Ultra-Rare Circulating Tumor DNA Detection with PAP & Digital Bead Technologies

描述（由申请人提供）：癌症的诊断和预后越来越多地受益于分子标志物和分子方法的使用。越来越多的证据表明，在各种癌症患者的外周血中循环着肿瘤DNA。多项研究表明，循环DNA携带在相应的原发性肿瘤中发现的遗传和表观遗传改变。因此，血液中循环DNA的存在是一个强有力的诊断和预后指标，使血液成为非侵入性和预防性癌症诊断以及监测积极接受癌症治疗的患者的有价值的实体肿瘤替代品。检测血液中肿瘤DNA最常用的靶标之一是p53突变。p53抑癌基因产物（TP 53）在早期肿瘤发生中起重要作用。麦克斯韦传感器公司与City of Hope国家医疗中心合作，建议开发一种用于早期癌症筛查的“TP 53-TYPER”系统。原型TP 53-TYPER设计用于（i）扩增血液中循环的过量正常基因组DNA中的超罕见TP 53突变，（ii）通过杂交将扩增的等位基因捕获到数字条形码微珠上，以及（iii）鉴定、检测和定量扩增的等位基因。该技术将双向PAP等位基因特异性扩增（Bi-PAP）与数字微珠上Bi-PAP扩增的TP 53体细胞突变的捕获沿着等位基因丰度的识别和荧光扫描相结合，我们能够以可靠，经济高效和节省时间的方式进行高通量和高分辨率基因分型。Bi-PAP是一种具有超高灵敏度（1个突变DNA分子）和选择性（109至1011个野生型分子中有1个突变体）的扩增技术，与可寻址微珠微流控检测相结合，将使血液中生物标志物的常规使用能够用于早期临床癌症诊断。TP 53-TYPER系统适合于商业系统集成和自动化，用于高效的大规模分析。第一阶段的工作将集中在系统制造和可行性测试。

DESCRIPTION (provided by applicant): Diagnosis and prognosis of cancer have increasingly benefited from the use of molecular markers and molecular approaches. Accumulating lines of evidence have shown that there is tumor DNA circulating in the peripheral blood of patients with a variety of cancers. Multiple studies have shown that circulating DNA carries genetic and epigenetic alterations that are found in the corresponding primary tumors. Therefore, the presence of circulating DNA in blood is a strong diagnostic and prognostic indicator, making blood a valuable solid tumor surrogate for non-invasive and preventive cancer diagnostics as well as monitoring of patients who are actively under cancer therapy. One of the most commonly used targets for detecting tumor DNA in blood are p53 mutations. The p53 tumor suppressor gene product (TP53) is playing an important role in early cancer initiation. Maxwell Sensors Inc. together with City of Hope National Medical Center proposes to develop a "TP53-TYPER" system for early cancer screening. The prototype TP53-TYPER will be designed to (i) amplify ultra-rare TP53 mutations within the excess of normal genomic DNA circulating in blood, (ii) capture the amplified alleles onto digitally bar-coded beads by hybridization, and (iii) identify, detect and quantify the amplified alleles. The technology combining Bi-directional PAP allele specific amplification (Bi-PAP) with the capture of Bi-PAP amplified TP53 somatic mutations on digital microbeads along with identification and fluorescent scanning of allele abundance, we are enabling high-throughput and high resolution genotyping to be performed in a reliable, cost-effective and time-saving manner. The combination of Bi-PAP, an amplification technology with ultra-high sensitivity (1 mutant DNA molecule) and selectivity (1 mutant in 109 to 1011 wild-type molecules), with addressable bead-based microfluidic detection will enable the routine use of biomarkers in blood in early clinical cancer diagnostics. The TP53-TYPER system is amenable to commercial system integration and automation for efficient large scale analyses. The Phase I work will focus on system fabrication and feasibility testing.