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

项目摘要 这项研究的首要目标是利用基于血浆的无细胞DNA（cfDNA）基因组生物标志物 以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 使用计算流水线（ichorCNA）的“全基因组测序”（ULP-WGS）测定来确定 循环中肿瘤DNA的量（“肿瘤分数”; TFx）。TFx的变化可以作为早期识别符， 对治疗有反应或没有反应的患者和cfDNA ULP-WGS提供了一种成本有效的， 微创方法来确定TFX。我们的ULP-WGS cfDNA检测可实现快速、精确的定量 在没有肿瘤突变的先验知识的情况下，从单个血液样本中提取TFx。虽然大多数液体活检 迄今为止的方法都集中在跟踪癌症中已知的改变或常见的突变基因上，我们的 这种方法是突变不可知的，并且广泛适用于晚期癌症。 这项提议是通过三位主要研究人员在音乐会上的深入合作而提出的。 无细胞DNA检测的开发和应用，以指导晚期癌症的预后和治疗， 癌的迄今为止，我们已经对超过3000个患者样本进行了该测定的研究版本， 到目前为止，我们已经发表了三篇文章，证明了转移性乳腺癌和前列腺癌的临床实用性， 多发性骨髓瘤我们在临床乳腺癌、测序检测开发和CLIA方面拥有专业知识 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 致力于使这一建议取得成功，其作用和责任各不相同，但相辅相成。 在本提案中，我们将分析验证我们的cfDNA ULP-WGS测定法（UH 2部分），然后建立临床试验。 有效性和前瞻性评价治疗性临床试验中的性能（UH 3部分）。在UH 2部分中， 我们将使用患者样本的系列稀释液确定ULP-WGS的灵敏度和特异性，然后 评估再现性、重复性和可报告范围。然后，我们将在上下文中确定性能 “真实的世界”分析前变异性，包括采血管类型、血浆量和检测 相对于DNA输入量的阈值。在UH 3部分，我们将通过以下方式确定临床有效性： 在700例转移性乳腺癌患者的临床血浆标本中评估TFx，然后推进我们的研究。 通过评价TFX与患者对治疗的反应和生存期的相关性来了解cfDNA 结果。最后，我们将在两项转移性三重肿瘤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.