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10.1245/ASO.2003.10.014
Annals of Surgical Oncology 10:792-800 (2003)
© 2003 Society of Surgical Oncology
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ORIGINAL ARTICLES

Cell-Cycle Regulators and the Ki-67 Labeling Index Can Predict the Response to Chemoradiotherapy and the Survival of Patients With Locally Advanced Squamous Cell Carcinoma of the Esophagus

Hiroya Takeuchi, MD, Soji Ozawa, MD, Nobutoshi Ando, MD, Yuko Kitagawa, MD, Masakazu Ueda, MD and Masaki Kitajima, MD

From the Department of Surgery, School of Medicine, Keio University, Tokyo, Japan.

Correspondence: Address correspondence and reprint requests to: Soji Ozawa, MD, Department of Surgery, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan; Fax: 81-3-3355-4707; E-mail: ozawa{at}sc.itc.keio.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: We investigated whether aberrant p53 and p16 expression, the Ki-67 labeling index (LI), and int-2/cyclin D1 gene amplification predict the response to chemoradiotherapy (CRT) in patients with locally advanced esophageal squamous cell carcinoma (ESCC).

Methods: p53 and p16 expression status, the Ki-67 LI, and int-2/cyclin D1 amplification were assessed by immunohistochemical staining and slot blot analysis in pretreatment endoscopic biopsy specimens of 41 patients with T4 or M1 Lym (distant lymph node metastasis) ESCC. All patients received a course of chemotherapy (5-fluorouracil and cisplatin) with radiotherapy.

Results: The CRT therapeutic response rate was 71%, and resection after CRT was successful in 15 of the cases in which the CRT effect was significant. The cumulative survival rate after CRT in the p53-negative patients was significantly higher than in the p53-positive patients (P = .037). The mean Ki-67 LI in the CRT response cases was significantly higher than in the CRT no-response cases (P = .023). Multivariate regression analysis revealed high Ki-67 LI to be an independent variable linked to a pathologic complete response to CRT (P = .033). The cumulative survival rate after CRT in the group that was p53-negative and int-2/cyclin D1 amplification-positive was significantly higher than in the other groups (P = .008).

Conclusions: Evaluating predictive factors in pretreatment endoscopic biopsy specimens may allow selection of more suitable multimodal treatment for ESCC patients and improve their quality of life.

Key Words: Chemoradiotherapy • Esophageal cancer • p53 • p16 • Ki-67 • int-2/Cyclin D1 amplification


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Esophageal squamous cell carcinoma (ESCC) has one of the highest malignant potentials of any tumor. The 5-year survival rate of ESCC patients after surgery is low and ranges from 24% to 45%.1–4 The recent progress in long-term survival can be attributed to technical improvements in the diagnosis of T1 ESCC, extended lymph node dissection, and advances in perioperative management.4 Because curative resection is impossible, however, the prognosis of T4 (direct invasion to adjacent organs) and M1 Lym (distant lymph node metastasis) ESCC is still unfavorable, and multimodal treatment is required.2 We have been performing neoadjuvant chemoradiotherapy (CRT) for patients with T4 or M1 Lym ESCC since 1992.

Recent studies have demonstrated that gene abnormalities of cell-cycle regulators that function in the transition from the G1 to S phase are associated with clinicopathologic features of ESCC. Amplification of cyclin D1/PRAD-1 and cyclin D1 overexpression have been reported as prognostic markers associated with lymph node metastasis and distant organ metastasis of ESCC.5,6 The cyclin D1/PRAD-1 gene, which maps to the 11q13 chromosomal region and encodes the cyclin D1 protein, accelerates S phase entry from the G1 phase by complexing with cyclin-dependent kinase-4 (CDK4) and activating CDK4-mediated phosphorylation of the retinoblastoma (RB) protein.7,8 The int-2 gene, which belongs to the fibroblast growth factor family, also maps to 11q13, approximately 150 kilobases distant from the cyclin D1/PRAD-1 gene. We have reported amplification of the int-2 gene as another prognostic marker associated with distant organ metastasis in ESCC.9 The p16/CDKN2 gene, a tumor-suppressor gene, controls S phase entry by inhibiting CDK4-mediated phosphorylation of the RB protein, and we previously reported that loss of p16 expression is associated with the prognosis of ESCC.6

Abnormalities of the p53 gene are associated with oncogenesis and tumor progression in ESCC10,11; however, the association between p53 mutation (aberrant p53 expression) and survival remains unclear and is still being assessed.12–14 DNA-damaging therapy, such as irradiation or chemotherapy, induces wild-type p53 protein, which regulates the cell cycle via p21 expression; this arrests the cell cycle at the G1 phase. Furthermore, because wild-type p53 protein induces apoptosis in cells with gene abnormalities, some investigations have suggested that p53 gene status is a useful indicator for predicting the radiosensitivity or chemosensitivity of various tumors.15,16

The Ki-67 labeling index (LI) has been identified as a parameter of tumor proliferation. ESCC patients with a high Ki-67 LI have lower postoperative survival rates; thus, a high Ki-67 LI is one of the prognostic factors of ESCC.17,18 Ki-67 is a proliferation-associated nuclear antigen expressed in all cycling cells except resting cells in the G0 phase, and it reflects cells in the S/G2+M phase, in particular. The association between Ki-67 LI and response to CRT for ESCC, however, is still unclear.

A response to CRT (cisplatin, 5-fluorouracil, and radiotherapy) cannot be expected with certainty in all patients with T4 or M1 Lym ESCC; thus, CRT is frequently ineffective, valuable time is wasted, and patients experience severe toxicity as a result of CRT.2 Although no biological factors that predict a response to CRT in patients with locally advanced ESCC have ever been identified, recent investigations of the effects of cytotoxic agents and irradiation on the cell cycle suggest that the response of patients with advanced ESCC to CRT may be associated with dysfunction of cell-cycle regulators and Ki-67 LI.19 We hypothesized that alterations in genes encoding cell-cycle regulators or the Ki-67 LI in endoscopic biopsy specimens of primary tumors in untreated patients with advanced-stage ESCC predict sensitivity to neoadjuvant CRT and patient survival.

To test this hypothesis, in this study we investigated the status of p53 and p16 expression, int-2/cyclin D1 amplification, and the Ki-67 LI in endoscopic biopsy specimens of untreated ESCC and investigated whether these gene alterations and Ki-67 LI predicted the response to CRT and survival.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
The subjects of this study were 41 patients (mean age, 61years) with clinically diagnosed T4 and/or M1a (Lym) thoracic ESCC without distant organ metastasis. The patients were evaluated by esophagography, esophagoscopy, bronchoscopy, computed tomography (CT), and endoscopic ultrasonography, and staging was performed according to the tumor-node-metastasis classification proposed by the International Union Against Cancer. Patients with an esophagobronchial fistula were excluded from the study. The eligibility requirements were that the subjects be < 80 years old, have an Eastern Cooperative Oncology Group performance status of 0 to 2, and have a life expectancy of at least 8 weeks. Patients with serious complications or active carcinoma at another site were also excluded. After signing an informed human subject institutional review board consent form, all patients underwent CRT at Keio University Hospital (Tokyo, Japan) between 1993 and 1997. The clinicopathologic background factors and clinical stage estimates are listed in Table 1. Endoscopic biopsy specimens were obtained from all patients before the start of CRT and were used to detect aberrant p53 and p16 expression, to determine the Ki-67 LI, and to investigate int-2/cyclin D1 gene amplification. Surgical resection (transthoracic esophagectomy) was performed in the patients whose lesions became resectable as a result of a significant effect of CRT.


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  		TABLE 1. Patients and clinicopathologic background factors before chemoradiotherapy entry
 
Chemoradiotherapy
All patients received CRT consisting of continuous 5-fluorouracil administration at a dose of 800 mg/m2/day days 1 to 4 and 29 to 32, combined with (1) cisplatin administration at a dose of 70 mg/m2/day day 1 and 29 or 20 mg/m2/day days 1 to 4 and days 29 to 32 and (2) concomitant radiotherapy (daily dose of 2.0 Gy to a total dose of 50 Gy over 5 weeks).

The responses to CRT were evaluated by three clinicians on the basis of the results of esophagography, esophagoscopy, bronchoscopy, CT, and endoscopic ultrasonography. Effectiveness was classified into four categories according to the Guidelines for the Clinical and Pathologic Studies on Carcinoma of the Esophagus of the Japanese Society for Esophageal Diseases20: complete response (CR), partial response (PR), no change (NC), and progression of disease (PD). CR was defined as 100% regression of all tumors, PR as a >=50% reduction in the sum of the products of the longest diameter of measurable disease for 4 weeks, NC as <50% reduction of the tumor, and PD as a >=25% enlargement of the tumor or the appearance of a new tumor. The indications for surgical resection after CRT were judged by the three clinicians, and the only objective was potentially curative resection. If it was concluded that the stage might remain T4 or M1 Lym after CRT, surgery was considered not to be indicated, even if a PR had been achieved. Surgery was performed approximately 4 weeks after the final dose of radiotherapy, and on the basis of the pathologic findings, the resected specimens were classified into three categories by two pathologists according to the following guidelines20: grade 3, no viable cancer cells in the resected specimens; grade 2, viable cancer cells account for less than one third of the tumor tissue; grade 1, viable cancer cells account for more than one third of the tumor tissue; grade 0, no effect of CRT on the cancer cells and tissues. The patients were followed up in outpatient clinics, where diagnostic examinations consisting of chest x-ray, esophagography, endoscopy, CT, and ultrasonography were performed every 3 months to detect recurrence. The longest follow-up period was 57 months, and the median observation period was 28 months.

Immunohistochemistry
Expression of p53, p16, and Ki-67 was assessed by immunohistochemistry. Untreated endoscopic tumor biopsy specimens were fixed in 10% formalin and embedded in paraffin by conventional techniques. Freshly cut 4-µm sections were deparaffinized in xylene, and the slides were subjected to an antigen-retrieval step in Target Unmasking Fluid purchased from PharMingen (San Diego, CA) at 90°C for 10 minutes.21 The sections were reacted with the monoclonal mouse antihuman p53 antibody DO-7 (DAKO, Glostrup, Denmark) 5 µg/mL at 4°C overnight for p53 staining, with the monoclonal mouse antihuman p16 antibody PMG175-405 (PharMingen) 5 µg/mL at room temperature for 1 hour, and with monoclonal mouse antihuman MIB-1 (Immunotech, Marseille, France) .5 µg/mL at 4°C overnight for Ki-67 staining. Negative control slides were treated with nonspecific mouse immunoglobulin G1 under equivalent conditions. Secondary reagents were included in the DAKO labeled streptavidin-biotin kit. Slides were developed with diaminobenzaminidine and counterstained with hematoxylin.

We used the criteria for assessing the immunohistochemical results as previously described.6,22–24 p53 staining was considered positive if >10% of the tumor cells showed nuclear staining and negative if <10% of the tumor cells showed nuclear staining. p16 staining was considered positive if >80% of the tumor cells showed nuclear staining.6 The reliability of the anti-p16 antibody PMG175-405 was confirmed by Western blot analysis in studies on ESCC cell lines.6 Parabasal cells of the normal esophageal epithelium, inflammatory cells, and the ESCC cell line TE1 were used as a positive control for p16 staining. The Ki-67 LI was calculated as the percentage of Ki-67-positive cancer cells that showed nuclear staining among 1000 cancer cells counted in >3 fields of a specimen. The nuclei of the parabasal cells in normal epithelium were used as positive controls for Ki-67 staining.

Slot Blot Analysis
int-2/Cyclin D1 amplification was assessed by slot blot analysis as previously described.5,9,25 Untreated endoscopic biopsy specimens of the tumors and adjacent normal mucosa were frozen at -80°C, and the DNA was extracted by conventional techniques.5,9,25 DNA concentrations were estimated spectrophotometrically, and DNA (10 µg) was dissolved in .4 N NaOH. The samples were applied to a nylon membrane (Hybond-N; Amersham Life Science, Buckinghamshire, UK) and incubated for 30 minutes at room temperature. The filters were then rinsed in 5x sodium chloride-sodium phosphate-ethylene diamine tetraacetic acid (SSPE) and dried at room temperature. Probes were prepared by multiprime radioactive labeling with [32P]deoxycytidine triphosphate and an oligolabeling kit (Pharmacia, Uppsala, Sweden). Filters were prehybridized in 5x Denhardt’s solution, 50% formamide, 5x SSPE, .5% sodium dodecyl sulfate (SDS), and 100 µg of denatured salmon sperm DNA at 42°C for 2 hours and hybridized to the labeled probe at 42°C for 12 hours in the same solution. The probes used were SS6, a .9-kilobase pair SacI/SacI fragment of the int-2 gene provided by the Japanese Cancer Research Bank, and DRD2, a 1.6-kilobase pair BamHI fragment of the dopamine receptor D2 gene obtained from the American Type Culture Collection. The cyclin D1 probe was the 888-base pair polymerase chain reaction product containing the entire open reading frame region. The primers used for polymerase chain reaction were as follows: upper primer, 5'-ATG GAA CAC CAG CTC CTG TG-3'; and lower primer, 5'-TCA GAT GTC CAC GTC CCG CA-3'.5 The filters were washed twice at 65°C for 10 minutes in 2x SSPE and .5% SDS, once for 15 minutes in 1x SSPE and .5% SDS, and once for 10 minutes in .1x SSPE and .5% SDS. Autoradiograms were evaluated quantitatively with a bioimage analyzer (BAS 2000; Fujix, Tokyo, Japan). The number of gene copies was calculated as follows5: Go


(1)

Specimens in which more than 2.5-fold the gene copy number of int-2 or cyclin D1 was detected were judged to have undergone gene amplification.5,9,25

Statistical Analysis
Patient groups were compared by using the {chi}2 test, the Fisher exact probability test, and the Mann-Whitney U-test. The cumulative survival rates for patient groups were calculated by the Kaplan-Meier method and compared by using the Mantel-Cox test. A multiple regression model was used for multivariate analysis of variables predicting the CRT response. P values of <.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CRT Response Rate
Of the 41 cases that received CRT, 29 were CR or PR, and the therapeutic response rate (CR + PR) was 71% (CR, 0 cases; PR, 29 cases; NC, 8 cases; PD, 4 cases). Among the 29 cases in which the CRT effect was significant, complete surgical resection after CRT was successful in 15 cases (resectability rate, 37%). A pathologic CR (grade 3) both in the primary lesion and lymph nodes in the resected specimens was achieved in 4 cases. Three cases were grade 2, and eight cases were grade 1. In 6 of the 15 resected cases, including the 3 grade 3 cases, the patients were alive without recurrence of ESCC, with the longest survival being 57 months. One patient among the four with grade 3 resected specimens died of postoperative pyothorax. Postoperative respiratory complications occurred in 4 patients (27%), and the operative mortality rate was 7% (1 in 15). The patients with a CRT response (PR) had a significantly longer survival than the CRT no-response (NC + PD) patients (Fig. 1A). Moreover, the cumulative survival rate among the CRT response patients in whom surgery was subsequently performed was significantly higher than among the CRT response patients who did not undergo resection (Fig. 1A). The cumulative survival rate in the 7 patients with grade 3 or grade 2 resected specimens was significantly higher than that of the other 34 patients (P = .005; Fig. 1B).



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  		FIG. 1. Cumulative survival curves after chemoradiotherapy (CRT). (A) CRT response (partial response; PR) cases with or without surgical resection and no response (no change [NC] + progressive disease [PD]). PR with resection versus PR without resection, P = .001; PR without resection versus NC + PD, P = .008; PR with resection versus NC + PD, P < .001. (B) Grade 3 or grade 2 cases versus other cases.

 
p53 and p16 Expression
There were no significant differences in the clinicopathologic background factors between the p53-positive and p53-negative (normal expression) groups according to the {chi}2 test (Table 1). There were 5 (33%) p53-positive (aberrant expression) cases among the 15 CRT response (PR) cases in which surgery was successfully performed, 10 patients (71%) among the 14 CRT response cases without resection, and 8 (67%) among the 12 CRT no-response cases (NC + PD; P = .081; Table 2). Being p53-positive tended to be associated with poorer CRT response, but the association did not reach statistical significance. The cumulative survival rate after CRT in the p53-negative cases was significantly higher than among the p53-positive cases (P = .037; Fig. 2A). p16 expression was not correlated with any of the clinicopathologic background factors, the CRT response, or survival (Table 2). However, 8 (67%) of 12 no-response cases were p53 positive and p16 negative (loss of p16 expression), as opposed to only 9 (31%) of the 29 CRT response cases (P = .096; Table 2).


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  		TABLE 2. Association between cell-cycle regulators and CRT response
 


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  		FIG. 2. Cumulative survival curves after chemoradiotherapy (CRT). (A) Cases with and without p53 expression. (B) Cases with and without int-2/cyclin D1 amplification. (C) Cases with p53(-) and int-2/cyclin D1 amplification (+) versus cases in the other groups.

 
Ki-67 LI
The Ki-67 LIs of the 41 patients ranged from 2% to 65% (mean, 29%), and they were not correlated with any of the clinicopathologic background factors assessed. The mean Ki-67 LI in the seven cases in which grade 3 or grade 2 was achieved in the resected specimens was significantly higher than in the CRT no-response (NC + PD) cases (P = .023; Table 3). The Ki-67 LI in the grade 3 cases was particularly high (mean, 46.5% ± 10.2%). The 3-year survival rate after CRT of the 5 patients with a Ki-67 LI >50% was significantly higher than among the 15 patients with a Ki-67 LI <20% (3-year survival, 60% vs. 7%; P = .032).


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  		TABLE 3. Ki-67 labeling index and CRT response
 
Multivariate regression analysis revealed high Ki-67 LI to be an independent variable linked to a pathologic CR (grade 3) to CRT (P = .033; Table 4).


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  		TABLE 4. Regression coefficients of variables associated with pathologic complete response (grade 3) on multivariate regression analysis
 
int-2/Cyclin D1 Amplification
int-2/Cyclin D1 amplification was assessed by slot blot analysis in 32 of the 41 cases, after 9 cases were excluded because the amount of DNA in the endoscopic biopsy specimens was insufficient. Amplification of the int-2/cyclin D1 gene was detected in 5 (21%) of the 24 CRT response (PR) cases and in 3 (38%) of the 8 CRT no-response (NC + PD) cases. There were no significant differences in clinicopathologic background factors or CRT response between the group with int-2/cyclin D1 gene amplification and the group without int-2/cyclin D1 gene amplification. The cumulative survival rate after CRT in the patients with int-2/cyclin D1 amplification tended to be higher than in the patients without int-2/cyclin D1 amplification (P = .062; Fig. 2B).

Eleven (34%) of the 32 cases were p53-negative and int-2/cyclin D1 amplification-negative, 5 (16%) were p53-negative and int-2/cyclin D1 amplification-positive, 13 (41%) were p53-positive and int-2/cyclin D1 amplification-negative, and 3 (9%) were p53-positive and int-2/cyclin D1 amplification-positive. The cumulative survival rate after CRT in the group that was p53-negative and int-2/cyclin D1 amplification-positive was significantly higher than in the other groups (P = .008; Fig. 2C).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we investigated aberrant p53 and p16 expression, the Ki-67 LI, and int-2/cyclin D1 amplification by immunohistochemistry and slot blot analysis in endoscopic biopsy specimens of the 41 patients with untreated T4 and M1 Lym ESCC. The cumulative survival rate after CRT in the p53-negative patients was significantly higher than in the p53-positive patients. The mean Ki-67 LI in the CRT response cases (grade 2 and grade 3) was significantly higher than in the CRT no-response cases, and the cumulative survival rate after CRT in the p53-negative and int-2/cyclin D1 amplification-positive group was significantly higher than in the other groups.

In a preliminary study, we optimized the immunohistochemistry and slot blot assays to detect each molecular marker accurately in the pretreatment endoscopic biopsy specimens, because we needed to determine whether the results for these molecular markers in small biopsy specimens reflected the characteristics of the whole tumor. Because intratumor heterogeneity and technical issues regarding the assays may prevent the results in the biopsy specimens from reflecting those of the whole tumors, we obtained at least five biopsy specimens for immunohistochemistry and another two biopsy specimens for slot blot analysis with a large forceps to obtain an adequate sample of endoscopic biopsy specimens from various sites of the tumor. For the slot blot assay, we verified the absence of necrotic tissues and normal epithelium in the specimens on the basis of endoscopists’ observations and hematoxylin and eosin staining. We also compared the molecular markers in endoscopic biopsy specimens with those in primary ESCC tumors resected without neoadjuvant CRT in a preliminary study. The results of p53 staining of the in-biopsy specimens coincided with the results of staining in the resected tumors in 11 (92%) of the 12 cases, and the results of p16 staining of the biopsy specimens coincided with those in the resected tumors in 9 (75%) of the 12 cases. The Ki-67 LIs in the biopsy specimens showed a trend similar to that in the resected tumors (Spearman correlation coefficient, .705; P = .02; data not shown). The int-2/cyclin D1 amplification in the biopsy specimens coincided with that in the tumor resected without CRT in 42 (88%) of 48 cases. We still need to bear in mind the presence of intratumor heterogeneity; however, these preliminary investigations verified the efficacy of the assays and usefulness of pretreatment endoscopic biopsy specimens as a reflection of the tumor as a whole when used in the study.

p53-positive staining tended to correlate with a poorer response to CRT, and the cumulative survival rate after CRT in the p53-negative patients was significantly higher than in the p53-positive patients. Wild-type p53 protein is rapidly degraded and not detected by immunohistochemical staining; however, mutation of the p53 gene results in accumulation of aberrant p53 proteins in ESCC that is identified immunohistochemically as p53-positive staining.26–28 Sarbia et al.13 found no correlation between p53 status and survival among patients with surgically treated T1 to T3 ESCC,14 and our findings suggest that the wild p53 gene (p53-negative expression) may induce cell-cycle arrest and apoptosis in response to CRT in ESCC. CRT in patients with the wild p53 gene allows tumor downstaging and curative resection, which significantly improves survival. In particular, demonstration of a correlation between the p53 status and prognosis after CRT in this study suggests that the wild p53 gene may enhance sensitivity to chemotherapy, which suppresses micrometastasis to distant organs, in addition to enhancing radiosensitivity, which is associated with greater probability of local control. Other investigations have reported that p53 gene insertion enhances chemosensitivity or radiosensitivity in various cell lines, and the wild p53 gene has been found to be associated with a favorable response to CRT in breast cancer.15,16,29–31 Our results are consistent with these reports.

We immunohistochemically evaluated the other cell-cycle regulator that has been studied as one of the prognostic factors of ESCC: p16.6,14,32 Loss of p16 expression is unlikely to serve as a clinically useful predictor of CRT response and survival, but the combination of p53 and p16 expression was related to the CRT response. These results suggest that the combination of these two cell-cycle regulators may enable prediction of the response to CRT.

The Ki-67 LI was significantly correlated with the local response to CRT and the survival of patients with T4/M1 Lym ESCC in this study. The CRT response cases had a high Ki-67 LI, which means a higher population of cycling cells than cases with a low Ki-67 LI. Several studies have demonstrated that cycling cells are more radiosensitive than quiescent cells.33–35 Okuno et al.36 have reported results showing a correlation between high Ki-67 LI and radiosensitivity to ESCC similar to our own. Our results indicated that CRT can increase the probability of local control and result in longer survival of patients with a high Ki-67 LI in pretreatment biopsy specimens.

We previously reported that the cumulative survival rate of T1 to T3 ESCC patients with int-2/cyclin D1 amplification was significantly lower than that of patients without int-2/cyclin D1 amplification.5,6,9 In this study, however, after CRT, the survival of the T4/M1 Lym patients with int-2/cyclin D1 amplification was better than that of the patients without int-2/cyclin D1 amplification, and the p53-negative and int-2/cyclin D1 amplification-positive group, in particular, had a significantly longer survival. These results may indicate that the survival of the T4/M1 Lym patients with int-2/cyclin D1 amplification was improved by the CRT. The mechanism linking the int-2/cyclin D1 gene abnormality and chemo- or radiosensitivity has not yet been defined. Some investigations have shown that cyclin D1 overexpression is related to resistance to cisplatin or methotrexate in vitro.37,38 Martin et al.,39 however, found that cyclin D1 overexpression enhanced radiation-induced apoptosis and radiosensitivity in a breast cancer cell line. In their study and others, radiation treatment resulted in a rapid increase in p53 and p21 and induced apoptosis in the cyclin D1-overexpression cells,39–41 and those findings are consistent with our results showing better survival in the p53-negative (wild-type p53) and int-2/cyclin D1 amplification-positive group. Cyclin D1 gene amplification and overexpression is thought to accelerate the cell cycle, and this is correlated with an increased cell proliferative index in ESCC.42,43 Chemotherapy and radiotherapy are generally effective against tumor cells that have a high proliferative index (including Ki-67 LI).33–36,39,44 Recent reports have demonstrated a correlation between cyclin D1 overexpression and both increased tumor chemo-/radiosensitivity and better survival in patients with squamous cell carcinoma of the head and neck, similar to our own results.45–48

To verify our results, we should observe changes in response to CRT by the insertion of missing molecular markers or the antisense oligonucleotide into ESCC in vivo, and a randomized study based on information about molecular markers is needed to verify the clinical significance of molecular markers obtained from pretreatment biopsy specimens in ESCC patients treated with neoadjuvant CRT. Although no randomized studies have confirmed the clinical benefits of neoadjuvant CRT for patients with ESCC,49–51 the subjects of our neoadjuvant CRT were limited to T4 and/or M1 Lym ESCC patients, and its purpose was downstaging and improving the resectability of the whole tumor. The patients in this study whose tumors became surgically resectable after CRT had a significantly better survival than the patients with inoperable (unresectable) tumors after CRT. Long-term survival was achieved after surgery, particularly in the pathologic CR (grade 3) cases.51–55 The pathologic response to CRT has been determined only on the basis of the microscopic findings in the resected specimens. However, grade 3 patients may not require surgery, because esophagectomy after CRT often results in failure, along with various postoperative complications and mortality.51,56,57 However, the patients in whom CRT was ineffective also experienced severe toxicity because of the CRT, such as nausea, esophagitis, and leukopenia,51 and these issues make it very important to be able to predict the response of ESCC patients to CRT.

This study showed a significant correlation between high Ki-67 LI and local response to CRT, and wild p53 (p53-negative) expression predicted a good prognosis in patients treated with CRT. CRT may be useful for ESCC with int-2/cyclin D1 amplification, and the p53-negative and int-2/cyclin D1 amplification-positive group, in particular, showed a significantly longer survival. Larger multicentric randomized studies are needed to confirm the predictive values of these factors; however, evaluating these predictive factors in endoscopic biopsy specimens may allow the selection of more suitable multimodal therapy for ESCC patients and improve their quality of life.


   ACKNOWLEDGMENTS
 
The acknowledgments are available online at www.annalssurgicaloncology.org.

The authors thank Dr. M. Mukai (Department of Pathology, School of Medicine, Keio University, Tokyo) for technical assistance.


   FOOTNOTES
 
We investigated p53 and p16 expression, the Ki-67 labeling index, and int-2/cyclin D1 amplification in pretreatment biopsy specimens. p53 status predicted the survival of T4/M1 Lym (distant lymph node metastasis) esophageal squamous cell carcinoma patients treated with chemoradiotherapy, and the Ki-67 labeling index was significantly correlated with the local response.

Received for publication October 11, 2002. Accepted for publication April 23, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Iizuka T, Isono K, Kakegawa T, Watanabe H. Parameter linked to ten-year survival in Japan of resected esophageal carcinoma. Chest 1989; 96: 1005–11.[Abstract/Free Full Text]
  2. Ohtsu A, Boku N, Muro K, et al. Definitive chemoradiotherapy for T4 and/or M1 lymph node squamous cell carcinoma of the esophagus. J Clin Oncol 1999; 17: 2915–21.[Abstract/Free Full Text]
  3. Ide H, Nakamura T, Hayashi K, et al. Esophageal squamous cell carcinoma: pathology and prognosis. World J Surg 1994; 18: 321–30.[CrossRef][Medline]
  4. Ando N, Ozawa S, Kitagawa Y, Shinozawa Y, Kitajima M. Improvement in the results of surgical treatment of advanced squamous esophageal carcinoma during 15 consecutive years. Ann Surg 2000; 232: 225–32.[CrossRef][Medline]
  5. Shinozaki H, Ozawa S, Ando N, et al. Cyclin D1 amplification as a new predictive classification for squamous cell carcinoma of the esophagus, adding gene information. Clin Cancer Res 1996; 2: 1155–61.[Abstract]
  6. Takeuchi H, Ozawa S, Ando N, et al. Altered p16/MTS1/CDKN2 and cyclin D1/PRAD-1 gene expression is associated with the prognosis of squamous cell carcinoma of the esophagus. Clin Cancer Res 1997; 3: 2229–36.[Abstract/Free Full Text]
  7. Matsushime H, Roussel MF, Ashumun RA, Sherr CJ. Colony-stimulating factor 1 regulates novel cyclins during the G1 phase of the cell cycle. Cell 1991; 65: 701–13.[CrossRef][Medline]
  8. Motokura T, Bloom T, Kim HG, et al. A novel cyclin encoded by a bcl1-linked candidate oncogene. Nature (Lond) 1991; 350: 512–5.[CrossRef][Medline]
  9. Kitagawa Y, Ueda M, Ando N, Shinozawa Y, Shimizu N, Abe O. Significance of int-2/hst-1 coamplification as a prognostic factors in patients with esophageal squamous cell carcinoma. Cancer Res 1991; 51: 1504–8.[Abstract/Free Full Text]
  10. Wagata T, Shibagaki I, Imamura M, et al. Loss of 17p, mutation of the p53 gene, and overexpression of p53 protein in esophageal squamous cell carcinomas. Cancer Res 1993; 53: 846–50.[Abstract/Free Full Text]
  11. Gao H, Wang LD, Zhou Q, Hong JY, Huang TY, Yang CS. p53 tumor suppressor gene mutation in early esophageal precancerous lesions and carcinoma among high-risk populations in Henan, China. Cancer Res 1994; 54: 4342–6.[Abstract/Free Full Text]
  12. Shimaya K, Shiozaki H, Inoue M, et al. Significance of p53 expression as a prognostic factor in esophageal squamous cell carcinoma. Virchows Arch A Pathol Anat Histopathol 1993; 422: 271–6.[CrossRef][Medline]
  13. Sarbia M, Porschen R, Borchard F, Horstmann O, Willers R, Gabbert HE. p53 protein expression and prognosis in squamous cell carcinoma of the esophagus. Cancer 1994; 74: 2218–23.[CrossRef][Medline]
  14. Sturm I, Petrowsky H, Volz R, et al. Analysis of p53/BAX/p16ink4a/CDKN2 in esophageal squamous cell carcinoma: high BAX and p16ink4a/CDKN2 identifies patients with good prognosis. J Clin Oncol 2001; 19: 2272–81.[Abstract/Free Full Text]
  15. Spitz FR, Giacco GG, Hess K, et al. p53 immunohistochemical staining predicts residual disease after chemoradiotherapy in patients with high-risk rectal cancer. Clin Cancer Res 1997; 3: 1685–90.[Abstract]
  16. Bergh J, Norberg T, Sjogren S, Lindgren A, Holmberg L. Complete sequencing of the p53 gene provides prognostic information in breast-cancer patients, particularly in relation to adjuvant systemic therapy and radiotherapy. Nat Med 1995; 1: 1029–34.[CrossRef][Medline]
  17. Youssef EM, Matsuda T, Takada N, et al. Prognostic significance of the MIB-1 proliferative index for patients with squamous cell carcinoma of the esophagus. Cancer 1995; 76: 358–66.[CrossRef][Medline]
  18. Lam KY, Law SYK, So MKP, Fok M, Ma LT, Wong J. Prognostic implication of proliferative markers MIB-1 and PC10 in esophageal squamous cell carcinoma. Cancer 1996; 77: 7–13.[CrossRef][Medline]
  19. Deisseroth AB, DeVita VT Jr. The cell cycle. Probing new molecular determinants of resistance and sensitivity to cytotoxic agents. Cancer J 1995; 1: 15–21.
  20. Japanese Society for Esophageal Diseases. Guidelines for the Clinical and Pathologic Studies on Carcinoma of the Esophagus. 9th ed. Tokyo: Kanehara Public Co, 1999.
  21. van den Berg FM, Baas IO, Polak MM, Offerhaus GJA. Detection of p53 overexpression in routinely paraffin-embedded tissue of human carcinomas using a novel target unmasking fluid. Am J Pathol 1993; 142: 381–5.[Abstract]
  22. Casey G, Lopez ME, Ramos JC, et al. DNA sequence analysis of exon 2 through 11 and immunohistochemical staining are required to detect all known p53 alternations in human malignancies. Oncogene 1996; 13: 1971–81.[Medline]
  23. Sgambato A, Migaldi M, Leocata P, et al. Loss of p27kip1 expression is a strong independent prognostic factor of reduced survival in N0 gastric carcinomas. Cancer 2000; 89: 2247–57.[CrossRef][Medline]
  24. Takeuchi H, Ozawa S, Ando N, et al. Further evidence that altered p16/CDKN2 gene expression is associated with lymph node metastasis in squamous cell carcinoma of the esophagus. Oncol Rep 2001; 8: 627–32.[Medline]
  25. Ikeda Y, Ozawa S, Ando N, Kitagawa Y, Ueda M, Kitajima M. Meanings of c-erb B and int-2 amplification in superficial esophageal squamous cell carcinomas. Ann Thorac Surg 1996; 62: 835–8.[Abstract/Free Full Text]
  26. Levine AJ, Momand J, Finlay CA. The p53 tumor suppressor gene. Nature 1991; 351: 453–6.[CrossRef][Medline]
  27. Finlay CA, Hinds PW, Tan TH, Eliyahu D, Oren M, Levine AJ. Activating mutations for transformation by p53 produce a gene product that forms an hsc70-p53 complex with an altered half life. Mol Cell Biol 1988; 8: 531–9.[Abstract/Free Full Text]
  28. Scott N, Sagar P, Stewart J, Blair GE, Dixon MF, Quirke P. p53 in colorectal cancer: clinicopathological correlation and prognostic significance. Br J Cancer 1991; 63: 317–9.[Medline]
  29. Lowe SW, Ruley HE, Jacks T, Housman DE. p53-dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell 1993; 74: 957–67.[CrossRef][Medline]
  30. McIlwrath AJ, Vasey PA, Ross GM, Brown R. Cell cycle arrests and radiosensitivity of human tumor cell lines: dependent on wild-type p53 for radiosensitivity. Cancer Res 1994; 54: 3718–72.[Abstract/Free Full Text]
  31. Fujiwara T, Grimm EA, Mukhopadhyay T, Zhang WW, Owen-Schaub LB, Roth JA. Induction of chemosensitivity in human lung cancer cells in vivo by adenovirus-mediated transfer of the wild-type p53 gene. Cancer Res 1994; 54: 2287–91.[Abstract/Free Full Text]
  32. Maesawa C, Tamura G, Nishizuka S, et al. Inactivation of the CDKN2 gene by homozygous deletion and de novo methylation is associated with advanced stage esophageal squamous cell carcinoma. Cancer Res 1996; 56: 3875–8.[Abstract/Free Full Text]
  33. Mendonca MS, Rodriguez A, Alpen EL. Quiescence in pL cells and correlation with radiosensitivity and PLD repair. Radiat Res 1989; 117: 433–47.[Medline]
  34. Masunaga S, Ono K, Abe M. A method for the selective measurement of the radiosensitivity of quiescent cells in solid tumors—combination of immunofluorescence staining to BrdU and micronucleus assay. Radiat Res 1991; 125: 243–7.[Medline]
  35. Nakano T, Oka K. Differentiation values of Ki-67 index and mitotic index of proliferating cell population, an assessment of cell cycle and prognosis in radiation therapy for cervical cancer. Cancer 1993; 72: 2401–8.[CrossRef][Medline]
  36. Okuno Y, Nishimura Y, Kashu I, Ono K, Hiraoka M. Prognostic values of proliferating cell nuclear antigen (PCNA) and Ki-67 for radiotherapy of oesophageal squamous cell carcinomas. Br J Cancer 1999; 80: 387–95.[CrossRef][Medline]
  37. Warenius HM, Seabra LA, Maw P. Sensitivity to cis-diamminedichloroplatinum in human cancer cells is related to expression of cyclin D1 but not c-raf-1 protein. Int J Cancer 1996; 67: 224–31.[CrossRef][Medline]
  38. Hochhauser D, Schnieders B, Ercikan-Abali E, et al. Effect of cyclin D1 overexpression on drug sensitivity in a human fibrosarcoma cell line. J Natl Cancer Inst 1996; 88: 1269–75.[Abstract/Free Full Text]
  39. Martin JMC, Balkenende A, Verschoor T, Lallemand F, Michalides R. Cyclin D1 overexpression enhances radiation-induced apoptosis and radiosensitivity in a breast tumor cell line. Cancer Res 1999; 59: 1134–40.[Abstract/Free Full Text]
  40. Kranenburg O, van der Eb AJ, Zantema A. Cyclin D1 is an essential mediator of apoptotic neuronal cell death. EMBO J 1996; 15: 46–54.[Medline]
  41. Sofer-Levi Y, Resnitzky D. Apoptosis induced by ectopic expression of cyclin D1 but not cyclin E. Oncogene 1996; 13: 2431–7.[Medline]
  42. Shamma A, Doki Y, Shiozaki H, et al. Effect of cyclin D1 and associated proteins on proliferation of esophageal squamous cell carcinoma. Int J Oncol 1998; 13: 455–60.[Medline]
  43. Musgrove EA, Lee CS, Buckley MF, Sutherland RL. Cyclin D1 induction in breast cancer cell lines shortens G1 and is sufficient for cells arrested in G1 to complete the cell cycle. Proc Natl Acad Sci U S A 1994; 91: 8022–6.[Abstract/Free Full Text]
  44. Konishi T, Hiraishi M, Mafune K, et al. The therapeutic efficacy and toxicity of sequential methotrexate and 5-fluorouracil in gastric cancer. Anticancer Res 1994; 14: 1277–9.[Medline]
  45. Shintani S, Mihara M, Ueyama Y, Matsumura T, Wong DTW. Cyclin D1 overexpression associates with radiosensitivity in oral squamous cell carcinoma. Int J Cancer 2001; 96: 159–65.[CrossRef][Medline]
  46. Yoo SS, Carter D, Turner BC, et al. Prognostic significance of cyclin D1 protein levels in early-stage larynx cancer treated with primary radiation. Int J Cancer 2000; 90: 22–8.[CrossRef][Medline]
  47. Akervall J, Brun E, Dictor M, Wennerberg J. Cyclin D1 overexpression versus response to induction chemotherapy in squamous cell carcinoma of the head and neck—preliminary report. Acta Oncol 2001; 40: 505–11.[Medline]
  48. Hwang CF, Cho CL, Huang CC, et al. Loss of cyclin D1 and p16 expression correlates with local recurrence in nasopharyngeal carcinoma following radiotherapy. Ann Oncol 2002; 13: 1246–51.[Abstract/Free Full Text]
  49. Ajani JA. Preoperative therapy for patients with resectable squamous cell carcinoma of the esophagus: are we still confused? Ann Surg Oncol 2002; 9: 605–6.[Free Full Text]
  50. Chan ACW, Lee DWH, Griffith JF, et al. The clinical efficacy of neoadjuvant chemotherapy in squamous esophageal cancer: a prospective nonrandomized study of pulse and continuous-infusion regimens with cisplatin and 5-fluorouracil. Ann Surg Oncol 2002; 9: 617–24.[Abstract/Free Full Text]
  51. Geh JI, Crellin AM, Glynne-Jones R. Preoperative (neoadjuvant) chemoradiotherapy in oesophageal cancer. Br J Surg 2001; 88: 338–56.[CrossRef][Medline]
  52. Jones DR, Detterbeck FC, Egan TM, Parker LA Jr, Bernard SA, Tepper JE. Induction chemoradiotherapy followed by esophagectomy in patients with carcinoma of the esophagus. Ann Thorac Surg 1997; 64: 185–92.[Abstract/Free Full Text]
  53. Ancona E, Ruol A, Santi S, et al. Only pathologic complete response to neoadjuvant chemotherapy improves significantly the long term survival of patients with resectable esophageal squamous cell carcinoma: final report of a randomized, controlled trial of preoperative chemotherapy versus surgery alone. Cancer 2001; 91: 2165–74.[CrossRef][Medline]
  54. Lew JI, Gooding WE, Ribeiro U Jr, Safatle-Ribeiro AV, Posner MC. Long-term survival following induction chemoradiotherapy and esophagectomy for esophageal carcinoma. Arch Surg 2001; 136: 737–42.[Abstract/Free Full Text]
  55. Heath EI, Burtness BA, Heitmiller RF, et al. Phase II evaluation of preoperative chemoradiation and postoperative adjuvant chemotherapy for squamous cell and adenocarcinoma of the esophagus. J Clin Oncol 2000; 18: 868–76.[Abstract/Free Full Text]
  56. Slater MS, Holland J, Faigel DO, Sheppard BC, Deveney CW. Does neoadjuvant chemoradiation downstage esophageal carcinoma? Am J Surg 2001; 181: 440–4.[CrossRef][Medline]
  57. Ikeda K, Ishida K, Sato N, et al. Chemoradiotherapy followed by surgery for thoracic esophageal cancer potentially or actually involving adjacent organs. Dis Esophagus 2001; 14: 197–201.[CrossRef][Medline]



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