Japanese Journal of Clinical Oncology 33:613-619 (2003)
© 2003 Foundation for Promotion of Cancer Research
Intratumor Microvessel Density in Biopsy Specimens Predicts Local Response of Hypopharyngeal Cancer to Radiotherapy
1 Pathology Division, National Cancer Center Research Institute East, Kashiwa, Chiba, 2 Department of Head and Neck Surgery, National Cancer Center Hospital East, Kashiwa, Chiba, 3 Pathology Division, Graduate School of Medicine, University of Tokyo, Tokyo and 4 Laboratory of Cancer Biology, Graduate School of Frontier Science, University of Tokyo, Kashiwa, Chiba, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Background: The aim of this retrospective study was to identify reliable predictive factors for local control of hypopharyngeal cancer (HPC) treated by radiotherapy.
Methods: A cohort of 38 patients with HPC treated by radical radiotherapy at the National Cancer Center Hospital East between 1992 and 1999 were selected as subjects for the present study. Paraffin-embedded pre-therapy biopsy specimens from these patients were used for immunostaining to evaluate the relationships between local tumor control and expression of the following previously reported predictive factors for local recurrence of head and neck cancer treated by radiotherapy: Ki-67, Cyclin D1, CDC25B, VEGF, p53, Bax and Bcl-2. The predictive power of microvessel density (MVD) in biopsy specimens and of clinicopathologic factors (age, gender and clinical tumor-node-metastasis stage) was also statistically analyzed.
Results: Twenty-five patients developed tumor recurrence at the primary site. Univariate analysis indicated better local control of tumors with high microvessel density [MVD 
median (39 vessels/field)] than with low MVD (< median, P = 0.042). There were no significant associations between local control and expression of Ki-67 (P = 0.467), Bcl-2 (P = 0.127), Bax (P = 0.242 ), p53 (P = 0.262), Cyclin D1 (P = 0.245), CDC25B (P = 0.511) or VEGF (P = 0.496). Clinicopathologic factors were also demonstrated to have no significant influence on local control (age, P = 0.974; gender, P = 0.372; T factor, P = 0.602; N factor, P = 0.530; Stage, P = 0.499).
Conclusion: Microvessel density in biopsy specimens was closely correlated with local control of HPC treated by radiotherapy.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Radical radiotherapy or chemoradiotherapy of hypopharyngeal cancer (HPC) has the potential advantage over surgery of less cosmetic and functional loss. However, if it fails, the alternative curative surgery will be delayed and the complications of treatment will increase. Although it is important for clinicians to determine if an individual with HPC should be treated by radiotherapy or chemoradiotherapy as initial therapy or the main modality, there is no generally applicable indicator of the radiocurability of HPC available as yet.
One potential biological marker for radiocurability is surviving fraction at 2 Gy (SF2), which reflects intrinsic tumor radiosensitivity, but studies in this area have not led to any consensus that this can be clinically used as a predictor of tumor recurrence (1,2). As a result of a recent increase in our knowledge of tumor molecular biology, interest is now being focused on molecules involved in cell signal transduction, cell-cycle control, tumor angiogenesis and apoptosis pathways, and there is a long list of candidate molecules that includes p53, Bax, Bcl-2, EGFR, VEGF, Cyclin D1 and CDC25B (3–8).
Tumor hypoxia accounts for the major portion of the radioresistance mechanism in tumors treated by irradiation with low energy transfer (LET), including X-rays and 
-rays, and indicators of tumor hypoxia should predict the radioresponse. However, only a few papers have assessed the hypoxia indicators, hypoxia induced factor-
(HIF-) or tumor PO2, as prognostic factors after radiotherapy (9–11).
Since tumor radiosensitivity is thought to be influenced by multiple factors involved in several cell survival pathways, we investigated the predictive power of the following factors for local control rate of HPC treated by radiotherapy or chemoradiotherapy: Ki-67 as a proliferative factor; Cyclin D1 and CDC25B as cell cycle modifiers; p53, Bcl-2 and Bax as apoptosis factors; and vascular endothelial growth factor (VEGF) as an angiogenesis factor, all of which were previously reported to be predictive for radiosensitivity of head and neck cancer. In addition to these markers, we also investigated whether tumor microvessel density (MVD) can be used as a potential hypoxia marker to predict tumor local recurrence in HPC. We also assessed the clinicopathologic factors of patients which might affect local tumor control by radiotherapy.
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PATIENTS AND METHODS |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Patients
A cohort of 38 patients with HPC treated by radical radiotherapy or chemoradiotherapy at the National Cancer Center Hospital East between 1992 and 1999 were selected as subjects for this study. Required inclusion criteria included: (i) previously untreated, (ii) local control followed up for more than 6 months, (iii) no death from intermittent disease, (iv) biopsy specimen adequate for immunohistological analysis and (v) no further treatment at the primary site during the follow-up period. The characteristics of all 38 cases are listed in Table 1. A total of 43 specimens were used for analysis (two specimens each were taken from five cases).
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All 38 patients received curative dose of 60–72 Gy at the primary site. TNM classification was performed according to the criteria of the Union International Contre le Cancer (UICC). All biopsy specimens were taken from the primary sites before treatment and diagnosed as squamous cell carcinoma. Twenty-five patients developed recurrence at the primary site. The mean local disease-free interval was 14.9 months (range, 1–94.2 months).
Treatment
Of the 38 patients, 17 were treated by conventional fractionated radiotherapy and 21 were given two courses of 30 Gy radiation with concurrent administration of cisplatin (bolus injection of 40 mg/m2 on days 1 and 8) and 5-FU (continuous infusion of 200 mg/m2 on days 1–4 and 8–11) with 2 weeks rest. The dose in the second course could be raised to a maximum of 42 Gy according to the tumor response.
Immunohistochemical Staining
The immunohistochemical staining method has been described elsewhere (12). Formalin-fixed, paraffin-embedded specimens were cut into 4 µm-thick sections, then deparaffinized in xylene and dehydrated through graded alcohol. Endogenous peroxidase activity was inhibited by immersion in 0.3% H2O2 for 20 min. For antigen retrieval, the sections were heated in a microwave oven (750 W, 20 min in citrate buffer) or autoclaved at 121°C for 10 min (for anti-Bax). Non-specific conjugation was blocked with 2% normal bovine serum. The slides were incubated overnight at 4°C with the following different antibodies: (i) an anti-Ki-67 antibody (MIB-1; 1:50 dilution; DAKO, Glostrup, Denmark), (ii) an anti-Bax antibody (1:50 dilution; Calbiochem, Cambridge, MA), (iii) an anti-Bcl-2 antibody (1:40 dilution; DAKO), (iv) an anti-Cyclin D1 antibody (1:400 dilution; Santa Cruz, Santa Cruz, CA), (v) an anti-CDC25B antibody (1:100 dilution; Santa Cruz), (vi) an anti-VEGF antibody (1:100 dilution; Santa Cruz), (vii) an anti-p53 antibody (1:20 000 dilution; Nichirei, Chiba, Japan) and (viii) an anti-CD31 antibody (1:50 dilution; DAKO). After washing in PBS, tissues were incubated with biotin-labeled anti-rabbit or anti-mouse second antibodies (Vector Laboratories Inc., Burlingame, CA) for 30 min at room temperature and then reacted with streptavidin–biotin horseradish peroxidase complex (DAKO) for 30 min. The reaction products were visualized by immersing the slides in freshly prepared diaminobenzidine solution for 5–20 min and counterstained with hematoxylin before dehydration and mounting.
Evaluation for Immunostaining
All immunostained tissue sections were evaluated in a coded manner without knowledge of the clinical and pathological parameters. To evaluate Ki-67, p53 and Cyclin D1, the number of brown-stained tumor nuclei per total number of tumor cells in the most highly stained area on each slide was counted in the selected microscopic field at x200. A total of 571–1000 tumor cells were examined in each specimen. As in previous studies (12), when >40% of tumor cells were stained positive Ki-67, the tumor was evaluated as positive. For p53 and Cyclin D1, the tumor was considered positive when 10% and 30%, respectively, of the cells were stained positive. Bcl-2 and CDC25B staining was localized to the cell cytoplasm, and sections in which >10% of the tumor cells stained positive were categorized as positive. VEGF and Bax immunostaining was semi-quantitatively rated on a 4-grade scale (0, +, ++, +++) according to the intensity of the reaction and extent of staining and patients were divided into two groups (0, + versus ++, +++) for statistical analysis. CD31-stained sections were scanned at low magnification (x40) to determine areas with the highest number of microvessels (hot spots). Microvessels were counted at a magnification of x200 in two hot spots on each section and MVD was calculated as the average of the two measurements. For the five cases with two specimens, hot spots were counted in each specimen and the average of the highest two was recorded as MVD.
Statistical Analysis
Univariate analysis for tumor-free survival was performed using log-rank tests. The survival curves were calculated by the Kaplan–Meier method. P < 0.05 was considered significant. Statistical analysis was performed with the Statistica package (Statsoft, Tulsa, OK).
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RESULTS |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Clinicopathologic Factors and Local Control of HPC Treated by Radiotherapy Alone or by Chemoradiotherapy
The cohort was composed of 35 men (92%) and three women (8%) with a mean age at the time of diagnosis of 62.8 years (range 41–83, Table 1). In 31 cases the tumor was located in pyriform, in six cases in posterior wall (15.8%) and in one case in post cricoid (2.6%). Twenty-one patients had an early T classification (T1 and T2: 55.3%). Approximately two thirds of the patients had a positive N classification (N1–N3: 71.1%), and the majority of cases were in an advanced clinical stage (stage III stage IV: 81.6%). Univariate analysis showed no significant associations between local control and any of the clinicopathologic parameters (Table 2) and no relation between local control and concurrent administration of low dose of 5-FU and cisplatin with radiotherapy (Table 2).
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Immunohistological Factors and Local Control by Radiotherapy or Chemoradiotherapy
Positive staining for Ki-67, p53 (Fig. 1A), Cyclin D1 (Fig. 1B), CDC25B, Bax, Bcl-2 (Fig. 1C) and VEGF was observed in 26, 16, 21, 24, 20, 10 and 16, respectively, of the 38 cases (68%, 42%, 55%, 61%, 53%, 26% and 42%, respectively). In the univariate analysis, none of these factors was significantly related to local control by radiotherapy/chemoradiotherapy (Table 3).
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MVD and Local Control by Radiotherapy or Chemoradiotherapy
After immunostaining with anti-CD31 antibody, brown-stained lumens in the tumor tissue were counted as blood vessels (Fig. 1D). MVD ranged from 5 to 74 microvessels per field (= 0.391 mm2). Since the median MVD was 39 microvessels/field, the tumors were divided into a high MVD group (MVD
median) and a low MVD group (MVD < median). Univariate analysis showed a significant relation between higher MVD and better local control by radiotherapy (P = 0.042). Considering that five cases were analyzed with two specimens each there may be bias of MVD count between the two specimens. We randomly divided these specimens into two groups and performed the univariate analysis for local tumor-free survival of 38 cases, respectively. The results from these two groups were the same as we had initially found (P = 0.042). In fact, both groups had the same median MVD count as the first, which will not influence the result of primary statistical analysis (Table 4).
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Survival Curves According to MVD by Kaplan–Meier Analysis
Since univariate analysis showed that only MVD was correlated with local control, we performed the Kaplan–Meier analysis to assess the predictive value of MVD for local control of HPC treated by radical radiotherapy or by chemoradiotherapy. The results are shown in Figure 2. The local control rate 3 years after treatment was 58% among those with high MVD, as opposed to only 21% among those with low MVD (P = 0.042).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Few studies have specifically assessed the radiosensitivity of HPC. Our knowledge about predictive factors for the radioresponse of HPC basically comes from previous studies on various sites of head and neck cancers. In the current study, we assessed the predictive value of factors that have been reported to be related to the radiosensitivity of head and neck cancer in the area of HPC. An important result of this study was that local control of HPC after radiotherapy or chemoradiotherapy is strongly associated with the intratumor MVD of biopsy specimens, suggesting that the MVD of biopsy specimens may be of help in predicting the radioresistance of HPC.
It is well known that tumor hypoxia causes radioresistance of solid tumors, and indicators of tumor hypoxia have long been thought of as predictive factors of tumor radioresistance. Measurement of PO2 obtained with polargraphic electrode has been reported to be a predictor of the therapeutic response of head and neck cancers to radiotherapy (10,11). However, its invasive nature restricts measurements to easily accessible tumors and may impede their clinical application. HIF1- is a key transcription factor induced by hypoxia and regulates the pathways of angiogenesis, glycolysis, growth-factor signaling and other essential adaptive responses to hypoxia. Aebersold et al. reported expression of HIF-1 in oropharyngeal cancer to be an independent predictive factor of tumor radioresponse (9). In addition, a study on cervical cancer xenografts suggested that the spatial distribution of HIF-1 colocalized with the nitroimidazole hypoxia marker EF5 and warranted further study of the relationship between HIF-1 and radiosensitivity (13). Tumor cells obtain oxygen and nutrients from neighboring blood flows. As early as 1955, Thomlinson and Gray found that oxygen diffusion from blood vessels to tumor tissues has a distance limitation of about 150 µm (14), which suggested tumor oxygen profile is determined by its vasculature. In fact, we have shown that intratumor MVD is correlated with local control rate in laryngeal cancer treated by radiotherapy (12). In the present study, we also showed that MVD is a predictor for the local control of radiation-treated HPCs.
Although many studies, including the present one, have shown that higher MVD in tumor biopsy specimen significantly correlates with better radioresponse, some investigators have argued that, since angiogenesis is induced by hypoxia, neovasculature should be taken as an indicator of hypoxia (15). In the present study, newly-formed vessels, which appeared as single endothelial cells in the section, were excluded from the vessel count. An area with active angiogenesis may not have a high vessel count in this study. Although the tumor vascular network of solid tumors holds more non-functional vessels than that of normal tissue, for a given type of tumor, we should be able to assume that a highly vascularized one comprises more oxygenated cells in a given area than a poorly vascularized one. However, little data available to date can provide direct evidence for this postulation. Study focusing on the relation of MVD and tumor virtual oxygen profile is needed to clarify this problem. MVD heterogeneity in tumors is another important issue awaiting further study. In the present study, although analyses using different groups with one of two alternative specimens yielded the same result, MVD hotspots between two specimens from the same tumor did show differing counts (Table 4). This raised the question of MVD heterogeneity between tumors and their biopsy specimens. In fact, the problem is not only about MVD. It will remain a crucial issue for pathologists to decide whether histological characteristics and molecular markers in biopsy specimen are able to represent the original tumor. Surgically treated cases should be examined in future study to analyze the differences of these factors in tumors and their biopsy specimens. In the present study, we also compared MVD in different T classifications as well as clinical stages and found there was no significant difference between each group (data not shown). Since this study only analyzed a cohort of 38 patients of which 31 patients were in stage III and stage IV, a larger cohort for investigation is needed to elucidate the relation between MVD and tumor progression.
In addition to hypoxia, radioresistance of tumor cells also influences the results of radiotherapy, and their surviving fraction at 2 Gy (SF2) has been demonstrated to be useful in comparing radiosensitivity between different tumor types (16,17). However, the results for predicting the radioresponse of an individual patient are not so convincing (1,2). In addition, low cell plating efficiency is a well-known problem that compromises its clinical application as in vitro assay. Several studies have assessed the predictive value of Tpot, an assay for tumor proliferative potential, but it failed to predict the outcome of radiotherapy (18,19).
In the present study, detection of several molecules in pretreatment biopsy specimens by immunohistochemical methods was found to be unrelated to local control by radiotherapy or chemoradiotherapy. The molecules tested were the proliferation marker Ki-67, the apoptosis factors p53, Bax and Bcl-2, the cell-cycle modifiers Cyclin D and CDC25B, and vascular endothelial growth factor. These findings were inconsistent with the results reported in earlier studies on head and neck cancers. However, this is not the only differing voice. The associations between the expression of these factors and tumor radiosensitivity are not well established among various studies.
Numerous reports on such clinical factors as tumor stage, T classification and N classification as prognostic factors or predictors in head and neck cancer treated by radiotherapy have yielded conflicting results. The present study showed no significant relation between tumor local control and any of the clinical factors assessed. A large cohort of patients treated by radiotherapy alone may be needed to clarify this problem. Again, because of the limited number of patients, we did not investigate whether the histological differentiation of the tumors correlated with their radioresponse.
In summary, our present study showed that the MVD of pretreated biopsy specimens may be used as a predictor for the local control of HPC treated by radiotherapy alone or chemoradiotherapy. Compared with other predictive assays, MVD count may be a simple and convenient method for clinicians to stratify patients for treatment alternatives. Nevertheless, since the vessel count is a subjective method and results may vary with the observer, we tried to determine the oxygenated area of whole biopsy specimens with an image analysis apparatus as an objective method of predicting tumor hypoxia and the outcome of radiotherapy. The results of a pioneer study on early stage laryngeal cancer are exciting and further study is in progress for its application to prediction of the radiosensitivity of HPC as well as cancers in other areas (20).
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Acknowledgments |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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This work was supported in part by Japan–China Medical Association and Grant for Scientific Research Expenses for Health Labor and Welfare Programs, by the Foundation for the Promotion for Cancer Research, and by 2nd-Term Comprehensive 10-year Strategy for Cancer Control. We thank Dr Tomoyuki Hanaoka and Dr Takashi Sangai for their statistical assistance. We also thank Yoko Okuhara and Chie Okumura for their technical assistance.
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FOOTNOTES |
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+ For reprints and all correspondence: Atsushi Ochiai, Pathology Division, National Cancer Center Research Institute East, Laboratory of Cancer Biology, Graduate School of Frontier Science, University of Tokyo, 6-5-1, Kashiwanoha, Kashiwa, Chiba 277-8577, Japan. E-mail: aochiai{at}east.ncc.go.jp
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REFERENCES |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Received August 26, 2003; accepted November 18, 2003
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