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

项目总结 这项研究的首要目标是利用基于血浆的无细胞dna(Cfdna)基因组生物标记物。 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的超低通 全基因组测序(ULP-WGS)分析和计算流水线(IchorCNA)以确定 循环中的肿瘤DNA数量(肿瘤分数；TFX)。TFX中的变化可以作为 对治疗有反应或无反应的患者和cfDNA ULP-WGS提供了一种经济高效的， 微创方法测定TFX。我们的ULP-WGS cfDNA分析可以快速、准确地定量 在事先不知道肿瘤突变的情况下，从单个血样中提取TFX。虽然大多数液体活组织检查 到目前为止，我们的方法主要集中在追踪癌症中已知的变化或常见的突变基因 该方法是突变不可知性的，广泛适用于晚期癌症。 这项提议是通过三名主要调查人员之间的深度合作而提出的 无细胞DNA检测在指导晚期患者预后和治疗中的应用 癌症。到目前为止，我们已经在3000多名患者样本上进行了这项测试的研究版本，在 到目前为止，我们发表了三篇论文，展示了转移性乳腺癌和前列腺癌以及 多发性骨髓瘤。我们带来了临床乳腺癌、测序分析开发和CLIA方面的专业知识 大规模测序，与多个共享出版物合作的良好记录。所有三个PI 都致力于这项提案的成功，具有不同但相辅相成的作用和责任。 在这项建议中，我们将分析验证我们的cfDNA ULP-WGS分析(UH2部分)，然后建立临床 有效性和前瞻性评估治疗临床试验的表现(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 评估重复性、可重复性和可报告的范围。然后，我们将在上下文中确定性能 “真实世界”分析前的可变性，包括血液采集管类型、血浆数量和检测 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 700例转移性乳腺癌患者临床血浆TFX检测结果分析 通过评估TFX与患者治疗反应和生存的关系来了解cfDNA 结果。最后，我们将评估ULP-WGS在两个前瞻性治疗转移性三重肿瘤的临床试验中的应用。 阴性乳腺癌，评估与治疗反应的相关性。

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.