Deep sequencing for minimal residual disease detection in Acute Lymphoblastic Leu

描述（由申请人提供）：急性淋巴细胞白血病（ALL）是一种侵袭性未成熟淋巴样肿瘤。在过去的几十年里，这些患者的疾病结局有了实质性的改善[3]。在某种程度上，这种改善是由于基于微小残留病（MRD）评估，加强了对需要额外治疗的患者的识别。目前，通过使用患者特异性分子测定（主要在欧洲）和/或多参数流式细胞术（mpFC）（通常在美国）来评估MRD。目前，以统一的方式实施这些方法是复杂和具有挑战性的。MRD的个体化分子评估受到大型机构基础设施要求的限制，因为每个患者的分子测定必须单独设计和验证。相比之下，多参数流式细胞术难以标准化，导致不同的质量。虽然很明显，MRD是重要的指导患者的具体护理，目前的方法是不健全的，以实现这一目标一致。 在一个涉及正在进行的T-ALL儿童肿瘤组（COG）试验AALL 0434 [1]的43对治疗前和治疗后样本的初步项目中，我们证明了T细胞受体基因重排的高通量测序增强了极低水平MRD的检测，提高了多参数流式细胞术的灵敏度和特异性，而不需要复杂的机构基础设施，所述机构基础设施是对微小残留疾病进行个体化高灵敏度分子评价所必需的。我们发现，高通量测序可以在多参数流式细胞术确定疾病的所有患者中确定MRD。然而，测序也允许以约10倍的更高灵敏度检测MRD。此后，我们完成了对来自COG试验AALL 0932（手稿正在编写中）的另一个99例B系急性淋巴细胞白血病患者队列的分析。在这项研究中，我们还发现，下一代免疫球蛋白受体基因重排测序可以提供增强的MRD检测。 这些数据提出了几个问题。首先，这些发现的临床特异性和敏感性如何？第二，下一代测序在多大程度上适用于临床实验室的常规实施？在这项提议中，我们应用高通量测序技术敏感和全面地评估B和T细胞ALL患者的MRD。

DESCRIPTION (provided by applicant): Acute lymphoblastic leukemia (ALL) is an aggressive immature lymphoid neoplasm. Over the last couple of decades, there has been substantial improvement in disease outcomes for these patients [3]. In part, this improvement is due to enhanced identification of patients requiring additional therapy based on the assessment of minimal residual disease (MRD). Currently, assessment of MRD is achieved through the use of either patient-specific molecular assays (predominantly in Europe), and/or multi-parametric flow cytometry (mpFC), (commonly in the US). At present, implementation of these approaches in a uniform manner is complex and challenging. Individualized molecular assessment of MRD is limited by requirements for a large institutional infrastructure, as molecular assays for each patient must be individually-designed and validated. By contrast, multi-parametric flow cytometry is difficult to standardize, resulting in disparate quality. While it is clear that MRD i important for guiding patient-specific care, current approaches are not robust to achieve this goal consistently. In a preliminary project involving 43 paired pre- and post-treatment samples from an ongoing T-ALL Children's Oncology Group (COG) trial AALL0434 [1], we demonstrate that high-throughput sequencing of T-cell receptor gene rearrangements enhanced detection of very low-level MRD, improving upon the sensitivity and specificity of multi-parametric flow cytometry, without requiring the complex institutional infrastructure required to individualize high-sensitivity molecular evaluation for minimal residual disease. We found that high-throughput sequencing could identify MRD in all patients in whom multi-parametric flow cytometry identified disease. However, sequencing also permitted detection of MRD at a higher sensitivity of approximately 10-fold. We have since completed analysis of another cohort of 99 patients with B-lineage acute lymphoblastic leukemia derived from COG trial AALL0932 (manuscript in preparation). In this study, we also found that next-generation sequencing of immunoglobulin receptor gene rearrangements could provide enhanced detection of MRD. Several questions are raised by these data. First, what is the clinical specificity and sensitivityof these findings? Second, to what extent is next-generation sequencing suitable for routine implementation in the clinical laboratory? In this proposal, we apply high-throughput sequencing to sensitively and comprehensively assess MRD in patients with B- and T-lineage ALL.