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)约占西方世界所有NHL的12%-15%，是 与悲观的预后有关。此外，诊断具有挑战性，因为30%-50%的PTCL病例不能诊断 分配给特定实体，并被归类为PTCL-未另行指定(PTCL-NOS)。我们已经定义了 可区分五种常见PTCL实体的强健基因表达特征：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)、 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可分为两种不同的生物学和预后 亚组(PTCL-Tbx21和PTCL-GATA3亚组)。我们翻译了基于RNA的诊断和预后 用于临床广泛使用的福尔马林固定石蜡包埋(FFPE)组织的算法 专一性。我们还使用相应的DNA鉴定了PTCL亚型中的不同遗传损伤，以及 证明这种损伤可以用浅层全基因组分析(SWGA)在相应的 无浆细胞DNA，因此液体活检有助于诊断和疾病监测。 由于在常规的临床病理实验室中，生物显微镜的加工过程和质量有很大的不同， 基于RNA或DNA的签名的可靠性需要在不同的环境下进行评估。这是至关重要的 在新的诊断之前确定分析前变量如何影响分析的稳健性 这些工具可以应用于大型研究或常规临床实践。我们假设，对 生物样品分析前变量将导致优化的生物标本采购框架，从而导致 提高了组织和液体活组织检查设置中的诊断准确性和重复性，并可在 常规临床实践/试验的跨CLIA实验室设置。该提案旨在建立标准化的、以证据为基础的 生物标本(RNA/DNA)加工、储存和运输的程序，以确保准确、可重复性 化验性能。确定的条件和参数将在预期的样本上进行验证，最好是在 临床试验环境，因此研究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的分析的可靠性的分析前变量 FFPE组织 具体目标2：确定影响循环肿瘤DNA(ct-DNA)检测的分析前因素和 PTCL患者的定量研究 具体目标3：验证分析前变量在改进PTCL诊断或 CLIA(临床实验室改进修正案)实验室间环境中的预后分析 这些研究将产生强有力的方案，优化组织活检或血浆中的生物分子保存，以 确保分子检测的准确性和重复性，改进PTCL分类和预测。

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.