The study design was a prospective case-control study conducted on 80 BAL samples obtained from 80 adult patients admitted to the Respiratory Department, sixty of whom had a suspicious lung mass on chest CT, which was later confirmed to be malignant by BAL cytology and/or histopathology of lung biopsy obtained by different techniques, e.g., CT-guided biopsy, ultrasound-guided biopsy, trans-bronchial biopsy, or open biopsy. Patients had neither co-exiting malignancies, lung metastases, history of chemotherapy nor previous lung malignancy. Twenty patients with benign lung lesions (e.g., foreign body aspiration, tracheal web, etc.), matched for age and gender, served as a control group, all had underwent bronchoscopy and BAL but did not show any evidence of lung cancer by CT or cytology/histopathology. Detailed clinical history including smoking habits, drug use was documented and full clinical examination, routine laboratory, and radiological investigations (chest X-ray or CT) were also performed. SHOX2 DNA methylation levels were detected using methylation analysis by restriction endonuclease digestion and real-time PCR.
The study was approved by Institutional Research Ethics Committee (#616.07) and a written informed consent was obtained from all participants before enrolment in the study. Patients were all coded and detailed information was stored confidentially. This work has been carried out in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki and its later amendments) for experiments involving humans.
Specimen collection
All enrolled patients underwent flexible bronchoscopic examination to visualize the airways, assess the presence of endo-bronchial lesion, surgical accessibility for resection, obtain BAL for cytological analysis, and obtain tissue biopsy for histopathological examination. Broncho-alveolar lavage (BAL) samples were collected during bronchoscopy by aspiration from the region of the suspicious lesion after injecting 10–20 ml of isotonic saline solution. Cytology from BAL was done to confirm or exclude malignancy.
DNA Extraction
DNA extraction was performed from the BAL using QIAamp DNA blood mini kit 50 (QIAGEN, Germany, cat. # 51104) and measurement of DNA quantity and purity was determined using Nanodrop 2000 spectrophotometer.
Digestion by Methylation Sensitive Restriction Enzymes
The DNA samples were subjected to restriction digestion by EpiTect Methyl II DNA Restriction Kit (Qiagen cat. # 335452), which prepares genomic DNA samples for DNA methylation analysis using EpiTect Methyl II PCR Assay. The kit contains: restriction digestion buffer, enzyme A (methylation-sensitive) and enzyme B (methylation-dependent). Using the enzymes and buffer provided in the kit, 4 digests were performed in order to detect different methylated DNA fractions. The product of a mock digest (Mo) contains all of the input genomic DNA. The product of the methylation-sensitive (Ms) restriction enzyme mixture (enzyme A) digest contains methylated DNA sequences, while the product of the methylation-dependent (Md) restriction enzyme mixture (enzyme B) digest contains unmethylated DNA sequences. The product of a double digest (Msd) measures the background and the success of both enzymatic digestions.
Equal amounts of a genomic DNA (125 ng) were added to 4 separate tubes into which buffer (13 μl/tube) and the appropriate restriction enzyme combinations (0.5 μl/tube) were added. The mock digest (Mo) had no enzymes added, enzyme A was added to the methylation-sensitive restriction digest (Ms), enzyme B was added to the methylation-dependent restriction digest (Md), and both enzyme mixtures were added to the double digest (Msd). All digests were incubated at 37 °C for 6 h in a thermal cycler (Thermo Scientific Arkitik Thermocycler) and the enzymes were heat inactivated at 65 °C, for 20 min.
Quantification of SHOX2 gene methylation percentage by real-time PCR
After DNA samples were digested by restriction enzymes, SHOX2 gene methylation status was assessed using EpiTect Methyl II PCR Assay. The EpiTect Methyl II PCR Assay (Cat #/ID: 335452) was used to analyze the amount of DNA in each digest of each sample to determine the methylation status of CpG islands in SHOX2 gene. The kit included the following: (1) RT2 SYBR Green ROX qPCR Mastermix (Qiagen catalogue number 330520) and (2) EpiTect Methyk II qPCR primer Assay (Human SHOX2) (Qiagen catalogue number 335002).
The method employed by the EpiTect Methyl II PCR kit is based on the detection of the remaining input DNA after cleavage with a methylation-sensitive and/or a methylation-dependent restriction enzyme. These enzymes digest unmethylated and methylated DNA, respectively. Following digestion, the remaining DNA in each individual enzyme reaction was quantified by real-time PCR using primers that flank a promoter (gene) region of interest. The relative fractions of methylated and unmethylated DNA were subsequently determined by comparing the amount in each digest with that of a mock (no enzymes added) digest using a ΔCT method.
Briefly, input genomic DNA is aliquoted into four equal portions and subjected to mock (no enzyme), methylation-sensitive (MSRE), methylation-dependent (MDRE), and double (MSRE and MDRE) restriction endonuclease digestion (5 μl of each enzyme). After digestion, the enzyme digests (5 μl/tube) were mixed directly with qPCR master mix (12.5 μl/tube) and dispensed into a PCR tubes containing pre-aliquoted primer mixes (1 μl/tube). Real-time PCR (Stratagene MX 3000P) was carried out using specified cycling conditions, (Supplementary material).
The product of the mock (no enzyme) digestion represents the total amount of input DNA for real-time PCR detection. In the methylation-sensitive digestion (Ms) reaction, the MSRE will digest unmethylated and partially methylated DNA. The remaining hypermethylated DNA (DNA in which all CpG sites are methylated) was detected by real-time PCR. In the methylation-dependent digestion (Md) reaction, the MDRE preferentially digested methylated DNA. The remaining unmethylated DNA was detected by real-time PCR. In the double digestion (Msd) reaction, both enzymes were present, and all DNA molecules (both methylated and unmethylated) were digested. This reaction measures the background and the fraction of input DNA refractory to enzyme digestion.
EpiTect Methyl II PCR assays provide gene methylation status as percentage unmethylated (UM) and percentage methylated (M) fraction of input DNA. Unmethylated represents the fraction of input genomic DNA containing no methylated CpG sites in the amplified region of a gene. Methylated represents fraction of input genomic DNA containing two or more methylated CpG sites in the targeted region of a gene.
The raw ΔCT values were inserted into the data analysis spreadsheet- provided by Qiagen (www.sabiosciences.com/dna_methylation_data_analysis.php), which automatically calculated the relative amount of methylated and unmethylated DNA fractions.
Statistical analysis of the data
Data was fed into a computer and analyzed using IBM SPSS software package version 20.0. (Armonk, NY: IBM Corp). Qualitative data was described using absolute values and percent. The Kolmogorov-Smirnov test was used to test the normality of distribution. Quantitative data was described as range (minimum and maximum), mean, standard deviation and median. Significance of the obtained results was judged at the 5% level.
Receiver operating characteristic (ROC) curve was generated by plotting sensitivity (true positive) on Y-axis versus 1-specificity (false positive) on X-axis at different cut-off values. The area under the ROC curve (AUC) denotes the diagnostic performance of the test. Area more than 50% gives acceptable performance with respect to 100%, as perfect accuracy [16].