Cobas cfdna sample preparation kit

Venous blood samples were collected from patients using 21G needles into one 10-mL Cell-Free DNA BCT tube (Streck) per patient. For the 1-step centrifugation group, within 4 hours after room-temperature blood collection, blood samples were centrifuged at 1,600g for 10 minutes, dispensed at 2 mL each into Eppendorf tubes, and stored at  ‒70°C until analysis Figure 1 . For the 2-step centrifugation group, a second centrifugation step was performed at 13,200g for 10 minutes in a benchtop microcentrifuge just before analysis Figure 1 . Second centrifugation was performed at 1 week to 1.5 years after first centrifugation. All plasma specimens were analyzed after only 1 freeze-thaw cycle. The plasma cfDNA was extracted from a 2-mL starting volume and eluted in 100 μL of elution buffer provided with the cobas cfDNA sample preparation kit (Roche Diagnostics) according to the manufacturer’s instructions.

Plasma or PE-SUP cfDNA was isolated using the cobas cfDNA sample preparation kit (Roche Molecular Systems), according to the manufacturer's instructions. For all clinical plasma samples, plasma cfDNA was extracted from a starting volume of 2 mL and eluted in 100 µL of elution buffer. Two equal volumes of eluted cfDNA samples were homogenized (200 µL of cfDNA was extracted from 4 mL of each plasma sample) to ensure evenly matched comparative conditions between the two kits. For PE-SUP cfDNA extraction, 0.5 mL (25% of the plasma sample volume) of PE-SUP was used as the starting volume (the volume was determined by preliminary assessment). Except for the starting volume of PE-SUP, all other extraction processes were conducted in the same manner for all samples.
Each cfDNA of 47 SDC samples was prepared as per the clinical sample extraction (100/110 µL of cfDNA extracted from a 2.0/2.2 mL starting volume), duplication (final volume of 200/220 µL from a 4.0/4.4 mL starting volume), and homogenizing processes.

At the time of histologic diagnosis, mutational analysis of EGFR gene exons 18 to 21 was performed according to standard clinical practice.
At disease progression, LB was performed to assess the EGFR T790M mutational status in ctDNA. In T790M negative patients at LB, a tissue biopsy was carried out, when feasible. If test was still T790M negative, new plasma samples were collected.
In case of LB, cell free-DNA was isolated from 2 mL of plasma using the cobas^® cf-DNA Sample Preparation kit (Roche, Basel, Switzerland) or the Helix^® circulating Nucleic Acid assay (Diatech Pharmacogenetics, Jesi, Italy), and the EGFR mutational analysis was performed by polymerase chain reaction (PCR)-based methods (cobas^® EGFR Mutation Test v2, Roche, Basel, Switzerland, or Easy EGFR kit, Diatech Pharmacogenetics, Jesi, Italy).
In case of tissue biopsy, DNA was extracted from formalin-fixed paraffin-embedded tissue sections using the QIAamp DNA Mini kit (Qiagen, Hilden, Germany) and analyzed by mass spectrometry-based methods (Sequenom MassARRAY, Diatech Pharmacogenetics, Jesi, Italy).
Results of liquid and tissue biopsies were classified as: positive (when EGFR T790M mutation was present); negative (when neither T790M nor activating mutations were found); other (when only EGFR activating mutations but not T790M were reported).

Archived formalin-fixed, paraffin-embedded (FFPE) tissue specimens obtained at the time of diagnosis were collected. Blood samples (14 ml) were collected into tubes containing EDTA just before the first and second cycle and at the end of the protocol treatment; the samples were then centrifuged at 1200 × g for 10 min within 1 h after collection. Tumor tissue DNA in FFPE was isolated using the Allprep DNA/RNA FFPE Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Plasma ctDNA was isolated using the cobas® cfDNA Sample Preparation Kit (Roche Diagnostics Ltd., Penzberg, Germany) according to the manufacturer’s instructions. The quality and quantity of the nucleic acid were verified using PicoGreen dsDNA Reagent (all from Thermo Scientific, Wilmington, DE).