The EMT Status in the Primary Tumor Does Not Predict Postoperative Recurrence or Disease-free Survival in Lung Adenocarcinoma

Patients and Methods

Patients, clinical features, and follow-up. The Institutional Review Board of our university approved this study and informed consent for the use of the tumor specimens was obtained from all the patients or their legal guardians. Tumor samples were obtained from 296 patients with primary lung adenocarcinoma who had undergone a surgical resection between 2003 and 2007 in our department. Nine of these patients had stage IV disease, and 25 underwent an incomplete resection. The tumor samples from 79 patients were too small to evaluate by immunohistochemical (IHC) staining for the E-cadherin, gamma-catenin, vimentin and fibronectin status. As a result, 113 patients were excluded from further analysis. Therefore, 183 tumor specimens were evaluated. All of the patients were Japanese, consisting of 102 males and 81 females in this series, with a median age of 70 years (range 23-88 years). The tumor stage was classified according to the TNM Classification for Lung Cancer (8). According to the pathological stage, 106 patients had tumors of stage IA, 39 of IB, 13 of IIA, 6 of IIB, 16 of IIIA and 3 of stage IIIB.

The patients were followed-up every month for the first postoperative year and at approximately 2- to 4-month intervals thereafter. The evaluations included a physical examination, chest roentgenography, a chemical blood analysis, and measurements of tumor markers. Chest and abdominal computed tomography, brain magnetic resonance imaging, and a bone scintiscan were performed every 6 months for 3 years after surgery. Additional examinations were performed if any symptoms or signs of recurrence were detected. Follow-up was conducted for all patients. The median follow-up period was 53.7 months. Twenty-seven (15.3%) patients had received adjuvant chemotherapy as follows: carboplatin plus paclitaxel (n=18), carboplatin plus gemcitabine (n=7), and tegafur–uracil (UFT) (n=2) (9, 10). At the last follow-up (in September 2011), 144 patients were alive and free of cancer, 11 patients had died of other causes without evidence of cancer, 8 patients were alive with recurrent cancer, and 20 patients had died of cancer. In total, 28 (15.3%) our of the 183 patients demonstrated disease recurrence after surgery. The majority of the sites of tumor recurrence were hematogenous metastases. Twenty-five cases had hematogenous (9 brain, 10 lung, 5 bone, and 1 adrenal metastasis) and six had locoregional (4 lymph node metastasis and 2 pleural dissemination) recurrences. Recurrent tumors in both the brain and bone in two cases, brain and adrenal gland in one, and bone and lymph nodes in one, respectively.

IHC staining of paraffin-embedded tumor samples. IHC staining was conducted using serial sections from the same paraffin-embedded blocks by previously described methods (11, 12). Briefly, all tissue specimens were formalin-fixed and processed similarly, according to the standard histology practices. A 3 μm thick formalin-fixed, paraffin-embedded tissue section was prepared from each specimen. All specimens were stained with hematoxylin–eosin for the histological diagnosis. The sections were briefly immersed in citrate buffer [0.01 mol/l citric acid (pH 6.0)] and then were incubated twice for 10 min at 121°C in a high-pressure sterilization oven for antigen retrieval. They were then incubated with antibody against E-cadherin (DAKO, Denmark) diluted at 1:2000; gamma-catenin (Dako) diluted at 1:200; vimentin (DAKO) diluted at 1:200; and fibronectin (DAKO) diluted at 1:100; in phosphate-buffered saline 60 min at room temperature (13). Thereafter, IHC staining was performed by the labeled polymer method (Histofine Simple Stain MAX-PO kit, Nichirei, Tokyo, Japan) according to the manufacturer's instructions (14). The positive controls for E-cadherin, and gamma-catenin were samples of the normal bronchial epithelium adjacent to the tumor, and the controls for vimentin and fibronectin were samples of malignant mesothelioma (7, 15). The negative controls used mouse IgG (Dako) instead of the primary antibody.

Evaluation of the stained specimens. Following the IHC detection of protein expression in each specimen, the percentage of immunoreactive tumor cells in five fields at ×400 selected randomly on one slide was recorded, and then the final proportion of positive tumor cells was determined as the average. To evaluate any correlations with the clinicopathological characteristics, these protein expression scores were divided into positive or negative groups by subdividing according to the percentage of nuclear staining using a cutoff of 50% (7). The slides were independently examined by two of the investigators (Y.C. and H.S.) who were blinded to the clinicopathological data. When a discrepancy was found between the two investigators, a consensus was reached via their simultaneous examination using a double-headed microscope.

Statistical analyses. Statistical significance was evaluated using the chi-square test or Fisher's exact test. The Kaplan-Meier method was used to estimate the probability of survival, and survival differences were analyzed by the log-rank test. A multivariate analysis was then performed according to Cox's proportional hazards model. The odds ratio (OR) and 95% confidence interval (95% CI) were calculated for each variable. Differences were considered to be statistically significant for p-values <0.05. The data were analyzed using the Stat View software package (Abacus Concepts, Inc., Berkeley, CA, USA).