Super-sensitive detection of universal markers of allograft rejection

同种异体移植排斥反应通用标志物的超灵敏检测机密主要研究者：Shafer，大卫A.，博士 项目摘要 美国每年进行超过35，000例器官移植，包括肾脏，肝脏，心脏和肺。 移植是最常见的。移植受者通常会服用免疫抑制剂， 皮质类固醇或其他药物，以防止同种异体移植排斥反应;然而，排斥反应最终发生在高达 三分之一的收件人。监测排斥反应有助于及时治疗，因为可以换用一种新药， 药物，或调整当前药物的剂量。组织活检是诊断的金标准 同种异体移植物排斥，尽管它们是侵入性的，但可能导致医学并发症，并且通常产生不充分的 标本目前，AlloMap®检测（CareDx）是FDA批准的唯一一种用于 识别移植受者中急性细胞排斥的风险。AlloMap®测试不直接测量 供体来源的无细胞DNA（ddcfDNA），具有较低的阳性预测值，并且不能检测抗体- 调解拒绝。因此，真正的临床需要的准确的方法，使廉价，非- 通过血浆样本进行侵入性“液体活检”生物标志物检测，以快速识别有排斥风险的受试者。 本项目的总体目标是开发一种用于检测ddcfDNA的实时PCR SNP分析测试 作为排斥反应的早期指标。我们的主要系统，内部DNA- 检测开关（iDDS）探针系统包括两个相互作用的组件：荧光标记探针和荧光标记探针。 猝灭剂标记的反探针，其与探针几乎互补。这种独特的探头系统显示出上级 在比普通方法宽得多的退火温度范围（10-30ºC）内进行单碱基鉴别。 最近，我们将iDDS探针技术与Wild Terminator（WTx）方法相结合， 通过阻断野生型序列的扩增来检测罕见突变体（0.1-0.01%频率）。将合并的 方法在检测肺癌患者的循环EGFR变异体方面显示出更高的灵敏度， FDA批准的cobas® EGFR突变检测，v2。我们更高的灵敏度和特异性将使开发 独立于平台的液体活检检测。我们的具体目标是：1）开发基于双iDDS探针的SNP 2）开发WTx-iDDS探针测定法，用于定量供体来源的SNP， ddcfDNA。在目标1中，我们将在20个基因座中的每个基因座上开发两种SNP变体的双iDDS探针分析法， 总体次要等位基因频率接近0.5的位点。选择具有几乎相等的主要/次要等位基因的SNP 频率将大大简化检测移植物血液中ddcfDNA水平尖峰的过程 在拒绝发生之前接收者。在目标2中，我们将开发选择性扩增20种靶向的WTx检测方法， SNP等位基因，并验证关键测试特征（线性、分析灵敏度、与NGS的比较和精密度） 510（k）提交所需的。我们将要求每个WTx-iDDS探针检测试剂盒可以在以下条件下检测目标SNP： 频率为0.1%，低于同种异体移植排斥反应前患者观察到的阈值。成功 第一阶段将证明扩大的第二阶段试验验证研究的合理性，以准备提交FDA批准。

Super-sensitive detection of universal markers of allograft rejection Confidential Principal Investigator: Shafer, David A., PhD PROJECT SUMMARY Over 35,000 organ transplants are performed annually in the US, with kidney, liver, heart, and lung transplants being most common. Transplant recipients are normally prescribed immunosuppressants, corticosteroids, or other medications to prevent allograft rejection; however, rejection eventually occurs in up to one third of recipients. Monitoring rejection facilitates timely therapy by switching to a new drug, adding another drug, or adjusting the dose of the current medications. Tissue biopsies are the gold standard for diagnosing allograft rejection, although they are invasive, may result in medical complications, and often yield inadequate specimens. Currently, the AlloMap® test (CareDx) is the only FDA-approved molecular diagnostics test for identifying the risk of acute cell rejection in transplant recipients. The AlloMap® test does not directly measure donor-derived cell-free DNA (ddcfDNA), has a low positive-predictive value, and does not detect antibody- mediated rejection. Thus, a genuine clinical need exists for accurate methods that enable inexpensive, non- invasive “liquid biopsy” biomarker testing via plasma samples to quickly identify subjects at risk of rejection. The overall objective of this project is to develop a real-time PCR SNP profiling test for detecting ddcfDNA in the plasma of transplant recipients, as an early indicator of rejection. Our primary system, the internal DNA- Detection Switch (iDDS) probe system, comprises two interacting components: a fluor-labeled probe, and a quencher-labeled antiprobe that is nearly complementary to the probe. This unique probe system shows superior single-base discrimination over a much wider annealing-temperature range (10–30ºC) than common methods. Recently, we merged our iDDS probe technology with our Wild Terminator (WTx) methods that enable detection of rare mutants (0.1–0.01% frequency) by blocking amplification of the wild-type sequence. The combined method showed greater sensitivity in detecting circulating EGFR variants from lung cancer patients than the FDA-approved cobas® EGFR Mutation Test, v2. Our higher sensitivity and specificity will enable development of platform-independent, liquid biopsy assays. Our Specific Aims are: 1) to develop dual-iDDS probe-based SNP profiling assays for donor-derived SNPs, and 2) to develop WTx-iDDS probe assays for quantitating SNPs in ddcfDNA. In Aim 1, we will develop dual-iDDS probe profiling assays for two SNP variants at each of 20 loci, at sites where the global minor allele frequency is nearly 0.5. Selecting SNPs with nearly equal major/minor allele frequencies will greatly simplify the process of detecting a spike in ddcfDNA levels in the blood of transplant recipients before rejection occurs. In Aim 2, we will develop WTx assays that selectively amplify the 20 targeted SNP alleles, and validate key test features (linearity, analytical sensitivity, comparison with NGS, and precision) required for a 510(k) submission. We will require that each WTx-iDDS probe assay can detect the target SNP at a frequency of 0.1%, which is below the threshold observed in patients before allograft rejection. Success in Phase I will justify expanded Phase II assay-validation studies in preparation for filing for FDA approval.