Hematological Malignancies CEBPA Mutation Testing in Acute Myeloid Leukemia (AML)

Mutations in the CCAAT/enhancer binding protein-alpha (CEBPA) gene are associated with a relatively favorable prognosis in adult and pediatric patients with normal cytogenetic AML, similar to that of patients with mutant NPM1 without FLT3-ITD.  Approximately 5-10% of AML patients possess CEBPA mutations, mainly in the M1 and M2 sub-types.  The CEBPA protein is a member of the leucine zipper (LZ) transcription factor family and is a critical regulator of granulopoiesis.  Two main types of mutations have been identified in AML – nonsense mutations in the N-terminal region that prevent expression of full-length protein resulting in a truncated isoform which acts as a dominant negative inhibitor, and in-frame mutations in the C-terminal leucine zipper domain with decreased DNA binding and dimerization properties.  The CEBPA mutations characterized involve a heterogeneous array of insertions, deletions, and point mutations. A minority of cases of AML without CEBPA mutations show silencing of the gene by promoter hypermethylation.

Clinical Utility:

AML patients with a mutation in the CCAAT/enhancer binding protein-alpha (CEBPA) gene (without a corresponding FLT3 gene mutation) have a relatively favorable prognosis.  Thus, a knowledge of the CEBPA mutation status, in combination with cytogenetics, the status of other gene mutations (FLT3, NPM, DNMT3A, IDH, etc.), and clinical parameters, together allow a comprehensive assessment of AML prognostic risk and help inform appropriate management strategy.

CEBPA mutations are heterogeneous in nature and have widespread localization.  Therefore, we have developed an assay based on the work of Pabst et al. (2001) to sequence the entire coding region in all AML patients.  The CEBPA gene does not contain introns, making sequencing of the full gene more easily accomplished.  Additionally, we will continue to monitor the literature and compile a database of known polymorphisms to aid in their distinction from mutations.

Analytical Sensitivity:  Approximately 20% mutant allele.

• 4-7 mL in purple (EDTA) or yellow (ACD) tube (unspun) whole blood or bone marrow.
• If sending DNA, please send 200ng at minimum of 25ng/µL (DNA must be extracted in a CLIA-certified laboratory or a laboratory meeting equivalent requirements as determined by the CAP and/or CMS).
• Deliver to lab at shipping address on the provided FedEx priority overnight label within 24 hours of collection; if sample cannot arrive within 24 hours, refrigerate until sample can be transported.

A REQUISITION FORM MUST ACCOMPANY ALL SAMPLES.  Please include detailed clinical information.

Weekly

7 – 10 Days

• Keep specimen cold during transit, but do not ship on dry ice. 
• Please contact Client Services at (855) 535-1522 for shipping kits and instructions. 
• Use the cold pack provided in the KDL shipping kit. 
• Ship the specimen overnight express, using the FedEx priority overnight label provided.

1. Benthaus et al., 2008.  Rapid and sensitive screening for CEBPA mutations in acute myeloid leukemia.  British J. Haemotology 143:230-239.
2. Dohner and Dohner, 2008.  Molecular characterization of acute myeloid leukemia.  Haematologica 93:976-982.
3. Ho et al., 2009.  Prevalence and prognostic implications of CEBPA mutations in pediatric acute myeloid leukemia (AML): a report from the Children’s Oncology Group.  Blood 25:6558-6566.
4. Leecharendkeat et al., 2008.  CCAAT/enhancer binding protein-alpha polymorphisms occur more frequently than mutations in acute myeloid leukemia and exist across all cytogenetic risk groups and leukemia subtypes.  Int. J. Cancer 123:2321-2326.
5. Marcucci et al., 2008.  Prognostic significance of, and gene and microRNA expression signatures associated with CEBPA mutations in cytogenetically normal acute myeloid leukemia with high-risk molecular features; a cancer and leukemia group B study.  J. of Clinical Oncology (online version 10.1200/JCO.2008.17.5554) 1-13.
6. Pabst et al., 2001.  Dominant-negative mutations of CEBPA, encoding CCAAT/enhancer binding protein alpha (C/EBPalpha), in acute myeloid leukemia.  Nature Genetics 27:263-270.
7. Preudhomme et al., 2002.  Favorable prognostic significance of CEBPA mutations in patients with de novo acute myeloid leukemia: a study from the Acute Leukemia French Association (ALFA).  Blood 100:2717-2723.
8.  Renneville et al., 2008. Cooperating gene mutations in acute myeloid leukemia: a review of the literature.  Leukemia 22:915-931.
9.  Wouters et al., 2007.  Distinct gene expression profiles of acute myeloid/T-lymphoid leukemia with silenced CEBPA and mutations in NOTCH1.  Blood 110:3706-3714.