Coexpression of Hypoxia-inducible Factors 1 and 2 , Carbonic Anhydrase IX, and Vascular Endothelial Growth Factor in Nasopharyngeal Carcinoma and Relationship to Survival

Purpose: Tumor hypoxia is known to be associated with resistance to chemotherapy, radiotherapy, and poorer survival. Recently, it is shown that hypoxia induces the expression of hypoxia-inducible factor-1 and 2 (HIF-1 and HIF-2 ), which then up-regulates the expression of downstream genes such as carbonic anhydrase IX (CA IX) and vascular endothelial growth factor (VEGF). Experimental Design: We examined the expression of HIF-1 , HIF-2 , CA IX, and VEGF by immunohistochemistry in nasopharyngeal carcinoma (NPC) biopsies from 90 consecutive patients recruited between 1994 and 1997 in a randomized controlled trial of chemoradiation in locally advanced NPC and investigated their relationship with survival. Results: HIF-1 was expressed in 52 of 90 (58%), HIF-2 in 6 of 89 (7%), CA IX in 51 of 90 (57%), and VEGF in 54 of 90 (60%) of tumors. Tumor HIF-1 expression correlated significantly with that of CA IX (P 0.008) and VEGF (P 0.003). High tumor HIF-1 expression was associated with a trend for poor overall survival (P 0.06). Tumors with a positive hypoxic profile (defined as high expression of both HIF-1 and CA9) were associated with worse progression-free survival (P 0.04). Tumors with both hypoxic and angiogenic profile (defined as high VEGF expression) were associated with a worse progression-free survival (P 0.0095). Conclusion: Overexpression of HIF-1 , CA IX, and VEGF is common in NPC, which is probably related to hypoxia up-regulated expression involving a HIF-dependent pathway, and is associated with poor prognosis. Targeting the hypoxia pathway may be useful in the treatment of NPC. INTRODUCTION In the West, NPC occurs sporadically and, histologically, usually belongs to the WHO type I squamous cell carcinoma, which is associated with alcohol and smoking. In many parts of Asia, including Southern China and Southeast Asia, NPC is an endemic disease with incidence rates of 15–50 per 100,000 (1). Histologically, these usually belong to WHO types II and III, nonkeratinizing and undifferentiated carcinoma. Unlike WHO type I, types II and III have no association with alcohol and smoking but are strongly associated with the EBV. The primary treatment for nonmetastatic NPC is radical external RT. More accurate tumor localization by computed tomography and better RT technique have contributed to improvement in local control of this disease, with a local failurefree rate of 80% (2). However, patients with locoregionally advanced disease have significant rates of both local recurrences and particularly distant metastases after RT alone. Hence, new therapeutic strategies are urgently needed. Recent evidence from two prospective randomized trials supports the routine use of concurrent cisplatin-radiotherapy in these patients (3, 4). Tumor hypoxia has long been known to be associated with resistance to chemotherapy and radiotherapy as well as a more malignant tumor phenotype with increased invasiveness, metastases, and poorer survival (5–7). The development of simple and reliable tests to estimate tumor hypoxia would be of clinical importance for the identification of subgroups of patients that could benefit from hypoxia targeting therapeutic strategies. Previous clinical studies of tumor hypoxia concentrated on direct measurement with a polarographic electrode or by injection of a hypoxia labeling marker, such as pimonidazole, into the patient’s blood before biopsy and subsequent detection of the marker by immunohistochemistry. The recent development of scintigraphic and magnetic resonance imaging of hypoxia-labeling markers are promising new approaches. In head and neck Received 1/9/02; revised 4/29/02; accepted 5/6/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 The study was supported in part by the Direct Grants scheme and the Institute of Molecular Oncology of the Chinese University of Hong Kong. 2 To whom requests for reprints should be addressed, at Department of Clinical Oncology, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong. Phone: 852-2632-2989; Fax: 852-26487097; E-mail: [email protected]. 3 The abbreviations used are: NPC, nasopharyngeal carcinoma; HIF, hypoxia-inducible factor; CA IX, carbonic anhydrase IX; VEGF, vascular endothelial growth factor; RT, radiotherapy; OS, overall survival; PFS, progression-free survival. 2595 Vol. 8, 2595–2604, August 2002 Clinical Cancer Research Research. on April 15, 2017. © 2002 American Association for Cancer clincancerres.aacrjournals.org Downloaded from squamous cell cancer, using direct measurement of tumor pO2, a low pretreatment pO2 has been shown to predict poor response to radiation and shorter survival (8, 9). In NPC, using the hypoxia imaging agent fluorine-18 fluoromisonidazole with positron emission tomography system, tumor hypoxia was demonstrated in 100% of primary tumor and 58% of cervical lymph nodes metastases (10). However, these techniques can only be applied in vivo and on a prospective basis. In search of a simple test that would detect evidence of hypoxia even on archival tissue material, immunohistochemical detection of proteins induced by clinical relevant levels of hypoxia represents an appealing option. Such intrinsic marker of hypoxia would have the advantage of being assessable on routine clinical biopsies without the need for specialist equipment or administration of exogenous hypoxia markers. The transcriptional complex HIF-1 plays a pivotal role in essential adaptive responses to hypoxia, and its expression increases exponentially with decreases in levels of cellular oxygen. HIF-1 has emerged recently as an important mediator of gene expression patterns in many tumors (11). HIF-1 is a heterodimer composed of HIF-1 and HIF-1 subunits. HIF-1 is constitutively expressed, whereas HIF-1 is protected from ubiquitination and proteasomal degradation under hypoxic conditions (12). Recently, another member of the family showing close sequence homology and similar properties to HIF-1 has been described and is named HIF-2 (also known as endothelial PAS domain protein-1; Ref. 13). CA IX is a novel member of the CA family that codes for a transmembrane glycoprotein with a suggested function in maintaining the acid-base balance and intercellular communication (14). CA IX can confer a variety of features of the transformed phenotype when transfected into NIH 3T3 cells (15). VEGF is one of the most well-studied markers of tumor angiogenesis, and its expression has been shown to be of prognostic significance in most human tumors studied. Recently, it has been shown that hypoxia induces the expression of HIF-1 , which then up-regulates the expression of downstream genes CA IX and VEGF (16–18). In cervical cancer, CA IX expression was demonstrated to correlate with tumor hypoxia as measured by needle electrode (19). HIF-1 expression was shown to overlap with the hypoxia-labeling marker EF5 in cervical cancer xenograft (20). HIF-1 and CA IX are thus potential endogenous markers of tissue hypoxia. This study was undertaken with the aim to establish the expression pattern of HIF-1 , HIF-2 , CA IX, and VEGF in NPC biopsies and to correlate the level of expression with clinicopathological characteristics and survival outcome. MATERIALS AND METHODS Patients and Tissues. Formalin-fixed, paraffin-embedded biopsy tissues of 90 consecutive NPC patients recruited between 1994 and 1997 in a randomized controlled trial of chemoradiation in locally advanced NPC were retrieved. Patients with biopsy-proven, previously untreated NPC with Ho’s N2 or N3 stage, or N1 stage with lymph node size 4 cm (Ho’s staging; Ref. 1), and without distant metastases (M0), were eligible for the trial. Eligible patients were randomized to receive either standard radiotherapy alone or the same radiotherapy given concurrently with weekly 40 mg/m cisplatin for up to 8 weeks (4). After completion of treatment, patients were followed up every 8 weeks during the first year, every 12 weeks for the second year and third years, and every 16 to 24 weeks thereafter. Patients who developed local or distant recurrence were subjected to any treatment considered appropriate in the opinion of the attending physician including surgery, chemotherapy, or radiotherapy. Informed consent for immunohistochemical study of biological markers was obtained at the time of biopsy. All biopsies were taken via a nasopharyngeal endoscope for diagnostic purposes before the start of treatment. Flanking sections from each tumor biopsy were studied by immunohistochemistry for the expression of HIF-1 , HIF-2 , CA IX, and VEGF. Immunohistochemistry. The HIF-1 , HIF-2 , CA IX, and VEGF proteins in tissue sections were detected using the murine monoclonal antibodies ESEE 122 (21), EP 190b (21), M75 (22), and VG1 (23), respectively, according to methodology described previously (16, 21, 23, 24). HIF-1 and HIF-2 . Staining was performed on 4–5 m paraffin sections. The slides were first placed in a 60°C oven for 15 min. They were then deparaffinized in Citroclear twice for 10 min and rehydrated through a series of graded alcohols and distilled water. After being placed in Tris buffered saline (TBS) buffer for 5 min, the slides were transferred to a jar containing 1 mM EDTA (pH 8.0) buffer and placed in a preheated 60°C water bath for overnight incubation to achieve antigen retrieval. To block endogenous peroxidase activity, DAKO peroxidase block solution was applied for 5 min. After two washes in TBS buffer, 0.2% Triton X-100 in TBS was applied for 10 min. For HIF-1 , the primary antibody ESEE 122 was applied at 1:40 dilution for 30 min. For HIF-2 , the primary antibody EP 190b was applied without dilution for 30 min. Secondary labeled polymer from the Envision HRP Kit (DAKO) was applied for 30 min. The peroxidase reaction was developed using diaminobenzidine chromogen kit from DAKO for 10 min. After washing, the slides were lightly counterstained with Hematoxylin and mounted. CA IX. Sections were deparaffinized and rehydrated. Endogenous peroxidase was quenched with DAKO peroxidase block solution applied for 5 min. Normal human serum at 10% in TBS was then applied for 15 min. After knocking off excess blocking serum from the slides, the primary antibody M75 was applied at 1:50 for 30 min. Secondary polymer from the Envision HRP kit (DAKO) was applied for 30 min. Diaminobenzidine (DAKO) was applied for 8 min. The slides were then counterstained with hematoxylin and mounted. VEGF. The paraffin sections were baked in a 60°C oven for 15 min before deparaffinization. Antigen retrieval was achieved by pressure cooking in Tris-EDTA (pH 9.0) buffer (preparation: 12.2 g of Tris base, 1.48 g of EDTA in 2 liters of distilled water) for 3 min. DAKO peroxidase block solution was applied for 5 min. Primary antibody VG1 was applied in 1:10 dilution for 30 min. Secondary polymer from the Envision HRP kit (DAKO) was applied for 30 min, followed by diaminobenzidine (DAKO) for 5 min. The slides were counterstained with hematoxylin and mounted. For all of the above staining, positive control slides from a tissue block with known positive staining for the respective 2596 Expression of HIF-1 , HIF-2 , CA IX, and VEGF in NPC Research. on April 15, 2017. © 2002 American Association for Cancer clincancerres.aacrjournals.org Downloaded from primary antibody was included with each run. Negative control was achieved by substituting the primary antibody with TBS. In the case of VEGF, the universal presence of serum (which contains VEGF) in the blood vessel lumens of each section also served as a good internal positive control. Scoring Method. All slides were evaluated independently by two investigators (E. P. H. and F. P.) who were blinded to the patient’s clinical data. The difference in scores between the two observers was resolved at a conference microscopy. Each slide was examined at low ( 40) and high ( 250) power to study both the staining pattern and distribution. The staining pattern of individual cells was classified into membranous, nuclear, or cytoplasmic. The percentage of tumor cells showing positive staining for each antibody under study was scored. Staining intensity was not incorporated in our scoring method because we noted that it was more or less constant. The staining pattern of stromal cells and normal epithelium in the same tissue section was also assessed. The following grading system was adopted to score the number of positive stained macrophages in the tumor or stroma with HIF-2 : 0, none seen in section; / , very occasional single cell positive: , few positive cells either in foci or scattered; , moderate numbers either in foci or scattered; , large numbers of positive cells. Statistical Analysis. All statistical analysis and graphs were performed with the statistical package SPSS Release 9.0.0 (SPSS, Inc., Chicago, IL). Correlation among the markers was analyzed using Spearman’s correlation. Association between HIF-1 , HIF-2 , CA IX, and VEGF expression and the various clinicopathological parameters was analyzed using 2 test. Overall survival was defined from the day of randomization to the day of death or last follow-up. Progression-free survival was defined from the day of randomization to the day of progression or last follow-up. Univariate analysis of overall survival and progression-free survival was performed by the Kaplan-Meier method and log-rank test. The Cox proportional hazards model was used for multivariate analysis. For all tests, a two-sided P 0.05 was considered significant.