A Clinical Grade Sequencing-Based Assay for CEBPA Mutation Testing Report of a Large Series of Myeloid Neoplasms
A Clinical Grade Sequencing-Based Assay for CEBPA Mutation Testing Report of a Large Series of Myeloid Neoplasms
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DOI：
10.1016/j.jmoldx.2014.09.007
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发表时间：
2015-01-01
影响因子：
4.1
通讯作者：
Betz, Bryan L.
中科院分区：
文献类型：
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作者：
Behdad, Amir;Weigelin, Helmut C.;Betz, Bryan L.
CEBPA 突变的诊断测试是细胞遗传学正常的急性髓系白血病的标准治疗方法。该测试的广泛实施受到与基因 GC 含量、突变变异性以及序列分析和变异解释的复杂性相关的技术挑战的阻碍。我们开发了一种强大的桑格测序测试来检测诊断性急性髓系白血病样本中的 CEBPA 突变。介绍了我们对 2393 例疑似骨髓肿瘤病例进行检测的经验。并行确定 NPM1、FLT3-内部串联重复 (ITD) 和 FLT3-D835 突变状态； 160例（6.7%）病例存在CEBPA突变，其中单突变86例，双突变74例。 19 例单突变病例和 3 例双突变病例仅显示核苷酸取代，而基于片段分析的测试无法检测到该核苷酸取代。一部分病例存在双突变，突变等位基因频率不均匀，由于可能存在白血病异质性，需要仔细解释。与双突变病例相比，NPM1 和 FLT3-ITD 突变在单突变病例中更为常见（NPM1 为 31%，FLT3-ITD 为 5%，FLT3-ITD 为 28%，为 16%）。这种基于测序的检测提供了高效可靠的 CEBPA 突变检测平台，可以检测所有突变，并立即区分单突变和双突变病例。考虑到技术挑战，尽管多基因下一代测序检测不断发展，但强大的桑格测序检测在临床 CEBPA 检测中继续保持重要作用。
Diagnostic testing for CEBPA mutations is the standard of care for cytogenetically normal acute myeloid Leukemia. Widespread implementation of this testing is hampered by technical challenges associated with the GC content of the gene, the variability of the mutations, and the complexity of the sequence analysis and variant interpretation. We developed a robust Sanger-sequencing test to detect CEBPA mutations in diagnostic acute myeloid leukemia specimens. Our experience with testing 2393 cases of suspected myeloid neoplasms is presented. NPM1, FLT3-internal tandem duplication (ITD), and FLT3-D835 mutation status were determined in parallel; 160 (6.7%) cases harbored CEBPA mutations, including 86 with a single mutation and 74 with double mutations. Nineteen single-mutant cases and 3 double-mutant cases showed only nucleotide substitutions that could not be detected by fragment-analysis based tests. A subset of cases harbored double mutations with uneven mutant allele frequency and required careful interpretation because of possible Leukemic heterogeneity. NPM1 and FLT3-ITD mutations were more frequent in single- compared with double-mutation cases (31% versus 5% for NPM1, and 28% versus 16% for FLT3-ITD). This sequencing-based assay provides an efficient and reliable CEBPA mutation testing platform, permitting detection of all mutations with immediate distinction of single- and double-mutation cases. Given the technical challenges, robust Sanger-sequencing assays continue to maintain an important role in clinical CEBPA testing despite the development of multigene next-generation sequencing assays.