Design and Testing of a Custom Melanoma Next Generation Sequencing Panel for Analysis of Circulating Tumor DNA
Design and Testing of a Custom Melanoma Next Generation Sequencing Panel for Analysis of Circulating Tumor DNA
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DOI：
10.3390/cancers12082228
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发表时间：
2020-08-01
期刊：
影响因子：
5.2
通讯作者：
Rizos, Helen
中科院分区：
文献类型：
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作者：
Diefenbach, Russell J.;Lee, Jenny H.;Rizos, Helen
使用来自血浆的循环肿瘤DNA（ctDNA）检测黑素瘤相关突变是使用来自侵入性组织活检的基因组DNA的潜在替代方案。在这项研究中，我们开发了一种定制的黑色素瘤下一代测序（NGS）面板，其中包括30个基因中的123个扩增子，涵盖驱动和靶向突变以及与治疗耐药性相关的改变。对74例III期和IV期初治黑色素瘤患者的队列分析显示，ctDNA检测的灵敏度受循环游离DNA（cfDNA）输入量和黑色素瘤分期的影响。在推荐的cfDNA输入量为20 ng（74例患者中有28例可用）时，在84%的IV期患者和47%的III期患者的ctDNA中检测到至少一种癌症相关突变，突变等位基因频率（MAF）的检测限为0.2%。这种定制的黑色素瘤组显示出与液滴数字PCR（ddPCR）的显著相关性，并提供了更全面的黑色素瘤突变谱。我们的定制面板可以通过替换跨越TERT启动子的扩增子来进一步优化，由于高GC含量，扩增子表现不佳。为了将IV期黑色素瘤的检出率提高至90%并降低对0.1%MAF的灵敏度，我们建议将血浆体积增加至8 mL，以实现最小推荐cfDNA输入和对表现不佳的扩增子的优化。我们的小组还可以扩展到包括新的靶向和治疗耐药突变，以改善对接受全身药物治疗的黑色素瘤患者的治疗反应和耐药性的跟踪。
Detection of melanoma-associated mutations using circulating tumor DNA (ctDNA) from plasma is a potential alternative to using genomic DNA from invasive tissue biopsies. In this study, we developed a custom melanoma next-generation sequencing (NGS) panel which includes 123 amplicons in 30 genes covering driver and targetable mutations and alterations associated with treatment resistance. Analysis of a cohort of 74 stage III and IV treatment-naive melanoma patients revealed that sensitivity of ctDNA detection was influenced by the amount of circulating-free DNA (cfDNA) input and stage of melanoma. At the recommended cfDNA input quantity of 20 ng (available in 28/74 patients), at least one cancer-associated mutation was detected in the ctDNA of 84% of stage IV patients and 47% of stage III patients with a limit of detection for mutant allele frequency (MAF) of 0.2%. This custom melanoma panel showed significant correlation with droplet digital PCR (ddPCR) and provided a more comprehensive melanoma mutation profile. Our custom panel could be further optimized by replacing amplicons spanning theTERTpromoter, which did not perform well due to the high GC content. To increase the detection rate to 90% of stage IV melanoma and decrease the sensitivity to 0.1% MAF, we recommend increasing the volume of plasma to 8 mL to achieve minimal recommended cfDNA input and the refinement of poorly performing amplicons. Our panel can also be expanded to include new targetable and treatment resistance mutations to improve the tracking of treatment response and resistance in melanoma patients treated with systemic drug therapies.