Patients and tissue samples
A total of 86 consecutive ESCC patients, who underwent curative surgery without neoadjuvant treatment at Tohoku University Hospital (Sendai, Japan) between January 2000 and December 2005, were selected. All patients underwent thoracoscopic esophagectomy with two- or three-field node dissection, except for four patients who underwent pharyngo-laryngo-esophagectomy with one-field node dissection, six patients who underwent transhiatal esophagectomy with one-field node dissection, and eight patients who underwent esophagectomy by right thoracotomy.
The resected specimens and lymph nodes were fixed in 10% formalin, and representative sections were embedded in paraffin wax. The sections were histologically examined according to the Union for International Cancer Control (UICC) TNM (tumor, node, metastasis) classification (7th edition) system. Patient survival time was determined from the date of surgery until death, recurrence, or the last follow-up examination. This study was approved by the ethical committee of the Tohoku University Hospital (Accession number 2011–596).
Immunohistochemical staining and evaluation
Serial sections (4 μm thick), including the deepest area of the tumors, were deparaffinized in xylene, rehydrated in graded alcohol, and immersed in 3.0% hydrogen peroxide in methanol for 10 min at room temperature (RT) to inhibit endogenous peroxidase activity. For antigen retrieval, the slides for p53 were heated by microwave irradiation at 95°C for 15 min in 0.01 M citrate buffer (pH 6.0). The slides for p16, p27, MDM2, and Ki-67 were heated by autoclave at 121°C for 5 min in 0.01 M citrate buffer (pH 6.0). The slides for CD133 were autoclaved at 121°C for 5 min in Histofine antigen retrieval solution (pH 9.0, Nichirei Biosciences Inc., Tokyo, Japan). The slides for EGFR were incubated in 0.05% protease in Tris–HCl buffer (pH 7.6) at 37°C for 10 min. After washing three times for 5 min each in phosphate-buffered saline (PBS), the slides were incubated in 1% normal rabbit serum for 30 min at RT to reduce nonspecific antibody binding and were subsequently incubated at 4°C overnight with mouse monoclonal antibody against p53 (DO-7, Leica Microsystems, Bannockburn, IL, USA, diluted 1/100), p16 (G175-1239, BD Biosciences, Franklin Lakes. NJ, USA, diluted 1/100), p27 (SX53G8, Dako, Glostrup, Denmark, diluted 1/800), MDM2 (SMP14, Santa Cruz Biotechnology Inc., CA, USA, diluted 1/1000), Ki-67 (MIB-1, Dako, diluted 1/300), EGFR (31G7, Nichirei Biosciences Inc., dilution unknown, product code 413701), CD133 (AC133, Miltenyi Biotec, Auburn, CA, USA, diluted 1/10). The following day, the sections were washed three times for 5 min each in PBS, incubated with biotinylated anti-mouse immunoglobulin (Nichirei Biosciences Inc.), washed three times for 5 min each in PBS, and incubated with peroxidase-labeled streptavidin (Nichirei Biosciences Inc.) for 30 min at RT. The immunohistochemical signal was visualized with 3,3′-diaminobenzidine, and the slides were counterstained with Mayer’s hematoxylin, dehydrated in graded alcohol, and cleared in xylene. For CD133, omission of the primary antibody and substitution by nonspecific immunoglobulin (Mouse IgG1, Dako) at the same concentration were used as negative and isotype controls, respectively.
The sections were examined by two independent observers (HO and FF) who were blinded to patients’ clinical information. The proportion of positive nuclei in more than 1,000 tumor cells of more than three fields under a ×400 magnification microscope (Leica DM LB2) at the deepest area of each tumor was calculated for p53, p27, MDM2, and Ki-67. The proportion of nuclei and cytoplasm of tumor cells positive for p16 was evaluated. The proportion of membranes of tumor cells positive for EGFR was evaluated. The cut-off values for abnormal expression were as follows: p53, ≥10%; p16, ≤5%; p27, ≥10%; MDM2, ≥20%; and Ki-67, ≥30%. An immunoreactive score (IRS) was used for the scoring of EGFR. The IRS is 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 to 50%; 3, >50 to 80%; 4, >80%) and ranges from 1 to 12. An IRS of ≥6 was defined as positive for EGFR expression. When evaluating CD133, the tumors were defined as positive and negative when >1% and ≤1% of the membranes and cytoplasm of all tumor cells were immunostained, respectively[13, 30].