TempO-Seq for Preserved Tissues in Toxicity Testing Phase II

项目摘要 我们已经达到或超过了第一阶段的成功标准;开发了基于捕获自由连接的克里思- Seq基因表达检测;实施、验证并提供NIEHS TempO-Seq的抢先体验 用于分析RNA、细胞裂解物和福尔马林固定、石蜡包埋（FFPE）裂解物的大鼠替代试验 组织小至0.01mm3;并显示NIEHS人S1500+ TempO-Seq测定测量了 与RNAseq高度相关的差异表达和鉴定的前列腺癌生物标志物， 来自存档前列腺FFPE的治疗靶点。该第二阶段计划将解决NIEHS领域， 特别兴趣主题（A.）“开发和验证替代测试方法，以保护人类和 动物健康，同时减少，改进或取代动物试验”。我们将验证大鼠整体的使用 转录组测定和人全转录组测定，其中NIEHS S1500速率替代物是 用于分析存档的大鼠和临床FFPE样本。我们将确认替代试验 数据可以在计算机中用于准确预测整个转录组中的基因表达变化， 尽管只有几千个基因被直接测量。我们将证明TempO-Seq整体 转录组测定可以以相同的成本分析比RNAseq更多的样品， 替代测定甚至进一步降低了成本。因此，FFPE的TempO-Seq测定法将允许对FFPE进行测序。 动物FFPE档案被有效地挖掘，提供体内分子表型， 过去测试的化合物，而不必重复动物研究。我们将通过分析 NIEHS提供的存档FFPE来自几项毒性和饮食研究， RNAseq、微阵列和/或组织学，以与TempO-Seq结果相关联。我们在第一阶段表明， TempO-Seq测定的精确度提供了<10%的“动物间”平均CV，并且这一点是重要的。 精密度使得能够鉴定差异ED 50的分子特征。这将提高 药物化学家，以增加药物的治疗安全窗口，并将有助于验证在 使用体外试验，以避免动物研究。它还将使毒性能够在较低剂量下检测到 和更早的时间点，从而允许减少动物研究的规模或停止化合物 在投资长期动物模型之前，这大大减少了临床安全性测试。按给药 大鼠与参考化合物和相关的组织学TempO-Seq数据，我们将证明， 可以建立基因和途径标记的精确ED 50。将证明临床效用 来自一项对乳腺癌患者存档FFPE的转化研究，其5年、10年、20年结局 是已知的，并且从其中乳腺癌亚型的分子表型和预后生物标志物将 确认和扩展今天所知道的。

PROJECT SUMMARY We have met or exceeded the Phase I success criteria; developed the capture free ligation-based TempO- Seq gene expression assay; implemented, validated, and offered early access to the NIEHS TempO-Seq rat surrogate assay for profiling RNA, cell lysates, and lysates of formalin fixed, paraffin embedded (FFPE) tissue as small as 0.01mm3; and shown that the NIEHS human S1500+ TempO-Seq assay measures differential expression that is highly correlated to RNAseq and identified prostate cancer biomarkers and therapeutic targets from archived prostate FFPE. This Phase II program will address an NIEHS area of special interest Topic (A.) “Development and validation of alternative test methods to protect human and animal health while reducing, refining, or replacing animal tests”. We will validate the use of the rat whole transcriptome assay and a human whole transcriptome assay in which the NIEHS S1500 rate surrogate is embedded for profiling archived rat and clinical FFPE samples. We will confirm that the surrogate assay data can be used in silico to accurately predict gene expression changes across the whole transcriptome, though only a few thousand genes are directly measured. We will demonstrate that the TempO-Seq whole transcriptome assays can profile many more samples than RNAseq for the same cost, and that the surrogate assay lowers cost even further. Consequently, the TempO-Seq assay of FFPE will permit the archives of animal FFPE to be mined cost effectively, providing in vivo molecular phenotypes for compounds tested in the past, without having to repeat animal studies. We will demonstrate this by profiling archived FFPE provided by NIEHS from several toxicity and dietary studies for which there is associated RNAseq, microarray, and/or histology to correlate with TempO-Seq results. We showed in Phase I that the precision of the TempO-Seq assay provides “between animal” average CVs of <10%, and that this precision enables identification of molecular signatures for differential ED50s. This will improve the ability of medicinal chemists to increase the therapeutic safety window of drugs, and will be useful for validating in vitro assays for use in order to spare animal studies. It will also enable toxicity to be detected at lower doses and earlier time points, thus permitting the size of animal studies to be reduced or discontinuing compounds early before investment in long term animal models, significantly reducing clinical safety testing. By dosing rats with reference compounds and correlating histology to TempO-Seq data, we will demonstrate that precise ED50s for genes and pathway signatures can be established. Clinical utility will be demonstrated from a translational study of archived FFPE from breast cancer patients for which 5-, 10-, 20-year outcomes are known, and from which molecular phenotypes of breast cancer subtypes and prognostic biomarkers will be identified, confirming and extending what is known today.