1 amino acid sequences share 100% homology [15]. ISL-1 has previously been described to play crucial roles in development of the heart, motor neurons, and pancreas [16–18]. Recently, ISL-1 was implicated in the development of cancer, mainly based on the fact that expression of ISL-1 is dysregulated in a range of tumor types [19–22]. In a pilot study, we examined the expression of ISL-1 in a variety of human tumor types including breast, prostate, colon, liver, lung, and esophageal cancer using immunohistochemical staining. Markedly higher expression of ISL-1 was observed in human gastric carcinoma specimens compared with the tumor specimens from other organs [23]. However, no comprehensive immunohistochemical or clinicopathological studies of ISL-1 in gastric carcinoma have been performed. An accurate evaluation of the expression of ISL-1 in gastric cancer is required to investigate its prognostic value in this tumor type. In the present study, we investigated the expression of ISL-1 in gastric cancer tissues and normal tissues. We found that an abnormally high expression of ISL-1 was significantly associated with the depth of invasion, lymph node metastasis, TNM stage, and histological grade, as well as poorer overall survival, in patients with gastric cancer. The findings of this study indicate that ISL-1 has potential as a diagnostic and prognostic biomarker for gastric carcinoma.
2. Materials and methods 2.1. Patients and tissue microarray Human gastric cancer tissues and normal gastric tissue microarray sections with complete stage and grade information were purchased from Biomax (T012A, T014, ST1001, ST2091; Rockville, MD) and Shanghai Biochip Co Ltd (HStm-Ade180Sur-04; Shanghai, People's Republic of China). In total, ISL-1 protein expression was analyzed in
C. Guo et al. 262 gastric cancer tissues, 144 noncancerous tissues (including 9 gastric ulcers, 17 gastric inflammations, 6 hyperplasias, and 112 normal gastric tissues), and 50 gastric adenocarcinoma lymph node metastases. In addition, tissue specimens from 90 patients with gastric cancer (Shanghai Outdo Biotech Co Ltd, HStm-Ade180Sur-04) for whom complete overall survival data were available (from the date of surgery until death or August 2013) were analyzed. The clinicopathological characteristics of these patients are shown in Table 1; the patients were staged according to the sixth edition of the American Joint Committee on Cancer manual [24]. Patients were only included in the study if they had provided written consent to participate in the study after receiving oral and written information regarding its nature and purpose. Approval for the study was received from the ethics committee of the host institutions in addition to the company that provided the samples.
2.2. Animal experiments All animals were purchased from the Department of Laboratory Animal Science, Peking University; and all protocols were approved by the Animal Care and Use Committee of Peking University (LA 2010-066). The plasmid pcDNA3-ISL1 and control vector pcDNA3 were separately transfected into NIH3T3 cells to establish stable ISL-1–overexpressing cells (pcDNA3-ISL-1) and control cells (pcDNA3), as previously described [22]. Five-week-old female nude mice were subcutaneously injected on the right side of the dorsum (pcDNA3-ISL-1) and left armpit (pcDNA3) with 1 × 107 cells suspended in 200 μL phosphate-buffered saline (PBS). Tumors were measured at the indicated time points using calipers, and tumor volumes were calculated using 1/2 × length × width2. The significance of the differences between groups was determined using 2-way analysis of variance.
2.3. Immunohistochemistry Five-micrometer-thick tissue sections were cut from each tissue microarray block. All subsequent procedures were carried out at room temperature, unless otherwise stated. Tissue sections were rehydrated; and antigen retrieval was performed by submerging the slides in sodium citrate buffer (10 mmol/L sodium citrate, 0.05% Tween 20, pH 6.0) in a pressure cooker, boiling for 3 minutes, followed by cooling for 30 minutes at room temperature. Afterward, the tissue sections were incubated in 3% H2O2 in PBS-T (0.01% Tween 20 in PBS) for 10 minutes to block endogenous peroxidase activity; and a circular hydrophobic barrier was drawn around each specimen using a Pap pen (ZLI-9305; ZSGB-Bio, Beijing, China). The sections were blocked in 5% bovine serum albumin in PBS-T for 30 minutes and, incubated with mouse polyclonal anti-ISL-1 antibody (1:400; ab86472; Abcam, Hong Kong, China) in 5% bovine serum albumin in PBS-T in a humidified chamber overnight,
Abnormal expression of ISL-1 in gastric cancer
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Table 1 Demographic and clinicopathological features of the 262 patients with gastric cancer Feature Age, y ≤60 N60 Sex Male Female Histological type Adenocarcinoma Mucinous adenocarcinoma Signet ring cell adenocarcinoma Others a TNM stage I II III IV Depth of invasion T1 T2 T3 T4 Lymph node metastasis N0 N1 N2 N3 Distant metastasis M0 M1 Histological grade b G1 G2 G3 G4
n (%) 141 (53.8%) 121 (46.2%) 188 (71.8%) 74 (28.2%) 234 16 4 8
(89.3%) (6.1%) (1.5%) (3.1%)
34 115 95 18
(13.0%) (43.9%) (36.3%) (6.8%)
8 35 191 28
(3.1%) (13.4%) (72.9%) (10.6%)
136 56 38 32
(51.9%) (21.4%) (14.5%) (12.2%)
257 (98.1%) 5 (1.9%) 17 87 136 22
(6.5%) (33.2%) (51.9%) (8.4%)
a
Eight cases are classified into this category, which include squamous cell carcinoma (1), adenosquamous carcinoma (2), lowgrade interstitialoma (1), carcinoid (2), undifferentiated carcinoma (1), and papillary adenocarcinoma (1). b G1, well-differentiated adenocarcinoma; G2, moderately differentiated adenocarcinoma; G3, poorly differentiated adenocarcinoma; G4, unassessed differentiation status and undefined cases.
and the sites of immunoreactivity were detected using the Envision Detection Kit/DAB staining kit (GK500705; DAKO A/S, Glostrup, Denmark) according to the manufacturer's protocol. The slides were counterstained with hematoxylin (ZLI-9609, ZSGB-Bio) for 1 minute at room temperature and then dehydrated and sealed using crystal mounting solution (ZLI-9516, ZSGB-Bio).
2.4. Image analysis and scoring The detection of ISL-1 expression was performed in the whole lesion from mucosal to subserosal or serosal in the
same slides, and the lymph nodes (LNs) were also from the same patients. The staining scores were parallel to the patients' information. ISL-1 immunoreactivity was quantified using Image-Pro Plus software version 6 (Media Cybernetics, Silver Spring, MD). Briefly, digital images (TIFF format) close to the center of each tumor core were captured using a ×20 objective (Leica DM25000B microscope; Leica, Ernst-Leitz-Strasse, Germany). Images of all tissue cores were acquired in the same session using a constant set of microscope and imaging software parameters. The images were subjected to optical density analysis using Image-Pro Plus software. Intensity range selection was based on the image histogram, with intensity and saturation set at maximum and hue set at a range where most of the brown diaminobenzidine tetrahydrochloride hydrate color was selected, whereas the blue counterstained nuclei were excluded. These settings were saved and then subsequently applied to all of the images analyzed. On opening each image, a region of interest was drawn to include most of the malignant epithelial cells in the image and exclude stroma, muscle, necrosis, and other nontumor components. After defining the region of interest, the mean optical density of the selected area (integrated optical density [IOD]/unit area) was determined by the software. The IOD/unit area represents the immunoreactivity of the candidate protein within the tumor tissue. To facilitate statistical analysis, the IOD/unit area values were manually categorized as negative immunoreactivity (values ≤40) or positive immunoreactivity (values N40), as described in the “Results” section [25].
2.5. Statistical analysis All statistical analyses were performed using SPSS (version 20; SPSS, Chicago, IL). The association between ISL-1 immunoreactivity and the clinicopathological features of the patients was assessed using the χ2 test. The impact of ISL-1 on patient survival was examined using Kaplan-Meier survival curves; statistical significance was determined using the log-rank test. Multivariate analysis based on the Cox proportional hazards regression model was applied to identify independent prognostic factors. For all statistical tests, P values less than .05 were considered statistically significant.
3. Results 3.1. The function of ISL-1 in tumor growth in vivo We established a xenograft model in nude mice to investigate whether ISL-1 could promote the tumorigenicity of NIH-3T3 cells in vivo. Overexpression of ISL-1 (pcDNA3-ISL-1) enhanced the formation and growth of tumor xenografts in vivo. In contrast, no tumors formed after injection of the control cells (pcDNA3; Fig. 1).
4
3.2. Expression of ISL-1 in gastric carcinoma and noncancerous gastric tissues The expression of ISL-1 was analyzed by immunohistochemistry in 262 primary gastric carcinoma specimens, 112 cancer-adjacent normal gastric tissues, 32 gastric disease tissues (including 9 gastric ulcers, 17 gastric inflammations, and 6 hyperplasias), and 50 gastric adenocarcinoma lymph node metastases. ISL-1 was mainly localized to the nucleus of the gastric lesions, with different staining intensities observed in different stages of gastric cancer (Fig. 2). There was a clear trend that higher levels of ISL-1 were expressed in gastric cancer compared with normal gastric mucosa. Positive expression of ISL-1 was detected in 58.4% (153/ 262) of the primary cancer tissues and 4.5% (5/112) of the
C. Guo et al. noncancer gastric tissues; the rate of ISL-1 expression in primary gastric cancer and noncancer tissues was significantly different (P = .001; χ2 test). Likewise, the rate of ISL-1 expression in metastatic gastric adenocarcinoma lymph node specimens (76.0%, 38/50) and primary cancer (58.4%, 153/262) was also significantly different (P b .01, χ2 test; data not shown).
3.3. Correlation between ISL-1 expression and the clinicopathological features of primary gastric carcinoma The clinicopathological characteristics of the 262 patients with gastric cancer analyzed in this study are summarized in Table 1. The correlations between ISL-1 expression and the clinicopathological features of the patients are summarized
Fig. 1 ISL-1 promotes tumor growth in vivo. A and B, Images of the 5 mice (A) and the excised tumors (B) on day 21. C and D, Representative hematoxylin and eosin staining of the tumors formed by control cells (C) and ISL-1–overexpressing cells (D). Control tumor cells had clear cytoplasm and pale and round nuclei; ISL-1–positive tumor cells had irregular and long spindle-shaped nuclei. E, ISL-1 expression was detected by Western blot analysis in stably transfected NIH3T3 cells. Glyceraldehyde 3-phosphate dehydrogenase served as an internal control. F, Tumor growth curves. Tumor volume (mm3) was measured using calipers and calculated using the following standard formula: (shortest diameter) 2 × (longest diameter) × 0.5 (P = .002 versus control tumors at the same time, Pearson χ2 test). Abbreviation: GADPH, glyceraldehyde 3-phosphate dehydrogenase.
Abnormal expression of ISL-1 in gastric cancer
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Fig. 2 Representative images of immunohistochemical staining for ISL-1 in human gastric carcinoma tissues and noncancerous gastric tissues. A, Normal gastric mucosa; negative ISL-1 expression. B, Early stage gastric carcinoma (moderately differentiated adenocarcinoma); weak ISL-1 expression in nucleus and cytoplasm of gastric lesions. C, Gastric signet ring cell carcinoma; strong ISL-1 expression in nucleus of signet ring cell. D, Stage IV gastric carcinoma (moderately differentiated adenocarcinoma); strong ISL-1 expression in nucleus and cytoplasm of gastric cancer cell. Images were captured using a 40× objective lens (scale bar = 100 μm).
in Table 2. Positive expression of ISL-1 was potentially associated with moderately to poorly differentiated histological type of gastric cancer (P = .022; χ2 test); we should expand the samples to validate its clinical significance. Positive expression of ISL-1 was also significantly associated with the depth of invasion, lymph node metastasis, and TNM stage (P = .001, P = .002, and P b .001, respectively; χ2 test). However, there was no significant association between the expression of ISL-1 and age, sex, tumor location, tumor size, or distant metastasis (P N .05; t test or χ2 test).
3.4. Relationship between expression of ISL-1 and overall survival in primary gastric carcinoma Kaplan-Meier survival curves were constructed to evaluate whether expression of ISL-1 correlated with prognosis in primary gastric carcinoma. During the follow-up period, 62 (68.9%) of the 90 patients died. Only 1 patient was lost to follow-up, and this did not have any significant impact on the survival analysis. The 5-year overall survival rates for patients with positive and negative expression of ISL-1 were 62.5% and 35.3%, respectively; this difference was significantly different (P b .001, log-rank test; Fig. 2A). In addition, subgroup analysis revealed that positive expression of ISL-1 was associated with significantly poorer overall survival in both patients with stage I to II disease and patients with stage III-IV disease (P b.001 and P = .001, respectively; log-rank test; Fig. 2B and C). Therefore, ISL-1 is associated with prognosis in every stage of gastric cancer.
Furthermore, univariate analysis using a Cox proportional hazards model indicated that depth of invasion, advanced clinical stage, and high ISL-1 expression were associated with a poorer prognosis in gastric cancer. After adjustment for ISL-1 immunoreactivity and other known prognostic factors including tumor size, tumor site, depth of invasion, cancer stage, and cancer grade in multivariate Cox regression analysis, depth of invasion and ISL-1 expression remained independent prognostic factors for gastric cancer (P = .001 and P = .047, respectively; Cox regression analysis; Fig. 3 and Table 3). As shown in Table 2, the ISL-1 expression level was significantly positively correlated with the depth of invasion, which increases the reliability of ISL-1 as a prognostic factor.
4. Discussion Although early screening and diagnosis methods and treatments have been improved to a certain extent in recent years, the prognosis and clinical outcome of patients with gastric cancer remain poor. At present, immunohistochemical staining of formalin-fixed and paraffin-embedded tissues is widely used in diagnostic surgical pathology to provide information of the biological behavior of a tumor and patient prognosis [11]. However, no single marker has an adequate level of accuracy or specificity in all stages of gastric carcinoma. In addition, as the majority of gastric cancers are sporadic and it is also a multifactorial disease characterized by both genetic and environmental factors, the prognostic
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C. Guo et al. Table 2 Comparison of the clinicopathological features of patients with ISL-1–negative and ISL-1–positive gastric carcinoma Characteristic
Age, y Sex Male Female Depth of invasion T1-T2 T3-T4 Lymph node metastasis N0 N1-N3 Distant metastasis M0 M1 TNM stage I II III IV Histological grade G1 G2 G3 G4
Expression of ISL-1 Negative (%)
Positive (%) P value
(n = 109)
(n = 153)
58 ± 26
60 ± 26
.932
78 (71.6%) 31 (28.4%)
110 (71.9%) 43 (28.1%)
.953
28 (25.7%) 81 (74.3%)
15 (9.8%) 138 (90.2%)
.001
69 (63.3%) 40 (36.7%)
67 (43.8%) 86 (56.2%)
.002
106 (97.3%) 3 (2.7%)
151 (98.7%) 2 (1.3%)
.701
25 (22.9%) 48 (44.0%) 31 (28.4%) 5 (4.6%)
9 (5.9%) 67 (43.8%) 64 (41.8%) 13 (8.5%)
b.001
7 (6.4%) 35 (32.1%) 52 (47.7%) 15 (13.8%)
10 (6.5%) 52 (34.0%) 84 (54.9%) 7 (4.6%)
.022
value of many molecular markers such as vascular endothelial growth factor remains controversial [9]. Therefore, identification of novel diagnostic or prognostic markers may help to improve prognostication and the 5-year survival rate in gastric cancer.
ISL-1 was initially functionally characterized in 1990 and is a member of the LIM/homeodomain family of transcription factors that are related to the regulation of insulin gene expression [14]. Hansel et al [26] used global gene expression analysis and reported that ISL-1 was overexpressed in human biliary cancer. ISL-1 has also been found to participate in metastasis in pancreatic neuroendocrine neoplasms, pancreatic endocrine tumors, non–muscle-invasive bladder cancer, and non-Hodgkin lymphoma [19–22]. In addition, the level of ISL-1 protein expression is closely associated with the development of cancer, cancer cell growth, and metastasis, as well as the prognosis of patients with pancreatic endocrine tumors [21]. To examine the role of ISL-1 in the pathogenesis of gastric carcinoma, we created a xenograft model in nude mice to study the impact of ISL-1 on tumor growth in vivo. Overexpression of ISL-1 in NIH3T3 cells significantly promoted the establishment and growth of xenograft tumors in vivo. This indicates that ISL-1 plays a role in tumor initiation and progression. However, NIH 3T3 is a mouse fibroblast cell line; and the tumorigenicity role of ISL-1 demonstrated in this model needs to be further validated in appropriate human gastric normal cells. This is the first report to indicate that ISL-1 is overexpressed in gastric cancer. In a previous immunohistochemical study, we found that ISL-1 was significantly upregulated in gastric cancer but not in other types of gastrointestinal tumors, such as colon cancer, esophageal cancer, and pancreatic cancer, which suggests that high expression levels of ISL-1 may be a specific marker for gastric cancer [23]. Therefore, we examined the expression of ISL-1 using immunohistochemical analysis in 456 gastric lesions at different disease stages including normal mucosa, gastric diseases, primary gastric carcinoma, and distant metastases. ISL-1 protein was expressed at high levels in gastric adenocarcinoma tissues compared with the other gastric tissues. Interestingly, in normal and benign tumor tissues,
Fig. 3 Association between ISL-1 expression and overall survival in primary gastric carcinoma. Kaplan-Meier overall survival curves for all 90 patients (A; P b .001), patients with stage I to II gastric carcinoma (B; P = .001), and patients with stage III to IV gastric carcinoma (C; P b .001; log-rank test). Patients are stratified by positive and negative expression of ISL-1.
Abnormal expression of ISL-1 in gastric cancer
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Table 3 Univariate and multivariate Cox regression analysis of the association of ISL-1 expression and other clinicopathologic features with overall survival in primary gastric carcinoma Factor
Age Sex Size Site Depth of invasion Stage Grade ISL-1 expression
Univariate
Multivariate
OR
95% CI
P value
1.024 1.069 0.602 1.030 2.183 2.051 1.171 0.193
0.998-1.050 0.589-1.940 0.354-1.022 0.749-1.415 1.408-3.383 1.391-3.024 0.771-1.779 0.100-0.375
.065 .827 .060 .857 b.001 b.001 .460 b.001
OR
95% CI
P value
4.818 1.992
2.050-11.321 0.942-4.211
.001 .071
0.335
0.114-0.987
.047
Abbreviations: OR, odds ratio; CI, confidence interval.
ISL-1 staining was distributed throughout the tumor cells and mesenchymal cells, whereas ISL-1 immunoreactivity was only observed in the nuclei of advanced-stage gastric adenocarcinoma, indicating its transcriptional activation [27]. The growth and development of tumors are a complex, continuous process. The regulatory mechanisms are also diversified. It has been reported that caudal-related homeobox transcription factor can inhibit invasion and migration, and promote differentiation and the multidrug resistance of gastric cancer cells [28–30]. Ectopic expression of caudalrelated homeobox transcription factor is speculated to be involved in gastric cancer development. Overexpression of ISL-1 in MGC803, a human gastric cancer cell line, could promote the cell proliferation and cell migration (unpublished data). Although the mechanism of ISL-1 in gastric cancer still remains unclear, we believe that it may have multimechanisms to promote gastric cancer development. It should be pointed out that ISL-1 expression is involved in human gastric cancer invasion, TNM stage, and lymph node metastasis; but because the sample size is too small (only 5 cases), we cannot make a conclusion for its correlation with distant metastasis. Notably, the markers commonly used to detect gastric cancer in the clinic are also used for other digestive system cancers. However, the present study indicates that ISL-1 is overexpressed in gastric cancer but not in other gastrointestinal tumors. These results imply that ISL-1 may be a sensitive and specific biomarker for gastric cancer and that ISL-1 may have a competitive advantage as a novel therapeutic target for gastric adenocarcinoma. Next, we investigated the relationship between ISL-1 expression and the prognosis and clinical features of patients with gastric cancer. The overall survival time for ISL-1– positive patients was significantly shorter than that of ISL-1– negative patients. In particular, multivariate analysis confirmed that ISL-1 was an independent prognostic factor for poor overall survival in gastric cancer. No similar results exist for other types of gastrointestinal cancer. In recent years, the presence of tumor budding (TB) at the tumor-host interface of gastrointestinal cancers has been recognized as a hallmark of unfavorable disease biology. Tumor budding is defined as the presence of dedifferentiated cells or
small clusters of up to 5 cells at the tumor invasive front [31]. Although the Union for International Cancer Control has officially recognized TB as an additional independent prognostic factor in colon and rectum cancers [32], the evidence for prognostic impact of TB in gastric adenocarcinoma is still insufficient. We found that highly expressed ISL-1 promoted metastasis of MGC803 (a gastric cancer cell line) cells (data not shown), which is consistent with the observation that the high-grade TB could predict the first step toward metastasis and early disease recurrence in solid tumor [33]. However, whether the high level of ISL-1 expression has a possible connection with an increased frequency of TB in patients at the tumor invasive front remains to be elucidated in gastric cancer. In conclusion, this study demonstrates that ISL-1 is expressed at high levels in gastric carcinoma compared with the adjacent noncancerous tissues and that high-level expression of ISL-1 was significantly associated with advanced TNM stage. Moreover, positive expression of ISL-1 was associated with significantly poorer overall survival in gastric cancer. Furthermore, both univariate and multivariate Cox proportional hazards regression revealed that high ISL-1 expression was an independent prognostic factor for poorer overall survival in gastric cancer. Based on these results, we suggest that ISL-1 represents a potentially useful prognostic marker in gastric cancer. The clinical value of ISL-1 as a biomarker and prognostic factor and the mechanisms by which ISL-1 regulates the progression of gastric carcinoma require further evaluation.
Acknowledgments We thank Dr Fei Pei, Department of Pathology, Peking University, for her kind help in the image analysis and scoring of tissue microarray.
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Original contribution
An abnormally high expression of ISL-1 represents a potential prognostic factor in gastric cancer☆,☆☆ Chen Guo MS, Weiping Wang PhD, Qiong Shi BS, Ping Chen BS, Chunyan Zhou MD, PhD ⁎ Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Key Laboratory of Molecular Cardiovascular Sciences (Ministry of Education), Key Laboratory of Protein Posttranslational Modifications and Cell Function (Beijing), Peking University,38 Xue Yuan Rd, Beijing 100191, China Received 14 February 2015; revised 6 May 2015; accepted 8 May 2015
Keywords: ISL-1; Prognostic biomarker; Gastric cancer; Immunohistochemistry; Overall survival
Summary Insulin gene enhancer binding protein–1 (ISL-1) is a transcription factor involved in development of the heart, motor neurons, and pancreas. Our previous study indicated that ISL-1 was overexpressed in gastric cancer but not in other gastrointestinal tumors. However, no immunohistochemical or clinicopathological studies of ISL-1 in gastric carcinoma have been performed. The aim of this study was to determine the expression and prognostic value of ISL-1 in gastric carcinoma. A nude mouse xenograft model was established to study the role of ISL-1 on cancer genesis and development in vivo. Overexpression of ISL-1 significantly enhanced the tumorigenicity of NIH3T3 cells in vivo. ISL-1 expression was evaluated using immunohistochemistry in 456 human gastric carcinoma and normal tissues. ISL-1 was significantly overexpressed in gastric adenocarcinoma compared with normal gastric tissues. ISL-1 expression was significantly associated with depth of invasion, lymph node metastasis, TNM stage, and histological grade (P b .05, χ2 test). Positive ISL-1 expression was associated with poorer 5-year overall survival in gastric cancer (P = .001, log-rank test). Multivariate Cox regression analysis demonstrated that ISL-1 expression (P = .047) could be an independent prognostic factor for overall survival in gastric carcinoma. This study suggests that ISL-1 may be a useful prognostic biomarker and may represent a novel therapeutic target for gastric adenocarcinoma. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Although its incidence has been declining for several decades, gastric cancer was the leading cause of cancer☆
Conflict of interest: The authors declare no conflict of interest. This work was supported by the National Natural Science Foundation of China, Beijing (81071675, 81170713, 8137023, 81472022), the Natural Science Foundation of Beijing (5122021), the Leading Academic Discipline Project of Beijing Education Bureau, and the 111 Project of China (B07001). ⁎ Corresponding author. E-mail addresses: [email protected] (C. Guo), [email protected] (W. Wang), [email protected] (Q. Shi), [email protected] (P. Chen), [email protected] (C. Zhou). ☆☆
http://dx.doi.org/10.1016/j.humpath.2015.05.006 0046-8177/© 2015 Elsevier Inc. All rights reserved.
related deaths worldwide until the mid-1990s. A total of 989 600 new cases and 738 000 deaths due to gastric cancer were estimated to have occurred in 2008, accounting for 8% and 10% of all cases and cancer-related deaths, respectively, with two-thirds of these cases in developing countries [1]. Gastric cancer is now relatively rare in North America and most of Northern and Western Europe; however, the incidence is higher in Eastern Europe, Russia, and selected areas of Central and South America and East Asia [2,3]. In China, gastric cancer is the most common type of cancer and the second leading cause of cancer deaths after lung cancer [4]. The overall morbidity and mortality of gastric cancer are doubled in male patients than female patients in China [1,4].
2 Disease stage is the most important factor for predicting the treatment outcome of patients [5,6]. Despite the fact that most cases of early stage gastric carcinoma can be cured by surgical resection, metastasis contributes to the high rate of mortality in gastric carcinoma [7]. Although numerous immunohistochemical markers such as human epidermal growth factor receptor 2, matrix metallopeptidase–2, and epithelium cadherin have been recognized as biomarkers in other types of cancer, these markers are not commonly used to assess biological behavior and prognosis in gastric cancer because of low levels of sensitivity and specificity [8–13]. Therefore, there is an urgent need to identify novel prognostic and predictive biomarkers to improve the diagnosis and clinical management of patients with gastric cancer, which in turn may help to develop more effective treatment strategies. Insulin enhancer binding protein–1 (ISL-1), a LIMhomeodomain transcription factor, was originally isolated as an insulin-responsive protein that binds to the islet β-cell–specific enhancer element [14]. The rat, hamster, and human ISL-1 amino acid sequences share 100% homology [15]. ISL-1 has previously been described to play crucial roles in development of the heart, motor neurons, and pancreas [16–18]. Recently, ISL-1 was implicated in the development of cancer, mainly based on the fact that expression of ISL-1 is dysregulated in a range of tumor types [19–22]. In a pilot study, we examined the expression of ISL-1 in a variety of human tumor types including breast, prostate, colon, liver, lung, and esophageal cancer using immunohistochemical staining. Markedly higher expression of ISL-1 was observed in human gastric carcinoma specimens compared with the tumor specimens from other organs [23]. However, no comprehensive immunohistochemical or clinicopathological studies of ISL-1 in gastric carcinoma have been performed. An accurate evaluation of the expression of ISL-1 in gastric cancer is required to investigate its prognostic value in this tumor type. In the present study, we investigated the expression of ISL-1 in gastric cancer tissues and normal tissues. We found that an abnormally high expression of ISL-1 was significantly associated with the depth of invasion, lymph node metastasis, TNM stage, and histological grade, as well as poorer overall survival, in patients with gastric cancer. The findings of this study indicate that ISL-1 has potential as a diagnostic and prognostic biomarker for gastric carcinoma.
2. Materials and methods 2.1. Patients and tissue microarray Human gastric cancer tissues and normal gastric tissue microarray sections with complete stage and grade information were purchased from Biomax (T012A, T014, ST1001, ST2091; Rockville, MD) and Shanghai Biochip Co Ltd (HStm-Ade180Sur-04; Shanghai, People's Republic of China). In total, ISL-1 protein expression was analyzed in
C. Guo et al. 262 gastric cancer tissues, 144 noncancerous tissues (including 9 gastric ulcers, 17 gastric inflammations, 6 hyperplasias, and 112 normal gastric tissues), and 50 gastric adenocarcinoma lymph node metastases. In addition, tissue specimens from 90 patients with gastric cancer (Shanghai Outdo Biotech Co Ltd, HStm-Ade180Sur-04) for whom complete overall survival data were available (from the date of surgery until death or August 2013) were analyzed. The clinicopathological characteristics of these patients are shown in Table 1; the patients were staged according to the sixth edition of the American Joint Committee on Cancer manual [24]. Patients were only included in the study if they had provided written consent to participate in the study after receiving oral and written information regarding its nature and purpose. Approval for the study was received from the ethics committee of the host institutions in addition to the company that provided the samples.
2.2. Animal experiments All animals were purchased from the Department of Laboratory Animal Science, Peking University; and all protocols were approved by the Animal Care and Use Committee of Peking University (LA 2010-066). The plasmid pcDNA3-ISL1 and control vector pcDNA3 were separately transfected into NIH3T3 cells to establish stable ISL-1–overexpressing cells (pcDNA3-ISL-1) and control cells (pcDNA3), as previously described [22]. Five-week-old female nude mice were subcutaneously injected on the right side of the dorsum (pcDNA3-ISL-1) and left armpit (pcDNA3) with 1 × 107 cells suspended in 200 μL phosphate-buffered saline (PBS). Tumors were measured at the indicated time points using calipers, and tumor volumes were calculated using 1/2 × length × width2. The significance of the differences between groups was determined using 2-way analysis of variance.
2.3. Immunohistochemistry Five-micrometer-thick tissue sections were cut from each tissue microarray block. All subsequent procedures were carried out at room temperature, unless otherwise stated. Tissue sections were rehydrated; and antigen retrieval was performed by submerging the slides in sodium citrate buffer (10 mmol/L sodium citrate, 0.05% Tween 20, pH 6.0) in a pressure cooker, boiling for 3 minutes, followed by cooling for 30 minutes at room temperature. Afterward, the tissue sections were incubated in 3% H2O2 in PBS-T (0.01% Tween 20 in PBS) for 10 minutes to block endogenous peroxidase activity; and a circular hydrophobic barrier was drawn around each specimen using a Pap pen (ZLI-9305; ZSGB-Bio, Beijing, China). The sections were blocked in 5% bovine serum albumin in PBS-T for 30 minutes and, incubated with mouse polyclonal anti-ISL-1 antibody (1:400; ab86472; Abcam, Hong Kong, China) in 5% bovine serum albumin in PBS-T in a humidified chamber overnight,
Abnormal expression of ISL-1 in gastric cancer
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Table 1 Demographic and clinicopathological features of the 262 patients with gastric cancer Feature Age, y ≤60 N60 Sex Male Female Histological type Adenocarcinoma Mucinous adenocarcinoma Signet ring cell adenocarcinoma Others a TNM stage I II III IV Depth of invasion T1 T2 T3 T4 Lymph node metastasis N0 N1 N2 N3 Distant metastasis M0 M1 Histological grade b G1 G2 G3 G4
n (%) 141 (53.8%) 121 (46.2%) 188 (71.8%) 74 (28.2%) 234 16 4 8
(89.3%) (6.1%) (1.5%) (3.1%)
34 115 95 18
(13.0%) (43.9%) (36.3%) (6.8%)
8 35 191 28
(3.1%) (13.4%) (72.9%) (10.6%)
136 56 38 32
(51.9%) (21.4%) (14.5%) (12.2%)
257 (98.1%) 5 (1.9%) 17 87 136 22
(6.5%) (33.2%) (51.9%) (8.4%)
a
Eight cases are classified into this category, which include squamous cell carcinoma (1), adenosquamous carcinoma (2), lowgrade interstitialoma (1), carcinoid (2), undifferentiated carcinoma (1), and papillary adenocarcinoma (1). b G1, well-differentiated adenocarcinoma; G2, moderately differentiated adenocarcinoma; G3, poorly differentiated adenocarcinoma; G4, unassessed differentiation status and undefined cases.
and the sites of immunoreactivity were detected using the Envision Detection Kit/DAB staining kit (GK500705; DAKO A/S, Glostrup, Denmark) according to the manufacturer's protocol. The slides were counterstained with hematoxylin (ZLI-9609, ZSGB-Bio) for 1 minute at room temperature and then dehydrated and sealed using crystal mounting solution (ZLI-9516, ZSGB-Bio).
2.4. Image analysis and scoring The detection of ISL-1 expression was performed in the whole lesion from mucosal to subserosal or serosal in the
same slides, and the lymph nodes (LNs) were also from the same patients. The staining scores were parallel to the patients' information. ISL-1 immunoreactivity was quantified using Image-Pro Plus software version 6 (Media Cybernetics, Silver Spring, MD). Briefly, digital images (TIFF format) close to the center of each tumor core were captured using a ×20 objective (Leica DM25000B microscope; Leica, Ernst-Leitz-Strasse, Germany). Images of all tissue cores were acquired in the same session using a constant set of microscope and imaging software parameters. The images were subjected to optical density analysis using Image-Pro Plus software. Intensity range selection was based on the image histogram, with intensity and saturation set at maximum and hue set at a range where most of the brown diaminobenzidine tetrahydrochloride hydrate color was selected, whereas the blue counterstained nuclei were excluded. These settings were saved and then subsequently applied to all of the images analyzed. On opening each image, a region of interest was drawn to include most of the malignant epithelial cells in the image and exclude stroma, muscle, necrosis, and other nontumor components. After defining the region of interest, the mean optical density of the selected area (integrated optical density [IOD]/unit area) was determined by the software. The IOD/unit area represents the immunoreactivity of the candidate protein within the tumor tissue. To facilitate statistical analysis, the IOD/unit area values were manually categorized as negative immunoreactivity (values ≤40) or positive immunoreactivity (values N40), as described in the “Results” section [25].
2.5. Statistical analysis All statistical analyses were performed using SPSS (version 20; SPSS, Chicago, IL). The association between ISL-1 immunoreactivity and the clinicopathological features of the patients was assessed using the χ2 test. The impact of ISL-1 on patient survival was examined using Kaplan-Meier survival curves; statistical significance was determined using the log-rank test. Multivariate analysis based on the Cox proportional hazards regression model was applied to identify independent prognostic factors. For all statistical tests, P values less than .05 were considered statistically significant.
3. Results 3.1. The function of ISL-1 in tumor growth in vivo We established a xenograft model in nude mice to investigate whether ISL-1 could promote the tumorigenicity of NIH-3T3 cells in vivo. Overexpression of ISL-1 (pcDNA3-ISL-1) enhanced the formation and growth of tumor xenografts in vivo. In contrast, no tumors formed after injection of the control cells (pcDNA3; Fig. 1).
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3.2. Expression of ISL-1 in gastric carcinoma and noncancerous gastric tissues The expression of ISL-1 was analyzed by immunohistochemistry in 262 primary gastric carcinoma specimens, 112 cancer-adjacent normal gastric tissues, 32 gastric disease tissues (including 9 gastric ulcers, 17 gastric inflammations, and 6 hyperplasias), and 50 gastric adenocarcinoma lymph node metastases. ISL-1 was mainly localized to the nucleus of the gastric lesions, with different staining intensities observed in different stages of gastric cancer (Fig. 2). There was a clear trend that higher levels of ISL-1 were expressed in gastric cancer compared with normal gastric mucosa. Positive expression of ISL-1 was detected in 58.4% (153/ 262) of the primary cancer tissues and 4.5% (5/112) of the
C. Guo et al. noncancer gastric tissues; the rate of ISL-1 expression in primary gastric cancer and noncancer tissues was significantly different (P = .001; χ2 test). Likewise, the rate of ISL-1 expression in metastatic gastric adenocarcinoma lymph node specimens (76.0%, 38/50) and primary cancer (58.4%, 153/262) was also significantly different (P b .01, χ2 test; data not shown).
3.3. Correlation between ISL-1 expression and the clinicopathological features of primary gastric carcinoma The clinicopathological characteristics of the 262 patients with gastric cancer analyzed in this study are summarized in Table 1. The correlations between ISL-1 expression and the clinicopathological features of the patients are summarized
Fig. 1 ISL-1 promotes tumor growth in vivo. A and B, Images of the 5 mice (A) and the excised tumors (B) on day 21. C and D, Representative hematoxylin and eosin staining of the tumors formed by control cells (C) and ISL-1–overexpressing cells (D). Control tumor cells had clear cytoplasm and pale and round nuclei; ISL-1–positive tumor cells had irregular and long spindle-shaped nuclei. E, ISL-1 expression was detected by Western blot analysis in stably transfected NIH3T3 cells. Glyceraldehyde 3-phosphate dehydrogenase served as an internal control. F, Tumor growth curves. Tumor volume (mm3) was measured using calipers and calculated using the following standard formula: (shortest diameter) 2 × (longest diameter) × 0.5 (P = .002 versus control tumors at the same time, Pearson χ2 test). Abbreviation: GADPH, glyceraldehyde 3-phosphate dehydrogenase.
Abnormal expression of ISL-1 in gastric cancer
5
Fig. 2 Representative images of immunohistochemical staining for ISL-1 in human gastric carcinoma tissues and noncancerous gastric tissues. A, Normal gastric mucosa; negative ISL-1 expression. B, Early stage gastric carcinoma (moderately differentiated adenocarcinoma); weak ISL-1 expression in nucleus and cytoplasm of gastric lesions. C, Gastric signet ring cell carcinoma; strong ISL-1 expression in nucleus of signet ring cell. D, Stage IV gastric carcinoma (moderately differentiated adenocarcinoma); strong ISL-1 expression in nucleus and cytoplasm of gastric cancer cell. Images were captured using a 40× objective lens (scale bar = 100 μm).
in Table 2. Positive expression of ISL-1 was potentially associated with moderately to poorly differentiated histological type of gastric cancer (P = .022; χ2 test); we should expand the samples to validate its clinical significance. Positive expression of ISL-1 was also significantly associated with the depth of invasion, lymph node metastasis, and TNM stage (P = .001, P = .002, and P b .001, respectively; χ2 test). However, there was no significant association between the expression of ISL-1 and age, sex, tumor location, tumor size, or distant metastasis (P N .05; t test or χ2 test).
3.4. Relationship between expression of ISL-1 and overall survival in primary gastric carcinoma Kaplan-Meier survival curves were constructed to evaluate whether expression of ISL-1 correlated with prognosis in primary gastric carcinoma. During the follow-up period, 62 (68.9%) of the 90 patients died. Only 1 patient was lost to follow-up, and this did not have any significant impact on the survival analysis. The 5-year overall survival rates for patients with positive and negative expression of ISL-1 were 62.5% and 35.3%, respectively; this difference was significantly different (P b .001, log-rank test; Fig. 2A). In addition, subgroup analysis revealed that positive expression of ISL-1 was associated with significantly poorer overall survival in both patients with stage I to II disease and patients with stage III-IV disease (P b.001 and P = .001, respectively; log-rank test; Fig. 2B and C). Therefore, ISL-1 is associated with prognosis in every stage of gastric cancer.
Furthermore, univariate analysis using a Cox proportional hazards model indicated that depth of invasion, advanced clinical stage, and high ISL-1 expression were associated with a poorer prognosis in gastric cancer. After adjustment for ISL-1 immunoreactivity and other known prognostic factors including tumor size, tumor site, depth of invasion, cancer stage, and cancer grade in multivariate Cox regression analysis, depth of invasion and ISL-1 expression remained independent prognostic factors for gastric cancer (P = .001 and P = .047, respectively; Cox regression analysis; Fig. 3 and Table 3). As shown in Table 2, the ISL-1 expression level was significantly positively correlated with the depth of invasion, which increases the reliability of ISL-1 as a prognostic factor.
4. Discussion Although early screening and diagnosis methods and treatments have been improved to a certain extent in recent years, the prognosis and clinical outcome of patients with gastric cancer remain poor. At present, immunohistochemical staining of formalin-fixed and paraffin-embedded tissues is widely used in diagnostic surgical pathology to provide information of the biological behavior of a tumor and patient prognosis [11]. However, no single marker has an adequate level of accuracy or specificity in all stages of gastric carcinoma. In addition, as the majority of gastric cancers are sporadic and it is also a multifactorial disease characterized by both genetic and environmental factors, the prognostic
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C. Guo et al. Table 2 Comparison of the clinicopathological features of patients with ISL-1–negative and ISL-1–positive gastric carcinoma Characteristic
Age, y Sex Male Female Depth of invasion T1-T2 T3-T4 Lymph node metastasis N0 N1-N3 Distant metastasis M0 M1 TNM stage I II III IV Histological grade G1 G2 G3 G4
Expression of ISL-1 Negative (%)
Positive (%) P value
(n = 109)
(n = 153)
58 ± 26
60 ± 26
.932
78 (71.6%) 31 (28.4%)
110 (71.9%) 43 (28.1%)
.953
28 (25.7%) 81 (74.3%)
15 (9.8%) 138 (90.2%)
.001
69 (63.3%) 40 (36.7%)
67 (43.8%) 86 (56.2%)
.002
106 (97.3%) 3 (2.7%)
151 (98.7%) 2 (1.3%)
.701
25 (22.9%) 48 (44.0%) 31 (28.4%) 5 (4.6%)
9 (5.9%) 67 (43.8%) 64 (41.8%) 13 (8.5%)
b.001
7 (6.4%) 35 (32.1%) 52 (47.7%) 15 (13.8%)
10 (6.5%) 52 (34.0%) 84 (54.9%) 7 (4.6%)
.022
value of many molecular markers such as vascular endothelial growth factor remains controversial [9]. Therefore, identification of novel diagnostic or prognostic markers may help to improve prognostication and the 5-year survival rate in gastric cancer.
ISL-1 was initially functionally characterized in 1990 and is a member of the LIM/homeodomain family of transcription factors that are related to the regulation of insulin gene expression [14]. Hansel et al [26] used global gene expression analysis and reported that ISL-1 was overexpressed in human biliary cancer. ISL-1 has also been found to participate in metastasis in pancreatic neuroendocrine neoplasms, pancreatic endocrine tumors, non–muscle-invasive bladder cancer, and non-Hodgkin lymphoma [19–22]. In addition, the level of ISL-1 protein expression is closely associated with the development of cancer, cancer cell growth, and metastasis, as well as the prognosis of patients with pancreatic endocrine tumors [21]. To examine the role of ISL-1 in the pathogenesis of gastric carcinoma, we created a xenograft model in nude mice to study the impact of ISL-1 on tumor growth in vivo. Overexpression of ISL-1 in NIH3T3 cells significantly promoted the establishment and growth of xenograft tumors in vivo. This indicates that ISL-1 plays a role in tumor initiation and progression. However, NIH 3T3 is a mouse fibroblast cell line; and the tumorigenicity role of ISL-1 demonstrated in this model needs to be further validated in appropriate human gastric normal cells. This is the first report to indicate that ISL-1 is overexpressed in gastric cancer. In a previous immunohistochemical study, we found that ISL-1 was significantly upregulated in gastric cancer but not in other types of gastrointestinal tumors, such as colon cancer, esophageal cancer, and pancreatic cancer, which suggests that high expression levels of ISL-1 may be a specific marker for gastric cancer [23]. Therefore, we examined the expression of ISL-1 using immunohistochemical analysis in 456 gastric lesions at different disease stages including normal mucosa, gastric diseases, primary gastric carcinoma, and distant metastases. ISL-1 protein was expressed at high levels in gastric adenocarcinoma tissues compared with the other gastric tissues. Interestingly, in normal and benign tumor tissues,
Fig. 3 Association between ISL-1 expression and overall survival in primary gastric carcinoma. Kaplan-Meier overall survival curves for all 90 patients (A; P b .001), patients with stage I to II gastric carcinoma (B; P = .001), and patients with stage III to IV gastric carcinoma (C; P b .001; log-rank test). Patients are stratified by positive and negative expression of ISL-1.
Abnormal expression of ISL-1 in gastric cancer
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Table 3 Univariate and multivariate Cox regression analysis of the association of ISL-1 expression and other clinicopathologic features with overall survival in primary gastric carcinoma Factor
Age Sex Size Site Depth of invasion Stage Grade ISL-1 expression
Univariate
Multivariate
OR
95% CI
P value
1.024 1.069 0.602 1.030 2.183 2.051 1.171 0.193
0.998-1.050 0.589-1.940 0.354-1.022 0.749-1.415 1.408-3.383 1.391-3.024 0.771-1.779 0.100-0.375
.065 .827 .060 .857 b.001 b.001 .460 b.001
OR
95% CI
P value
4.818 1.992
2.050-11.321 0.942-4.211
.001 .071
0.335
0.114-0.987
.047
Abbreviations: OR, odds ratio; CI, confidence interval.
ISL-1 staining was distributed throughout the tumor cells and mesenchymal cells, whereas ISL-1 immunoreactivity was only observed in the nuclei of advanced-stage gastric adenocarcinoma, indicating its transcriptional activation [27]. The growth and development of tumors are a complex, continuous process. The regulatory mechanisms are also diversified. It has been reported that caudal-related homeobox transcription factor can inhibit invasion and migration, and promote differentiation and the multidrug resistance of gastric cancer cells [28–30]. Ectopic expression of caudalrelated homeobox transcription factor is speculated to be involved in gastric cancer development. Overexpression of ISL-1 in MGC803, a human gastric cancer cell line, could promote the cell proliferation and cell migration (unpublished data). Although the mechanism of ISL-1 in gastric cancer still remains unclear, we believe that it may have multimechanisms to promote gastric cancer development. It should be pointed out that ISL-1 expression is involved in human gastric cancer invasion, TNM stage, and lymph node metastasis; but because the sample size is too small (only 5 cases), we cannot make a conclusion for its correlation with distant metastasis. Notably, the markers commonly used to detect gastric cancer in the clinic are also used for other digestive system cancers. However, the present study indicates that ISL-1 is overexpressed in gastric cancer but not in other gastrointestinal tumors. These results imply that ISL-1 may be a sensitive and specific biomarker for gastric cancer and that ISL-1 may have a competitive advantage as a novel therapeutic target for gastric adenocarcinoma. Next, we investigated the relationship between ISL-1 expression and the prognosis and clinical features of patients with gastric cancer. The overall survival time for ISL-1– positive patients was significantly shorter than that of ISL-1– negative patients. In particular, multivariate analysis confirmed that ISL-1 was an independent prognostic factor for poor overall survival in gastric cancer. No similar results exist for other types of gastrointestinal cancer. In recent years, the presence of tumor budding (TB) at the tumor-host interface of gastrointestinal cancers has been recognized as a hallmark of unfavorable disease biology. Tumor budding is defined as the presence of dedifferentiated cells or
small clusters of up to 5 cells at the tumor invasive front [31]. Although the Union for International Cancer Control has officially recognized TB as an additional independent prognostic factor in colon and rectum cancers [32], the evidence for prognostic impact of TB in gastric adenocarcinoma is still insufficient. We found that highly expressed ISL-1 promoted metastasis of MGC803 (a gastric cancer cell line) cells (data not shown), which is consistent with the observation that the high-grade TB could predict the first step toward metastasis and early disease recurrence in solid tumor [33]. However, whether the high level of ISL-1 expression has a possible connection with an increased frequency of TB in patients at the tumor invasive front remains to be elucidated in gastric cancer. In conclusion, this study demonstrates that ISL-1 is expressed at high levels in gastric carcinoma compared with the adjacent noncancerous tissues and that high-level expression of ISL-1 was significantly associated with advanced TNM stage. Moreover, positive expression of ISL-1 was associated with significantly poorer overall survival in gastric cancer. Furthermore, both univariate and multivariate Cox proportional hazards regression revealed that high ISL-1 expression was an independent prognostic factor for poorer overall survival in gastric cancer. Based on these results, we suggest that ISL-1 represents a potentially useful prognostic marker in gastric cancer. The clinical value of ISL-1 as a biomarker and prognostic factor and the mechanisms by which ISL-1 regulates the progression of gastric carcinoma require further evaluation.
Acknowledgments We thank Dr Fei Pei, Department of Pathology, Peking University, for her kind help in the image analysis and scoring of tissue microarray.
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