A clinical grade sequencing-based assay for CEBPA mutation testing: report of a large series of myeloid neoplasms.

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.