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

摘要和特定目的福尔马林固定是临床实践和临床试验中用于保存皮肤活检的常用方法。福尔马林固定石蜡包埋（FFPE）组织具有良好的组织形态，但DNA和RNA被福尔马林固定降解1。重排T细胞受体基因（HTS）的高通量测序已经改变了皮肤T细胞淋巴瘤（CTCL）患者的诊断、护理和治疗反应评估2-5。冷冻CTCL皮肤活检的HTS结果可高度重现，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，可能导致患者诊断、反应评估和治疗选择方面的错误。U01基金资助的首要目标是为研究CTCL的小型皮肤活检确定一种统一的最佳组织处理方法，该方法将提供准确和可重复的DNA， RNA和蛋白质测量，保持良好的组织学，并保存剩余组织以用于未来新出现的生物标志物的测量。假设：非交联组织固定剂将在不牺牲组织病理学质量的情况下保持核酸和蛋白质的完整性，并且在保存的福尔马林固定标本中对DNA降解进行定量校正将允许在现有CTCL患者的皮肤活检中准确测量HTS。

ABSTRACT AND SPECIFIC AIMS Formalin fixation is a common method used to preserve skin biopsies in both clinical practice and clinical trials. Formalin-fixed, paraffin-embedded (FFPE) tissues have excellent tissue morphology, but DNA and RNA are degraded by formalin fixation 1 . 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)2-5. 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. The overarching goal of the funded parent U01 grant is to identify a single uniform optimal tissue processing approach for small skin biopsies to study CTCL that will give accurate and reproducible DNA, RNA and protein measurements, maintain excellent histology, and preserve remaining tissue for future measurement of emerging biomarkers. Hypothesis: Non-cross-linking tissue fixatives will maintain nucleic acid and protein integrity without sacrificing histopathologic quality and quantitative corrections for DNA degradation in banked formalin-fixed specimens will allow accurate HTS measurements in existing skin biopsies from patients with CTCL.