Tumor samples were obtained from 293 patients (mean age, 66 years) with primary ESCC who underwent surgical resection at the Department of Gastroenterological Surgery, Osaka City University Hospital, between 2011 and 2014. In this study, the following cases were excluded: 7 patients undergone endoscopic submucosal dissection, 9 cases of non-radical resection, 31 patients of preoperative radiotherapy, 4 patients who died during the perioperative period, 4 patients with intramural metastasis in the stomach, and 2 patients with pathological complete response by NAC. A total of 236 patients (mean age, 67 years) who had undergone esophagectomy with reconstruction for ESCC were enrolled in this study. The median follow-up time was 51 months (range 0–100 months). Indications for preoperative chemotherapy and details of the regimens were based on the esophageal cancer treatment guidelines. The preoperative regimen was 5-FU + cisplatin (FP), 5-FU + nedaplatin (FGP), or 5-FU + cisplatin + docetaxel (DCF). The chemotherapy dose was reduced as needed according to patient condition and adverse events. None of the patients received immunotherapy prior to surgery. Tumors were diagnosed histologically based on the 11th Edition of the Japanese Classification of Esophageal Cancer [10, 11]. Upper gastrointestinal endoscopy, CT scan and fluorodeoxyglucose-position emission tomography imaging were performed in all patients for clinical diagnosis. The diagnosis was based on the consensus of all esophageal surgeons in our hospital. Informed consents were obtained from all patients. The differentiation of squamous cell carcinoma was classified as follows: well-differentiated type (n = 20), moderately differentiated type (n = 118), poorly differentiated type (n = 87), and others (n = 11). Patients received adjuvant chemotherapy in case with pathologically diagnosed lymph node metastasis.
Immunohistochemical of tissue sections
Formalin-fixed paraffin-embedded (FFPE) tumor blocks with representative tumor areas were collected from 236 consecutive patients with ESCC. Immunohistochemical staining was performed according to previous reports from our department . In briefly, immunohistochemical staining was performed on 4-μm-thick sections of paraffin-embedded tumor blocks from ESCC patients. After incubation at 60 ˚C for 10 min, the sections were deparaffinized in xylene and rehydrated with a graded ethanol series (70%, 80%, 90% and 100%). Endogenous peroxidase activity was blocked using absolute methanol containing 3% hydrogen peroxidase for 15 min. After washing the sections in PBS, antigens were retrieved by microwave treatment. Nonspecific binding was subsequently blocked using nonspecific staining blocking reagent (DAKO, Kyoto, Japan). The sections were then reacted with the primary antibody listed below, standing at 4 ˚C overnight. All reactions were performed using appropriate positive and negative controls. Antibodies used for immunohistochemical analyses were as follows: a mouse monoclonal anti-CD3 antibody (clone: F7.2.38, 1:50, high pH retrieval; Abcam, Cambridge, UK), a mouse monoclonal anti-CD8 antibody (clone: F7.2.38, 1:100, high pH retrieval; Abcam, Cambridge, UK), a mouse monoclonal anti-CD20 antibody (clone: IR604, prediluted, low pH, Merck, Darmstadt, Germany), a mouse monoclonal anti-CD21 antibody (clone: 2G9, prediluted, low pH, Nichirei, Tokyo, Japan), a rabbit monoclonal anti-CD23 antibody (clone: SP23, prediluted, low pH, Nichirei, Tokyo, Japan), and a rat monoclonal anti-PNAd (clone: 120,801, 1:25, high pH, Nichirei, Tokyo, Japan). Sections were incubated with secondary antibody histofine reagent (Nichirei, Tokyo, Japan), and the signal was visualized using 3–3’-diaminobenzidine (DAB). Sections were counterstained with hematoxylin before mounting.
Evaluation of immunohistochemistry
Tumor slides stained with anti-CD20 and anti-CD23 antibodies were scanned at low magnification to select three fields (9.1 mm2; original magnification 20 × ; 1600 × 1200 resolution) with the greatest number of intratumoral and peritumoral CD20+ or CD23+ cells. The microscopic images were imported from the digital photo filing system DP-73 (Olympus, Tokyo, Japan). The area (mm2) of CD20+ cells was measured, and the percentage area (%) of each field that was covered by the CD20+ or CD23+ area was calculated with Image J software (NIH, Bethesda, MD). For the CD20+ and CD23+ areas, clusters occupying ≥ 0.04% and ≥ 0.01%, respectively, were counted as positive. CD20 and CD23 densities were determined as the mean values of the percentage areas in the three fields. Tumor slides stained with anti-CD8 antibody were scanned at high magnification, and five fields (original magnification 400 ×) were selected with staining of intratumoral and peritumoral CD8+ cells. The mean numbers of positively immunostained cells per field were counted using Image J software. To determine the cutoff value, each histogram was generated, and the quartile was calculated. A histogram of the obtained numbers was created to set the cutoff values for CD20, CD23, and CD8, respectively. The cutoff value was set to 0.863 for the first quartile for CD20, 0.128 for the second quartile for CD23, and 64 for the first quartile for CD8.
The association with relapse-free survival (RFS) was analyzed initially by a Kaplan–Meier curve and the log-rank test. RFS curves were drawn using the Kaplan–Meier method, and the log-rank test was used to assess the significance of differences in survival. RFS was defined as the time between the day of surgery and recurrence. P-values < 0.05 were considered significant. Each statistical analysis was performed using the JMP software program (SAS Institute, Cary, NC, USA).
The “T.T” ESCC cell lines were used in this study. T.T human esophageal squamous cell carcinoma cells were obtained from the Health Science Research Resources Bank (Osaka, Japan). The cells were cultured at 37˚C and in 5% CO2. The media used were DMEM (FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan) and RPMI-1640 (FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan), supplemented with 10% fetal bovine serum (FBS; Gibco, NY, USA), 100 IU/ml penicillin (ICN Biomedicals, CA, USA), 100 mg/ml streptomycin (ICN Biomedicals, CA, USA), and 0.5 mM sodium pyruvate (Bioproducts).
In vitro cell co-culture model
T.T cells (1 × 106/ml) were plated in 10 ml of full medium treated with or without treatment with 5-FU and CDDP (30 µM) for 24 h. PBMCs were isolated from total 10 healthy donors using Ficoll density gradient centrifugation with Ficoll-Paque™ PLUS (Cytiva, Tokyo, Japan) at 1,025 × g for 30 min at 20 ˚C, with the brakes off. The PBMCs (1 × 107/ml) were then co-cultured with naïve or chemical-treated T.T cells (1 × 106/ml) for 24 h (n = 7 and 3, respectively). The ratio of CD20+ B cells, CD23+ B cells, and CD8+ T cells in the PBMCs after co-culture with T.T cells was examined by flow cytometry. In brief, after saturation with BD Fetal Bovine Serum Stain Buffer (BD Biosciences, NJ, USA), mononuclear cells were incubated with the primary antibodies and Fc-receptor blocking buffer (cat. no. 564220, 2% HS in PBS) for 30 min at 4 ˚C in the dark. The following monoclonal directly labeled anti-human antibodies were used: PE-labeled anti-CD20 (cat. no. 590961), BV421-labeled anti-CD23 (cat. no. 562707) and FITC- labeled anti-CD8 (cat. no. 555634). The evaluation of dead cells was performed with 7-Amino-Actinomycin D (cat. no. 559925). All antibodies used for flow cytometry were purchased from BD Biosciences. Flow cytometric analyses were performed with the BD LSRFortessa™ X-20 (BD Biosciences).
Chi-square test and Fisher’s exact test were used to assess the associations of the expressions of CD20+ B cells, CD23+ B cells, and CD8+ T cells with clinicopathological features. The degree of infiltration between the density of CD20+/CD23+ B cells and the number of CD8+ T cells was compared by Spearman’s correlation coefficient. The regression line was determined by plotting the number of each cell, and the correlation was examined. A Cox proportional hazard model was used for univariate and multivariate analyses of prognostic factors. P-values < 0.05 were considered significant. Correlation strength was defined as weak (0.2 < r ≤ 0.4) and strong (r > 0.7). The Wilcoxon rank sum test was used to compare the expressions of CD20+ B cells, CD23+ B cells, and CD8+ T cells between the two groups.