Detection of minimal residual disease in NPM1-mutated acute myeloid leukemia by next-generation sequencing.
Detection of minimal residual disease in NPM1-mutated acute myeloid leukemia by next-generation sequencing.
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DOI：
10.1038/modpathol.2014.57
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发表时间：
2014-11
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影响因子：
--
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检测微小残留病可预测急性髓系白血病患者的不良结局。目前，荧光定量聚合酶链式反应(RQ-PCR)或流式细胞仪检测微小残留病均有一定的实用价值和诊断局限性。在这里，我们描述了一种用于检测NPM1突变的急性髓系白血病微小残留病的新一代测序方法，该方法包括60%的正常核型急性髓系白血病患者的∼。使用测序接头连接的引物对NPM1的第12外显子进行聚合酶链式反应(PCR)扩增，并进行深度测序，以检测低发病率的急性髓系白血病特异性激活突变。我们将我们的结果与流式细胞术进行了比较，流式细胞术是我们机构诊断急性髓细胞白血病微小残留病的标准。使用NPM1突变阳性细胞系的特定稀释度和来自急性髓系白血病患者的纵向临床样本来评估这两种方法的性能。使用确定的对照材料，我们发现该检测方法对大约0.001%的突变细胞敏感，比流式细胞仪高出一个数量级。下一代测序是精确和半定量的，超过4个数量级。在来自6名急性髓系白血病患者的22个纵向样本中，下一代测序在所有被流式细胞仪认定为阴性的样本中检测到微小的残留疾病。此外，在三分之一的患者中，测序除了患者的索引突变外，还检测到了替代的NPM1突变，这与肿瘤的异质性一致。下一代测序提供的信息不需要事先了解NPM1突变亚型或验证RQ-PCR分析所需的等位基因特异性探针，也不需要生成和解释复杂的多维流式细胞仪数据。这种方法可能会补充现有的技术，以增强患者特定的临床决策。
Detection of minimal residual disease predicts adverse outcome in patients with acute myeloid leukemia. Currently, minimal residual disease may be detected by RQ-PCR or flow cytometry, both of which have practical and diagnostic limitations. Here, we describe a next-generation sequencing assay for minimal residual disease detection in NPM1-mutated acute myeloid leukemia, which encompasses ∼60% of patients with normal karyotype acute myeloid leukemia. Exon 12 of NPM1 was PCR amplified using sequencing adaptor-linked primers and deep sequenced to enable detection of low-prevalence, acute myeloid leukemia-specific activating mutations. We benchmarked our results against flow cytometry, the standard-of-care for acute myeloid leukemia minimal residual disease diagnosis at our institution. The performance of both approaches was evaluated using defined dilutions of an NPM1-mutation positive cell line and longitudinal clinical samples from acute myeloid leukemia patients. Using defined control material, we found this assay sensitive to approximately 0.001% mutant cells, outperforming flow cytometry by an order-of-magnitude. Next-generation sequencing was precise and semi-quantitative over 4 orders-of-magnitude. In 22 longitudinal samples from 6 acute myeloid leukemia patients, next-generation sequencing detected minimal residual disease in all samples deemed negative by flow cytometry. Further, in one-third of patients, sequencing detected alternate NPM1-mutations in addition to the patient's index mutation, consistent with tumor heterogeneity. Next-generation sequencing provides information without prior knowledge of NPM1 mutation subtype or validation of allele-specific probes as required for RQ-PCR assays, and without generation and interpretation of complex multi-dimensional flow cytometry data. This approach may complement current technologies to enhance patient-specific clinical decision-making.
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影响因子：
158.5
作者：
Falini, B;Mecucci, C;Martelli, MF
通讯作者：
Martelli, MF
DOI：
10.1038/ismej.2012.8
发表时间：
2012-08
期刊：
The ISME journal
影响因子：
--
作者：
通讯作者：
--
影响因子：
4.1
作者：
Pritchard, Colin C.;Salipante, Stephen J.;Walsh, Tom
通讯作者：
Walsh, Tom
影响因子：
2.6
作者：
Bacher, Ulrike;Badbaran, Anita;Kroeger, Nicolaus
通讯作者：
Kroeger, Nicolaus
DOI：
10.1073/pnas.2335924100
发表时间：
2003-11-25
影响因子：
11.1
作者：
Ley, TJ;Minx, PJ;Wilson, RK
通讯作者：
Wilson, RK