【佳学基因检测】胰腺癌液体活检之循环肿瘤DNA(ctDNA)基因检测-佳学基因官网：基因检测 | 基因解码 | 遗传病 | 肿瘤靶向药物 | 阻断 | 北京

1948年，Mandel和Metais报道了人类血液中存在无细胞核酸片段的证据。值得注意的是，早在1977年，León及其同事就对癌症患者的循环DNA发表了有趣的声明。细胞外DNA，也称为循环游离DNA（cfDNA），包括从细胞释放的细胞核和/或线粒体DNA，存在于各种生理循环液中。肿瘤细胞释放的cfDNA通常称为ctDNA，是癌症的高度特异性标志物。基因解码研究表明，ctDNA通常携带胰腺导管腺癌（PDAC）组织中常见的致癌突变，涉及基因，如Kristen大鼠肉瘤（KRAS）、细胞周期依赖性激酶抑制剂2A（CDKN2A）、肿瘤蛋白53（TP53）和SMAD家族成员4（SMAD4）/Delete in Pancrec Cancer-4（DPC4）。值得注意的是，根据基因检测大数据机构佳学基因的统计，在可检测到ctDNA的胰腺导管腺癌（PDAC）病例中，分别有超过90%和73%的病例到KRAS突变或p53失活。

各种技术，如等位基因特异性聚合酶链反应（PCR）、数字PCR（dPCR）、液滴数字PCR（ddPCR）、珠乳液扩增磁学（BEAMing）和下一代测序（NGS），可用于检测液体活检（LB）样本中的ctDNA。虽然检测ctDNA可能带来挑战，但结合各种技术可以提高这一过程的准确性和效率。

一些研究发现，与胰腺神经内分泌肿瘤或CP患者相比，从胰腺导管腺癌（PDAC）患者中检测到的cfDNA水平更高，并且与较差的疾病特异性生存率有关。在胰腺导管腺癌（PDAC）患者ctDNA检测到的所有突变基因中，KRAS是最常见的（50-90％）。虽然突变也可以在健康对照和CP患者中发现，但其突变水平在胰腺导管腺癌（PDAC）中明显更高。在他们的综述中，朱和他的同事强调，虽然ctDNA的灵敏度略低于CTC，但ctDNA提供了更高的特异性。值得注意的是，虽然ctDNA的检测被认为适用于诊断胰腺导管腺癌（PDAC），但并不适合筛查。ctDNA在早期胰腺导管腺癌（PDAC）中的灵敏度有限，这是由于该阶段细胞坏死极少，导致只有少量ctDNA释放到外周血液中。

Table 1：Comparison between biomarkers of pancreatic ductal adenocarcinoma ctDNA

CTCs	EVs
Target	KRAS, TP53, CDKN2A, SMAD4, BRAF, PIK3CA, ADAMTS1, BNC1, 5MC, H2AZ, H2A1.1, H3K4me2, h2ak119ub	CD45, CEP8, CK, EpCAM	KRAS, TP53, RNA: miRNA, longRNA Proteins markers: EFGR, EPCAM, MUC-1, GPC-1, WNT2
Isolation	Blood	Blood	Body fluids
Tumor information	Epigenetic information	DNA, RNA, Protein	DNA, RNA, Protein
Technological approaches	qPCR, dPCR, ddPCR, NGS, commercial kits	Immunoaffinity, Physical methods (size and density)	Density-based, size-based, affinity-based, commercial kits
Advantages	qPCR: Fast and low-cost dPCR: High sensitivity/Specificity NGS: capability to screen for a broad range of genetic variants using high DNA input	Immunoaffinity: Specific, label-free obtained Physical methods: Fast, simple, Low-cost, label-free obtained	Density-based: low cost. Independent of marker expression. Size-based: Low-cost, fast, Independent of marker expression. Affinity-based: Specificity. High purity. Commercial kits: Simple, fast.
Disadvantages	General: No early stages qPCR: Low sensitivity. Only points mutations. dPCR: High cost. Only points mutations. NGS: Variable sensitivity. High cost.	General: Isolation complex and expensive. Technical variability Immunoaffinity: capture only one subpopulation. Low purity. Physical methods: Needs immuno-labeling techniques to distinguish CTCs	General: Isolation complex by contamination and expensive Density-based: Time, high volume sample, can damage EVs. Size based: contamination. Affinity-based: low sample yield. Commercial kits: High cost.
Sensitivity (S) (%)	34–71% KRAS mutations: codons 12, 13, 61, in different stages.	73–76% CD45/CEP8 100% Mt, 58% resectable Anti-EpCAM portal vein Blood	67% ES, 80% LA, 85% Mt KRAS mutations in exoDNA 50% ES GPC1 miRNAs Increased expression
Specificity (Sp) (%)	75–81% Mutations KRAS exon 2	68% CD45/CEP8	90% ES GPC1
Combined techniques (%)	S: 85–98%, Sp: 77–81% ctDNA (KRAS exon 2) with CA19.9 S: 47% ctDNA (KRAS MAFs) with CA19.9	S: 100%, Sp: 80% CTCs.with EVs	NR
Application	No suitable for screening of PDAC Monitoring postoperative minimal residual disease Predictor of disease recurrence and prognosis	Not present in healthy controls Variable sensitivity in early diagnosis Excellent specificity. Follow-up of disease recurrence and prognosis Functional analysis drug resistance	The highest sensitivity and specificity in early detection Evaluated response of resection or any therapy Biotherapeutic application

BEAMing: beads-emulsion-amplification-magnetics; CEP8: chromosome 8 centromere; ctDNA: circulating tumor DNA; CTC: circulating tumor cells; dPCR: digital polymerase chain reaction; ddPCR: droplet digital PCR; EpCAM: epithelial cell adhesion molecule; ES: early stages; EVs: extracellular vesicles; GPC1: glypican-1; LA: locally advanced; miRNA: micro-RNA; Mt: metastatic; NGS: next-generation sequencing; NR: not reported.

支持本文的科学文献

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10855073/