© The Author (2008). Published by Oxford University Press. All rights reserved
Prognostic Implications of Glucose Transporter Protein-1 (Glut-1) Overexpression in Bone and Soft-Tissue Sarcomas
1 Division of Orthopaedic Oncology, National Cancer Center Hospital, Tokyo
2 Division of Diagnostic Radiology, National Cancer Center Hospital, Tokyo, Japan
3 Division of Clinical Laboratory, National Cancer Center Hospital, Tokyo, Japan
For reprints and all correspondence: Ukihide Tateishi, Division of Diagnostic Radiology, National Cancer Center Hospital, 5-1-1, Tsukiji, Chuo-ku, 104-0045, Tokyo, Japan. E-mail: utateish{at}ncc.go.jp
Received June 28, 2007; accepted August 13, 2007
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Abstract |
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Background: The glucose transporter protein 1 (Glut-1) overexpression is associated with poor overall survival (OS) in various malignant tumors. The aim of this study was to investigate prognostic significance of Glut-1 overexpression in patients with bone and soft-tissue sarcomas.
Methods: A total of 67 patients (mean age, 43 years; range, 8–79 years) with bone and soft tissue sarcomas were analyzed. Pathologic confirmation was observed from surgical specimens in all patients. Pathologic variables including tumor differentiation, necrosis, mitotic index, MIB-1 (Ki-67) grade and Glut-1 expression were assessed. Clinical characteristics and pathologic variables were determined by Kaplan–Meyer curve of OS after treatment.
Results: Glut-1 overexpression was found in 56 patients (83%). The patients with Glut-1 overexpression showed significantly poor OS compared with those without Glut-1 overexpression (P = 0.029). The presence of metastasis, treatment without surgical resection, tumor differentiation, necrosis, mitotic index and MIB-1 grade were also significantly negative prognostic factors. The presence of metastasis was independently associated with poor OS (P = 0.031).
Conclusions: Assessment of Glut-1 expression prior to treatment has a predictive potential effect in patients with bone and soft-tissue sarcomas.
Key Words: sarcoma • glucose trasnsporter protein • prognosis
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INTRODUCTION |
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Bone and soft-tissue sarcomas are classified according to their grade, which represents the most important prognostic factors. The presence of necrosis has been shown to be an independent parameter for predicting prognosis (1). The size and the location of the tumor are other important prognostic factors (2).
The glucose transporter protein-1 (Glut-1) is one of the proteins upregulated in hypoxic conditions. The presence of hypoxia in tumors is leading to resistance to radiotherapy and chemotherapy and is associated with an increased potential for metastases (3–5). This latter finding is thought to be related to the promotion of genomic instability associated with an carcinogenesis and malignant progression (6). Glut-1 is also associated with an increased expression of some proteins that can change tumor cells to survive the severe microenvironment. Glut-1 also promotes glucose metabolism and is overexpressed in several tumors (7–9). The level of Glut-1 expression might be a suitable marker of hypoxia and glucose metabolism, which could be measured simply and inexpensively as part of the routine histologic assessment of tumors (10,11). Increased expression of Glut-1 has been shown to be correlated with a poor prognosis in a variety of tumors (12–14). However, little is known about its expression in bone and soft-tissue sarcomas.
In the present study, immunohistochemical staining of Glut-1 was performed prospectively in the patients with bone and soft-tissue sarcomas. The aim of the present study was to test the hypothesis that Glut-1 overexpression is related to the clinical outcome in patients with bone and soft-tissue sarcomas.
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PATIENTS AND METHODS |
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Patients
Sixty-seven patients aged 8–79 years and having histologically proven sarcomas arising from bone (n = 22, 33%) or soft tissue (n = 55, 67%) since July 2001 until July 2006 were included in this study. All patients underwent initial staging based on a review of the medical history, physical examinations and imaging studies. This study was conducted in accordance with the Helsinki declaration, and all the patients had provided their informed consent for the review of their records.
Treatment and Follow-up
Surgical resection was performed in 54 patients (81%). Surgical procedures were wide resection (n = 33, 61%), marginal resection (n = 15, 22%), radical resection (n = 3, 4%) and intralesional resection (n = 3, 4%). Adjuvant chemotherapy was performed in 46 patients (69%). Radiotherapy was performed in 24 patients (36%), mean dose was 46.5 Gy (range, 30–70.4 Gy). The follow-up period was dated from the time of diagnosis, and the mean follow-up period was 25 months (range, 6–60 months). During the follow-up, 39 patients (58%) developed metastases. Metastatic sites included lung (n = 26, 39%), bone (n = 7, 10%), lymph node (n = 6, 9%), soft tissue (n = 4, 6%), adrenal gland (n = 2, 3%), liver (n = 1, 1%), heart (n = 1, 1%) and pancreas (n = 1, 1%). Seventeen patients (25%) died with disease, 19 patients (28%) were alive with disease and 31 patients (46%) were no evidence of disease.
Histological Examination
Pathology specimens of all the patients' tumors were obtained by incisional biopsy or surgical resection after the imaging studies, and histologic slides were prepared by two expert pathologists for diagnosis. Each tumor was staged according to the TNM classification of UICC and AJCC staging protocol for bone and soft-tissue sarcomas (15,16). Whenever necessary, immunohistochemical staining was carried out to confirm the diagnosis or tumor type according to the WHO classification system (17). The histologic grade of the tumor was determined using a three-grade system in which tumor differentiation, tumor necrosis and MIB-1 LI were given a score of 0, 1, 2 or 3, respectively, and the scores were added together (18). Lesions with MIB-1 LI of 0–9, 10–29 and >30% were assigned MIB-1 scores of 1, 2 and 3, respectively. The three separate scores were added together to produce a combined grade: lesions whose total score was 2 or 3 were classified as Grade 1, those whose total score was 4 or 5 were clarified as Grade 2 and those whose total score was 6, 7 or 8 were clarified as Grade 3. According to this MIB-1 system, tumors were assigned Grades 1–3.
Immunohistochemical Analysis of Glut-1
Immunohistochemical analysis was performed using the labeled streptavidin–biotin method and tissue sections from paraffin blocks. The sections were dewaxed, rehydrated and moistened, then pretreated in an autoclave before being incubated with an affinity-purified goat polyclonal anti-Glut-1 antibody (A3536; diluted 1:500; DakoCytomation). The intensity of Glut-1 staining was quantified with regard to the percentage of cells stained. It was scored as 0 (0%), 1 (1–9%), 2 (10–29%) or 3 (>30%). The results were evaluated by an expert pathologist who was unaware of the clinical status of the patients. The sections were examined using a multi-head microscope, and a consensus judgment was adopted based on Glut-1 staining score of the tumor. The staining scores of 2 and 3 were regarded as indicators of the overexpression of Glut-1.
Statistical Analysis
For the study of the prognostic value, overall survival (OS) was chosen as an end point. OS was defined as the time from diagnosis to death from any cause. Univariate regression analysis was performed to assess the value of all the prognostic factors for the prediction of OS by comparing Kaplan–Meier os curves and carrying out log-rank tests. Kruskal–Wallis test was performed to compare the relations between MIB-1 grade and Glut-1 intensity. Multivariate proportional hazards (Cox) regression analysis was used to test the independency of established prognostic factors for the prediction of OS. Differences and correlations at a P-value of <0.05 was considered statistically significant. All data analyses were performed using SPSS 12.0J (SPSS, Chicago, IL, USA).
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RESULTS |
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The characteristics of 67 patients (mean age, 43 years; range, 8–79 years) are shown in Table 1. Most frequent histologic types were osteosarcoma (n = 16, 24%) followed by pleomorphic malignant fibrous histiocytoma (n = 12, 18%). The tumors were located in the trunk in 37 patients (55%) and in the extremity in 30 patients (45%). The largest diameter of the tumor is 9.4 cm (range, 1.2–24.0 cm). Twenty-two bone sarcomas (33%) and 45 soft-tissue sarcomas (67%) were also included in Table 1. Tumor stage was IA (n = 2, 3%), IB (n = 4, 6%), IIA (n = 14, 21%), IIB (n = 9, 13%), III (n = 12, 18%), IV (n = 18, 27%), IVA (n = 4, 6%) and IVB (n = 4, 6%).
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The univariate analysis results are summarized in Table 2. The presence of metastasis (P = 0.0030) and the treatment without surgical resection (P = 0.0010) were significantly associated with poor OS. Age, gender, tumor type, anatomical site (trunk or extremity), tumor size and treatment modality had no prognostic value.
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Mitotic grades were Grade 1 (n = 8, 12%), Grade 2 (n = 11, 16%) and Grade 3 (n = 48, 72%). The median MIB-1 index of tumor was 32.5%. MIB-1 grades were Grade 1 (n = 7, 10%), Grade 2 (n = 10, 15%) and Grade 3 (n = 50, 75%). On the basis of pathologic examinations, it was found that tumor differentiation (P = 0.0165), necrosis (P = 0.0399), mitotic index (P = 0.0071) and MIB-1 grade (P = 0.0311) were associated with a trend toward poor OS.
Glut-1 expression (Figs 1
–3) was found in 64 patients (96%) and their intensity was 1 (n = 8, 12%), 2 (n = 15, 22%) and 3 (n = 41, 61%). Glut-1 immunostaining was absent in three tumors (4%): two well-differentiated liposarcomas and one clear cell chondrosarcoma. Along with Glut-1 expression in cytoplasm of tumor cells, specific Glut-1 expression was also seen in erythrocytes, perineurium of the peripheral nerves and lymphocytes in the germinal zone. Glut-1 overexpression was significantly associated with the histologic grade by Kruskal–Wallis test (P < 0.0001) (Table 3). No significant difference in Glut-1 staining intensity was found among the other variables, including age, gender, anatomical site (trunk or extremity), tumor size and the presence of local recurrence or metastasis and treatment modality. On the basis of univariate analysis, it was found that Glut-1 overexpression was significantly associated with poor OS (P = 0.029, Fig. 4).
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Multivariate analysis shows that the presence of metastasis is associated with significantly poor OS (P = 0.031, Table 4) than those without metastasis. It also shows that the anatomical site (trunk or extremity), the presence of metastasis, treatment without surgical resection, tumor size, treatment modality, tumor differentiation, necrosis, mitotic index, MIB-1 grade and Glut-1 overexpression were not independently associated with OS. When we excluded the presence of metastasis from variables, mitotic index was significant indicator of poor OS [hazard ratio, 8.709; 95% confidence interval 1.980–38.319; P = 0.004).
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DISCUSSION |
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This study suggests three notable features. First, Glut-1 overexpression is a possible adverse prognostic factor similar to the presence of metastasis, treatment without surgical resection, tumor differentiation, necrosis, mitotic index and MIB-1 grade. Secondly, the presence of metastasis is independently associated with poor prognosis in multivariate analysis. Thirdly, there is significant correlation between Glut-1 intensity and MIB-1 grade. Our study is the first that focusing on the relations between Glut-1 expression and prognosis in patients with bone and soft-tissue sarcomas.
Glut-1 expression has been investigated in a variety of tumors (12–14), however, its expression in bone and soft-tissue sarcomas has rarely studied. The aim of this study was to determine whether the intensity of Glut-1 expression in a tumor could serve as a surrogate marker of the survival. On the basis of this study, it was found that Glut-1 overexpression is associated with OS in the univariate analysis. However, it is unknown whether Glut-1 overexpression is more predictive of OS than other variables. Therefore, the immunohistochemical results support the significance of Glut-1 expression as a biomarker of poor prognosis in patients with bone and soft-tissue sarcomas. Glut-1 antibody is achieved easily and inexpensively, and Glut-1 staining can be done as a part of routine pathologic procedure.
Our results suggested that glucose transport by Glut-1 plays an important role in progression of bone and soft-tissue sarcoma. Besides, glucose transport and metabolism provide crucial prognostic information, they have the potential of being future treatment targets. Inhibition of glucose transport is investigated in some cancer cells, for example, inhibition of glucose transport by cytochalasin-B presented increased gemcitabine-induced apoptosis in hepatoma cells (30). Investigation about the inhibition of glucose transport in bone and soft-tissue sarcomas is challenges for the future.
Glut-1 expression is the common mediator of glucose uptake in malignant tumors (12–14). However, Glut-1 immunostaining was absent in three tumors (4%) in our study: two well-differentiated liposarcomas and a clear cell chondrosarcoma. Glucose transporter proteins other than Glut-1 exist and expresses in various histologic kinds of malignant tumor (31). It is possible that Glut-1 negative tumors would have been positive for other glucose transporters.
The present study had limitations. Follow-up duration of our study is relatively short to calculate patient OS. Whether Glut-1 overexpression adds original information to several prognostic variables requires a further evaluation in an ongoing long-term study. Glut-1 overexpression has a poor prognostic significance in the univariate analysis. This finding to some extent validates our study population because Glut-1 is an excellent indicator of tumor grade and one of the most important prognostic factors in patients with soft-tissue sarcomas treated with combination therapy. However, Glut-1 overexpression was not independently associated with poor prognosis in our study. Short duration of follow-up in our study may affect the results of multivariate analysis. Treatment regimens and duration were not the same for all the patients. Since combination therapy in our study is eligible for patients with bone and soft-tissue sarcomas, this might be biased with the study analysis.
In conclusion, Glut-1 overexpression could be a negative prognostic factor in patients with bone and soft-tissue sarcomas. These findings support the concept of pretherapeutic stratification with Glut-1 immunostaining to identify high-risk patients and propose a more risk-adapted approach of treatment in patients with bone and soft-tissue sarcomas.
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FUNDING |
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This work was supported in part by grants from Scientific Research Expenses for Health and Welfare Programs and the Grant-in-Aid for Cancer Research from the Ministry of Health, Labour and Welfare.
Conflict of interest statement
None declared.
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References |
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