ResearchDx, Inc., 5 Mason, Irvine, CA, USA.
PacificDx Clinical Laboratory, 5 Mason, Irvine, CA, USA.
BMC Cancer. 2020 Oct 1;20(1):945. doi: 10.1186/s12885-020-07445-5.
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.
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).
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.
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.
临床实验室进行的循环肿瘤（ct）DNA 检测为生物制药临床试验提供了肿瘤生物标志物检测支持。然而，对于许多临床实验室来说，内部开发自己的液体活检检测既不切实际，也不经济可行。商业上可用的 ctDNA 试剂盒是寻求将液体活检纳入其测试菜单的实验室的潜在解决方案。然而，患者样本的稀缺性和购买验证参考标准的成本给进入市场带来了障碍。在本研究中，我们评估了 AVENIO ctDNA 液体活检平台（罗氏测序解决方案）在我们临床实验室中的分析性能。
根据美国病理学家学院（CAP）关于使用购买的参考标准、去识别的人类无细胞（cf）血浆游离 DNA 样本和商业购买的正常和癌症人类血浆衍生的合成样本验证靶向下一代测序检测的验证指南，对 AVENIO ctDNA 靶向、扩展和监测试剂盒（仅供研究使用）进行了实验室内部性能评估。所有样本均使用 NextSeq High Output 试剂盒（Illumina）在与临床设置相关的读取深度下进行测序。
在临床相关的读取深度下，Avenio ctDNA 试剂盒在检测≥0.5%等位基因频率（AF）的单核苷酸变异（SNV）时，100%的灵敏度为检测 0.1% AF 的 SNV 的灵敏度为 50%，使用 20-40ng 样本输入量。该检测无缝集成到我们实验室的 NGS 工作流程中，输入 DNA 质量、目标等位基因频率（TAF）、多重性和读取次数进行了优化，以支持适用于生物制药试验的高通量检测。
我们的研究表明，AVENIO ctDNA 液体活检平台为将液体活检检测高效地纳入临床实验室，以检测低至 0.5%的体细胞变异提供了可行的替代方案。在临床需要和经济可行的情况下，通过更深的测序可以更准确地检测低于 0.5%的变异。
