Molecular Characterization of PML/RARO Fusion Gene in Thai patients with Acute Promyelocytic Leukemia (APL) by nested RT-PCR
Keywords:
Acute promyelocytic leukemia, Chromosome translocation, PML-RARα gene variant, Nested PCRAbstract
Background: Patients with acute promyelocytic leukemia (ArL) generally cary a unique transtocation between chromosomes 15 and 17 which generates a characteristic PML/RAR fusion gene. Chromosome 15 breakpoints fall within the three breakpoint cluster regions (bcr1, bcr2 and bcr3) and chromosome 17 breakpoints occur within intron 2. The bcr1 and bcr3 correspond to PML introns 6 and 3, respectively, whereas bcr2 is located within PML exon 6. Objective: To develop a molecular assay for the detection of PML/RAROl fusion gene and to determine the frequency and type of PML-RAR
gene variants (bcr1, bcr2 or bcr 3) and their association with leukemia karyotypes in Thai APL patients. Materials and Methods: APL was diagnosed using morphologic and immunophenotypic analysis of leukemic cells. Flow cytometry was performed using a cocktail of monoclonal antibodies and CD45/
SSC selection gates. Karyotypic analysis was done by conventional G- and Q- banding methods. The breakpoint sites of the PML/RAR fusion gene were identified by reverse transcriptase-polymerase chain reaction (RT-PCR) with a nested approach. NB4 leukemic cell line was used as a positive control for PML-RAR
gene. Results: The bcr1, bcr2, and bcr3 variants, were detected in 81.25%, 3.125%, and 0%, respectively, of Thai APL patients diagnosed by flow cytometry (n = 64). The majority of cytogenetically confirmed APL cases had bcr1 (10 out of 12 cases) and bcr2 (2 out of 12 cases). In APL patients with a normal karyotype (n = 36) or insufficient metaphases (n = 16), PML-RAR
gene was detected in 40 cases (76.29%). Conclusion: bcr1 is the most frecuent PML-RAR
gene variant detected in Thai APL patients. Molecular analysis of PML-RAR
appears to be more sensitive than conventional karyotypic analysis for the confirmation of the diagnosis of APL.
Downloads
References
Warrell RP Jr, de The H, Wang ZY, Degos L. Acute promyelocytic leukemia. N Engl J Med 1993;329:177-89.
Lowenberg B, Downing JR, Bumnett A. Acute myeloid leukemia. N Engl J Med 1999;341:1051-62.
Grignani F, Fagioli M, Alcalay M, Longo L, Pandolfi PP, Donti E, et al. Acute promyelocytic leukemia: from genetics to treatment. Blood 1994;83:10-25.
Melnick A, Licht JD. Deconstructing a disease: RARalpha, its fusion partners, and their roles in the pathogenesis of acute promyelocytic leukemia. Blood 1999:93.3167-215.
Ruggero D, Wang ZG, Pandolfi PP.The puzzling multiple lives of PML and its role in the genesis of cancer. Bioessays 2000;22:827-35.
Pandolfi PP. Oncogenes and tumor suppressors in the molecular pathogenesis of acute promyelocytic leukemia. Hum Mol Genet 2001;10:769-75.
Slack JL, Arthur DC, Lawrence D, Mrozek K, Mayer RJ, Davey FR, et al. Secondary cytogenetic changes in acute promyelocytic leukemia-prognostic importance in patients treated with chemotherapy alone and association with the intron 3 breakpoint of the PML gene: a Cancer and Leukemia Group B study. J Clin Oncol 1997;15:1786-95.
Lo Coco F, Diverio D, Falini B, Biondi A, Nervi C, Pelicci PG. Genetic diagnosis and molecular monitoring in the management of acute promyelocytic leukemia. Blood 1999:94:12-22.
Lo-Coco F, Breccia M, Diverio D. The importance of molecular monitoring in acute promyelocytic leukaemia. Best Pract Res Clin Haematol 2003;16:503-20.
Basso G, Buldini B, De Zen L, Orfao A. New methodologic approaches for immunophenotyping acute leukemias. Haematologica 2001;86:675-92.
Kaleem 2, Crawford E, Pathan MH, Jasper L, Covinsky MA, Johnson LR, et al. Flow cytometric analysis of acute leukemias. Diagnostic utility and critical analysis of data. Arch Pathol Lab Med 2003;127:42-8.
Orfao A, Ortuno F, de Santiago M, Lopez A, San Miguel J. Immunophenotyping of acute leukemias and myelodysplastic syndromes. Cytometry 2004;58A:62-71.
Biondi A, Rambaldi A, Pandolfi PP, Rossi V, Giudici G, Alcalay M, et al. Molecular monitoring of the myl/retinoic acid receptor-alpha fusion gene in acute promyelocytic leukemia by polymerase chain reaction. Blood 1992;80:492-7.
van Dongen JJ, Macintyre EA, Gabert JA, Delabesse E, Rossi V, Saglio G, et al. Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease. Report of the BIOMED-1 Concerted Action: investigation of minimal residual disease in acute leukemia. Leukemia 1999;13:1901-28.
Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol chloroform extraction. Anal Biochem 1987;162:156-9.
Degos L. The history of acute promyelocytic leukaemia. Br J Haematol 2003;122:539-53.
Lanotte M, Martin-Thouvenin V, Najman S, Balerini P, Valensi F, Berger R. NB4, a maturation inducible cell line with t(15;17) marker isolated from a human acute promyelocytic leukemia (M3). Blood 1991;77:1080-6.
Harris NL, Jaffe ES, Diebold J, Flandrin G, Muller-Hermelink HK, Vardiman J, et al. World Health Organization classification of neoplastic diseases of the hematopoietic and lymphoid tissues: report of the Clinical Advisory Committee meeting-Airlie House, Virginia, November 1997. J Clin Oncol 1999;17:3835-49.
Vardiman JW, Harris NL, Brunning RD. The World Health Organization (WHO) classification of the myeloid neoplasms. Blood 2002;100:2292-302.
Paietta E. Comments on the 2001 WHO proposal for the classification of haematopoietic neoplasms. Best Pract Res Clin Haematol 2003;16:547-59.
Hiorns LR, Min T, Swansbury GJ, Zelent A, Dyer MJ, Catovsky D. Interstitial insertion of retinoic acid receptor-alpha gene in acute promyelocytic leukemia with normal chromosomes 15 and 17. Blood 1994;83:2946-51.
Naoe T, Kudo K, Yoshida H, Horibe K, Ohno R. Molecular analysis of the t(15;17) translocation in de novo and secondary acute promyelocytic leukemia. Leukemia 1997;11(Suppl 3):287-8.
Chillon CM, Garcia-Sanz R, Balanzategui A, Ramos F, Fernandez-Calvo J, Rodriguez MJ, et al. Molecular characterization of acute myeloblastic leukemia according to the new WHO classification: a different distribution in Central-West Spain. Haematologica
:162-6.
Sucic M, Zadro R, Burazer B, Labar B, Nemet D, Mrsic M, et al. Acute promyelocytic leukemia M3: cytomorphologic, immunophenotypic, cytogenetic, and molecular variants. J Hematother Stem Cell Res 2002;11:941-50.
Douer D, Santillana S, Ramezani L, Samanez C, Slovak ML, Lee MS, et al. Acute promyelocytic leukaemia in patients originating in Latin America is associated with an increased frequency of the bcr1 subtype of the PML/RARalpha fusion gene. Br J Haematol 2003;122:563-70.
Benedetti L, Levin AA, Scicchitano BM, Grignani F, Allenby G, Diverio D, et al. Characterization of the retinoid binding properties of the major fusion products present in acute promyelocytic leukemia cells. Blood 199790:1175-85.
Burnett AK, Grimwade D, Solomon E, Wheatley K, Goldstone AH. Presenting white blood cell count and kinetics of molecular remission predict prognosis in acute promyelocytic leukemia treated with all-transretinoic acid: result of the Randomized MRC Trial. Blood 1999;93:4131-43.
Downloads
Published
Issue
Section
License
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
