CXCR2 expression and postoperative complications affect long-term survival in patients with esophageal cancer

Eighty-two patients who underwent transthoracic esophagectomy for ESCC at Keio University Hospital (Tokyo, Japan) between 1997 and August 2002 were examined. Patients were included in the study if they meet the following criteria: (1) histologically proven squamous cell carcinoma of the thoracic esophagus, (2) no microscopic residual tumor (R0), (3) no past history of neoadjuvant chemotherapy or chemoradiotherapy for ESCC, (4) no other cancer, and (5) no steroid use during the perioperative period. Informed consent was obtained from each patient enrolled in this study. All patients underwent two- or three-field lymph node dissection through right thoracotomy, either video-assisted thoracic surgery or open thoracotomy [2, 28]. Physical examinations before surgery did not detect high-risk factors for transthoracic esophagectomy under general anesthesia in any of the patients. Informed consent was obtained from each patient enrolled in this study. The study was approved by Keio University School of Medicine Ethics Committee.

The incidence of postoperative complications was determined by inspection of patients’ medical records. Vocal cord palsy was defined as hoarseness at the time of discharge. Pneumonia was defined as an abnormal shadow on a chest radiograph with fever (>38 °C), positive sputum, and/or a white blood cell count of ≥12,000/mm³. Postoperative complications were defined as complications of a grade equal to or higher than grade II according to the Clavien–Dindo classification [29]: grade I, any deviation from the normal postoperative course without the need for pharmacologic treatment or surgical, endoscopic, or radiologic intervention; grade II, requiring pharmacologic treatment with drugs; grade III, requiring surgical, endoscopic, or radiologic intervention; grade IV, life-threatening complication requiring intensive care unit management; and grade V, death.

All patients were surveyed after esophagectomy in order to detect recurrences. Physical examinations and serum CEA and squamous cell carcinoma antigen tests were performed every 3 months. CT scans from the neck to the upper abdomen and upper gastrointestinal endoscopy were performed from every 6 months.

Tumor recurrence was classified as locoregional or distant. Locoregional recurrence was defined as recurrence at the primary site including the anastomosis and regional lymph nodes. It was described as distant recurrence when located in the liver, lungs, or other distant organs. OS was measured from the date of surgery to the date of death, or last follow-up. Recurrence-free survival (RFS) was measured from the date of surgery to the date of first evidence of relapse. For patients who had not relapsed or died, RFS was censored at the last date at which the absence of relapse was confirmed.

For this study, tissue samples were obtained from esophageal specimens following surgical resection. For immunohistochemistry (IHC) analyses, slides were deparaffinized with xylene and rehydrated in a graded ethanol series staining. Antigen retrieval was performed using an enzyme solution (Proteinase K DAKO®, Glostrup, Denmark). Endogenous peroxidase activity was quenched with 0.5 % periodic acid for 10 min at room temperature. CXCR2 rabbit anti-human polyclonal antibodyLS-A803 (diluted at 1:250, LifeSpan BioSciences®, Seattle, WA, USA) was used as a primary antibody. Prepared sections were incubated overnight at 4 °C. Slides were subsequently incubated with labeled secondary antibody for 30 min at room temperature. We used biotinylated goat anti-rabbit IgG antibody BA-1000 (Vector®, CA, USA) as the secondary antibody. Peroxidase activity was detected with the enzyme substrate 3,3-diaminobenzidine tetrachloride solution. On the final section, the slide was lightly counterstained with hematoxylin and mounted. For each test case, a negative control was also included, which received the same treatment as the test slide, except that the primary antibody was omitted. We also used esophageal squamous epithelial cells without inflammation that lacked CXCR2 expression as negative controls. Positive controls (neutrophils and monocytes in the spleen for CXCR2 antibody, according to Emadi et al. [30]) were obtained to optimize the conditions. We evaluated CXCR2 expression in viable ESCC cells on slides that contained the most invasive tumor lesion. CXCR2 expression was assessed by two investigators (T. N. and H. T.) with no knowledge of the clinicopathological factors. Cases were considered as CXCR2-positive if more than 10 % of cancer cells exhibited cell wall staining.

We previously confirmed that CXCR2 protein is overexpressed in an ESCC cell line, TE4, by Western blot analysis. We also confirmed CXCR2 mRNA expression in TE4 using reverse transcriptase polymerase chain reaction [13].

All data are presented as medians. Statistical significance was determined by a non-parametric Mann–Whitney U test or Fisher’s exact test. Cumulative survival rates for patient groups were calculated using the Kaplan–Meier method and were compared using the Mantel–Cox log-rank test. Cox proportional hazard models were used for multivariate analysis of variables for predicting postoperative survival. A p value of <0.05 was considered statistically significant. Statistical analyses were performed with SPSS v18.0 software (SPSS, Chicago, IL, USA).