Murine Double Minute 2 and Its Association with Chemoradioresistance of Esophageal Squamous Cell Carcinoma

Patients and Methods

Patients. We performed 68 salvage esophagectomies following dCRT at the Tohoku University Hospital (Sendai, Japan) between September 2001 and November 2008. Two cases of distant metastasis, three cases of initial endoscopic treatment, and one case of previous CRT for head and neck cancer were excluded. Therefore, 62 patients with ESCC were included in the study, out of whom 38 had persistent disease and 24 had recurrent disease. The definitions of persistent and recurrent disease used in this study are described in the next section.

dCRT and salvage esophagectomy. The dCRT protocol in our study basically followed that of the Japan Clinical Oncology Group (JCOG) trial 9906 (2). In brief, the protocol comprised of two cycles of intravenous cisplatin (40 mg/m²) infusion on days 1 and 8 and continuous 5-fluorouracil (400 mg/m²) infusion over 24 h on days 1-5 and 8-12 every five weeks with concurrent radiotherapy (60 Gy administered in 30 fractions over a period of eight weeks, including a 2-week rest period after the administration of 30 Gy). The radiotherapy administered is three-dimensional. Gross tumour volume (GTV) included the primary tumour and metastatic lymph nodes evaluated by endoscopy, computed tomography (CT), and 2-[¹⁸F]fluoro-2-deoxy-D-glucose positron-emission tomography (FDG-PET), if necessary. The clinical target volume included the GTV and the supraclavicular, mediastinal, and celiac axis lymph node regions. When the tumours were located in the upper third of the esophagus, the celiac axis lymph node region was excluded. After 40 Gy, an extra boost of radiation was administered to GTV via an oblique approach (20 Gy administered in 10 fractions). Additional chemotherapy was administered until May 2004. This comprised of two cycles of 80 mg/m² of cisplatin on day 1 and a continuous infusion of 800 mg/m² of 5-fluorouracil on days 1-5, every four weeks. Clinical evaluation by endoscopy with biopsy, CT, and FDG-PET (if necessary) were performed one month after treatment completion. Patients who were evaluated as having an incomplete response at this time and who subsequently underwent salvage esophagectomy were defined as persistent cases. Patients who were evaluated as having a complete response (CR) at this time, but whose tumours subsequently recurred in the same location and who underwent salvage esophagectomy later were defined as recurrent cases. Recurrence was confirmed by biopsy. A total of 24 out of the 38 patients with persistent disease and 15 out of the 24 patients with recurrent disease underwent the JCOG 9906 protocol. The other patients underwent treatment according to different protocols followed by other hospitals or other Departments (Radiation Oncology, Clinical Oncology, or Otolaryngology) of the Tohoku University Hospital. The treatments that the patients received are summarized in Table I. Some patients required a dose decrease or a delay in chemotherapy because of adverse side-effects. All patients received the complete scheduled radiation dose.

Salvage esophagectomy was usually performed under thoracoscopic guidance. One- or two-field lymph node dissection was performed for tumours of the cervical esophagus or the middle or lower thirds of the esophagus, and 3-field lymph node dissection was performed for tumours of the upper third of the esophagus.

Immunohistochemical staining and pathological evaluation. Surgical specimens were fixed in 10% formalin and representative sections were embedded in paraffin wax. Excluding specimens in which no residual tumour cells were observed microscopically, the specimens of 33 patients with persistent disease and 22 patients with recurrent disease were evaluated immunohistochemically. Immunohistochemical staining was performed using the streptavidin–biotin complex method, as follows. Serial 4-μm-thick sections from the most representative area of each specimen were de-paraffinized in xylene, rehydrated in a graded ethanol series, and then immersed in 3.0% hydrogen peroxide in methanol for 10 min at room temperature (RT) to block endogenous peroxidase activity. For antigen retrieval, the slides for p53 were heated in a microwave at 95°C for 15 min in 0.01 M citrate buffer (pH 6.0). The slides for p16, p27, MDM2, cyclin D1, and Ki-67 were heated for 5 min in 0.01 M citrate buffer (pH 6.0) using an autoclave at 121°C. The slides for EGFR were incubated in 0.05% protease in Tris-HCL buffer (pH 7.6) at 37°C for 10 min. The slides were incubated in 1% normal rabbit (for mouse monoclonal antibody) or goat (for rabbit monoclonal antibody) serum for 30 min at RT to reduce non-specific antibody binding. Subsequently, the slides were incubated at 4°C overnight with mouse monoclonal antibody against p53 (DO-7, diluted 1/100; Nichirei Biosciences Inc., Tokyo, Japan), p16 (G175-1239, diluted 1/100; BD Biosciences, Franklin Lakes, NJ, USA), p27 (SX53G8, diluted 1/800; Dako, Glostrup, Denmark), MDM2 (SMP14 diluted 1/1000; Santa Cruz Biotechnology Inc., CA, USA), Ki-67 (MIB-1, diluted 1/300; Dako), EGFR (31G7, used as delivered, product code 413701; Nichirei Biosciences Inc.), and rabbit monoclonal antibody against cyclin D1 (SP4, used as delivered, product code 413521; Nichirei Biosciences Inc.). The next day, the sections were incubated with biotinylated anti-mouse or anti-rabbit immunoglobulin (Nichirei Biosciences Inc.) as secondary antibodies and incubated with peroxidase-labeled streptavidin (Nichirei Biosciences Inc.) for 30 min at RT. The antigen–antibody complexes were visualized with 3,3’-diaminobenzidine, and the slides were counterstained with Mayer's haematoxylin, dehydrated in a graded ethanol series, and cleared in xylene.

The staining and pathological findings were evaluated independently by two of the authors (HO and FF) who were blinded to the patients' clinical data. The histopathological findings were classified according to the seventh edition of the Union for International Cancer Control system (17). The percentage of p53-, p27-, MDM2-, Ki-67-, and cyclinD1-positive nuclei was determined for more than three regions of the deepest area of the tumour and 1000 viable tumour cells were evaluated at a magnification of ×400 by microscopy. For p16, the percentage of cells with positive nuclei and positive cytoplasm was determined, and for EGFR, the percentage of cells with positive membranes was determined. The cutoff values for abnormal expression were as follows: p53, ≥10% (13); p16, ≤5% (16); p27, ≥10% (15); MDM2, ≥20% (18); cyclin D1, ≥10% (14); Ki-67, ≥39% (12). Scoring for EGFR was performed using the immunoreactive score (IRS) obtained by multiplying the intensity score (0=no staining, 1=faint staining, 2=moderate staining, 3=strong staining) by the extent score (0=none, 1=<10%, 2=10%-50%, 3=>50%-80%, 4=>80%), and ranged from 1 to 12. It was decided that an IRS ≥6 was indicative of abnormal expression (10). Histopathological tumour regression was classified into five categories according to the Japanese Classification of Esophageal Cancer, tenth edition (19) as follows: grade 3, markedly effective (no viable residual tumour cells); grade 2, moderately effective (less than one-third residual tumour cells); grade 1, slightly effective (1b, one-third to two-thirds residual tumour cells; 1a, more than two-thirds residual tumour cells); grade 0, ineffective (no therapeutic effect observed).

Statistical analysis. Continuous data were analysed using the Student's t-test or the Mann–Whitney U-test. Categorical data were evaluated using Pearson's chi-square test, Fisher's exact test, or the Mann–Whitney U-test as appropriate. Normality was assessed using the Shapiro–Wilk test. Equality of variances was evaluated using the F test. Overall curves were determined by the Kaplan–Meier method, and a log-rank test was used to compare the survival curves. The patient survival time was determined from the date of salvage surgery until death or the last follow-up examination. All statistical analyses were performed using JMP Pro Version 9.0.2 (SAS Institute Inc., Cary, NC, USA). Two-tailed p-values <0.05 were considered statistically significant.

This study was approved by the Ethical Committee of Tohoku University Hospital (accession number 2011-596).