Main Article Content
Background: The mutations of CCAAT/enhancer binding protein-alpha (CEBPA) gene are evaluated as favorable prognostic tools for acute myeloid leukemia (AML) patients. The gold standard method for detection of CEBPA gene mutations is direct sequencing. This method has some disadvantages, and CEBPA mutations can occur across the whole gene, and there should be a screening test before designating the type of mutation by direct sequencing.
Objective: This study was to evaluate the ability of denaturing high-performance liquid chromatography (DHPLC) for screening CEBPA mutations.
Method: The coding region of CEBPA gene in 114 AML patients and 40 normal controls were screened by DHPLC and confirmed by direct sequencing.
Results: Our results demonstrated that DHPLC is a useful screening test to detect CEBPA gene mutations in AML patients. Fifteen types of CEBPA gene mutations including insertion, duplication, deletion, and substitution were also detected by DHPLC.
Conclusion: A combination of DHPLC and direct sequencing is an appropriate approach for detecting CEBPA mutations.
Pabst T, Mueller BU. Complexity of CEBPA dysregulation in human acute myeloid leukemia. Clin Cancer Res. 2009;15(17):5303-5307. doi:10.1158/1078-0432.CCR-08-2941.
Koschmieder S, Halmos B, Levantini E, Tenen DG. Dysregulation of the C/EBPalpha differentiation pathway in human cancer. J Clin Oncol. 2009;27(4):619-628. doi:10.1200/JCO.2008.17.9812.
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. Nat Genet. 2001;27(3):263-270.
Watkins PJ, Condreay JP, Huber BE, Jacobs SJ, Adams DJ. Impaired proliferation and tumorigenicity induced by CCAAT/enhancer-binding protein. Cancer Res. 1996;56(5):1063-1067.
Timchenko NA, Wilde M, Nakanishi M, Smith JR, Darlington GJ. CCAAT/enhancer-binding protein alpha (C/EBP alpha) inhibits cell proliferation through the p21 (WAF-1/CIP-1/SDI-1) protein. Genes Dev. 1996;10(7):804-815.
Hendricks-Taylor LR, Darlington GJ. Inhibition of cell proliferation by C/EBP alpha occurs in many cell types, does not require the presence of p53 or Rb, and is not affected by large T-antigen. Nucleic Acids Res. 1995;23(22):4726-33.
Leroy H, Roumier C, Huyghe P, Biggio V, Fenaux P, Preudhomme C. CEBPA point mutations in hematological malignancies. Leukemia. 2005;19(3):329-334.
Nerlov C. C/EBPalpha mutations in acute myeloid leukaemias. Nat Rev Cancer. 2004;4(5):394-400.
Radomska HS, Huettner CS, Zhang P, Cheng T, Scadden DT, Tenen DG. CCAAT/enhancer binding protein alpha is a regulatory switch sufficient for induction of granulocytic development from bipotential myeloid progenitors. Mol Cell Biol. 1998;18(7):4301-4314.
Scott LM, Civin CI, Rorth P, Friedman AD. A novel temporal expression pattern of three C/EBP family members in differentiating myelomonocytic cells. Blood. 1992;80(7):1725-1735.
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. Proc Natl Acad Sci U S A. 1997;94(2):569-574.
El-Sharnouby JA, Ahmed LM, Taha AM, Kamal O. Prognostic Significance of CEBPA Mutations and BAALC Expression in Acute Myeloid Leukemia Patients with Normal Karyotype. Eur J Gen Med. 2008;7(1):17-28.
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. J Clin Oncol. 2010;28(16):2739-2747. doi:10.1200/JCO.2009.26.2501.
Wouters BJ, Lowenberg B, Erpelinck-Verschueren CA, van Putten WL, Valk PJ, Delwel R. Double CEBPA mutations, but not single CEBPA mutations, define a subgroup of acute myeloid leukemia with a distinctive gene expression profile that is uniquely associated with a favorable outcome. Blood. 2009;113(13):3088-3091. doi:10.1182/blood-2008-09-179895.
Pabst T, Mueller BU. Transcriptional dysregulation during myeloid transformation in AML. Oncogene. 2007;26(47):6829-6837.
Preudhomme C, Sagot C, Boissel N, et al. Favorable prognostic significance of CEBPA mutations in patients with de novo acute myeloid leukemia: a study from the Acute Leukemia French Association (ALFA). Blood. 2002;100(8):2717-2723.
Marcucci G, Maharry K, Radmacher MD, et al. 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 Clin Oncol. 2008;26(31):5078-5087. doi:10.1200/JCO.2008.17.5554.
Bienz M, Ludwig M, Leibundgut EO, et al. Risk assessment in patients with acute myeloid leukemia and a normal karyotype. Clin Cancer Res. 2005;11(4):1416-1424.
Pabst T, Eyholzer M, Fos J, Mueller BU. Heterogeneity within AML with CEBPA mutations; only CEBPA double mutations, but not single CEBPA mutations are associated with favourable prognosis. Br J Cancer. 2009;100(8):1343-1346. doi:10.1038/sj.bjc.6604977.
Ahn JY, Seo K, Weinberg O, Boyd SD, Arber DA. A comparison of two methods for screening CEBPA mutations in patients with acute myeloid leukemia. J Mol Diagn. 2009;11(4):319-323. doi:10.2353/jmoldx.2009.080121.
Mitchell M, Cutler J. Denaturing HPLC for mutation screening. Methods Mol Biol. 2011;688:17-33. doi:10.1007/978-1-60761-947-5_3.
Xiao W, Oefner PJ. Denaturing high-performance liquid chromatography: A review. Hum Mutat. 2001;17(6):439-474.
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):2321-2326. doi:10.1002/ijc.23796.
Fuster O, Barragan E, Bolufer P, et al. Fragment length analysis screening for detection of CEBPA mutations in intermediate-risk karyotype acute myeloid leukemia. Ann Hematol. 2012;91(1):1-7. doi:10.1007/s00277-011-1234-z.
Kim S, Kim DH, Jang JH, et al. Novel mutations in CEBPA in Korean Patients with acute myeloid leukemia with a normal karyotype. Ann Lab Med. 2012;32(2):153-157. doi:10.3343/alm.2012.32.2.153.
Ahmad F, Rajput S, Mandava S, Das BR. Molecular evaluation of CEBPA gene mutation in normal karyotype acute myeloid leukemia: a comparison of two methods and report of novel CEBPA mutations from Indian acute myeloid leukemia patients. Genet Test Mol Biomarkers. 2012;16(7):707-715. doi:10.1089/gtmb.2011.0317.
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. J Clin Oncol. 2004;22(4):624-633.
Wouters BJ, Louwers I, Valk PJ, Lowenberg 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):389-390.
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):2493-2494.
Lin LI, Chen CY, Lin DT, 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 Cancer Res. 2005;11(4):1372-1379.
Lin LI, Lin TC, Chou WC, Tang JL, Lin DT, Tien HF. A novel fluorescence-based multiplex PCR assay for rapid simultaneous detection of CEBPA mutations and NPM mutations in patients with acute myeloid leukemias. Leukemia. 2006;20(10):1899-1903.
Szankasi P, Ho AK, Bahler DW, Efimova O, Kelley TW. Combined testing for CCAAT/enhancer-binding protein alpha (CEBPA) mutations and promoter methylation in acute myeloid leukemia demonstrates shared phenotypic features. Leuk Res. 2011;35(2):200-207. doi:10.1016/j.leukres.2010.09.018.
Dufour A, Schneider F, Hoster E, et al. Monoallelic CEBPA mutations in normal karyotype acute myeloid leukemia: independent favorable prognostic factor within NPM1 mutated patients. Ann Hematol. 2012;91(7):1051-1063. doi:10.1007/s00277-012-1423-4.
Benthaus T, Schneider F, Mellert G, et al. Rapid and sensitive screening for CEBPA mutations in acute myeloid leukaemia. Br J Haematol. 2008;143(2):230-239. doi:0.1111/j.1365-2141.2008.07328.x.
Wongboonma W, Thongnoppakhun W, Auewarakul CU. A single-tube allele specific-polymerase chain reaction to detect T315I resistant mutation in chronic myeloid leukemia patients. J Hematol Oncol. 2011;4:7. doi:10.1186/1756-8722-4-7.