Liquid biopsy approaches to inform neuroblastoma prognosis and disease monitoring

摘要 在所有患有高危神经母细胞瘤的儿童中，只有不到一半的人成为长期幸存者。目前，它不是 有可能预测一个孩子是会用标准疗法治愈还是注定会复发。此外，标准 临床评估缺乏检测最终导致复发的微小残留病(MRD)的敏感性。 因此，开发新的精确生物标记物来识别患者是一个关键的挑战和未得到满足的需求。 世卫组织最终将对高强度治疗反应不佳，并可能从替代方法中受益。 我们将开发新的生物标记物来指导治疗决策，使用无细胞DNA(CfDNA)和一种新的， 基于表观遗传学的方法论，将确定导致侵袭性神经母细胞瘤的潜在生物学因素。在许多 癌症类型，从外周血中分离的cfDNA分析显示了希望，揭示了癌症的生物标志物 诊断、预测和肿瘤监测。DNA中的胞嘧啶可以是未经修饰的、甲基化的(5- 甲基胞嘧啶(5mC)，或含有氧化形式的5mC，5-羟甲基胞嘧啶(5hmC)。与5mC不同， 基因体上5hmC沉积的增加标志着转录的活跃。在这项提案中，我们将使用纳米技术 HMC-SEAL，一种用于分析cfDNA中5hmC修饰的全基因组方法学。最近，我们评估了 连续采集的神经母细胞瘤患儿cfDNA中的5hmC谱 与疾病负担和患者预后相关。重要的是，我们还发现了一种cfDNA 5hmC衍生 生物标记物可以区分治疗反应更好的患者和复发风险较高的患者。5hmC CfDNA与诊断高危原发肿瘤的比较表明cfDNA来源于 临床上具有侵袭性的恶性细胞，在复发肿瘤中常见的网络激活。对未来而言 确定基于5hmC的cfDNA生物标志物的预后强度，我们将使用纳米HMC-Seal来生成 来自400名入选患者的有临床注释的系列血液样本(液体活检)的5hmC谱 关于正在进行的儿童肿瘤组高危神经母细胞瘤III期研究(ANBL1531， NCT03126916)。我们假设来自神经母细胞瘤儿童的cfDNA5hmC图谱将作为 与目前的临床方法相比，在反应和生存方面具有更好的生物标志物，将揭示转录 网络导致了旧病复发。其具体目的是：1)进化和验证诊断时反应差的生物标志物； 2)根据系列cfDNA 5hmC图谱前瞻性地识别微小残留病(MRD)并预测复发； 3)实验确认复发时cfDNA含量丰富的候选网络。这项提议的成功将 导致：1)史无前例的诊断生物标志物，以改善治疗决策；2)早期发现和 对导致MRD的复发患者的干预；3)确定推动 旧病复发。这项工作将对识别从早期引进中受益的患者产生革命性的影响 替代疗法，改善那些患有侵袭性疾病的人的结果。

Abstract Fewer than half of all children with high-risk neuroblastoma become long-term survivors. Currently, it is not possible to predict if a child will be cured with standard therapy or is destined to relapse. Furthermore, standard clinical evaluations lack sensitivity to detect minimal residual disease (MRD) that ultimately leads to recurrence. Thus, there is a critical challenge and an unmet need to develop new precision biomarkers to identify patients who will ultimately have a poor response to the high-intensity therapy and may benefit from alternate approaches. We will develop new biomarkers to guide treatment decisions using cell-free DNA (cfDNA) and a novel, epigenetic-based methodology that will identify underlying biology driving aggressive neuroblastoma. In many cancer types, analysis of cfDNA isolated from peripheral blood has shown promise, revealing biomarkers for diagnosis, prognostication, and tumor surveillance. Cytosines in DNA can either be unmodified, methylated (5- methylcytosine, 5mC), or contain an oxidized form of 5mC, 5-hydroxymethylcytosine (5hmC). Unlike 5mC, elevated 5hmC deposition across a gene body marks active transcription. In this proposal, we will use nano- hmC-seal, a whole-genome methodology for analyzing 5hmC modifications in cfDNA. Recently, we evaluated 5hmC in cfDNA collected serially from children with neuroblastoma and demonstrated that 5hmC profiles correlated with disease burden and patient outcome. Importantly, we also found a cfDNA 5hmC derived biomarker can distinguish patients with superior response to treatment from those at high risk for relapse. 5hmC profiles from cfDNA compared to diagnostic high-risk primary tumors demonstrated cfDNA is derived from clinically aggressive, malignant cells with activation of networks common in relapsed tumors. To prospectively determine the prognostic strength of 5hmC-based cfDNA biomarkers, we will use nano-hmC-seal to generate 5hmC profiles from clinically annotated serial blood samples (liquid biopsies) collected from 400 patients enrolled on the ongoing Children’s Oncology Group High-Risk Neuroblastoma Phase III study (ANBL1531, NCT03126916). We hypothesize that cfDNA 5hmC profiles from children with neuroblastoma will serve as superior biomarkers for response and survival compared to current clinical methods and will reveal transcriptional networks driving relapse. The specific aims are: 1) Evolve and validate biomarkers of poor response at diagnosis; 2) Prospectively identify minimal residual disease (MRD) and predict relapse from serial cfDNA 5hmC profiles; 3) Experimentally confirm candidate networks enriched in cfDNA at relapse. The success of this proposal will lead to: 1) unprecedented diagnostic biomarkers to improve therapeutic decisions; 2) early detection and interventions for patients with relapse causing MRD; 3) identification of epigenetic mechanisms which drive relapse. This work will have a transformative impact by to identifying patients who benefit from early introduction of alternate therapy, improving outcomes for those with aggressive disease.