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Clinical Cancer Research Vol. 8, 124-130, January 2002
© 2002 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Biopsy Specimen Microvessel Density Is a Useful Prognostic Marker in Patients with T2–4M0 Esophageal Cancer Treated with Chemoradiotherapy1

Shuichi Hironaka, Takahiro Hasebe, Tomoyuki Kamijo, Atsushi Ohtsu, Narikazu Boku, Shigeaki Yoshida, Hiroki Saitoh and Atsushi Ochiai2

Pathology Division, National Cancer Center Research Institute East, Chiba 277-8577, Japan [S. H., T. H., T. K., A. Oc.]; Division of Digestive Endoscopy and Gastrointestinal Oncology, National Cancer Center Hospital East, Chiba 277-8577, Japan [S. H., A. Oh., N. B., S. Y.]; and Second Department of Internal Medicine, Asahikawa Medical College, Asahikawa 078-8510, Japan [H. S.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: The purpose of this study was to identify prognostic markers for chemoradiotherapy (CRT) in T2–4M0 esophageal cancer.

Experimental Design: We investigated clinicopathological and biological markers in biopsy specimens from 73 T2–4M0 esophageal cancer patients treated with CRT (5-fluorouracil plus cisplatin and 60 Gy of radiation). Expressions of p53 gene product, Ki-67 labeling index, epidermal growth factor receptor, cyclin D1, vascular endothelial growth factor, microvessel density (MVD), thymidylate synthase, dihydropyrimidine dehydrogenase, and glutathione S-transferase {pi} in formalin-fixed biopsy samples of primary tumors before CRT were examined immunohistochemically. Clinicopathological and biological marker expressions were compared in terms of survival.

Results: Univariate analysis revealed that performance status and T stage in clinicopathological features had a significant association with survival (P = 0.007 and 0.04, respectively) and that patients whose tumors showed high MVD [>median (19.7 vessels)] in biological markers had significantly better survival than those with low MVD (<=median, P = 0.02). Also, there were weak associations of p53 and Ki-67 with survival (P = 0.08 and 0.07, respectively). Multivariate analysis, using both clinicopathological and biological markers, showed that MVD, T stage, and performance status became independent variables (P = 0.002, 0.02, and 0.02, respectively). Kaplan-Meier analysis showed that the patients with high MVD tumors survived longer than those with low MVD tumors (median survival time, not reached and 13 months, respectively; 3-year survival rate, 61% and 33%, respectively), with a significant difference of P = 0.02.

Conclusions: These results indicate that MVD using pretreatment biopsy specimens is a potentially useful prognostic marker for CRT in patients with T2–4M0 esophageal cancer who are treated with CRT.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Esophageal cancer is one of the most lethal malignancies among gastrointestinal neoplasms. Although surgery had been performed as the standard treatment for esophageal cancer, a variety of combined modality approaches have been investigated in efforts to improve long-term survival. Chemotherapy combined with radiotherapy for the treatment of esophageal cancer has been investigated since the 1980s, and the combination of 5-FU3 and CDDP has been regarded as being active and enhancing radiosensitivity (1, 2, 3) . Recent reports on CRT as a definitive and preoperative treatment have indicated various advantages in managing carcinoma of the esophagus (4, 5, 6) . We have reported that definitive CRT has curative potential for locally advanced esophageal carcinoma (7) , and Chan and Wong (8) reported that combined chemotherapy and radiation appeared to be as effective as esophagectomy in localized esophageal cancer. Thus, CRT is potentially an alternative to surgery, and investigating prognostic factors for esophageal cancer treated with CRT is very important.

Because of recent advances in basic research, many biological markers detected immunohistochemically have been reported in esophageal cancer. For p53, it has been postulated that tumors with p53 mutations may be more susceptible to CRT than tumors with wild-type p53 because of a lack of wild-type p53-induced arrest at G1-S and reduced time for DNA repair (9 , 10) . For proliferation-associated markers, such as Ki-67 and EGFR, tumors that respond best to DNA-damaging stimuli such as radiotherapy have been known to display a high proliferation rate (11) . For cyclin D1, recent identification of genes involved in cell cycle regulation has also led to an understanding that altered expression of these genes in cancer cells may be important in determining drug or radiation sensitivity (12) . VEGF induces mitogenesis of vascular endothelial cells, and vascular permeabilization and microvessel formation in a tumor are associated with tumor nutrition and oxygenation. They are associated with drug delivery and radiosensitivity because a well-oxygenated cell is fully radiosensitive (13 . TS is a target substrate of 5-FU, and it is reported that resistance to 5-FU is related in part to insufficient inhibition of TS (14) . The action of DPD is a critical and rate-limiting step in the catabolism of 5-FU, and intratumoral levels correlate with the response to 5-FU-based regimens (15) . GST-{pi} is an enzyme that plays an important role in cellular detoxification, and increases in this enzyme have been associated with resistance to antineoplastic agents such as CDDP (16) . However, the precise mechanism of tumor response to CRT is not fully understood. Some markers, including p53 (17 , 18) , Ki-67 (19) , EGFR (20) , cyclin D1 (21) , MVD (22) , and GST-{pi} (23) are reportedly prognostic in patients given CRT for esophageal cancer. However, most of these studies were performed using a small number of subjects, and their results were also controversial because of differences among treatment regimens.

This study was designed to identify useful prognostic markers in T2–4M0 esophageal cancer patients given a combination of 5-FU and CDDP with radiotherapy. We examined the expressions of p53, Ki-67, EGFR, cyclin D1, VEGF, MVD, TS, DPD, and GST-{pi} using an immunohistochemical staining method in biopsy specimens before CRT, and we investigated useful prognostic markers between clinicopathological and biological markers by multivariate analysis.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects.
A total of 209 esophageal cancer patients received CRT between August 1992 and April 1999 at the National Cancer Center Hospital East. Seventy-three of these patients met or fulfilled the following criteria and were included in this study: (a) sufficient biopsy specimens obtainable before treatment; (b) no previous treatment had been received; (c) age <= 75 years; (d) PS on the Eastern Cooperative Oncology Group scale <= 2; (e) adequate bone marrow, hepatic, and renal functions; and (f) stage T2–4, any N, M0 on the International Union against Cancer tumor-node-metastasis (TNM) classification. We excluded patients with M1a disease because of its very poor prognosis (24) . All patients were given the same regimen of concurrent chemotherapy and radiotherapy.

Treatment Schedule.
Chemotherapy consisted of a protracted infusion of 5-FU (400 mg/m2/day) on days 1–5 and 8–12, combined with CDDP (40 mg/m2/day) with adequate hydration and antiemetic coverage on days 1 and 8 (6) . This schedule was repeated twice every 5 weeks. Radiation therapy using megavoltage X-rays was started on day 1 concomitantly with chemotherapy. There was a 2-week break after a dose of 30 Gy. Radiation therapy was restarted on day 36, along with the same chemotherapy schedule used before. For patients who showed an objective response to treatment, additional chemotherapy was administered and consisted of a protracted infusion of 5-FU (800 mg/m2/day) on days 1–5 and a 2-h infusion of CDDP (80 mg/m2/day) on day 1. This treatment was repeated every 4 weeks for two courses. Additional courses of chemotherapy were optional but limited to a total of four courses. No further treatment was administered if no disease progression was observed.

Survival time was counted from the initiation of the first course of treatment to the date of death or to the final date of confirmation of survival.

Immunohistochemical Examination.
One to five biopsies, 1–5 mm in diameter, were taken for each tumor (one specimen, 7 cases; two specimens, 23 cases; three specimens, 33 cases; four specimens, 9 cases; and five specimens, 1 case). All of the biopsies were taken at the initial time of the diagnosis.

Immunohistochemical staining was carried out using the avidin-biotin-peroxidase complex method. Formalin-fixed, paraffin-embedded biopsy materials were cut into 3-µm sections, which were then deparaffinized in xylene, dehydrated in a graded ethanol series, and finally immersed in methanol with 0.3% H2O2 for 20 min to inhibit endogenous peroxidase activity. The sections for VEGF and CD31 staining were treated with 0.05% pepsin in 0.01 N HCl for 20 and 5 min at room temperature, respectively. The sections for p53, anti-Ki-67 antibody (MIB-1), EGFR, CD31, TS, DPD, and GST-{pi} staining were heated to 95°C by microwave irradiation twice for 10 min in 10 mM citrate buffer solution (pH 6.0), and the sections for cyclin D1 were immersed in EDTA retrieval fluid (pH 8.0). The sections were then cooled for 30 min at room temperature. After washing in PBS, all sections were blocked from nonspecific binding by preincubation with 5% skim milk (7.5 mg) and 2% BSA (3 mg) in PBS (150 ml) for 30 min. Next, the sections were incubated overnight at 4°C with the primary antibodies listed in Table 1Citation . After washing five times in PBS with 0.1% Tween 20 (washing buffer), slides were incubated with biotinylated secondary antimouse antibodies for p53, Ki-67, CD31, cyclin D1, and TS and antirabbit antibodies for VEGF, DPD, and GST-{pi} diluted 1:200 with blocking buffer for 30 min. After being washed five times with washing buffer, the sections were incubated with avidin-biotin complex (ABC) reagent (DAKO, Glostrup, Denmark), and a color reaction was developed using 2% 3,3'-diaminobenzidine in 50 mM Tris buffer (pH 7.6) containing 0.3% hydrogen peroxide for 5–10 min. The sections were counterstained with Meyer’s hematoxylin. In negative controls, the primary antibody solutions were replaced by the blocking buffer.


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  		Table 1 Panel of primary antibodies

 
Evaluation of Immunostaining.
The percentages of p53-, Ki-67-, and cyclin D1-positive tumor cells were calculated by counting the number of brown-stained tumor nuclei/total number of cancer cells in the most highly stained area on a high-power view (x400; 0.196 mm2/field). In each specimen, more than 400 cells for p53, cyclin D1, and Ki-67 were counted. According to the intensity of cell membrane EGFR staining in the whole tumor (median area, 3.14 mm2), all patients could be divided into the following four groups: (a) group ±, fainter staining than normal esophageal epithelium; (b) group +, the same staining as normal epithelium; (c) group ++, moderately stronger staining; and (d) group +++, markedly stronger staining. Groups ± and + were defined as negative for EGFR expression, and groups ++ and +++ were defined as positive for EGFR expression. The staining of VEGF was graded as follows: (a) +, the staining intensity in cancer cells was stronger than that in the stromal cells; (b) ±, the staining intensity in cancer cells was equal to that in stromal cells; and (c) -, the staining intensity in cancer cells was weaker than that in stromal cells. The cases graded as + were defined as positive, as described in previous reports (25) . The microvessel count was assessed by light microscopy in three of the most extensive areas of neovascularization (termed "hot spots") at a high-power view (x400; x40 objective and x10 ocular; 0.196 mm2/field), and we calculated the average number of vessels. We counted intratumoral and stromal vessels with actual lumens around the tumor nests but did not count a single endothelial cell (or cluster) and vessels that existed far from the tumor nests. For TS and DPD staining, positivity was based on a subjective estimation of the intensity (0–3) and extent of tumor staining. Positive staining was defined if >30% of the tumor was stained, and negative staining was defined as staining in <=30% of the tumor. Intensity levels 0 and 1 were grouped together and considered negative, whereas staining intensities 2 and 3 were considered positive (18) . The intensity of GST-{pi} staining was graded as follows: (a) ++, strong; (b) +, faint; and (c) -, no visible staining. For this marker, cases were defined as positive when >20% of all cancer cells in each section showed ++ or + staining (26) . Immunohistochemical staining was evaluated independently by two investigators who were blind to the clinical outcomes of the patients. When the evaluation for each antibody differed between investigators, the investigators discussed it, with or without reevaluation, until an agreement was reached.

Statistical Analysis.
Subjects were categorized as positive or negative according to the immunohistochemical results. Univariate analysis for survival was performed by using log-rank tests. The influence of each biological variable on patient survival was assessed by the Cox proportional hazards model. The survival curves were calculated by the Kaplan-Meier method. P < 0.05 was considered significant. Statistical calculations were performed using the Statistica package (Statsoft, Tulsa, OK).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Patient Characteristics.
Clinicopathological features of the patients in this study are shown in Table 2Citation . In terms of T stage, 10 patients had T2 disease, 41 patients had T3 disease, and 22 patients had T4 disease. In terms of N stage, 26 patients had N0 disease, and 47 patients had N1 disease. A total of 71 patients (97%) completed at least the CRT segment with a total radiation dose of 60 Gy, and the other 2 patients received 40 and 45 Gy, respectively. Twenty patients (16%) received one additional course of chemotherapy, and 32 patients (44%), 2 patients (3%), and 3 patients (4%) received an additional two, three, and four courses, respectively.


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  		Table 2 Characteristics of patients with T2–4 M0 esophageal cancer

 
Immunoreactivity.
All 73 specimens were immunohistochemically evaluated for p53, Ki-67, EGFR, cyclin D1, VEGF, CD31, TS, DPD, and GST-{pi}. Representative immunohistochemical p53, EGFR, CD31, and DPD stainings are shown in Fig. 1Citation . Positive p53, Ki-67, and cyclin D1 immunoreactivities were detected in nuclei, whereas VEGF, TS, DPD, and GST-{pi} reactivities were observed in the cytoplasm. EGFR expression was seen both on the cell membrane and in the cytoplasm. Microvessels were detected immunohistochemically using anti-CD31 antibody. We made histograms of the p53, Ki-67, and cyclin D1 results, and they showed bimodal distribution. Then, we decided that the trough of the histogram was the cutoff value for positive versus negative or high versus low marker levels. Expressions of p53, Ki-67, and cyclin D1 varied from 0–95%, 28–88%, and 0–100% with median values of 71%, 72%, and 56%, respectively. We categorized the samples as positive if >20%, >64%, and >40% of the tumor nuclei were stained by anti-p53, anti-Ki-67, and anti-cyclin D1 antibodies, respectively. With regard to MVD, the level varied from 3.7 to 43.7 vessels with a median value of 19.7. We classified tumors into two groups, high MVD and low MVD, based on the number of median value.



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  		Fig. 1. Representative immunohistochemical p53, EGFR, CD31, and DPD stainings in biopsy specimens before CRT. A, p53. p53 immunoreactivity is detected in the nuclear region of tumor cells, as was that for Ki-67 and cyclin D1. B, EGFR positive. EGFR immunostaining is seen in the cell membranes of tumor cells. C, EGFR negative. Immunostaining is not seen in any cell membranes. D, CD31 for microvessels. Microvessels are most numerous at the tumor periphery. E, DPD positive. DPD immunostaining was detected in the tumor cell cytoplasm, as was immunostaining for VEGF, TS, and GST-{pi}. F, DPD negative. Immunostaining is not seen in any tumor cells.

 
Positivity for p53, EGFR, cyclin D1, VEGF, TS, DPD, and GST-{pi} staining was observed in 51, 42, 45, 36, 46, 44, and 46 of 73 cases (70%, 58%, 62%, 49%, 63%, 60%, and 63%), respectively. Fifty-four cases (74%) were determined to have a Ki-67 labeling index of >64, and 36 of 73 (49%) were categorized into the high MVD group.

Univariate Analysis for Survival.
Table 3Citation presents the clinicopathological features of patients and survival. PS and T stage had a significant association with survival (P = 0.007 and 0.04, respectively). The relations of other clinicopathological factors, including age, sex, tumor location, histological type, and N stage, with survival were negligible.


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  		Table 3 Summary of relation between clinicopathological markers and survival

 
Table 4Citation summarizes biological markers and survival. MVD had the strongest association with survival (P = 0.02), and p53 and Ki-67 had weak associations (P = 0.08 and 0.07, respectively) with survival. Negligible relations were observed for EGFR, cyclin D1, VEGF, TS, DPD, and GST-{pi} expressions.


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  		Table 4 Summary of relation between biological markers and survival

 
Multivariate Analysis for Survival.
The effects of clinicopathological and biological variables, including T stage, N stage, PS, MVD, p53, and Ki-67, were examined by multivariate analysis using the Cox proportional hazards model (Table 5)Citation . MVD, T stage, and PS were identified as significant and independent variables (P = 0.002, 0.02, and 0.02, respectively). A low MVD value yielded a hazard ratio of 3.23, with 95% confidence interval ranging from 1.54–6.78. No other variables were significantly associated with survival on multivariate analysis.


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  		Table 5 Multivariate analysis of clinicopathological and biological markers in survival of 73 T2–4 M0 patients treated with CRT

 
Survival Curves According to MVD by Kaplan-Meier Analysis.
The median follow-up period was 34 months. The MST of all patients with T2–4M0 esophageal cancer given CRT was 34 months, and the 3-year survival rate was 44%. Fig. 2Citation shows the survival curves according to MVD using Kaplan-Meier analysis. The patients with high MVD tumors survived significantly longer than those with low MVD tumors (P = 0.02). The MST of the former group was not reached, and their 3-year survival rate after treatment was 61%; the MST and 3-year survival rate of the latter group were 13 months and 33%, respectively.



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  		Fig. 2. The survival curves for 73 patients with T2–4M0 esophageal squamous cell carcinoma given CRT, according to MVD. Patients with high MVD tumor survived longer than those with low MVD tumor (MST, not reached and 13 months; 3-year survival rate, 61% and 33%, respectively), with a statistically significant difference (P = 0.02).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we examined the usefulness of prognostic factors including clinicopathological and immunohistochemical biological markers in patients with T2–4M0 esophageal cancer who were given CRT. We found MVD, as well as PS, to be an independent prognostic factor for CRT on multivariate survival analysis. We have recently reported that high MVD laryngeal squamous cell carcinomas are more radiosensitive than those with low MVD, and we found that patients with the former survived significantly longer than those with low MVD (27) . Basically, tissue oxygen status has been demonstrated to be a very important factor determining radiosensitivity both in vitro and in vivo (28 , 29) . 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. On the other hand, the tumor microcirculation is clearly an important factor in drug delivery to cancer cells, and the efficacy of drug delivery can be high in richly vascularized tumors. These data are consistent with our present observation that MVD is a favorable prognostic factor in patients who have undergone CRT. In contrast to the present study, it was reported that cancers showing high angiogenesis had a poor prognosis and that this was related to local invasion and distant metastasis (30) . There was a statistically significant difference in survival rate in favor of the hypovascular group in surgically resected cases (31 , 32) . This discrepancy in the usefulness of the MVD of tumor tissues between surgically and CRT-treated cases is potentially interesting. Furthermore, anticancer drugs, especially the 5-FU and CDDP used in our CRT regimen, are regarded as radiosensitizers (1, 2, 3) . Therefore, a combination of chemotherapy and radiotherapy is a more effective strategy and improved the survival of patients with high MVD tumors more than the survival of those with low MVD tumors because of the point of oxidation and drug delivery to cancer cells. This is in contrast to the previous studies involving surgical cases.

There is another difference in microvessel characterization during microscopic examination from previous reports (22 , 31, 32, 33) . Others counted any brown-stained endothelial cell or endothelial cluster as a single vessel; a vessel lumen was not required for identification of a microvessel. Eric Hall (34) stated that the oxygen diffusion distance in tumor tissue around vessels was approximately 150 µm; i.e., that is to say that cancer cells within a diameter of approximately 150 µm around vessels are involved in the oxygenation area. For this reason, we counted intratumoral and stromal vessels with actual lumens around the tumor nests in consideration of tumor oxidation. We did not count a single endothelial cell (or cluster), and we did not count vessels that existed far from the tumor nests. This evaluation of MVD is thought to be reasonable for studying chemoradiosensitivity and investigating a prognostic marker in patients treated with radiotherapy and/or chemotherapy. Additional studies are needed to confirm the efficacy of this evaluation.

Recently, the effectiveness of CRT for locally advanced esophageal cancer has become clear, and its curative potential is as high as that of surgery (7 , 8) . In the RTOG 85-01 trial, the MST was 14.1 months, and the 5-year survival rate was 27% after combined CDDP, 5-FU infusion, and radiation (35 , 36) . In a literature review on the surgical management of esophageal cancer by Müller et al. (37) , however, the mean respective 3-year and 5-year survival rates after esophagectomy were 25% and 20%. There is no remarkable difference in survival benefit between CRT and surgery for localized esophageal cancer. Esophageal cancer patients may be able to select between CRT and surgery according to the individual characteristics of their tumors. Therefore, it is important to clarify prognostic markers in esophageal cancer patients with operable stage tumors. In this context, our results show MVD to be a potentially useful prognostic marker in patients treated with CRT. We also found that the prognostic significance of MVD is opposite that for surgery as described in previous reports. These observations suggest MVD to be a potential marker for choosing between CRT and surgery. Using pretreatment biopsy samples, we are currently investigating clinicopathological and biological prognostic markers including MVD in esophageal cancer patients with T2–3M0 disease who underwent surgery. We hope that MVD will be beneficial for selecting the optimal treatment regimen, thereby prolonging survival for all esophageal cancer patients in the near future.


   ACKNOWLEDGMENTS
 
We thank Dr. Masakazu Fukushima (Taiho Pharmaceutical Co., Ltd.) for providing TS monoclonal antibody and DPD monoclonal antibody. We also thank Mari Nakane and Yuki Yanagisawa for 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 Supported in part by Grant-in-Aid 11-12 for Cancer Research from the Ministry of Health and Welfare and by a Grant-in-Aid for the Second Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare, 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: 81-471-33-1111; Fax: 81-471-31-4724; E-mail: aochiai{at}east.ncc.go.jp Back

3 The abbreviations used are: 5-FU, 5-fluorouracil; CRT, chemoradiotherapy; CDDP, cisplatin; DPD, dihydropyrimidine dehydrogenase; EGFR, epidermal growth factor receptor; GST-{pi}, glutathione S-transferase {pi}; MST, median survival time; MVD, microvessel density; PS, performance status; TS, thymidylate synthase; VEGF, vascular endothelial growth factor. Back

Received 1/29/01; revised 10/ 8/01; accepted 10/ 8/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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