CellMax Life U.S., 1271 Oakmead Parkway, Sunnyvale, CA, 94085, USA.
Department of Clinical Pathology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA, 94305, USA.
Mol Diagn Ther. 2019 Aug;23(4):521-535. doi: 10.1007/s40291-019-00406-0.
Comprehensive genetic cancer profiling using circulating tumor DNA has enabled the detection of National Comprehensive Cancer Network (NCCN) guideline-recommended somatic alterations from a single, non-invasive blood draw. However, reliably detecting somatic variants at low variant allele fractions (VAFs) remains a challenge for next-generation sequencing (NGS)-based tests. We have developed the single-molecule sequencing (SMSEQ) platform to address these challenges.
The OncoLBx assay utilizes the SMSEQ platform to optimize cell-free DNA extraction and library preparation with variant type-specific calling algorithms to improve sensitivity and specificity. OncoLBx is a pan-cancer panel for solid tumors targeting 75 genes and five microsatellite sites analyzing five classes of NCCN-recommended somatic variants: single-nucleotide variants (SNVs), insertions and deletions (indels), copy number variants (CNVs), fusions and microsatellite instability (MSI). Circulating DNA was extracted from plasma, followed by library preparation using SMSEQ. Analytical validation was performed according to recently published American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) guidelines and established the limit of detection (LOD), sensitivity, specificity, accuracy and reproducibility using 126 gold-standard reference samples, healthy donor samples verified by whole-exome sequencing by an external College of American Pathologists (CAP) reference lab and cell lines with known variants. Results were analyzed using a locus-specific modeling algorithm.
We have demonstrated that OncoLBx detects VAFs of ≥ 0.1% for SNVs and indels, ≥ 0.5% for fusions, ≥ 4.5 copies for CNVs and ≥ 2% for MSI, with all variant types having specificity ≥ 99.999%. Diagnostic performance of paired samples displays 80% sensitivity and > 99.999% clinical specificity. Clinical utility and performance were assessed in 416 solid tumor samples. Variants were detected in 79% of samples, for which 87.34% of positive samples had available targeted therapy.
使用循环肿瘤 DNA 进行全面的癌症基因分析，使得能够从单次非侵入性血液采集检测到国家综合癌症网络 (NCCN) 指南推荐的体细胞改变。然而，对于基于下一代测序 (NGS) 的检测，可靠地检测低变异等位基因分数 (VAF) 的体细胞变体仍然是一个挑战。我们已经开发了单分子测序 (SMSEQ) 平台来解决这些挑战。
OncoLBx 检测利用 SMSEQ 平台优化无细胞 DNA 提取和文库制备，采用具有变体类型特异性的调用算法来提高灵敏度和特异性。OncoLBx 是一种针对实体瘤的泛癌面板，针对 75 个基因和五个微卫星位点进行分析，分析 NCCN 推荐的五类体细胞变体：单核苷酸变体 (SNVs)、插入和缺失 (indels)、拷贝数变体 (CNVs)、融合和微卫星不稳定性 (MSI)。从血浆中提取循环 DNA，然后使用 SMSEQ 进行文库制备。根据最近发表的美国医学遗传学与基因组学学院 (ACMG)/分子病理学协会 (AMP) 指南进行分析验证，并使用 126 个金标准参考样本、经外部美国病理学家学院 (CAP) 参考实验室全外显子测序验证的健康供体样本和具有已知变体的细胞系来建立检测限 (LOD)、灵敏度、特异性、准确性和重复性。结果使用基于基因座的建模算法进行分析。
我们已经证明，OncoLBx 可以检测到 SNVs 和 indels 的 VAF≥0.1%、融合的 VAF≥0.5%、CNVs 的 VAF≥4.5 拷贝和 MSI 的 VAF≥2%，所有变体类型的特异性≥99.999%。配对样本的诊断性能显示 80%的灵敏度和>99.999%的临床特异性。在 416 个实体瘤样本中评估了临床实用性和性能。在 79%的样本中检测到了变体，其中 87.34%的阳性样本有可用的靶向治疗药物。
