Ferredoxin Reductase Is Useful for Predicting the Effect of Chemoradiation Therapy on Esophageal Squamous Cell Carcinoma

Patients and Methods

Study group. The present study involved 50 consecutive patients with advanced ESCC who underwent CRT at the Department of Surgical Oncology and Digestive Surgery of the Kagoshima University Hospital between 1997 and 2010. They underwent CRT followed by esophagectomy with lymph node dissection 4-6 weeks after completing CRT. After patients gave their informed consent, biopsy specimens of primary tumors were endoscopically collected. Classifications of the specimen were determined according to the International Union against Cancer tumor-node-metastasis (TNM) classification system (13). No patients received adjuvant therapy until they developed recurrent disease. Follow-up data after surgery were available for all patients with a median follow-up period of 34 months (range=3-136 months). The clinicopathological features of the study group are summarized in Table I. The cT4 tumors in this study were resectable tumors that invaded the lung, pleura, or the recurrent nerve. All M1 tumors were due to distant lymph node metastases. The study was approved by the Institutional Review Board of the Kagoshima University and performed according to the Helsinki Declaration.

Chemoradiation therapy. A total radiation dose of 40 Gy were applied; 2-Gy fractions were delivered 5 days per week, for 4 weeks, to the mediastinum, neck and/or upper abdomen including clinical metastatic lymph node areas. In the same period, patients received intravenous chemotherapy with cisplatin (7 mg/m² over 2 h) and 5-fluorouracil (350 mg/m² over 24 h). The clinical criteria for the response to CRT against the primary ESCC site were evaluated by endoscopic examination (14, 15). A complete response (CR) was defined as the disappearance of tumor lesions, disappearance of ulceration, and absence of cancer cells in biopsy specimens. Existence of erosion, a granular protruded lesion, ulcer scar, and unstained lesions by iodine did not prevent a CR evaluation. Progressive disease (PD) was defined as obvious enlargement of the tumor lesion or progression of esophageal stenosis by tumor enlargement. Incomplete response/stable disease (IR/SD) was defined as not satisfying CR criteria without obvious enlargement of the tumor lesion. The patients whose clinical effect was CR were considered susceptible to CRT, whereas the patients with IR/SD or PD were considered not susceptible.

The histological criteria for the response to CRT were as follows (14, 15): Grade 0, neither necrosis nor cellular or structural changes can be seen throughout the lesion; Grade 1, necrosis or disappearance of the tumor is present in no more than two-thirds of the whole lesion; Grade 2, necrosis or disappearance of the tumor is present in more than two-thirds of the whole lesion, but viable tumor cells are still remaining; and Grade 3, the whole lesion contains necrosis and/or is replaced by fibrosis, with or without granulomatous changes, and no viable tumor cells are observed. In patients whose histological response was Grade 2 or 3, the CRT was considered effective. On the other hand, in patients whose histological response was Grade 0 or 1, the CRT was considered ineffective.

Immunohistochemical staining and evaluation of Fdxr in ESCC. Tumor samples were fixed with 10% formaldehyde in phosphate-buffered saline (PBS), embedded in paraffin, and sectioned into 4-mm–thick slices. The sections were washed with PBS for 5 min three times and then blocked by PBS containing 3% skim milk for 10 min at room temperature. The blocked sections were incubated overnight at 4°C with primary antibody against Fdxr (sc-365949, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) diluted in PBS, followed by staining with a streptavidin-biotin-peroxidase kit (Nichirei, Tokyo, Japan). Sections were counter-stained with hematoxylin, and mounted. Fdxr expression was determined by counting the number of cancer cells in which the cytoplasm was stained with the anti-Fdxr antibody. The Fdxr-expressing tumor tissues that we previously reported were used as positive controls, and negative controls were prepared by replacing the primary antibody with PBS (12). Fdxr expression was assessed by determining the proportion of positive cells and intensity. For cytoplasmic expression of Fdxr, immunostaining was scored on a 3-tiered scale for both intensity (absent/weak, 1; moderate, 2; strong, 3) and percentage (<10%, 1; 10%-50%, 2; >50%, 3), and then the two scores were multiplied to give a combination score ranging from 1 to 9 for each section. Cytoplasmic protein expression was defined as positive when the combination scores were >6 and negative when the combination scores were <6. This evaluation method is an improved version of previous methods (12).

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Figure 1.

Expression of Fdxr in clinical samples by immunostaining (original magnification, ×400). (A) Positive expression of Fdxr in ESCC. (B) Negative expression of ESCC. Positive staining was detected in the cytoplasm.

Statistical analysis. Statistical analysis of group differences was performed using the χ² test or the Mann-Whitney U test. The Kaplan-Meier method was used for survival analysis, and differences in survival were estimated by the log-rank test. Prognostic factors were examined by univariate and multivariate analyses (Cox proportional hazards regression model). p-Values <0.05 were considered statistical significant. All statistical analyses were performed with StatView™ version 5.0 (Abacus Concepts, Berkeley, CA, USA).