Deep sequencing for minimal residual disease detection in Acute Lymphoblastic Leu

描述（申请人提供）：急性淋巴母细胞白血病（ALL）是一种侵袭性的未成熟淋巴样肿瘤。在过去的几十年里，这些患者的疾病结果有了实质性的改善。在某种程度上，这种改善是由于基于最小残留疾病（MRD）评估的患者需要额外治疗的增强识别。目前，MRD的评估是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）来实现的。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的个性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分析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，导致质量参差不齐。虽然MRD对于指导患者特异性护理很重要，但目前的方法并不足以始终如一地实现这一目标。在一项涉及正在进行的T-ALL儿童肿瘤组（COG）试验AALL0434[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对极低水平MRD的检测，提高了多参数流式细胞术的灵敏度和特异性。不需要复杂的机构基础设施，需要个性化的高灵敏度分子评估最小残留疾病。我们发现高通量测序可以在所有多参数流式细胞术鉴定疾病的患者中鉴定MRD。然而，测序也允许以大约10倍的更高灵敏度检测MRD。我们已经完成了另一组来自COG试验AALL0932的99例b系急性淋巴细胞白血病患者的分析（手稿正在准备中）。在本研究中，我们还发现免疫球蛋白受体基因重排的下一代测序可以增强MRD的检测。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是什么？第二，下一代测序在多大程度上适合在临床实验室常规实施？在这个提议中，我们应用高通量测序来敏感和全面地评估B和t谱系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.