Analytical performance evaluation of a commercial next generation sequencing liquid biopsy platform using plasma ctDNA, reference standards, and synthetic serial dilution samples derived from normal plasma

Abstract

Background Circulating tumor (ct) DNA assays performed in clinical laboratories provide tumor biomarker testing support for biopharmaceutical clinical trials. Yet it is neither practical nor economically feasible for many of these clinical laboratories to internally develop their own liquid biopsy assay. Commercially available ctDNA kits are a potential solution for laboratories seeking to incorporate liquid biopsy into their test menus. However, the scarcity of characterized patient samples and cost of purchasing validation reference standards creates a barrier to entry. In the current study, we evaluated the analytical performance of the AVENIO ctDNA liquid biopsy platform (Roche Sequencing Solutions) for use in our clinical laboratory. Method Intra-laboratory performance evaluation of AVENIO ctDNA Targeted, Expanded, and Surveillance kits (Research Use Only) was performed according to College of American Pathologists (CAP) guidelines for the validation of targeted next generation sequencing assays using purchased reference standards, de-identified human plasma cell-free (cf) DNA samples, and contrived samples derived from commercially purchased normal and cancer human plasma. All samples were sequenced at read depths relevant to clinical settings using the NextSeq High Output kit (Illumina). Results At the clinically relevant read depth, Avenio ctDNA kits demonstrated 100% sensitivity in detecting single nucleotide variants (SNVs) at ≥0.5% allele frequency (AF) and 50% sensitivity in detecting SNVs at 0.1% AF using 20–40 ng sample input amount. The assay integrated seamlessly into our laboratory’s NGS workflow with input DNA mass, target allele frequency (TAF), multiplexing, and number of reads optimized to support a high-throughput assay appropriate for biopharmaceutical trials. Conclusions Our study demonstrates that AVENIO ctDNA liquid biopsy platform provides a viable alternative for efficient incorporation of liquid biopsy assays into the clinical laboratory for detecting somatic alterations as low as 0.5%. Accurate detection of variants lower than 0.5% could potentially be achieved by deeper sequencing when clinically indicated and economically feasible.

Co-occurring MET Amplification Predicts Inferior Clinical Response to First-Line Erlotinib in Advanced Stage EGFR-Mutated NSCLC Patients

“…The isolated cfDNA was quantified using the Qubit dsDNA High Sensitivity Kit (Life Technologies, Carlsbad, CA). Libraries were prepared with the AVENIO ctDNA Surveillance kit (Roche Sequencing Solutions) 20 and a median input cfDNA of 46 ng (7-82 ng). Multiplexed libraries of 16 samples were sequenced on a NextSeq 500 High Output lane (Illumina, San Diego, CA).…”

TP53 mutation and MET amplification in circulating tumor DNA analysis predict disease progression in patients with advanced gastric cancer

“…Liquid biopsy techniques, such as ctDNA, can not only avoid these limitations, but have also been integrated into daily clinical practice (Ilie & Hofman, 2016). Multigene NGS-panel technology for assessing ctDNA can be used to study a variety of genes, while only a small amount of ctDNA is obtained from even smaller samples, eventually including circulating tumour cells and free serum DNA (Meyerson, Gabriel & Getz, 2010;Verma et al, 2020). Comparing with traditional technology (dPCR), NGS sequencing technology has high sensitivity and accuracy for detecting rare ctDNA mutations, thus detecting very rare mutations with AF <0.001% and representing the original DNA population (Kukita et al, 2015).…”

Next-Generation Sequencing with Liquid Biopsies from Treatment-Naïve Non-Small Cell Lung Carcinoma Patients

“…In this context, different options can be considered: (i) increasing the volume of the blood sample taken from the patient to obtain a higher quantity of nucleic acid after extraction. However, it does not seem possible to get more than 20 mL from patients with metastatic lung cancer (the average volume is 10 mL of blood in daily practice); (ii) optimize the pre-analytical steps by using an efficient buffer that limits degradation of leucocytes and thus the release of germinal DNA from these cells into the blood; (iii) reduce as much as possible the time between blood puncture and the centrifugation steps and (iv) use some new reagents that increase nucleic acid extraction from plasma and thus optimize the ratio between the available plasmatic volume and the amount of extracted nucleic acid [ 111 , 112 , 113 , 114 , 115 , 116 , 117 , 118 , 119 , 120 , 121 ].…”