Prognostic Impact of Tumor IL-6 Expression after Preoperative Chemoradiotherapy in Patients with Advanced Esophageal Squamous Cell Carcinoma

Patients and Methods

Patients. Data regarding 41 patients with clinical T3-T4 esophageal squamous cell carcinoma, who underwent induction CRT followed by esophagectomy (CRT group) and 60 patients with clinical T1-T4 esophageal squamous cell carcinoma, who underwent esophagectomy without any preoperative therapy (Surgery group) at Kyoto Prefectural University of Medicine Hospital between 2001 and 2010 were analyzed in this retrospective study. An immunohistochemical study for tumor IL-6 expression was performed using resected specimens from consecutive patients between 2001 and 2009 in the CRT group, and those from consecutive patients between 2006 and 2010 in the Surgery group. Induction CRT was indicated for unresectable or marginally-resectable tumors, i.e. T4 or bulky T3 tumors that were considered difficult for complete resection if any induction therapy had not been performed. If a clinical response was observed, and complete resection was thus considered possible, the patient was scheduled for surgery. Clinical and pathological staging was performed according to the tumor-node-metastasis (TNM) classification of the International Union Against Cancer (UICC, sixth edition) (8). Esophagography, endoscopy, computed tomography, and/or bronchoscopy were routinely performed to determine pre-treatment clinical staging and treatment responses. Endoscopic ultrasonography was occasionally performed. From 2004 onward, positron emission tomography scans were performed before and after CRT. Written informed consent was obtained from all patients.

Induction CRT. The induction CRT regimen consisted of radiation and concurrent administration of 5-fluorouracil (5-FU) and cisplatin as described previously (7). Briefly, 5-FU was administered intravenously at 200-250 mg/m²/day on days 1-5, 8-12, 15-19, and 22-26, and cisplatin was administered at a dose of 5-7 mg/m²/day by drip infusion for 1 h on days 1-5, 8-12, 15-19, and 22-26. In total, 40 Gy of radiation was delivered for four weeks at 2 Gy daily (five days/week). Treatment responses were evaluated 2-3 weeks after the completion of CRT. Surgery was scheduled 4-6 weeks after the last day of CRT in patients for whom complete resection was considered possible.

Surgery. All patients underwent en bloc esophagectomy with regional lymphadenectomy through right thoracotomy and laparotomy with reconstruction using the stomach. Regional lymphadenectomy included two-field lymph node dissection (mediastinal and abdominal regions) for carcinomas of the lower third of the esophagus and three-field lymph node dissection (cervical, mediastinal, and abdominal regions) for carcinoma of the upper two-thirds of the esophagus.

Evaluation of the pathological response to CRT. The pathological response to CRT was evaluated by grading the response of the primary tumor. The grade of the response to CRT was as follows (9): Grade 3, complete disappearance of viable cancer cells in the tumor bed; grade 2, more than two-thirds disappearance of viable cancer cells; and grade 1, less than two-thirds disappearance of viable cancer cells. CRT was considered effective in patients with a histological response of grade 2 or 3. CRT was judged ineffective in patients with a histological response of grade 1.

Immunohistochemical staining for IL-6. Tumor specimens were fixed in 4% paraformaldehyde and embedded in paraffin wax. Five-micrometer-thick sections were cut, placed on glass slides, and sequentially deparaffinized and rehydrated through xylene and graded alcohol solutions. These sections were immunostained using a mouse monoclonal antibody against IL-6 (MAB2061; dilution 1:50; R&D Systems, Abingdon, UK). Antigen retrieval was carried out at 98°C in citric acid buffer (10 mmol/l, pH 6.0) for 40 min. Endogenous peroxidase was blocked by immersing the slides in methanol containing 3% hydrogen peroxide for 30 min. After washing, non-specific binding was blocked at room temperature for 60 min with 1% bovine serum albumin in phosphate-buffered saline. These sections were incubated overnight at 4°C with the antibody. After washing, the sections were treated with an EnVision kit (Dako, Glostrup, Denmark) according to the manufacturer's instructions. The immunoproducts were examined by treating slides with diaminobenzidine tetrahydrochloride followed by counterstaining with hematoxylin. Negative controls were performed in all cases by omitting the first antibody.

Evaluation of tumor IL-6 expression. The immunoreactivity of IL-6 was evaluated according to the extent and intensity of staining. The extent of staining was graded as follows: grade 0, no specific staining; grade 1, <25% of the tumor cells stained; grade 2, 25% to <50% of the tumor cells; grade 3, 50% to <75% of the tumor cells; and grade 4, ≥75% of the tumor cells. The staining intensity was graded as follows: 0, none (not stained); 1, mild (between 0 and 2); and 2, strong. An immunoreactive score was calculated by the addition of the percentage score of positively stained cells and the score of staining intensity (0-6). Tumors with an immunoreactive score of 0-1 were designated as ‘negative’, and tumors with an immunoreactive score of 2-6 were designated as ‘positive’.

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Figure 1.

Immunohistochemical detection of tumor interleukin-6 (IL-6) expression. Diffuse cytoplasmic staining of IL-6 protein was detected in tumor cells (magnification, ×100).

Statistical analysis. Comparisons between groups of patients were carried out using the χ² or Fisher's exact test, as appropriate. Cumulative survival was calculated by the Kaplan–Meier method and survival curves were tested by the log-rank method. Univariate survival analysis was used to estimate the dependence of survival on each variable. Multivariate survival analysis was calculated according to a Cox proportional hazard model in a forward stepwise manner. All statistical analyses were carried out using JMP software (version 8 for Macintosh; SAS Institute Inc., Cary, NC, USA). Significance was defined as a p-value less than 0.05.