Biologic Factors Associated with Tumor Oxygenation Are Prognostic in Patients with Stage III Esophageal Cancer: Long-term Results

Patients and Methods

Patients. A total of 64 patients, who received radio-chemotherapy for stage III esophageal cancer, were included in this study. In this cohort of patients, twelve potential prognostic factors were investigated with respect to survival and loco-regional control up to 10 years following treatment. These potential prognostic factors included the tumor cell expression of EPO and EPO-R. Low expression (0-20%) was compared to higher expression (>20%).

Immunohistochemistry. Esophagus tissue samples were fixed in 10% buffered formalin/pH 7.0 (J.T. Baker, Griesheim, Germany), embedded in paraffin and 4-μm-thick serial sections were prepared. Four-micrometer-thick serial sections were de-paraffinized in xylene and rehydrated in graded alcohols. Antigen retrieval was carried out in 0.01 mol/L sodium citrate buffer/pH 6.0 (Sigma-Aldrich, Hamburg, Germany) for 5 minutes in a steamer-cooker. Endogenous peroxidase was blocked with 0.3% hydrogen peroxidase for 5 min. The sections were incubated with the anti-human EPO rabbit polyclonal antibody (clone H-162; 1/50 dilution; Santa Cruz Biotechnologies, Inc., Santa Cruz, CA, USA) and anti-human EPO-R rabbit polyclonal antibody (clone C-20, 1/450 dilution; Santa Cruz Biotechnologies). Anti-human EPO rabbit polyclonal antibody was raised against a recombinant protein corresponding to amino acids 28 to 189, representing mature EPO of human origin, whereas the anti-human EPO-R antibody was raised against a peptide mapping at the carboxyl terminal of EPO-R of human origin. Sections were then washed with tris-buffered saline (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) containing 0.1% Tween 20 (pH 7.0) and subsequent reaction was performed with the biotin-free horseradish peroxidase enzyme-labelled polymer of Envision+ detection system (DakoCytomation, Carpinteria, CA, USA); diaminobenzidine (DAB) complex (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) was used as chromogene. Sections were counterstained with hematoxylin. Negative controls were done for each tumor section, first by omission of the primary antibody and secondly by incubation with the normal rabbit IgG (dilution 1/200, 2 μg/mL concentration, Santa Cruz Biotechnologies) instead of the primary antibody.

Treatment. Radiotherapy was delivered with 6-16 MV photons from a linear accelerator (Siemens Medical Solutions, Concord, CA, USA) with daily doses of 1.8 or 2.0 Gy, 5 days per week. Initial radiotherapy fields (to 50-50.4 Gy) had superior and inferior margins of 5 cm beyond the primary gross tumor volume. The lateral, anterior and posterior margins were a minimum of 2 cm. Regional lymph nodes were included. For definitive treatment, a boost of 9-10 Gy was delivered to the primary tumor with 2 cm margins and enlarged lymph nodes with a margin ≥1 cm. Two courses of chemotherapy were administered concurrently with radiotherapy. 5-fluorouracil (5-FU) (TEVA GmbH, Ulm, Germany) was administered as continuous infusion of 1,000 mg/m²/day for 120 h (days 1-5 of each course) every four weeks. Cisplatin (medac GmbH, Wedel, Germany) was administered as intravenous bolus of 75-80 mg/m² over 1 hour on day 1 of each course. Surgery for tumors of the upper and middle third was radical en-bloc resection of the esophagus and two field lymphadenectomy. For tumors of the lower third, a trans-hiatal esophagectomy was performed. Esophageal continuity was restored by gastric tube.

Hemoglobin levels during radiotherapy. Because hemoglobin levels were monitored weekly during 5-6.5 weeks of radiotherapy, 5-6 hemoglobin levels were obtained. Two groups were formed with respect to the majority (3 of 5 or 4 of 6 levels) of hemoglobin levels during radiotherapy, <12 g/dl versus ≥12 g/dl. No patient treated for 6 weeks had three hemoglobin levels <12 g/dl and three levels ≥12 g/dl.

Additional potential prognostic factors. In addition to tumor cell expression of EPO and EPO-R and hemoglobin levels during radiotherapy, the following potential prognostic factors were investigated: age (≤60 vs. >60 years), gender, Karnofsky Performance Score (KPS) (≤80 vs. >80), tumor length (≤6 vs. >6 cm), histology (squamous cell carcinoma (SCC) vs. adenocarcinoma), histologic grade (G1-2 vs. G3), T-stage (T3 vs. T4), N-stage (N0 vs. N+) and additional surgery (no vs. yes). In the 20 patients receiving surgery, the impact of the resection margin (R0=no residual tumor, R1/R2=microscopic or macroscopic residual tumor) was investigated. Patient characteristics are shown in Table I.

Statistical analyses. Loco-regional control was defined as absence of loco-regional progression based on findings of endoscopy, endoscopic ultrasound, or computed tomography. Survival and loco-regional control were calculated with the Kaplan-Meier method (3) and measured from the last day of radiotherapy. Differences between the Kaplan-Meier curves were evaluated with the log-rank test. Results were considered significant if p<0.05. Those factors being significant or showing a trend (p≤0.08) in the univariate analysis were additionally included in a multivariate analysis, performed with the Cox proportional hazard model.