用于肺癌无创筛查的多重数字甲基化特异性 PCR,Advanced Science

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用于肺癌无创筛查的多重数字甲基化特异性 PCR
Advanced Science ( IF 14.3 ) Pub Date : 2023-04-11 , DOI: 10.1002/advs.202206518
Yang Zhao ₁ , Christine M O'Keefe ₁ , Kuangwen Hsieh ₂ , Leslie Cope ₃ , Sonali C Joyce _{4,

5} , Thomas R Pisanic _{3,

6} , James G Herman _{4,

5} , Tza-Huei Wang _{1,

2,

6}

Affiliation

人们仍然对开发液体活检检测方法以检测通过无创抽血获得的无细胞 DNA (cfDNA) 中的癌症特异性改变（例如突变和 DNA 甲基化）有着极大的兴趣。然而，由于循环肿瘤 DNA (ctDNA) 的比例极低，液体活检分析通常具有挑战性，因此需要使用扩展的肿瘤生物标志物组。虽然多重 PCR 策略具有更高通量等优势，但它们的实施往往受到引物二聚体和 PCR 竞争等挑战的阻碍。或者，数字 PCR (dPCR) 方法通常提供卓越的性能，但具有受限的复用能力。本文介绍了第一个多重数字甲基化特异性 PCR (mdMSP) 平台的开发和验证，该平台用于同时分析四种甲基化生物标志物，用于基于液体活检的非小细胞肺癌 (NSCLC) 检测。mdMSP 采用微流体装置，包含四个独立但相同的模块，总共容纳 40 160 个纳米孔。mdMSP 平台的分析验证表明多重检测的分析特异性低至 0.0005%。mdMSP 的临床效用也在 72 个来自计算机断层扫描 (CT) 扫描不确定肺结节患者的低体积液体活检标本临床样本队列中得到证实，与传统的 MSP 检测无创检测相比，表现出更优越的临床性能早期非小细胞肺癌。

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Multiplex Digital Methylation-Specific PCR for Noninvasive Screening of Lung Cancer

There remains tremendous interest in developing liquid biopsy assays for detection of cancer-specific alterations, such as mutations and DNA methylation, in cell-free DNA (cfDNA) obtained through noninvasive blood draws. However, liquid biopsy analysis is often challenging due to exceedingly low fractions of circulating tumor DNA (ctDNA), necessitating the use of extended tumor biomarker panels. While multiplexed PCR strategies provide advantages such as higher throughput, their implementation is often hindered by challenges such as primer-dimers and PCR competition. Alternatively, digital PCR (dPCR) approaches generally offer superior performance, but with constrained multiplexing capability. This paper describes development and validation of the first multiplex digital methylation-specific PCR (mdMSP) platform for simultaneous analysis of four methylation biomarkers for liquid-biopsy-based detection of non-small cell lung cancer (NSCLC). mdMSP employs a microfluidic device containing four independent, but identical modules, housing a total of 40 160 nanowells. Analytical validation of the mdMSP platform demonstrates multiplex detection at analytical specificities as low as 0.0005%. The clinical utility of mdMSP is also demonstrated in a cohort of 72 clinical samples of low-volume liquid biopsy specimens from patients with computed tomography (CT)-scan indeterminant pulmonary nodules, exhibiting superior clinical performance when compared to traditional MSP assays for noninvasive detection of early-stage NSCLC.

更新日期：2023-04-11

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