Avenio ctdna surveillance kit

Purified plasma, 3–5 ml (n = 62), was thawed prior to enrichment of cell-free DNA by the AVENIO ctDNA Analysis Kit (Roche) according to the protocol provided by the producers (Supplementary material). Sequencing libraries were prepared from 13–50 ng cell-free DNA using the AVENIO ctDNA Analysis Kit paired with the AVENIO ctDNA Surveillance Kit (Roche) as described by the manufacturers. The libraries for both ctDNA and tumour were sequenced on a NextSeq550 (Illumina) using the NextSeq 500/550 High Output v2 kit (300 cycles) (Illumina) and results were analysed using AVENIO ctDNA Analysis Software version 2.0.0 (Roche) as recommended by the suppliers, with hg38 as reference genome (Supplementary material). The detection threshold was 0.1% for single nucleotide variants.

Peripheral blood samples were collected from patients into K2 EDTA vacutainers (Becton Dickinson, Franklin Lakes, NJ, USA) before administering the combination therapy. Plasma was isolated immediately after drawing blood and stored at − 80 °C until use. Subsequently, cfDNA was extracted from the plasma samples and used for next-generation sequencing (NGS) library construction with the AVENIO ctDNA Surveillance Kit (Roche Diagnostics, Basel, Switzerland) to assess genetic alterations in 197 cancer-related genes. Sequencing was performed using the NextSeq 500 System (Illumina, San Diego, CA, USA), followed by mutation analyses using the Avenio Oncology Analysis Server (Roche Diagnostics).

CAPP-seq is based on hybridization-capture NGS [2 (link)]. The sequencing library was prepared using the AVENIO ctDNA surveillance kit (Roche Sequencing Solutions, Mannheim, Germany), covering 198 kb in 197 hypermutated genes [38 (link)]. The gene fragments were sequenced using a NextSeq 500 (Illumina, San Diego, CA, USA), and the data were analyzed using the AVENIO Oncology Analysis Software (v. 2.0.0). Deduplicated BAM files based on unique molecular identifiers (UMIs) were used for a cfChIP enrichment analysis, whereas position-deduplicated BAM files were used to estimate fragment lengths and FEMs.

Potential biomarkers in the plasma proteins were evaluated at baseline, 6 weeks, and 12 weeks after the initiation of combination therapy and when disease progressed. Fresh blood samples (14 mL) were collected in EDTA tubes and centrifuged for 10 min at 1400× g at room temperature to separate plasma and buffy coats. All samples were stored at −80 °C until the analysis. CtDNA was subsequently extracted from the plasma using an AVENIO cell-free DNA isolation kit (Roche Diagnostics, Mannheim, Germany). Sample plasma analysis was carried out for the following panel of circulating angiogenesis markers and cytokines using Bio-Plex 200 (Bio–Rad, Hercules, CA, USA) and the Human Angiogenesis/Growth Factor Magnetic Bead Panel (Merck Millipore, Burlington, VT, USA): angiopoietin-2, bone morphogenetic protein-9, EGF, endoglin, fibroblast growth factor-2, placental growth factor (PLGF), granulocyte colony-stimulating factor, heparin-binding EGF-like growth factor, endothelin-1, hepatocyte growth factor, follistatin, IL-8, leptin, VEGF-A, VEGF-C, and VEGF-D. The ctDNA samples were analyzed with NextSeq 500 (Illumina, San Diego, CA, USA) and the AVENIO ctDNA Surveillance Kit, which can detect mutations in 197 genes (Roche Diagnostics).