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Japanese Journal of Clinical Oncology Advance Access published online on January 19, 2007
Japanese Journal of Clinical Oncology, doi:10.1093/jjco/hyl137
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© 2007 Foundation for Promotion of Cancer Research

Overexpression of Hypoxia-Inducible Factor-1{alpha} in Human Osteosarcoma: Correlation with Clinicopathological Parameters and Survival Outcome

Qing-Cheng Yang1, Bing-Fang Zeng1,, Yang Dong1, Zhong-Min Shi1, Zhi-Ming Jiang2 and Jin Huang2

1 Department of Orthopedics
2 Department of Pathology, Shanghai Sixth People's Hospital of Shanghai JiaoTong University, Shanghai, China

For reprints and all correspondence: Bing-Fang Zeng, Department of Orthopedics, Shanghai Sixth People's Hospital of Shanghai JiaoTong University, Yishan Road No. 600, Shanghai, 200233, China. E-mail: bingfangzeng{at}yahoo.com.cn

Received May 4, 2006; accepted September 29, 2006


   Abstract
TOP
 Abstract
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 Conflict of interest statement
 References
 
BACKGROUND: Hypoxia is a common feature of many solid cancers and linked to malignant transformation, metastases and treatment resistance. Hypoxia is known to induce hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) expression. The aim of this study is to investigate the impact of overexpression of HIF-1{alpha} on prognosis and the relationship with clinicopathological characteristics in human osteosarcoma.

METHODS: Immunochemistry with digital image analysis was used to determine the HIF-1{alpha} protein expression in histologic sections from 39 treated patients.

RESULTS: According to our study, expression of HIF-1{alpha} protein were detected in 31 of 39 cases (79%), with signal concentrated primarily within the nuclei of tumor cell. In contrast, non-cancerous adjacent tissues showed no HIF-1{alpha} immunoreactivity. HIF-1{alpha} expression was significantly associated with surgical stage, percentage of dead cells and microvessel density (MVD). Surgical stage, percentage of dead cells and HIF-1{alpha} expression showed significant influence on overall survival (OS) and disease-free survival (DFS) in univariate analysis. In multivariate analysis, surgical stage (IIA versus IIB/III) and percentage of dead cells (<90% versus ≥ 90%) were significant for DFS and OS. Those patients with HIF-1{alpha} moderate/strong expression showed significantly shorter OS and DFS compared with HIF-1{alpha} negative/weak expression.

CONCLUSIONS: Overexpression of HIF-1{alpha} is predictive of a poor outcome and might be a novel therapeutic target in human osteosarcoma.

Key Words: osteosarcoma • HIF-1{alpha} • prognosis • immunochemistry


   INTRODUCTION
TOP
 Abstract
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 Conflict of interest statement
 References
 
Osteosarcoma is the most common malignant bone tumor in adolescents and young adults. Although the use of neoadjuvant chemotherapy and improvement in surgical technology have increased the survival rate to 65–75% (1), pulmonary metastasis occurs in approximately 40% of patients with osteosarcoma and remains a major cause of fatal outcome (2).

Hypoxia is a common feature in various human solid cancers. Although the importance of hypoxia in osteosarcoma has not been studied previously, 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 (3–6). It is well known that growth of malignant tumors is limited to several mm2 in the absence of neoangiogenesis because of insufficient oxygen and glucose diffusion from blood vessels (7). Neoangiogenesis and cellular adaptation to hypoxia are therefore recognized as essential events for cancer progression. There is also evidence that hypoxia may be responsible for a variety of growth-modulating effects that could confer a growth advantage upon the malignant cells (8,9).

A newly described transcription factor, hypoxia-inducible factor-1{alpha} (HIF-1{alpha}), appears to be an important and highly specific regulator of the cellular response to hypoxia in many different systems (10,11). Hypoxia-inducible factor-1 is a heterodimeric DNA-binding complex, of which the ß subunit is responsible for its translocation into the nucleus and the {alpha} subunit for its oxygen sensitivity (12,13). HIF-1{alpha} influences a number of genes that may in part play roles in tumor progression, including erythropoietin, transferrin, endothelin-1, inducible nitric oxide synthetase, hemeoxygenase 1, vascular endothelial growth factor (VEGF), insulin-like growth factor-2, insulin-like growth factor-binding proteins and different glucose transporters and glycolytic enzymes (14).

Many recent studies revealed that overexpression of HIF-1{alpha} were implicated with the poor prognosis of ovarian cancer, pancreatic carcinoma, breast cancer, nasopharyngeal carcinoma, cervix cancer, head and neck cancer, among others (15–22). Until now, the effect of HIF-1{alpha} expression on the prognosis of osteosarcoma had not been studied. The present study was undertaken with the purpose of investigating the expression pattern of HIF-1{alpha} in patients with osteosarcoma and to correlate the level of expression with clinicopathological characteristics and survival outcome.


   PATIENTS AND METHODS
TOP
 Abstract
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 Conflict of interest statement
 References
 
Patients
A total of 39 unselected cases (22 males and 17 females; mean age, 19 years; age range 5–56 years) with primary osteosarcoma were eligible for the study. Before the study all patients underwent systemic neoadjuvent chemotherapy (methotrexate, adriamycin, cisplatin, isofosfamide). Closed biopsies of all cases were performed by fine-needle aspiration or trephine for diagnosis and then surgical treatment. The biopsy samples with bone tissue were decalcified. Non-cancerous adjacent tissues were available in 25 of 39 patients. The clinicopathological characteristics of the patient cohort are summarized in Table 1. Tumor size data were available based on a review of the imaging studies and the pathology reports. Tumor volume was calculated on the basis of an ellipsoid formula, using the measurements: height x width x depth x 0.52. Surgical staging was done according to the method of Enneking et al. (23), with differentiation among highly malignant intracompartmental osteogenic sarcomas (IIA), extracompartmental lesions (IIB), and osteogenic sarcomas with manifestation of metastases present on recognition of the disease (III). The effect of pre-operative chemotherapy as well as the degree of histological regression of the tumor, which was defined as percentage of dead cells, were studied by means of semi-quantitative methods obtained from the literature (24). According to the percentage of dead cells, all cases were divided into two groups: tumors showing good response to pre-operative chemotherapy (dead cells > 90%, surviving cells < 10%), tumors showing poor response (surviving cells > 10%). The mean follow-up time was 50 months (range, 13–86 months).


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  		Table 1. Distribution of clinicopathological parameters and cross-tabulations with hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) expression

 
Immunohistochemistry
Formalin-fixed, paraffin embedded tissues were cut into 4 µm sections consecutively. Hematoxylin-eosin staining was performed for histological diagnosis. The immunostaining for HIF-1{alpha} was performed utilizing the streptavidin- biotin-peroxidase method, according to the manufacturer's instructions. In brief, histological slides were deparaffinized in xylol. Slides were heated in 0.01 M citrate buffer for 10 min in a microwave oven. After cooling for 20 min and washing in PBS, endogenous peroxidase was blocked with methanol containing 0.3% hydrogen peroxide for 30 min, followed by incubation with PBS for 30 min. For immunohistochemical detection of HIF-1{alpha}, specimens were incubated overnight at 4°C with the primary antibody HIF-1{alpha} H1{alpha} 67 (Santa Cruz Biotech) at a dilution of 1 : 50. Nuclei were lightly counterstained with hematoxylin.

As a positive control for HIF-1{alpha} expression, immunostaining was performed on a sample of breast cancer tissue with known strong expression of HIF-1{alpha}, which had been used in a previous study (20). For a negative control, primary antibodies were replaced by PBS. As additional negative control, immunostaining for HIF-1{alpha} was also performed in 25 samples of non-cancerous adjacent tissues.

The quality (number, intensity and pattern) of every staining procedure for HIF-1{alpha} has been comparatively evaluated using consecutive control sections. Tumor cell immunoreactivity was scored according to both the extent of nuclear staining (relative number of HIF-1{alpha} positive cells) and the intensity of the reaction: –, not detected; (+), < 1% positive cells; +, 1–10% weakly to moderately stained cells; ++, 1–10% intensively stained cells or 10–50% weakly stained cells; +++, 10–50% positive cells with moderate to strong staining; ++++, > 50% positive cells. For the statistical analysis, the six grades of staining were reduced to four groups: I, negative, (–); II, weak, [(+)/+]; III, moderate, [++/+++]; and IV, strong, [++++]. The assessments were performed independently by two experienced investigators without prior knowledge of the clinical and pathological data.

In the case of mouse anti-CD31, the antibody reaction is preceded by treatment with trypsin for 10 min at 37°C. After the blockage of non-specific binding by immersion in TBS containing 1% casein for 10 min, sections were incubated with the first antibody diluted in TBS: mouse anti-CD31 (Novus Co.) 1 : 100. Microvessel density (MVD) within the tumor was assessed by light microscopy after immunostaining for CD31. Based on the criteria of Weidner et al. (25), a vessel lumen was not required for identification of a microvessel. The three ‘hot’ areas with the highest number of discrete microvessels were identified, photographs of each area were taken at x 200 to count accurate microvessel numbers and the average counts of the three areas were recorded as the MVD.

Statistcs
Association between HIF-1{alpha} expression and various clinicopathological characteristics were analyzed using the {chi}2 test. The results were considered significant at P < 0.05. Correlation between HIF-1{alpha} expression and MVD was analyzed using Spearman's rank correlation analysis.

Two endpoints were examined for survival analyses: disease-free survival (DFS) and overall survival (OS). OS was defined from the day of surgery until death of the patient. Data on patients who had survived until the end of observation period were censored at their last follow-up visit. Death from a cause other than osteosarcoma or survival until the end of the observation period was considered a censoring event. DFS was defined from the end of primary therapy until first evidence of local, regional or distant tumor progression of disease, if the patient showed no evidence of disease after primary therapy. DFS and OS curves were plotted according to the Kaplan–Meier method, the Log-rank test being used to determine the significance of differences between clinicopathological parameters. The Cox proportional hazards model was used for multivariate analysis. For all tests, P < 0.05 was considered as significant.


   RESULTS
TOP
 Abstract
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 Conflict of interest statement
 References
 
Expression of HIF-1{alpha} Protein and MVD in Osteosarcoma
Expression of HIF-1{alpha} protein was detected in 31 of 39 cases (79%), with the signal concentrated primarily within the nuclei of the tumor cell. In contrast, non-cancerous adjacent tissues showed no HIF-1{alpha} immunoreactivity.

Eight patients (21%) qualified for the ‘negative’ HIF-1{alpha} expression group, 14 patients (36%) qualified for the ‘weak’ HIF-1{alpha} expression group, nine patients (23%) qualified for the ‘moderate’ HIF-1{alpha} expression group and eight patients (20%) qualified for the ‘strong’ HIF-1{alpha} expression group. Four patterns of staining were encountered in our study: focal expression, tumor cell expression with no HIF-1{alpha} immunoreactivity around normal tissue (Fig. 1A); at the border of a necrotic region (Fig. 1B) or with the most intense reaction occurring distal to the closest vessels (Fig. 1C, D); or diffuse expression, which was independent of vessel proximity (Fig. 1E, F). In most patients, there was no exclusivity for one or the other staining pattern.


Figure 1371
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  		Figure 1. Immunohisochemical staining of hypoxia-inducible factor-1{alpha} (A,B,C,E) and CD31 (D,F) expression in human osteosarcoma. A, HIF-1{alpha} strong expression is detectable in tumor cells and absent in para-tumor tissue; B, HIF-1{alpha} expression appears at the border of a necrotic region; C, D, HIF-1{alpha} and CD31 expression appear distal to the closest vessels; E, F, HIF-1{alpha} and CD31 expression diffuse throughout the entire tumor. (P, para-tumor tissue; N, necrotic region; T, tumor tissue; Arrows, microvessel.)

 
Correlation of HIF-1{alpha} Protein Expression with Clinicopathological Parameters
The cross-tabulations for clinicopathological parameters are summarized in Table 1. HIF-1{alpha} expression was significantly associated with surgical stage (P = 0.024) and percentage of dead cells (P = 0.046). However, other clinicopathological parameters, including gender, age, anatomic location, tumor size and surgical treatment were not associated with HIF-1{alpha} expression.

We assessed the association of HIF-1{alpha} expression and MVD in all patients. The number of vessels counted in tumor tissues (24.45 ± 2.37) was significantly greater than that in the adjacent normal tissues (15.63 ± 2.85) (P < 0.05). The data analysis revealed that MVD in HIF-1{alpha}-negative, weak, moderate and strong tumors were 20.68 ± 1.52, 21.28 ± 3.09, 27.98 ± 2.01 and 29.92 ± 3.82, respectively. HIF-1{alpha} expression was significantly correlated with microvessel density by Spearman's rank correlation analysis (P = 0.005, r = 0.813) (Fig. 2).


Figure 1372
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  		Figure 2. Spearman's rank correlation analysis between HIF-1{alpha} expression and microvessel density.

 
Survival Analysis
During the observation period, 21 patients (54%) developed recurrent disease (five showed regional recurrence, 13 showed distant metastases and three showed regional recurrence and distant metastases), and 29 patients (74%) died from their osteosarcoma. The surgical stage of the patient, percentage of dead cells and HIF-1{alpha} expression significantly influenced DFS (P = 0.011, P = 0.0000 and P = 0.003, respectively) and OS (P = 0.011, P = 0.0001 and P = 0.004, respectively) in univariate analysis (Log-rank test, Table 2).


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  		Table 2. Univariate survival analysis (Log-rank tests) of clinicopathological parameters

 
Surgical treatment also influenced DFS significantly (P = 0.044). A multivariate analysis (Cox regression model, Table 3) including age, surgical stage, percentage of dead cells and HIF-1{alpha} expression was performed for OS and DFS. The surgical stage (IIA versus IIB/III) and percentage of dead cells (<90% versus ≥ 90%) were significant for DFS (P = 0.015, P = 0.017) and for OS (P = 0.041, P = 0.031), respectively.


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  		Table 3. Multivariate survival analysis (Cox regression model) of clinicopathological parameters

 
Whereas age and HIF-1{alpha} expression did not attain significance, patients with a moderate/strong expression of HIF-1{alpha} showed significantly shorter OS and DFS compared with negative/weak expression. The Kaplan–Meier curves for OS and DFS and HIF-1{alpha} expression are shown in Fig. 3 (panel A, panel B) for all patients.


Figure 1373
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  		Figure 3. Panel A: overall survival (OS) of 39 patients with osteosarcoma, as a function of ‘negative/weak’ versus ‘moderate/strong’ hypoxia-inducible factor-1{alpha} expression. Panel B: disease-free survival (DFS) of 39 patients with osteosarcoma, as a function of ‘negative/weak’ versus ‘moderate/strong’ hypoxia-inducible factor-1{alpha} expression.

 

   DISCUSSION
TOP
 Abstract
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
Conflict of interest statement
 References
 
Expression of HIF-1{alpha} has been demonstrated recently to be associated with a more aggressive phenotype of cancer cells and with impaired clinical outcome in a variety of human tumors, including cervical, ovarian, breast cancer, pancreatic cancer and cerebral oligodendrogliomas (16–18,20,21,26). This study was undertaken because there were no data available on the prognostic relevance of HIF-1{alpha} expression in human osteosarcoma.

This is, to our knowledge, the first study determining the influence of HIF-1{alpha} expression on the prognosis of human osteosarcoma. Expression of HIF-1{alpha} protein, determined by immunohistochemistry, was observed in 79% of specimens. Non-cancerous adjacent tissues were shown to be HIF-1{alpha} negative. The staining for HIF-1{alpha} protein reflects two different expression patterns consistent with the research results in other human tumors (20,27,28). The first depends on the distance from blood vessels and on the perinecrotic regions, which may accord with a decrease in oxygen concentration in tumor tissues. The second expression pattern is manifested as a diffuse immunoreactivity throughout the entire tumor, indicating that HIF-1{alpha} expression can be influenced by factors other than hypoxia. This suggests that expression of HIF-1{alpha} in osteosarcoma cannot be attributed to hypoxia alone. Some tumor suppressor genes and oncogenes, e.g. P53 and von Hippel Lindau (VHL), may play a crucial role in the up-regulation of HIF-1{alpha} expression irrespective of tumor hypoxia (11,29–31).

In our study, we demonstrated a strong correlation between HIF-1{alpha} expression and increased MVD in human osteosarcoma. Also, it is well known that significant increased MVD and expression of VEGF are associated with poor prognosis in human osteosarcoma (32). Neoangiogenesis is considered essential for tumor growth and the development of metastases, as well as for the progression of invasive cancer. Because cancer cell proliferation may outpace the rate of angiogenesis, thus causing hypoxia, the adaptation of tumor cells to tissue hypoxia is known to be associated with resistance to chemotherapy, radiotherapy and poorer survival (3,5,33). Hypoxia-inducible factor-1{alpha} plays a key role in the cellular adaptation to hypoxia and the activation of several genes, e.g. transactivation of the VEGF gene that have been implicated in tumor growth (34). Zagzag et al. (35) demonstrated a correlation between HIF-1{alpha} overexpression and induction of angiogenesis in human brain tumors by assessment of the formation of blood vessels. This finding can be revealed that angiogenesis was promoted by HIF-1{alpha} induced expression of VEGF. Therefore, overexpression of HIF-1{alpha} may contribute to increased MVD and tumor progression.

HIF-1{alpha} has been suggested to be endogenous marker of tumor hypoxia (36), correlation of HIF-1{alpha} expression with treatment resistance and overall poor prognosis appeared to explain the prognostic implications of tumor hypoxia (21,27,35,37). Our statistical analysis revealed that HIF-1{alpha} expression is significantly correlated with surgical stage and percentage of dead cells, which have been reported as prognostic factors in osteosarcoma (38). Both surgical stage and percentage of dead cells are significant for DFS and OS in univariate analysis and multivariate analysis, whereas HIF-1{alpha} expression differed significantly in the univariate analysis, but not in the multivariate analysis for DFS and OS. This means that overexpression of HIF-1{alpha} has a significant influence on the prognosis, but is not the sole factor. This result is consistent with Guber's study in high-risk breast cancer (20). In univariate analysis, surgical treatment is significant for DFS but not for OS. This may explain why patients who have required amputation had higher stage and larger tumors compared with those patients who have had limb salvage, and hence local recurrence and distant metastases have occurred in patients with amputation.

In conclusion, HIF-1{alpha} was expressed in the majority of osteosarcomas. Overexpression of HIF-1{alpha} is predictive of a poor outcome, although its impact is less evident than surgical stage, percentage of dead cells and MVD. It is possible that HIF-1{alpha} could be a novel therapeutic target in human osteosarcoma in patients with amputation.


   Conflict of interest statement
TOP
 Abstract
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 Conflict of interest statement
References
 
None declared.


   Acknowledgments
 
We thank Dr JiaQiang Ren for technical assistance with immunohistochemistry and Dr Xu Zhang, Tong Ji University, for the data statistics.


   References
TOP
 Abstract
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 Conflict of interest statement
 References
 
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