Institute of Precision Medicine, The First Affiliated Hospital, Sun Yat-Sen University, No. 58, Zhongshan Second Road, Guangzhou, 510080, China.
Department of Endocrinology, The First Affiliated Hospital, Sun Yat-Sen University, No. 58, Zhongshan Second Road, Guangzhou, 510080, China.
BMC Cancer. 2023 Apr 20;23(1):363. doi: 10.1186/s12885-023-10852-z.
Thyroid cancer is the most frequent malignancy of the endocrine system, of which papillary thyroid cancer (PTC) is the predominant form with a rapid increasing incidence worldwide. Rearranged during transfection (RET) fusions are common genetic drivers of PTC and the potent RET inhibitor selpercatinib has been recently approved for treating advanced or metastatic RET fusion-positive thyroid cancer. In this study we aimed to develop a droplet digital PCR (ddPCR) system to accurately detect RET fusion in PTC samples.
The frequency and distribution of RET fusions in PTC were analyzed using genomic data of 402 PTC patients in The Cancer Genome Atlas (TCGA) database. To establish the ddPCR system for detecting CCDC6::RET fusion, a plasmid containing CCDC6::RET infusion fragment was constructed as standard template, the annealing temperature and concentrations of primers and probe were optimized. The analytical performance of ddPCR and quantitative reverse transcription PCR (qRT-PCR) were assessed in standard templates and tissue samples from 112 PTC patients. Sanger sequencing was performed in all the RET fusion-positive samples identified by ddPCR.
RET fusions were observed in 25 (6.2%) of the 402 TCGA samples, and 15 (60%) of the RET fusion-positive patients had the CCDC6::RET fusion. Compared with qRT-PCR, the ddPCR method showed a lower limit of detection (128.0 and 430.7 copies/reaction for ddPCR and qRT-PCR, respectively). When applying the two methods to 112 tissue samples of PTC, eleven (9.8%) CCDC6::RET fusion-positive samples were detected by qRT-PCR, while ddPCR identified 4 additional positive samples (15/112, 13.4%). All the CCDC6::RET fusion-positive cases identified by ddPCR were confirmed by Sanger sequencing except for one case with 0.14 copies/uL of the fusion.
The accurate and sensitive ddPCR method reported here is powerful to detection CCDC6::RET fusion in PTC samples, application of this method would benefit more RET fusion-positive patients in the clinic.
甲状腺癌是内分泌系统最常见的恶性肿瘤，其中甲状腺乳头状癌（PTC）是最主要的形式，其发病率在全球范围内呈快速上升趋势。重排基因在转染期间（RET）融合是 PTC 的常见遗传驱动因素，最近已批准强力 RET 抑制剂塞尔帕替尼用于治疗晚期或转移性 RET 融合阳性甲状腺癌。本研究旨在开发一种液滴数字 PCR（ddPCR）系统，以准确检测 PTC 样本中的 RET 融合。
使用 TCGA 数据库中 402 名 PTC 患者的基因组数据分析 PTC 中 RET 融合的频率和分布。为了建立用于检测 CCDC6::RET 融合的 ddPCR 系统，构建了一个包含 CCDC6::RET 输注片段的质粒作为标准模板，优化了引物和探针的退火温度和浓度。在 112 名 PTC 患者的标准模板和组织样本中评估了 ddPCR 和定量逆转录 PCR（qRT-PCR）的分析性能。在所有通过 ddPCR 鉴定为 RET 融合阳性的样本中进行了 Sanger 测序。
在 402 个 TCGA 样本中观察到 25 个（6.2%）RET 融合，在 RET 融合阳性患者中，15 个（60%）有 CCDC6::RET 融合。与 qRT-PCR 相比，ddPCR 方法的检测下限更低（ddPCR 和 qRT-PCR 分别为 128.0 和 430.7 拷贝/反应）。当将两种方法应用于 112 个 PTC 组织样本时，qRT-PCR 检测到 11 个（9.8%）CCDC6::RET 融合阳性样本，而 ddPCR 则鉴定出 4 个额外的阳性样本（15/112，13.4%）。除了一个融合物为 0.14 拷贝/μL 的病例外，ddPCR 鉴定的所有 CCDC6::RET 融合阳性病例均通过 Sanger 测序得到证实。
本研究报道的准确灵敏的 ddPCR 方法可有效检测 PTC 样本中的 CCDC6::RET 融合，该方法的应用将使更多的 RET 融合阳性患者受益于临床治疗。
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