无细胞 DNA (cfDNA) 研究能够对神经母细胞瘤患者的肿瘤细胞特异性遗传改变进行连续分析。已确定 18 名接受第三代 ALK 抑制剂 Lorlatinib 治疗的复发性神经母细胞瘤患者（SACHA 国家登记处和/或机构） ）。通过执行 WGS 文库构建，然后对热点突变（F1174L、R1275Q、I1170N）（变异等位基因分数 (VAF)）进行 ALK 靶向 ddPCR，对 9 名患者复发时的 cfDNA 进行分析，并依次对 5 名患者（血液/骨髓血浆）进行分析检测限 0.1%）和 WES 来评估疾病负担和克隆进化，与肿瘤/种系 WES 进行比较。Lorlatinib 的总体缓解率为 33%（CI 13-59%），其中 6/10 例未使用 Lorlatinib 的患者观察到缓解，而 0 例则为 0 /8 例具有 MYCN 扩增（MNA）。 ALK VAF 与总体临床疾病状态相关，临床缓解时 VAF <0.1%，而进展时 VAF 较高 (>30%)。重要的是，序贯 ALK ddPCR 比临床成像更早检测到复发。 cfDNA WES 揭示了 Lorlatinib 治疗后所有疾病进展情况中未见于原发肿瘤的新 SNV，表明克隆进化，包括与肿瘤侵袭性 (TP53) 或新靶标 (EGFR) 相关的基因的改变。基因通路分析揭示了新兴克隆中针对细胞分化的基因和持久性克隆中针对细胞粘附的基因的富集。在后续样本中可以观察到克隆造血的证据。我们证明了结合 ALK cfDNA ddPCR 用于疾病监测和 cfDNA WES 用于研究接受 ALK 靶向治疗的神经母细胞瘤患者的克隆进化和耐药机制的临床实用性。

The study of cell free DNA (cfDNA) enables sequential analysis of tumor cell-specific genetic alterations in neuroblastoma patients.Eighteen patients with relapsing neuroblastoma having received Lorlatinib, a 3rd generation ALK inhibitor, were identified (SACHA national registry and/or in the institution). cfDNA was analyzed at relapse for 9 patients, and sequentially for 5 patients (blood/bone marrow plasma) by performing WGS library construction followed by ALK-targeted ddPCR of the hotspot mutations (F1174L, R1275Q, I1170N) (variant allele fraction (VAF) detection limit 0.1%) and WES to evaluate disease burden and clonal evolution, following comparison with tumor/germline WES.Overall response rate to Lorlatinib was 33% (CI 13-59%), with response observed in 6/10 cases without versus 0/8 cases with MYCN amplification (MNA). ALK VAFs correlated with the overall clinical disease status, with a VAF<0.1% in clinical remission, versus higher VAFs (>30%) at progression. Importantly, sequential ALK ddPCR detected relapse earlier than clinical imaging. cfDNA WES revealed new SNVs, not seen in the primary tumor, in all instances of disease progression after Lorlatinib treatment, indicating clonal evolution, including alterations in genes linked to tumor aggressivity (TP53) or novel targets (EGFR). Gene pathway analysis revealed an enrichment for genes targeting cell differentiation in emerging clones, and cell adhesion in persistent clones. Evidence of clonal hematopoiesis could be observed in follow-up samples.We demonstrate the clinical utility of combining ALK cfDNA ddPCR for disease monitoring and cfDNA WES for the study of clonal evolution and resistance mechanisms in neuroblastoma patients receiving ALK targeted therapy.