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

摘要/摘要 重排的T细胞受体基因(HTS)的高通量测序改变了诊断、护理 和评估皮肤T细胞淋巴瘤(CTCL)患者的治疗反应，这项检测是 成为CTCL临床试验的金标准。HTS结果在冷冻的CTCL皮肤中具有高度的重复性 活组织检查，但在许多临床试验中用于保存皮肤活检的福尔马林会降解DNA并影响HTS 测量，可能会在患者诊断、反应评估和治疗选择方面造成错误。 我们寻求为小型CTCL皮肤活检确定一种单一的统一组织处理方法，该方法将给出 准确和可重复性的HTS结果，支持DNA、RNA和蛋白质测量，保持卓越 组织学，并保存剩余组织，以便将来测量新出现的生物标记物。在目标1中，我们确定 最佳的组织传输条件并测试非交联型固定剂对核酸的支持能力， 小样本皮肤活检的蛋白质和组织学研究。目的2研究储藏时间和温度的影响 对核酸完整性、组织学性能和HTS读数进行测试，并测试两种方法以缓解 DNA降解对高温超导测量的影响Aim 3提供了对我们的优化样本的真实测试 处理程序，用它们来研究在行业中获得的皮肤活检，随机，安慰剂- 外用利奎莫特凝胶剂在CTCL中的III期对照试验。我们的总体目标是建立新的最好的组织 处理未来临床试验的实践，并建立校正，以允许对现有的 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 对免疫疗法的反应。因此，我们确定的优化的组织处理程序具有 可能对许多癌症类型有用。

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.