Detection of recurrent balanced translocations in Acute Myeloid Leukemia by Multiplex RT-PCR
Keywords:
acute myeloid leukemia, multiplex, polymerase chain reaction
Abstract
Background: Diagnosis of acute myeloid leukemia (AML) has to be established by immunophenotyping and molecular testing for presence of recurrent genetic aberrations. This helps clinician to plan therapeutic protocol as AMLs are a heterogenous group of malignancies having varied response to treatment and prognosis. Multiplex Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) is method capable of detecting 28 known fusion-transcripts of acute leukemia including cryptic translocations which would otherwise be missed on conventional cytogenetics.Methods: In this case series, a total of 19 consecutive patients diagnosed as AML were included. Morphology and cytochemical character of blasts were ascertained. Immunophenotying was carried out by flowcytometry. Messenager RNA (mRNA) was extracted from peripheral blood or bone marrow aspirates and converted to Complimentary DNA (cDNA). The cDNA was then subjected to multiplex RT-PCR for presence of fusion-transcripts using primers from Hemavision kit, DNA technologies, Denmark. The amplified product was correlated with the morphological, cytochemical and immunophenotypic character of AML.Result: Of the 19 cases of AML, 12 cases showed amplification of a fusion transcript. Most cases with fusion-transcripts showed an expected blast morphology and immunophenotype. Multiplex RT-PCR picked up presence of rare translocations such as t(16,21), t(9;22), t(6;9) and t(3;5), with unique clinicopathological features.Conclusion: Multiplex RT-PCR is a unique assay to identify multiple known recurrent genetic aberrations, including many splice variants. This aids in accurate subtyping of AML and helps the clinician to formulate a specific treatment plan for the patient. DOI: 10.21276/apalm.2017.1071References
1. Vardiman JW, Brunning RD, Arber DA et al. Introduction and overview of classification of the myeloid neoplasms. In: Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H et al, editors. WHO Classification of Tumors of Haematopoietic and Lymphoid Tissues. Lyon, France: IARC Press, 2008; p. 18-30
2. Olesen LH, Clausen N, Dimitrijevic A, Kerndrup G, Kjeldsen E, Hokland P. Prospective application of a multiplex reverse transcription-polymerase chain reaction assay for the detection of balanced translocations in leukaemia: a single-laboratory study of 390 paediatric and adult patients. Br J Haematol. 2004; 127(1):59-66
3. Hutchings HM, Wirenfeldt KT, Hasle H, Schmiegelow K, Brondum-Nielsen K, Johnsen HE. Multiplex reverse transcriptase polymerase chain reaction screening in acute myeloid leukemia detects cytogenetically unrevealed abnormalities of prognostic significance. Haematologica. 2005; 90(7):984-986
4. Salto-Tellez M, Shelat SG, Benoit B et al. Multiplex RT-PCR for the detection of leukemia-associated translocations: validation and application to routine molecular diagnostic practice. J Mol Diagn. 2003; 5(4):231-236
5. Strehl S, König M, Mann G, Haas OA. Multiplex reverse transcriptase-polymerase chain reaction screening in childhood acute myeloblastic leukemia. Blood. 2001 ;97(3):805-808
6. Khalidi HS, Medeiros LJ, Chang KL, Brynes RK, Slovak ML, Arber DA. The immunophenotype of adult acute myeloid leukaemia: high frequency of lymphoid antigen expression and comparison of immunophenotype, FAB classification, and karyotypic abnormalities. Am J Clin Pathol 1998; 109(2):211–220
7. Head DR. Diagnosis and classification of the acute leukemia and myelodysplastic syndromes. In: Greer JP, Foerster J, Rodgers GM, Paraskevas F, Glader B, Arber DA et al editors. Wintrobe’s clinical hematology. 12th ed: Lippincott Williams; 2009.p.1809-1819
8. Sandler DP, Collman GW. Cytogenetic and environmental factors in the etiology of the acute leukaemias in adults. Am J Epidemiol 1987; 126:1017–1032
9. Kotwal J, Manoj MG, Malik A, Sinha K, Singh D, Dutta V. Role of multiplex reverse-transcriptase PCR for diagnosis of Acute myelosis in an infant with Down's syndrome. Med J Armed Forces India. 2015;71:S12-5
10. Choi HJ, Kim HR, Shin MG, Kook H, Kim HJ, Shin JH, et al. Spectra of Chromosomal Aberrations in 325 Leukemia Patients and Implications for the Development of New Molecular Detection Systems. J Korean Med Sci. 2011; 26(7):886-92
11. Arber DA, Brunning RD, Le Beau MM, Falini B. Acute myeloid leukemia with recurrent genetic abnormalities In: Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW (Eds): WHO classification of Tumors of haematopoietic and Lymphoid Tissues. IARC: Lyon; 2008.p.110-123
12. Licht JD, Chomienne C, Goy A, Chen A, Scott AA, Head DR, et al. Clinical and molecular characterization of a rare syndrome of acute promyelocytic leukemia associated with translocation (11;17). Blood 1995; 85:1083
13. Guidez F, Ivins S, Zhu J, Söderström M, Waxman S, Zelent A. Reduced retinoic acid-sensitivities of nuclear receptor co-repressor binding to PML-and PLZF-RARα underlie molecular pathogenesis and treatment of acute promyelocytic leukemia. Blood.1998 ;91(8):2634-2642
14. Gilliland DG, Griffin JD. The roles of FLT3 in hematopoiesis and leukemia. Blood 2002;100:1532–1542
15. Paschka P, Marcucci G, Ruppert AS, Mrozek K, Chen H, Kittles R A et al (2006). Adverse prognostic significance of KIT mutations in adult myeloid leukemia with inv(16) and t(8;21): a Cancer and Leukemia Group B study. J Clin Oncol 24:3904-3911
16. Mrózek K, Heinonen K, Lawrence D et al. Adult patients with de novo acute myeloid leukemia and t (9; 11)(p22; q23) have a superior outcome to patients with other translocations involving band 11q23: a cancer and leukemia group B study. Blood. 1997;90(11):4532-8
17. Sadamori N, Yao E, Tagawa M et al. 16;21 translocation in acute non lymphocytic leukemia with abnormal eosinophils: a unique subtype. Acta Haematol. 1990; 84(4):212-6
18. Gopal MM, Kotwal J, Kapoor R. Acute Myeloid Leukemia with BCR/ABL Fusion Chimera. Indian Journal of Hematology and Blood Transfusion. 2014;30(1):280-2
19. Oyarzo MP, Lin P, Glassman A, Bueso-Ramos CE, Luthra R, Medeiros LJ. Acute myeloid leukemia with t (6; 9)(p23; q34) is associated with dysplasia and a high frequency of flt3 gene mutations. American journal of clinical pathology. 2004;122(3):348-58
20. Delaunay J, Vey N, Leblanc T et al. Prognosis of inv (16)/t (16; 16) acute myeloid leukemia (AML): a survey of 110 cases from the French AML Intergroup. Blood. 2003;102(2):462-9
21. Raimondi SC, Dubé ID, Valentine MB et al. Clinicopathologic manifestations and breakpoints of the t(3;5) in patients with acute non lymphocytic leukemia. Leukemia. 1989; 3(1):42-7
22. Song MJ, Kim HJ, Park CH et al. Diagnostic utility of a multiplex RT-PCR assay in detecting fusion transcripts from recurrent genetic abnormalities of acute leukemia by WHO 2008 classification. Diagnostic molecular pathology. 2012;21(1):40-4
2. Olesen LH, Clausen N, Dimitrijevic A, Kerndrup G, Kjeldsen E, Hokland P. Prospective application of a multiplex reverse transcription-polymerase chain reaction assay for the detection of balanced translocations in leukaemia: a single-laboratory study of 390 paediatric and adult patients. Br J Haematol. 2004; 127(1):59-66
3. Hutchings HM, Wirenfeldt KT, Hasle H, Schmiegelow K, Brondum-Nielsen K, Johnsen HE. Multiplex reverse transcriptase polymerase chain reaction screening in acute myeloid leukemia detects cytogenetically unrevealed abnormalities of prognostic significance. Haematologica. 2005; 90(7):984-986
4. Salto-Tellez M, Shelat SG, Benoit B et al. Multiplex RT-PCR for the detection of leukemia-associated translocations: validation and application to routine molecular diagnostic practice. J Mol Diagn. 2003; 5(4):231-236
5. Strehl S, König M, Mann G, Haas OA. Multiplex reverse transcriptase-polymerase chain reaction screening in childhood acute myeloblastic leukemia. Blood. 2001 ;97(3):805-808
6. Khalidi HS, Medeiros LJ, Chang KL, Brynes RK, Slovak ML, Arber DA. The immunophenotype of adult acute myeloid leukaemia: high frequency of lymphoid antigen expression and comparison of immunophenotype, FAB classification, and karyotypic abnormalities. Am J Clin Pathol 1998; 109(2):211–220
7. Head DR. Diagnosis and classification of the acute leukemia and myelodysplastic syndromes. In: Greer JP, Foerster J, Rodgers GM, Paraskevas F, Glader B, Arber DA et al editors. Wintrobe’s clinical hematology. 12th ed: Lippincott Williams; 2009.p.1809-1819
8. Sandler DP, Collman GW. Cytogenetic and environmental factors in the etiology of the acute leukaemias in adults. Am J Epidemiol 1987; 126:1017–1032
9. Kotwal J, Manoj MG, Malik A, Sinha K, Singh D, Dutta V. Role of multiplex reverse-transcriptase PCR for diagnosis of Acute myelosis in an infant with Down's syndrome. Med J Armed Forces India. 2015;71:S12-5
10. Choi HJ, Kim HR, Shin MG, Kook H, Kim HJ, Shin JH, et al. Spectra of Chromosomal Aberrations in 325 Leukemia Patients and Implications for the Development of New Molecular Detection Systems. J Korean Med Sci. 2011; 26(7):886-92
11. Arber DA, Brunning RD, Le Beau MM, Falini B. Acute myeloid leukemia with recurrent genetic abnormalities In: Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW (Eds): WHO classification of Tumors of haematopoietic and Lymphoid Tissues. IARC: Lyon; 2008.p.110-123
12. Licht JD, Chomienne C, Goy A, Chen A, Scott AA, Head DR, et al. Clinical and molecular characterization of a rare syndrome of acute promyelocytic leukemia associated with translocation (11;17). Blood 1995; 85:1083
13. Guidez F, Ivins S, Zhu J, Söderström M, Waxman S, Zelent A. Reduced retinoic acid-sensitivities of nuclear receptor co-repressor binding to PML-and PLZF-RARα underlie molecular pathogenesis and treatment of acute promyelocytic leukemia. Blood.1998 ;91(8):2634-2642
14. Gilliland DG, Griffin JD. The roles of FLT3 in hematopoiesis and leukemia. Blood 2002;100:1532–1542
15. Paschka P, Marcucci G, Ruppert AS, Mrozek K, Chen H, Kittles R A et al (2006). Adverse prognostic significance of KIT mutations in adult myeloid leukemia with inv(16) and t(8;21): a Cancer and Leukemia Group B study. J Clin Oncol 24:3904-3911
16. Mrózek K, Heinonen K, Lawrence D et al. Adult patients with de novo acute myeloid leukemia and t (9; 11)(p22; q23) have a superior outcome to patients with other translocations involving band 11q23: a cancer and leukemia group B study. Blood. 1997;90(11):4532-8
17. Sadamori N, Yao E, Tagawa M et al. 16;21 translocation in acute non lymphocytic leukemia with abnormal eosinophils: a unique subtype. Acta Haematol. 1990; 84(4):212-6
18. Gopal MM, Kotwal J, Kapoor R. Acute Myeloid Leukemia with BCR/ABL Fusion Chimera. Indian Journal of Hematology and Blood Transfusion. 2014;30(1):280-2
19. Oyarzo MP, Lin P, Glassman A, Bueso-Ramos CE, Luthra R, Medeiros LJ. Acute myeloid leukemia with t (6; 9)(p23; q34) is associated with dysplasia and a high frequency of flt3 gene mutations. American journal of clinical pathology. 2004;122(3):348-58
20. Delaunay J, Vey N, Leblanc T et al. Prognosis of inv (16)/t (16; 16) acute myeloid leukemia (AML): a survey of 110 cases from the French AML Intergroup. Blood. 2003;102(2):462-9
21. Raimondi SC, Dubé ID, Valentine MB et al. Clinicopathologic manifestations and breakpoints of the t(3;5) in patients with acute non lymphocytic leukemia. Leukemia. 1989; 3(1):42-7
22. Song MJ, Kim HJ, Park CH et al. Diagnostic utility of a multiplex RT-PCR assay in detecting fusion transcripts from recurrent genetic abnormalities of acute leukemia by WHO 2008 classification. Diagnostic molecular pathology. 2012;21(1):40-4
Published
2017-02-19
Issue
Section
Original Article
Authors who publish with this journal agree to the following terms:
- Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution License that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.
- Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.
- Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work (See The Effect of Open Access at http://opcit.eprints.org/oacitation-biblio.html).
