A next-generation sequencing-based assay for minimal residual disease assessment in AML patients with FLT3-ITD mutations
A next-generation sequencing-based assay for minimal residual disease assessment in AML patients with FLT3-ITD mutations
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DOI：
10.1182/bloodadvances.2018015925
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发表时间：
2018-04-24
期刊：
影响因子：
7.5
通讯作者：
Miller, Jeffrey E.
中科院分区：
文献类型：
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作者：
Levis, Mark J.;Perl, Alexander E.;Miller, Jeffrey E.
fms样酪氨酸激酶3 （FLT3-ITDs）的内部串联重复在急性髓性白血病（AML）中很常见，并导致预后不良。在FLT3-ITD突变的AML中，一种检测微小残留病（MRD）的敏感和特异性检测方法可以指导治疗决策。由于敏感性有限，现有的FLT3-ITD AML MRD检测方法并不是特别有用。我们利用下一代测序技术开发了一种针对FLT3-ITD突变的敏感和特异性MRD检测方法。该试验的初始验证是通过将固定数量的突变DNA插入野生型DNA中，以建立相当于1万个含有bbbb1 flt3 - itd的细胞的检测灵敏度，最少输入10万个细胞当量的DNA。随后，我们在缓解期FLT3-ITD AML患者的骨髓样本中验证了该分析。最后，我们分析了80名FLT3- itd复发/难治性AML患者的骨髓样本，这些患者参加了一种新型FLT3抑制剂gilteritinib的试验，并证明了突变负担（通过该试验检测）与总生存率之间的关系。这种新型MRD检测具有特异性，比目前可用的聚合酶链反应或下一代基于测序的FLT3-ITD检测灵敏2个数量级。该分析正在进行的随机临床试验中进行前瞻性验证。
Internal tandem duplications in fms-like tyrosine kinase 3 (FLT3-ITDs) are common in acute myeloid leukemia (AML) and confer a poor prognosis. A sensitive and specific assay for the detection of minimal residual disease (MRD) in FLT3-ITD mutated AML could guide therapy decisions. Existing assays for MRD in FLT3-ITD AML have not been particularly useful because of limited sensitivity. We developed a sensitive and specific MRD assay for FLT3-ITD mutations using next-generation sequencing. The initial validation of this assay was performed by spiking fixed amounts of mutant DNA into wild-type DNA to establish a sensitivity of detection equivalent to >1 FLT3-ITD-containing cell in 10 000, with a minimum input of 100 000 cell equivalents of DNA. We subsequently validated the assay in bone marrow samples from patients with FLT3-ITD AML in remission. Finally, we analyzed bone marrow samples from 80 patients with FLT3-ITD relapsed/refractory AML participating in a trial of a novel FLT3 inhibitor, gilteritinib, and demonstrated a relationship between the mutation burden, as detected by the assay, and overall survival. This novel MRD assay is specific and 2 orders of magnitude more sensitive than currently available polymerase chain reaction-or next-generation sequencing-based FLT3-ITD assays. The assay is being prospectively validated in ongoing randomized clinical trials.