Liquid biopsy analysis, mainly based on circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), provides an extremely powerful tool for the molecular profiling of cancer patients in real time. In this study, we directly compared PIK3CA hotspot mutations (E545K, H1047R) in EpCAM-positive CTCs and paired plasma-ctDNA in breast cancer (BrCa). PIK3CA hotspot mutations in CTCs and ctDNA were analyzed using our previously developed highly sensitive (0.05%), specific, and validated assay in plasma-ctDNA from 77 early and 73 metastatic BrCa patients and 40 healthy donors. We further analyzed and directly compared PIK3CA hotspot mutations in DNAs isolated from CellSearch® cartridges (CTCs) and paired plasma-ctDNA, in 56 cases of early and 27 cases of metastatic breast cancer, and 16 corresponding primary tumors. In plasma-ctDNA, PIK3CA hotspot mutations were identified in 30/77(39.0%) early and 35/73(47.9%) metastatic BrCa cases; none (0/40, 0%) of the healthy donors' plasma-ctDNA samples were positive. Our direct comparison study in DNAs isolated from CellSearch® cartridges (CTCs) and paired plasma-ctDNA from the same blood draws has shown a lack of concordance in early BrCa (27/56, 48.2%), while the concordance in the metastatic setting was higher (18/27, 66.6%). Our results were validated by ddPCR methodology, and the concordance between our assay and ddPCR for PIK3CA E545K hotspot mutation was 30/37 (81.1%). In many cases, PIK3CA hotspot mutations were detected in samples found to be negative for CTCs in CellSearch® . Our data demonstrated for the first time that (a) PIK3CA hotspot mutations are present at high frequencies in CTCs isolated from CellSearch® cartridges and paired plasma-ctDNA both in early and metastatic BrCa, (b) the detection and concordance of PIK3CA hotspot mutations between plasma-ctDNA and CTCs are higher in the metastatic setting, (c) PIK3CA mutational status significantly changes after therapeutic intervention, and (d) PIK3CA mutation detection in CTCs and plasma-ctDNA provides complementary information.

中文翻译：

---

乳腺癌中循环肿瘤细胞中PIK3CA热点突变和配对循环肿瘤DNA的直接比较研究。

液体活检分析主要基于循环肿瘤细胞（CTC）和循环肿瘤DNA（ctDNA），为癌症患者的实时分子谱分析提供了极为强大的工具。在这项研究中，我们直接比较了EpCAM阳性CTC中PIK3CA热点突变（E545K，H1047R）和乳腺癌（BrCa）中成对的血浆ctDNA。使用我们先前开发的高灵敏度（0.05％），特异性和经过验证的血浆ctDNA中的CTC和ctDNA中的PIK3CA热点突变，分析了77名早期和73名转移性BrCa患者和40名健康供者的血浆ctDNA。我们进一步分析和直接比较了从CellSearch®药筒（CTC）和成对的血浆ctDNA分离的DNA中PIK3CA热点突变在56例早期转移癌和27例转移性乳腺癌以及16种相应的原发性肿瘤中的发生。在血浆ctDNA中 在30/77（39.0％）早期和35/73（47.9％）转移性BrCa病例中发现了PIK3CA热点突变；健康捐献者的血浆ctDNA样本均无阳性（0 / 40，0％）。我们对从CellSearch®药筒（CTC）分离的DNA和来自同一血样的成对血浆ctDNA的直接比较研究表明，早期BrCa缺乏一致性（27/56，48.2％），而转移环境的一致性为更高（18/27，66.6％）。通过ddPCR方法验证了我们的结果，我们的检测与ddPCR之间PIK3CA E545K热点突变的一致性为30/37（81.1％）。在许多情况下，在CellSearch®中发现CTC阴性的样品中检测到PIK3CA热点突变。