【广东会GDH基因检测】胰腺癌液体活检之循环肿瘤DNA(ctDNA)基因检测
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广东会GDH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正确性和效率。
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是贼常见的（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
| 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 evalsuated response of resection or any therapy Biotherapeutic application |
| CTCs | EVs |
|---|
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.
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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10855073/
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