Pre-analytical variables of bioanalytes affecting the accuracy of PTCL diagnostic and prognostic genetic signatures

摘要 外周T细胞淋巴瘤（PTCL）占西方世界所有NHL的约12-15%，并且是 与预后不佳有关。此外，诊断是具有挑战性的，因为30-50%的PTCL病例不能被诊断。 分配给特定的实体，并被分类为PTCL-未另外指定（PTCL-NOS）。我们已经定义 强大的基因表达特征，可以区分五种常见的PTCL实体：血管免疫母细胞T细胞 淋巴瘤（AITL）、间变性淋巴瘤激酶阳性间变性大细胞淋巴瘤（ALK（+）ALCL）、ALK- 阴性间变性大细胞淋巴瘤（ALK（-）ALCL）、成人T细胞白血病/淋巴瘤（ATLL）和结外 自然杀伤/T细胞淋巴瘤（ENKTCL）。PTCL-NOS可分为两个不同的生物学和预后 亚组（PTCL-TBX 21和PTCL-GATA 3亚组）。我们翻译了基于RNA的诊断和预后 用于福尔马林固定石蜡包埋（FFPE）组织的算法，用于具有高灵敏度的广泛临床用途， 的特异性我们还使用相应的DNA鉴定了PTCL亚型中不同的遗传病变， 证明这种损伤可以使用相应的浅层全基因组分析（sWGA）进行验证。 血浆细胞游离DNA，因此液体活检可以帮助诊断和疾病监测。 由于生物标本的处理以及质量在常规临床病理学实验室中差异很大， 基于RNA或DNA的标记的可靠性需要在可变的情况下进行评估。有必要 在新诊断试剂盒之前，确定分析前变量如何影响测定的稳健性 工具可以应用于大型研究或常规临床实践。我们假设，全面评估 生物样本的预分析变量将导致优化的生物样本采购框架， 在组织和液体活检设置中提高诊断准确性和再现性， 常规临床实践/试验的CLIA间实验室设置。该提案旨在建立标准化的、基于证据的 生物样本（RNA/DNA）处理、储存和运输程序，以确保准确、可重现 测定性能。确定的条件和参数将在前瞻性样本上进行验证，最好在 临床试验设置，因此结果可以与临床数据相关联。因此，提出了三个具体目标： 具体目标1：确定影响基于RNA的检测可靠性的分析前变量， FFPE组织 具体目的2：确定影响循环肿瘤DNA（ct-DNA）检测的分析前因素， PTCL患者的定量 具体目标3：验证分析前变量在改善PTCL诊断或 CLIA（临床实验室改进修正案）间实验室环境中的预后测定 这些研究将产生强大的方案，优化组织活检或血浆中的生物分子保存， 确保分子检测的准确性和重现性，改善PTCL分类和鉴定。

Abstract Peripheral T-cell lymphomas (PTCL) represent approximately 12-15% of all NHL in the western world and are associated with dismal prognosis. Furthermore, the diagnosis is challenging as 30-50% of PTCL cases cannot be assigned to a specific entity and are categorized as PTCL-not otherwise specified (PTCL-NOS). We have defined robust gene expression signatures that can differentiate the five common PTCLs entities: angioimmunoblastic T-cell lymphoma (AITL), anaplastic lymphoma kinase positive anaplastic large-cell lymphoma (ALK (+) ALCL), ALK- negative anaplastic large-cell lymphoma (ALK (-) ALCL), adult T-cell leukemia/lymphoma (ATLL), and extra-nodal natural killer/T-cell lymphoma (ENKTCL). PTCL-NOS can be divided into two distinct biological and prognostic subgroups (PTCL-TBX21 and PTCL-GATA3 subgroups). We translated the RNA based diagnostic and prognostic algorithms for formalin fixed paraffin embedded (FFPE) tissues for widespread clinical usage with high sensitivity and specificity. We also identified distinguishing genetic lesions in PTCL subtypes using corresponding DNA , and demonstrated that such lesion can be validated using shallow whole genome analysis (sWGA) in corresponding plasma cell-free DNA, thus liquid biopsy can aid in diagnosis and disease monitoring. Since the biospecimen processing, and hence quality, varies significantly in routine clinical pathology laboratories, the reliability of RNA or DNA based signatures need to be evaluated under variable circumstances. It is essential to determine how the robustness of the assay may be affected by pre-analytical variables before the novel diagnostic tools can be applied to large studies or routine clinical practice. We hypothesize that a comprehensive evaluation of pre-analytical variables of biospecimen will lead to optimized bio-specimen procurement framework leading to improved diagnostic accuracy and reproducibility in tissue and liquid biopsy setting and can be standardized in an inter-CLIA lab setting for routine clinical practice/trials. This proposal aims to establish standardized, evidence-based procedures on bio-specimen (RNA/DNA) processing, storage and transportation to ensure accurate, reproducible assay performance. The identified conditions and parameters will be validated on prospective samples, preferably in a clinical trial setting, so findings can be correlated with clinical data. Thus, three specific aims are proposed: Specific Aim 1: To determine pre-analytical variables that affects the reliability of RNA-based assays in FFPE tissue Specific Aim 2: To identify pre-analytical factors affecting circulating tumor DNA (ct-DNA) detection and quantification in patients with PTCL Specific Aim 3: To validate harmonization of the pre-analytical variables in improving PTCL diagnostic or prognostic assay in an inter-CLIA (Clinical Laboratory Improvement Amendments) lab setting The studies will lead to robust protocols that optimize the preservation biomolecules in tissue biopsies or plasma to ensure accuracy and reproducibility of molecular assays, improving PTCL classification and prognostication.