The Use of DNA Methylation Profiling to Assess the Significance of Different Types of Mutations in CEBPA-Mutated AML

• Published in: Blood 2014-12, Vol.124 (21), p.2328-2328
• Main Authors: El-Sharkawi, Dima, Green, Claire L, Sproul, Duncan, Jenkinson, Sarah, Feber, Andrew, Linch, David C, Gale, Rosemary E
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V124.21.2328.2328

In AML, the favorable prognosis associated with mutations in the CEBPA gene is restricted to those cases with double CEBPA mutations (CEBPADM), consistent with the loss of normal CEBP/alpha activity from both alleles. Current recommendations are that CEBPADM-mutated patients should not receive a stem cell transplant in first remission. In general, these cases have 2 ‘classical’ mutations, an N-terminal out-of-frame insertion/deletion that leads to loss of the full-length p42 protein and increased levels of the p30 isoform translated from an internal start site, coupled with a C-terminal in-frame insertion/deletion in the DNA binding domain (DBD) or leucine zipper domain (LZD) that interferes with DNA binding or dimerization. However, in our study of 1427 younger adult patients, 26% of mutations did not fit this classical description due to either the location or type of mutation. Furthermore, of the CEBPADM cases, 20% had a classical plus a ‘non-classical’ mutation or a homozygous non-classical mutation. It will be important to understand the functional consequences of these atypical mutations if CEBPA genotype is to be used to determine patient management. As methylation profiling has shown that CEBPADM cases form a distinct hypermethylated cluster, we investigated whether this can provide information about non-classical cases. A test set of 40 diagnostic samples were analyzed on the Illumina Infinium 27K Human Methylation Array, all normal karyotype with wild type (WT) NPM1, FLT3ITD and FLT3TKD; 10 were CEBPADM, 30 CEBPAWT. Unsupervised cluster analysis showed that the 10 CEBPADM cases clustered within a group of 16 hypermethylated cases that separated from 24 hypomethylated cases. A methylation signature was created from the 25 most-differentially methylated CpG sites between the CEBPADM and CEBPAWT cases and used to examine a validation set of 95 samples analyzed on the Illumina Infinium 450K Human Methylation Array (31 CEBPADM, 38 single-mutated CEBPA [CEBPASM], 26 CEBPAWT). This included 38 cases with non-classical mutations, 14 of them CEBPADM. On unsupervised cluster analysis, most CEBPADM cases (81%) fell in a hypermethylated group that was distinct from CEBPASM and CEBPAWT cases, with no segregation between the latter. We derived a genotype predictor by comparing the % methylation in a sample at each of the 25 CpG sites with that in the CEBPADM and CEBPAWT signatures to determine which signature the sample data most approximated. This correctly p