Molecular Diagnosis of MACC1 Status in Lung Adenocarcinoma by Immunohistochemical Analysis

Patients and Methods

Patients, clinical features, and follow-up. Tumor samples were obtained from 494 patients with primary lung adenocarcinoma who had undergone surgical resection between 1998 and 2007, in our department. Fifty-six patients had had an incomplete resection. The tumor samples from 59 patients were too small to extract sufficient amounts of DNA for the analyses. One hundred and sixty-eight had cancer overlapping other organs or had multiple lung tumors, and 13 patients died due to other reasons without recurrence. As a result, 297 patients were excluded from the analysis. Finally, a total of 197 patients were included in the present series.

The patients were followed-up every month within the first postoperative year and at approximately 2- to 4-month intervals thereafter. The evaluations included a physical examination, chest roentgenography, blood chemistry analyses, and the measurement of tumor markers. Chest and abdominal computed tomography, brain magnetic resonance imaging, and a bone scintiscan were performed every 6 months for 3 years following surgery. Additional examinations were performed if any symptoms or signs of recurrence were detected.

All of the patients were Japanese, including 107 males and 90 females in this series, with a mean age of 67.8 years (range: 23-91 years). No patients had received either chemotherapy or radiotherapy prior to the resection. There were 78 patients who had never smoked, 51 former smokers, and 68 current smokers. Former smokers were defined as those who had quit smoking at least 3 years before the time of surgery. The tumor stage was classified according to the new TNM (7th edition) classification for Lung Cancer (5). According to the pathological stage, 104 patients were at stage IA, 40 at IB, 13 at IIA, 8 with IIB, 25 at IIIA, 6 at IIIB, and 1 at IV. Twenty-nine (14.7%) patients received adjuvant chemotherapy: 22 received carboplatin plus paclitaxel, 5 received carboplatin plus gemcitabine, and 2 received tegafur-uracil. Follow-up was conducted for all patients and the median follow-up period was 46.7 months. One hundred and fifty-seven patients were alive and free of cancer at the last follow-up, 2 patients had died of other causes with recurrent cancer, 17 patients were alive with recurrent cancer, and 21 patients had died of cancer.

Immunohistochemical staining and evaluation of the MACC1 expression. The study was approved by the Institutional Review Board and informed consent for the use of the tumor specimens was obtained from either the patient or from the patient's legal guardian. IHC staining was conducted using serial sections from the same paraffin-embedded blocks, according to the previously described methods (6, 7). All specimens were stained with hematoxylin-eosin for the histological diagnosis. Briefly, the sections were placed in 0.01 mol/L citrate buffer (pH 6.0) and autoclaved at 121°C for 10 min. They were treated with 3% H₂O₂ for 5 min to block the endogenous peroxidase activity. The primary antibodies used were a rabbit polyclonal antibody against human MACC1 (HPA020103; SIGMA, St, Louis, USA), diluted 1:100 in PBS and incubated for 18 hours at 4°C. 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 (8-10). The positive and the negative controls were a metastatic lung tumor arising from colon cancer specimens expressing MACC1, and the exclusion of the primary antibody, respectively. IHC was considered to be positive only when distinct cytoplasmic staining was evident (Figure 1). An average of 1,500 cells were evaluated per section. The stained specimens were then categorized into 6 degree classes according to the quantitative score (11). Initially, 4 degrees of the proportional score (PS) for the positive staining cells were assigned according to the frequency of positive tumor cells (0, none; 1, 1/100 to 1/4; 2, 1/4 to 1/2; and 3, >1/2). Thereafter, 4 degrees of the intensity score (IS) were assigned according to the intensity of the staining (0, none; 1, weak; 2, intermediate; and 3, strong). The proportional score and the intensity score were then added to obtain a total score (TS), which ranged from 0 to 6. According to the TS, the MACC1 expression of the tumor was categorized as negative when the score was 0-3 and as positive when the score was 4-6. The slides were independently examined by two of the investigators (H. S. and T. O.) who were blinded to the clinicopathological data. When a discrepancy was found between the two investigators, a consensus was reached via simultaneous examination using a double-headed microscope. The correlation of MACC1 protein and mRNA (described below) was also analyzed.

Detection of MACC1 mRNA. The expression of MACC1 mRNA was analyzed in all samples by quantitative real-time PCR, performed on a StepOnePlus Real-Time PCR System (Applied Biosystems, CA, USA) by using a Fast SYBR Green Master Mix (Applied Biosystems) as previously described (11). We quantified gene expression by comparing the levels of the target gene to the levels of beta-actin, used as an internal control. The quantification was based on a standard curve generated from human normal genomic DNA. The relative MACC1 copy number was also normalized to human genomic DNA. PCR for each primer set was performed in triplicate, and the means were reported (1).

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