Assay Validation of Cell-Free DNA Shallow Whole Genome Sequencing To Determine 'Tumor Fraction' in Advanced Cancers

项目概要 这项研究的总体目标是利用基于血浆的游离 DNA (cfDNA) 基因组生物标志物 指导转移性乳腺癌和其他癌症的治疗。在我们的方法中，我们使用 cfDNA“超低通 全基因组测序（ULP-WGS）分析与计算管道（ichorCNA）以确定 循环中肿瘤 DNA 的量（“肿瘤分数”；TFx）。 TFx 的变化可以作为早期标识符 患者对治疗有反应或没有反应，而 cfDNA ULP-WGS 提供了一种经济高效、 确定 TFx 的微创方法。我们的 ULP-WGS cfDNA 检测可实现快速、精确的定量 在事先不了解肿瘤突变的情况下，从单个血液样本中提取 TFx。虽然大多数液体活检 迄今为止的方法主要集中于追踪癌症中已知的改变或常见突变基因，我们的 该方法与突变无关，广泛适用于晚期癌症。 该提案是通过三名主要研究人员在音乐会上的深入合作而制定的 开发和应用无细胞 DNA 检测来指导晚期预后和治疗 癌症。迄今为止，我们已经对 3000 多名患者样本进行了该测定的研究版本，并且 迄今为止，我们已经发表了三篇论文，证明了其在转移性乳腺癌和前列腺癌以及 多发性骨髓瘤。我们带来临床乳腺癌、测序分析开发和 CLIA 方面的专业知识 大规模测序，具有与多个共享出版物合作的良好记录。所有三个 PI 致力于该提案的成功，具有独特但互补的角色和责任。 在此提案中，我们将分析验证我们的 cfDNA ULP-WGS 测定（UH2 部分），然后建立临床 有效性并前瞻性评估治疗性临床试验中的表现（UH3 部分）。在UH2部分， 我们将使用患者样本的连续稀释来确定 ULP-WGS 的敏感性和特异性，然后 评估再现性、重复性和可报告范围。然后我们将确定上下文中的性能 “真实世界”的分析前变异性，包括采血管类型、血浆量和检测 关于 DNA 输入量的阈值。在 UH3 部分，我们将通过以下方式建立临床有效性： 评估来自转移性乳腺癌患者的 700 份临床血浆样本中的 TFx，然后推进我们的研究 通过评估 TFx 与患者对治疗的反应和生存的关系来了解 cfDNA 结果。最后，我们将在两项转移性三重治疗的前瞻性治疗临床试验中评估 ULP-WGS 阴性乳腺癌，评估与治疗反应的关联。

PROJECT SUMMARY The overarching goal of this research is to utilize a plasma-based genomic biomarker of cell-free DNA (cfDNA) to guide therapy in metastatic breast and other cancers. In our approach, we use a cfDNA ‘ultra low pass whole genome sequencing’ (ULP-WGS) assay with computational pipeline (ichorCNA) to determine the amount of tumor DNA in circulation (‘tumor fraction’; TFx). Changes in TFx may serve as an early identifier for patients responding – or failing to respond – to therapy and cfDNA ULP-WGS provides a cost-effective, minimally-invasive approach to determine TFx. Our ULP-WGS cfDNA assay allows rapid, precise quantitation of TFx from a single blood sample without prior knowledge of tumor mutations. While most liquid biopsy approaches to date have focused on tracking known alterations or commonly mutated genes in cancer, our approach is mutation agnostic and broadly applicable across advanced cancers. This proposal developed through deep collaboration between three primary investigators working on concert on the development and application of a cell-free DNA assay to guide prognosis and therapy in advanced cancers. To date, we have performed the research version of this assay on over 3000 patients samples and, in three publications to date, we demonstrate clinical utility in metastatic breast and prostate cancer as well as multiple myeloma. We bring expertise in clinical breast cancer, sequencing assay development, and CLIA sequencing at scale, with a strong track record of collaboration with multiple shared publications. All three PIs are dedicated to the success of this proposal, with distinct yet complementary roles and responsibilities. In this proposal, we will analytically validate our cfDNA ULP-WGS assay (UH2 portion) then establish clinical validity and prospectively evaluate performance in therapeutic clinical trials (UH3 portion). In the UH2 portion, we will determine the sensitivity and specificity of ULP-WGS using serial dilutions of patient samples, then assess reproducibility, repeatability, and reportable range. We will then determine performance in the context of ‘real world’ pre-analytic variability including blood collection tube type, amount of plasma, and detection thresholds with respect to DNA input quantity. In the UH3 portion, we will establish clinical validity by evaluating TFx in 700 clinical plasma specimens from patients with metastatic breast cancer then advance our understanding of cfDNA by evaluating the association of TFx with patient response to therapy and survival outcomes. Finally, we will evaluate ULP-WGS in two prospective therapeutic clinical trials of metastatic triple- negative breast cancer, evaluating association with response to therapy.