Department of Medicine, Memorial Sloan Kettering Cancer Center, New York.
Department of Investigational Cancer Therapeutics, MD Anderson Cancer Center, Houston.
Ann Oncol. 2019 Apr 1;30(4):597-603. doi: 10.1093/annonc/mdz046.
Noninvasive genotyping using plasma cell-free DNA (cfDNA) has the potential to obviate the need for some invasive biopsies in cancer patients while also elucidating disease heterogeneity. We sought to develop an ultra-deep plasma next-generation sequencing (NGS) assay for patients with non-small-cell lung cancers (NSCLC) that could detect targetable oncogenic drivers and resistance mutations in patients where tissue biopsy failed to identify an actionable alteration.
Plasma was prospectively collected from patients with advanced, progressive NSCLC. We carried out ultra-deep NGS using cfDNA extracted from plasma and matched white blood cells using a hybrid capture panel covering 37 lung cancer-related genes sequenced to 50 000× raw target coverage filtering somatic mutations attributable to clonal hematopoiesis. Clinical sensitivity and specificity for plasma detection of known oncogenic drivers were calculated and compared with tissue genotyping results. Orthogonal ddPCR validation was carried out in a subset of cases.
In 127 assessable patients, plasma NGS detected driver mutations with variant allele fractions ranging from 0.14% to 52%. Plasma ddPCR for EGFR or KRAS mutations revealed findings nearly identical to those of plasma NGS in 21 of 22 patients, with high concordance of variant allele fraction (r = 0.98). Blinded to tissue genotype, plasma NGS sensitivity for de novo plasma detection of known oncogenic drivers was 75% (68/91). Specificity of plasma NGS in those who were driver-negative by tissue NGS was 100% (19/19). In 17 patients with tumor tissue deemed insufficient for genotyping, plasma NGS identified four KRAS mutations. In 23 EGFR mutant cases with acquired resistance to targeted therapy, plasma NGS detected potential resistance mechanisms, including EGFR T790M and C797S mutations and ERBB2 amplification.
Ultra-deep plasma NGS with clonal hematopoiesis filtering resulted in de novo detection of targetable oncogenic drivers and resistance mechanisms in patients with NSCLC, including when tissue biopsy was inadequate for genotyping.
利用血浆无细胞游离 DNA（cfDNA）进行非侵入性基因分型有可能避免癌症患者进行某些侵入性活检，同时阐明疾病异质性。我们试图为非小细胞肺癌（NSCLC）患者开发一种超深度血浆下一代测序（NGS）检测方法，该方法能够在组织活检未能确定可操作改变的情况下，检测到靶向致癌驱动基因和耐药突变。
前瞻性收集晚期进展性 NSCLC 患者的血浆。我们使用从血浆中提取的 cfDNA 以及与匹配的白细胞进行超深度 NGS，使用杂交捕获面板覆盖 37 个与肺癌相关的基因，测序至 50000×原始靶覆盖过滤归因于克隆性造血的体细胞突变。计算血浆检测已知致癌驱动基因的临床敏感性和特异性，并与组织基因分型结果进行比较。在部分病例中进行了正交 ddPCR 验证。
在 127 例可评估患者中，血浆 NGS 检测到的驱动突变的变异等位基因分数范围为 0.14%至 52%。在 22 例患者中的 21 例中，EGFR 或 KRAS 突变的血浆 ddPCR 揭示了与血浆 NGS 几乎相同的发现，变异等位基因分数的一致性很高（r=0.98）。在对组织基因型进行盲法检测的情况下，血浆 NGS 对新发现的已知致癌驱动基因的血浆检测的敏感性为 75%（68/91）。在组织 NGS 为阴性的患者中，血浆 NGS 的特异性为 100%（19/19）。在 17 例肿瘤组织认为不足以进行基因分型的患者中，血浆 NGS 鉴定出 4 个 KRAS 突变。在 23 例接受针对 EGFR 突变的靶向治疗后获得耐药的患者中，血浆 NGS 检测到潜在的耐药机制，包括 EGFR T790M 和 C797S 突变以及 ERBB2 扩增。
经过克隆性造血过滤的超深度血浆 NGS 导致 NSCLC 患者新发现的靶向致癌驱动基因和耐药机制，包括组织活检不足以进行基因分型的情况。
