Pediatr Blood Cancer 2009;53:1035–1039
VEGF Expression as a Prognostic Marker in Osteosarcoma Jyoti Bajpai, MD,1 Meharchand Sharma, MD,2 Vishnubhatla Sreenivas, PhD,3 Rakesh Kumar, Shivanand Gamnagatti, MD,5 Shah Alam Khan, MS,6 Shishir Rastogi, MS,6 Arun Malhotra, MD,4 and Sameer Bakhshi, MD1* Background. The vascular endothelial growth factor (VEGF) pathway is the key regulator of angiogenesis. In osteosarcoma baseline VEGF is of proven prognostic value but prognostic potential of post-NACT VEGF expression is largely unexplored. Procedure. Treatment naive patients with osteosarcoma were subjected to initial staging workup followed by three cycles of neoadjuvant chemotherapy (NACT) and surgery; resected tumors were assessed for histological necrosis by Huvos grading. Initial biopsy and resected tumor specimens post-NACT were examined for VEGF expression by immunohistochemistry. Positive VEGF expression was considered when intensive positive staining was observed in >10% of the tumor cells. VEGF expression at baseline was compared with grade of tumor; pre-NACT and post-NACT VEGF expression were compared with histological necrosis. Receiver operating characteristic curves were generated to assess best
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threshold and predictability. Results. A total of 31 patients were recruited with median age of 17 years (range 5–66 years); male/ female ratio was 25:6; 23 patients (74%) were non-metastatic. At baseline, there was 90% concordance between positive VEGF expression and higher histological grade (28/31); baseline VEGF expression did not correlate well with stage and histological necrosis. Twenty-one (67%) were poor and 10 (33%) were good histologic responders; post-NACT VEGF expression as well as VEGF change following NACT significantly correlated with histological necrosis. Conclusion. Positive VEGF expression in surviving tumor cells post-NACT in resected tumors appears to be an important negative prognostic factor in osteosarcoma which may help future therapies to be identified according to the angiogenic potential of the disease. Pediatr Blood Cancer 2009;53:1035–1039. ß 2009 Wiley-Liss, Inc.
angiogenesis; necrosis; neoadjuvant chemotherapy; osteosarcoma; vascular endothelial growth factor (VEGF)
INTRODUCTION New blood vessel formation (angiogenesis) is a fundamental event in the process of tumor growth and metastatic dissemination. The vascular endothelial growth factor (VEGF) pathway is well established as one of the key regulators of this process. Activation of the VEGF-receptor pathway triggers a network of signaling processes that promote endothelial cell growth, migration, and survival from pre-existing vasculature. In addition, VEGF mediates vessel permeability, and has been associated with malignant effusions. More recently, an important role for VEGF has emerged in mobilization of endothelial progenitor cells from the bone marrow to distant sites of neovascularization. Due to its central role in tumor angiogenesis, the VEGF/VEGF-receptor pathway has become a major focus of research and antiangiogenic drug development in oncology [1]. Increased VEGF production has been shown to be important in the growth of various solid tumors in humans including osteosarcoma, gastric, esophageal, colorectal, renal, lung, and breast carcinomas [2–4]. In osteosarcoma, patients with VEGF positive tumors have poorer disease-free and overall survival compared with those with VEGF negative tumors [3,5]. VEGF expression in pretreated osteosarcoma specimens is predictive of eventual development of pulmonary metastasis; further circulating VEGF levels by ELISA were found to be significantly higher in patients with osteosarcoma who had pulmonary metastasis [5]. Prognostic value of post-neoadjuvant chemotherapy (NACT) VEGF expression is largely unexplored. Therefore, in the present study we investigated the prognostic potential of VEGF expression at baseline as well as in post-NACT surviving tumor cells in relation with histologic necrosis, an established robust prognostic factor in osteosarcoma [6,7].
MATERIALS AND METHODS This is a prospective, diagnostic study conducted at our institute from January 2006 to December 2008. Treatment naive osteosarcoma
ß 2009 Wiley-Liss, Inc. DOI 10.1002/pbc.22178 Published online 20 July 2009 in Wiley InterScience (www.interscience.wiley.com)
patients with adequate organ function for receiving NACT and adequate biopsy sample for analysis were eligible for the study. After a detailed history and examination, patients were subjected to initial investigations of MRI and staging workup, which included CT chest and bone scan. Patients were staged as per AJCC staging system [8]. NACT included three cycles of cisplatin (40 mg/m2) and doxorubicin (25 mg/m2), both for 3 days every 3 weeks. Following NACT, the patient was reevaluated, underwent radical resection of tumor (limb salvage or amputation) and assessment of histologic necrosis in the resected specimen. The initial biopsy block was reviewed for the grade, histologic subtype, and VEGF expression by immunohistochemistry (IHC). The resected tumor specimen post-NACT was examined for histopathologic necrosis and VEGF expression. All the slides were coded and evaluated by a pathologist, who was blinded with regard to the clinical status and results of VEGF staining or histopathologic necrosis of the patient. This study was approved by the ethics committee and the institutional review board.
IHC Analysis After an initial review of all the available hematoxylin and eosin (H&E) stained slides of the biopsy and surgical specimens, one — ————— 1
Department of Medical Oncology, Dr. B. R. A. Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi, India; 2Department of Pathology, All India Institute of Medical Sciences, New Delhi, India; 3Department of Biostatistics, All India Institute of Medical Sciences, New Delhi, India; 4Department of Nuclear Medicine, All India Institute of Medical Sciences, New Delhi, India; 5Department of Radiodiagnosis, All India Institute of Medical Sciences, New Delhi, India; 6Department of Orthopedics, All India Institute of Medical Sciences, New Delhi, India *Correspondence to: Sameer Bakhshi, Associate Professor of Pediatric Oncology, Department of Medical Oncology, Dr. B. R. A. Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi 110029, India. E-mail:
[email protected] Received 4 February 2009; Accepted 2 June 2009
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paraffin-embedded tissue block was selected from each case in which viable tumor cells were present. Five microns thick sections were recut and routine H&E stained sections of each case were reviewed and diagnosis reconfirmed. IHC was done by streptavidin–biotin peroxidase complex method using monoclonal antibodies to anti-human VEGF rabbit monoclonal immunoglobulin G antibody (dilution 1:100) (BIO SB, Santa Barbara, CA). For the negative controls we used pancytokeratin (M/s; Dako, Glostrup, Denmark) and human glioblastoma multiforme was taken as positive controls. The cell types with positive staining for VEGF were defined morphologically by using H&E staining. Our analysis was semiquantitative wherein we counted 100 surviving tumor cells and positive VEGF expression was considered when intensive positive staining of VEGF was observed in >10% of the tumor cells. Further subdivision included grade I as 11–25%, grade II as 26– 50%, and grade III as 51–100% cells showing positive staining of VEGF [9,10].
Histopathologic Response Assessment Tumor necrosis was graded as per Huvos pathologic tumor response grading wherein grade I is 10%) I 11–25 II 26–50 III 51–100
Number of patients baseline
Number of patients postNACT
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1 5 22
5 10 10
(% of tumor cells positive) Variable Mean VEGF SD Mean VEGF change SD
Baseline
Post-NACT
78.38 34.65 48.7 36 29.7 47.5
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DISCUSSION
Fig. 1. Good responder: Pre-chemotherapy biopsy (H&E) slide (A) and pre-chemotherapy biopsy slide showing 100% VEGF expression (B); post-chemotherapy resection specimen of same patient (H&E) slide showing extensive areas of necrosis (>90%) (C) and postchemotherapy resection specimen of same patient showing no VEGF expression of surviving cells (D). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
Fig. 2. Poor responder: Pre-chemotherapy biopsy (H&E) slide (A) and pre-chemotherapy biopsy slide showing 100% VEGF expression (B); post-chemotherapy resection specimen of same patient (H&E) slide showing focal areas of necrosis (10%) with extensive viable areas (C) and post-chemotherapy resection specimen of same patient showing 75% VEGF expression of surviving cells (D). [Color figure can be viewed in the online issue, which is available at www.interscience. wiley.com.] Pediatr Blood Cancer DOI 10.1002/pbc
The importance of VEGF in cancer progression has been validated in other organ cancers [12,13]; in sarcoma also VEGF expression was found to have association with stage and grade of tumors, as well as survival and pulmonary metastasis [3,14]. In the present study, we hypothesized that the cells surviving after NACT to be more important because the surviving population of the cells may be the one that results in recurrence or subsequent metastases. Our approach differs from those in previous studies, which have examined the prognostic influence of VEGF expression in diagnostic biopsy specimens before any NACT is given [3]. The result in our patient population suggests that baseline VEGF expression had significant association with histologic grades of tumor with a concordance of 90% between the VEGF expression and histologic grade. However, there was no significant association of baseline VEGF expression with stage of the disease, which may have been due to the disproportionate stage distribution in our cohort. Similar lack of association with stage was found in a study by Kaya et al. [3] in which disproportionate stage distribution was proposed as an explanation. Previous investigators have identified various factors associated with a poor prognosis in patients with osteosarcoma; the most consistent factor identified is a poor response (30% VEGF expression. This difference, however, was not statistically significant. The basic demographic features of osteosarcoma in our center revealed that approximately one-fourth of our patients were metastatic at presentation which is in contrast with the metastatic rate of 11.4–20% reported by other investigators [15,16]. This may be the result of referral patterns as our center is a major tertiary care center in Northern India and an increased proportion of advanced cases maybe be referred. It is, however, difficult to comment on whether the disease biology in this part of the country results in a more aggressive disease at the outset.
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Fig. 3. Association of VEGF expression in surviving tumor cells post-NACT with histologic necrosis (A); association of change in VEGF expression following NACT from baseline with histologic necrosis (B); and association of VEGF Expression in tumor cells pre-NACT with histologic necrosis (C).
Fig. 4. Kaplan–Meier Survival curves with respect to post-chemotherapy VEGF expression of surviving tumor cells (median followup ¼ 9.4 months) showing event free survival (A) and overall survival (B). Y-axis ¼ Survival proportion; VEGF 0 ¼ post-chemotherapy 30%VEGF expression of surviving tumor cells; VEGF 1 ¼ post-chemotherapy >30%VEGF expression of surviving tumor cells.
High VEGF expression of surviving tumor cells in postchemotherapy resected tumors appears to be an important negative prognostic factor in osteosarcoma. Suppression of tumor angiogenesis, for example, by inhibition of the action of VEGF, has shown promise in animal models as a potential new therapeutic strategy for treatment of osteosarcoma [17]. If the results obtained in the present study can be reproduced in a larger cohort of the patients then it will further establish role of post-NACT VEGF as a prognostic factor in osteosarcoma and may serve as a platform to Pediatr Blood Cancer DOI 10.1002/pbc
build future therapies according to the angiogenic potential of the disease and provide means to test antiangiogenic molecules in osteosarcoma.
REFERENCES 1. Hicklin DJ, Ellis LM. Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis. J Clin Oncol 2005;23: 1011–1027.
VEGF in Osteosarcoma 2. Handa A, Tokunaga T, Tsuchida T, et al. Neuropilin-2 expression affects the increased vascularization and is a prognostic factor in osteosarcoma. Int J Oncol 2000;17:291–295. 3. Kaya M, Wada T, Akatsuka T, et al. Vascular endothelial growth factor expression in untreated osteosarcoma is predictive of pulmonary metastasis and poor prognosis. Clin Cancer Res 2000; 6:572–577. 4. Voest EE, D’Amore PA. Tumor angiogenesis and microcirculation. New York, NY: Marcel Dekker; 2001. 5. Kaya M, Wada T, Kawaguchi S, et al. Increased pre-therapeutic serum vascular endothelial growth factor in patients with early clinical relapse of osteosarcoma. Br J Cancer 2002;86:864– 869. 6. Saeter G, Hoie J, Stenwig AE, et al. Systemic relapse of patients with osteogenic sarcoma: Prognostic factors for long term survival. Cancer 1995;75:1084–1093. 7. Enneking WF, Spanier SS, Goodman MA. A system for the surgical staging of musculoskeletal sarcoma. Clin Orthop 1980;153:106– 120. 8. Greene FL, Page DL, Fleming ID, et al. AJCC Cancer Staging Manual, 6th edition. New York: Springer; 2002. 9. Charity RM, Foukas AF, Deshmukh NS, et al. Vascular endothelial growth factor expression in osteosarcoma. Clin Orthop Relat Res 2006;448:193–198. 10. Choi JY, Jang KT, Shim YM, et al. Prognostic significance of vascular endothelial growth factor expression and microvessel
Pediatr Blood Cancer DOI 10.1002/pbc
11.
12.
13.
14. 15.
16.
17.
1039
density in sophageal squamous cell carcinoma: Comparison with positron emission tomography. Ann Surg Oncol 2006;13:1054– 1062. Rosen G, Marcove RC, Huvos AG, et al. Primary osteogenic sarcoma: Eight years experience with adjuvant chemotherapy. J Cancer Res Clin Oncol 1983;106:55–67. Knopp MV, Weiss E, Sinn HP, et al. Pathophysiologic basis of contrast enhancement in breast tumors. J Magn Reson Imaging 1999;10:260–266. George ML, Dzik-Jurasz AS, Padhani AR, et al. Non-invasive methods of assessing angiogenesis and their value in predicting response to treatment in colorectal cancer. Br J Surg 2001;88:1628– 1636. DuBois S, Demetri G. Markers of angiogenesis and clinical features in patients with sarcoma. Cancer 2007;109:813–819. Kager L, Zoubek A, Po¨tschger U, et al. Primary metastatic osteosarcoma: Presentation and outcome of patients treated on neoadjuvant Cooperative Osteosarcoma Study Group protocols. J Clin Oncol 2003;21:2011–2018. Bacci G, Bertoni F, Longhi A, et al. Neoadjuvant chemotherapy for high-grade central osteosarcoma of the extremity: Histologic response to preoperative chemotherapy correlates with histologic subtype of the tumor. Cancer 2003;97:3068–3075. Tsunemi T, Nagoya S, Kaya M, et al. Postoperative progression of pulmonary metastasis in osteosarcoma. Clin Orthop Relat Res 2003;407:159–166.
