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Test Code CEBPAB or CEBPAM CEBPA Mutation Analysis, PB or BM

Specimen Type

Blood or Bone Marrow

Container

Lavender Top (EDTA)

Preferred Volume

5 mL

Offsite Collection Instructions

Peripheral blood/Bone Marrow samples collected in EDTA (lavender top) transported at room temperature to Lab Services Center. Frozen cells from blood or bone marrow from other University of Chicago CLIA laboratories may come in on ice. This sample type may be used if no other sample is available.  

UCMC Collection Instructions

Peripheral blood/Bone Marrow samples collected in EDTA (lavender top), stored at room temperature or refrigerator. Samples should be sent no later than 48 hours from the time of collection to UCM Lab Services Center at ambient temperature.

Availability

Monday – Friday, 8:00am – 5:00pm

Turnaround Time

10-12 business days

STAT Availability

Not Available

Test Usage

CEBPA (CCAAT/enhancer-binding protein-Alpha) is a single-exon gene, mapped to 19q13.1, which encodes a leucine zipper transcription factor with an important role in myeloid differentiation. CEBPA mutations are encountered in approximately 8% to 15% of AML, most commonly in cytogenetically normal AML (CN-AML)3-5. Patients with CEBPA mutated AML (CEBPA-AML) may carry one (single-mutated CEPBA) or two mutated alleles (double-mutated CEPBA). CEBPA mutations are highly variable and can occur across the entire coding region of the gene. Mutations in CEBPA single-mutant cases are distributed in the entire coding region, with a greater portion in the midregion. CEBPA mutation status has gained utility as an independent prognostic factor for CN-AML, which is categorized as an intermediate risk group. Recent studies have shown that double mutations in the CEBPA gene is associated with lower relapse rates and improved survival, and therefore predicts an improved prognosis for patients with CN-AML. Given its prognostic importance, AML with mutated CEBPA represents a provisional entity in the 2008 World Health Organization classification of tumors of the hematopoietic and lymphoid tissues. Consequently, testing for CEBPA mutations may assist in the prognostic stratification of these patients, which ultimately may facilitate the selection of individualized and risk adapted therapies.

Test Methodology

DNA is isolated from peripheral blood or bone marrow specimens using the QIAamp DNA Blood Mini Kit (Qiagen). Following extraction, DNA is quantified using the Qubit fluorometric assay (Thermo Fisher Scientific). The entire coding region of CEBPA is amplified via a single long-range polymerase chain reaction (PCR) reaction. The resulting amplicon is quantified using the Qubit fluorometric assay (Thermo Fisher Scientific) and is fragmented (with incorporation of partial adapter sequences) using Nextera XT reagents (Illumina Inc.). A subsequent PCR amplification step is performed to complete the adapter sequences and to incorporate patient-specific index sequences to allow for pooled sequencing. Final libraries are quantified using a quantitative PCR assay (Library Quantification Kit, KAPA Biosystems), and are then pooled and sequenced via the Illumina MiSeq system to produce paired end reads of at least 151 bp in length.
 

Additional Information

This is no longer a sendout test and is now offered in house at the UCMC Molecular Pathology Laboratory.
Specimen requirements:

Formalin-fixed paraffin-embedded (FFPE) tissues cannot be used. Appropriate specimens contains >20% tumor cells, as the limit of detection for mutant allelic frequency is 10% for this assay. Specimens with 10-20% tumor cells may be tested at the discretion of the attending molecular pathologist.
Reporting:

Mutations with known or possible pathogenic or clinical significance are reported. Variants of unknown significance are reported as such.
 

REFERENCES 1. Tenen DG, Hromas R, Licht JD, Zhang DE. Transcription factors, normal myeloid development, and leukemia. Blood 1997; 90(2): 489-519.
2. Zhang DE, Zhang P, Wang ND, Hetherington CJ, Darlington GJ, Tenen DG. Absence of granulocyte colony-stimulating factor signaling and neutrophil development in CCAAT enhancer binding protein alpha-deficient mice. Proceedings of the National Academy of Sciences of the United States of America 1997; 94(2): 569-74.
3. Pabst T, Mueller BU, Zhang P, et al. Dominant-negative mutations of CEBPA, encoding CCAAT/enhancer binding protein-alpha (C/EBPalpha), in acute myeloid leukemia. Nature genetics 2001; 27(3): 263-70.
4. Schlenk RF, Dohner K, Krauter J, et al. Mutations and treatment outcome in cytogenetically normal acute myeloid leukemia. The New England journal of medicine 2008; 358(18): 1909-18.
5. Frohling S, Schlenk RF, Stolze I, et al. CEBPA mutations in younger adults with acute myeloid leukemia and normal cytogenetics: prognostic relevance and analysis of cooperating mutations. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2004; 22(4): 624-33.
6. Green CL, Koo KK, Hills RK, Burnett AK, Linch DC, Gale RE. 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. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2010; 28(16): 2739-47.
7. Dufour A, Schneider F, Metzeler KH, et al. Acute myeloid leukemia with biallelic CEBPA gene mutations and normal karyotype represents a distinct genetic entity associated with a favorable clinical outcome. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2010; 28(4): 570-7.
8. Fasan A, Haferlach C, Alpermann T, et al. The role of different genetic subtypes of CEBPA mutated AML. Leukemia 2014; 28(4): 794-803.
9. Behdad A, Weigelin HC, Elenitoba-Johnson KS, Betz BL. A Clinical Grade Sequencing-Based Assay for CEBPA Mutation Testing: Report of a Large Series of Myeloid Neoplasms. The Journal of molecular diagnostics : JMD 2015; 17(1): 76-84.
10. Ho PA, Alonzo TA, Gerbing RB, Pollard J, Stirewalt DL, Hurwitz C, Heerema NA, Hirsch B, Raimondi SC, Lange B, Franklin JL, Radich JP, Meshinchi S. Prevalence and prognostic implications of CEBPA mutations in pediatric AML: a report from the Children’s Oncology Group. Blood. 2009;113(26):6558-66.
11. Lin L, Chen C et al. Characterization of CEBPA Mutations in Acute Myeloid Leukemia: Most Patients with CEBPA Mutations Have Biallelic Mutations and Show a Distinct Immunophenotype of the Leukemic Cells. Clin Can Res. 2005;11:1372–1379.
12. Fuchs O, Provaznikova D et al. CEBPA polymorphisms and mutations in patients with acute myeloid
leukemia, myelodysplastic syndrome, multiple myeloma and non-Hodgkin’s lymphoma. Blood Cells, Molecules, and Diseases. 2008; 40:401–405.
13. Resende C, Regalo G, Durães C, Carneiro F, Machado JC. Genetic changes of CEBPA in cancer: mutations or polymorphisms? J Clin Oncol. 2007; 25(17):2493-4. UCM Clinical Genomics Laboratory 5841 S Maryland Ave, MC1089 Chicago, IL 60637  Page 25 of 26
14. Wouters BJ, Louwers I, Valk PJ, Löwenberg B, Delwel R. A recurrent in-frame insertion in a CEBPA transactivation domain is a polymorphism rather than a mutation that does not affect gene expression profiling-based clustering of AML. Blood. 2007; 109(1):389-90.
15. Biggio V, Renneville A, Nibourel O, Philippe N, Terriou L, Roumier C, Amouyel P, Cottel D, Castaigne S, Dombret H, Thomas X, Fenaux P, Preudhomme C; French alfa group. Recurrent in-frame insertion in C/EBPalpha TAD2 region is a polymorphism without prognostic value in AML. Leukemia. 2008;22(3):655-7.
16. Leecharendkeat A, Tocharoentanaphol C, Auewarakul CU. 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. 2008; 123(10):2321-6

CPT Code

81403

STAT Turnaround Time

N/A

Reference Range

Interpretive report will be provided.

Critical Results

None.

Test Limitations

This test will not detect mutations outside the targeted genomic region (hg19 coordinates chr19:33792220-33793332).Copy number alterations, large-scale insertions/deletions, gene arrangements and translocations will not be detected.  Limit of detection for this assay is 10% mutant alleles (~20% tumor cellularity), thus it is not intended to detect minimal residual disease.  Assay sensitivity is 10% mutant allele frequency (MAF), thus false-negative results may occur when there is a lower mutant allele burden.

Minimum Volume

200 uL

Test Includes

The assay is intended to complement the OncoHeme panel, and covers the whole coding sequence of CEBPA (CCAAT/enhancer-binding protein-Alpha), a single-exon gene mapping to 19q13.1. CEBPA mutations are encountered in 8-15% of AML and represent one of the most common mutations in cytogenetically normal (CN) AML. The CEBPA assay is clinically validated to detect mutations at a level of >10% mutant allelic frequency (MAF), suitable for diagnostic specimens from CN-AML patients (>20% leukemic blasts by definition).

Fee Code

79832

Synonyms

CEBPAPB,
CEBPABM