Abstract
Background: Identification of specific chromosomal translocations is essential for the diagnosis and prognosis of leukemia. In this study, we employ DNA microarray technology to detect chromosomal aberrations in patients with chronic myeloid leukemia (CML) and acute myeloid leukemia (AML), as well as in leukemic cell lines.
Methods: Reverse transcription using a random 9-mer primer was performed with total RNA from patients and leukemic cells lines. Multiplex PCR reactions using four groups of primer sets were then performed for amplification of cDNA from reverse-transcribed total RNA samples. Normal and fusion sequences were distinguished by hybridization of the amplified cDNA to a selective oligonucleotide array (SOA) containing 20-30mer synthetic probes. A total of 23 sets of oligomers were fabricated on glass slides for the detection of normal and fusion genes, as follows: BCR/ABL, AML/EAP, AML/ETO, AML/MDS, PML/RARA, NUMA1/RARA, PLZF/RARA, and CBFB/MYH.
Results: Gene translocation in leukemia was effectively identified with the SOA containing various leukemiaspecific fusion and normal control sequences. Leukemic fusion sequences from patients and cell lines hybridized specifically to their complementary probes. The probe sets differing by ≈50% at their 5′ or 3′ ends could distinguish between normal and fusion sequences. The entire process of detection was completed within 8 hours using the SOA method.
Conclusions: Probe sets on SOA can effectively discriminate between leukemia-specific fusion and normal sequences with a chip hybridization procedure. The oligonucleotide array presents several advantages in identifying leukemic gene translocations, such as multiplex screening, relatively low cost, and speed.
References
Look AT. Oncogenic transcription factors in the human acute leukemias. Science 1997; 278: 1059–64
Cox MC, Maffei L, Buffolino S, et al. A comparative analysis of FISH, RT-PCR, and cytogenetics for the diagnosis of bcr-abl-positive leukemia. Am J Clin Pathol 1998; 109: 24–31
Lee S, Kim DW, Cho B, et al. Risk factors for adults with Philadelphia-chromosome-positive acute lymphoblastic leukemia in remission treated with allogenic bone marrow transplantation: the potential of real-time quantitative reverse-transcription polymerase chain reaction. Br J Haematol 2003; 120(14): 145–53
Pallisgaard N, Hoklan P, Riishoj DC, et al. Multiplex reverse transcription-polymerase chain reaction for simultaneous screening of 29 translocations and chromosomal aberrations in acute leukemia. Blood 1998; 92(2): 574–88
Elia L, Mancini M, Moleti L, et al. A multiplex reverse transcription-polymerase chain reaction strategy for the diagnostic molecular screening of chimeric genes: a clinical evaluation on 170 patients with acute lymphoblastic leukemia. Haematologica 2003; 88: 275–9
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): 231–6
Nashed AL, Rao KW, Margart LG. Clinical application of BCR-ABL molecular testing in acute leukemia. J Mol Diagn 2003; 5(2): 63–72
Schnittger S, Weisser M, Schoch C, et al. New score predicting for prognosis in PML-RARA-, AML1-ETO-, or CBFB-MYH11-positive acute myeloid leukemia based on quantification of fusion transcripts. Blood 2003; 102: 2746–55
Pekova S, Markova J, Pajer P, et al. Touch-down reverse transcriptase-PCR detection of IgVH Rearrangement and syber-green-based real-time RT-PCR quantitation of minimal residual disease in patients with chronic lymphocytic leukemia. Mol Diagn 2005; 9(1): 23–34
van der Velden VHJ, Hochhaus A, Cazzaniga G, et al. Detection of minimal residual disease hematologic malignancies by real-time quantatitative PCR: principles, approaches, and laboratory aspects. Leukemia 2003; 17: 1013–34
Beillard E, Pallisgaard N, van der Velden VHJ, et al. Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patient using ‘real-time’ quantitative reverse-transcriptase polymerase chain reaction (RQ-PCR): a Europe Against Cancer program. Leukemia 2003; 17: 2474–86
Larramendy M, Niini T, Elonen E, et al. Over expression of translocation-associate fusion genes of FGFR1, MYC, NPM1, and DEK, but absence of the translocations in acute myeloid leukemia: a microarray analysis. Haematologica 2002; 87: 569–77
Zou TT, Selaru FM, Xu Y, et al. Application of cDNA microarray to generate a molecular taxonomy capable of distinguishing between colon cancer and normal colon. Oncogen 2002; 21: 4855–62
Nasedkina T, Domer P, Zharinov V, et al. Identification of chromosomal translocations in leukemias by hybridization with oligonucleotide microarrays. Haematologica 2002; 87: 363–72
Yershov G, Basky V, Belogovskiy A, et al. DNA analysis and diagnostics on oligonucleotide microarray. Proc Natl Acad Sci USA 1996; 93: 4913–8
Lee Y, Lee C-S, Kim Y-J, et al. Development of DNA Chip for the simultaneous detection of various b-lactam antibiotic-resistant genes. Mol Cells 2002; 14: 192–7
Lindroos K, Lijedahl U, Raitio M, et al. Minisequencing on oligonucleotide microarrays: comparison of immobilization chemistries. Nucleic Acids Res 2001; 29(13): e69
Patinen T, Kurg A, Metspalu A, et al. Minisequencing: a specific tool for DNA analysis and diagnostics on oligonucleotide arrays. Genome Res 1997; 7: 606–14
Maroc N, Morel A, Beillard E, et al. A diagnostic biochip for the comprehensive analysis of MLL tranlocations in acute leukemia. Leukemia 2004; 18(9): 1522–30
Dupont M, Goldsborough A, Levayer T, et al. Multiplex fluorescent RT-PCR to quantify leukemic fusion transcript. BioTechniques 2002; 33: 158–64
Fabian I, Halpein D, Lefter S, et al. Alteration of actin organization by jaspamide inhibits ruffing, but not phagocytosis or oxidative burst, in HL-60 cells and human minocytes. Blood 1999; 93: 3994–4005
Higuchi RG, Ochman H. Production of single-stranded DNA templates by exonuclease digestion following the polymerase chain reaction. Nucleic Acids Res 1989; 17: 5865–71
Curry J, McHale C, Smith M. Low efficiency of the moloney murine leukemia virus reverse transcriptase during reverse transcription of rare t(8;21) fusion gene transcripts. Biotechniques 2002; 32: 768–75
Baxter EJ, Hochhaus A, Bolufer P, et al. The t(4;22)(ql2;ql 1) in atypical chronic myeloid leukemia fuses BCR to PDGFRA. Human Mol Gen 2002; 11: 1391–7
Acknowledgments
This study was partly supported by a grant from the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (grant no. 01-PJ10-PG6-01GN16-0005).
B-D Han and Y-T Oh are employees of Koma Biotech.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Chun, SM., Kim, YL., Choi, H.B. et al. Identification of Leukemia-Specific Fusion Gene Transcripts with a Novel Oligonucleotide Array. Mol Diag Ther 11, 21–28 (2007). https://doi.org/10.1007/BF03256220
Published:
Issue Date:
DOI: https://doi.org/10.1007/BF03256220