Available online at www.sciencedirect.com
ScienceDirect EJSO xx (2014) 1e7
www.ejso.com
VEGF and Id-1 in pancreatic adenocarcinoma: Prognostic significance and impact on angiogenesis D. Georgiadou a,*, T.N. Sergentanis b, S. Sakellariou c, G.M. Filippakis d, F. Zagouri d, D. Vlachodimitropoulos e, T. Psaltopoulou b, A.C. Lazaris c, E. Patsouris c, G.C. Zografos d b
a 3rd Surgical Clinic of George Gennimatas General Hospital, Mesogeion Ave 154, 156 69 Athens, Greece Department of Hygiene, Epidemiology and Medical Statistics, School of Medicine, University of Athens, M. Asias 75, Goudi, 11527 Athens, Greece c 1st Department of Pathology, National and Kapodistrian University of Athens, Mikras Asias 75, 11527 Athens, Greece d 1st Department of Propaedeutic Surgery, Hippokratio Hospital, Vassilissis Sophias Avenue 114, 115 27 Athens, Greece e Department of Forensic Pathology and Toxicology, Medical School, University of Athens, Mikras Asias 75, 11527 Athens, Greece
Accepted 4 January 2014 Available online - - -
Abstract Objectives: The significance of vascular endothelial growth factor (VEGF) and inhibitor of differentiation/DNA synthesis (Id-1) in tumor neoangiogenesis and tumor progression in pancreatic ductal adenocarcinoma (PDAC) is still unclear. Given the central role of VEGF in cancer angiogenesis and the inconclusive results on Id-1 expression in PDAC, it is of great interest to investigate whether Id-1 and VEGF expression are associated with angiogenesis and prognosis in PDAC. Methods: Paraffin-embedded specimens from 60 consecutive patients with PDAC were immunostained for VEGF, Id-1 and CD34 and staining quantification was assessed by Image analysis system. The correlations among the expression of individual angiogenic factors and microvessel density (MVD), clinicopathologic features and clinical prognosis were analyzed. Results: Id-1 and VEGF Positive Activity Indices (PAIs) closely correlated with each other. MVD positively correlated with both Id-1 and VEGF expression. More advanced T and N status correlated with more intense expression of Id-1, VEGF and higher MVD. With regard to prognostic significance higher Id-1 PAI (adjusted HR ¼ 1.69, 95%CI: 1.10e2.59, p ¼ 0.017), higher VEGF PAI (adjusted HR ¼ 2.66, 95% CI: 1.09e6.50, p ¼ 0.032), and MVD (adjusted HR ¼ 1.55, 95%CI: 1.27e1.88, p < 0.001) were associated with poorer survival. Conclusions: VEGF and Id-1 overexpression were found to be associated with high MVD and emerged as adverse prognostic factors in terms of patient survival in PDAC. The potential of selective anti-angiogenic targeting therapy for pancreatic malignancies should prompt further validation of the present findings in studies encompassing larger samples and more elaborate techniques. Ó 2014 Elsevier Ltd. All rights reserved. Keywords: VEGF; Id-1; MVD; Pancreatic adenocarcinoma; Image analysis
Introduction Pancreatic ductal adenocarcinoma (PDAC) is the fifth leading cause of cancer-related death in Western countries and remains a challenge to clinicians. The anatomical * Corresponding author. Tel.: þ30 697 643 5861; fax: þ30 210 770 6915, þ30 210 776 8634. E-mail addresses: [email protected], [email protected] (D. Georgiadou), [email protected] (T.N. Sergentanis), [email protected] (S. Sakellariou), [email protected] (G.M. Filippakis), florazagouri@ yahoo.co.uk (F. Zagouri), [email protected] (D. Vlachodimitropoulos), [email protected] (T. Psaltopoulou), alazaris@ med.uoa.gr (A.C. Lazaris), [email protected] (E. Patsouris), [email protected] (G.C. Zografos).
complexity and late diagnosis have led to a disappointingly low resectability rate of around 10e20%, even in specialized pancreatic disease centers. PDAC’s propensity toward aggressive growth, early metastasis and resistance to cytotoxic agents and radiation results in an overall 5-year survival rate of less than 5%.1 New therapeutic approaches based on the biologic characteristics of this disease may improve response rates and survival. One promising approach is the inhibition of angiogenesis. Like other solid neoplasms, PDAC depends on the process of angiogenesis, namely the formation of tumor blood vessels, for both local and metastatic growth. In general, the induction of neoangiogenesis is the result of an imbalance between pro- and antiangiogenic molecules.
0748-7983/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejso.2014.01.004 Please cite this article in press as: Georgiadou D, et al., VEGF and Id-1 in pancreatic adenocarcinoma: Prognostic significance and impact on angiogenesis, Eur J Surg Oncol (2014), http://dx.doi.org/10.1016/j.ejso.2014.01.004
2
D. Georgiadou et al. / EJSO xx (2014) 1e7
Among the identified proangiogenic regulators, vascular endothelial growth factor (VEGF) has been identified as key mediator of the regulation of pathologic blood vessel growth and maintenance,2 inducing also endothelial cell proliferation and enhancing vascular permeability.2 Blocking the effects of VEGF on endothelial cells by receptor antagonists3 or neutralizing anti-VEGF antibodies4 inhibits the spreading of a variety of neoplasms. However, the significance of VEGF in tumor neoangiogenesis and progression in PADC is still unclear. The inconsistency in the available literature about the prognostic value of VEGF in PDAC5e8 and recent evidence suggesting only transient effects of anti-VEGF strategies,9 indicate that further investigation into the mechanistic basis of VEGF is needed in order to elucidate its therapeutic role in targeting tumor angiogenesis. Id-1 (inhibitor of differentiation/DNA synthesis) protein belongs to the Id family of helix-loop-helix (HLH) proteins.10 Id 1 has been shown to play a pivotal role in cell proliferation, differentiation and senescence.10 Overexpression of Id-1, in either transcriptional or translational levels, has been reported in over 20 types of human cancers including breast, prostate, cervical, colorectal, liver and pancreatic cancers10 correlating with highly aggressive breast, prostate and ovarian cancer.11 Loss of Id-1 has been shown to lead to downregulation of several proangiogenic genes in tumor endothelial cells12; in parallel, a positive role of Id-1 in tumor metastasis through promoting angiogenesis was suggested.11 According to experimental studies,13 Id-1induced VEGF expression may be a critical molecular event prerequisite in promoting angiogenesis; however, the exact regulatory mechanisms are still unclear. Given the scarce research on Id-1 expression in PDAC, it seems of great interest to elucidate whether overexpression of Id-1 promotes PDAC angiogenesis assessing also any positive correlation with VEGF expression. Microvessel density (MVD) is an established method for quantitating angiogenesis in tumors.14 The assessment of tumor MVD has been reported as a prognostic indicator in several human malignancies. Specifically, studies have shown that higher tumor vascularity correlates with worse patient outcomes in breast, prostate and ovarian cancers.15 A number of endothelial surface markers (CD34, CD31 and factor VIII) have been used to examine MVD in PDAC. CD34 was the marker more frequently associated with survival in this malignancy through multivariate analysis.7,16 To date, its prognostic value remains controversial, as other studies have not demonstrated any relationship with survival.6,8 In this study, we report the results of automated digital assessment of VEGF, Id-1 as well as MVD in PDAC. As the prognostic value of those markers in PDAC remains inconclusive, the aim of the present survey is to determine whether their expression is related to clinicopathological prognostic factors and patient survival.
Materials and methods Patient and tissue samples After approval from the ethics board, 60 consecutive cases of surgically removed pancreatic adenocarcinoma between December 1999 and July 2004 were obtained from the Division of Pathology at “Eugenidio” General Hospital (Athens, Greece). Whipple procedure (n ¼ 26), whipple pylorus preserving procedure (n ¼ 18) and distal pancreatectomy (n ¼ 16) for adenocarcinoma of the head or body-tail of the pancreas were performed by a single surgeon. Any other pathology of the pancreas, treated during the same time period, as well as palliative bypass operations, was excluded from the study. All but four patients included in the study had a preoperative evaluation that was in concordance with intraoperative findings. Those four patients (two with tumor in the head and two in the body-tail) exhibited one single liver metastasis
ScienceDirect EJSO xx (2014) 1e7
www.ejso.com
VEGF and Id-1 in pancreatic adenocarcinoma: Prognostic significance and impact on angiogenesis D. Georgiadou a,*, T.N. Sergentanis b, S. Sakellariou c, G.M. Filippakis d, F. Zagouri d, D. Vlachodimitropoulos e, T. Psaltopoulou b, A.C. Lazaris c, E. Patsouris c, G.C. Zografos d b
a 3rd Surgical Clinic of George Gennimatas General Hospital, Mesogeion Ave 154, 156 69 Athens, Greece Department of Hygiene, Epidemiology and Medical Statistics, School of Medicine, University of Athens, M. Asias 75, Goudi, 11527 Athens, Greece c 1st Department of Pathology, National and Kapodistrian University of Athens, Mikras Asias 75, 11527 Athens, Greece d 1st Department of Propaedeutic Surgery, Hippokratio Hospital, Vassilissis Sophias Avenue 114, 115 27 Athens, Greece e Department of Forensic Pathology and Toxicology, Medical School, University of Athens, Mikras Asias 75, 11527 Athens, Greece
Accepted 4 January 2014 Available online - - -
Abstract Objectives: The significance of vascular endothelial growth factor (VEGF) and inhibitor of differentiation/DNA synthesis (Id-1) in tumor neoangiogenesis and tumor progression in pancreatic ductal adenocarcinoma (PDAC) is still unclear. Given the central role of VEGF in cancer angiogenesis and the inconclusive results on Id-1 expression in PDAC, it is of great interest to investigate whether Id-1 and VEGF expression are associated with angiogenesis and prognosis in PDAC. Methods: Paraffin-embedded specimens from 60 consecutive patients with PDAC were immunostained for VEGF, Id-1 and CD34 and staining quantification was assessed by Image analysis system. The correlations among the expression of individual angiogenic factors and microvessel density (MVD), clinicopathologic features and clinical prognosis were analyzed. Results: Id-1 and VEGF Positive Activity Indices (PAIs) closely correlated with each other. MVD positively correlated with both Id-1 and VEGF expression. More advanced T and N status correlated with more intense expression of Id-1, VEGF and higher MVD. With regard to prognostic significance higher Id-1 PAI (adjusted HR ¼ 1.69, 95%CI: 1.10e2.59, p ¼ 0.017), higher VEGF PAI (adjusted HR ¼ 2.66, 95% CI: 1.09e6.50, p ¼ 0.032), and MVD (adjusted HR ¼ 1.55, 95%CI: 1.27e1.88, p < 0.001) were associated with poorer survival. Conclusions: VEGF and Id-1 overexpression were found to be associated with high MVD and emerged as adverse prognostic factors in terms of patient survival in PDAC. The potential of selective anti-angiogenic targeting therapy for pancreatic malignancies should prompt further validation of the present findings in studies encompassing larger samples and more elaborate techniques. Ó 2014 Elsevier Ltd. All rights reserved. Keywords: VEGF; Id-1; MVD; Pancreatic adenocarcinoma; Image analysis
Introduction Pancreatic ductal adenocarcinoma (PDAC) is the fifth leading cause of cancer-related death in Western countries and remains a challenge to clinicians. The anatomical * Corresponding author. Tel.: þ30 697 643 5861; fax: þ30 210 770 6915, þ30 210 776 8634. E-mail addresses: [email protected], [email protected] (D. Georgiadou), [email protected] (T.N. Sergentanis), [email protected] (S. Sakellariou), [email protected] (G.M. Filippakis), florazagouri@ yahoo.co.uk (F. Zagouri), [email protected] (D. Vlachodimitropoulos), [email protected] (T. Psaltopoulou), alazaris@ med.uoa.gr (A.C. Lazaris), [email protected] (E. Patsouris), [email protected] (G.C. Zografos).
complexity and late diagnosis have led to a disappointingly low resectability rate of around 10e20%, even in specialized pancreatic disease centers. PDAC’s propensity toward aggressive growth, early metastasis and resistance to cytotoxic agents and radiation results in an overall 5-year survival rate of less than 5%.1 New therapeutic approaches based on the biologic characteristics of this disease may improve response rates and survival. One promising approach is the inhibition of angiogenesis. Like other solid neoplasms, PDAC depends on the process of angiogenesis, namely the formation of tumor blood vessels, for both local and metastatic growth. In general, the induction of neoangiogenesis is the result of an imbalance between pro- and antiangiogenic molecules.
0748-7983/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejso.2014.01.004 Please cite this article in press as: Georgiadou D, et al., VEGF and Id-1 in pancreatic adenocarcinoma: Prognostic significance and impact on angiogenesis, Eur J Surg Oncol (2014), http://dx.doi.org/10.1016/j.ejso.2014.01.004
2
D. Georgiadou et al. / EJSO xx (2014) 1e7
Among the identified proangiogenic regulators, vascular endothelial growth factor (VEGF) has been identified as key mediator of the regulation of pathologic blood vessel growth and maintenance,2 inducing also endothelial cell proliferation and enhancing vascular permeability.2 Blocking the effects of VEGF on endothelial cells by receptor antagonists3 or neutralizing anti-VEGF antibodies4 inhibits the spreading of a variety of neoplasms. However, the significance of VEGF in tumor neoangiogenesis and progression in PADC is still unclear. The inconsistency in the available literature about the prognostic value of VEGF in PDAC5e8 and recent evidence suggesting only transient effects of anti-VEGF strategies,9 indicate that further investigation into the mechanistic basis of VEGF is needed in order to elucidate its therapeutic role in targeting tumor angiogenesis. Id-1 (inhibitor of differentiation/DNA synthesis) protein belongs to the Id family of helix-loop-helix (HLH) proteins.10 Id 1 has been shown to play a pivotal role in cell proliferation, differentiation and senescence.10 Overexpression of Id-1, in either transcriptional or translational levels, has been reported in over 20 types of human cancers including breast, prostate, cervical, colorectal, liver and pancreatic cancers10 correlating with highly aggressive breast, prostate and ovarian cancer.11 Loss of Id-1 has been shown to lead to downregulation of several proangiogenic genes in tumor endothelial cells12; in parallel, a positive role of Id-1 in tumor metastasis through promoting angiogenesis was suggested.11 According to experimental studies,13 Id-1induced VEGF expression may be a critical molecular event prerequisite in promoting angiogenesis; however, the exact regulatory mechanisms are still unclear. Given the scarce research on Id-1 expression in PDAC, it seems of great interest to elucidate whether overexpression of Id-1 promotes PDAC angiogenesis assessing also any positive correlation with VEGF expression. Microvessel density (MVD) is an established method for quantitating angiogenesis in tumors.14 The assessment of tumor MVD has been reported as a prognostic indicator in several human malignancies. Specifically, studies have shown that higher tumor vascularity correlates with worse patient outcomes in breast, prostate and ovarian cancers.15 A number of endothelial surface markers (CD34, CD31 and factor VIII) have been used to examine MVD in PDAC. CD34 was the marker more frequently associated with survival in this malignancy through multivariate analysis.7,16 To date, its prognostic value remains controversial, as other studies have not demonstrated any relationship with survival.6,8 In this study, we report the results of automated digital assessment of VEGF, Id-1 as well as MVD in PDAC. As the prognostic value of those markers in PDAC remains inconclusive, the aim of the present survey is to determine whether their expression is related to clinicopathological prognostic factors and patient survival.
Materials and methods Patient and tissue samples After approval from the ethics board, 60 consecutive cases of surgically removed pancreatic adenocarcinoma between December 1999 and July 2004 were obtained from the Division of Pathology at “Eugenidio” General Hospital (Athens, Greece). Whipple procedure (n ¼ 26), whipple pylorus preserving procedure (n ¼ 18) and distal pancreatectomy (n ¼ 16) for adenocarcinoma of the head or body-tail of the pancreas were performed by a single surgeon. Any other pathology of the pancreas, treated during the same time period, as well as palliative bypass operations, was excluded from the study. All but four patients included in the study had a preoperative evaluation that was in concordance with intraoperative findings. Those four patients (two with tumor in the head and two in the body-tail) exhibited one single liver metastasis
