Archives of Medical Research 45 (2014) 394e399
ORIGINAL ARTICLE
Reduced Kr€uppel-like Factor 17 (KLF17) Expression Correlates with Poor Survival in Patients with Gastric Cancer Jing-jing Peng,a Biao Wu,b Xin-bo Xiao,b Yong-sheng Shao,b Yan Feng,c and Meng-xin Yinc a
Department of Gastroenterology, General Hospital of the Yangtze River Shipping, Wuhan, Hubei Province, China b Department of Gastrointestinal Surgery, cDepartment of Pathology, The First Hospital of Wu Han City, Wuhan, Hubei Province, China Received for publication February 16, 2014; accepted June 6, 2014 (ARCMED-D-14-00102).
Background and Aims. To investigate the expression and prognostic significance of Kr€ uppel-like factor 17 (KLF17) in human gastric cancer. Methods. KLF17 expressions in 158 paraffin-embedded gastric cancer samples were analyzed using immunohistochemistry. In addition, KLF17 expressions patterns in three fresh gastric cancer tissues and noncancerous gastric mucosa were examined by Western blotting. The correlation between KLF17 expression and clinicopathological factors as well as patient survival was investigated. Results. Immunohistochemical staining data indicated that KLF17 expression was significantly decreased in 98 of 158 gastric adenocarcinoma cases. Reduced KLF17 expression in fresh gastric cancer tissues was confirmed by Western blotting. Reduced expression of KLF17 was strongly correlated with tumor size, pN stage and lymphovascular invasion. Multivariate Cox regression analysis identified KLF17 expression as an independent prognostic factor for both overall survival (HR 5 0.481, 95% CI 5 0.225e0.665, p 5 0.009) and disease-free survival (HR 5 0.438, 95% CI 5 0.254e0.758, p 5 0.003). Conclusion. The reduced expression of KLF17 protein in gastric cancer was correlated with tumor size, pN stage and lymphovascular invasion and was an independent predictor for poor survival in patients undergoing surgery for gastric cancer. Ó 2014 IMSS. Published by Elsevier Inc. Key Words: KLF17, Stomach neoplasms, Prognosis, Immunohistochemistry, Western blotting.
Introduction Gastric cancer is currently the second most common cause of death from cancer worldwide (1). Despite the introduction of multimodal treatments, the survival of gastric cancer patients remains poor due to recurrence or metastasis. The American Joint Committee on Cancer TNM staging system for gastric cancer is considered valid for prognostic stratification, but different survival probabilities have been observed even in the same stage (2). Therefore, identification of specific biomarkers for prognostic evaluation is important in the management of gastric cancer patients.
Address reprint requests to: Xin-bo Xiao, MD, Department of Gastrointestinal Surgery, The First Hospital of Wu Han City, No. 215 Zhongshan Avenue, Wuhan City, Hubei Provence 430022, PR China; Phone and FAX: þ86 02785332697; E-mail: [email protected]
Kr€uppel-like factor 17 (KLF17) is a new member of the Kr€uppel-like factor family of transcription factors and encoded by human KLF17 gene, which is located on chromosome 1p34.1 (3,4). A previous study indicates that KLF17 expression was decreased in invasive breast cancer cell lines and in breast cancer tissues with lymph node metastasis (5). KLF17, as a new metastasis suppressor gene, acts through regulation of the epithelial-tomesenchymal transition (5e8). Recent studies found that reduced expression of KLF17 protein was related to poor survival in human lung adenocarcinoma and hepatocellular carcinoma (9,10). However, little information is available regarding the expression and clinical/prognostic significance of KLF17 protein in human gastric adenocarcinoma. In the current study we investigated the expression of KLF17 protein in human gastric adenocarcinoma by
0188-4409/$ - see front matter. Copyright Ó 2014 IMSS. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.arcmed.2014.06.005
KLF17 Expression in Gastric Cancer
immunohistochemistry and Western blotting and evaluated its clinical/prognostic significance.
395
Table 1. Relationship between KLF17 expression and clinicopathologic characteristics of 158 gastric cancer patients KLF17 expression Variables
Materials and Methods Patients and Tissue Samples Paraffin-embedded tumor and matched adjacent non-tumor tissues were obtained from 158 patients who underwent curative (R0) resection and D2 lymphadenectomy for primary gastric adenocarcinoma at the First Hospital of Wu Han City between January 2002 and June 2007. Three pairs of fresh cancer tissues and noncancerous gastric mucosa tissues were obtained from patients who underwent curative (R0) resection and D2 lymphadenectomy for poorly differentiated adenocarcinoma of the stomach at the First Hospital of Wu Han City in 2012. The cases were selected consecutively based on the following criteria: (1) primary gastric adenocarcinoma confirmed by postoperative histopathology, (2) availability of both of resection tissue and follow-up data, (3) no neoadjuvant chemotherapy or radiotherapy, and (4) no other previous or synchronous malignancy. A pathologist reassessed all the primary hematoxyline eosin (HE)-stained slides to confirm the previous diagnosis, to select representative cancer specimens, and to reevaluate the presence of lymphovascular invasion. Lymphovascular invasion was defined as the presence of tumor cell emboli within spaces surrounded by a clearly visualized endothelial lining in the periphery of the tumor on slides (11). Tumor location was classified according to the Japanese classification of gastric carcinoma, 3rd English edition (12), and tumor-node-metastasis (TNM) classification was reclassified according to the 7th edition cancer staging manual for carcinoma of the stomach (13). Histotypes were divided into intestinal and diffuse-mixed types according to Lauren’s classification (14). Clinicopathological characteristics of the patients are described in Table 1. The patients in the current study included 47 women and 111 men with a mean age of 61 years (range 27e81 years). The median follow-up time for all the patients was 60 months at the time of analysis and ranged from 3e118 months. Patients were followed up from the date of surgery to emigration, death, or June 2012, whichever came first. Survival data were collected prospectively based on both the outpatients’ records and telephone follow-up. Overall survival (OS) was defined as the time from the surgery to the patient’s death or the last follow-up. Disease-free survival (DFS) was defined as the period from the completion of curative gastrectomy to first recurrence of gastric cancer or death from any cause. The study was approved by the Ethics Committee of the First Hospital of Wu Han City, Hubei Province, China, and written informed consent was obtained from all patients.
Gender Female Male Age at diagnosis (years) #65 O65 Tumor size (cm) #5.0 O5.0 Tumor location Upper or middle third Low third Lauren histotype Intestinal Diffuse-mixed lymphovascular invasion Present Absent pT stage T1 T2 T3 T4 pN stage N0 N1 N2 N3
No. of cases
Negative
Positive
47 111
25 73
22 38
94 64
56 42
38 22
111 47
62 36
49 11
57 101
38 60
19 41
69 89
39 59
30 30
42 116
32 66
10 50
18 29 14 97
8 15 10 65
10 14 4 32
47 38 39 34
24 20 26 28
23 18 13 6
p 0.137
0.442
0.014a
0.366
0.209
0.027a
0.157
0.017a
KLF17, Kr€uppel-like factor 17. Statistically significant ( p !0.05).
a
Immunohistochemistry (IHC) Staining and Assessment IHC staining was performed on 5-mm sections from paraffin-embedded specimens. Monoclonal rabbit antihuman EIF5A2 antibody (Epitomics, Burlingame, CA) and PV-6000 Polymer Detection System (ZSBG-BIO, China) were used for staining in accordance with the manufacturer’s instructions. In brief, slides with paraffin sections were deparaffinized with xylene and rehydrated with ethanol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 min. For antigen retrieval, slides were microwave-treated and boiled in a 0.01 M citrate buffer (pH 6.0) for 10 min at 37 C. The slides were incubated with polyclonal rabbit anti-human KLF17 antibody (1:100 dilution; Abcam, Hong Kong) for 1.5 h at 37 C in a humidified chamber. As a negative staining control, primary antibody was replaced with non-specific rabbit IgG. Two independent pathologists who were blinded to the clinicopathological information evaluated the immunohistochemical staining of KLF17. A semiquantitative scoring method was used with reference to the previous study in which both the proportion of cells stained and staining intensity was assessed (9). The total KLF17 immunostaining
396
Peng et al./ Archives of Medical Research 45 (2014) 394e399
Figure 1. Immunohistochemical staining of KLF17 expression in normal gastric epithelia and gastric cancer tissues. (A) KLF17-positive staining was observed in non-cancerous gastric mucosa epithelial cells. (B) KLF17-positive staining was observed in non-cancerous gastric glands epithelial cells. (C) KL17-positive expression in moderately differentiated adenocarcinoma. (D) KLF17-negative expression in poorly differentiated adenocarcinoma (magnification 200).
values were calculated with the value of the intensity (no staining 5 0, weak staining 5 1, moderate staining 5 2, or strong staining 5 3) the value of percentage of immunostaining-positive cells (!25% 5 1, 25e50% 5 2, O50e!75% 5 3, 75e100% 5 4 scores). The total immunostaining values ranged from 0e12. Western Blotting Total proteins were extracted from frozen gastric cancer and normal tissues using T-PER Tissue Protein Extraction Reagent (Thermo #78510) and quantified using Thermo Pierce BCA Protein Assay kit according to the manufacturer’s instructions. Proteins were separated by 12% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA) using standard protocols. The membranes were blocked in 5% fat-free milk solution for 2.5 h at room temperature followed by incubation with a rabbit anti-human KLF17 polyclonal antibody (1:1000; Abcam) or GAPDH antibody (1:500; Santa Cruz Biotechnology, Santa Cruz, CA) at 4 C overnight. After washing with TBST buffer, blots were incubated with a goat anti-rabbit IgG HRP-conjugated secondary antibody (1:5000; Santa Cruz Biotechnology) for 1 h. The bound antibodies were detected using enhanced chemiluminescence and Alpha Ease FC Imaging System. Statistical Analysis All analyses were performed using SPSS 12.0 (SPSS, Chicago, IL). Pearson’s c2 tests or Fisher’s exact tests were
used to compare differences between categorical variables. The difference of KLF17 expression between tumor and matched adjacent non-tumor tissues was compared by McNemar test. Survival curves were calculated using the KaplaneMeier method and compared by the log-rank test. The Cox proportional hazards model was used to investigate the independent prognostic factors. All p values were two-sided; p values !0.05 were considered statistically significant.
Results Downregulation of KLF17 Expression in Primary Gastric Cancer Tissues Compared with Matched Adjacent Nontumor Tissues Positive signals of KLF17 were predominantly located in the cytoplasm of cells. The immunostaining values of KLF17 in 87.34% (138) matched adjacent non-tumor tissues were $4; therefore, we defined immunostaining values of 4e12 as positive KLF17 expression, whereas immunostaining values of 0e3 were defined as negative expression of KLF17. Positive expression of KLF17 protein was detected in 60/158 (37.97%) tumor specimens compared with 138/158 (87.34%) matched adjacent non-tumor mucosal tissues ( p !0.0001, McNemar test). Representative IHC staining of KLF17 in gastric adenocarcinoma and adjacent nontumor tissues is shown in Figure 1. We further validated the KLF17 protein expression in three pairs of fresh gastric
KLF17 Expression in Gastric Cancer
397
KLF17 expression ( p 5 0.009), AJCC pN stage ( p 5 0.001), lymphovascular invasion ( p 5 0.017), and tumor location (p 5 0.003) (Table 2).
Figure 2. KLF17 is downregulated in fresh gastric adenocarcinoma tissues. Western blotting analysis of KLF17 expression in gastric adenocarcinoma tissues and paired distant non-tumor tissues from the same patients.
Uni- and Multivariate Analysis of Factors Associated with DFS of Patients Receiving Curative Resection for Gastric Cancer
Pearson’s c2 tests indicated that KLF17 protein expression was inversely correlated with tumor size, lymphovascular invasion and pN stage (Table 1). No correlation was found between the decreased expression of KLF17 protein and gender, age, tumor location, Lauren histotype and pT stage (Table 1).
KaplaneMeier survival curves showed that the 5-year DFS for the negative KLF17 expression group (n 5 98) was shorter than that for the positive KLF17 expression group (n 5 60) (39.7 vs. 73.2%, log rank p !0.001; Figure 3B). Univariate Cox regression analyses demonstrated tumor size ( p 5 0.002), tumor location ( p !0.001), lymphovascular invasion ( p !0.001), AJCC pT stage ( p 5 0.001), AJCC pN stage ( p !0.001) and KLF17 expression ( p ! 0.001) as being significantly associated with DFS (Table 2). Variables with significance in univariate analysis were included in the multivariate Cox regression analyses, which identified KLF17 expression ( p 5 0.003), AJCC pN stage ( p 5 0.001), lymphovascular invasion ( p 5 0.025), and tumor location (p 5 0.015) as independent factors influencing DFS (Table 3).
Uni- and Multivariate Analysis of Factors Associated with Overall Survival of Patients after Curative Surgery
Discussion
KaplaneMeier survival curves showed that the 5-year OS for the negative KLF17 expression group (n 5 98) was shorter than for the positive KLF17 expression group (n 5 60) (42.5% vs. 73.2%, log rank p !0.001; Figure 3A). Univariate Cox regression analyses identified tumor size ( p !0.001), tumor location ( p !0.001), lymphovascular invasion ( p !0.001), AJCC pT stage ( p 5 0.001), AJCC pN stage ( p !0.001) and KLF17 expression ( p !0.001) as being significantly associated with OS (Table 2). Variables with significance in univariate analysis were included in multivariate Cox regression analyses, which identified the following as independent factors influencing OS:
Human KLF17 was identified as a new member of the Sp/ KLF family of transcription factors (4). Recently, researchers found that downregulated KLF17 expression was involved in human cancer cell invasion or growth and associated with poor patients’ survival (5,8e10). However, the expression and clinical/prognostic significance of KLF17 in human gastric adenocarcinoma still remains unknown. The current study demonstrated that KLF17 expression was reduced in human gastric cancer tissues compared with normal tissues, consistent with previous studies in other human cancers (9,10). Our results showed that KLF17 protein expression was inversely correlated with tumor size,
adenocarcinoma and distant nontumor tissues by Western blotting (Figure 2). Association of KLF17 Immunohistochemical Staining with Patients’ Clinicopathological Features
Figure 3. Kaplan-Meier survival curves of gastric cancer patients after curative surgery. Patients with KLF17-negative gastric cancer showed both significantly poor overall survival (A) and disease-free survival (B) than those with KLF17-positive gastric cancer (log-rank test, both p !0.001).
398
Peng et al./ Archives of Medical Research 45 (2014) 394e399
Table 2. Uni- and multivariate Cox regression analyses of factors associated with overall survival of patients with gastric cancer Univariate analysis Variables Gender Male vs. female Age (years) O65 vs. #65 Lauren histotype Diffuse-mixed vs. intestinal Tumor size (cm) O5.0 vs. #5.0 Tumor location Upper or middle vs. low Lymphovascular invasion Present vs. absent pT stage pT3-4 vs. pT1-2 pN stage N1-3 vs. N0 KLF17 expression Positive vs. negative
HR (95% CI)
Multivariate analysis p
HR (95% CI)
p
1.172 (0.702e1.956)
0.544
1.227 (0.776e1.939)
0.381
1.488 (0.931e2.378)
0.096
2.295 (1.449e3.634)
!0.001
2.752 (1.744e4.341)
!0.001
2.039 (1.267e3.282)
0.003
3.480 (2.193e5.524)
!0.001
1.836 (1.116e3.021)
0.017
2.746 (1.507e5.003)
0.001 !0.001
3.489 (1.623e7.500)
0.001
0.001
0.481 (0.278e0.833)
0.009
5.000 (2.397e10.428) 0.387 (0.225e0.665)
HR, hazard ratio; CI, confidence interval; KLF17, Kr€uppel-like factor 17.
lymphovascular invasion, and pN stage. These results implied that KLF17 may play an important role in the progression of human gastric cancer. Previous studies showed that KLF17 also was inversely correlated with lymph node metastasis in other human cancer such as breast cancer, hepatocellular carcinoma and lung cancer (5,9,10). KLF17, as a tumor suppressor gene, suppressed tumor invasion and metastasis by inhibiting epithelial-to-mesenchymal transition (5,8). Furthermore, the current study investigated the prognostic significance of KLF17 protein in patients with gastric cancer.
Cox regression analyses showed that KLF17 was an independent factor influencing survival, consistent with previous studies performed in other human cancers (9,10). To the best of our knowledge, the current study is the first report on the prognostic value of KLF17 protein for human gastric cancer. These results supported the hypothesis that KLF17 may be a novel prognostic biomarker for human gastric cancer patients after curative surgery. We also found that lymphovascular invasion, pN stage and tumor location were independent predictors for both OS and DFS, confirming the findings of previous studies
Table 3. Uni- and multivariate Cox regression analyses of factors associated with DFS of patients with gastric cancer Univariate analysis Variables Gender Male vs. female Age (years) O65 vs. #65 Lauren histotype Diffuse-mixed vs. intestinal Tumor size (cm) O5.0 vs. #5.0 Tumor location Upper or middle vs. low Lymphovascular invasion Present vs. absent pT stage pT3-4 vs. pT1-2 pN stage N1-3 vs. N0 KLF17 expression Positive vs. negative
HR (95% CI)
Multivariate analysis p
HR (95% CI)
p
1.107 (0.677e1.811)
0.686
1.174 (0.750e1.863)
0.482
1.428 (0.906e2.251)
0.125
2.057 (1.309e3.231)
0.002
2.481 (1.592e3.867)
!0.001
1.787 (1.120e2.851)
0.015
3.407 (2.164e5.364)
!0.001
1.753 (1.072e2.865)
0.025
2.661 (1.491e4.749)
0.001
5.319 (2.555e11.073)
!0.001
3.759 (1.773e8.121)
0.001
0.352 (0.206e603)
!0.001
0.438 (0.254e0.758)
0.003
DFS, disease-free survival; HR, hazard ratio; CI, confidence interval; KLF17, Kr€uppel-like factor 17.
KLF17 Expression in Gastric Cancer
(15e18). However, the incidence of lymphovascular invasion was lower than that of previous studies (16,17). One of the most plausible reasons for the difference was that neither special staining such as Elastica staining, Victoria blue staining nor immunohistochemical staining was used for evaluating the lymphovascular invasion in this study. On the basis of our findings, we conclude that reduced KLF17 expression in gastric cancer is correlated with tumor size, lymph node metastasis and lymphovascular invasion and may be an independent biomarker for predicting poor survival, but further studies are required to validate our results and to investigate the possible role and mechanisms of KLF17 in gastric cancer.
Conflict of Interest None declared.
References 1. Jemal A, Bray F, Center MM, et al. Global cancer statistics. CA Cancer J Clin 2011;61:69e90. 2. Sun Z, Wang ZN, Zhu Z, et al. Evaluation of the seventh edition of American Joint Committee on Cancer TNM staging system for gastric cancer: results from a Chinese monoinstitutional study. Ann Surg Oncol 2012;19:1918e1927. 3. McConnell BB, Ang VW. Mammalian Kruppel-like factors in health and diseases. Physiol Rev 2010;90:1337e1381. 4. van Vliet J, Crofts LA, Quinlan KG, et al. Human KLF17 is a new member of the Sp/KLF family of transcription factors. Genomics 2006;87:474e482. 5. Gumireddy K, Li A, Gimotty PA, et al. KLF17 is a negative regulator of epithelial-mesenchymal transition and metastasis in breast cancer. Nat Cell Biol 2009;11:1297e1304.
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6. Iwanicki MP, Brugge JS. Transcriptional regulation of metastatic (Id) entity by KLF17. Genome Biol 2009;10:244. 7. Ismail IA, Kang HS, Lee HJ, et al. 2-Hydroxycinnamaldehyde inhibits the epithelial-mesenchymal transition in breast cancer cells. Breast Cancer Res Treat 2013;137:697e708. 8. Sun Z, Han Q, Zhou N, et al. MicroRNA-9 enhances migration and invasion through KLF17 in hepatocellular carcinoma. Mol Oncol 2013;7:884e894. 9. Cai XD, Zhou YB, Huang LX, et al. Reduced expression of Kruppellike factor 17 is related to tumor growth and poor prognosis in lung adenocarcinoma. Biochem Biophys Res Commun 2012;418:67e73. 10. Liu FY, Deng YL, Li Y, et al. Down-regulated KLF17 expression is associated with tumor invasion and poor prognosis in hepatocellular carcinoma. Med Oncol 2013;30:425. 11. Hyung WJ, Lee JH, Choi SH, et al. Prognostic impact of lymphatic and/or blood vessel invasion in patients with node-negative advanced gastric cancer. Ann Surg Oncol 2002;9:562e567. 12. Japanese Gastric Cancer Association. Japanese classification of gastric carcinoma: 3rd English ed. Gastric Cancer 2011;14:101e112. 13. Washington K. 7th ed. of the AJCC Cancer Staging Manual: Stomach. Ann Surg Oncol 2010;17:3077e3079. 14. Lauren P. The two histological main types of gastric carcinoma: diffuse and so-called intestinal-type carcinoma. An attempt at a histo-clinical classification. Acta Pathol Microbiol Scand 1965;64: 31e49. 15. Dicken BJ, Graham K, Hamilton SM, et al. Lymphovascular invasion is associated with poor survival in gastric cancer: an application of gene-expression and tissue array techniques. Ann Surg 2006;243: 64e73. 16. del Casar JM, Corte MD, Alvarez A, et al. Lymphatic and/or blood vessel invasion in gastric cancer: relationship with clinicopathological parameters, biological factors and prognostic significance. J Cancer Res Clin Oncol 2008;134:153e161. 17. Kim JH, Park SS, Park SH, et al. Clinical significance of immunohistochemically-identified lymphatic and/or blood vessel tumor invasion in gastric cancer. J Surg Res 2010;162:177e183. 18. Selcukbiricik F, Buyukunal E, Tural D, et al. Clinicopathological features and outcomes of patients with gastric cancer: a single-center experience. World J Gastroenterol 2013;19:2154e2161.
ORIGINAL ARTICLE
Reduced Kr€uppel-like Factor 17 (KLF17) Expression Correlates with Poor Survival in Patients with Gastric Cancer Jing-jing Peng,a Biao Wu,b Xin-bo Xiao,b Yong-sheng Shao,b Yan Feng,c and Meng-xin Yinc a
Department of Gastroenterology, General Hospital of the Yangtze River Shipping, Wuhan, Hubei Province, China b Department of Gastrointestinal Surgery, cDepartment of Pathology, The First Hospital of Wu Han City, Wuhan, Hubei Province, China Received for publication February 16, 2014; accepted June 6, 2014 (ARCMED-D-14-00102).
Background and Aims. To investigate the expression and prognostic significance of Kr€ uppel-like factor 17 (KLF17) in human gastric cancer. Methods. KLF17 expressions in 158 paraffin-embedded gastric cancer samples were analyzed using immunohistochemistry. In addition, KLF17 expressions patterns in three fresh gastric cancer tissues and noncancerous gastric mucosa were examined by Western blotting. The correlation between KLF17 expression and clinicopathological factors as well as patient survival was investigated. Results. Immunohistochemical staining data indicated that KLF17 expression was significantly decreased in 98 of 158 gastric adenocarcinoma cases. Reduced KLF17 expression in fresh gastric cancer tissues was confirmed by Western blotting. Reduced expression of KLF17 was strongly correlated with tumor size, pN stage and lymphovascular invasion. Multivariate Cox regression analysis identified KLF17 expression as an independent prognostic factor for both overall survival (HR 5 0.481, 95% CI 5 0.225e0.665, p 5 0.009) and disease-free survival (HR 5 0.438, 95% CI 5 0.254e0.758, p 5 0.003). Conclusion. The reduced expression of KLF17 protein in gastric cancer was correlated with tumor size, pN stage and lymphovascular invasion and was an independent predictor for poor survival in patients undergoing surgery for gastric cancer. Ó 2014 IMSS. Published by Elsevier Inc. Key Words: KLF17, Stomach neoplasms, Prognosis, Immunohistochemistry, Western blotting.
Introduction Gastric cancer is currently the second most common cause of death from cancer worldwide (1). Despite the introduction of multimodal treatments, the survival of gastric cancer patients remains poor due to recurrence or metastasis. The American Joint Committee on Cancer TNM staging system for gastric cancer is considered valid for prognostic stratification, but different survival probabilities have been observed even in the same stage (2). Therefore, identification of specific biomarkers for prognostic evaluation is important in the management of gastric cancer patients.
Address reprint requests to: Xin-bo Xiao, MD, Department of Gastrointestinal Surgery, The First Hospital of Wu Han City, No. 215 Zhongshan Avenue, Wuhan City, Hubei Provence 430022, PR China; Phone and FAX: þ86 02785332697; E-mail: [email protected]
Kr€uppel-like factor 17 (KLF17) is a new member of the Kr€uppel-like factor family of transcription factors and encoded by human KLF17 gene, which is located on chromosome 1p34.1 (3,4). A previous study indicates that KLF17 expression was decreased in invasive breast cancer cell lines and in breast cancer tissues with lymph node metastasis (5). KLF17, as a new metastasis suppressor gene, acts through regulation of the epithelial-tomesenchymal transition (5e8). Recent studies found that reduced expression of KLF17 protein was related to poor survival in human lung adenocarcinoma and hepatocellular carcinoma (9,10). However, little information is available regarding the expression and clinical/prognostic significance of KLF17 protein in human gastric adenocarcinoma. In the current study we investigated the expression of KLF17 protein in human gastric adenocarcinoma by
0188-4409/$ - see front matter. Copyright Ó 2014 IMSS. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.arcmed.2014.06.005
KLF17 Expression in Gastric Cancer
immunohistochemistry and Western blotting and evaluated its clinical/prognostic significance.
395
Table 1. Relationship between KLF17 expression and clinicopathologic characteristics of 158 gastric cancer patients KLF17 expression Variables
Materials and Methods Patients and Tissue Samples Paraffin-embedded tumor and matched adjacent non-tumor tissues were obtained from 158 patients who underwent curative (R0) resection and D2 lymphadenectomy for primary gastric adenocarcinoma at the First Hospital of Wu Han City between January 2002 and June 2007. Three pairs of fresh cancer tissues and noncancerous gastric mucosa tissues were obtained from patients who underwent curative (R0) resection and D2 lymphadenectomy for poorly differentiated adenocarcinoma of the stomach at the First Hospital of Wu Han City in 2012. The cases were selected consecutively based on the following criteria: (1) primary gastric adenocarcinoma confirmed by postoperative histopathology, (2) availability of both of resection tissue and follow-up data, (3) no neoadjuvant chemotherapy or radiotherapy, and (4) no other previous or synchronous malignancy. A pathologist reassessed all the primary hematoxyline eosin (HE)-stained slides to confirm the previous diagnosis, to select representative cancer specimens, and to reevaluate the presence of lymphovascular invasion. Lymphovascular invasion was defined as the presence of tumor cell emboli within spaces surrounded by a clearly visualized endothelial lining in the periphery of the tumor on slides (11). Tumor location was classified according to the Japanese classification of gastric carcinoma, 3rd English edition (12), and tumor-node-metastasis (TNM) classification was reclassified according to the 7th edition cancer staging manual for carcinoma of the stomach (13). Histotypes were divided into intestinal and diffuse-mixed types according to Lauren’s classification (14). Clinicopathological characteristics of the patients are described in Table 1. The patients in the current study included 47 women and 111 men with a mean age of 61 years (range 27e81 years). The median follow-up time for all the patients was 60 months at the time of analysis and ranged from 3e118 months. Patients were followed up from the date of surgery to emigration, death, or June 2012, whichever came first. Survival data were collected prospectively based on both the outpatients’ records and telephone follow-up. Overall survival (OS) was defined as the time from the surgery to the patient’s death or the last follow-up. Disease-free survival (DFS) was defined as the period from the completion of curative gastrectomy to first recurrence of gastric cancer or death from any cause. The study was approved by the Ethics Committee of the First Hospital of Wu Han City, Hubei Province, China, and written informed consent was obtained from all patients.
Gender Female Male Age at diagnosis (years) #65 O65 Tumor size (cm) #5.0 O5.0 Tumor location Upper or middle third Low third Lauren histotype Intestinal Diffuse-mixed lymphovascular invasion Present Absent pT stage T1 T2 T3 T4 pN stage N0 N1 N2 N3
No. of cases
Negative
Positive
47 111
25 73
22 38
94 64
56 42
38 22
111 47
62 36
49 11
57 101
38 60
19 41
69 89
39 59
30 30
42 116
32 66
10 50
18 29 14 97
8 15 10 65
10 14 4 32
47 38 39 34
24 20 26 28
23 18 13 6
p 0.137
0.442
0.014a
0.366
0.209
0.027a
0.157
0.017a
KLF17, Kr€uppel-like factor 17. Statistically significant ( p !0.05).
a
Immunohistochemistry (IHC) Staining and Assessment IHC staining was performed on 5-mm sections from paraffin-embedded specimens. Monoclonal rabbit antihuman EIF5A2 antibody (Epitomics, Burlingame, CA) and PV-6000 Polymer Detection System (ZSBG-BIO, China) were used for staining in accordance with the manufacturer’s instructions. In brief, slides with paraffin sections were deparaffinized with xylene and rehydrated with ethanol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 min. For antigen retrieval, slides were microwave-treated and boiled in a 0.01 M citrate buffer (pH 6.0) for 10 min at 37 C. The slides were incubated with polyclonal rabbit anti-human KLF17 antibody (1:100 dilution; Abcam, Hong Kong) for 1.5 h at 37 C in a humidified chamber. As a negative staining control, primary antibody was replaced with non-specific rabbit IgG. Two independent pathologists who were blinded to the clinicopathological information evaluated the immunohistochemical staining of KLF17. A semiquantitative scoring method was used with reference to the previous study in which both the proportion of cells stained and staining intensity was assessed (9). The total KLF17 immunostaining
396
Peng et al./ Archives of Medical Research 45 (2014) 394e399
Figure 1. Immunohistochemical staining of KLF17 expression in normal gastric epithelia and gastric cancer tissues. (A) KLF17-positive staining was observed in non-cancerous gastric mucosa epithelial cells. (B) KLF17-positive staining was observed in non-cancerous gastric glands epithelial cells. (C) KL17-positive expression in moderately differentiated adenocarcinoma. (D) KLF17-negative expression in poorly differentiated adenocarcinoma (magnification 200).
values were calculated with the value of the intensity (no staining 5 0, weak staining 5 1, moderate staining 5 2, or strong staining 5 3) the value of percentage of immunostaining-positive cells (!25% 5 1, 25e50% 5 2, O50e!75% 5 3, 75e100% 5 4 scores). The total immunostaining values ranged from 0e12. Western Blotting Total proteins were extracted from frozen gastric cancer and normal tissues using T-PER Tissue Protein Extraction Reagent (Thermo #78510) and quantified using Thermo Pierce BCA Protein Assay kit according to the manufacturer’s instructions. Proteins were separated by 12% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA) using standard protocols. The membranes were blocked in 5% fat-free milk solution for 2.5 h at room temperature followed by incubation with a rabbit anti-human KLF17 polyclonal antibody (1:1000; Abcam) or GAPDH antibody (1:500; Santa Cruz Biotechnology, Santa Cruz, CA) at 4 C overnight. After washing with TBST buffer, blots were incubated with a goat anti-rabbit IgG HRP-conjugated secondary antibody (1:5000; Santa Cruz Biotechnology) for 1 h. The bound antibodies were detected using enhanced chemiluminescence and Alpha Ease FC Imaging System. Statistical Analysis All analyses were performed using SPSS 12.0 (SPSS, Chicago, IL). Pearson’s c2 tests or Fisher’s exact tests were
used to compare differences between categorical variables. The difference of KLF17 expression between tumor and matched adjacent non-tumor tissues was compared by McNemar test. Survival curves were calculated using the KaplaneMeier method and compared by the log-rank test. The Cox proportional hazards model was used to investigate the independent prognostic factors. All p values were two-sided; p values !0.05 were considered statistically significant.
Results Downregulation of KLF17 Expression in Primary Gastric Cancer Tissues Compared with Matched Adjacent Nontumor Tissues Positive signals of KLF17 were predominantly located in the cytoplasm of cells. The immunostaining values of KLF17 in 87.34% (138) matched adjacent non-tumor tissues were $4; therefore, we defined immunostaining values of 4e12 as positive KLF17 expression, whereas immunostaining values of 0e3 were defined as negative expression of KLF17. Positive expression of KLF17 protein was detected in 60/158 (37.97%) tumor specimens compared with 138/158 (87.34%) matched adjacent non-tumor mucosal tissues ( p !0.0001, McNemar test). Representative IHC staining of KLF17 in gastric adenocarcinoma and adjacent nontumor tissues is shown in Figure 1. We further validated the KLF17 protein expression in three pairs of fresh gastric
KLF17 Expression in Gastric Cancer
397
KLF17 expression ( p 5 0.009), AJCC pN stage ( p 5 0.001), lymphovascular invasion ( p 5 0.017), and tumor location (p 5 0.003) (Table 2).
Figure 2. KLF17 is downregulated in fresh gastric adenocarcinoma tissues. Western blotting analysis of KLF17 expression in gastric adenocarcinoma tissues and paired distant non-tumor tissues from the same patients.
Uni- and Multivariate Analysis of Factors Associated with DFS of Patients Receiving Curative Resection for Gastric Cancer
Pearson’s c2 tests indicated that KLF17 protein expression was inversely correlated with tumor size, lymphovascular invasion and pN stage (Table 1). No correlation was found between the decreased expression of KLF17 protein and gender, age, tumor location, Lauren histotype and pT stage (Table 1).
KaplaneMeier survival curves showed that the 5-year DFS for the negative KLF17 expression group (n 5 98) was shorter than that for the positive KLF17 expression group (n 5 60) (39.7 vs. 73.2%, log rank p !0.001; Figure 3B). Univariate Cox regression analyses demonstrated tumor size ( p 5 0.002), tumor location ( p !0.001), lymphovascular invasion ( p !0.001), AJCC pT stage ( p 5 0.001), AJCC pN stage ( p !0.001) and KLF17 expression ( p ! 0.001) as being significantly associated with DFS (Table 2). Variables with significance in univariate analysis were included in the multivariate Cox regression analyses, which identified KLF17 expression ( p 5 0.003), AJCC pN stage ( p 5 0.001), lymphovascular invasion ( p 5 0.025), and tumor location (p 5 0.015) as independent factors influencing DFS (Table 3).
Uni- and Multivariate Analysis of Factors Associated with Overall Survival of Patients after Curative Surgery
Discussion
KaplaneMeier survival curves showed that the 5-year OS for the negative KLF17 expression group (n 5 98) was shorter than for the positive KLF17 expression group (n 5 60) (42.5% vs. 73.2%, log rank p !0.001; Figure 3A). Univariate Cox regression analyses identified tumor size ( p !0.001), tumor location ( p !0.001), lymphovascular invasion ( p !0.001), AJCC pT stage ( p 5 0.001), AJCC pN stage ( p !0.001) and KLF17 expression ( p !0.001) as being significantly associated with OS (Table 2). Variables with significance in univariate analysis were included in multivariate Cox regression analyses, which identified the following as independent factors influencing OS:
Human KLF17 was identified as a new member of the Sp/ KLF family of transcription factors (4). Recently, researchers found that downregulated KLF17 expression was involved in human cancer cell invasion or growth and associated with poor patients’ survival (5,8e10). However, the expression and clinical/prognostic significance of KLF17 in human gastric adenocarcinoma still remains unknown. The current study demonstrated that KLF17 expression was reduced in human gastric cancer tissues compared with normal tissues, consistent with previous studies in other human cancers (9,10). Our results showed that KLF17 protein expression was inversely correlated with tumor size,
adenocarcinoma and distant nontumor tissues by Western blotting (Figure 2). Association of KLF17 Immunohistochemical Staining with Patients’ Clinicopathological Features
Figure 3. Kaplan-Meier survival curves of gastric cancer patients after curative surgery. Patients with KLF17-negative gastric cancer showed both significantly poor overall survival (A) and disease-free survival (B) than those with KLF17-positive gastric cancer (log-rank test, both p !0.001).
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Peng et al./ Archives of Medical Research 45 (2014) 394e399
Table 2. Uni- and multivariate Cox regression analyses of factors associated with overall survival of patients with gastric cancer Univariate analysis Variables Gender Male vs. female Age (years) O65 vs. #65 Lauren histotype Diffuse-mixed vs. intestinal Tumor size (cm) O5.0 vs. #5.0 Tumor location Upper or middle vs. low Lymphovascular invasion Present vs. absent pT stage pT3-4 vs. pT1-2 pN stage N1-3 vs. N0 KLF17 expression Positive vs. negative
HR (95% CI)
Multivariate analysis p
HR (95% CI)
p
1.172 (0.702e1.956)
0.544
1.227 (0.776e1.939)
0.381
1.488 (0.931e2.378)
0.096
2.295 (1.449e3.634)
!0.001
2.752 (1.744e4.341)
!0.001
2.039 (1.267e3.282)
0.003
3.480 (2.193e5.524)
!0.001
1.836 (1.116e3.021)
0.017
2.746 (1.507e5.003)
0.001 !0.001
3.489 (1.623e7.500)
0.001
0.001
0.481 (0.278e0.833)
0.009
5.000 (2.397e10.428) 0.387 (0.225e0.665)
HR, hazard ratio; CI, confidence interval; KLF17, Kr€uppel-like factor 17.
lymphovascular invasion, and pN stage. These results implied that KLF17 may play an important role in the progression of human gastric cancer. Previous studies showed that KLF17 also was inversely correlated with lymph node metastasis in other human cancer such as breast cancer, hepatocellular carcinoma and lung cancer (5,9,10). KLF17, as a tumor suppressor gene, suppressed tumor invasion and metastasis by inhibiting epithelial-to-mesenchymal transition (5,8). Furthermore, the current study investigated the prognostic significance of KLF17 protein in patients with gastric cancer.
Cox regression analyses showed that KLF17 was an independent factor influencing survival, consistent with previous studies performed in other human cancers (9,10). To the best of our knowledge, the current study is the first report on the prognostic value of KLF17 protein for human gastric cancer. These results supported the hypothesis that KLF17 may be a novel prognostic biomarker for human gastric cancer patients after curative surgery. We also found that lymphovascular invasion, pN stage and tumor location were independent predictors for both OS and DFS, confirming the findings of previous studies
Table 3. Uni- and multivariate Cox regression analyses of factors associated with DFS of patients with gastric cancer Univariate analysis Variables Gender Male vs. female Age (years) O65 vs. #65 Lauren histotype Diffuse-mixed vs. intestinal Tumor size (cm) O5.0 vs. #5.0 Tumor location Upper or middle vs. low Lymphovascular invasion Present vs. absent pT stage pT3-4 vs. pT1-2 pN stage N1-3 vs. N0 KLF17 expression Positive vs. negative
HR (95% CI)
Multivariate analysis p
HR (95% CI)
p
1.107 (0.677e1.811)
0.686
1.174 (0.750e1.863)
0.482
1.428 (0.906e2.251)
0.125
2.057 (1.309e3.231)
0.002
2.481 (1.592e3.867)
!0.001
1.787 (1.120e2.851)
0.015
3.407 (2.164e5.364)
!0.001
1.753 (1.072e2.865)
0.025
2.661 (1.491e4.749)
0.001
5.319 (2.555e11.073)
!0.001
3.759 (1.773e8.121)
0.001
0.352 (0.206e603)
!0.001
0.438 (0.254e0.758)
0.003
DFS, disease-free survival; HR, hazard ratio; CI, confidence interval; KLF17, Kr€uppel-like factor 17.
KLF17 Expression in Gastric Cancer
(15e18). However, the incidence of lymphovascular invasion was lower than that of previous studies (16,17). One of the most plausible reasons for the difference was that neither special staining such as Elastica staining, Victoria blue staining nor immunohistochemical staining was used for evaluating the lymphovascular invasion in this study. On the basis of our findings, we conclude that reduced KLF17 expression in gastric cancer is correlated with tumor size, lymph node metastasis and lymphovascular invasion and may be an independent biomarker for predicting poor survival, but further studies are required to validate our results and to investigate the possible role and mechanisms of KLF17 in gastric cancer.
Conflict of Interest None declared.
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