Optimizing pre-analytic sample handling for high throughput TCR sequencing in cutaneous T cell lymphoma

摘要/摘要 重排T细胞受体基因（HTS）的高通量测序已经改变了诊断、护理和治疗方法。 和评估皮肤T细胞淋巴瘤（CTCL）患者的治疗反应， 成为CTCL临床试验的金标准。HTS结果在冷冻CTCL皮肤中具有高度重现性 但是在许多临床试验中用于保存皮肤活检组织的福尔马林会降解DNA并影响HTS 测量结果，潜在地导致患者诊断、反应评估和治疗选择中的错误。 我们试图确定一个单一的统一的组织处理方法，小CTCL皮肤活检， 准确和可重复的HTS结果，支持DNA、RNA和蛋白质测量，保持优异的 组织学，并保存剩余的组织用于将来测量新出现的生物标志物。在目标1中，我们确定 最佳的组织运输条件和测试非交联固定剂支持核酸的能力， 蛋白质和组织学研究的小皮肤活检。目的2研究了贮存时间和温度对大豆蛋白的影响 对核酸完整性，组织学性能和HTS读数，并测试两种方法，以减轻 DNA降解对HTS测量的影响。Aim 3提供了对我们优化样本的真实的测试 处理程序，使用它们来研究在行业赞助的、随机的、安慰剂中获得的皮肤活检， CTCL患者外用瑞喹莫特凝胶的III期对照试验。我们的总体目标是建立新的最佳组织 处理未来临床试验的实践，并建立纠正措施，以便准确分析现有的 标本HTS现在经常用于许多癌症类型，以测量肿瘤T细胞数量、多样性和免疫原性。 对免疫疗法的反应。因此，我们确定的优化组织处理程序具有 可能在许多癌症类型中有用。

Summary/Abstract High throughput sequencing of the rearranged T cell receptor genes (HTS) has transformed the diagnosis, care and assessment of therapeutic responses in patients with cutaneous T cell lymphoma (CTCL) and this assay is becoming the gold standard in CTCL clinical trials. HTS results are highly reproducible in frozen CTCL skin biopsies but the formalin used to preserve skin biopsies in many clinical trials degrades DNA and affects HTS measurements, potentially causing errors in patient diagnosis, assessment of responses and choices of therapy. We seek to identify a single uniform tissue processing approach for small CTCL skin biopsies that will give accurate and reproducible HTS results, support DNA, RNA and protein measurements, maintain excellent histology, and preserve remaining tissue for future measurement of emerging biomarkers. In Aim 1, we identify optimal tissue transport conditions and test non-cross-linking fixatives for their ability to support nucleic acid, protein and histologic studies on small skin biopsies. Aim 2 studies the effects of storage time and temperature on nucleic acid integrity, histologic performance and HTS readings, and tests two approaches to mitigate the effects of DNA degradation on HTS measurements. Aim 3 provides real world testing of our optimized sample handling procedures, using them to study skin biopsies obtained in the industry sponsored, randomized, placebo- controlled phase III trial of topical resiquimod gel in CTCL. Our overall goals are to establish new best tissue handling practices for future clinical trials and to establish corrections that allow accurate analyses of existing specimens. HTS is now frequently used in many cancer types to measure tumor T cell numbers, diversity and responses to immune therapies. The optimized tissue handling procedures we identify therefore have the potential to be useful in many cancer types.