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：
10247804
负责人：
Rachael Ann Clark
金额：
$ 40.51万
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/10247804
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 Gold High-Throughput Nucleotide Sequencing Histologic Histology Human Immunooncology Immunotherapy Industrialization Industry Lead Malignant - descriptor Measurement Measures Non-Hodgkin&apos s Lymphoma Nucleic Acids Pathology Patients Performance Placebos Procedures Proteins RNA Randomized Reading 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 base 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 T 细胞受体基因 (HTS) 的高通量测序改变了诊断、护理皮肤 T 细胞淋巴瘤 (CTCL) 患者的治疗反应评估，该测定是成为 CTCL 临床试验的金标准。 HTS 结果在冷冻 CTCL 皮肤中具有高度可重复性但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，可能会导致患者诊断、反应评估和治疗选择方面的错误。我们寻求为小型 CTCL 皮肤活检确定一种统一的组织处理方法，从而提供准确且可重复的HTS结果，支持DNA、RNA和蛋白质测量，保持优异的组织学，并保存剩余组织以供将来测量新兴生物标志物。在目标 1 中，我们确定最佳组织运输条件并测试非交联固定剂支持核酸的能力，小皮肤活检的蛋白质和组织学研究。目标 2 研究储存时间和温度的影响核酸完整性、组织学性能和 HTS 读数，并测试了两种减轻影响的方法 DNA 降解对 HTS 测量的影响。 Aim 3 为我们优化的样品提供真实世界的测试处理程序，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 中局部雷西莫德凝胶的对照 III 期试验。我们的总体目标是建立新的最佳组织未来临床试验的处理实践，并建立纠正措施，以便准确分析现有的临床试验标本。 HTS 现在经常用于多种癌症类型，以测量肿瘤 T 细胞数量、多样性和对免疫疗法的反应。因此，我们确定的优化组织处理程序具有可能对多种癌症类型有用。