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Intratumoral microvessel density predicts local treatment failure of radically irradiated squamous cell cancer of the oropharynx
Int. J. Radiation Oncology Biol. Phys., Vol. 48, No. 1, pp. 17–25, 2000 Copyright © 2000 Elsevier Science Inc. Printed in the USA. All rights reserved 0360-3016/00/$–see front matter
PII S0360-3016(00)00573-3
CLINICAL INVESTIGATION
Head and Neck
INTRATUMORAL MICROVESSEL DENSITY PREDICTS LOCAL TREATMENT FAILURE OF RADICALLY IRRADIATED SQUAMOUS CELL CANCER OF THE OROPHARYNX
STEFAN
DANIEL M. AEBERSOLD, M.D.,* KARL T. BEER, M.D.,* JEAN LAISSUE, M.D.,† HUG,‡ ATTILA KOLLAR,* RICHARD H. GREINER, M.D.,* AND VALENTIN DJONOV, M.D.‡
*Department of Radiation Oncology, †Institute of Pathology, and ‡Institute of Anatomy, University of Berne, Berne, Switzerland Purpose: To determine the predictive value of intratumoral microvessel density (IMD), and of the expression of p53, vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1) for the radiocurability of patients with squamous cell cancer of the oropharynx. Materials and Methods: 139 patients with squamous cell cancer of the oropharynx were radically irradiated (median dose, 74 Gy) between 1991 and 1997. Biopsies from 100 patients were processed for immunohistochemistry. IMD was determined in hot spot areas of tissue stained with anti-CD31 at a magnification of ⴛ200. Staining for p53 was considered positive if more than 10% of the cell nuclei overexpressed the protein. Immunostaining of VEGF and TSP-1 was assessed semiquantitatively. Results: Increasing IMD (range, 54 –282) was strongly correlated with incomplete remission of both the primary tumors (p ⴝ 0.01) and lymph node metastases (p ⴝ 0.02). Moreover, multivariate Cox regression analysis revealed local failure-free survival to decline with increasing IMD (IMD continuous: risk ratio ⴝ 1.01 per increase of 1 microvessel, p ⴝ 0.0001; IMD categorical: < 80: baseline, 81–110: risk ratio ⴝ 2.71, 111–130: risk ratio ⴝ 4.55, > 130: risk ratio ⴝ 13.01). Neither the expression of p53, nor that of VEGF or TSP-1 was associated with the treatment outcome or IMD, but VEGF and TSP-1 expression were positively correlated (p ⴝ 0.02). Conclusion: IMD represents a powerful and independent predictive factor for local treatment failure in radically irradiated patients with squamous cell cancer of the oropharynx. © 2000 Elsevier Science Inc. Head and neck cancer, Radiotherapy, Predictive factor, Angiogenesis, Microvessel density.
INTRODUCTION
radiation effect, either in terms of tumor oxygenation (5), of the vasculotoxic effects of X-rays (6) or of the anti-apoptotic properties of angiogenic factors (7–9). Nonetheless, few clinical reports have assessed angiogenic activity as a predictive factor in radiation therapy and these are contradictory (10 –14). Angiogenesis is closely regulated by a broad range of stimulators and inhibitors (15). p53, which is essentially involved in cell cycle control, DNA repair and the regulation of apoptosis (16), also contributes to the regulation of angiogenesis: Loss of functional p53 leads to reduced expression of thrombospondin-1 (TSP-1) (17), a key inhibitor of angiogenesis (18). Mutant p53 has been shown to upregulate the expression of vascular endothelial growth factor (VEGF) (19), which has a pivotal role in triggering angiogenesis (20). To investigate whether or not the angiogenic phenotype
It is now generally accepted that angiogenesis is crucial both for the growth of a primary tumor and for the development of distant metastases (1). Accordingly, parameters of angiogenic activity, such as intratumoral microvessel density (IMD) and the expression of angiogenic factors, have been linked to the prognosis of a broad spectrum of cancers (2), including squamous cell cancer of the head and neck (3). In addition to the assessment of prognosis, prediction of the response probability to a certain therapy would also be of value, in helping to individualize, and hence to optimize, the therapeutical approach. This applies particularly to advanced cancer of the head and neck, the treatment success of which is difficult to predict, morbidity from both surgery and radiation being disconcertingly high (4). A considerable body of evidence points toward the existence of an interaction between tumor angiogenesis and
This work was supported by the Bernese Cancer League and the Bernese Radium Foundation. Presented in part at the 41st Annual Meeting of ASTRO, San Antonio, Texas, 1999, and at ECCO 10, Vienna, Austria, 1999. Accepted for publication 1 November 1999.
Reprint requests to: Dr. V. Djonov, Institute of Anatomy, University of Berne, Bu¨hlstrasse 26, 3000 Berne 9, Switzerland. E-mail: [email protected] Acknowledgment—We would like to thank Dr. P. H. Burri, Chairman of the Institute of Anatomy, University of Berne, for his generous support and B. de Breuyn for her excellent technical assistance. 17
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Table 1. Clinicopathologic characteristics and therapy results No. of patients (total ⫽ 100)
Parameter T stage
N stage
Histological grade Site of primary tumor
Chemotherapy Complete remission
1 2 3 4 0 1 2 3 1 2 3 Tonsillar fossa Base of tongue Faucial arch Lateral/posterior wall Vallecula epiglottica No Yes Primary tumor Lymph nodes
Local treatment failure Progression after of non-CR* Relapse after CR† Distant metastases
4 8 32 56 34 14 42 10 11 63 26 40 29 21 6 4 73 27 81 34 of 66 42 19 23 14
* Non-CR ⫽ incomplete remission (includes partial remission, stable disease and progressive disease). † CR ⫽ Complete remission.
of head and neck squamous cell cancer relates to radiocurability, we performed a retrospective study on a well-defined series of patients with squamous cell carcinoma of the oropharynx who had undergone radical irradiation (21). IMD, as well as the immunoreactivity for VEGF, TSP-1, and p53, were correlated with the response to therapy and locoregional recurrence. PATIENTS AND METHODS Patients Between October 1991 and December 1997, 139 patients with biopsy-proven squamous cell cancer of the oropharynx were radically irradiated at the Department of Radiation Oncology, the Inselspital, Berne. 39 patients were excluded from the present study due to the small size of the biopsy (16 patients), previous or synchronous malignancies (12 patients), irradiation after neck dissection (7 patients), intercurrent death during therapy (2 patients), lack of follow-up (1 patient), and presence of distant metastases at the onset of treatment (1 patient). The main clinical characteristics of the series of patients are shown in Table 1. Most of the patients manifested an advanced stage of disease. The detailed UICC-TNM clinical stages in patients were: T1N0 in 3, T1N1 in 1, T2N0 in 6, T2N1 in 1, T2N2 in 1, T3N0 in 13, T3N1 in 4, T3N2 in 11, T3N3 in 1, T4N0 in 12, T4N1 in 8, T4N2 in 30, and T4N3 in 9. Tumors of the tonsillar
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fossa (40%), base of tongue (29%) and faucial arch (21%) predominated. All biopsies were taken before radiotherapy had started. Histopathological diagnosis and grading of biopsies were reviewed by an experienced pathologist (J.L.). Most of the patients had Grade II tumors (Grade I in 11 cases, Grade II in 63, and Grade III in 26). Therapy and response assessment All patients were prepared for radiotherapy by placement of a thermoplast mask, planning CT, two- or three-dimensional-based planning and control by simulator and portal vision imaging device. Megavoltage radiation therapy was administered by a linear accelerator in daily fractions, 5 times a week for 5– 8 weeks. A median total dose of 74 Gy (range, 54 – 80.5) was delivered, 97% of patients receiving at least 66 Gy. Twenty-seven patients underwent concomitant chemotherapy, which consisted mainly of a cisplatin (CDDP) -containing regimen: 12 patients were subjected to CDDP monotherapy, 13 patients had a combination with CDDP and 5-fluorouracil, 1 patient received methotrexate, and 1 carboplatin. In patients with complete remission of the primary tumor and with clinical or radiological evidence of persistent lymph node metastasis, a salvage neck dissection was performed. Baseline studies included physical examination, chest X-rays, panendoscopy of the upper aerodigestive tract, and magnetic resonance imaging or computed tomography of the neck. During treatment, patients were examined on a weekly basis. The response to treatment was assessed 2– 4 weeks after the end of therapy, primary tumor and the lymph node metastases being evaluated separately. Two categories were defined: complete remission (CR: complete disappearance of tumor manifestation) and incomplete remission (non-CR: including partial remission, stable disease, or progressive disease). After treatment, all patients underwent clinical examination and imaging on a regular basis. Immunohistochemistry A total of 151 paraffin-embedded biopsies from the 100 patients were processed for immunohistochemistry; 3-mthick sections were transferred to gelatinized micro-slides and air-dried overnight at 37°C. They were dewaxed in xylene (three changes), rehydrated in ethanol, and rinsed in Tris-buffered saline (TBS: 50 mM Tris/HCl, pH 7.4, containing 100 mM sodium chloride [two changes]). Endogenous peroxidase activity was suppressed by treatment with 0.3% hydrogen peroxide for 10 min. Prior to incubation with antibodies, sections were bathed in 0.01 M sodium citrate, pH 6.0 (or TrisHCl, pH 1.0, in the case of antiTSP-1) and heated in a microwave oven (180 W) for 15 min. In the case of mouse anti-CD31, this step was preceded by treatment with trypsin ([Difco Lab., Detroit, MI, USA] 0.2 mg/mL in TBS/CaCl2 buffer) for 10 min at 37°C. After blocking of unspecific binding in TBS containing 1% casein (SIGMA 8654) for 10 min, sections were incubated with the first antibody diluted in TBS: mouse anti-CD31 1:20 (JC/
Microvessel density and failure of radiation therapy in oropharyngeal cancer
70A, M-0823; Dako, Glostrup, Denmark), mouse anti-p53 1:200 (DO-7, M7001; DAKO, Glostrup, Denmark), rabbit anti-VEGF 1:200 (sc-152; Santa Cruz Biotechnology, Santa Cruz, CA, USA), or mouse anti-TSP-1 1:25 (Ab-7/MS 590, NeoMarkers, Fremont, CA, USA) for 15 h at 4°C. Sections were exposed to an affinity-purified biotinylated second antibody ([anti-mouse EO 433, anti-rabbit EO 353, Dako, Glostrup, Denmark] diluted 1:200 in TBS) for 45 min at ambient temperature, washed three times in TBS, then treated with the avidin-biotin-complex/horseradish peroxidase (P355, Dako, Glostrup, Denmark) for a similar period at the same temperature. The reaction product was visualized by exposing sections to 3-amino-9-ethylcarbazole or 3.3-diaminobenzidine (Sigma Chemicals Company, St. Louis, Missouri, USA), which were then mounted in Aquatex (Merck, Darmstadt, Germany). Negative controls were performed using nonspecific mouse and rabbit sera. Sections were counterstained with haematoxylin. For a better identification of microvessels, counterstaining of CD31 stained sections was purposely rendered weak. In TSP-1 stained sections, counterstaining was weak due to pretreatment of sections with TrisHCl buffer at low pH.
Quantification IMD. The IMD was assessed according to the standard method defined in a consensus paper (22): CD31-stained sections were scanned at low magnification (⫻10 and ⫻40) for areas with the highest numbers of microvessels (‘hot spots’). Microvessels within three such hot spots were then counted at a magnification of ⫻200 with the aid of an ocular grid (area size: 0.64 mm2). Biopsies with fewer than three evaluable hot spots were excluded. Any endothelial cell or endothelial cell cluster positive for CD31 and clearly separate from an adjacent cluster was considered as a single, countable microvessel. Counting was performed by two independent investigators (D.M.A., V.D). Disagreements (more than 10% difference) were resolved at a discussion microscope. The highest number of vessels per hot spot was selected for further evaluation. For the statistical analysis, IMD was used both as a continuous and as a categorical variable (4 groups: IMD ⱕ 80, 81–110, 111–130, ⬎ 130). p53. Occasional positive staining of tumor cells is interpreted as an accumulation of p53 wild-type protein in response to DNA damage, whereas intense staining of most cells is usually indicative of a mutation (23). In our study, staining for p53 was considered to be positive if more than 10% of the 500 cells counted expressed the protein. VEGF and TSP-1. Immunostaining was assessed semiquantitatively (0, ⫹, ⫹⫹, ⫹⫹⫹). Both the extent of staining (relative number of VEGF-positive cells; extent of extracellular matrix staining with TSP-1) and the intensity of the reaction were taken into account. For the statistical analysis, patients were assigned to one of two categories (0/⫹ vs. ⫹⫹/⫹⫹⫹).
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Statistics The correlation between IMD (as a continuous variable) and each of the other parameters was statistically assessed using the Wilcoxon two-sample test. Correlations between clinical and angiogenesis parameters were evaluated by means of Fisher’s exact test (two-tailed). Variables were correlated with the response of the primary tumor and of the lymph node metastases to treatment (CR vs. non-CR), as well as with local relapse-free survival, local failure-free survival, and overall survival. For the analysis of local relapse-free survival, only patients with CR of the primary tumor were included. Lymph node metastases had also to be in CR or removed by neck dissection for inclusion. Analysis of local failure-free survival considered local tumor progression after non-CR and local relapse after CR as adverse events. Analysis of overall survival embraced any kind of death. Survival was measured from the time when therapy was initiated to that when the first adverse event was detected or to the date of the last check-up. Deaths due to non-tumor-related causes were censored, except for analysis of overall survival. Correlations between variables and the response to treatment were analyzed by implementing the logistic regression method in both the univariate and multivariate fashion, including a backward elimination procedure to remove variables with a p value greater than 0.05. Survival curves were plotted according to the Kaplan–Meier method, the log– rank test being used to determine significant differences between the survival curves. A Cox regression was performed to calculate the risk ratios. For multivariate analyses, all clinical and angiogenesis-related variables were included in a Cox regression model. A backward elimination procedure was performed to remove nonsignificant variables (p ⬎ 0.05). Correlations between variables and the development of distant metastases were calculated according to the logistic regression method. Statistical analyses were performed using the SAS package (Version 6.12; SAS Institute, Cary, NC).
RESULTS Clinical outcome The median follow-up time was 654 days (range, 58 – 2036). CR of the primary tumor was achieved in 81 (81%) patients, and in 34 of the 66 (51.5%) with nodal spread, CR of lymph node metastases was accomplished (Table 1). In 42 of the 100 patients, local failure occurred, this being due either to relapse at the same site after CR (23 patients) or to progression after non-CR (19 patients). 14 patients without CR of cervical lymph node metastases underwent a neck dissection with complete removal of remaining tumor manifestations; they were included in the analysis of local relapse-free survival. 14 patients developed distant metastases.
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Fig. 1. Photomicrographs of immunostained tumoral tissue derived from patients with squamous cell cancer of the oropharynx. (A): Microvessels within a hot spot area, revealing the endothelial cells to be heavily stained with anti-CD31 (magnification ⫻200). Counterstaining with hematoxylin was purposely rendered weak. (B): Overexpression of p53 in ⬎ 10% of tumor cell nuclei (magnification ⫻300). (C): High expression of vascular endothelial growth factor in tumor cells, endothelial cells (arrow heads) and fibroblasts (arrows) (magnification ⫻200). (D): Intense immunostaining for thrombospondin-1 within the extracellular matrix (magnification ⫻300).
Clinical and angiogenesis-related variables IMD was evaluable in 100 patients and ranged from 54 to 282 microvessels per high-power (⫻200) optical field (median, 104; mean 107.7; SD ⫾ 30.1). In more detail, 14 carcinomas had an IMD ⱕ 80, 46 had an IMD between 81 and 110, 21 had an IMD between 111 and 130 and 19 had an IMD ⬎ 130. The pattern of staining for anti-CD31 was similar to that already reported (24): staining was confined to vascular endothelial cells (Fig. 1A); occasionally, inflammatory cells were immunoreactive as well. In each of the 100 evaluable patients, the expression of p53, VEGF, and TSP-1 could be readily assessed. In 67 of the patients, more than 10% of the nuclei were p53-positive (Fig. 1B). VEGF was expressed by tumor cells in 98% of cases (35% ⫹, 56% ⫹⫹, 7% ⫹⫹⫹) and was also observed in endothelial cells and fibroblasts (Fig. 1C). Immunostaining for TSP-1 was detected in 47% of patients (20% ⫹, 21% ⫹⫹, 6% ⫹⫹⫹), the reaction being confined exclusively to the tumoral stroma (Fig. 1D). No obvious correlation existed between IMD and either
the clinical stage (T or N) or the histological grade (Table 2). Likewise, there was no apparent relationship between IMD and either VEGF, TSP-1, or p53 expression. Overexpression of p53 did not correlate with that of either TSP-1 or VEGF. However, tumors with high levels of VEGF immunoreactivity manifested significantly higher amounts of TSP-1 (p ⫽ 0.02; Table 2). Prediction of response to treatment In the univariate analysis, a significant correlation between IMD and the response to radiation was found: the higher the IMD, the lower the probability of CR. This relationship held true both for the response of the primary tumor (p ⫽ 0.01; IMD continuous) and for that of the lymph node metastases (p ⫽ 0.02; IMD continuous). The odds ratio for CR of the primary tumor and of lymph node metastases was 0.97 per increase of 1 microvessel. When IMD was subdivided into four groups, the odds ratios decreased from 0.43 to 0.10 for CR of the primary tumor and from 0.11 to 0.06 for CR of the lymph node metastases in
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Table 2. Correlation between angiogenesis-related variables and patient variables ( p values) IMD* continuous
p53 ⱕ10% vs. ⬎10%
VEGF† 0/⫹ vs. ⫹⫹/⫹⫹⫹
TSP-1‡ 0/⫹ vs. ⫹⫹/⫹⫹⫹
0.83 0.86 0.41 0.45 0.63 0.74
1.00 1.00 0.48 — — —
1.00 1.00 0.16 0.27 — —
0.73 0.64 0.80 0.47 0.02 —
T stage, T1–2 vs. T3–4 N stage, N0 vs. N1–3 Grade, G1 vs. G2–3 p53, ⱕ10% vs. ⬎10% VEGF, 0/⫹ vs. ⫹⫹/⫹⫹⫹ TSP-1, 0/⫹ vs. ⫹⫹/⫹⫹⫹
* IMD ⫽ intratumoral microvessel density. VEGF ⫽ vascular endothelial growth factor. ‡ TSP-1 ⫽ thrombospondin-1. †
the group with the highest microvessel counts (Table 3). No obvious relationship between the response to treatment and either the clinical stages (T or N), histological grade, chemotherapy, p53, VEGF, or TSP-1 was apparent. In the multivariate analysis, only the IMD remained significant during the backward elimination procedure (Table 4). Survival analysis In the univariate analysis, only IMD was significantly correlated to local relapse-free survival, the risk ratio being 1.02 per increase of 1 microvessel (p ⫽ 0.03). When subdivided into four groups, the risk ratio for local relapse increased from 1.56 to 2.89 to 8.91 in the high-risk group (Table 5). Although not significant, a trend toward correlation was also apparent for the T stage of the disease, the risk ratio being 4.89 (p ⫽ 0.12). Local failure-free survival, which considered all kinds of local treatment failures, namely, local relapse and local tumor progression after non-CR, was, likewise, significantly correlated only with IMD, the risk ratio being 1.01 per microvessel. But in this
instance, the level of significance was much higher (p ⫽ 0.0001). When subdivided into four groups, the risk ratio for local failure increased from 2.71 or 4.55 to 13.01 in that with the highest number of vessels (Table 5, Fig. 2). Once again, a trend toward a correlation was observed for the T stage of the disease, the risk ratio being 3.73 (p ⫽ 0.07). In the multivariate Cox regression analysis, which included the backward elimination, IMD remained the only significant variable (Table 4). No significant correlation was apparent between the development of distant metastases and either clinicopathological or angiogenesis-related parameters (Table 6). However, both the univariate and multivariate analysis with backward elimination of nonsignificant variables revealed IMD and, in addition, T stage to be correlated with overall survival (Table 4 and 7). DISCUSSION Intratumoral microvessel density is known to be of prognostic value in many human malignancies, this circum-
Table 3. Univariate analysis (logistic regression) of variables for predicting complete remission of the primary tumor and lymph node metastases CR* of primary tumor Variable Category
OR†
Confidence interval (95%)
T stage, T1–2 vs. T3–4 N stage, N0 vs. N1–3 Grade, G1 vs. G2–3 Chemotherapy, 0 vs. ⫹ IMD‡ continuous IMD categorical ⱕ80 81–110 111–130 ⬎130 p53, ⱕ10% vs. ⬎10% VEGF§, 0/⫹ vs. ⫹⫹/⫹⫹⫹ TSP-1储, 0/⫹ vs. ⫹⫹/⫹⫹⫹
0.35 0.64 1.40 1.49 0.97 – 0.43 0.26 0.10 2.14 0.74 0.76
0.04–2.92 0.21–1.96 0.42–4.67 0.45–4.96 0.95–0.99 – 0.05–3.82 0.03–2.38 0.01–1.08 0.77–5.92 0.26–2.16 0.26–2.25
CR of lymph node metastases p
OR
Confidence interval (95%)
p
0.33 0.44 0.57 0.52 0.01 – 0.45 0.24 0.06 0.14 0.59 0.62
0.00 NA 1.05 1.36 0.97 – 0.11 0.11 0.06 1.44 0.85 1.07
0.00–999.00 NA 0.37–2.97 0.49–3.77 0.95–0.99 – 0.01–0.99 0.01–1.08 0.004–0.92 0.52–4.03 0.31–2.31 0.37–3.09
0.98 NA 0.92 0.55 0.02 – 0.05 0.06 0.04 0.49 0.75 0.91
* CR ⫽ complete remission. OR ⫽ odds ratio. ‡ IMD ⫽ intratumoral microvessel density; OR per increase of 1 microvessel. § VEGF ⫽ vascular endothelial growth factor. 储 TSP-1 ⫽ thrombospondin-1. †
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Table 4. Remaining variables after backward elimination of nonsignificant variables in the multivariate analysis of response to treatment and survival Endpoint
Remaining variable
p
OR/RR*
Confidence interval (95%)
CR† of primary tumor CR of lymph node metastases Local relapse-free survival Local failure-free survival Overall survival
IMD‡ continuous IMD continuous IMD continuous IMD continuous IMD continuous T stage, T1–2 vs. T3–4
0.01 0.02 0.03 0.0001 ⬍0.0001 0.02
0.97 0.97 1.02 1.01 1.02 3.11
0.95–0.99 0.95–0.99 1.00–1.04 1.01–1.02 1.01–1.03 1.17–8.26
* OR/RR ⫽ odds ratio/risk ratio. † CR ⫽ complete remission. ‡ IMD ⫽ intratumoral microvessel density; OR/RR per increase of 1 microvessel.
stance being mainly attributable to the involvement of angiogenesis in the development of distant metastases. However, there exists but little information concerning the importance of angiogenesis-related parameters in predicting the probability of treatment success. In the present study, we have demonstrated IMD to be a powerful tool for predicting the success probability of radiotherapy for squamous cell cancer of the oropharynx. The probabilities for controlling the primary tumor and lymph node metastases were analyzed separately. This was undertaken with a view to focusing on the primary tumor, control of which by radiation is generally deemed essential for a curative treatment strategy in advanced oropharyngeal cancer. Lymph node metastases, on the other hand, can often be approached surgically. The different rates of CR for primary tumors (81%) and lymph node metastases (51.5%) indicate the existence of distinct radiosensitivities at different sites. However, IMD assessed in biopsies from the primary tumor proved to be of predictive value in determining the response to treatment at both sites, the odds ratio being the same in each case (Table
3). Furthermore, the risk of local relapse after CR could also be predicted by IMD (Table 5). A higher level of significance for IMD as a correlating variable was attained in the analysis of failure-free survival, which considered both tumor progression after non-CR and local relapse as adverse events. In a previous study of patients with squamous cell cancer at various sites in the head and neck, low vascularity was likewise revealed to correlate with a higher probability of complete remission (12), but no association between vascularization and local recurrence was demonstrated. This discrepancy between our own findings and those of Gasparini et al. may derive from the large diversity of tumors included in the latter study, our own cohort being strictly confined to individuals with oropharyngeal cancer of identical histological type. Heterogeneity in tumor entities could likewise account for at least some of the discrepant results by Giatromanolaki et al. (14); although the highest local failure rate was observed in patients with the largest number of microvessels, the best treatment results were achieved in individuals with intermediate vessel counts. We, however, report a
Table 5. Univariate analysis (Cox regression) of variables for predicting local relapse-free survival and local failure-free survival Local failure-free survival†
Local relapse-free survival* Variable Category
RR‡
Confidence interval (95%)
p
RR
Confidence interval (95%)
p
T stage, T1–2 vs. T3–4 N stage, N0 vs N1–3 Grade, G1 vs G2–3 Chemotherapy, 0 vs ⫹ IMD§ continuous IMD categorical ⱕ 80 81–110 111–130 ⬎30 p53, ⱕ10% vs. ⬎ 10% VEGF储, 0/⫹ vs. ⫹⫹/⫹⫹⫹ TSP-1#, 0/⫹ vs. ⫹⫹/⫹⫹⫹
4.89 0.79 1.68 0.94 1.02 – 1.56 2.89 8.91 2.03 0.64 0.71
0.65–36.62 0.34–1.84 0.71–4.01 0.37–2.40 1.00–1.04 – 0.34–7.22 0.63–13.40 1.22–65.06 0.69–6.02 0.28–1.48 0.24–2.10
0.12 0.59 0.24 0.90 0.03 – 0.57 0.17 0.03 0.20 0.30 0.54
3.73 1.04 1.11 0.89 1.01 – 2.71 4.55 13.01 0.79 0.78 0.98
0.90–15.54 0.56–1.97 0.57–2.18 0.45–1.77 1.01–1.02 – 0.63–11.75 1.05–19.81 2.68–63.70 0.42–1.49 0.42–1.44 0.48–2.00
0.07 0.89 0.75 0.74 0.0001 – 0.18 0.04 0.002 0.48 0.42 0.96
* considers local relapse after complete remission. considers tumor progression after incomplete remission (partial remission, stable disease and progressive disease) and local relapse after complete remission. ‡ RR ⫽ risk ratio. § IMD ⫽ intratumoral microvessel density; RR per increase of 1 microvessel. 储 VEGF ⫽ vascular endothelial growth factor. # TSP-1 ⫽ thrombospondin-1. †
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Fig. 2. Local failure-free survival (Kaplan–Meier plot) after subdividing intratumoral microvessel density into 4 groups (IMD ⱕ 80, 81–110, 111–130, and ⬎ 130). Local failure considers both tumor progression after incomplete remission (partial remission, stable disease, and progressive disease) and local relapse after complete remission of the primary tumor as adverse events. Risk ratios (RR) for local failure and p values were calculated by performing a Cox regression analysis.
linear increase in the risk for local failure. Such a relationship has also been documented for prostate cancer (11). Beside IMD, T stage was the most important variable for both local relapse-free survival, local failure free-survival and overall survival in terms of risk ratio (4.89, 3.73, and 2.68, respectively; Table 5 and 7). This corresponds well with the fact that T stage has been recognized as decisive for local disease control and survival in head and neck tumors (4). However, the level of significance was not reached in our study for the correlation of T stage with local relapse-free (p ⫽ 0.12)
Table 6. Univariate analysis (logistic regression) of variables for predicting distant metastasis
T stage, T1–2 vs. T3–4 N stage, N0 vs. N1–3 Grade, G1 vs. G2–3 Chemotherapy, 0 vs. ⫹ IMD† continuous p53, 0 vs. ⫹ VEGF‡, 0/⫹ vs. ⫹⫹/⫹⫹⫹ TSP-1§, 0/⫹ vs. ⫹⫹/⫹⫹⫹
RR*
p
1.53 2.07 1.16 1.10 0.99 1.27 0.54 0.41
0.97 0.29 0.81 0.89 0.48 0.70 0.28 0.26
* RR ⫽ risk ratio. IMD ⫽ intratumoral microvessel density; RR per increase of 1 microvessel. ‡ VEGF ⫽ vascular endothelial growth factor. § TSP-1 ⫽ thrombospondin-1. †
and failure-free survival (p ⫽ 0.07), the p value for correlation with overall survival being 0.04. The relatively high p values despite impressively elevated risk ratios reflect most probably the very low number of patients with T1 and T2 tumors (12%) in the study population (Table 1). Table 7. Univariate analysis (Cox regression) of variables for predicting overall survival Overall survival Variable category
RR*
Confidence interval (95%)
p
T stage, T1–2 vs. T3–4 N stage, N0 vs. N1–3 Grade, G1 vs. G2–3 Chemotherapy, 0 vs. ⫹ IMD† continuous IMD categorical ⱕ80 81–110 111–130 ⬎130 p53, ⱕ 10% vs. ⬎10% VEGF‡, 0/⫹ vs. ⫹⫹/⫹⫹⫹ TSP-1§, 0/⫹ vs. ⫹⫹/⫹⫹⫹
2.68 1.46 1.43 1.14 1.02 – 1.43 1.91 3.60 0.65 1.20 1.27
1.03–7.01 0.83–2.55 0.57–3.58 0.63–2.05 1.01–1.02 – 0.54–3.74 0.68–5.38 1.29–10.06 0.38–1.11 0.69–2.09 0.70–2.31
0.04 0.19 0.45 0.66 0.0001 – 0.47 0.22 0.01 0.11 0.52 0.43
* RR ⫽ risk ratio. IMD ⫽ intratumoral microvessel density; RR per increase of 1 microvessel. ‡ VEGF ⫽ vascular endothelial growth factor. § TSP-1 ⫽ thrombospondin-1. †
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Beside its predictive power for local control by radiotherapy, IMD proved to be a prognostic factor too as it was significantly associated with overall survival. However, no correlation of IMD with distant metastasis was found (Table 6). Thus, the prognostic power of IMD must derive from its prediction of locoregional control which is known to be crucial for survival in head and neck cancer (4). Association with locoregional control may also account for the prognostic power of T stage. The multivariate Cox regression analysis revealed the predictive power of IMD to be independent of all other variables: IMD remained the only significant variable during the backward elimination of nonsignificant variables in the analysis both of the response to treatment and of the local relapse-free and failure-free survival. The independence of IMD as a predictive factor was reflected in the absence of significant correlations between clinicopathologic variables, angiogenesis-related parameters and IMD (Table 2). The association between IMD and the risk for lymphatic spread of squamous cell cancer of the head and neck described by Gasparini et al. (25) was not observed in our study. Furthermore, overexpression of p53, which has been shown by the same authors to be correlated with IMD (25) as well as by other investigators in the cases of colorectal (26) and non–small-cell lung cancers (27), was not associated either with IMD or with VEGF or TSP-1 expression in our patients. As previously shown for head and neck cancer in general (28 –30), we found VEGF to be expressed in most patients with squamous cell cancer of the oropharynx. Despite its key role in regulating angiogenesis, we observed no correlation between this factor and IMD (Table 2). Its contribution to vascularization in squamous cell cancer of the head and neck thus remains to be clarified. The significant correlation between VEGF and TSP-1 expression apparent in our series of patients may reflect a negative feedback mechanism, since a VEGF-mediated induction of TSP-1 expression has been observed in bovine retinal endothelial cells (31). And in retinal pigmented epithelial cells, TSP-1 has been shown to upregulate VEGF (32). Whether the mutual regulation of VEGF and TSP-1 represents a general mechanism has yet to be determined. The predictive power of IMD for radiocurability may be explained in several ways. Hypoxia is a major cause of resistance to radiation (5), and because oxygen delivery
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depends upon blood flow, measurement of tumoral vascularity has been deemed to represent a direct means of assessing the tissue’s oxygenation state (10, 33). However, since hypoxia is a key trigger for the induction of angiogenesis (34), the number of microvessels could—theoretically—reflect the degree of tumor hypoxia. This view takes into account the circumstance that tumor vasculature is often dysfunctional, being characterized by sluggish blood flow or shunting (35). Moreover, high-resolution intravital measurements have revealed the absence of correlation between blood flow and perivascular pO2 (36). Hence, the relationship between vascularity and the degree of oxygenation in clinical tumor samples remains to be clarified. Apart from the link between angiogenesis and hypoxia, angiogenic factors per se may interfere with radiosensitivity by inhibiting X-ray-induced apoptosis: VEGF is known to suppress ␥ ray-induced apoptosis in both CMK86 cells and normal haematopoietic stem cells (7). Furthermore, basic fibroblast growth factor has been shown to protect endothelial cells from radiation-induced apoptosis via a protein kinase C mediated mechanism (8), and hepatocyte growth factor/scatter factor protects breast cancer cells from radiation-induced apoptosis by targeting the anti-apoptotic mitochondrial membrane protein Bcl-XL (9). In our study, VEGF expression did not interfere with radiocurability. However, blockage of the VEGF stress response has been recently shown to enhance the tumor-destroying effect of ionizing radiation (37). Hence, VEGF signaling may be a target not only for anti-angiogenesis but also for radiosensitization. In conclusion, our results demonstrate the potential of IMD to predict the radiocurability of oropharyngeal cancer. Its reliability has to be further validated in prospective studies. In addition to their application in individualizing treatment strategies, predictive factors may represent an attractive target for modifying the response to treatment. Indeed, several anti-angiogenic strategies have been successfully explored for their radiosensitizing effect in animal models (38 – 41). The strong predictive power of IMD for radiocurability detected in the present study strengthens the rationale for performing clinical trials that combine anti-angiogenic drugs with radiotherapy.
REFERENCES 1. Folkman J. The role of angiogenesis in tumor growth. Semin Cancer Biol 1992;3:65–71. 2. Fox SB, Harris AL. Markers of tumor angiogenesis: Clinical applications in prognosis and anti-angiogenic therapy. Invest New Drugs 1997;15:15–28. 3. Albo D, Granick MS, Jhala N, et al. The relationship of angiogenesis to biological activity in human squamous cell carcinomas of the head and neck. Ann Plast Surg 1994;32: 588 –594. 4. Vokes EE, Weichselbaum RR, Lippman SM, et al. Head and neck cancer. N Engl J Med 1993;328:184 –194.
5. Horsman MR, Overgaard J. The oxygen effect. In: Steel GG, ed. Basic clinical radiobiology. 2nd ed. London: Arnold; 1997. p. 132–140. 6. Baker DG, Krochak RJ. The response of the microvascular system to radiation: A review. Cancer Invest 1989;7:287– 294. 7. Katoh O, Tauchi H, Kawaishi K, et al. Expression of the vascular endothelial growth factor (VEGF) receptor gene, KDR, in hematopoietic cells and inhibitory effect of VEGF on apoptotic cell death caused by ionizing radiation. Cancer Res 1995;55:5687–5692.
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8. Haimovitz-Friedman A, Balaban N, McLoughlin M, et al. Protein kinase C mediates basic fibroblast growth factor protection of endothelial cells against radiation-induced apoptosis. Cancer Res 1994;54:2591–2597. 9. Fan S, Wang JA, Yuan RQ, et al. Scatter factor protects epithelial and carcinoma cells against apoptosis induced by DNA-damaging agents. Oncogene 1998;17:131–141. 10. Revesz L, Siracka E, Siracky J, et al. Variation of vascular density within and between tumors of the uterine cervix and its predictive value for radiotherapy. Int J Radiat Oncol Biol Phys 1989;16:1161–1163. 11. Hall MC, Troncoso P, Pollack A, et al. Significance of tumor angiogenesis in clinically localized prostate carcinoma treated with external beam radiotherapy. Urology 1994;44:869 – 875. 12. Gasparini G, Bevilacqua P, Bonoldi E, et al. Predictive and prognostic markers in a series of patients with head and neck squamous cell invasive carcinoma treated with concurrent chemoradiation therapy. Clin Cancer Res 1995;1:1375–1383. 13. Zatterstrom UK, Brun E, Willen R, et al. Tumor angiogenesis and prognosis in squamous cell carcinoma of the head and neck. Head Neck 1995;17:312–318. 14. Giatromanolaki A, Koukourakis MI, Georgoulias V, et al. Angiogenesis vs. response after combined chemoradiotherapy of squamous cell head and neck cancer. Int J Cancer 1999; 80:810 – 817. 15. Pepper MS. Positive and negative regulation of angiogenesis: From cell biology to the clinic. Vasc Med 1996;1:259 –266. 16. Agarwal ML, Taylor WR, Chernov MV, et al. The p53 network. J Biol Chem 1998;273:1– 4. 17. Dameron KM, Volpert OV, Tainsky MA, et al. Control of angiogenesis in fibroblasts by p53 regulation of thrombospondin-1. Science 1994;265:1582–1584. 18. Tuszynski GP, Nicosia RF. The role of thrombospondin-1 in tumor progression and angiogenesis. Bioessays 1996;18:71– 76. 19. Kieser A, Weich HA, Brandner G, et al. Mutant p53 potentiates protein kinase C induction of vascular endothelial growth factor expression. Oncogene 1994;9:963–969. 20. Ferrara N, Keyt B. Vascular endothelial growth factor: Basic biology and clinical implications. In: Goldberg ID, Rosen EM, editors. Regulation of angiogenesis (EXS; 79). Basel, Boston, Berlin: Birkha¨user; 1997. p. 209 –232. 21. Beer KT, von Briel C, Lampret T, et al. Predictive significance of reflex otalgia in local radical radiotherapy of oropharyngeal carcinomas. Strahlenther Onkol 1998;174:306 –310. 22. Vermeulen PB, Gasparini G, Fox SB, et al. Quantification of angiogenesis in solid human tumours: An international consensus on the methodology and criteria of evaluation. Eur J Cancer 1996;32A:2474 –2484. 23. Levine AJ, Momand J, Finlay CA. The p53 tumour suppressor gene. Nature 1991;351:453– 456. 24. Parums DV, Cordell JL, Micklem K, et al. JC70: a new monoclonal antibody that detects vascular endothelium associated antigen on routinely processed tissue sections. J Clin Pathol 1990;43:752–757. 25. Gasparini G, Weidner N, Maluta S, et al. Intratumoral microvessel density and p53 protein: Correlation with metastasis in head-and-neck squamous-cell carcinoma. Int J Cancer 1993;55:739 –744.
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26. Vermeulen PB, Roland L, Mertens V, et al. Correlation of intratumoral microvessel density and p53 protein overexpression in human colorectal adenocarcinoma. Microvasc Res 1996;51:164 –174. 27. Fontanini G, Vignati S, Lucchi M, et al. Neoangiogenesis and p53 protein in lung cancer: their prognostic role and their relation with vascular endothelial growth factor (VEGF) expression [see comments]. Br J Cancer 1997;75:1295–1301. 28. Salven P, Heikkila P, Anttonen A, et al. Vascular endothelial growth factor in squamous cell head and neck carcinoma: Expression and prognostic significance. Mod Pathol 1997;10: 1128 –33. 29. Eisma RJ, Spiro JD, Kreutzer DL. Vascular endothelial growth factor expression in head and neck squamous cell carcinoma. Am J Surg 1997;174:513–517. 30. Denhart BC, Guidi AJ, Tognazzi K, et al. Vascular permeability factor/vascular endothelial growth factor and its receptors in oral and laryngeal squamous cell carcinoma and dysplasia. Lab Invest 1997;77:659 – 664. 31. Suzuma K, Takagi H, Otani A, et al. Expression of thrombospondin-1 in ischemia-induced retinal neovascularization. Am J Pathol 1999;154:343–354. 32. Mousa SA, Lorelli W, Campochiaro PA. Role of hypoxia and extracellular matrix-integrin binding in the modulation of angiogenic growth factors secretion by retinal pigmented epithelial cells. J Cell Biochem 1999;74:135–143. 33. Awwad HK, el Naggar M, Mocktar N, et al. Intercapillary distance measurement as an indicator of hypoxia in carcinoma of the cervix uteri. Int J Radiat Oncol Biol Phys 1986;12: 1329 –1333. 34. Dachs GU, Chaplin DJ. Microenvironmental control of gene expression: Implications for tumor angiogenesis, progression, and metastasis. Semin Radiat Oncol 1998;8:208 –216. 35. Jain RK. Barriers to drug delivery in solid tumors. Sci Am 1994;271:58 – 65. 36. Helmlinger G, Yuan F, Dellian M, et al. Interstitial pH and pO2 gradients in solid tumors in vivo: High- resolution measurements reveal a lack of correlation. Nat Med 1997;3:177– 182. 37. Gorski DH, Beckett MA, Jaskowiak NT, et al. Blockage of the vascular endothelial growth factor stress response increases the antitumor effects of ionizing radiation. Cancer Res 1999; 59:3374 –3378. 38. Teicher BA, Holden SA, Ara G, et al. Comparison of several antiangiogenic regimens alone and with cytotoxic therapies in the Lewis lung carcinoma. Cancer Chemother Pharmacol 1996;38:169 –177. 39. Mauceri HJ, Hanna NN, Beckett MA, et al. Combined effects of angiostatin and ionizing radiation in antitumour therapy. Nature 1998;394:287–291. 40. Wilson WR, Li AE, Cowan DS, et al. Enhancement of tumor radiation response by the antivascular agent 5,6-dimethylxanthenone-4-acetic acid. Int J Radiat Oncol Biol Phys 1998;42: 905–908. 41. Li L, Rojiani A, Siemann DW. Targeting the tumor vasculature with combretastatin A-4 disodium phosphate: Effects on radiation therapy. Int J Radiat Oncol Biol Phys 1998;42:899 – 903.
PII S0360-3016(00)00573-3
CLINICAL INVESTIGATION
Head and Neck
INTRATUMORAL MICROVESSEL DENSITY PREDICTS LOCAL TREATMENT FAILURE OF RADICALLY IRRADIATED SQUAMOUS CELL CANCER OF THE OROPHARYNX
STEFAN
DANIEL M. AEBERSOLD, M.D.,* KARL T. BEER, M.D.,* JEAN LAISSUE, M.D.,† HUG,‡ ATTILA KOLLAR,* RICHARD H. GREINER, M.D.,* AND VALENTIN DJONOV, M.D.‡
*Department of Radiation Oncology, †Institute of Pathology, and ‡Institute of Anatomy, University of Berne, Berne, Switzerland Purpose: To determine the predictive value of intratumoral microvessel density (IMD), and of the expression of p53, vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1) for the radiocurability of patients with squamous cell cancer of the oropharynx. Materials and Methods: 139 patients with squamous cell cancer of the oropharynx were radically irradiated (median dose, 74 Gy) between 1991 and 1997. Biopsies from 100 patients were processed for immunohistochemistry. IMD was determined in hot spot areas of tissue stained with anti-CD31 at a magnification of ⴛ200. Staining for p53 was considered positive if more than 10% of the cell nuclei overexpressed the protein. Immunostaining of VEGF and TSP-1 was assessed semiquantitatively. Results: Increasing IMD (range, 54 –282) was strongly correlated with incomplete remission of both the primary tumors (p ⴝ 0.01) and lymph node metastases (p ⴝ 0.02). Moreover, multivariate Cox regression analysis revealed local failure-free survival to decline with increasing IMD (IMD continuous: risk ratio ⴝ 1.01 per increase of 1 microvessel, p ⴝ 0.0001; IMD categorical: < 80: baseline, 81–110: risk ratio ⴝ 2.71, 111–130: risk ratio ⴝ 4.55, > 130: risk ratio ⴝ 13.01). Neither the expression of p53, nor that of VEGF or TSP-1 was associated with the treatment outcome or IMD, but VEGF and TSP-1 expression were positively correlated (p ⴝ 0.02). Conclusion: IMD represents a powerful and independent predictive factor for local treatment failure in radically irradiated patients with squamous cell cancer of the oropharynx. © 2000 Elsevier Science Inc. Head and neck cancer, Radiotherapy, Predictive factor, Angiogenesis, Microvessel density.
INTRODUCTION
radiation effect, either in terms of tumor oxygenation (5), of the vasculotoxic effects of X-rays (6) or of the anti-apoptotic properties of angiogenic factors (7–9). Nonetheless, few clinical reports have assessed angiogenic activity as a predictive factor in radiation therapy and these are contradictory (10 –14). Angiogenesis is closely regulated by a broad range of stimulators and inhibitors (15). p53, which is essentially involved in cell cycle control, DNA repair and the regulation of apoptosis (16), also contributes to the regulation of angiogenesis: Loss of functional p53 leads to reduced expression of thrombospondin-1 (TSP-1) (17), a key inhibitor of angiogenesis (18). Mutant p53 has been shown to upregulate the expression of vascular endothelial growth factor (VEGF) (19), which has a pivotal role in triggering angiogenesis (20). To investigate whether or not the angiogenic phenotype
It is now generally accepted that angiogenesis is crucial both for the growth of a primary tumor and for the development of distant metastases (1). Accordingly, parameters of angiogenic activity, such as intratumoral microvessel density (IMD) and the expression of angiogenic factors, have been linked to the prognosis of a broad spectrum of cancers (2), including squamous cell cancer of the head and neck (3). In addition to the assessment of prognosis, prediction of the response probability to a certain therapy would also be of value, in helping to individualize, and hence to optimize, the therapeutical approach. This applies particularly to advanced cancer of the head and neck, the treatment success of which is difficult to predict, morbidity from both surgery and radiation being disconcertingly high (4). A considerable body of evidence points toward the existence of an interaction between tumor angiogenesis and
This work was supported by the Bernese Cancer League and the Bernese Radium Foundation. Presented in part at the 41st Annual Meeting of ASTRO, San Antonio, Texas, 1999, and at ECCO 10, Vienna, Austria, 1999. Accepted for publication 1 November 1999.
Reprint requests to: Dr. V. Djonov, Institute of Anatomy, University of Berne, Bu¨hlstrasse 26, 3000 Berne 9, Switzerland. E-mail: [email protected] Acknowledgment—We would like to thank Dr. P. H. Burri, Chairman of the Institute of Anatomy, University of Berne, for his generous support and B. de Breuyn for her excellent technical assistance. 17
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Table 1. Clinicopathologic characteristics and therapy results No. of patients (total ⫽ 100)
Parameter T stage
N stage
Histological grade Site of primary tumor
Chemotherapy Complete remission
1 2 3 4 0 1 2 3 1 2 3 Tonsillar fossa Base of tongue Faucial arch Lateral/posterior wall Vallecula epiglottica No Yes Primary tumor Lymph nodes
Local treatment failure Progression after of non-CR* Relapse after CR† Distant metastases
4 8 32 56 34 14 42 10 11 63 26 40 29 21 6 4 73 27 81 34 of 66 42 19 23 14
* Non-CR ⫽ incomplete remission (includes partial remission, stable disease and progressive disease). † CR ⫽ Complete remission.
of head and neck squamous cell cancer relates to radiocurability, we performed a retrospective study on a well-defined series of patients with squamous cell carcinoma of the oropharynx who had undergone radical irradiation (21). IMD, as well as the immunoreactivity for VEGF, TSP-1, and p53, were correlated with the response to therapy and locoregional recurrence. PATIENTS AND METHODS Patients Between October 1991 and December 1997, 139 patients with biopsy-proven squamous cell cancer of the oropharynx were radically irradiated at the Department of Radiation Oncology, the Inselspital, Berne. 39 patients were excluded from the present study due to the small size of the biopsy (16 patients), previous or synchronous malignancies (12 patients), irradiation after neck dissection (7 patients), intercurrent death during therapy (2 patients), lack of follow-up (1 patient), and presence of distant metastases at the onset of treatment (1 patient). The main clinical characteristics of the series of patients are shown in Table 1. Most of the patients manifested an advanced stage of disease. The detailed UICC-TNM clinical stages in patients were: T1N0 in 3, T1N1 in 1, T2N0 in 6, T2N1 in 1, T2N2 in 1, T3N0 in 13, T3N1 in 4, T3N2 in 11, T3N3 in 1, T4N0 in 12, T4N1 in 8, T4N2 in 30, and T4N3 in 9. Tumors of the tonsillar
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fossa (40%), base of tongue (29%) and faucial arch (21%) predominated. All biopsies were taken before radiotherapy had started. Histopathological diagnosis and grading of biopsies were reviewed by an experienced pathologist (J.L.). Most of the patients had Grade II tumors (Grade I in 11 cases, Grade II in 63, and Grade III in 26). Therapy and response assessment All patients were prepared for radiotherapy by placement of a thermoplast mask, planning CT, two- or three-dimensional-based planning and control by simulator and portal vision imaging device. Megavoltage radiation therapy was administered by a linear accelerator in daily fractions, 5 times a week for 5– 8 weeks. A median total dose of 74 Gy (range, 54 – 80.5) was delivered, 97% of patients receiving at least 66 Gy. Twenty-seven patients underwent concomitant chemotherapy, which consisted mainly of a cisplatin (CDDP) -containing regimen: 12 patients were subjected to CDDP monotherapy, 13 patients had a combination with CDDP and 5-fluorouracil, 1 patient received methotrexate, and 1 carboplatin. In patients with complete remission of the primary tumor and with clinical or radiological evidence of persistent lymph node metastasis, a salvage neck dissection was performed. Baseline studies included physical examination, chest X-rays, panendoscopy of the upper aerodigestive tract, and magnetic resonance imaging or computed tomography of the neck. During treatment, patients were examined on a weekly basis. The response to treatment was assessed 2– 4 weeks after the end of therapy, primary tumor and the lymph node metastases being evaluated separately. Two categories were defined: complete remission (CR: complete disappearance of tumor manifestation) and incomplete remission (non-CR: including partial remission, stable disease, or progressive disease). After treatment, all patients underwent clinical examination and imaging on a regular basis. Immunohistochemistry A total of 151 paraffin-embedded biopsies from the 100 patients were processed for immunohistochemistry; 3-mthick sections were transferred to gelatinized micro-slides and air-dried overnight at 37°C. They were dewaxed in xylene (three changes), rehydrated in ethanol, and rinsed in Tris-buffered saline (TBS: 50 mM Tris/HCl, pH 7.4, containing 100 mM sodium chloride [two changes]). Endogenous peroxidase activity was suppressed by treatment with 0.3% hydrogen peroxide for 10 min. Prior to incubation with antibodies, sections were bathed in 0.01 M sodium citrate, pH 6.0 (or TrisHCl, pH 1.0, in the case of antiTSP-1) and heated in a microwave oven (180 W) for 15 min. In the case of mouse anti-CD31, this step was preceded by treatment with trypsin ([Difco Lab., Detroit, MI, USA] 0.2 mg/mL in TBS/CaCl2 buffer) for 10 min at 37°C. After blocking of unspecific binding in TBS containing 1% casein (SIGMA 8654) for 10 min, sections were incubated with the first antibody diluted in TBS: mouse anti-CD31 1:20 (JC/
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70A, M-0823; Dako, Glostrup, Denmark), mouse anti-p53 1:200 (DO-7, M7001; DAKO, Glostrup, Denmark), rabbit anti-VEGF 1:200 (sc-152; Santa Cruz Biotechnology, Santa Cruz, CA, USA), or mouse anti-TSP-1 1:25 (Ab-7/MS 590, NeoMarkers, Fremont, CA, USA) for 15 h at 4°C. Sections were exposed to an affinity-purified biotinylated second antibody ([anti-mouse EO 433, anti-rabbit EO 353, Dako, Glostrup, Denmark] diluted 1:200 in TBS) for 45 min at ambient temperature, washed three times in TBS, then treated with the avidin-biotin-complex/horseradish peroxidase (P355, Dako, Glostrup, Denmark) for a similar period at the same temperature. The reaction product was visualized by exposing sections to 3-amino-9-ethylcarbazole or 3.3-diaminobenzidine (Sigma Chemicals Company, St. Louis, Missouri, USA), which were then mounted in Aquatex (Merck, Darmstadt, Germany). Negative controls were performed using nonspecific mouse and rabbit sera. Sections were counterstained with haematoxylin. For a better identification of microvessels, counterstaining of CD31 stained sections was purposely rendered weak. In TSP-1 stained sections, counterstaining was weak due to pretreatment of sections with TrisHCl buffer at low pH.
Quantification IMD. The IMD was assessed according to the standard method defined in a consensus paper (22): CD31-stained sections were scanned at low magnification (⫻10 and ⫻40) for areas with the highest numbers of microvessels (‘hot spots’). Microvessels within three such hot spots were then counted at a magnification of ⫻200 with the aid of an ocular grid (area size: 0.64 mm2). Biopsies with fewer than three evaluable hot spots were excluded. Any endothelial cell or endothelial cell cluster positive for CD31 and clearly separate from an adjacent cluster was considered as a single, countable microvessel. Counting was performed by two independent investigators (D.M.A., V.D). Disagreements (more than 10% difference) were resolved at a discussion microscope. The highest number of vessels per hot spot was selected for further evaluation. For the statistical analysis, IMD was used both as a continuous and as a categorical variable (4 groups: IMD ⱕ 80, 81–110, 111–130, ⬎ 130). p53. Occasional positive staining of tumor cells is interpreted as an accumulation of p53 wild-type protein in response to DNA damage, whereas intense staining of most cells is usually indicative of a mutation (23). In our study, staining for p53 was considered to be positive if more than 10% of the 500 cells counted expressed the protein. VEGF and TSP-1. Immunostaining was assessed semiquantitatively (0, ⫹, ⫹⫹, ⫹⫹⫹). Both the extent of staining (relative number of VEGF-positive cells; extent of extracellular matrix staining with TSP-1) and the intensity of the reaction were taken into account. For the statistical analysis, patients were assigned to one of two categories (0/⫹ vs. ⫹⫹/⫹⫹⫹).
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Statistics The correlation between IMD (as a continuous variable) and each of the other parameters was statistically assessed using the Wilcoxon two-sample test. Correlations between clinical and angiogenesis parameters were evaluated by means of Fisher’s exact test (two-tailed). Variables were correlated with the response of the primary tumor and of the lymph node metastases to treatment (CR vs. non-CR), as well as with local relapse-free survival, local failure-free survival, and overall survival. For the analysis of local relapse-free survival, only patients with CR of the primary tumor were included. Lymph node metastases had also to be in CR or removed by neck dissection for inclusion. Analysis of local failure-free survival considered local tumor progression after non-CR and local relapse after CR as adverse events. Analysis of overall survival embraced any kind of death. Survival was measured from the time when therapy was initiated to that when the first adverse event was detected or to the date of the last check-up. Deaths due to non-tumor-related causes were censored, except for analysis of overall survival. Correlations between variables and the response to treatment were analyzed by implementing the logistic regression method in both the univariate and multivariate fashion, including a backward elimination procedure to remove variables with a p value greater than 0.05. Survival curves were plotted according to the Kaplan–Meier method, the log– rank test being used to determine significant differences between the survival curves. A Cox regression was performed to calculate the risk ratios. For multivariate analyses, all clinical and angiogenesis-related variables were included in a Cox regression model. A backward elimination procedure was performed to remove nonsignificant variables (p ⬎ 0.05). Correlations between variables and the development of distant metastases were calculated according to the logistic regression method. Statistical analyses were performed using the SAS package (Version 6.12; SAS Institute, Cary, NC).
RESULTS Clinical outcome The median follow-up time was 654 days (range, 58 – 2036). CR of the primary tumor was achieved in 81 (81%) patients, and in 34 of the 66 (51.5%) with nodal spread, CR of lymph node metastases was accomplished (Table 1). In 42 of the 100 patients, local failure occurred, this being due either to relapse at the same site after CR (23 patients) or to progression after non-CR (19 patients). 14 patients without CR of cervical lymph node metastases underwent a neck dissection with complete removal of remaining tumor manifestations; they were included in the analysis of local relapse-free survival. 14 patients developed distant metastases.
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Fig. 1. Photomicrographs of immunostained tumoral tissue derived from patients with squamous cell cancer of the oropharynx. (A): Microvessels within a hot spot area, revealing the endothelial cells to be heavily stained with anti-CD31 (magnification ⫻200). Counterstaining with hematoxylin was purposely rendered weak. (B): Overexpression of p53 in ⬎ 10% of tumor cell nuclei (magnification ⫻300). (C): High expression of vascular endothelial growth factor in tumor cells, endothelial cells (arrow heads) and fibroblasts (arrows) (magnification ⫻200). (D): Intense immunostaining for thrombospondin-1 within the extracellular matrix (magnification ⫻300).
Clinical and angiogenesis-related variables IMD was evaluable in 100 patients and ranged from 54 to 282 microvessels per high-power (⫻200) optical field (median, 104; mean 107.7; SD ⫾ 30.1). In more detail, 14 carcinomas had an IMD ⱕ 80, 46 had an IMD between 81 and 110, 21 had an IMD between 111 and 130 and 19 had an IMD ⬎ 130. The pattern of staining for anti-CD31 was similar to that already reported (24): staining was confined to vascular endothelial cells (Fig. 1A); occasionally, inflammatory cells were immunoreactive as well. In each of the 100 evaluable patients, the expression of p53, VEGF, and TSP-1 could be readily assessed. In 67 of the patients, more than 10% of the nuclei were p53-positive (Fig. 1B). VEGF was expressed by tumor cells in 98% of cases (35% ⫹, 56% ⫹⫹, 7% ⫹⫹⫹) and was also observed in endothelial cells and fibroblasts (Fig. 1C). Immunostaining for TSP-1 was detected in 47% of patients (20% ⫹, 21% ⫹⫹, 6% ⫹⫹⫹), the reaction being confined exclusively to the tumoral stroma (Fig. 1D). No obvious correlation existed between IMD and either
the clinical stage (T or N) or the histological grade (Table 2). Likewise, there was no apparent relationship between IMD and either VEGF, TSP-1, or p53 expression. Overexpression of p53 did not correlate with that of either TSP-1 or VEGF. However, tumors with high levels of VEGF immunoreactivity manifested significantly higher amounts of TSP-1 (p ⫽ 0.02; Table 2). Prediction of response to treatment In the univariate analysis, a significant correlation between IMD and the response to radiation was found: the higher the IMD, the lower the probability of CR. This relationship held true both for the response of the primary tumor (p ⫽ 0.01; IMD continuous) and for that of the lymph node metastases (p ⫽ 0.02; IMD continuous). The odds ratio for CR of the primary tumor and of lymph node metastases was 0.97 per increase of 1 microvessel. When IMD was subdivided into four groups, the odds ratios decreased from 0.43 to 0.10 for CR of the primary tumor and from 0.11 to 0.06 for CR of the lymph node metastases in
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Table 2. Correlation between angiogenesis-related variables and patient variables ( p values) IMD* continuous
p53 ⱕ10% vs. ⬎10%
VEGF† 0/⫹ vs. ⫹⫹/⫹⫹⫹
TSP-1‡ 0/⫹ vs. ⫹⫹/⫹⫹⫹
0.83 0.86 0.41 0.45 0.63 0.74
1.00 1.00 0.48 — — —
1.00 1.00 0.16 0.27 — —
0.73 0.64 0.80 0.47 0.02 —
T stage, T1–2 vs. T3–4 N stage, N0 vs. N1–3 Grade, G1 vs. G2–3 p53, ⱕ10% vs. ⬎10% VEGF, 0/⫹ vs. ⫹⫹/⫹⫹⫹ TSP-1, 0/⫹ vs. ⫹⫹/⫹⫹⫹
* IMD ⫽ intratumoral microvessel density. VEGF ⫽ vascular endothelial growth factor. ‡ TSP-1 ⫽ thrombospondin-1. †
the group with the highest microvessel counts (Table 3). No obvious relationship between the response to treatment and either the clinical stages (T or N), histological grade, chemotherapy, p53, VEGF, or TSP-1 was apparent. In the multivariate analysis, only the IMD remained significant during the backward elimination procedure (Table 4). Survival analysis In the univariate analysis, only IMD was significantly correlated to local relapse-free survival, the risk ratio being 1.02 per increase of 1 microvessel (p ⫽ 0.03). When subdivided into four groups, the risk ratio for local relapse increased from 1.56 to 2.89 to 8.91 in the high-risk group (Table 5). Although not significant, a trend toward correlation was also apparent for the T stage of the disease, the risk ratio being 4.89 (p ⫽ 0.12). Local failure-free survival, which considered all kinds of local treatment failures, namely, local relapse and local tumor progression after non-CR, was, likewise, significantly correlated only with IMD, the risk ratio being 1.01 per microvessel. But in this
instance, the level of significance was much higher (p ⫽ 0.0001). When subdivided into four groups, the risk ratio for local failure increased from 2.71 or 4.55 to 13.01 in that with the highest number of vessels (Table 5, Fig. 2). Once again, a trend toward a correlation was observed for the T stage of the disease, the risk ratio being 3.73 (p ⫽ 0.07). In the multivariate Cox regression analysis, which included the backward elimination, IMD remained the only significant variable (Table 4). No significant correlation was apparent between the development of distant metastases and either clinicopathological or angiogenesis-related parameters (Table 6). However, both the univariate and multivariate analysis with backward elimination of nonsignificant variables revealed IMD and, in addition, T stage to be correlated with overall survival (Table 4 and 7). DISCUSSION Intratumoral microvessel density is known to be of prognostic value in many human malignancies, this circum-
Table 3. Univariate analysis (logistic regression) of variables for predicting complete remission of the primary tumor and lymph node metastases CR* of primary tumor Variable Category
OR†
Confidence interval (95%)
T stage, T1–2 vs. T3–4 N stage, N0 vs. N1–3 Grade, G1 vs. G2–3 Chemotherapy, 0 vs. ⫹ IMD‡ continuous IMD categorical ⱕ80 81–110 111–130 ⬎130 p53, ⱕ10% vs. ⬎10% VEGF§, 0/⫹ vs. ⫹⫹/⫹⫹⫹ TSP-1储, 0/⫹ vs. ⫹⫹/⫹⫹⫹
0.35 0.64 1.40 1.49 0.97 – 0.43 0.26 0.10 2.14 0.74 0.76
0.04–2.92 0.21–1.96 0.42–4.67 0.45–4.96 0.95–0.99 – 0.05–3.82 0.03–2.38 0.01–1.08 0.77–5.92 0.26–2.16 0.26–2.25
CR of lymph node metastases p
OR
Confidence interval (95%)
p
0.33 0.44 0.57 0.52 0.01 – 0.45 0.24 0.06 0.14 0.59 0.62
0.00 NA 1.05 1.36 0.97 – 0.11 0.11 0.06 1.44 0.85 1.07
0.00–999.00 NA 0.37–2.97 0.49–3.77 0.95–0.99 – 0.01–0.99 0.01–1.08 0.004–0.92 0.52–4.03 0.31–2.31 0.37–3.09
0.98 NA 0.92 0.55 0.02 – 0.05 0.06 0.04 0.49 0.75 0.91
* CR ⫽ complete remission. OR ⫽ odds ratio. ‡ IMD ⫽ intratumoral microvessel density; OR per increase of 1 microvessel. § VEGF ⫽ vascular endothelial growth factor. 储 TSP-1 ⫽ thrombospondin-1. †
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Table 4. Remaining variables after backward elimination of nonsignificant variables in the multivariate analysis of response to treatment and survival Endpoint
Remaining variable
p
OR/RR*
Confidence interval (95%)
CR† of primary tumor CR of lymph node metastases Local relapse-free survival Local failure-free survival Overall survival
IMD‡ continuous IMD continuous IMD continuous IMD continuous IMD continuous T stage, T1–2 vs. T3–4
0.01 0.02 0.03 0.0001 ⬍0.0001 0.02
0.97 0.97 1.02 1.01 1.02 3.11
0.95–0.99 0.95–0.99 1.00–1.04 1.01–1.02 1.01–1.03 1.17–8.26
* OR/RR ⫽ odds ratio/risk ratio. † CR ⫽ complete remission. ‡ IMD ⫽ intratumoral microvessel density; OR/RR per increase of 1 microvessel.
stance being mainly attributable to the involvement of angiogenesis in the development of distant metastases. However, there exists but little information concerning the importance of angiogenesis-related parameters in predicting the probability of treatment success. In the present study, we have demonstrated IMD to be a powerful tool for predicting the success probability of radiotherapy for squamous cell cancer of the oropharynx. The probabilities for controlling the primary tumor and lymph node metastases were analyzed separately. This was undertaken with a view to focusing on the primary tumor, control of which by radiation is generally deemed essential for a curative treatment strategy in advanced oropharyngeal cancer. Lymph node metastases, on the other hand, can often be approached surgically. The different rates of CR for primary tumors (81%) and lymph node metastases (51.5%) indicate the existence of distinct radiosensitivities at different sites. However, IMD assessed in biopsies from the primary tumor proved to be of predictive value in determining the response to treatment at both sites, the odds ratio being the same in each case (Table
3). Furthermore, the risk of local relapse after CR could also be predicted by IMD (Table 5). A higher level of significance for IMD as a correlating variable was attained in the analysis of failure-free survival, which considered both tumor progression after non-CR and local relapse as adverse events. In a previous study of patients with squamous cell cancer at various sites in the head and neck, low vascularity was likewise revealed to correlate with a higher probability of complete remission (12), but no association between vascularization and local recurrence was demonstrated. This discrepancy between our own findings and those of Gasparini et al. may derive from the large diversity of tumors included in the latter study, our own cohort being strictly confined to individuals with oropharyngeal cancer of identical histological type. Heterogeneity in tumor entities could likewise account for at least some of the discrepant results by Giatromanolaki et al. (14); although the highest local failure rate was observed in patients with the largest number of microvessels, the best treatment results were achieved in individuals with intermediate vessel counts. We, however, report a
Table 5. Univariate analysis (Cox regression) of variables for predicting local relapse-free survival and local failure-free survival Local failure-free survival†
Local relapse-free survival* Variable Category
RR‡
Confidence interval (95%)
p
RR
Confidence interval (95%)
p
T stage, T1–2 vs. T3–4 N stage, N0 vs N1–3 Grade, G1 vs G2–3 Chemotherapy, 0 vs ⫹ IMD§ continuous IMD categorical ⱕ 80 81–110 111–130 ⬎30 p53, ⱕ10% vs. ⬎ 10% VEGF储, 0/⫹ vs. ⫹⫹/⫹⫹⫹ TSP-1#, 0/⫹ vs. ⫹⫹/⫹⫹⫹
4.89 0.79 1.68 0.94 1.02 – 1.56 2.89 8.91 2.03 0.64 0.71
0.65–36.62 0.34–1.84 0.71–4.01 0.37–2.40 1.00–1.04 – 0.34–7.22 0.63–13.40 1.22–65.06 0.69–6.02 0.28–1.48 0.24–2.10
0.12 0.59 0.24 0.90 0.03 – 0.57 0.17 0.03 0.20 0.30 0.54
3.73 1.04 1.11 0.89 1.01 – 2.71 4.55 13.01 0.79 0.78 0.98
0.90–15.54 0.56–1.97 0.57–2.18 0.45–1.77 1.01–1.02 – 0.63–11.75 1.05–19.81 2.68–63.70 0.42–1.49 0.42–1.44 0.48–2.00
0.07 0.89 0.75 0.74 0.0001 – 0.18 0.04 0.002 0.48 0.42 0.96
* considers local relapse after complete remission. considers tumor progression after incomplete remission (partial remission, stable disease and progressive disease) and local relapse after complete remission. ‡ RR ⫽ risk ratio. § IMD ⫽ intratumoral microvessel density; RR per increase of 1 microvessel. 储 VEGF ⫽ vascular endothelial growth factor. # TSP-1 ⫽ thrombospondin-1. †
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Fig. 2. Local failure-free survival (Kaplan–Meier plot) after subdividing intratumoral microvessel density into 4 groups (IMD ⱕ 80, 81–110, 111–130, and ⬎ 130). Local failure considers both tumor progression after incomplete remission (partial remission, stable disease, and progressive disease) and local relapse after complete remission of the primary tumor as adverse events. Risk ratios (RR) for local failure and p values were calculated by performing a Cox regression analysis.
linear increase in the risk for local failure. Such a relationship has also been documented for prostate cancer (11). Beside IMD, T stage was the most important variable for both local relapse-free survival, local failure free-survival and overall survival in terms of risk ratio (4.89, 3.73, and 2.68, respectively; Table 5 and 7). This corresponds well with the fact that T stage has been recognized as decisive for local disease control and survival in head and neck tumors (4). However, the level of significance was not reached in our study for the correlation of T stage with local relapse-free (p ⫽ 0.12)
Table 6. Univariate analysis (logistic regression) of variables for predicting distant metastasis
T stage, T1–2 vs. T3–4 N stage, N0 vs. N1–3 Grade, G1 vs. G2–3 Chemotherapy, 0 vs. ⫹ IMD† continuous p53, 0 vs. ⫹ VEGF‡, 0/⫹ vs. ⫹⫹/⫹⫹⫹ TSP-1§, 0/⫹ vs. ⫹⫹/⫹⫹⫹
RR*
p
1.53 2.07 1.16 1.10 0.99 1.27 0.54 0.41
0.97 0.29 0.81 0.89 0.48 0.70 0.28 0.26
* RR ⫽ risk ratio. IMD ⫽ intratumoral microvessel density; RR per increase of 1 microvessel. ‡ VEGF ⫽ vascular endothelial growth factor. § TSP-1 ⫽ thrombospondin-1. †
and failure-free survival (p ⫽ 0.07), the p value for correlation with overall survival being 0.04. The relatively high p values despite impressively elevated risk ratios reflect most probably the very low number of patients with T1 and T2 tumors (12%) in the study population (Table 1). Table 7. Univariate analysis (Cox regression) of variables for predicting overall survival Overall survival Variable category
RR*
Confidence interval (95%)
p
T stage, T1–2 vs. T3–4 N stage, N0 vs. N1–3 Grade, G1 vs. G2–3 Chemotherapy, 0 vs. ⫹ IMD† continuous IMD categorical ⱕ80 81–110 111–130 ⬎130 p53, ⱕ 10% vs. ⬎10% VEGF‡, 0/⫹ vs. ⫹⫹/⫹⫹⫹ TSP-1§, 0/⫹ vs. ⫹⫹/⫹⫹⫹
2.68 1.46 1.43 1.14 1.02 – 1.43 1.91 3.60 0.65 1.20 1.27
1.03–7.01 0.83–2.55 0.57–3.58 0.63–2.05 1.01–1.02 – 0.54–3.74 0.68–5.38 1.29–10.06 0.38–1.11 0.69–2.09 0.70–2.31
0.04 0.19 0.45 0.66 0.0001 – 0.47 0.22 0.01 0.11 0.52 0.43
* RR ⫽ risk ratio. IMD ⫽ intratumoral microvessel density; RR per increase of 1 microvessel. ‡ VEGF ⫽ vascular endothelial growth factor. § TSP-1 ⫽ thrombospondin-1. †
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Beside its predictive power for local control by radiotherapy, IMD proved to be a prognostic factor too as it was significantly associated with overall survival. However, no correlation of IMD with distant metastasis was found (Table 6). Thus, the prognostic power of IMD must derive from its prediction of locoregional control which is known to be crucial for survival in head and neck cancer (4). Association with locoregional control may also account for the prognostic power of T stage. The multivariate Cox regression analysis revealed the predictive power of IMD to be independent of all other variables: IMD remained the only significant variable during the backward elimination of nonsignificant variables in the analysis both of the response to treatment and of the local relapse-free and failure-free survival. The independence of IMD as a predictive factor was reflected in the absence of significant correlations between clinicopathologic variables, angiogenesis-related parameters and IMD (Table 2). The association between IMD and the risk for lymphatic spread of squamous cell cancer of the head and neck described by Gasparini et al. (25) was not observed in our study. Furthermore, overexpression of p53, which has been shown by the same authors to be correlated with IMD (25) as well as by other investigators in the cases of colorectal (26) and non–small-cell lung cancers (27), was not associated either with IMD or with VEGF or TSP-1 expression in our patients. As previously shown for head and neck cancer in general (28 –30), we found VEGF to be expressed in most patients with squamous cell cancer of the oropharynx. Despite its key role in regulating angiogenesis, we observed no correlation between this factor and IMD (Table 2). Its contribution to vascularization in squamous cell cancer of the head and neck thus remains to be clarified. The significant correlation between VEGF and TSP-1 expression apparent in our series of patients may reflect a negative feedback mechanism, since a VEGF-mediated induction of TSP-1 expression has been observed in bovine retinal endothelial cells (31). And in retinal pigmented epithelial cells, TSP-1 has been shown to upregulate VEGF (32). Whether the mutual regulation of VEGF and TSP-1 represents a general mechanism has yet to be determined. The predictive power of IMD for radiocurability may be explained in several ways. Hypoxia is a major cause of resistance to radiation (5), and because oxygen delivery
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depends upon blood flow, measurement of tumoral vascularity has been deemed to represent a direct means of assessing the tissue’s oxygenation state (10, 33). However, since hypoxia is a key trigger for the induction of angiogenesis (34), the number of microvessels could—theoretically—reflect the degree of tumor hypoxia. This view takes into account the circumstance that tumor vasculature is often dysfunctional, being characterized by sluggish blood flow or shunting (35). Moreover, high-resolution intravital measurements have revealed the absence of correlation between blood flow and perivascular pO2 (36). Hence, the relationship between vascularity and the degree of oxygenation in clinical tumor samples remains to be clarified. Apart from the link between angiogenesis and hypoxia, angiogenic factors per se may interfere with radiosensitivity by inhibiting X-ray-induced apoptosis: VEGF is known to suppress ␥ ray-induced apoptosis in both CMK86 cells and normal haematopoietic stem cells (7). Furthermore, basic fibroblast growth factor has been shown to protect endothelial cells from radiation-induced apoptosis via a protein kinase C mediated mechanism (8), and hepatocyte growth factor/scatter factor protects breast cancer cells from radiation-induced apoptosis by targeting the anti-apoptotic mitochondrial membrane protein Bcl-XL (9). In our study, VEGF expression did not interfere with radiocurability. However, blockage of the VEGF stress response has been recently shown to enhance the tumor-destroying effect of ionizing radiation (37). Hence, VEGF signaling may be a target not only for anti-angiogenesis but also for radiosensitization. In conclusion, our results demonstrate the potential of IMD to predict the radiocurability of oropharyngeal cancer. Its reliability has to be further validated in prospective studies. In addition to their application in individualizing treatment strategies, predictive factors may represent an attractive target for modifying the response to treatment. Indeed, several anti-angiogenic strategies have been successfully explored for their radiosensitizing effect in animal models (38 – 41). The strong predictive power of IMD for radiocurability detected in the present study strengthens the rationale for performing clinical trials that combine anti-angiogenic drugs with radiotherapy.
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