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

优化皮肤 T 细胞淋巴瘤高通量 TCR 测序的分析前样品处理

基本信息

批准号：
10688079
负责人：
Rachael Ann Clark
金额：
$ 39.71万
依托单位：
BRIGHAM AND WOMEN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-01 至 2025-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10688079
关键词：
Affect Age Biological Assay Biological Markers Biometry Biopsy Biopsy Specimen Blood Caring Cell Count Clinical Clinical Trials Clinical effectiveness Collection Cutaneous Lymphoma Cutaneous T-cell lymphoma DNA DNA Damage Data Dermatology Diagnosis Disease Disease Progression Epitopes Fixatives Formalin Freezing Future Gel Gene Expression Genes Goals High-Throughput Nucleotide Sequencing Histologic Histology Human Immunology Immunotherapy Industrialization Industry Link Malignant - descriptor Measurement Measures Non-Hodgkin&apos s Lymphoma Nucleic Acids Oncology Pathology Patients Performance Placebo Control Procedures Proteins RNA Randomized Reproducibility Reverse Transcriptase Polymerase Chain Reaction Sampling Skin Specimen T cell receptor repertoire sequencing T-Cell Receptor T-Cell Receptor Genes T-Lymphocyte Techniques Temperature Testing Time Tissue Banks Tissues cancer type disorder risk histological studies indexing innovation lymph nodes next generation phase III trial preservation resiquimod response skin lesion tissue processing treatment response tumor

项目摘要

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.

摘要/摘要重排的T细胞受体基因（HTS）的高吞吐量测序已改变了诊断，护理以及评估皮肤T细胞淋巴瘤（CTCL）患者的治疗反应，此测定是成为CTCL临床试验的黄金标准。 HTS结果在冷冻CTCL皮肤中高度可重现活检，但福尔马林在许多临床试验中用于保留皮肤活检的福尔马林降低了DNA并影响HTS 测量结果，可能导致患者诊断错误，评估治疗的反应和选择。我们试图确定小型CTCL皮肤活检的单一均匀组织加工方法准确且可再现的HTS结果，支持DNA，RNA和蛋白质测量值，保持出色组织学，并保留剩余的组织，以未来测量新兴的生物标志物。在AIM 1中，我们确定最佳组织运输条件并测试非交叉固定剂的支持核酸的能力，蛋白质和组织学研究小型皮肤活检。 AIM 2研究存储时间和温度的影响关于核酸完整性，组织学性能和HTS读数，并测试了两种减轻方法 DNA降解对HTS测量的影响。 AIM 3提供了我们优化样本的现实世界测试处理程序，利用它们研究在该行业中获得的皮肤活检，随机，随机，安慰剂 - CTCL中局部resiquimod凝胶的控制期III期试验。我们的总体目标是建立新的最好的纸巾处理未来临床试验的实践，并建立校正，以允许对现有的准确分析标本。 HTS现在经常在许多癌症类型中使用，以测量肿瘤T细胞数量，多样性和对免疫疗法的反应。我们确定的优化组织处理程序具有潜力在许多癌症类型中有用。