Cytokine production in advanced head and neck cancer patients Conti-Freitas LC et al. treatment. We found that BCG is able to modulate the immune response of these patients (6). In this study, we evaluated the production of IFN-c and IL-10 by cultured PBMCs isolated from patients with advanced supraglottic laryngeal cancer. PBMCs were isolated during the pre- operative period (PREOP) and the late postoperative (LP) period. METHODS Fourteen patients with stage III or IV supraglottic laryngeal carcinoma were selected at the outpatient clinic of the University Hospital of the Ribeira˜o Preto School of Medicine, Sa˜o Paulo University. Each tumor was staged according to the TNM classification. Patients with infectious and/or inflammatory diseases detected by blood and urine tests and chest X-rays and patients using immunosuppres- sive medications were excluded. Surgical treatment con- sisted of total or partial laryngectomy according to lesion stage. T3 stage patients underwent total or supraglottic laryngectomy, and T4 stage patients were treated with total laryngectomy. Blood samples were collected from all patients during the PREOP period and at 236¡18 days after surgery (LP). Thirteen healthy age- and sex-matched individuals were recruited as the control group. The presence of neoplasms in the control volunteers was ruled out by detailed physical investigation. The study was approved by the Ethics Committee of the University Hospital of the Ribeira˜o Preto School of Medicine, Sa˜o Paulo University, and all patients gave written informed consent prior to participation in the study. Measurement of the cytokine concentrations Blood samples were collected from the peripheral vein under sterile conditions during both the PREOP and LP periods from patients with laryngeal cancer. Blood staples were collected only once from the control subjects. Peripheral blood mononuclear cells (PBMCs) were isolated with a Ficoll- Hypaque gradient, and their viability was determined by 2% Trypan Blue exclusion. Cells were kept at a concentration of 2.0x105 cells/mL in 12.5% bovine fetal serum. Non-stimu- lated and stimulated cultures were evaluated; stimulated cells were exposed to 10 mg/mL of concanavalin A (ConA) or 20 mg/mL of BCG. After 72 h of culture at 37˚C in a humid environment containing nearly 5% CO2, the supernatant was collected, and the IL-10 and IFN-c levels were measured (7). Human monoclonal IL-10 and IFN-c antibodies were used [Pharmingen International (Life Science Research), San Diego, CA, USA] as the capture antibodies, and biotinylated antihuman antibodies against the analyzed cytokines (Pharmingen) were used as the detecting antibodies. Binding was detected with peroxidase-labeled streptavidin (DAKO, Glostrup, Denmark) and O-phenylenediamine- 2HCl/substrate (OPD, Sigma, St. Louis, MO, USA). The intra-assay and inter-assay variation levels were below 10%. The detection limits of the cytokine ELISAs were 78 pg/mL and 39 pg/mL for IFN-c and IL-10, respectively. The results are reported as the median (M), mean (x) and standard deviation (SD). GraphPad Prism (San Diego, CA, USA) was used for statistical analysis. Between-group comparisons were performed using the Mann-Whitney and Wilcoxon tests, and p,0.05 was considered statistically significant. 588 CLINICS 2012;67(6):587-590 RESULTS Demographic data for the patients and controls are shown in Table 1. All patients smoked an average of 20¡8.5 (x¡DP) cigarettes/day for 44.5¡10 years. Eight patients received adjuvant radiotherapy 27.1¡8 days after surgery, and the post-operative evaluation was conducted at least six months after the completion of the radiotherapy. No patient exhibited evidence of tumor recurrence at the time of blood sample collection for LP evaluation, as determined by endoscopy and radiography. All patients had stopped smoking by the time of the postoperative evaluation. Thirteen healthy control subjects were selected, ten men and three women. The control subjects ranged in age from 40 to 67 years (52¡9.3). No significant differences were observed between the controls and the patients. The IFN-c levels in the PREOP and LP BCG-stimulated cultures were similar to those in the control group cultures, whereas, the IFN-c levels were lower in the non-stimulated PREOP and LP cultures than in the non-stimulated control cultures (Figure 1). For the control cultures, the IFN-c level was 154.86 (32.32 – 192.8) pg/ml in the non-stimulated cultures and 239.9 (139.83 – 1,718.08) pg/ml in the BCG- stimulate cultures. A higher elevation index was observed in patients at both the PREOP and LP timepoints, with increases from 27.63 (1.12 – 140.25) pg/ml to 116.19 (5.5 – 3,030.86) pg/ml and from 41.47 (30.03 – 218.09) pg/ml to 186.47 (27.74 – 1,459.73) pg/ml, respectively (Table 2). The IFN-c levels in the supernatants of non-stimulated PBMC cultures were lower for the PREOP and LP cultures than for the control cultures (p = 0.001, p = 0.04, respectively). However, when the cultures were stimulated with ConA, there were no significant differences between the control cultures and either the PREOP or LP cultures. Higher levels of IFN-c were observed in the supernatants of the ConA- stimulated LP cultures than in those of the ConA-stimulated PREOP cultures (p = 0.005) (Figure 1). The IL-10 levels in the supernatants of the ConA- and BCG-stimulated cultures of cells from patients in the PREOP and LP periods were also similar to in the levels for the control group cultures. The IL-10 level was undetectable in non-stimulated cultures (Table 2). DISCUSSION The impairment of immunological defense mechanisms may play an important role in cancer pathogenesis. Decreased in vitro lymphocytic function, lower numbers and percentages of lymphocytes and impaired lymphopro- liferation have been reported in many cancer studies (8-12). Many researchers have examined different aspects of the immune behavior of head and neck cancer; however, few studies have examined supraglottic laryngeal cancer speci- fically. This attention is justified by the local behavior, Table 1 - Sex, age and tumor stage (TNM classification; American Joint Committee on Cancer) of the patients and controls. Sex Age (years) Stage (TNM) M: male, F: female, SD: Standard Deviation. Patients 12M:2F 58.5 (SD = 9) 7 stage III/ 7 stage IV Control Group 10M:3F 52 (SD = 9.3) -