Patients and samples
Frozen primary lung tumor and corresponding non-neoplastic lung specimens of 83 consecutive NSCLC patients who underwent radical surgery at the San Luigi Hospital, Division of Thoracic Surgery, between December 2003 and March 2005, were analyzed.
Patients (64 males and 19 females) had a median age of 67 years (range 40 to 82 years) and no patient received either pre-operative or post-operative chemo and/or radio-therapy according to the institutional treatment policy for resectable rescue in those years. Histological examination was performed on formalin-fixed tissues in all cases and tumors were diagnosed and classified according to the WHO classification [13] as follows:
40 adenocarcinomas (ADC); 30 squamous cell carcinomas (SQC); 4 large cell carcinomas (LCC); and 9 bronchiolo-alveolar carcinoma/adenocarcinoma in situ (BAC/AIS). Differentiation grade (grade 1: 16, grade 2: 29, grade 3: 38), pT status (pT1: 7, pT2: 59, pT3: 9, pT4: 8) and pN status (pN0: 52, pN1: 13, pN2: 18) were also recorded. According to the TNM classification for solid tumors [14], 41 cases had a pathological stage I; 15 stage II; 24 stage III; and 3 stage IV. Follow up data was available for all cases. Informed consent was obtained from each patient and the study was approved by the Institutional Review Board of the San Luigi Hospital. All samples were de-identified and cases anonymized by a pathology staff member not involved in the study. Clinical parameters were compared and analyzed through coded data.
RNA extraction, cDNA synthesis and Qpcr
RNA was extracted from 15-25 mg and 60-80 mg of tumor and normal lung tissue specimens, respectively. Genomic DNA contamination was removed by DNAseI treatment (Promega). TotRNA was then quantified with an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) and stored at -80°C. Two μg totRNA were retro-transcribed with random hexamer primers and Multiscribe Reverse transcriptase (High Capacity cDNA Archive Kit, Applied Biosystems, Foster City, CA), in accordance with manufacturer's suggestions.
Expression levels of AURKA and of reference genes POLR2B and ESD were evaluated with SYBR technology with optimized PCR conditions and primer concentrations. Primer sequences were as follows: AURKA.FW:GAGATTTTGGGTGGTCAGTAGATG, AURKA.RW:TAGTCCAGCGTGCCACAGAGA, ESD.FW:TGTTGTCATTGCTCCAGATACCA, ESD.RW:CCCAGCTCTCATCTTCACCTTT, POLR2B.FW:CCTGATCATAACCAGTCCCCTAGA,OLR2B.RW:GTAAACTCCCATAGCCTGCTTACC.
Melting curve analysis and efficiency evaluations were performed for all the amplicons. Quantitative PCR (qPCR) was carried-out on an ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems) in 384-well plates assembled by Biorobot 8000 (Qiagen, Germantown, ML). Reactions were performed in a final volume of 20 μl. All qPCR mixtures contained 1 μl of cDNA template, 1Х SYBR Universal PCR Master Mix (2×) (Applied Biosystems). Cycle conditions were as follows: after an initial 2-min hold at 50°C to allow AmpErase-UNG activity, and 10 minutes at 95°C, the samples were cycled 40 times at 95°C for 15 seconds and 60°C for 1 minute. Baseline and threshold for Ct calculation were set-up manually with the ABI Prism SDS 2.1 software.
Immunohistochemistry
Formalin-fixed paraffin-embedded tissues were cut into 4 μm thick sections and collected onto charged slides for immunohistochemical staining. After de-paraffination and rehydration through graded alcohols and phosphate-buffered saline (pH 7.5), the endogenous peroxidase activity was blocked by incubation with absolute methanol and 0.3% hydrogen peroxide for 15 minutes. Sections were incubated at the optimal conditions with the following primary antibodies:
(1) mouse monoclonal antibody anti-Ki67 (1:300; MIB-1, DakoCytomation, Glostrup, Denmark); (2) Mouse monoclonal AURKA (1:200; H00006790-M01, Abnova, Taipei, Taiwan).
Immunoreaction was revealed by a dextran-chain (biotin-free) detection system (EnVision; DakoCytomation), using 3,3'-diaminobenzidine (DAB; DakoCytomation) as a chromogen. The sections were lightly counterstained with haematoxylin. Negative control reactions were obtained by omitting the primary antibody. Ki67 proliferation index was calculated as the percentage of positive nuclei amongst at least 200 nuclei counted at high magnification in areas of highest labeling.
Statistical analysis
AURKA mRNA Ct values, calculated by Applied Biosystems SDS2.1 software, were normalized by subtraction of the geometric mean obtained between Ct for two internal controls, POLR2B and ESD, generating ΔCt values. Differential AURKA transcript expression between ΔCt values for tumor and corresponding normal tissue samples were evaluated using t-test for paired data and expressed by the formula: ΔΔCt = -(ΔCt cancer - ΔCt normal) corresponding to log2[fold change]. Protein and mRNA expression levels have been dichotomized into two groups of "high" and "low" expression using median value as threshold cut-off. For AURKA staining intensity, percentage of cells with nuclear expression and H score (intensity x % cells positive) were evaluated. The association between ΔΔCt and clinico-pathological variables was evaluated using the Kruskal-Wallis test. Overall survival time was calculated from the date of surgery to death or last follow-up date. Cox regression was used in the univariate survival analysis to determine the association of AURKA modulation with overall survival. Statistical analysis was performed using R statistical software [15].