Author Affiliations: Department of General Surgery (Zhou L, Li J, Zhao YP, Guo JC, Zhang TP, Wu WM, You L and Shu H) and Department of Pathology (Cui QC and Zhou WX), Peking Union Medical College Hospital, Chinese Academy of Medical Sciences/Peking Union Medical College, Beijing 100730, China

Corresponding Author: Yu-Pei Zhao, MD, PhD, Department of General Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences/Peking Union Medical College, Beijing 100730, China (Tel: +86-10-69156007; Fax: +86-10-65124875; Email: zhao8028@263.net)

 

© 2014, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(14)60272-1

Published online June 23, 2014.

 

Contributors: ZL proposed the study and wrote the first draft. ZL and LJ performed the experiments. CQC and ZWX evaluated staining results. ZL, LJ, ZYP and GJC analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. ZL and LJ contributed equally to this work. ZYP is the guarantor.

Funding: This study was supported by a grant from the Research Special Fund for Public Welfare Industry of Health (201202007).

Ethical approval: This study has been approved by the Institutional Ethics Committee.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

BACKGROUND: Recent studies have shown the clinical significance of epidermal growth factor-like domain 7 (EGFL7) in a variety of cancers. However, the relationship between EGFL7 and the prognosis of pancreatic cancer (PC) remains unclear. The present study was undertaken to investigate the role of EGFL7 in the prognosis of PC.

 

METHODS: The expression of EGFL7 in nine PC cell lines was first determined by Western blotting analysis. Tissue microarray-based immunohistochemical staining was performed in paired formalin-fixed paraffin-embedded tumor and non-tumor samples from 83 patients with PC. Finally, correlations between EGFL7 expression and clinicopathological variables as well as overall survival were evaluated.

 

RESULTS: EGFL7 was widely expressed in all PC cell lines tested. EGFL7 expression in tumor tissues was significantly higher than that in non-tumor tissues (P=0.040). In addition, univariate analysis revealed that high EGFL7 expression in tumor tissues was significantly associated with poor overall survival, accompanied by several conventional clinicopathological variables, such as gender, histological grade and lymph node metastasis. In a multivariate Cox regression test, EGFL7 expression was identified as an independent marker for long-term outcome of PC.

 

CONCLUSION: Our data showed that EGFL7 is extensively expressed in PC and that EGFL7 is associated with poor prognosis.

 

(Hepatobiliary Pancreat Dis Int 2014;13:523-528)

 

KEY WORDS: epidermal growth factor-like domain 7; pancreatic cancer; prognosis; Western blotting; immunohistochemistry; tissue microarray

Pancreatic cancer (PC), has long been recognized as a lethal malignancy with extremely poor prognosis.^[1,2] In addition, the long-term survival of PC has not been improved within two decades, despite widely-performed surgical resections.^[3] Therefore, factors predictive for poor prognosis of the disease are of interest. Among these factors, conventional clinical and pathological parameters such as lymph node metastasis, neural/perineural invasion and resection margin were previously identified as significant variables.^[4-8] Recently, much attention has been paid to many tumor initiation/development-associated molecules that were associated with PC prognosis.^{[9, 10]} However, further clues and evidences are required.

 

Epidermal growth factor-like domain 7 (EGFL7), a secreted protein containing two EGF-like domains, was initially identified as a modulator of smooth muscle cell migration.^[11] The biological roles of EGFL7 in the vascular system have been extensively investigated.^[12-15] In human cancer cells, EGFL7 accelerates migration through the focal adhesion kinase (FAK)-associated pathway,^[16] and promotes tumor progression by reducing the expression of endothelial molecules that mediate immune cell infiltration.^[17] A study^[18] on human tissues revealed that EGFL7 is overexpressed in ten human epithelial tumor types, including hepatocellular carcinoma, lung cancer, breast cancer, prostate cancer, colorectal cancer, gastric cancer, esophageal cancer, malignant glioma, ovarian cancer and renal cancer. In addition, correlations between EGFL7 expression and unfavorable biological behaviors as well as poor prognosis were also suggested in many malignancies.^{[16, 18-21]} However, controversy still remains because EGFL7 expression was associated with better prognosis and with the absence of lymph node invasion in human breast cancer.^[22] So far, there is no study on the expression and prognostic role of EGFL7 in PC.

 

The present study was to explore the expression of EGFL7 in PC, its association with clinicopathological variables, and the role of EGFL7 in the prognosis of PC.

 

 

Nine human PC cell lines (AsPC-1, BxPC-3, Capan-1, Colo357, MIAPaCa-2, PANC-1, Su86.86, SW1990 and T3M4), gifts from Professor Helmut Friess, Heidelberg University, Germany, were cultured in Dulbecco's modified Eagle's medium (DMEM) or RPMI-1640 medium (Hyclone, Thermo Fisher Scientific Inc., Waltham, MA, USA), supplemented with 10% fetal bovine serum (FBS, Hyclone), as previously reported.^[23]

 

Western blotting detection of EGFL7 in PC cell lines was performed according to the method of Liu et al,^[24] using a rabbit anti-human EGFL7 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

 

Eighty-three patients with PC after radical surgical resection (R0) were recruited, 53 males and 30 females, with a mean age of 61.8±10.6 years (mean±SD). The clinicopathological features of the patients are shown in Table 1. Standard lymphadenectomy (including resection of nodes of No. 12, 13, 17, 8 and 14 groups) was performed. Post-surgical gemcitabine-based adjuvant chemotherapy/chemoradiotherapy was recommended for all patients. The study was approved by the Institutional Ethics Committee.

 

A tissue microarray was constructed using formalin-fixed paraffin-embedded blocks of PC. Diagnosis was proven by hematoxylin-eosin staining-based routine pathologic examination. After careful review and screening of representative tumor and non-tumor regions, two cores of corresponding tissues for each patient were sampled from typical areas using a 1.5-mm punch. The TMA was constructed with a manual tissue arrayer (Beecher Instruments, Sun Prairie, WI, USA).

 

TMA-based immunohistochemical staining for EGFL7 was performed in tissues from 83 patients with PC. A rabbit anti-human EGFL7 polyclonal antibody (Santa Cruz) and a two-step staining kit (EnVision+kit, Dako, Glostrup, Denmark) were used. The tissues were cross-sectioned in 4 µm thickness and mounted, deparaffinized and rehydrated. An autoclave was used to retrieve antigen. Slides were subsequently incubated with 3% hydrogen peroxide to block the endogenous peroxidase. Then, the slides were incubated with the primary antibody (dilution: 1:20) overnight at 4 ��. Following washing in PBS, the slides were incubated with horseradish peroxidase (HRP)-labeled secondary antibody, and after washing, the slides were stained with diaminobenzidine and then counterstained with hematoxylin. Non-immune rabbit serum at the same dilution was applied as the negative control.

 

Two pathologists with no prior information of clinicopathological and survival data (CQC and ZWX) performed staining evaluation, reaching a consensus after discussion when there was a controversy. The brown color located in the cytoplasm was defined as the positive signal. The initial scoring was given using previously introduced criteria.^[20] Then, a final decision for EGFL7 expression was made according to the standard (low: scores 0-4; high: scores 5-12).

 

Post-operative follow-up, with a median term of 15 months (range 2-87), was given to all patients. Among the patients, 54 died and 29 were alive at the time of follow-up.

 

EGFL7 staining between tumor and non-tumor tissues was compared using the Mann-Whitney U test. The Chi-square test was used to show the association between EGFL7 expression and clinicopathological variables. Overall survival was calculated by the Kaplan-Meier method and analyzed by the log-rank test. Cox regression (proportional hazard model) was used for multivariate analysis of prognostic factors. Statistical software package SPSS11.5 (SPSS Inc., Chicago, IL, USA) was employed for all the analyses. A P value of less than 0.05 was defined as statistical significance.

 

 

Western blotting analysis showed that EGFL7 was widely expressed in all PC cell lines tested (Fig. 1). The expression was relatively higher in SW1990 and T3M4 and lower in PANC-1 cells.

 

Positive EGFL7 staining was present in both tumor and non-tumor tissues (Fig. 2A, 2B, respectively). The Mann-Whitney U test showed that the staining score in tumor tissues was statistically higher than that in non-tumor samples (Fig. 2C, P=0.040). No significant association between tumoral EGFL7 expression and estimated clinicopathological variables was observed (Table 1, P>0.05).

 

Univariate analysis showed that patients with high EGFL7 expression in tumor tissues carried poor overall survival (Fig. 3, Table 2, P=0.032). In addition, other conventional clinical and pathological parameters including gender, histological grade and lymph node metastasis were also predictive for prognosis (Table 2, P<0.05). In a multivariate Cox regression test, EGFL7 expression in tumor tissues was identified as an independent prognostic marker (Table 2, P=0.001), in addition to the aforementioned significant univariate variables (Table 2, P<0.05).

 

 

It was shown that EGFL7 affects smooth muscle cell migration- and vessel-associated phenotypes, such as vascular tubulogenesis, elastogenesis, endothelial integrity and cell death.^[11-15] In 2009, Wu et al^[16] found that EGFL7 accelerated migration through the EGFL7-EGFR-FAK axis in hepatocellular carcinoma cells, thus providing the first evidence of the effect of EGFL7 on cancer cell behavior. An observation focusing on EGFL7 and immune cells, based on experiments in breast and lung carcinoma cells, indicated that the protein contributes to tumor escape from immunity.^[17] Therefore, EGFL7 might play important roles in cancer development. Recently, EGFL7 was found to be overexpressed in ten human epithelial tumor tissues.^[18] In addition, EGFL7 expression was also associated with unfavorable clinicopathological features and poorer prognosis in the majority of malignant tumors tested,^{[16, 18-21]} although the adverse clue could also be seen.^[22] Thus, EGFL7 might be a cancer promoter. However, the expression of EGFL7 in PC remains unknown. In the present study, EGFL7 was widely expressed in all of the 9 PC cell lines, suggesting its possible role in PC. Furthermore, determination of the baseline level of EGFL7 expression lays a foundation for further investigations. Immunohistochemical staining showed that the expression of EGFL7 in PC tissues was significantly higher than that in non-tumor samples. The data were consistent with those in most cancers,^{[16, 18-21]} and suggested that EGFL7 might be involved in pancreatic carcinogenesis. However, unlike previous reports,^{[16, 18-21]} no positive associations existed between EGFL7 and clinicopathological variables in patients with PC. Our findings were consistent with those of studies on other proteins such as cyclin D1 Survivin and HUGL-1, all of which were associated with the survival in different cancers but none of them was correlated with clinical variables.^[25-27] The variations in sample size and patient characteristics as summarized by Knutsen et al^[26] might account, at least in part, for the observed phenomenon. Future prospective studies with more specimens might be necessary.

 

Nevertheless, EGFL7 was previously found to be associated with unfavorable outcome in some cancers.^[16,18,21] However, its prognostic significance in PC has not been clarified. Univariate analysis in our study showed that EGFL7 expression in tumor tissues was predictive for poor survival. Besides EGFL7, we also found the conventional clinicopathological variables, such as gender, differentiation and N status, were predictors of survival in patients with PC. Our results were consistent with other studies.^[4-6,28,29] More importantly, multivariate Cox regression test concluded that high tumor expression of EGFL7 was an independent prognostic factor of PC. This finding strengthened the potential of the protein as a novel biomarker of poor prognosis of PC, and might add a new promising candidate to the lists published.^{[9, 10]} Previously, some proteins that are mainly related to metastatic potential such as CD44v6, c-Met and EpCAM were found to have prognostic value in PC.^[30-35] In the future, combined estimation of EGFL7 and other clinicopathological or the aforementioned molecular factors, especially those associated with invasion/metastasis, could provide stronger predictive power and might be of interest to promote further investigations.

 

In conclusion, our findings suggest that EGFL7 is extensively expressed in PC and that EGFL7 may have prognostic implications in this malignancy.

 

 

1 Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, 2010. CA Cancer J Clin 2010;60:277-300. PMID: 20610543

2 Hidalgo M. Pancreatic cancer. N Engl J Med 2010;362:1605- 1617. PMID: 20427809

3 Witkowski ER, Smith JK, Tseng JF. Outcomes following resection of pancreatic cancer. J Surg Oncol 2013;107:97-103. PMID: 22991309

4 La Torre M, Cavallini M, Ramacciato G, Cosenza G, Rossi Del Monte S, Nigri G, et al. Role of the lymph node ratio in pancreatic ductal adenocarcinoma. Impact on patient stratification and prognosis. J Surg Oncol 2011;104:629-633. PMID: 21713779

5 Robinson SM, Rahman A, Haugk B, French JJ, Manas DM, Jaques BC, et al. Metastatic lymph node ratio as an important prognostic factor in pancreatic ductal adenocarcinoma. Eur J Surg Oncol 2012;38:333-339. PMID: 22317758

6 Golse N, Lebeau R, Lombard-Bohas C, Hervieu V, Ponchon T, Adham M. Lymph node involvement beyond peripancreatic region in pancreatic head cancers: when results belie expectations. Pancreas 2013;42:239-248. PMID: 23038054

7 Chatterjee D, Katz MH, Rashid A, Wang H, Iuga AC, Varadhachary GR, et al. Perineural and intraneural invasion in posttherapy pancreaticoduodenectomy specimens predicts poor prognosis in patients with pancreatic ductal adenocarcinoma. Am J Surg Pathol 2012;36:409-417. PMID: 22301497

8 Zhang Y, Frampton AE, Cohen P, Kyriakides C, Bong JJ, Habib NA, et al. Tumor infiltration in the medial resection margin predicts survival after pancreaticoduodenectomy for pancreatic ductal adenocarcinoma. J Gastrointest Surg 2012;16:1875-1882. PMID: 22878786

9 Ansari D, Rosendahl A, Elebro J, Andersson R. Systematic review of immunohistochemical biomarkers to identify prognostic subgroups of patients with pancreatic cancer. Br J Surg 2011;98:1041-1055. PMID: 21644238

10 Winter JM, Yeo CJ, Brody JR. Diagnostic, prognostic, and predictive biomarkers in pancreatic cancer. J Surg Oncol 2013;107:15-22. PMID: 22729569

11 Soncin F, Mattot V, Lionneton F, Spruyt N, Lepretre F, Begue A, et al. VE-statin, an endothelial repressor of smooth muscle cell migration. EMBO J 2003;22:5700-5711. PMID: 14592969

12 Parker LH, Schmidt M, Jin SW, Gray AM, Beis D, Pham T, et al. The endothelial-cell-derived secreted factor Egfl7 regulates vascular tube formation. Nature 2004;428:754-758. PMID: 15085134

13 Campagnolo L, Leahy A, Chitnis S, Koschnick S, Fitch MJ, Fallon JT, et al. EGFL7 is a chemoattractant for endothelial cells and is up-regulated in angiogenesis and arterial injury. Am J Pathol 2005;167:275-284. PMID: 15972971

14 Xu D, Perez RE, Ekekezie II, Navarro A, Truog WE. Epidermal growth factor-like domain 7 protects endothelial cells from hyperoxia-induced cell death. Am J Physiol Lung Cell Mol Physiol 2008;294:L17-23. PMID: 17934064

15 Lelièvre E, Hinek A, Lupu F, Buquet C, Soncin F, Mattot V. VE-statin/egfl7 regulates vascular elastogenesis by interacting with lysyl oxidases. EMBO J 2008;27:1658-1670. PMID: 18497746

16 Wu F, Yang LY, Li YF, Ou DP, Chen DP, Fan C. Novel role for epidermal growth factor-like domain 7 in metastasis of human hepatocellular carcinoma. Hepatology 2009;50:1839-1850. PMID: 19824075

17 Delfortrie S, Pinte S, Mattot V, Samson C, Villain G, Caetano B, et al. Egfl7 promotes tumor escape from immunity by repressing endothelial cell activation. Cancer Res 2011;71:7176-7186. PMID: 22037871

18 Fan C, Yang LY, Wu F, Tao YM, Liu LS, Zhang JF, et al. The expression of Egfl7 in human normal tissues and epithelial tumors. Int J Biol Markers 2013;28:71-83. PMID: 23558933

19 Díaz R, Silva J, García JM, Lorenzo Y, García V, Peña C, et al. Deregulated expression of miR-106a predicts survival in human colon cancer patients. Genes Chromosomes Cancer 2008;47:794-802. PMID: 18521848

20 Huang CH, Li XJ, Zhou YZ, Luo Y, Li C, Yuan XR. Expression and clinical significance of EGFL7 in malignant glioma. J Cancer Res Clin Oncol 2010;136:1737-1743. PMID: 20213100

21 Li JJ, Yang XM, Wang SH, Tang QL. Prognostic role of epidermal growth factor-like domain 7 protein expression in laryngeal squamous cell carcinoma. J Laryngol Otol 2011;125:1152-1157. PMID: 21899785

22 Philippin-Lauridant G, Baranzelli MC, Samson C, Fournier C, Pinte S, Mattot V, et al. Expression of Egfl7 correlates with low-grade invasive lesions in human breast cancer. Int J Oncol 2013;42:1367-1375. PMID: 23404186

23 Chen WY, Liu WJ, Zhao YP, Zhou L, Zhang TP, Chen G, et al. Induction, modulation and potential targets of miR-210 in pancreatic cancer cells. Hepatobiliary Pancreat Dis Int 2012;11:319-324. PMID: 22672828

24 Liu WJ, Zhao YP, Zhang TP, Zhou L, Cui QC, Zhou WX, et al. MLH1 as a direct target of MiR-155 and a potential predictor of favorable prognosis in pancreatic cancer. J Gastrointest Surg 2013;17:1399-1405. PMID: 23715647

25 Hui AM, Li X, Shi YZ, Takayama T, Torzilli G, Makuuchi M. Cyclin D1 overexpression is a critical event in gallbladder carcinogenesis and independently predicts decreased survival for patients with gallbladder carcinoma. Clin Cancer Res 2000;6:4272-4277. PMID: 11106243

26 Knutsen A, Adell G, Sun XF. Survivin expression is an independent prognostic factor in rectal cancer patients with and without preoperative radiotherapy. Int J Radiat Oncol Biol Phys 2004;60:149-155. PMID: 15337550

27 Biesterfeld S, Kauhausen A, Kost C, Gockel I, Schimanski CC, Galle PR. Preservation of HUGL-1 expression as a favourable prognostic factor in pancreatic carcinoma. Anticancer Res 2012;32:3153-3159. PMID: 22843887

28 David M, Lepage C, Jouve JL, Jooste V, Chauvenet M, Faivre J, et al. Management and prognosis of pancreatic cancer over a 30-year period. Br J Cancer 2009;101:215-218. PMID: 19568238

29 Hartwig W, Hackert T, Hinz U, Gluth A, Bergmann F, Strobel O, et al. Pancreatic cancer surgery in the new millennium: better prediction of outcome. Ann Surg 2011;254:311-319. PMID: 21606835

30 Gansauge F, Gansauge S, Rau B, Scheiblich A, Poch B, Schoenberg MH, et al. Low serum levels of soluble CD44 variant 6 are significantly associated with poor prognosis in patients with pancreatic carcinoma. Cancer 1997;80:1733-1739. PMID: 9351541

31 Zhou G, Chiu D, Qin D, Niu L, Cai J, He L, et al. Expression of CD44v6 and integrin-β1 for the prognosis evaluation of pancreatic cancer patients after cryosurgery. Diagn Pathol 2013;8:146. PMID: 24004467

32 Zhu GH, Huang C, Qiu ZJ, Liu J, Zhang ZH, Zhao N, et al. Expression and prognostic significance of CD151, c-Met, and integrin alpha3/alpha6 in pancreatic ductal adenocarcinoma. Dig Dis Sci 2011;56:1090-1098. PMID: 20927591

33 Park JK, Kim MA, Ryu JK, Yoon YB, Kim SW, Han HS, et al. Postoperative prognostic predictors of pancreatic ductal adenocarcinoma: clinical analysis and immunoprofile on tissue microarrays. Ann Surg Oncol 2012;19:2664-2672. PMID: 22395988

34 Fong D, Steurer M, Obrist P, Barbieri V, Margreiter R, Amberger A, et al. Ep-CAM expression in pancreatic and ampullary carcinomas: frequency and prognostic relevance. J Clin Pathol 2008;61:31-35. PMID: 16775119

35 Akita H, Nagano H, Takeda Y, Eguchi H, Wada H, Kobayashi S, et al. Ep-CAM is a significant prognostic factor in pancreatic cancer patients by suppressing cell activity. Oncogene 2011;30:3468-3476. PMID: 21399662