Japanese Journal of Clinical Oncology Advance Access originally published online on August 20, 2007
Japanese Journal of Clinical Oncology 2007 37(8):583-589; doi:10.1093/jjco/hym077
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© 2007 Foundation for Promotion of Cancer Research
Evaluation of Prognostic Factors of Esophageal Squamous Cell Carcinoma (Stage II–III) After Concurrent Chemoradiotherapy using Biopsy Specimens
Department of Internal Medicine II, Osaka Medical College, Takatsuki, Osaka, Japan
For reprints and all correspondence: Takayuki Kii, Department of Internal Medicine II, Osaka Medical College, 2-7 Daigakumachi, Takatsuki, Osaka 569-0801, Japan. E-mail: in2058{at}poh.osaka-med.ac.jp
Received October 25, 2006; accepted April 19, 2007
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Abstract |
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 TOP Abstract INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONConflict of interest statementReferences |
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Background: Recently, attention has been directed to concurrent chemoradiotherapy (CRT) for the treatment of squamous cell carcinoma of the esophagus with regard to efficacy, quality of life and functional preservation, and survival periods comparable to those after standard surgical therapy have been reported in responders to CRT. However, there are some non-responders to CRT, and the prediction of the outcome after CRT is an important subject for future studies. In this study, using biopsy specimens obtained before CRT, we evaluated the relationships between biological markers and the outcome after CRT in order to determine the prognostic factors of CRT.
Methods: The subjects were 51 patients (42 males and nine females: median age 68 years). who were histologically confirmed to have squamous cell carcinoma of the esophagus at stage II or III (UICC). Concurrent CRT consisting of chemotherapy using 5FU and CDDP and radiation therapy (60 Gy) was performed as the initial treatment, and the relationships of overexpression of EGFR, p53, VEGF, PCNA and CyclinD1 were examined immunohistochemically in biopsy specimens collected before treatment. Overall survival was estimated by multivariate analysis.
Results: The percentages of patients overexpressing p53, VEGF, PCNA, CyclinD1, and EGFR were 33, 31, 37, 31 and 29%, respectively. On multivariate analysis, T stage (P = 0.0393) and PCNA (P = 0.0302) were found to be significant prognostic factors.
Conclusions: PCNA overexpression appears to be a prognostic factor for squamous cell carcinoma of the esophagus after CRT.
Key Words: esophageal squamous cell carcinoma • chemoradiotherapy • PCNA • overall survival
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INTRODUCTION |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONConflict of interest statementReferences |
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The mortality rate due to esophageal cancer in Japan has remained unchanged in males and has gradually increased in females during the past 20 years, and deaths due to the disease account for 3.6% of all deaths from malignant neoplasms (1). In Japan, more than 90% of esophageal cancers are squamous cell carcinomas, which are relatively sensitive to chemotherapy and/or radiation therapy. However, esophageal cancer is likely to develop lymph node metastases and distant metastases at an early stage, and many patients are treated after the disease has advanced with a poor prognosis. Therefore, an improvement in the therapeutic results is a major clinical target. Presently, the standard treatment for esophageal cancer is surgical resection in Japan. Although the results of surgical treatments such as three-field lymphadenectomy are improving, the 5-year survival rate in all surgically treated patients between 1988 and 1997 (11 642 patients) was only 36.1%, and esophageal cancer remains a disease with poor prognosis (2–4). In the 1980s, chemoradiotherapy (CRT) was introduced for stage I–IV esophageal cancer primarily in Western countries (5), and a subsequent phase III trial (RTOG85-01 study) comparing the outcomes of T1-3/N0-1/M0 esophageal cancers between a chemoradiotherapy group and radiotherapy alone group concluded chemoradiotherapy to be the standard non-surgical treatment on the basis of a significantly longer survival period (6,7). Then, in the 1990s, the therapeutic results of CRT as a non-surgical treatment for esophageal cancer began to be reported from various institutions to attract attention. Ohtsu et al. (8) reported the clinical results in T4 and/or M1 lymph node locally advanced esophageal cancers (JCOG9516) as follows: complete response (CR) rate 15%, response rate 68.3%, median survival period 8.4 months and 2-year survival period 31.5%. These were comparable to those of conventional surgical treatments in similar cases (9–12). Hironaka et al. retrospectively compared the results of CRT and surgery alone in UICC-stage II or III (T4 excluded) patients and reported no difference in the 5-year survival rate between the two groups (46 versus 51%) (13). In the present decade, the results of comparative studies between definitive CRT and surgery have been reported from Europe. In France, a randomized phase III trial was performed by treating the responders with introductory CRT by continued definitive CRT or surgery. In Germany, a randomized comparative trial of introductory chemotherapy followed by CRT + surgery or definitive CRT was performed. In both trials, the survival rate did not differ significantly in the responders to the introductory chemotherapy or CRT regardless of whether they were subsequently treated by CRT or surgery (14,15).
Thus, as the reported results of CRT in patients with esophageal cancer were comparable to those of conventional surgical therapy, evaluation of prognostic factors in patients undergoing CRT as well as those undergoing surgery has become important in order to set out the therapeutic strategy. Various factors have been evaluated as possible prognostic factors of esophageal cancer, primarily in surgically treated patients: p53, bax and bcl-2 related to apoptosis, cyclinD1, P16, P21 and PCNA related to the cell cycle, and EGFR, TGF-
, HER-2neu and KI-67 related to growth regulation, VEGF related to angiogenesis, and ERCC1 related to DNA repair (16). Among them, the expressions of p53 (17), EGFR (18), PCNA (19,20) VEGF have been reported to be associated with poor outcomes after surgery alone (21) and the expression of CyclinD1 has been reported to be associated with poor outcomes after pre-operative CRT (22,23).
In this study, we immnohistochemically examined biopsy specimens of stage II or III squamous cell carcinomas of the esophagus obtained before definitive CRT in order to find out if any prognostic factors that have been evaluated in surgical cases are overexpressed. We also retrospectively evaluated the relationships of their overexpression with the total survival time.
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PATIENTS AND METHODS |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONConflict of interest statementReferences |
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Subjects
The subjects were 51 patients who underwent chemoradiotherapy at Osaka Medical College Hospital between July 1994 and July 2003 who fulfilled the following criteria: (1) those histologically confirmed to have squamous cell carcinoma of the esophagus; (2) those previously untreated for the disease; (3) those aged 80 years or less; (4) those in whom the disease was stage II or III according to the International Union against Cancer Tumor-node-metastasis (TNM) Classification, 6th edn, 2002; (5) those in a 0–2 Eastern Cooperative Oncology Group Performance status; (6) those who retained functions of major organs (bone marrow, heart, liver, and kidney); and (7) those who submitted informed consent.
Treatment Schedule
Chemotherapy consisted of the protracted infusion of 5-FU 400 mg/m2/day on days 1–5 and 8–12 combined with CDDP 40 mg/m2 with adequate hydration and antiemetic coverage on days 1 and 8. This schedule was repeated twice every 5 weeks. Radiation therapy using megavoltage X-rays was started on day 1 concomitantly with chemotherapy. The planned target volume for carcinoma of the upper or middle third esophagus included the primary tumor with a 3 cm margin craniocaudally, metastatic nodes with a 1–1.5 cm margin, supraclavicular fossa and mediastinum. For carcinoma of the lower third esophagus, the field was extended to include the perigastric nodes, and the supraclavicular fossa was excluded if the cervical nodes tested negative. When the planned volume included both the supraclavicular fossa and upper abdominal nodes, a daily dose of 2.0 Gy was allowed. A 2-week interval took place after a dose of 30 Gy. Radiation therapy was restarted on day 36 along with the same schedule of chemotherapy as before. The irradiation techniques used were anterior- and posterior-opposed equally weighted beams up to a dose of 40 Gy. Then, the radiation portals were changed to shield the spinal cord and to craniocaudally encompass the primary tumor with a 2–3 cm margin. Metastatic nodes were encompassed with a 1–1.5 cm margin. The radiation dose to the spinal cord was kept at a maximum of 50 Gy. The homogeneity of the dose within the planned target volume was within ±10% of the prescribed dose. For patients treated with prophylactic filgrastim, a daily dose of 75 µg/total body was administered subcutaneously during the period between days 18 and 31. The treatment was discontinued when disease progression, patient refusal or delay of recovery from the toxicity in excess of 6 weeks from the initiation of the treatment occurred (8).
Clinical Response
Clinical responses were assessed by endoscopy, barium esophagogram and CT in accordance with the response criteria given by the Japan Society of Clinical Oncology: complete response (CR), the complete disappearance of clinical evidence of existing lesions for over 4 weeks; partial response (PR), a >50% reduction in the sum of the products of two perpendicular measurements taken of all measurable lesions lasting for over 4 weeks; no change (NC), change in tumor <50% over 4 weeks; progressive disease (PD), a >25% increase in the sum of the products of two perpendicular measurements taken of an evaluable lesion or the appearance of new lesions (24).
Immunohistochemical Staining Methods
Pretreatment endoscopic biopsy specimens from 51 patients were assessed for p53, EGFR, cyclin D1, PCNA and VEGF expression. Immunohistochemical staining was carried out with the labeled streptavidin biotin (LSAB) method using a Dako LSAB kit (Dako, Carpinteria, CA, USA). Primary antibodies used for the immunohistochemical staining were as follows: anti-human p53 protein mouse monoclonal antibody (DO-7; DAKO, Glostrup, Denmark, dilution 1 : 50); anti-EGFR rabbit polyclonal antibody (1005; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, dilution 1 : 100), anti-cyclin D1 mouse monoclonal antibody (DOS-6; Novocasta, dilution 1 : 50), anti- PCNA mouse monoclonal antibody (PC-10; DAKO, Glostrup, Denmark, dilution 1 : 200) and anti-VEGF rabbit polyclonal antibody (A-20; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, dilution 1 : 100).
Formalin-fixed, paraffin-embedded biopsy materials were cut into 4 µm sections. After deparaffinization, the sections were incubated in a microwave oven for 10 min three times, and incubated on 0.3% H2O2. Then these sections were incubated with the primary antibodies. After six rinses in phosphate-buffered saline (PBS), sections were incubated with the secondary biotinylated anti-human p53 protein mouse monoclonal antibody, anti-mouse antibodies for cyclin D1 and PCNA, and anti-rabbit antibodies for EGFR and VEGF for 20 min at room temperature. The primary antibodies were localized by the sequential application of biotylinated anti-mouse–rabbit IgG gout immunoglobins and streptavidin–peroxide conjugate (Dako, Carpinteria, CA, USA). Immunostaining was visualized by developing the slides in diaminobenzidine (DAB) and counterstaining with Meyer-hematoxylin. Finally, the sections were subjected to alcohol and xylene baths, and then mounted for examination. For negative controls, the primary antibody solutions were replaced by the blocking buffer.
Method for evaluation of immunohistochemical results
The immunoreactivity of EGFR was graded into four groups according to the intensity of cell membrane EGFR staining in the whole tumor: high (markedly stronger staining than normal esophageal epithelium), medium (moderately stronger staining), low (the same staining level as normal epithelium) and negative (fainter staining). Strong and moderate staining groups were defined as positive for EGFR expression, in agreement with previous interpretations of EGFR in esophageal squamous cell carcinoma (18,25,26). VEGF staining was graded as follows: (a) +, staining intensity in cancer cells was stronger than that in stromal cells; (b) ±, staining intensity in cancer cells was equal to that in stromal cells; and (c) –, 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 (27). The percentages of 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). Cut-off values were determined by the following estimation: cyclin D1-positive judgment was a more than 30% labeling index (28). PCNA was calculated as the percentage of PCNA-positive cancer cells by counting more than 1000 cancer cells in more than three fields of a specimen with x400 magnification microscopy without knowing any clinical information. For the endoscopic biopsy specimens, PCNA were counted at the site of the maximum number of positive nuclei in the whole tumor. Only strong nuclear staining was regarded as positive, and weak nuclear or cytoplasmic staining was regarded as negative (19,20,29). The PCNA index was the percentage of nuclei staining positive (30). A PCNA score greater than 40 was taken as PCNA-positive. Also, tumors in which positive nuclei were observed in 20% or more cells were considered to be overexpressing p53. The results of immunohistochemical staining were evaluated by two pathologists without being informed of endoscopic findings.
Statistical Analysis
The survival time was calculated from the date of treatment initiation to that of death from any cause or to the last date of confirmation of survival. We estimated survival curves using the Kaplan–Meier method and compared them with the log–rank test. Relative risks and their 95% confidence intervals (CIs) of chemoradiotherapy were estimated using the univariate Cox regression model adjusting for gender, age, performance status, tumor location, T stage, N stage, p53, EGFR, cyclin D1, PCNA and VEGF, and the multivariate Cox regression model adjusting for T stage, PCNA and VEGF. Statistical analyses were performed using Stat View software 5.0.
Statistical analysis concerning risk factors was performed by Student's t-test and the 
2-test.
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RESULTS |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONConflict of interest statementReferences |
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Characteristics of Patients and Results of Immunohistochemical Staining
The median age of the patients, comprising 42 males (82%) and nine females (18%), was 68 years (range 43–80 years). The performance status (PS) was 0/1 in 44 patients (84%), and the general condition was good in many patients. The location was the middle in 26 (50%). The T stage was T3/T4 in 40 (78%) and T1/T2 in 11 (22%). Lymph node metastasis was detected by CT or EUS in 39 (76%). The UICC stage was II in 18 and III in 33. The percentages of patients overexpressing various biological markers were 37% (19/51) for PCNA, 33% (17/51) for p53, 31% (16/51) for cyclinD1, 29% (15/51) for EGFR and 31% (16/51) for VEGF. Clinical response was CR in 55% (28/51), PR in 31% (16/51), SD in 8% (4/51) and PD in 6% (3/51; Table 1). It was PR, SD or PD in 23 patients, of whom five underwent surgery, three gastrostomy, eight chemotherapy and seven best supportive care. Of these patients, PCNA was positive in 48% (11/23) and the T stage was T3/4 in 91% (21/23).
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Overall Survival
The median survival time (MST) in all 51 patients with clinical stage II or III squamous cell carcinoma of the esophagus who underwent CRT was 553 days. The MST in clinical stage II patients was 807 days, and that in clinical stage III patients was 495 days (P = 0.1313), with no significant difference, but it was ‘not reached’ in T1/T2 patients and 485 days (P = 0.0125) in T3/T4 patients, with a significant difference. Concerning biological markers, the MSTs of patients with low and high VEGF expression were 669 and 352 days (P = 0.0474), and those of patients with low and high PCNA expression were 766 and 491 days (P = 0.0045), respectively, with significant differences (Figs 1 and 2). The MSTs of patients with low and high EGFR expression were 776 and 553 days (P = 0.9326), those of patients with low and high cyclinD1 expression were 553 and 669 days (P = 0.7275), and those of patients with low and high p53 expression were 491 and 669 days (P = 0.9368), respectively; no significant difference was observed.
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Univariate Analysis: the Cox Proportional Hazards Model
On univariate analyses, no difference was observed in the outcome according to sex, PS, location, N stage or clinical stage, but, according to the T stage, the outcome was better in T1/T2 patients than in T3/T4 patients (P = 0.0190, relative risk = 0.286, 95% CI = 0.101–0.814; Table 2). Concerning the biological markers, the outcome was better in the low VEGF expression group than the high expression group (P = 0.0515) and in the low PCNA expression group than in the high expression group (P = 0.0060; Table 3).
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Multivariate Analysis: yhe Cox Proportional Hazards Model
Multivariate analysis was performed using the T stage and PCNA, which showed significant differences on univariate analysis, and VEGF, which showed a slight change on univariate analysis. Among the T stage, PCNA and VEGF, T stage and PCNA (P = 0.0302, relative risk = 0.438, 95% CI = 0.208–0.924) were independent prognostic factors (Table 4).
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Relationships of Biological Markers and Clinical Response with Various Factors
Concerning the relationships of PCNA with various clinical factors, eight patients (8/19, 42%) showed high PCNA expression and were positive for lymph node metastasis and 28 (28/32, 87%) showed low PCNA expression and were positive for lymph node metastasis; a significant correlation (P = 0.0160) was observed between PCNA and lymph node metastasis. Immunohistochemically, the expression of both PCNA and p53 was high in 10 (10/19, 53%) and low in 25 (25/32, 78%), indicating a correlation between PCNA and p53 (P = 0.0243). No correlation was noted between PCNA and the T stage. p53, EGFR, VEGF, PCNA or CyclineD1 showed no correlation with clinical response. Similarly, no correlation was noted between the T stage and clinical response.
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DISCUSSION |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONConflict of interest statementReferences |
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As therapeutic results similar to those by surgical treatment were reported to have been obtained by definitive CRT in esophageal cancer (8,13), it has become of importance to examine prognostic factors in patients undergoing CRT as well as those undergoing surgery to evaluate the therapeutic strategies against the disease. In this study, we evaluated the relationships between clinical and immunohistochemical biological markers and the outcome in patients with stage II or III squamous cell carcinoma of the esophagus who underwent definitive CRT alone as the initial treatment. The relationship between the results of surgery and biological markers have already been evaluated, and the outcome of surgery alone has been reported to be poor in those expressing p53 (17). EGFR (18) and VEGF (21). The outcome of preoperative CRT was reported to be poor in those expressing cyclinD1 (22,23). Concerning patients showing high PCNA expression, Kinugasa et al. (20) reported that the outcome after surgery alone was poor, and Yasunaga et al. (19) reported that the outcomes after surgery alone and preoperative chemotherapy + surgery were poor. Also, Okuno et al. (31) reported that the outcome after radiation therapy alone was poor, and Hickey et al. (29) reported that the outcome of preoperative CRT + surgery was poor, in patients showing high PCNA expression. However, there has not been a report on the relationship between PCNA and the prognosis in patients with squamous cell carcinoma of the esophagus who underwent definitive CRT alone. In this study the outcome after definitive CRT was favorable in patients showing low PCNA expression, indicating that the T stage and PCNA were independent prognostic factors.
In definitive CRT for advanced esophageal cancer, the DNA of cancer cells is considered to be damaged by radiation and chemotherapy (5-FU/CDDP), p53 to be expressed, apoptosis to be induced by p53, and p21, which binds to PCNA, to be induced to protract the G1 period. The tumor suppression gene p53 induces apoptosis and regulation of the cell cycle by positively or negatively adjusting the expression of many genes as a transcription factor and causing arrests in the cell cycle in response to DNA damage. Also, PCNA is involved in DNA repair and replication as well as exhibiting other gene control functions and acts as a binding mechanism of other proteins requiring interactions with DNA. PCNA and p53 are considered to be interrelated in the cell cycle and to be associated with each other in the proliferation of cancer cells. In this study, a correlation (P = 0.0243) was observed between the expression of PCNA and that of p53. It has been reported that squamous cell carcinomas positive for p53 often show high PCNA expression (32). Since the prognosis is poor in patients showing high PCNA expression, our results are considered to be biologically plausible. While the PCNA expression has been related to the outcome of squamous cell carcinoma of the esophagus, Kinugawa et al. (20), who studied the outcomes of patients after surgery alone, reported a correlation between the T stage and PCNA, but Okuno et al. (31), who performed radiotherapy alone, reported no correlation between PCNA and the T stage. In this study, PCNA was an independent prognostic factor after definitive CRT, but no correlation was observed between PCNA and the T stage. Also, an inverse correlation was observed between PCNA and lymph node metastasis. This result may be explained by the fact that the N stage was determined not pathologically but clinically. However, the number of samples analyzed was small in this study, and our results need to be confirmed by increasing the number of patients.
In conclusion, the outcome was better in patients with low PCNA expression than those with high PCNA expression, indicating that the expression of PCNA affects the total survival time. Also from previous reports, the outcome is considered to be favorable in low PCNA expression patients by either surgery or definitive CRT. In contrast, the outcome of high PCNA expression patients is presently poor by surgery alone, preoperative CRT + surgery, or CRT alone, so that the development of new therapies, particularly the advent of new agents, is awaited.
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Conflict of interest statement |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONConflict of interest statementReferences |
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None declared.
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References |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONConflict of interest statementReferences |
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