Background and aims

Single-cell next-generation sequencing (scNGS) technology has been widely used in genomic profiling, which relies on whole-genome amplification (WGA). However, WGA introduces errors and is especially less accurate when applied to single nucleotide variant (SNV) analysis. Targeted scNGS for SNV without WGA has not been described. We aimed to develop a method to detect circulating tumor cells (CTCs) with DNA SNVs.

Methods

We tested this targeted scNGS method with three driver mutant genes (KRAS/TP53/SMAD4) on one pancreatic cancer cell line AsPC-1 and then applied it to patients with metastatic PDAC for the validation.

Results

All single-cell of AsPC-1 and spiked-in AsPC-1 cells in healthy donor blood, which were isolated by the filtration with size or by flow cytometry, were detected by targeted scNGS method. All blood samples from six patients with metastatic PDAC, for the validation of target scNGS method, showed CTCs with SNVs of KRAS/TP53/SMAD4 and the positive confirmation of immunofluorescent stainings with Pan-CK/Vimentin/CD45. Four patients with early stage disease, one patient with benign pancreatic cyst and a healthy control sample all showed concordant results between targeted scNGS and CTC enumeration.

Conclusions

The novel technique of targeted scNGS for SNV analysis, without pre-amplification, is a promising method for identifying and characterizing circulating tumor cells.

中文翻译：

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通过靶向单细胞下一代测序检测胰腺循环肿瘤细胞。

背景和目标

单细胞下一代测序（scNGS）技术已广泛应用于基因组分析中，该技术依赖于全基因组扩增（WGA）。但是，WGA会引入错误，并且在应用于单核苷酸变异（SNV）分析时尤其不准确。没有WGA的SNV的目标scNGS尚未描述。我们旨在开发一种检测带有DNA SNV的循环肿瘤细胞（CTC）的方法。

方法

我们在一个胰腺癌细胞系AsPC-1上用三个驱动突变基因（KRAS / TP53 / SMAD4）测试了这种靶向scNGS方法，然后将其应用于转移性PDAC患者进行验证。

结果

通过有针对性的scNGS方法检测健康供体血液中所有的AsPC-1单细胞和掺入的AsPC-1细胞，通过大小过滤或流式细胞术分离。为了验证靶标scNGS方法，从6例转移性PDAC患者的所有血液样本中，检测到的CTC具有SNV的KRAS / TP53 / SMAD4的SNV，并被Pan-CK / Vimentin / CD45的免疫荧光染色阳性。4名早期疾病患者，1名胰腺良性囊肿患者和健康对照样本均显示了靶向scNGS和CTC枚举之间的一致结果。

结论

无需预先扩增，用于SNV分析的靶向scNGS的新技术是一种鉴定和表征循环肿瘤细胞的有前途的方法。