CEBPA TARGETED MUTATION ANALYSIS
Label Name: CEBPA MUTATN
Lab Discipline: Molecular Diagnostics
Institution:  Duke University Health System 
EAP ID:  LAB6157 
Last Review:  3/16/2017 2:49:28 PM
Specimen Type
  Blood
Container & Volume
  Age Group   Container   Volume  
  0  - 18 Years LAVENDER TOP TUBE 2  ML
Label Reminders
  Be sure to include the patient's name, history #, date and time of collection, and collector's initials.

Collection Notes
  Adult:
  • Peripheral blood: One lavender-top EDTA tube (minimum of 3 ml) is required. Send unprocessed peripheral blood to the laboratory promptly at ambient temperature.

    Bone Marrow: One lavender-top EDTA tube (minimum of 1 ml) is required. Send unprocessed bone marrow to the laboratory promptly at ambient temperature.

    PERIPHERAL BLOOD AND BONE MARROW SPECIMENS MUST ARRIVE AT THE LABORATORY WITHIN 48 HOURS OF COLLECTION. THE SPECIMEN CANNOT BE FROZEN. GREEN-TOP (HEPARIN) TUBES ARE NOT ACCEPTABLE FOR TESTING
 
Transport
  Deliver to laboratory at ambient temperature. If there is a delay of more than 24 hours in delivery, please refrigerate the sample. Samples must arrive within 48 hours of collection. DO NOT FREEZE.

Turn Around Time -  Routine: 14 days   Stat: N/a
Reference Values
CEBPA MUTATN
NO MUTATION DETECTED
Methodology
  This assay uses PCR amplification followed by Sanger DNA sequencing to detect mutations in the CEBPA gene that confer a favorable prognosis in patients with acute myeloid leukemia (AML). The coding sequence and flanking UTR sequences (minimum of 20 base pairs) of the CEBPA gene are amplified from purified genomic DNA by PCR. Two overlapping primer sets with seven total PCR targets are used to amplify the entire coding region of CEBPA. The resulting PCR products are treated with an exonuclease/ phosphatase mixture (ExoSAP-IT, USB) to remove excess PCR primers and nucleotides. These purified DNA amplicons are then bi-directionally sequenced using the amplification primers and the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystem). These products are purified with the Big Dye XTerminator Purification Kit and resolved using the ABI 3130xl Genetic Analyzer. Data is analyzed using the ABI Data Collection software v3.0, Sequencing Analysis software 5.2 and SeqScape software v2.6 and compared to the reference sequence (GenBank NM 004364.3) for the CEBPA gene.

This test was developed and its performance characteristics determined by DUHS Clinical Molecular Diagnostic Laboratory. It has not been cleared or approved by the U.S. Food and Drug Administration. This test is used for clinical purposes. It should not be regarded as investigational or for research. This laboratory is certified under the Clinical Laboratory Improvement Amendments of 1988 ("CLIA") as qualified to perform high complexity clinical testing.
   
   
Clinical Significance and Interpretive Data
    BACKGROUND:

CEBPA is an intronless gene mapped to chromosome 19q13 that encodes the transcription factor C/EBP alpha (CCAAT/enhancer binding protein-alpha). CEBPA encodes two functional isoforms, a major 42kD transcript (p42kD), and a second truncated transcript (p30kD) normally transcribed at low levels. The full-length p42kD contains three trans-activation elements at the N-terminal, TE-I, TE-II, and TE-III which cooperatively function in transcriptional activation. The p30kD utilizes an alternate translational start codon at amino acid 120, leaving only TE-III. Both proteins house a basic leucine zipper motif (bZIP) at the C-terminus which mediates DNA binding and dimerization.

The CEBPA transcription factor is expressed mainly in myelomonocytic cells and plays an essential role in granulopoiesis. Upregulation of CEBPA occurs during myeloid differentiation and induces granulocytic development while blocking moncytic development. Loss of expression or activity, by results in a lack of granulocyte progenitor formation and an accumulation of immature myeloid blast cells. Additionally, CEBPA regulates the transcription of several cell cycle regulators that arrest mitotic growth in the terminally differentiated state. Thus, in the absence of normal CEBPA activity an increase in myeloid blast formation occurs, as is the case in AML.

CLINICAL SIGNIFICANCE

Approximately 40-50% of patients with AML will have a normal karyotype (NK) AML. Patients with NK-AML have an intermediate prognosis between patients with favorable recurrent cytogenetic abnormalities, such as t(8;21) or t(15;17), and patients with unfavorable cytogenetic abnormalities, such as deletion of 7q. The prognosis of NK-AML patients can be stratified based on gene specific mutational events, including mutations in the CCAAT/enhancer binding protein alpha (CEBPA). Mutations in CEBPA have been reported in 13%-15% of those with NK-AML, and have generally been associated with a favorable outcome with regard to increased remission duration and overall survival outcomes compared with wild-type CEBPA. The prognostic impact of CEBPA mutations appears to be influenced by the presence of single or double mutations, and by mutations in other genes, including NPM1 and FLT-3. The presence of a double (biallelic) mutation in CEBPA, or a single mutation in CEBPA with a mutation in NPM1, has been reported as favorable, whereas the presence of the FLT-3 ITD nullifies the apparent benefit. The prognostic significance of a single CEBPA mutation in isolation is uncertain. Additional chromosomal aberrations and patient-specific clinical factors may also modify the influence of CEBPA mutations. Therefore, this assay is intended for use as an aid in developing patient-specific prognoses and is not a substitute for a complete pathologic and clinical evaluation, or physician's judgment and clinical experience.

REFERENCES:

1.Nerlov C, Ziff EB. Three levels of functional interaction determine the activity of CCAAT/enhancer binding protein-alpha on the serum albumin promoter. Genes Dev. 1994;8(3):350-62.

2. Pabst T, et. al. Dominant-negative mutations of CEBPA encoding CCAAT/enhancer binding protein-á (C/EBPá), in acute myeloid leukemia. Nature Genetics. 2001;27:263-70.

3. Khanna-Gupta A. Sumoylation and the function of CCAAT enhancer binding protein alpha (C/EBPá). Blood Cells, Molecules, and Diseases. 2008;41:77-81.

4. Fuchs O, et. al. CEBPA polymorphisms and mutations in patients with acute myeloid leukemia, myelodysplastic syndrome, multiple myeloma and non-Hodgkin’s lymphoma. Blood Cells, Molecules, & Disease. 2008;40:401-5.

5. Green CL, et. al. Prognostic significance of CEBPA mutations in a large cohort of younger adult patients with acute myeloid leukemia: impact of double CEBPA mutations and the interaction with FLT3 and NPM1 mutations. J Clin Oncol. 2010; epub May 3rd.

6. Swerdlow SH, et. al. WHO classification of tumours of the haematopoietic and lymphoid tissues (ed 4). Geneva, Switzerland, WHO Press, 2008.

7. National Comprehensive Cancer Network. Acute Myeloid Leukemia Guidelines. NCCN 2013 March 1:Version 2.2013

8. Green CL, et al. Prognostic significance of CEBPA mutations in a large cohort of younger adult patients with acute myeloid leukemia: impact of double CEBPA mutations and the interaction with FLT3 and NPM1 mutations. J Clin Oncol 2010 Jun 1;28(16):2739-47.

9. Dufour A, et al. Acute myeloid leukemia with biallelic CEBPA gene mutations and normal karyotype represents a distinct genetic entity associated with a favorable clinical outcome. J Clin Oncol 2010 Feb 1;28(4):570-7.

10. Dufour A, et al. Monoallelic CEBPA mutations in normal karyotype acute myeloid leukemia: independent favorable prognostic factor within NPM1 mutated patients. Ann Hematol 2012;91:1051–1063.
Schlenk RF, et al. Mutations and treatment outcome in cytogenetically normal acute myeloid leukemia. N Engl J Med 2008; 358: 1909–1918.

11. ACMG Laboratory Practice Committeee Working Group. (2000) ACMG recommendations for standards for interpretation of sequence variations. Genetics in Medicine Volume 2.

12. Maddalena A et al. (2005) Technical standards and guidelines: molecular genetic testing for ultra-rare disorders. Genetics in Medicine 7:571-583.

Test Interpretation (11,12):

• DNA sequence data is compaired the reference sequence (GenBank NM 004364.3) for the CEBPA gene.

• A sample showing a clear heterozygous or homozygous change is evidence of a sequence difference and indicates either a possible mutation or a polymorphism.

• A sequence change is also reported for samples showing clear evidence of a deletion, insertion, duplication, or intron/exon border splice variant.

• Examination of CEBPA sequences from a patient’s buccal swab would allow for definitive assignment of any sequence changes as constitutional or acquired.

• Databases used in interpretation:

o The Human Gene Mutation Database (HGMD), http://www.hgmd.cf.ac.uk/ac/index.php

o Catalogue of somatic Mutations in Cancer (COSMIC database): http://www.sanger.ac.uk/genetics/CGP/cosmic/

o Polyphen: ACC# P49715
   
Indications
    Recent data suggests that AML cases with both a C-terminal and an N-terminal CEBPA mutation are associated with a better prognosis. However,the prognostic impact of CEBPA mutations may be further influenced by the subtype of AML, mutations in other genes (including FLT3 and NPM1), chromosomal aberrations or patient specific clinical factors. Thus, this assay is intended for use as an aid in developing patient specific prognostic predictions but is not a substitute for a complete pathologic and clinical evaluation, or physician's judgment and clinical experience.
   
Limitations
    The sensitivity and specificity of DNA sequencing is high for the detection of nucleotide base changes, small deletions and insertions in the regions analyzed. Only the coding regions of the CEBPA gene and immediate flanking intronic sequences were examined. Changes in the promoter region, farther into the introns, or in other non-coding regions of the gene, would not be detected. Mutations in genes other than CEBPA would not be identified. Large deletions, duplications, multiple exon insertions, sequence alterations adversely affecting primer binding, and complete deletion of one allele may not be identified using these methods. Mutations or polymorphisms in the DNA oligonucleotide primer binding regions, poor DNA quality, insufficient DNA quantity or the presence of PCR inhibitors can result in uninterpretable or (rarely) inaccurate results. This assay may not detect an acquired mutation that is present below the 15% detection limit (i.e., mutant cell population of < 30%). For additional information or for help interpreting the results of this test, clinicians should contact the DUHS Clinical Molecular Diagnostics Laboratory. Patients should contact their healthcare provider with any questions related to this report.
   
Test Synonyms
  Synonym(s):  AML
Synonym(s):  NORMAL KARYOTYPE
Synonym(s): CEBPA
Molecular Diagnostics Laboratory
(MDX)

Medical Director:
 Michael Datto, M.D., Ph.D.
 Phone: 919-684-6965
 Email: michael.datto@duke.edu
Lab Director:
 Catherine Rehder Ph.D, FACMG
 Phone: 919-613-8434
 Email: catherine.rehder@duke.edu
Lab Director:
 Siby Sebastian Ph.D., DABMG
 Phone: 919-613-8432
 Email: siby.s@duke.edu

Address: 
 Wadsworth Bldg, Cytogenetics, Rm 0220
 2351 Erwin Rd
 Durham,  NC  27705
 Phone: 919-684-2698
 FAX: 919-668-5424

Performing Times: