Non-small Cell Lung Cancer Detection Using MicroRNA Expression Profiling of Bronchoalveolar Lavage Fluid and Sputum

Materials and Methods

Patient selection. Eligible participants were adults with Zubrod performance status ≤2, with a life expectancy of ≥3 months, who were able to provide a sputum sample. Participants were categorized as NSCLC cases if they had a biopsy confirmed diagnosis of stage I or II NSCLC by the seventh edition of the American Joint Committee on Cancer (24) of any subtype. All NSCLC cases underwent staging investigations as indicated and received standard treatment for their NSCLC in accordance with local guidelines (25, 26). NSCLC cases were excluded if they had any prior or current history of malignancy other than non-melanomatous skin cancer. Participants were categorized as controls if clinical examination and diagnostic imaging [chest X-ray or computed tomography (CT) of the chest] within 12 months prior to study entry were negative for malignancy. Two types of controls were selected for inclusion: i) healthy patients without any active medical conditions, and ii) patients with chronic obstructive pulmonary disease (COPD) and smoking history at significant risk of developing lung cancer.

Sample collection. Sputum: Each participant was given an instructional session explaining how to provide a spontaneously expectorated sputum sample. Prior to sputum collection, patients rinsed their mouths thoroughly with water, took a deep inspiration, held their breath, and then coughed. All expectorated sputum was collected into a sterile sample container and was immediately refrigerated at 4°C. Upon delivery to the laboratory, sputum samples were visually inspected in order to ensure proper sputum sample volume (≥1.0 ml) and consistency. Samples deemed to contain only saliva were discarded and repeat sputum collection was performed.

BAL: For NSCLC cases, BAL samples were obtained on the same day as the sputum sample on the day of their surgery for their NSCLC. Sputum samples were obtained first in the preoperative waiting room, 30 to 60 minutes prior to the beginning of the surgical procedure. In the operating room, prior to lung resection, normal saline BAL samples were obtained by flexible bronchoscopy from the lobe in which the tumour was located (based on preoperative imaging). BAL fluids (approximately 40 cm³ per patient) were directly deposited into sterile bottles and were immediately refrigerated at 4°C.

Sample preparation. All sputum and BAL sample containers were labeled with coded identifiers that blinded laboratory personnel of the identity and disease status of the samples. A complete description of our sample handling and analytical methodology used to quantify individual miRNA levels and perform cluster analysis has been previously described in detail (20). In summary, samples were homogenized using a sputolysin solution (Sigma Aldrich, St. Louis, MO, USA) followed by high speed vortexing and incubation at 37°C. RNA was isolated using a TRIzol-based method, and was then quantified using a UV-spectrometer.

miRNA panel selection. The selection of the five miRNAs used for expression profiling (miR-21, miR-143, miR-155, miR-210, and miR-372) was based on an iterative process and prior studies at our institution (20) whereby a panel of 12 miRNAs was evaluated using cluster analysis of a retrospective training set of sputum from NSCLC cases and controls. This panel of 12 miRNAs was then validated and optimized for maximal sensitivity and specificity using a reserve stepwise selection process using receiver-operator curves. The diagnostic characteristics of the miRNA panel were optimal when five miRNAs (miR-21, miR-143, miR-155, miR-210, and miR-372) were used in this prior study and were thus selected for use in this study.

Each of these five miRNAs have been implicated in various aspects of the tumourigenesis of NSCLC. miR-21 overexpression in human lung cancer has been shown to inhibit the negative regulators of the rat sarcoma (RAS)/mitogen-activated protein kinase kinase (MEK)/extracellular Signal-regulated Kinases (ERK) pathway, apoptosis (27), and NSCLC growth and metastasis through modulation of the phosphatase and tensin homolog pathway (28). miR-143 down-regulation in lung cancer has been found to cause to dysregulation of apoptotic pathways (29). miR-155 has been found to be up-regulated in lung cancer, leading to tumourigenic changes such as promotion of cell survival, proliferation, and replicative immortality (30). miR-210 modulates hypoxia-inducible factor 1 (HIF-1) activity (31) and promotes a hypoxic phenotype in lung cancer (32). miR-372 has been found to down-regulate the large tumour suppressor, homolog 2 (LATS2) gene in a post-transcriptional manner (33).

Sample analysis. RNA reverse transcription was performed for each miRNA using the TaqMan Reverse Transcription Kit for individual miRNAs (Applied Biosystems, Carlsbad, CA, USA). Quantitative real-time Polymerase Chain Reaction (RT-qPCR) assays (Applied Biosystems) for each miRNA were performed in duplicate using the RT reaction derived from a single sputum sample for each patient using the StepOnePlus™ RT-PCR instrument (Applied Biosystems). Our previous experience using this experimental methodology demonstrated a high degree of analytic reproducibility (20), with an observed standard error range of 0.25 to 0.5 for threshold cycle (CT) (which was defined as the fractional cycle number at which the fluorescence passed the fixed threshold). SDS software (Applied Biosystems) was used to automatically identify C_T values. The comparative method (ΔΔC_T method) was used to quantify RT-qPCR data for miRNA expression whereby the fold-change in miRNA expression was normalized to that of the endogenous control (U6) and relative to the MRC-5 reference sample. The relative miRNA expression from a sample was expressed as follows: ΔΔRN=2^−ΔΔCT where RN is the amount of miRNA required to be tested and ΔΔCT=(C_Tm−C_Tec)_sample−(C_Tm−C_Tec)_reference, where C_Tm is the C_T for the measured miRNA, CTec is the CT for the endogenous control miRNA (U6) for samples to be tested (BAL fluid or sputum) and the reference sample is the MRC-5 normal lung fibroblast cell line.

Data collection. A medical history was obtained as per institutional standard of practice. Epidemiological data were also collected using a self-reported questionnaire which included information such as demographics, functional status, and social/occupational history. Relevant clinical data were obtained for each participant using electronic medical records including: diagnostic imaging scans, pathology reports, pulmonary function data, and previous medical and surgical history.

Statistical considerations. Three comparisons were carried-out for this study. Firstly, we assessed the ability of the panel of the five miRNAs to differentiate NSCLC cases, using their BAL samples, from controls, using their sputum samples. Secondly, we assessed the ability of the panel to differentiate cases from controls using sputum samples alone. These two comparisons were performed using the unsupervised hierarchical cluster analysis function of SPSS version 14 (IBM Corp., Armonk, NY, USA) of experimentally normalized miRNA expression profiles using within-group linkage and cosine correlation similarity.

A third analysis was performed in order to assess differences between matched pairs of BAL and sputum samples obtained from the same patient using a cosine similarity test of the ΔΔRn⁻¹ values for each miRNA tested. For cosine similarity, values close to 1 indicate a high degree of similarity, whereas those close to zero indicate a lack of similarity between vectors of an inner product space.

Ethical considerations. This study was approved by the Human Research Ethics Board of the University of Alberta (Edmonton, Canada) and the Alberta Cancer Research Ethics Committee (Alberta Health Services, Edmonton, Canada) (study approval number Pro00017473). Study participants provided their written informed consent prior to study entry.