Integrating Biospecimen Science Into The Development Of RNA-Based Clinical Assays For Patients With Metastatic Breast Cancer

项目摘要 我们开发了内分泌治疗敏感性(SETER/PR)基因表达指数来预测 转移性乳腺癌患者对内分泌治疗的敏感程度，以指导选择 疾病进展后的治疗。检测雌激素受体(ER)基因的表达 (ESR1)，在配体结合的热点区域发生激活突变的ESR1转录本的频率 结构域，以及表示与两者的表达最相关的基因表达的签名 雌激素(ER)和孕激素受体(PR)基因。因此，SETER/PR指数总结了18个选定的 转录本相对于代表基因表达范围的10个参考转录本。使用来自 高质量的乳腺癌新鲜样本，SETER/PR指数在不同的 分析和分析前条件，并预测PFS和总存活率(OS) 转移性乳腺癌患者的活检组织，然后接受内分泌治疗。我们最近做了 将该分析与常规福尔马林固定石蜡包埋(FFPE)组织切片一起使用 活组织检查。 我们知道，确定以RNA为基础的分析前条件将是重要的 可准确应用于临床常见的转移性乳腺癌标本。尽管这是一种常见的 转移性癌立即保存小活检的临床实践中，有重大的预分析 考虑的条件：使用细针吸取细胞学(几种样本保存方案是 可能)、稀释和污染效应来自宿主组织RNA内的细胞学或组织活检 转移，以及使用恶性积液样本进行检测的能力(更大的体积不是 立即修复)。我们设计了我们的研究，以提供对整个转录组分析的见解， 另外，我们为SETER/PR指数定制的、有针对性的RNA测序分析。 我们的第一个目标是通过对例行程序的影响进行稳健的测量来识别记录 在小的生物样本上的固定和加工，与从匹配的样品中提取的高质量的RNA相比 核糖核酸防腐剂。我们将使用整个转录组RNA测序(RNAseq)来测量每一个转录本 在保存肿瘤组织或细胞学活检的每个临床程序下，与高质量的RNA相比 作为参考。同样，我们将使用定制的RNAseq比较这些分析前条件 测定SETER/PR指数(基于液滴的靶标逆转录，然后测序)。这将是 确定基于RNA的细胞学和组织样本检测的最佳条件，并将为所有 在SETER/PR索引中，除了特定目标的记录外，还有其他的记录。 我们的第二个目标是定义一组宿主器官转录本，用来估计程度 从任何转移部位获得的临床活检样本中的宿主器官RNA。这涉及到 从RNAseq数据和现有的微阵列数据集识别特定器官的转录本，以便 转移性活检的SETER/PR指数的测量伴随着测量以估计 样本中包含的宿主器官RNA的范围，从而确定癌症的纯度。这将是 作为一种有用的质量控制措施，并有可能为肿瘤人群提供正确的报告。 我们的第三个目标是分析验证和测试SETER/PR的可行性和临床有效性 使用从临床试验收集的转移性乳腺癌活检样本的诊断分析的指数。 我们将最终确定SETER/PR指数分析，包括评估宿主器官范围的分析物 污染，完成分析验证研究，以确定这是符合CLIA的临床测试。 然后，我们将使用转移性乳腺癌的样本来测试SETER/PR指数测试的临床性能 来自转移性乳腺癌(MBC)试验，特别是在比较内分泌- 基于无进展和总存活率的治疗。MBC试验是随机试验的篮子试验 美国食品和药物管理局正在进行的治疗比较，学术肿瘤学 社区和国家癌症研究所。它的目标是协调预测性诊断开发与 新疗法的临床试验网络。 我们将完成一种准确测量表达、功能 转移性乳腺癌活检组织中ER序列完整性和转录产物的预测 转移性乳腺癌治疗过程中任一点对内分泌治疗的敏感性。

Project Summary We have developed the sensitivity to endocrine therapy (SETER/PR) index of gene expression to predict the degree of sensitivity to endocrine therapy for patients with metastatic breast cancer, to guide the selection of treatment after disease progression. The assay measures the expression of estrogen receptor (ER) gene (ESR1), the frequency of ESR1 transcripts with activating mutation in a hotspot region of the ligand-binding domain, and a signature representing the expression of genes most strongly correlated with expression of both estrogen (ER) and progesterone receptor (PR) genes. Therefore, the SETER/PR index summarizes 18 selected transcripts relative to 10 reference transcripts that represent the range of gene expression. Using RNA from high-quality fresh samples of breast cancer, the SETER/PR index was highly reproducible under diverse analytical and pre-analytical conditions, and was prognostic for PFS and overall survival (OS) when tested in biopsies of metastatic breast cancer from patients who then receive endocrine therapy. We have recently translated the assay for use with sections from routine formalin-fixed paraffin-embedded (FFPE) tissue biopsies. We know that it will be important to define the pre-analytical conditions under which an RNA-based assay can be accurately applied to usual clinical samples of metastatic breast cancer. Although it is usual to immediately preserve small biopsies of metastatic cancer in clinical practice, there are major pre-analytical conditions to consider: the use of fine needle aspiration cytology (several sample preservation protocols are possible), dilutive and contaminating effects from host tissue RNA within a cytologic or tissue biopsy of metastasis, and the ability to perform the assay using malignant effusion samples (larger volumes are not immediately fixed). We have designed our studies to provide insights into whole transcriptome assays and, separately, our customized, targeted RNA sequencing assay for the SETER/PR index. Our first objective is to identify the transcripts with measurement that is robust to the effects of routine fixation and processing on small biospecimens, compared to the high-quality RNA from matched samples in RNA preservative. We will use whole transcriptome RNA sequencing (RNAseq) to measure every transcript under every clinical procedure for preserving a tumor tissue or cytologic biopsy, compared to high-quality RNA as the reference. We will similarly, compare these pre-analytical conditions using our customized RNAseq assay for the SETER/PR index (droplet-based reverse transcription of targets, followed by sequencing). This will define the best conditions for RNA-based testing of cytology and tissue samples, and will provide data for all transcripts in addition to the specifically targeted transcripts in the SETER/PR index. Our second objective is to define a panel of host organ transcripts that will be used to estimate the extent of the host organ RNA within a clinical biopsy sample obtained from any metastatic site. This involves the identification of organ-specific transcripts from RNAseq data and existing microarray data sets, so that a measurement of the SETER/PR index from a metastatic biopsy is accompanied by a measurement to estimate the extent of host organ RNA that was included within the sample, and hence the purity of cancer. This will serve as a useful quality control measure, and potentially enable corrected reporting for the tumor population. Our third objective is to analytically validate and test the feasibility and clinical validity of the SETER/PR index using the diagnostic assay with biopsy samples of metastatic breast cancer collected from a clinical trial. We will finalize the SETER/PR index assay, including analytes to estimate the extent of host organ contamination, an complete the analytical validation studies to establish this as a CLIA-compliant clinical test. Then, we will test the clinical performance of the SETER/PR index test using samples of metastatic breast cancer from the Metastatic Breast Cancer (MBC) trial, specifically in the treatment arms that compared endocrine- based treatments for progression-free and overall survival. The MBC trial is a basket trial of randomized treatment comparisons that is being developed by the Food and Drug Administration, academic oncology community, and the National Cancer Institute. It aims to coordinate predictive diagnostic development with a network of clinical trials of new treatments. We will complete the development of an assay that accurately measures the expression, functional sequence integrity, and transcriptional product of ER within a biopsy of metastatic breast cancer – to predict the sensitivity to endocrine therapy at any point in the treatment course of metastatic breast cancer.