Patients
In this study, 98 ESCC patients were examined, all of whom underwent radical esophagectomy and regional lymph node dissection following NAC, according to the Japanese Clinical Oncology Group 9907 (JCOG9907) protocol at Tohoku University Hospital (Sendai, Japan) from April 2008 to December 2015 [3]. Among these 98 patients, biopsy specimens obtained prior to NAC were available in 42 cases. The specimens had been fixed in 10% neutral formalin for 36–48 h at room temperature and then embedded in paraffin wax. The sections were histologically examined according to the Eighth Edition of the Union for International Cancer Control tumor, node, and metastasis classification system [33]. The survival time of the patients was determined from the date of surgery until death, recurrence, or last censor. The current study protocol was approved by the Ethics Committee of the Tohoku University School of Medicine (Accession No. 2017-1-630), and informed consent was obtained from all patients prior to surgery.
NAC and esophagectomy
Preoperative chemotherapy was performed according to the JCOG 9907 protocol [3] as follows: continuous infusion of 80 mg/m2 of cisplatin on days 1 and 22 and 5-fluorouracil (5-FU) 800 mg/m2/day, 24 h per day on days 1–5 and 22–26. In addition, 29.7 mg dexamethasone was administered per course to prevent the potential side effects of chemotherapy.
The therapeutic effects of preoperative chemotherapy were evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 [34]. The patients were tentatively classified according to the new guidelines for determining the therapeutic effects of the solid tumors as complete response (CR), partial response (PR), progressive disease (PD), and stable disease (SD) [34]. According to the evaluation method reported in the JCOG 9907 protocol, the sum of the maximum diameter of the primary lesion and shortest diameter of lymph node lesions exceeding 1.5 cm were measured before and after treatment [3]. The maximum diameter of the primary lesion in CT following NAC corresponded to the slice measured by CT before treatment [3]. CR was defined as disappearance of the primary lesion, PR as reduction by 30% or more in maximum diameter of the primary lesion, PD as increase by 20% or more in maximum diameter of the primary lesion, and SD as other than CR, PD, and PR. A total of 13 cases were excluded from further evaluation because of difficulties in obtaining these parameters of clinical measurement. Histopathological tumor regression was tentatively classified into the following five categories according to the Japanese Classification of Esophageal Cancer, eleventh edition: grade 3, markedly effective (no viable residual tumor cells); grade 2, moderately effective (less than one-third residual tumor cells); grade 1, slightly effective (1b, one-third to two-thirds residual tumor cells; 1a, more than two-thirds residual tumor cells); grade 0, ineffective (no therapeutic effects detected) [35].
For esophagectomy, thoracoscopic esophageal subtotal excision, gastric tube reconstruction by hand-assisted laparoscopic technique or open laparotomy, and cervical esophagogastric anastomosis were performed with regional lymph node dissection.
Immunohistochemistry
Serial tissue sections of 4-μm thickness, containing the deepest area of the tumor invasion, were deparaffinized in xylene, rehydrated in graded alcohol, and immersed in 3.0% hydrogen peroxide in methanol for 10 min at room temperature to inhibit endogenous peroxidase activity. For antigen retrieval, the tissue slides for GR, Sgk1, and NDRG1 immunohistochemistry were heated in an autoclave at 121 °C for 5 min in 0.01 M citrate buffer (pH 6.0). After washing three times for 5 min each in phosphate-buffered saline (PBS), the reacted slides were incubated in 1% normal goat serum for 30 min at room temperature to reduce nonspecific antibody binding and then incubated at 4 °C overnight with rabbit monoclonal antibody against GR (D6H2L, Cell Signaling Technology, Danvers, MA, USA, diluted 1/400), Sgk1 (Y238, Abcam, Cambridge, UK, diluted 1/200), or NDRG1 (EPR5593, Abcam, diluted 1/400). The reacted sections were then washed three times for 5 min each in PBS, incubated with biotinylated anti-rabbit immunoglobulin (Nichirei Biosciences, Inc., Tokyo, Japan), washed three times for 5 min each in PBS, and incubated with peroxidase-labeled streptavidin (Nichirei Biosciences, Inc.) for 30 min at room temperature. Immunoreactivity was visualized with 3,3′-diaminobenzidine, and the slides were counterstained with Mayer’s hematoxylin, dehydrated in graded alcohol, and cleared in xylene.
Evaluation of immunoreactivity
GR immunoreactivity was evaluated in the nuclei of tumor cells and Sgk1 and NDRG1 in the cytoplasm of tumor cells. All immunostained slides were independently evaluated by two of the authors (SU and FF) without prior knowledge of any clinicopathological variables of the patients. GR immunoreactivity was semi-quantitatively assessed by H-score or calculating the percentage of nuclear-stained tumor cells multiplied by the relative immunointensity (0, negative; 1, weak; 2, moderate; 3, marked) resulting in a score in the range 0–300 [36]. Sgk1 and NDRG1 immunoreactivity was semi-quantitatively assessed by immunoreactive score, which was calculated as the percentage of cytoplasm-positive tumor cells (<10%: 0, 10–25%: 1, 25–50%: 2, 50–75%: 3, 75–100%: 4) multiplied by the intensity of immunoreactivity (0: negative, 1: weak, 2: moderate, 3: marked) resulting in a score in the range 0–12 [30]. We determined the optimal H-score and immunoreactive score cut-off values for the survival outcome of the patients using the receiver operating characteristic curve method [37] and established thresholds of 154 for GR, 5 for Sgk1, and 7 for NDRG1.
A score in the range of 0–154 was tentatively considered as low GR, while that in the range of 155–300 as high GR. A score in the range 0–5 was also tentatively classified as low Sgk1, and 6–12 as high Sgk1. A score in the range 0–7 was tentatively determined as low NDRG1 and 8–12 as high NDRG1.
Statistical analysis
JMP Pro version 13.2.0 software (SAS Institute, Inc., Cary, NC, USA) was used for all statistical analyses. Continuous data were analyzed using Student t-test or the Mann–Whitney U test. The relation and correlation between two variables were identified using the Pearson chi-square test, Fisher exact test, or Mann–Whitney U test and Wilcoxon test, as appropriate. Overall survival (OS) and disease-free survival (DFS) curves were constructed according to the Kaplan–Meier method and compared using the log-rank test. The Cox proportional hazard model was used for both univariate and multivariate analyses. When comparing paired pair values, the paired two-tailed t-test was used. A P value <0.05 was considered statistically significant.