Assessing circulating tumor DNA (ctDNA) is a promising method to evaluate somatic mutations from solid tumors in a minimally-invasive way. In a group of twelve metastatic colorectal cancer (mCRC) patients undergoing liver metastasectomy, from each patient DNA from cell-free DNA (cfDNA), the primary tumor, metastatic liver tissue, normal tumor-adjacent colon or liver tissue, and whole blood were obtained. Investigated was the feasibility of a targeted NGS approach to identify somatic mutations in ctDNA. This targeted NGS approach was also compared with NGS preceded by mutant allele enrichment using synchronous coefficient of drag alteration technology embodied in the OnTarget assay, and for selected mutations with digital PCR (dPCR). All tissue and cfDNA samples underwent IonPGM sequencing for a CRC-specific 21-gene panel, which was analyzed using a standard and a modified calling pipeline. In addition, cfDNA, whole blood and normal tissue DNA were analyzed with the OnTarget assay and with dPCR for specific mutations in cfDNA as detected in the corresponding primary and/or metastatic tumor tissue. NGS with modified calling was superior to standard calling and detected ctDNA in the cfDNA of 10 patients harboring mutations in APC, ATM, CREBBP, FBXW7, KRAS, KMT2D, PIK3CA and TP53. Using this approach, variant allele frequencies in plasma ranged predominantly from 1 to 10%, resulting in limited concordance between ctDNA and the primary tumor (39%) and the metastases (55%). Concordance between ctDNA and tissue markedly improved when ctDNA was evaluated for KRAS, PIK3CA and TP53 mutations by the OnTarget assay (80%) and digital PCR (93%). Additionally, using these techniques mutations were observed in tumor-adjacent tissue with normal morphology in the majority of patients, which were not observed in whole blood. In conclusion, in these mCRC patients with oligometastatic disease NGS on cfDNA was feasible, but had limited sensitivity to detect all somatic mutations present in tissue. Digital PCR and mutant allele enrichment before NGS appeared to be more sensitive to detect somatic mutations.

评估循环肿瘤 DNA (ctDNA) 是一种以微创方式评估实体瘤体细胞突变的有前途的方法。在一组接受肝转移切除术的 12 名转移性结直肠癌 (mCRC) 患者中，从每位患者的游离 DNA (cfDNA) 中提取 DNA、原发肿瘤、转移性肝组织、正常肿瘤相邻结肠或肝组织以及全血获得。研究了靶向 NGS 方法鉴定 ctDNA 体细胞突变的可行性。这种靶向 NGS 方法还与使用 OnTarget 测定中体现的同步阻力改变技术系数进行突变等位基因富集之前的 NGS 进行比较，并使用数字 PCR (dPCR) 进行选定的突变。所有组织和 cfDNA 样本均经过 IonPGM 测序，用于 CRC 特异性 21 基因组，并使用标准和修改的调用流程进行分析。此外，还使用 ​​OnTarget 检测和 dPCR 分析了 cfDNA、全血和正常组织 DNA，以检测在相应的原发性和/或转移性肿瘤组织中检测到的 cfDNA 特定突变。改良检出的 NGS 优于标准检出，并在 10 名携带APC、ATM、CREBBP、FBXW7、KRAS、KMT2D、PIK3CA和TP53突变的患者的 cfDNA 中检测到了 ctDNA 。使用这种方法，血浆中的变异等位基因频率主要在 1% 至 10% 之间，导致 ctDNA 与原发肿瘤 (39%) 和转移瘤 (55%) 之间的一致性有限。当通过 OnTarget 检测 (80%) 和数字 PCR (93%)评估 ctDNA 的KRAS、PIK3CA和TP53突变时，ctDNA 与组织之间的一致性显着提高。此外，使用这些技术在大多数患者形态正常的肿瘤邻近组织中观察到突变，而在全血中未观察到突变。总之，在这些患有寡转移性疾病的 mCRC 患者中，cfDNA 的 NGS 是可行的，但检测组织中存在的所有体细胞突变的敏感性有限。NGS 之前的数字 PCR 和突变等位基因富集似乎对检测体细胞突变更敏感。