Study participants
In total, 145 patients were selected by Professor Ying Wang from the Sichuan Cancer Hospital (Chengdu, China) between August 2006 and February 2008. The patients were recruited through oncologist referrals, having been diagnosed with NSCLC following presentation with lung cancer symptoms, such as chest pain, shortness of breath, hemoptysis and wheezing. The inclusion criteria were as follows: Patients with NSCLC; patients treated with surgery; and patients agreed to participate the present study. All the patients provided written informed consent for participation. The study experiments conformed to the ethical guidelines of the 1975 Declaration of Helsinki (20), and were approved by the Institutional Review Board of the Sichuan Cancer Hospital.
Of the 145 NSCLC patients, 51 patients were excluded due to having received prior radiation therapy or chemotherapy, having incomplete clinical information, or unavailability of paraffin-embedded tissue sections. The remaining 94 patients with primary NSCLC (male, 68; female, 26; age range, 29–82 years) were included in the present study (Fig. 1). These patients had not received prior radiation therapy or chemotherapy. Complete clinical information and paraffin-embedded sections were obtained. The pathological staging of the histological types was based on the standard histological classification of lung cancer, established by the World Health Organization in 2004 (21). Following the collection of clinical samples for the present study, the presence or absence of metastasis was assessed.
Patient follow-up
The patients were routinely followed-up on an outpatient basis, according to a standard protocol. Briefly, in the first year following treatment/surgery, visits were conducted every 3 months; in the second and third years, every 6 months; and until the end of the fifth year, once every 12 months. Patients who did not attend the outpatient program were contacted by telephone and/or letter in order to obtain follow-up data, until the visit cut-off date on August 30, 2012. The rate of missed visits was 4.6%. The period of survival was calculated from the date of diagnosis to the date of the final follow-up or the date of mortality due to recurrence or metastasis.
Immunohistochemical staining
AQP5 expression in the biopsy tissue specimens was detected by immunohistochemical analysis. The biopsy tissue specimens were collected from the NSCLC patients who had undergone surgical removal of the tumor. Tumor sections measuring 5 µm in thickness were initially deparaffinized and rehydrated in a graded series of alcohols: Slides were submerged in xylene two times for 5 min, before soaking in 100% ethanol two times for 5 min, 95% ethanol for 5 min and in 80% ethanol for 5 min. Following rehydration, an antigen retrieval process was performed under high pressure (22). The sections were then was hed twice in phosphate-buffered saline (PBS)-Tween 20 for 2 min each. The sections were allowed to cool prior to blocking with 20% goat serum in PBS for 15 min. Next, the sections were incubated at 37°C for 1 h, and then overnight at 4°C with a polyclonal rabbit antibody against AQP5 (dilution, 1:100; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; catalog no. SC-28628). Subsequent to was hing three times with PBS-Tween 20 for 2 min each, the sections were incubated for 10 min at room temperature in a peroxidase blocking solution. Next, the sections were rinsed three times with PBS-Tween 20 for 2 min each and incubated with a biotinylated goat anti-rabbit IgG secondary antibody (dilution, 1:100; Santa Cruz Biotechnology, Inc.; catalog no. SC-2040) in PBS. Subsequent to was hing, the sections were incubated in a horseradish peroxidase-streptavidin solution (dilution, 1:100; Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 37°C. The sections were again rinsed three times in PBS-Tween 20 for 2 min each and incubated in a peroxidase substrate solution (Sigma-Aldrich). Subsequently, the sections were rinsed in running tap water for 2–5 min, followed by dehydration with 95% ethanol for 1 min and twice with 100% ethanol for 3 min each. The sections were then cleared twice in 100% xylene for 5 min each and then covered with neutral gum (Bioworld Technology, Inc., St. Louis Park, MN, USA). The primary and secondary antibodies and 3,3′diaminobenzidine tetrahydrochloride (DAB) were obtained from Santa Cruz Biotechnology, Inc. All the steps were treated with deoxyribonuclease (Life Technologies, Grand lsland, NY, USA). This method was used to detect the expression of AQP5 in the lung cancer tissues. Microscopic immunohistochemical analysis of the tissue sections was performed using an Olympus BX41 microscope (Olympus Corporation, Tokyo, Japan).
Scoring of immunohistochemical staining results
Scoring of the AQP5 expression levels and analysis of all the specimens were performed by technicians who were blinded to the clinical information. The appearance of yellowish-brown granules in the cytoplasm was regarded to be a positive result for the expression of AQP5. The staining intensity of the AQP5-positive cells was scored based on the percentage of stained tumor cells as follows: i) 0, unstained; ii) 1, pale yellow staining; iii) 2, yellowish-brown staining; and iv) 3, brown staining. Scores were also assigned according to the percentage of AQP5-positive cells as follows: i) 4, >75%; ii) 3, 51–75%; iii) 2, 11–50%; iv) 1, ≤10%; and v) 0, negative. The two scores obtained for each case were then multiplied and assigned to one of the following groups: i) 0–2, (−); ii) 3–5, (+); iii) 5–8, (++); and iv) 9–12, (+++) (23).
Statistical analysis
To avoid bias, sample collection and data analysis were conducted without knowledge of the subject status. SPSS version 15.0 statistical software (SPSS, Inc., Chicago, IL, USA) was used for data analysis. The χ2 test was used to compare differences in AQP5 expression, the Kaplan-Meier method was used for the analysis of the survival data and the log-rank test was used to compare the survival curves. P<0.05 was considered to indicate a statistically significant difference.