Oncomine lung cfdna assay

Specimen preparation, cell-free DNA extraction, and next-generation sequencing library preparation, together with variant detection parameters, are detailed in Appendix A. Mutation detection was performed with Oncomine Lung cfDNA Assay using the Ion Torrent™ Ion S5™ system (Thermo Fisher Scientific (TFS), Waltham, MA, USA), while Ion Reporter Software v.5.12 (Ion Reporter Software, TFS) and the Integrative Genomics Viewer v.2.8.13 (Broad Institute, Cambridge, MA, USA) were used for mutation analysis.

Serum carcinoembryonic antigen (CEA) level was measured at baseline. For ctDNA analysis, blood samples were collected 1–2 weeks before initiation of preoperative therapy (baseline) and after treatment (just before surgery) for some patients. Blood samples were collected into BD Vacutainer® PPT™ plasma preparation tubes (2 × 5 mL) (BD Biosciences, Franklin Lakes, NJ, USA). Plasma was extracted by centrifugation at 1100 rcf for 10 min at room temperature and stored at −20 °C according to laboratory protocol. The samples were purified using PromegaInstrument Maxwell^® RSC (Promega Corporation, Madison, WI, USA). Libraries from cfDNA were prepared with Oncomine Lung cfDNA Assay (Thermo Fisher Scientific, Waltham, MA, USA), available in the laboratory for the most common mutations in rectal cancer. The cfDNA panel used in this study covered 11 genes (ALK, BRAF, EGFR, ERBB2, KRAS, MAP2K1, MET, NRAS, PIK3CA, ROS1 and TP53), with more than 150 hotspots (SNVs and short indels). We tracked the mutations with the highest mutation allele frequency (MAF) in each patient [63 (link)]. Patients with detectable mutations were categorized “ctDNA positive”, and the ones with undetectable mutations were characterized as “ctDNA negative”.

Targeted libraries were amplified using Oncomine™ Lung cfDNA Assay (ThermoFisher). The assay includes 35 amplicons covering 169 key hotspot mutations in 11 genes (ALK, BRAF, EGFR, ERBB2, KRAS, MAP2K1, MET, NRAS, PIK3CA, ROS1, and TP53) (Additional file 2: Table S2). Patients cfDNAs (range 6–52 ng per reaction) were employed to prepare manually targeted libraries following manufacturer’s instructions.
Briefly, each cfDNA molecule was assigned with unique molecular tag through a first PCR reaction in a Veriti thermal cycler (Applied Biosystems™, Foster City, CA) and subsequently, tagged library fragments were amplified in a second round of PCR to produce independent barcoded libraries. Libraries were purified using Agencourt™ AMPure™ XP beads (Beckman Coulter, Milano, Italy).
For library quantification, a qPCR (with Ion Library TaqMan Quantitation Kit, ThermoFisher) was performed and run on StepOne instrument (Applied Biosystems™).