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Clinical Cancer Research Vol. 6, 3159-3165, August 2000
© 2000 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Potential Role of Microvessel Density in Predicting Radiosensitivity of T1 and T2 Stage Laryngeal Squamous Cell Carcinoma Treated with Radiotherapy1

Tomoyuki Kamijo, Tomoyuki Yokose, Takahiro Hasebe, Hiroyuki Yonou, Satoshi Sasaki, Ryuichi Hayashi, Satoshi Ebihara, Hiroshi Miyahara, Hiroshi Hosoi and Atsushi Ochiai2

Pathology [T. K., T. Y., T. H., H. Y., A. O.] and Epidemiology [S. S.] Divisions, National Cancer Center Research Institute East, and Department of Head and Neck Surgery [R. H., S. E.], National Cancer Center Hospital East, Chiba 277-8577; and Department of Otolaryngology, Nara Medical University Hospital, Nara 634-8522 [H. M., H. H.], Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Curative radiotherapy is the first choice of therapy for T1 and T2 stage laryngeal squamous cell carcinoma (LSCC) patients to preserve their phonation. Patients with recurrent tumors who undergo salvage surgery require prolonged nasal feeding. Therefore, clinical interest has been focused on elucidating a predictive factor indicating which tumors are likely to be radiosensitive before radiotherapy. We analyzed the relations between radiosensitivity and clinicopathological factors (gender, tumor location, histological factors, and clinical tumor-node-metastasis stage), expression of apoptosis-related proteins (p53, bax, bcl-2), apoptotic index using the terminal deoxynucleotidyltransferase-mediated nick end labeling method, expression of cell proliferation-related proteins (Ki-67-labeling index and epidermal growth factor receptor overexpression) and microvessel density (MVD, vessels/field = 0.391 mm2) in biopsy specimens from 31 LSCC patients given radiotherapy (total radiotherapy dose of 52–70 Gy over 4–6.5 weeks). Univariate analysis revealed that tumors with a high MVD (>=35 vessels/field) showed better radiosensitivity than those with a low MVD (<35 vessels/field, P = 0.008) and that a high Ki-67-labeling index (>=40%) was weakly associated with radiosensitivity (P = 0.056). Multivariate analysis and Kaplan-Meier analysis showed that MVD alone had significant predictive power for radiosensitivity in T1 and T2 stage LSCCs after radiotherapy (P = 0.012, 0.0003, respectively). No significant association between clinicopathological factors, or of overexpression of p53, bax, bcl-2, epidermal growth factor receptor, or apoptotic index, with radiosensitivity was found. These results indicate that MVD is a potentially useful clinical factor predicting radiosensitivity for patients with early stage LSCCs before treatment.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Radiotherapy is generally considered to have a fair clinical outcome with organ preservation for patients with LSCC.3 LSCC patients who undergo radiotherapy and have nonrecurrent tumors after therapy can maintain a good quality of life with organ preservation. On the other hand, when a patient has residual or recurrent disease, total or partial laryngectomy is generally performed as a salvage therapy. Such patients require a prolonged treatment course and have a poor quality of life because radiotherapy causes delayed operative wound healing. Therefore, clinical interest has been focused on elucidating a predictive factor indicating which tumors are likely to be radiosensitive before radiotherapy.

Several biological markers have been analyzed for clinical significance as prognostic factors. Tumor kinetic and proliferative activities of tumor cells based on immunohistochemical staining with antibodies against proliferation-associated markers, such as Ki-67 and proliferating cell nuclear antigen, have proved to be useful parameters for predicting prognosis (1, 2, 3) . Overexpression of EGFR has also been reported to have prognostic value not only for cancers of the larynx but also those of the urinary bladder and esophagus (4 , 5) .

In addition to cell kinetics, induction of apoptosis through several pathways in tumor cells plays an important role in radiation effects (6) . The p53 tumor suppressor gene product, bcl-2 protein and bcl-2 family members including bax, bcl-x, and mcl-1 are well known to regulate apoptosis induced by radiotherapy (7) . p53 gene mutation and overexpression of bcl-2 in tumor cells result in an inhibition of apoptosis (8) , but overexpression of bax protein promotes apoptosis induced by irradiation (9) . However, controversial results regarding the significance of the expression of apoptosis-associated proteins, as predictions of apoptosis-associated factors, have been reported to date (10, 11, 12) .

The basic radiation biology of tumor tissues both in vivo and in vitro indicates that the well-oxygenated cell is fully radiosensitive (13) . Solid tumors, however, contain regions in which intermittent or persistent deficits in perfusion have rendered large numbers of tumor cells transiently or chronically hypoxic. Studies of radiosensitivity in transplanted tumors in mice and rats showed the presence of large numbers of hypoxic cells in radioresistant tumors (14 , 15) . Hulka et al. (16) reported the MVD of the tumor to be correlated with extraction flow products measured by magnetic resonance imaging. These data allow us to hypothesize that tumor MVD based on counting microvessel numbers represents blood flow volume and oxygenation of the tumor.

To elucidate predictive factors for radiosensitivity, we examined the expressions of p53, bax, bcl-2, Ki-67, EGFR, and CD68, the AI, and the MVD in addition to clinicopathological factors in patients with LSCC who had been given radiotherapy. We also attempted to determine which factors reflect the radiosensitivity of biopsy specimens before treatment.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and Tumors.
There were 34 patients who were treated with radical radiotherapy for T1 or T2 stage LSCCs at the National Cancer Center Hospital East between September 1992 and November 1995. However, a few patients have not enough outpatient follow period in the National Cancer Center Hospital East, or biopsy sample was very scanty, so, we retrospectively selected 31 patients. They received radiotherapy alone at a curative dose of 52–70 Gy (52 Gy, 1 case; 60 Gy, 4 cases; 66 Gy, 11 cases; 70 Gy, 15 cases). Clinicopathological characteristics of all cases are listed in Table 1Citation . It is difficult to measure precise tumor size through the laryngoscope. We tried to evaluate the tumor size by laryngoscopic figures and found that the tumor diameter was ~4–15 mm. From one to three biopsies 2–5 mm in diameter were taken for each tumor (1 specimen, 18 cases; 2 specimens, 11 cases; 3 specimens, 2 cases). All of the biopsies were taken at the initial time of diagnosis. All patients were habitual smokers. The mean regional disease-free interval was 15 months (range, 1–77 months). Clinical tumor-node-metastasis classification and determination of histological differentiation were performed according to the Union International Contre le Cancer tumor-node-metastasis clinical classification (17) . All recurrent tumors were histologically proved and occurred at the primary site of the laryngeal tumor. Eleven of the 31 patients (35.5%) developed recurrences at the primary site. A total of seven patients with recurrent tumors underwent total laryngectomy, whereas three underwent partial laryngectomy, and one patient refused salvage surgery. The patients who underwent total laryngectomy required prolonged gastric feeding (39–175 days). We categorized "radiosensitive" as the complete disappearance of a tumor from the primary region for at least 4 years and "radioresistant" as residual tumor or recurrent tumor within 4 years after irradiation.


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  		Table 1 Clinicopathological features of 31 LSCC patients

 
Immunohistochemical Staining.
Immunohistochemical staining were performed with the avidin-biotin-peroxidase complex method. Formalin-fixed, paraffin-embedded biopsy specimens were cut into 4-µm sections. They were then deparaffinized and dehydrated, and quench endogenous peroxidase activity was determined followed by autoclaving at 121°C for 10 min or microwaving (750 W, 20 min in citrate buffer). After treatment with 2.0% bovine serum albumin, 5% skim milk in PBS for 20 min and then incubated with 2% normal swine serum in PBS for 10 min to prevent nonspecific binding of the antibody. The slides were then incubated with seven different primary antibodies listed in Table 2Citation overnight at 4°C. After a washing in PBS, slides were incubated with biotin-labeled antimouse or antirabbit secondary antibodies (Vector Laboratories Inc., Burlingame, CA) at room temperature for 30 min, followed by washing in PBS and incubation with peroxidase-labeled streptavidin (DAKO, Glostrup, Denmark) for 30 min. The reaction products were visualized by immersing the slides in freshly prepared diaminobenzidine (Dojindo, Kumamoto, Japan) solution for 10–20 min, and the slides were counterstained with Meier’s hematoxylin before dehydration and mounting.


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  		Table 2 Panel of primary antibodies

 
Evaluation for Immunohistochemistry.
The percentages of p53- and Ki-67-positive tumor cells were calculated by counting the number of brown-stained tumor nuclei per total number of tumor cells in the most highly stained area on each slide in the selected microscopic field at x200. These microscope fields are 0.391 mm2. In each specimen, 277–799 tumor cells were examined. According to previous reports, we categorized the sample as positive if >=10 and 40% of the tumor nuclei were stained by anti-p53 and Ki-67 antibodies, respectively (10 , 18)

Bcl-2, bax, and CD68 immunoreactivities for macrophage infiltration were localized in the cell cytoplasm. The expression of bcl-2 proteins was categorized according to previous reports (12 , 18) as follows: negative, <30% of tumor cells were stained; positive, >=30% of tumor cells were stained. The expressions of bax proteins and CD68 were categorized as negative when there were no stained cells and positive when one or more cells were stained.

EGFR was expressed specifically on the cell membrane. The expression was defined as positive when membranous staining of >=10% of the cells was observed, as described in previous reports (19) .

Quantification of Apoptosis.
Apoptotic cells were visualized using an Apoptosis in situ Detection Kit (WAKO, Osaka, Japan). Briefly, deparaffinized and hydrated sections were treated with proteinase K (0.5 µg/ml). Sections were then subjected to 3'-end labeling of the DNA with terminal deoxynucleotidyltransferase, followed by washing in PBS and incubation with peroxidase-labeled streptavidin. The reaction products were visualized by immersing the slides in freshly prepared diaminobenzidine solution for 15 min. After the color reaction, sections were counterstained with methyl green. The addition of DNase I led to positive staining of all nuclei, whereas the omission of terminal deoxynucleotidyltransferase led to complete absence of staining. AI was defined as the percentage of apoptotic tumor cells and calculated for a x200 field. Each field contained at least 100 tumor cells, and at least 500 tumor cells were counted per specimen. Some necrotic cells that were also stained weakly were excluded for counting as apoptotic cells.

Measurement of MVD.
Small blood vessels were visualized by staining endothelial cell for CD31 antibody. Two or three high power fields were identified on each slide, and the MVD were calculated as the mean number of vessels on a x200 field. In calculating the MVD, areas of inflammation, sclerotic tumor, and adjacent benign tissue were excluded.

Statistical Analysis.
A Cox proportional hazard model was used to determine the statistical significance of the associations between all categorical variables. The probability of LRC rate was calculated by the Kaplan-Meier method and was compared by the log rank test. P < 0.05 was considered significant. LRC was expressed at 3 years after diagnosis. Statistical calculations were performed with the Stat View software package (version 5.0; Abacus Concepts, Inc., Berkeley, CA).

All procedures (immunohistochemical evaluation, quantification of apoptosis, and measurement of MVD) were performed independently in all cases by three investigators (T. Y., T. H., and T. K.) who had no previous knowledge of the clinical outcome.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Clinicopathological Parameters and Radiosensitivity.
Table 3Citation summarizes clinicopathological characteristics and radiosensitivity. Of the 31 patients, 26 (83.9%) underwent 66 or 70 Gy radiotherapy, whereas 5 received 60 Gy or less. The total irradiation dose was not associated with radiosensitivity (P > 0.999).


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  		Table 3 Univariate Cox proportional hazard analysis of relations between clinicopathological parameters and radiosensitivity in 31 LSCC

 
As shown in Table 3Citation , there were no significant associations between radiosensitivity and clinicopathological features such as gender, age, tumor location, T factor, N factor, and histological differentiation (P = 0.631, 0.716, >0.999, 0.707, 0.631, and >0.999, respectively). The number of macrophages infiltrating the tumor, as detected by anti-CD68 antibody, also showed no significant association with radiotherapy (P = 0.276). Recent reports have suggested that the pretreatment hemoglobin level is significantly associated with local control in patients with T1 and T2 LSCC (20) . Because we also investigated the patient hemoglobin level, the level was not associated with radiosensitivity in this study (radiosensitive, 13.74 ± 1.40 g/dl; radioresistant, 13.65 ± 2.51 g/dl).

Apoptosis-associated Factors, Proliferative Activity, and Radiosensitivity.
All 31 LSCC specimens were processed for p53, bcl-2, bax, Ki-67, and EGFR expression by immunohistochemistry. p53 and Ki-67 were detected in nuclei, whereas staining for bax and bcl-2 was observed in cytoplasm and that for EGFR was seen on the cell membrane.

The positive p53 immunostaining was observed in 14 of the 31 cases (45.2%; Fig. 1Citation A). Bcl-2 and bax immunostainings were both positive in 11 of 31 specimens (35.5%). Of the 31 tumors (41.9%), 13 were determined to be positive for EGFR antibody. No statistically significant relations were found among expressions of p53, bcl-2, bax, and EGFR proteins and radiosensitivity (Table 4)Citation .



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  		Fig. 1. Representative immunohistochemical stainings for p53, bcl-2, Ki-67, and CD31 in pretreated LSCC biopsy specimens. A, p53. p53 immunoreactivity is detected in nuclear of tumor cells. Original magnification, x200. B, Ki-67. In this case, nuclear Ki-67 staining is seen in the majority of tumor cells. x200. C, terminal deoxynucleotidyltransferase-mediated nick end labeling staining for AI. Apoptotic cells appeared in nuclear collapse, which produced a surrounding halo. x300. D, CD31 for microvessels. The microvessels are most numerous at the periphery of the tumor. x100.

 

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  		Table 4 Univariate Cox proportional hazard analysis of relations between apoptosis-related proteins, cell proliferation, and MVD, and radiosensitivity, in 31 LSCC

 
The Ki-67 immunoreactivity ranged from 4.55 to 54.5% (Fig. 1Citation B). Of the 31 patients, 5 (16.1%) were determined to have highly Ki-67-positive tumors. In univariate analysis, a low Ki-67-labeling index was weakly correlated with the radiosensitivity of the tumor (P = 0.056; Table 4Citation ).

Apoptosis and Radiosensitivity.
The median AI was 0.92% (mean, 1.63%; range, 0–4.85%). All cases were divided into high and low AI according to the median AI. Fifteen tumors were categorized as low AI; 16 tumors were categorized as high AI (Fig. 1Citation C). There was no significant difference in radiosensitivity between high and low AI tumors (P = 0.835; Table 4Citation ).

MVD and Radiosensitivity.
Fig. 1Citation D shows the microvessels immunohistochemically detected using anti-CD31 antibody. MVD ranged from 11 to 70 microvessels per field (=0.391 mm2). The median MVD was 35 vessels/field (mean, 38.6 vessels/field). Tumors were divided into two groups (high MVD and low MVD), i.e., more or less than the median MVD. Tumors with high MVD showed significantly better radiosensitivity than those with low MVD in univariate analysis (P = 0.008, Table 4Citation ).

Multivariate and Kaplan-Meier Analyses.
Multivariate analysis was performed and revealed the MVD of the tumor alone to be significantly associated with radiosensitivity, whereas the Ki-67 labeling index showed no association (Table 5)Citation . Furthermore, to assess the predictive value of MVD for radiosensitivity, Kaplan-Meier analysis was performed for patients with high and low MVD tumors. Fig. 2Citation shows the Kaplan-Meier analysis of MVD in LSCC after radiotherapy. The LRC rate 3 years after treatment was 93.8% for those with high MVD, but only 33.3% for those with low MVD. A significant association between MVD and the LRC rate was apparent (P = 0.0003).


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  		Table 5 Multivariate Cox proportional hazard analysis of Ki-67 labeling index and MVD affecting radiosensitivity in T1 and T2 stage LSCC

 


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  		Fig. 2. Kaplan-Meier LRC curve for 31 patients with T1 and T2 stage LSCCs treated with radiotherapy according to MVD (microvessel density). Median, 35 vessels in a x200 high power field. P = 0.0003.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Generally, biological factors predicting the radiosensitivity of tumors fall into three categories: cell proliferation; oxygen effect; and apoptosis (21) . The present study aimed to analyze the correlation between radiosensitivity and (a) histopathological factors, (b) representative of cell proliferation including Ki-67 and the EGFR labeling index, (c) oxygen effect as reflected by neovascularization of the tumor, and (d) apoptosis as measured by expression of apoptosis-related proteins as an apoptotic index. The results obtained led us to conclude that MVD might become a predictive factor for radiosensitivity in early stage LSCC.

Oxygen status in tissue has been proved to be a very important factor determining radiosensitivity both in vitro and in animal model studies (14 , 15) . Hockel et al. (22) reported oxygen status measured by oxygen probes to be highly correlated with radiosensitivity in cervical carcinoma of the uterus. However, few studies have focused on the relation between O2 status and radiosensitivity in clinical specimens. This is because of difficulties measuring O2 in human tissues. Oxygen delivery to tumor tissues appears to rely on a network of microvessels, based on in vivo observations of vascular geometry and blood flow in the tumor microcirculation. In addition, Eric (23) also calculated that the oxygen diffusion distance in tumor tissue around vessels is ~150 µm. These data indicate that tumor angiogenesis must correlate with the oxygenation of tumor tissue. In fact, Hulka et al. (16) and Secomb et al. (24) reported that MVD correlates well with blood flow in breast tumors. Because measurement of O2 status in LSCCs is difficult for anatomical reasons, we introduced tumor MVD in early stage LSCCs as a representative of O2 status in the present study and found MVD to be a useful predictive marker for evaluating radiosensitivity in T1 and T2 stage LSCC before radiotherapy. Although some studies have focused on relations between numbers of tumor microvessels and prognosis or metastasis in far advanced tumors (25 , 26) , there are no reports on the relation between MVD and radiosensitivity in early stage LSCCs.

MVD heterogeneity in tumors must be a crucial issue in this study. Because all tumors used in the present study were small, i.e., 15 mm in diameter at the largest, MVD heterogeneity may be limited in such small tumors and MVD in biopsy specimens must represent the MVD of T1 and T2 stage LSCCs. In fact, we measured MVD heterogeneity using both surgically resected T2 tumors and corresponding biopsy specimens in five cases and found that there was no significant difference between them (data not shown). Because the exact O2 level could not be measured in the present cases, measurement of oxygen status and comparison between MVD and O2 status in LSCCs await future studies.

Ki-67 monoclonal antibody selectively reacted with the antigen in cell nuclei in the proliferating phase of the cell cycle (27) . Previous studies demonstrated a correlation between the Ki-67 labeling index and the progression of tumors of several organs, such as gastric cancer, head and neck cancer, and breast cancer (1 , 18 , 28) . However, contrary results have also been reported in carcinoma of the cervix (29) and head and neck cancer (30) . We found the Ki-67 labeling index to be weakly associated with radiosensitivity in univariate analysis but to show no association in multivariate analysis. Expression of EGFR is reportedly related to the proliferation of surviving tumor cells after radiation therapy (5) . However, EGFR; apoptosis-related molecules including p53, bcl-2, and bax; and the AI in tumor cells showed no relation with radiosensitivity in the present study. These data suggest that the radiosensitivity of these small LSCCs is determined by O2 status around the tumor cells rather than by biological factors indicating tumor proliferative activity or apoptosis. To confirm the predicting value for the MVD in radiosensitivity of LSCCs, further study with larger numbers of LSCCs or other series is needed. If patient number increases, the other predicting factors, which have been reported previously to associate with radiosensitivity, may become a potent prognostic factor.

The patients with recurrent tumors who underwent salvage surgery required prolonged nasal feeding. In fact, those who underwent total laryngectomy after radiotherapy required nasal feeding for an average of 101.6 days. To achieve the aim of avoiding ineffective irradiation and improving the patient’s quality of life, evaluation of MVD in biopsy specimens is an efficient means of predicting radiosensitivity in early stage LSCC patients.


   ACKNOWLEDGMENTS
 
We thank M. Nakane, Y. Hirose, and M. Horino for their technical assistance.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by a Grant-in Aid for Cancer Research from the Ministry of Health and Welfare (11-12) and by a Grant-in-Aid for Second Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare in Japan. Back

2 To whom requests for reprints should be addressed, at Pathology Division, National Cancer Center Research Institute East, 6-5-1 Kashiwanoha, Kashiwa, Chiba 277-8577, Japan. Phone: 81-471-34-6855; Fax: 81-471-34-6865; E-mail: aochiai{at}east.ncc.go.jp Back

3 The abbreviations used are: LSCC, laryngeal squamous cell carcinoma; EGFR, epidermal growth factor receptor; MVD, microvessel density; AI, apoptotic index; LRC, local regional control. Back

Received 3/27/00; revised 5/30/00; accepted 5/31/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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S.-C. Zhang, S.-i. Miyamoto, T. Kamijo, R. Hayashi, T. Hasebe, G. Ishii, M. Fukayama, and A. Ochiai
Intratumor Microvessel Density in Biopsy Specimens Predicts Local Response of Hypopharyngeal Cancer to Radiotherapy
Jpn. J. Clin. Oncol., December 1, 2003; 33(12): 613 - 619.
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S. Hironaka, T. Hasebe, T. Kamijo, A. Ohtsu, N. Boku, S. Yoshida, H. Saitoh, and A. Ochiai
Biopsy Specimen Microvessel Density Is a Useful Prognostic Marker in Patients with T2-4M0 Esophageal Cancer Treated with Chemoradiotherapy
Clin. Cancer Res., January 1, 2002; 8(1): 124 - 130.
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T. Kamijo, T. Yokose, T. Hasebe, H. Yonou, R. Hayashi, S. Ebihara, and A. Ochiai
Image Analysis of Microvessel Surface Area Predicts Radiosensitivity in Early-Stage Laryngeal Carcinoma Treated with Radiotherapy
Clin. Cancer Res., September 1, 2001; 7(9): 2809 - 2814.
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