Is Class III β-Tubulin a True Predictive Marker of Sensitivity to Vinorelbine in Non-small Cell Lung Cancer? Chemosensitivity Data Evidence

Patients and Methods

Patients. All the study protocols were approved by the Institutional Review Board for Clinical Practice of our institution, and written informed consent was obtained from each patient.

Patients and specimens for the dose-response curve of vinorelbine in the HDRA. From March 2006 to August 2007, fresh surgically resected tumor specimens obtained from 9 patients were used to analyze the dose–response curve of vinorelbine in the HDRA and to measure the median effective dose (ED₅₀). The patients comprised 6 male patients and 3 female patients who ranged in age from 57 to 77 years (mean=70 years). The specimens examined were 6 adenocarcinomas, 2 squamous cell carcinomas and 1 carcinoid.

Patients and specimens for the inhibition rate of vinorelbine in the HDRA. After the ED₅₀ measurement, surgically resected fresh tumor specimens was obtained from 68 NSCLC patients at our institution from August 2007 to June 2010, and applied to the HDRA to measure the inhibition rate of vinorelbine. The patients comprised 47 male patients and 21 female patients who ranged in age from 43 to 83 years (mean=70 years). The specimens examined were 38 adenocarcinomas, 19 squamous cell carcinomas, 3 adenosquamous carcinomas, 2 pleomorphic carcinomas and 6 other histological types.

HDRA. The HDRA was used as an in vitro drug sensitivity test, and was carried out as described previously (17). Collagen sponge gels manufactured from pig skin were purchased from Sumitomo Pharma (Osaka, Japan). Cancerous portions of the specimens were minced into approximately 10-mg pieces and the pieces were placed on prepared collagen surfaces in 24-well microplates. The specimens were incubated in the presence of vinorelbine dissolved in RPMI-1640 medium (Sigma, St. Louis, MO, USA) containing 20% fetal calf serum (Invitrogen, Carlsbad, CA, USA) for 7 days at 37°C under a humidified atmosphere with 5% CO₂. Vinorelbine was provided by Kyowa Hakko Kirin Co. Ltd. (Tokyo, Japan).

After completion of the histoculture, 100 μl of Hanks' balanced salt solution containing 0.1 ml of type I collagenase (0.6 mg/ml) (Sigma) and 0.1 ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution (2 mg/ml) (Sigma) were added to each culture well and incubated with the specimens for 16 hours. After removal of the medium, formazan was extracted with 0.5 ml of 100% dimethylsulfoxide, and the absorbance of the solution in each well was measured at 540 nm (control: 630 nm) using a microplate reader (Model 680; Bio-Rad, Hercules, CA, USA). The absorbance per gram of cultured tumor tissue (OD/g) was calculated from the mean absorbance of the tissue specimens in 4 culture wells and the tumor tissue weight determined prior to the culture. The efficacy of the anticancer agent was calculated according to the inhibition rate (IR) using the following formula: IR (%)=(1–mean OD/g of cultured tumor/mean OD/g of control tumor) ×100. The HDRA was regarded as evaluable when the mean absorbance of the extracted formazan at 540 nm of the control tumor was ≥15 per gram. When the IR of the drug was negative, it was regarded as 0 and indicated absolutely no chemosensitivity.

Dose–response curve analysis. Dose–response curve analysis was performed as described previously for a cytotoxic anticancer agent (18, 19). The HDRA was performed for vinorelbine at concentrations of 3.375, 6.75, 12.5, 25, 50, 100, 200 and 400 μg/ml, in order to analyze the dose–response curves for individual patients. Each dose–response curve was calculated using the following formula: y=A [1–1/(1+exp{B (x–logC)}], where y is the inhibition rate, x is the logarithm of the drug concentration, A is the maximal response (IR at the maximum concentration), B is the slope factor (slope at ED₅₀) and C is the ED₅₀.

The OD/g values were directly applied to a nonlinear least squares analysis using the following formula for a simplified dose–response curve: y=α+β/{1+exp (γx–δ)}, where the values of α, β, γ and δ were obtained. Using these values, the dose–response curve of each specimen was reconstructed according to the following relationships: slope factor=γ, ED₅₀=10^(δ/γ) and maximal response=100×β/(α+β). The validity of the acquired dose-response curve was judged using the Akaike information criterion (AIC) (20). The dose–response curve was only adopted in specimens with an AIC that was smaller than the AIC of their completely nonresponsive equation (y=α).

Histopathologic analysis. Tissue sections were deparaffinized and rehydrated through 100%, 95% and 90% ethanol solutions. Heat-induced antigen retrieval was performed by a 40 min treatment with Target Retrieval Solution (code S2031; Dako, Tokyo, Japan) followed by a 20 min cool-down period at room temperature. The sections were immunostained using an automated staining system (AutostainerPlus; Dako) and incubated with an anti-neuronal class III β-tubulin monoclonal antibody (clone TUJ1; 1:500 dilution; code MMS-435P; Covance Inc., Berkeley, CA, USA) for 30 min at room temperature. Positive reactions were visualized by incubation with a labeled polymer (EnVision+ system; code K4001; Dako) for 30 min at room temperature, followed by incubation with 3,3’-diaminobenzidine (DAB) as a chromogen for 5 min. The sections were counterstained with hematoxylin for 5 min.

All of the sections were examined by an observer (Tatsuya Yoshimasu) who was blinded to the clinical information of the patients, and H-scores were calculated as previously reported (21). The staining intensity was graded on a scale of 0 to 3. The ratio of positively stained tumor cell cytoplasm was calculated for each specimen, and a proportion score was assigned. The proportion score was multiplied by the staining intensity to obtain a final semiquantitative H-score.

Statistical analysis. All the values were calculated as the mean±standard deviation (minimum–maximum). Dose–response relationships were calculated by a nonlinear least squares analysis using the Davidon–Fletcher–Powell algorithm. Relationships between immunohistochemical expression and the inhibition rate in the HDRA were examined using Spearman's correlation coefficient test by rank test and one-factor analyses of variance (ANOVA). Values of p<0.05 were considered to be significant.