Clinical samples
Three different groups of clinical samples were analyzed:
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(a)
Training group: consisting of primary NSCLC (fresh-frozen) tissues and corresponding adjacent non-neoplastic tissues of 35 patients, all diagnosed with operable (stage I-III) NSCLC; tumor histology was squamous cell carcinoma (SCC; n = 15), adenocarcinoma (ADC; n = 13) and undifferentiated (NOS; n = 7). In this group the majority of patients (65.7%) were smokers. All patients were treatment naïve when the samples were collected but after surgery all patients received standard chemotherapy protocols for adjuvant NSCLC. A clinical relapse was documented in 19/35 (54.3%) patients during the follow-up period.
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(b)
Validation group: consisting of 42 patients with operable NSCLC (stage IA–IIIA). Plasma-cfDNA samples and the corresponding CTC were analyzed from all patients however corresponding primary FFPEs tumor tissues were available for analysis only for 22 patients. Peripheral blood from the patients was obtained before surgery and before the initiation of any systemic treatment; 14 patients were diagnosed with ADC, 24 with SCC and 4 with NOS NSCLC. In this group the majority of patients (78.6%) were smokers.
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(c)
Control group: consisting of plasma-cfDNA and matched CTC samples isolated from 12 healthy blood donors (HD). All HD had no known illness or fever at the time of blood drawing and were ≥ 35 years old.
All patients gave a written informed consent to participate in the study, which was approved by the Ethics and Scientific Committee of the Thoracic Diseases General Hospital “Sotiria”.
Isolation of genomic DNA from fresh-frozen primary tumor tissues and formalin-fixed paraffin-embedded tissues
Genomic DNA (gDNA) from primary NSCLC fresh-frozen tissues and corresponding adjacent tissues was isolated using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. DNA extraction of formalin-fixed paraffin-embedded tissues (FFPEs) was performed using QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany). DNA concentration in all cases was measured with a NanoDrop 1000 Spectrophotometer (Thermo Scientific, USA).
Isolation of plasma-cfDNA
Plasma was isolated from peripheral blood (in EDTA) within 2 h to 4 h from sample collection by centrifugation at 530xg for 10 min at room temperature. Once isolated, plasma samples were centrifuged again at 2.000xg for 10 min, before transferring into clean 2-mL tubes and freezing at − 70 °C until time of processing. cfDNA was extracted from 2.00 mL plasma using the QIAamp® Circulating Nucleic Acid kit 50 (Qiagen®, Germany), as previously described [32].
CTCs enrichment using a size-based microfluidic device
The micro-fluidic device Parsortix (ANGLE plc, United Kingdom) [33] was used for the isolation of CTCs from 20 mL of peripheral blood in EDTA. A microscope slide sized disposable cassette was used for the division of blood components [34,35,36]. After this step, enriched CTCs were collected in a total volume of 200μL of PBS into Eppendorf tubes. The isolation of gDNA from enriched CTCs was performed using TRIZOL-LS (Thermo Fisher Scientific, United States) as previously described [19, 20].
Sodium bisulfite conversion
Up to 500 ng cfDNA were chemically modified with sodium bisulfite (SB) as previously described [20, 22, 32, 34]. In each SB-reaction negative and positive controls were included. The Universal Methylated Human DNA Standard (ZYMO Research) was used as fully methylated (100%) positive control. SB-converted DNA samples were stored at − 70 °C until further use.
Whole bisulfite amplification
Whole genome amplification (WGA) of SB-converted DNA was performed using the EpiTect Whole-Bisulfitome Kit (Qiagen) as previously described [37]. This protocol is optimized for the amplification of > 50 ng of SB-converted DNA, diluted with nuclease-free water to a final volume of 10μL. The amplification was performed in a thermal cycler (Mastercycler® pro, Eppendorf) (28 °C/8 h, 95 °C/5 min, and 4 °C until storage; lid temperature set to 70 °C).
Quality control
Positive and negative controls were included in all steps to ensure the quality and reproducibility of results. Human placental gDNA (Sigma-Aldrich) was used as a negative control after SB conversion. Universal Methylated Human DNA Standard (ZYMO Research) was used as fully methylated positive control. DNA integrity from all DNAs samples was checked by amplifying a region in exon 20 of the PIK3CA gene as previously described [38]. The quality of amplified SB-DNA was checked by a real-time PCR assay for β-actin (ACTB); only samples with positive amplification of ACTB were used for further analysis [39].
Real-time MSP assays
DNA methylation of RASSFIA, SLFN11 and FOXA1 was detected by using sensitive and specific real-time MSP assays based on specific primers pairs for methylated promoter sequences as previously described [40]. For the detection of APC and SHOX2 DNA methylation we designed in silico primers for using Primer Premier 5.00 software (Premier Biosoft) avoiding the formation of stable hairpin structures, primer dimers, cross dimers, and false priming sites. Optimization of experimental conditions including annealing temperature, time, concentrations of the primer pair, and finally buffer, MgCl2, dNTPs, and BSA concentrations (data not shown) was performed in order to develop sensitive and specific real-time MSP assays for APC and SHOX2 promoter methylation.
One microliter of SB-converted DNA was added to 9 μL reaction mixture containing 0.05 U/μL − 1 GoTaq® Hot Start Polymerase (Promega, Maddison, WI, USA), 0.2 × of the supplied PCR buffer, 2 mM of MgCl2, 0.2 mM of each dNTP (Thermo Fisher Scientific), 0.3 μg/μLBSA, 0.3 μM of the forward and reverse primers, and 1 × LCGreen Plus Dye (Idaho Technology, Salt Lake City, Utah, USA). Finally, deionized water was added to a final volume of 10μL. Real-time MSP protocol began with one cycle at 95 °C for 2 min followed by 45 cycles of 95 °C for 10 s, 63 °C for 20 s, and 72 °C for 20 s. Immediately after amplification, a rapid cooling cycle to 40 °C for 30 s was introduced in order to prepare the melting curve acquisition step. Real-time fluorescence acquisition was set at the elongation step (72 °C). The following melting curve analysis included the steps of 55 °C for 10 s, 92 °C for 0 s with a ramp rate 0.11 °C/s (acquisition mode: continuous), 92 °C for 1 min, and 40 °C for 1 min.
Statistical analysis
Correlations between methylation status and clinico-pathological features of the patients were assessed by using the Chi-square test. Disease-free interval (DFI) and overall survival (OS) curves were calculated by using the Kaplan–Meier method and comparisons were performed using the log-rank test. P-values < 0.05 were considered as statistically significant. Statistical analysis was performed by using the SPSS Windows version 17.0 (SPSS Inc., Chicago, IL, USA).