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c-erbB-2 oncoprotein expression related to chemoradioresistance in esophageal squamous cell carcinoma
Int. J. Radiation Oncology Biol. Phys., Vol. 57, No. 5, pp. 1323–1327, 2003 Copyright © 2003 Elsevier Inc. Printed in the USA. All rights reserved 0360-3016/03/$–see front matter
doi:10.1016/S0360-3016(03)00782-X
CLINICAL INVESTIGATION
Esophagus
c-erbB-2 ONCOPROTEIN EXPRESSION RELATED TO CHEMORADIORESISTANCE IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA MASAYUKI AKAMATSU, M.D.,* TOSHIHARU MATSUMOTO, M.D.,† KUNIYUKI OKA, M.D.,‡ SHIGETAKA YAMASAKI, M.D.,† HIROSHI SONOUE, M.T.,† YOSHIAKI KAJIYAMA, M.D.,§ MASAHIKO TSURUMARU, M.D.,§ AND KEISUKE SASAI, M.D.* *Department of Radiology, †First Department of Pathology, and §First Department of Surgery, Juntendo University School of Medicine, Tokyo, Japan; ‡Department of Pathology, Mito Saiseikai General Hospital, Mito, Japan Purpose: Esophageal carcinoma is a challenging target for radiotherapy. To improve treatment efficacy, an investigation of a predictive factor is desirable. In this study, we evaluated the significance of apoptosis and immunohistochemical staining for p53, Ki-67, c-erbB-2 (HER-2/neu), Ku (p70/p80), and DNA-PKcs for predictive markers of the responsiveness to chemoradiotherapy in esophageal squamous cell carcinoma. Methods and Materials: This retrospective analysis consisted of 34 patients with esophageal squamous cell carcinoma in whom tumor biopsy was performed before treatment. They were divided into chemoradiosensitive (n ⴝ 13) and chemoradioresistant (n ⴝ 21) groups according to the tumor response evaluated at a total radiation dose of 40 Gy. The biopsy samples were examined with immunohistochemical staining for various factors and with an in situ nick end labeling method for apoptosis. The examined data were compared between the two groups. Results: The difference in the Ki-67, p53, Ku (p70/p80), DNA-PKcs labeling indexes and the apoptosis index in tumor cells between the chemoradiosensitive and chemoradioresistant groups was not statistically significant. The expression of c-erbB-2 oncoprotein was statistically significant in the chemoradioresistant group (p ⴝ 0.02), although it did not correlate with survival. Conclusion: c-erbB-2 immunostaining is useful for the prediction of chemoradioresistance in esophageal squamous cell carcinoma. © 2003 Elsevier Inc. c-erbB-2, Immunostaining, Chemoradiotherapy, Esophagus, Squamous cell carcinoma.
studies have been limited to p53 and Ki-67 examinations (5– 8). In the current study, to determine the predictive factors for the responsiveness to chemoradiotherapy in esophageal squamous cell carcinoma, we performed immunohistochemical examinations of p53, Ki-67, c-erbB-2, Ku (p70/ p80), and DNA-PKcs on tumor tissue biopsies before chemoradiotherapy. Apoptosis in the tumor tissues was also examined.
INTRODUCTION Esophageal carcinoma is a challenging target for radiation therapy (RT). Recently, some investigators reported good treatment results with RT concurrently combined with chemotherapy. However, even with the improvement of these treatment modalities, the survival rates of patients with the advanced disease are not satisfactory. To improve treatment efficacy, an investigation of a predictive factor is desirable. Recently, Oka and colleagues (1–3) and Niibe et al. (4) performed detailed analyses of the predictive factors for the responsiveness to RT in cervical squamous cell carcinoma, using immunohistochemical examinations of proliferative markers, tumor suppressor oncogenes, and oncoproteins. However, in esophageal squamous cell carcinoma, such
METHODS AND MATERIALS Patients and methods Between August 1998 and February 2001, 84 consecutive patients with locally advanced esophageal carcinoma received RT with or without concurrent chemotherapy at the Japan Society for Promotion of Science (Grant 14570878). Acknowledgments—The authors thank D. Mrozek (Medical English Service, Kyoto, Japan) for the English revision. Received Feb 19, 2003, and in revised form Jun 11, 2003. Accepted for publication Jun 16, 2003.
Reprint requests to: Toshiharu Matsumoto, M.D., First Department of Pathology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 Japan. Tel: ⫹81-35802-1037; Fax: ⫹81-3-3812-1056; E-mail: machian@med. juntendo.ac.jp Supported in part by Grant-in-Aid for Scientific Research (C), 1323
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Table 1. Summary of clinical data and histologic diagnosis in chemoradiosensitive and chemoradioresistant groups Characteristic Age (y) Mean Range Gender (n) Male Female Primary tumor* (n) T1 T3 T4 Tumor size (mm) Mean Range Chemoradiotherapy (n) Radiotherapy only (n) SCC differentiation (n) Well Moderate Poor Stage (n)† I II III IV Prognosis (n) Alive DOD
Chemoradiosensitive group (n ⫽ 13)
Chemoradioresistant group (n ⫽ 21)
61 43–80
65 49–76
11 2
18 3
2 5 6
0 10 11
67 20–125 12 1
84 35–180 19 2
3 8 2
5 9 7
1 2 9 1
0 1 15 5
3 10
1 20
Abbreviations: SCC ⫽ squamous cell carcinoma; DOD ⫽ dead of disease. * International Union Against Cancer TNM clinical classification (9). † Guidelines for clinical and pathologic studies on carcinoma of the esophagus by Japanese Society of Esophageal Diseases (10).
Department of Radiology, Juntendo University Hospital, Tokyo. The patients with suspected invasion to adjacent major unresectable organs, including the trachea, bronchus, and aorta, at the preoperative examination received chemoradiotherapy. All patients were treated with preoperative intent. However, when the evaluation of the response to treatment at a total dose of 40 Gy revealed that the tumor was still unresectable, the patients received additional chemoradiotherapy. The good responders underwent esophagectomy and lymphadenectomy. For this study, formalinfixed, paraffin-embedded tissue biopsies from esophageal lesion were available from 34 patients, including good and poor responses to chemoradiotherapy. All 34 patients presented with histologic evidence of esophageal squamous cell carcinoma. Complete follow-up was obtained in all patients. Table 1 summarizes the patient characteristics. Of the 34 patients, 29 were men and 5 were women. The median age was 63 years (range 43– 80). They were divided into chemoradiosensitive (n ⫽ 13) and chemoradioresistant (n ⫽ 21) groups according to the clinical findings, which were obtained by a barium study of the esophagus and an endoscopic examination performed before treatment. The chemoradiosensitive group was defined as those showing a bi-dimensional ⱖ50% decrease or disappearance of the
tumor mass after chemoradiotherapy. All others were included in the chemoradioresistant group. The clinical and histologic findings in each group are described in Table 1. The tumor stage was determined according to the International Union Against Cancer TNM clinical classification (4th edition) (9). Clinical evaluation of the primary tumor was performed using the guidelines for the clinical and pathologic studies on carcinoma of the esophagus by the Japanese Society of Esophageal Diseases (10). Chemoradiotherapy protocol Of the 34 patients, 11 received preoperative chemoradiotherapy, 20 definitive chemoradiotherapy, and 3 RT alone. RT was usually delivered to the esophagus, mediastinum, and supraclavicular regions with opposite AP and PA ports of 6- or 10-MV X-rays. The dose per fraction ranged from 1.8 to 2.0 Gy. The tumor doses ranged from 39.6 to 40 Gy for preoperative RT and from 60 to 70 Gy for definitive treatment. In the definitive RT with or without concurrent chemotherapy, the treatment fields were reduced to the primary tumor and enlarged lymph nodes after a total dose of 40 Gy. Twenty-six patients received combination chemotherapy of cisplatinum and 5-fluorouracil (5 d/wk), three received
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cisplatinum, 5-fluorouracil, and leucovorin (5 d/wk), and two received cisplatinum alone (5 d/wk). The daily doses of the agents were as follows: cisplatinum 5–10 mg/m2, 5-fluorouracil 250 –500 mg/m2, and leucovorin 10 –30 mg/m2, for a maximum of 4 weeks. Histopathologic study All patients underwent biopsy during the endoscopic examination of the esophageal tumor before the start of treatment. The specimens were fixed with 10% formaldehyde for up to 24 h and embedded in paraffin for hematoxylin and eosin staining, immunohistochemical staining, and the terminal deoxynucleotidyl transferase-mediated dUTPbiotin nick end labeling (TUNEL) method. Immunohistochemical studies of Ki-67 (MIB-1, Immunotech International, Marseilles, France; 1:400), p53 (DO-7, Dakopatts, Glostrup, Denmark; 1:2000), c-erbB-2 (HER-2/neu; Dako rabbit antihuman polyclonal antibody to c-erbB-2 oncoprotein, code number A0485, Dakopatts; 1:300), Ku (p70/p80; NeoMarkers, Fremont, CA; 1:100), and DNA-PKcs (NeoMarkers; 1:100) were performed using the avidin-biotinperoxidase complex technique. Microwave epitope retrieval (0.01 mol/L citrate buffer, pH 6.0) was performed before the immunostaining for Ki-67, p53, and c-erbB-2. Enzyme (proteinase-K, 10 min) antigen retrieval was used for Ku (p70/p80) and DNA-PKcs immunostaining. Apoptosis was determined by morphology and TUNEL, as described elsewhere (4). The Ki-67, p53, Ku (p70/p80), and DNA-PKcs labeling indexes were calculated as the percentage of positive cells in a minimum of 1000 carcinoma cells. The apoptotic cell index was calculated by determining the percentage of apoptotic cells in a minimum of 1000 carcinoma cells. The primary antibody for c-erbB-2 immunostaining is a concentrate of the same anti-HER-2/neu antibody provided in a prediluted form in the Hercept Test kit. Therefore, its immunostaining in the tumor cells was evaluated as 0, 1⫹, 2⫹, and 3⫹ according to the U.S. Food and Drug Administration–approved scoring guidelines for the Hercep Test (11). Scores of 0 or 1⫹ were considered negative for c-erbB-2 overexpression, 2⫹ was weakly positive, and 3⫹ was strongly positive (11). To qualify for 2⫹ and 3⫹ scoring (i.e., positive), complete membrane staining of ⬎10% of tumor cells had to be observed. Statistical analysis Actuarial overall survival rates were calculated using the Kaplan-Meier method. A comparison of the overall survival between the two groups was performed using a log–rank test. Mann-Whitney’s U test and Fisher’s exact probability test were also used to compare the distributions of patient characteristics between the two groups. Statistical significance was considered as p ⬍0.05. RESULTS No statistically significant difference was found between the chemoradiosensitive and chemoradioresistant groups in
Fig. 1. Survival curves of chemoradiosensitive and chemoradioresistant groups.
the distributions of age, gender, clinical findings of the primary tumor, tumor size, and tumor stage. The grade of differentiation of squamous cell carcinoma and the doses of chemoradiotherapy did not differ in both groups. The survival rate in the chemoradiosensitive group was significantly better than that in the chemoradioresistant group (p ⬍0.01; Fig. 1). The results of the immunohistochemical studies and apoptosis examinations in the tumor cells of the chemoradiosensitive and chemoradioresistant groups are presented in Tables 2 and 3. No statistically significant difference was found in the Ki-67, p53, Ku (p70/p80), and DNA-PKcs labeling indexes and apoptosis index in the tumor cells between these two groups (Table 2). c-erbB-2 oncoprotein expression (Fig. 2) was more frequent in the chemoradioresistant group than in the chemoradiosensitive group (p ⫽ 0.02; Table 3). However, no difference was noted in the survival rate according to the expression of c-erbB-2 oncoprotein (p ⫽ 0.14; Fig. 3). DISCUSSION Although numerous efforts have been made to improve the outcome of esophageal carcinoma, the prognosis of patients with advanced disease is still poor. Recent studies have demonstrated that platinum-containing aggressive chemoradiotherapy was an effective treatment modality for this disease (12, 13). However, the regimens also produced significant toxicity, and therefore, it is important to predict the effect of the treatment before the start of therapy. Of the several factors we tested, only the c-erbB-2 oncogen product was predictive of the tumor response. It is a 185-kD transmembrane protein and is a member of the epidermal growth factor receptor (EGFR) family. The EGFR family
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Table 2. Ki-67, p53, Ku (p70/p80), DNA-PKcs, and apoptotic labeling index in tumor cells of the chemoradiosensitive and chemoradioresistant groups Labeling index
Chemoradiosensitive group (%)
Chemoradioresistant group (%)
p
Ki-67 p53 Ku (p70/p80) DNA-PKcs Apoptosis
64.2 (23.9) 70.9 (35.4) 90.2 (16.3) 74.5 (27.9) 0.30 (0.12)
53.6 (22.3) 53.5 (36.4) 87.2 (17.6) 80.8 (19.9) 0.37 (0.14)
0.26 0.07 0.14 0.80 0.16
Data presented as the mean, with the SD in parentheses.
protein has tyrosine kinase activity. erbB-2 is a constitutively active receptor and is the preferred co-receptor for other members of this family, erbB-3 and erbB-4. The heterodimers between erbB-2 and the other erbB receptors have relatively high ligand affinity. erbB-2 is, therefore, a master coordinator of the signaling network that functions as a shared co-receptor for erbB ligands (14). A major signaling route of the EGFR family is a Ras-Raf-MAP-kinase pathway involved in cell survival. Another important pathway is the one constituted by PI3 kinase and the downstream protein kinase Akt. After its activation, Akt transduces signals that regulate multiple biologic processes, including apoptosis, gene expression, and cellular proliferation. Akt is likely to send survival (antiapoptotic) signals by phosphorylating multiple targets, including the BCL-2 family member BAD and the cell death pathway enzyme caspase-9. Akt also has a prominent role in the regulation of cell cycle progression (15). Therefore, the activation of the EGFR family by ligands, ionizing radiation, or overexpression is strongly related to the radioresistance of the cell. Ionizing radiation indiscriminately activates all erbB molecules expressed by a cell, and the radiation response may primarily be determined by cellular erbB expression profiles. c-erbB-2 overexpression has been associated with the pathogenesis of several human malignancies and with a poor prognosis in breast carcinoma. Overexpression of the protein is also associated with ipsilateral breast carcinoma recurrence after RT. The results of laboratory experiments have suggested that this overexpression of c-erbB-2 is itself sufficient to confer radioresistance (14). In the current study, c-erbB-2 expression was related to local resistance to chemoradiotherapy, but it was not related to the prognosis. However, in uterine cervical squamous cell carcinoma, Oka et al. (2) and Nishioka et al. (16) showed the good relationship between c-erbB-2 expres-
sion and poor prognosis, as well as local resistance to RT. The reason for the difference between their observation and ours in esophageal carcinoma is unclear. The small number of patients in this study is a possible reason. Additional confirmation of the findings is needed in a large group of patients. Another possible reason is the difference in the characteristics of these two tumors. Uterine cervical carcinoma is, generally, locally invasive. Therefore, local control may be the most important factor in the overall survival of patients with uterine cervical carcinoma. As our study showed, cells with overexpression of c-erbB-2 are radioresistant. Because esophageal carcinoma is not only locally invasive, but also distantly metastatic, local control may not predict the prognosis of the patients well. Recently, Uno et al. demonstrated that a combined treatment of recombinant humanized anti-erbB-2 monoclonal antibody (rhuMab HER-2) and irradiation showed an additive effect in head-and-neck squamous cell carcinoma cells, as well as in breast carcinoma cells (17). In the current study, tumors with c-erbB-2 expression were resistant to cisplatinum-based chemoradiotherapy. Therefore, it may be possible to overcome this resistance using rhuMab HER-2 combined with cisplatinum-based chemoradiotherapy. The other factors tested here were not related to the responsiveness of esophageal carcinoma. p53 and Ki-67
Table 3. c-erbB-2 oncoprotein expression in tumor cells of chemoradiosensitive and chemoradioresistant groups c-erbB-2 oncoprotein expression Group*
Negative
Positive
Chemoradiosensitive Chemoradioresistant
9 6
4 15
* p ⫽ 0.02.
Fig. 2. Well-differentiated squamous cell carcinoma of esophagus in patient from chemoradioresistant group. Many tumor cells strongly positive for c-erbB-2 oncoprotein and showing a surface membrane staining pattern (c-erbB-2 immunostaining, original magnification ⫻400).
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ageal carcinoma was associated with a decreased responsiveness to treatment, others, including our group, did not observe such an association in patients with squamous cell carcinoma (5, 7, 8). Just as in our previous study (8), p53 expression was not related to the effect of chemoradiotherapy in the current study. Kitamura and colleagues (6) examined the relationship between the expression of Ki-67 in esophageal squamous cell carcinoma and prognosis after chemoradiotherapy. Although their study indicated that a high Ki-67 labeling index was related to the chemoradiosensitivity (6), we could not confirm this relationship. In other organs, different results for the relationship between Ki-67 and radiosensitivity of squamous cell carcinoma have been reported, including better prognosis with a high Ki-67 labeling index or no relationship (3, 18, 19).
Fig. 3. Survival curves of c-erbB-2–negative and c-erbB-2–positive groups.
immunostaining have been evaluated as predictive factors for the response of esophageal squamous cell carcinoma to chemoradiotherapy (5– 8). Although some investigators showed that the overexpression of the p53 protein in esoph-
CONCLUSION c-erbB-2 immunostaining is useful to predict the tumor response to RT concurrently combined with chemotherapy for esophageal squamous cell carcinoma. These results suggest that using rhuMab HER-2 combined with cisplatinumbased chemoradiotherapy may improve tumor response.
REFERENCES 1. Oka K, Nakano T, Arai T. p53CM1 expression is not associated with prognosis in uterine cervical carcinoma. Cancer 1993;72:160–164. 2. Oka K, Nakano T, Arai T. c-erbB-2 oncoprotein expression is associated with poor prognosis in squamous cell carcinoma of the cervix. Cancer 1994;73:668–671. 3. Oka K, Arai T. MIB1 growth fraction is not related to prognosis in cervical squamous cell carcinoma treated with radiotherapy. Int J Gynecol Pathol 1996;15:23–27. 4. Niibe Y, Nakano T, Ohno T, et al. Relationship between p21/WAF-1/CIP-1 and apoptosis in cervical cancer during radiation therapy. Int J Radiat Oncol Biol Phys 1999;44:297– 303. 5. Yang B, Rice TW, Adelstein DJ, et al. Overexpression of p53 protein associates decreased response to chemoradiotherapy in patients with esophageal carcinoma. Mod Pathol 1999;12: 251–256. 6. Kitamura K, Saeki H, Kawaguchi H, et al. Immunohistochemical status of the p53 protein and Ki-67 antigen using biopsied specimens can predict a sensitivity to neoadjuvant therapy in patients with esophageal cancer. Hepatogastroenterology 2000;47:419–423. 7. Ito T, Kaneko K, Makino R, et al. Prognostic value of p53 mutations in patients with locally advanced esophageal carcinoma treated with definitive chemoradiotherapy. J Gastroenterol 2001;36:303–311. 8. Kajiyama Y, Hattori K, Tomita N, et al. Histopathologic effects of neoadjuvant therapies for advanced squamous cell carcinoma of the esophagus: Multivariate analysis of predictive factors and p53 overexpression. Dis Esophagus 2002;15: 61–66. 9. Hermanek P, Hutter RVP, Sobin LH, et al., editors. UICC TNM Atlas: Illustrated guide to the TNM/pTNM classification of malignant tumours. 4th ed. Berlin: Springer-Verlag; 1997. 10. Japanese Society for Esophageal Diseases. Guidelines for the
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clinical and pathologic studies on carcinoma of the esophagus, 9th ed. Tokyo: Kanehara Ltd; 1999. Jacobs TW, Gown AM, Yaziji H, et al. Specificity of Hercep Test in determining HER-2/neu status of breast cancers using the United States Food and Drug Administration-approved scoring system. J Clin Oncol 1999;17:1983–1987. Forastiere AA, Orringer MB, Perez-Tamayo C, et al. Preoperative chemoradiation followed by transhiatal esophagectomy for carcinoma of the esophagus: Final report. J Clin Oncol 1993;11:1118–1123. Adelstein DJ, Rice TW, Becker M, et al. Use of concurrent chemotherapy, accelerated fractionation radiation, and surgery for patients with esophageal carcinoma. Cancer 1997;80: 1011–1120. Sartor CI. Biological modifiers as potential radiosensitizers: Targeting the epidermal growth factor receptor family. Semin Oncol 2000;27(Suppl. 11):15–20. Mendelsohn J, Baselga J. The EGF receptor family as targets for cancer therapy. Oncogene 2000;19:6550–6565. Nishioka T, West CML, Gupta N, et al. Prognostic significance of c-erbB-2 protein expression in carcinoma of the cervix treated with radiotherapy. J Cancer Res Clin Oncol 1999;125:96–100. Uno M, Otsuki T, Kurebayashi J, et al. Anti-HER2-antibody enhances irradiation-induced growth inhibition in head and neck carcinoma. Int J Cancer 2001;94:474–479. Pillai MR, Jayaprakash PG, Nair MK. Tumour-proliferative fraction and growth factor expression as markers of tumor response to radiotherapy in cancer of the uterine cervix. J Cancer Res Clin Oncol 1998;124:456–461. Bjo¨ rk-Eriksson T, West CML, Cvetskovska E, et al. The lack of correlation between proliferation (Ki-67, PCNA, LI, Tpot), p53 expression and radiosensitivity for head and neck cancers. Br J Cancer 1999;80:1400–1404.
doi:10.1016/S0360-3016(03)00782-X
CLINICAL INVESTIGATION
Esophagus
c-erbB-2 ONCOPROTEIN EXPRESSION RELATED TO CHEMORADIORESISTANCE IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA MASAYUKI AKAMATSU, M.D.,* TOSHIHARU MATSUMOTO, M.D.,† KUNIYUKI OKA, M.D.,‡ SHIGETAKA YAMASAKI, M.D.,† HIROSHI SONOUE, M.T.,† YOSHIAKI KAJIYAMA, M.D.,§ MASAHIKO TSURUMARU, M.D.,§ AND KEISUKE SASAI, M.D.* *Department of Radiology, †First Department of Pathology, and §First Department of Surgery, Juntendo University School of Medicine, Tokyo, Japan; ‡Department of Pathology, Mito Saiseikai General Hospital, Mito, Japan Purpose: Esophageal carcinoma is a challenging target for radiotherapy. To improve treatment efficacy, an investigation of a predictive factor is desirable. In this study, we evaluated the significance of apoptosis and immunohistochemical staining for p53, Ki-67, c-erbB-2 (HER-2/neu), Ku (p70/p80), and DNA-PKcs for predictive markers of the responsiveness to chemoradiotherapy in esophageal squamous cell carcinoma. Methods and Materials: This retrospective analysis consisted of 34 patients with esophageal squamous cell carcinoma in whom tumor biopsy was performed before treatment. They were divided into chemoradiosensitive (n ⴝ 13) and chemoradioresistant (n ⴝ 21) groups according to the tumor response evaluated at a total radiation dose of 40 Gy. The biopsy samples were examined with immunohistochemical staining for various factors and with an in situ nick end labeling method for apoptosis. The examined data were compared between the two groups. Results: The difference in the Ki-67, p53, Ku (p70/p80), DNA-PKcs labeling indexes and the apoptosis index in tumor cells between the chemoradiosensitive and chemoradioresistant groups was not statistically significant. The expression of c-erbB-2 oncoprotein was statistically significant in the chemoradioresistant group (p ⴝ 0.02), although it did not correlate with survival. Conclusion: c-erbB-2 immunostaining is useful for the prediction of chemoradioresistance in esophageal squamous cell carcinoma. © 2003 Elsevier Inc. c-erbB-2, Immunostaining, Chemoradiotherapy, Esophagus, Squamous cell carcinoma.
studies have been limited to p53 and Ki-67 examinations (5– 8). In the current study, to determine the predictive factors for the responsiveness to chemoradiotherapy in esophageal squamous cell carcinoma, we performed immunohistochemical examinations of p53, Ki-67, c-erbB-2, Ku (p70/ p80), and DNA-PKcs on tumor tissue biopsies before chemoradiotherapy. Apoptosis in the tumor tissues was also examined.
INTRODUCTION Esophageal carcinoma is a challenging target for radiation therapy (RT). Recently, some investigators reported good treatment results with RT concurrently combined with chemotherapy. However, even with the improvement of these treatment modalities, the survival rates of patients with the advanced disease are not satisfactory. To improve treatment efficacy, an investigation of a predictive factor is desirable. Recently, Oka and colleagues (1–3) and Niibe et al. (4) performed detailed analyses of the predictive factors for the responsiveness to RT in cervical squamous cell carcinoma, using immunohistochemical examinations of proliferative markers, tumor suppressor oncogenes, and oncoproteins. However, in esophageal squamous cell carcinoma, such
METHODS AND MATERIALS Patients and methods Between August 1998 and February 2001, 84 consecutive patients with locally advanced esophageal carcinoma received RT with or without concurrent chemotherapy at the Japan Society for Promotion of Science (Grant 14570878). Acknowledgments—The authors thank D. Mrozek (Medical English Service, Kyoto, Japan) for the English revision. Received Feb 19, 2003, and in revised form Jun 11, 2003. Accepted for publication Jun 16, 2003.
Reprint requests to: Toshiharu Matsumoto, M.D., First Department of Pathology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 Japan. Tel: ⫹81-35802-1037; Fax: ⫹81-3-3812-1056; E-mail: machian@med. juntendo.ac.jp Supported in part by Grant-in-Aid for Scientific Research (C), 1323
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Table 1. Summary of clinical data and histologic diagnosis in chemoradiosensitive and chemoradioresistant groups Characteristic Age (y) Mean Range Gender (n) Male Female Primary tumor* (n) T1 T3 T4 Tumor size (mm) Mean Range Chemoradiotherapy (n) Radiotherapy only (n) SCC differentiation (n) Well Moderate Poor Stage (n)† I II III IV Prognosis (n) Alive DOD
Chemoradiosensitive group (n ⫽ 13)
Chemoradioresistant group (n ⫽ 21)
61 43–80
65 49–76
11 2
18 3
2 5 6
0 10 11
67 20–125 12 1
84 35–180 19 2
3 8 2
5 9 7
1 2 9 1
0 1 15 5
3 10
1 20
Abbreviations: SCC ⫽ squamous cell carcinoma; DOD ⫽ dead of disease. * International Union Against Cancer TNM clinical classification (9). † Guidelines for clinical and pathologic studies on carcinoma of the esophagus by Japanese Society of Esophageal Diseases (10).
Department of Radiology, Juntendo University Hospital, Tokyo. The patients with suspected invasion to adjacent major unresectable organs, including the trachea, bronchus, and aorta, at the preoperative examination received chemoradiotherapy. All patients were treated with preoperative intent. However, when the evaluation of the response to treatment at a total dose of 40 Gy revealed that the tumor was still unresectable, the patients received additional chemoradiotherapy. The good responders underwent esophagectomy and lymphadenectomy. For this study, formalinfixed, paraffin-embedded tissue biopsies from esophageal lesion were available from 34 patients, including good and poor responses to chemoradiotherapy. All 34 patients presented with histologic evidence of esophageal squamous cell carcinoma. Complete follow-up was obtained in all patients. Table 1 summarizes the patient characteristics. Of the 34 patients, 29 were men and 5 were women. The median age was 63 years (range 43– 80). They were divided into chemoradiosensitive (n ⫽ 13) and chemoradioresistant (n ⫽ 21) groups according to the clinical findings, which were obtained by a barium study of the esophagus and an endoscopic examination performed before treatment. The chemoradiosensitive group was defined as those showing a bi-dimensional ⱖ50% decrease or disappearance of the
tumor mass after chemoradiotherapy. All others were included in the chemoradioresistant group. The clinical and histologic findings in each group are described in Table 1. The tumor stage was determined according to the International Union Against Cancer TNM clinical classification (4th edition) (9). Clinical evaluation of the primary tumor was performed using the guidelines for the clinical and pathologic studies on carcinoma of the esophagus by the Japanese Society of Esophageal Diseases (10). Chemoradiotherapy protocol Of the 34 patients, 11 received preoperative chemoradiotherapy, 20 definitive chemoradiotherapy, and 3 RT alone. RT was usually delivered to the esophagus, mediastinum, and supraclavicular regions with opposite AP and PA ports of 6- or 10-MV X-rays. The dose per fraction ranged from 1.8 to 2.0 Gy. The tumor doses ranged from 39.6 to 40 Gy for preoperative RT and from 60 to 70 Gy for definitive treatment. In the definitive RT with or without concurrent chemotherapy, the treatment fields were reduced to the primary tumor and enlarged lymph nodes after a total dose of 40 Gy. Twenty-six patients received combination chemotherapy of cisplatinum and 5-fluorouracil (5 d/wk), three received
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cisplatinum, 5-fluorouracil, and leucovorin (5 d/wk), and two received cisplatinum alone (5 d/wk). The daily doses of the agents were as follows: cisplatinum 5–10 mg/m2, 5-fluorouracil 250 –500 mg/m2, and leucovorin 10 –30 mg/m2, for a maximum of 4 weeks. Histopathologic study All patients underwent biopsy during the endoscopic examination of the esophageal tumor before the start of treatment. The specimens were fixed with 10% formaldehyde for up to 24 h and embedded in paraffin for hematoxylin and eosin staining, immunohistochemical staining, and the terminal deoxynucleotidyl transferase-mediated dUTPbiotin nick end labeling (TUNEL) method. Immunohistochemical studies of Ki-67 (MIB-1, Immunotech International, Marseilles, France; 1:400), p53 (DO-7, Dakopatts, Glostrup, Denmark; 1:2000), c-erbB-2 (HER-2/neu; Dako rabbit antihuman polyclonal antibody to c-erbB-2 oncoprotein, code number A0485, Dakopatts; 1:300), Ku (p70/p80; NeoMarkers, Fremont, CA; 1:100), and DNA-PKcs (NeoMarkers; 1:100) were performed using the avidin-biotinperoxidase complex technique. Microwave epitope retrieval (0.01 mol/L citrate buffer, pH 6.0) was performed before the immunostaining for Ki-67, p53, and c-erbB-2. Enzyme (proteinase-K, 10 min) antigen retrieval was used for Ku (p70/p80) and DNA-PKcs immunostaining. Apoptosis was determined by morphology and TUNEL, as described elsewhere (4). The Ki-67, p53, Ku (p70/p80), and DNA-PKcs labeling indexes were calculated as the percentage of positive cells in a minimum of 1000 carcinoma cells. The apoptotic cell index was calculated by determining the percentage of apoptotic cells in a minimum of 1000 carcinoma cells. The primary antibody for c-erbB-2 immunostaining is a concentrate of the same anti-HER-2/neu antibody provided in a prediluted form in the Hercept Test kit. Therefore, its immunostaining in the tumor cells was evaluated as 0, 1⫹, 2⫹, and 3⫹ according to the U.S. Food and Drug Administration–approved scoring guidelines for the Hercep Test (11). Scores of 0 or 1⫹ were considered negative for c-erbB-2 overexpression, 2⫹ was weakly positive, and 3⫹ was strongly positive (11). To qualify for 2⫹ and 3⫹ scoring (i.e., positive), complete membrane staining of ⬎10% of tumor cells had to be observed. Statistical analysis Actuarial overall survival rates were calculated using the Kaplan-Meier method. A comparison of the overall survival between the two groups was performed using a log–rank test. Mann-Whitney’s U test and Fisher’s exact probability test were also used to compare the distributions of patient characteristics between the two groups. Statistical significance was considered as p ⬍0.05. RESULTS No statistically significant difference was found between the chemoradiosensitive and chemoradioresistant groups in
Fig. 1. Survival curves of chemoradiosensitive and chemoradioresistant groups.
the distributions of age, gender, clinical findings of the primary tumor, tumor size, and tumor stage. The grade of differentiation of squamous cell carcinoma and the doses of chemoradiotherapy did not differ in both groups. The survival rate in the chemoradiosensitive group was significantly better than that in the chemoradioresistant group (p ⬍0.01; Fig. 1). The results of the immunohistochemical studies and apoptosis examinations in the tumor cells of the chemoradiosensitive and chemoradioresistant groups are presented in Tables 2 and 3. No statistically significant difference was found in the Ki-67, p53, Ku (p70/p80), and DNA-PKcs labeling indexes and apoptosis index in the tumor cells between these two groups (Table 2). c-erbB-2 oncoprotein expression (Fig. 2) was more frequent in the chemoradioresistant group than in the chemoradiosensitive group (p ⫽ 0.02; Table 3). However, no difference was noted in the survival rate according to the expression of c-erbB-2 oncoprotein (p ⫽ 0.14; Fig. 3). DISCUSSION Although numerous efforts have been made to improve the outcome of esophageal carcinoma, the prognosis of patients with advanced disease is still poor. Recent studies have demonstrated that platinum-containing aggressive chemoradiotherapy was an effective treatment modality for this disease (12, 13). However, the regimens also produced significant toxicity, and therefore, it is important to predict the effect of the treatment before the start of therapy. Of the several factors we tested, only the c-erbB-2 oncogen product was predictive of the tumor response. It is a 185-kD transmembrane protein and is a member of the epidermal growth factor receptor (EGFR) family. The EGFR family
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Table 2. Ki-67, p53, Ku (p70/p80), DNA-PKcs, and apoptotic labeling index in tumor cells of the chemoradiosensitive and chemoradioresistant groups Labeling index
Chemoradiosensitive group (%)
Chemoradioresistant group (%)
p
Ki-67 p53 Ku (p70/p80) DNA-PKcs Apoptosis
64.2 (23.9) 70.9 (35.4) 90.2 (16.3) 74.5 (27.9) 0.30 (0.12)
53.6 (22.3) 53.5 (36.4) 87.2 (17.6) 80.8 (19.9) 0.37 (0.14)
0.26 0.07 0.14 0.80 0.16
Data presented as the mean, with the SD in parentheses.
protein has tyrosine kinase activity. erbB-2 is a constitutively active receptor and is the preferred co-receptor for other members of this family, erbB-3 and erbB-4. The heterodimers between erbB-2 and the other erbB receptors have relatively high ligand affinity. erbB-2 is, therefore, a master coordinator of the signaling network that functions as a shared co-receptor for erbB ligands (14). A major signaling route of the EGFR family is a Ras-Raf-MAP-kinase pathway involved in cell survival. Another important pathway is the one constituted by PI3 kinase and the downstream protein kinase Akt. After its activation, Akt transduces signals that regulate multiple biologic processes, including apoptosis, gene expression, and cellular proliferation. Akt is likely to send survival (antiapoptotic) signals by phosphorylating multiple targets, including the BCL-2 family member BAD and the cell death pathway enzyme caspase-9. Akt also has a prominent role in the regulation of cell cycle progression (15). Therefore, the activation of the EGFR family by ligands, ionizing radiation, or overexpression is strongly related to the radioresistance of the cell. Ionizing radiation indiscriminately activates all erbB molecules expressed by a cell, and the radiation response may primarily be determined by cellular erbB expression profiles. c-erbB-2 overexpression has been associated with the pathogenesis of several human malignancies and with a poor prognosis in breast carcinoma. Overexpression of the protein is also associated with ipsilateral breast carcinoma recurrence after RT. The results of laboratory experiments have suggested that this overexpression of c-erbB-2 is itself sufficient to confer radioresistance (14). In the current study, c-erbB-2 expression was related to local resistance to chemoradiotherapy, but it was not related to the prognosis. However, in uterine cervical squamous cell carcinoma, Oka et al. (2) and Nishioka et al. (16) showed the good relationship between c-erbB-2 expres-
sion and poor prognosis, as well as local resistance to RT. The reason for the difference between their observation and ours in esophageal carcinoma is unclear. The small number of patients in this study is a possible reason. Additional confirmation of the findings is needed in a large group of patients. Another possible reason is the difference in the characteristics of these two tumors. Uterine cervical carcinoma is, generally, locally invasive. Therefore, local control may be the most important factor in the overall survival of patients with uterine cervical carcinoma. As our study showed, cells with overexpression of c-erbB-2 are radioresistant. Because esophageal carcinoma is not only locally invasive, but also distantly metastatic, local control may not predict the prognosis of the patients well. Recently, Uno et al. demonstrated that a combined treatment of recombinant humanized anti-erbB-2 monoclonal antibody (rhuMab HER-2) and irradiation showed an additive effect in head-and-neck squamous cell carcinoma cells, as well as in breast carcinoma cells (17). In the current study, tumors with c-erbB-2 expression were resistant to cisplatinum-based chemoradiotherapy. Therefore, it may be possible to overcome this resistance using rhuMab HER-2 combined with cisplatinum-based chemoradiotherapy. The other factors tested here were not related to the responsiveness of esophageal carcinoma. p53 and Ki-67
Table 3. c-erbB-2 oncoprotein expression in tumor cells of chemoradiosensitive and chemoradioresistant groups c-erbB-2 oncoprotein expression Group*
Negative
Positive
Chemoradiosensitive Chemoradioresistant
9 6
4 15
* p ⫽ 0.02.
Fig. 2. Well-differentiated squamous cell carcinoma of esophagus in patient from chemoradioresistant group. Many tumor cells strongly positive for c-erbB-2 oncoprotein and showing a surface membrane staining pattern (c-erbB-2 immunostaining, original magnification ⫻400).
Chemoradioresistance significance of c-erbB-2 expression
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ageal carcinoma was associated with a decreased responsiveness to treatment, others, including our group, did not observe such an association in patients with squamous cell carcinoma (5, 7, 8). Just as in our previous study (8), p53 expression was not related to the effect of chemoradiotherapy in the current study. Kitamura and colleagues (6) examined the relationship between the expression of Ki-67 in esophageal squamous cell carcinoma and prognosis after chemoradiotherapy. Although their study indicated that a high Ki-67 labeling index was related to the chemoradiosensitivity (6), we could not confirm this relationship. In other organs, different results for the relationship between Ki-67 and radiosensitivity of squamous cell carcinoma have been reported, including better prognosis with a high Ki-67 labeling index or no relationship (3, 18, 19).
Fig. 3. Survival curves of c-erbB-2–negative and c-erbB-2–positive groups.
immunostaining have been evaluated as predictive factors for the response of esophageal squamous cell carcinoma to chemoradiotherapy (5– 8). Although some investigators showed that the overexpression of the p53 protein in esoph-
CONCLUSION c-erbB-2 immunostaining is useful to predict the tumor response to RT concurrently combined with chemotherapy for esophageal squamous cell carcinoma. These results suggest that using rhuMab HER-2 combined with cisplatinumbased chemoradiotherapy may improve tumor response.
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