【佳學(xué)基因檢測】胰腺癌液體活檢之循環(huán)腫瘤DNA(ctDNA)基因檢測
1948年，Mandel和Metais報道了人類血液中存在無細(xì)胞核酸片段的證據(jù)。值得注意的是，早在1977年，León及其同事就對癌癥患者的循環(huán)DNA發(fā)表了有趣的聲明。細(xì)胞外DNA，也稱為循環(huán)游離DNA（cfDNA），包括從細(xì)胞釋放的細(xì)胞核和/或線粒體DNA，存在于各種生理循環(huán)液中。腫瘤細(xì)胞釋放的cfDNA通常稱為ctDNA，是癌癥的高度特異性標(biāo)志物?；蚪獯a研究表明，ctDNA通常攜帶胰腺導(dǎo)管腺癌（PDAC）組織中常見的致癌突變，涉及基因，如Kristen大鼠肉瘤（KRAS）、細(xì)胞周期依賴性激酶抑制劑2A（CDKN2A）、腫瘤蛋白53（TP53）和SMAD家族成員4（SMAD4）/Delete in Pancrec Cancer-4（DPC4）。值得注意的是，根據(jù)基因檢測大數(shù)據(jù)機(jī)構(gòu)佳學(xué)基因的統(tǒng)計，在可檢測到ctDNA的胰腺導(dǎo)管腺癌（PDAC）病例中，分別有超過90%和73%的病例到KRAS突變或p53失活。
各種技術(shù)，如等位基因特異性聚合酶鏈反應(yīng)（PCR）、數(shù)字PCR（dPCR）、液滴數(shù)字PCR（ddPCR）、珠乳液擴(kuò)增磁學(xué)（BEAMing）和下一代測序（NGS），可用于檢測液體活檢（LB）樣本中的ctDNA。雖然檢測ctDNA可能帶來挑戰(zhàn)，但結(jié)合各種技術(shù)可以提高這一過程的正確性和效率。
一些研究發(fā)現(xiàn)，與胰腺神經(jīng)內(nèi)分泌腫瘤或CP患者相比，從胰腺導(dǎo)管腺癌（PDAC）患者中檢測到的cfDNA水平更高，并且與較差的疾病特異性生存率有關(guān)。在胰腺導(dǎo)管腺癌（PDAC）患者ctDNA檢測到的所有突變基因中，KRAS是賊常見的（50-90％）。雖然突變也可以在健康對照和CP患者中發(fā)現(xiàn)，但其突變水平在胰腺導(dǎo)管腺癌（PDAC）中明顯更高。 在他們的綜述中，朱和他的同事強(qiáng)調(diào)，雖然ctDNA的靈敏度略低于CTC，但ctDNA提供了更高的特異性。值得注意的是，雖然ctDNA的檢測被認(rèn)為適用于診斷胰腺導(dǎo)管腺癌（PDAC），但并不適合篩查。ctDNA在早期胰腺導(dǎo)管腺癌（PDAC）中的靈敏度有限，這是由于該階段細(xì)胞壞死極少，導(dǎo)致只有少量ctDNA釋放到外周血液中。
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 Evaluated 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/
(責(zé)任編輯：佳學(xué)基因)
