Abstract
The MLL gene in chromosome band 11q23 is frequently rearranged in acute lymphoblastic and acute myeloid leukemias. To date, more than 50 different chromosomal regions are known to participate in translocations involving 11q23, many of which affect MLL. The pathogenetically important outcome of these rearrangements is most likely the creation of a fusion gene consisting of the 5′ part of the MLL gene and the 3′ end of the partner gene. Although abnormalities of the MLL gene as such are generally associated with poor survival, recent data suggest that the prognostic impact varies among the different fusion genes generated. Hence, detection of the specific chimeric gene produced is important for proper prognostication and clinical decision making. We have developed a paired multiplex reverse-transcriptase polymerase chain reaction analysis to facilitate a rapid and accurate detection of the most frequent MLL fusion genes in adult and childhood acute leukemias. To increase the specificity, two sets of primers were designed for each fusion gene, and these paired primer sets were run in parallel in two separate multiplex one-step PCR reactions. Using the described protocol, we were able to amplify successfully, in one single assay, the six clinically relevant fusion genes generated by the t(4;11)(q21;q23) [MLL/AF4], t(6;11)(q27;q23) [MLL/AF6], t(9;11)(p21–22;q23) [MLL/AF9], t(10;11)(p11–13;q23) [MLL/AF10], t(11;19)(q23;p13.1) [MLL/ELL], and t(11;19)(q23; p13.3) [MLL/ENL] in cell lines, as well as in patient material.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Super HJ, McCabe NR, Thirman MJ, Larson RA, Le Beau MM, Pedersen-Bjergaard J, Philip P, Diaz MO, Rowley JD . Rearrangements of the MLL gene in therapy-related acute myeloid leukemia in patients previously treated with agents targeting DNA-topoisomerase II Blood 1993 82: 3705–3711
Rowley JD . The critical role of chromosome translocations in human leukemias Annu Rev Genet 1998 32: 495–519
Look AT . Genes altered by chromosomal translocations in leukemias and lymphomas In: Kinzler K, Vogelstein B (eds) The Genetic Basis of Human Cancer first edn McGraw-Hill: New York 1998 pp 109–141
DiMartino JF, Cleary ML . Mll rearrangements in haematological malignancies: lessons from clinical and biological studies Br J Haematol 1999 106: 614–626
Nilson I, Lochner K, Siegler G, Greil J, Beck JD, Fey GH, Marschalek R . Exon/intron structure of the human ALL-1 (MLL) gene involved in translocations to chromosomal region 11q23 and acute leukaemias Br J Haematol 1996 93: 966–972
Djabali M, Selleri L, Parry P, Bower M, Young B, Evans GA . A trithorax-like gene is interrupted by chromosome 11q23 translocations in acute leukaemias Nat Genet 1993 2: 113–118
Gu Y, Nakamura T, Alder H, Prasad R, Canaani O, Cimino G, Croce CM, Canaani E . The t(4;11) chromosome translocation of human acute leukemias fuses the ALL-1 gene, related to Drosophila trithorax, to the AF-4 gene Cell 1992 71: 701–708
Tkachuk DC, Kohler S, Cleary ML . Involvement of a homolog of Drosophila trithorax by 11q23 chromosomal translocations in acute leukemias Cell 1992 71: 691–700
Thirman MJ, Gill HJ, Burnett RC, Mbangkollo D, McCabe NR, Kobayashi H, Ziemin-van der Poel S, Kaneko Y, Morgan R, Sandberg AA, Chaganti RSK, Larson RA, Le Beau MM, Diaz MO, Rowley JD . Rearrangement of the MLL gene in acute lymphoblastic and acute myeloid leukemias with 11q23 chromosomal translocations N Engl J Med 1993 329: 909–914
Mitelman F, Johansson B, Mertens F (eds) . Mitelman database of chromosome aberrations in cancer (2000): http://cgap.nci.nih.gov/Chromosomes/Mitelman
Johansson B, Moorman AV, Haas OA, Watmore AE, Cheung KL, Swanton S, Secker-Walker LM, on behalf of the European 11q23 Workshop participants . Hematologic malignancies with t(4;11)(q21;q23) – a cytogenetic, morphologic, immunophenotypic and clinical study of 183 cases Leukemia 1998 12: 779–787
Moorman AV, Hagemeijer A, Charrin C, Rieder H, Secker-Walker LM, on behalf of the European 11q23 Workshop participants . The translocations, t(11;19)(q23;p13.1) and t(11;19)(q23;p13.3): a cytogenetic and clinical profile of 53 patients Leukemia 1998 12: 805–810
Reaman GH, Sposto R, Sensel MG, Lange BJ, Feusner JH, Heerema NA, Leonard M, Holmes EJ, Sather HN, Pendergrass TW, Johnstone HS, O'Brien RT, Steinherz PG, Zeltzer PM, Gaynon PS, Trigg ME, Uckun FM . Treatment outcome and prognostic factors for infants with acute lymphoblastic leukemia treated on two consecutive trials of the Children's Cancer Group J Clin Oncol 1999 17: 445–455
Rubnitz JE, Camitta BM, Mahmoud H, Raimondi SC, Carroll AJ, Borowitz MJ, Shuster JJ, Link MP, Pullen DJ, Downing JR, Behm FG, Pui CH . Childhood acute lymphoblastic leukemia with the MLL-ENL fusion and t(11;19)(q23;p13.3) translocation J Clin Oncol 1999 17: 191–196
Martineau M, Berger R, Lillington DM, Moorman AV, Secker-Walker LM, on behalf of the European 11q23 Workshop participants . The t(6;11)(q27;q23) translocation in acute leukemia: a laboratory and clinical study of 30 cases Leukemia 1998 12: 788–791
Lillington DM, Young BD, Berger R, Martineau M, Moorman AV, Secker-Walker LM, on behalf of the European 11q23 Workshop participants . The t(10;11)(p12;q23) translocation in acute leukaemia: a cytogenetic and clinical study of 20 patients Leukemia 1998 12: 801–804
Swansbury GJ, Slater R, Bain BJ, Moorman AV, Secker-Walker LM, on behalf of the European 11q23 Workshop participants . Hematological malignancies with t(9;11)(p21–22;q23) – a laboratory and clinical study of 125 cases Leukemia 1998 12: 792–800
Mrózek K, Heinonen K, Lawrence D, Carroll AJ, Koduru PR, Rao KW, Strout MP, Hutchison RE, Moore JO, Mayer RJ, Schiffer CA, Bloomfield CD . 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: 4532–4538
Martinez-Climent JA, Espinosa R 3rd, Thirman MJ, Le Beau MM, Rowley JD . Abnormalities of chromosome band 11q23 and the MLL gene in pediatric myelomonocytic and monoblastic leukemias. Identification of the t(9;11) as an indicator of long survival J Pediatr Hematol Oncol 1995 17: 277–283
Huret JL, Brizard A, Slater R, Charrin C, Bertheas MF, Guilhot F, Hahlen K, Kroes W, van Leeuwen E, Schoot EV, Beishuizen A, Tanzer J, Hagemeijer A . Cytogenetic heterogeneity in t(11;19) acute leukemia: clinical, hematological and cytogenetic analyses of 48 patients – updated published cases and 16 new observations Leukemia 1993 7: 152–160
Dreyling MH, Schrader K, Fonatsch C, Schlegelberger B, Haase D, Schoch C, Ludwig W, Loffler H, Buchner T, Wormann B, Hiddemann W, Bohlander SK . MLL and CALM are fused to AF10 in morphologically distinct subsets of acute leukemia with translocation t(10;11): both rearrangements are associated with a poor prognosis Blood 1998 91: 4662–4667
van der Burg M, Beverloo HB, Langerak AW, Wijsman J, van Drunen E, Slater R, van Dongen JJ . Rapid and sensitive detection of all types of MLL gene translocations with a single FISH probe set Leukemia 1999 13: 2107–2113
Mathew S, Behm F, Dalton J, Raimondi S . Comparison of cytogenetics, Southern blotting, and fluorescence in situ hybridization as methods for detecting MLL gene rearrangements in children with acute leukemia and with 11q23 abnormalities Leukemia 1999 13: 1713–1720
Cimino G, Lo Coco F, Biondi A, Elia L, Luciano A, Croce CM, Masera G, Mandelli F, Canaani E . ALL-1 gene at chromosome 11q23 is consistently altered in acute leukemia of early infancy Blood 1993 82: 544–546
Marcucci G, Livak KJ, Bi W, Strout MP, Bloomfield CD, Caligiuri MA . Detection of minimal residual disease in patients with AML1/ETO-associated acute myeloid leukemia using a novel quantitative reverse transcription polymerase chain reaction assay Leukemia 1998 12: 1482–1489
Hochhaus A, Weisser A, La Rosee P, Emig M, Muller MC, Saussele S, Reiter A, Kuhn C, Berger U, Hehlmann R, Cross NC . Detection and quantification of residual disease in chronic myelogenous leukemia Leukemia 2000 14: 998–1005
Cassinat B, Zassadowski F, Balitrand N, Barbey C, Rain JD, Fenaux P, Degos L, Vidaud M, Chomienne C . Quantitation of minimal residual disease in acute promyelocytic leukemia patients with t(15;17) translocation using real-time RT-PCR Leukemia 2000 14: 324–328
Repp R, Borkhardt A, Haupt E, Kreuder J, Brettreich S, Hammermann J, Nishida K, Harbott J, Lampert F . Detection of four different 11q23 chromosomal abnormalities by multiplex-PCR and fluorescence-based automatic DNA-fragment analysis Leukemia 1995 9: 210–215
Pallisgaard N, Hokland P, Riishoj DC, Pedersen B, Jorgensen P . Multiplex reverse transcription-polymerase chain reaction for simultaneous screening of 29 translocations and chromosomal aberrations in acute leukemia Blood 1998 92: 574–588
Viehmann S, Borkhardt A, Lampert F, Harbott J . Multiplex PCR – a rapid screening method for detection of gene rearrangements in childhood acute lymphoblastic leukemia Ann Hematol 1999 78: 157–162
Cerveira N, Ferreira S, Doria S, Veiga I, Ferreira F, Mariz JM, Marques M, Castedo S . Detection of prognostic significant translocations in childhood acute lymphoblastic leukaemia by one-step multiplex reverse transcription polymerase chain reaction Br J Haematol 2000 109: 638–640
Morrissey J, Tkachuk DC, Milatovich A, Francke U, Link M, Cleary ML . A serine/proline-rich protein is fused to HRX in t(4;11) acute leukemias Blood 1993 81: 1124–1131
Tanabe S, Zeleznik-Le NJ, Kobayashi H, Vignon C, Espinosa R 3rd, LeBeau MM, Thirman MJ, Rowley JD . Analysis of the t(6;11)(q27;q23) in leukemia shows a consistent breakpoint in AF6 in three patients and in the ML-2 cell line Genes Chromosomes Cancer 1996 15: 206–216
Super HJ, Martinez-Climent J, Rowley JD . Molecular analysis of the MonoMac 6 cell line: detection of an MLL-AF9 fusion transcript Blood 1995 85: 855–856
Iida S, Seto M, Yamamoto K, Komatsu H, Akao Y, Nakazawa S, Ariyoshi Y, Takahashi T, Ueda R . Molecular cloning of 19p13 breakpoint region in infantile leukemia with t(11;19)(q23;p13) translocation Jpn J Cancer Res 1993 84: 532–537
Yamamoto K, Seto M, Komatsu H, Iida S, Akao Y, Kojima S, Kodera Y, Nakazawa S, Ariyoshi Y, Takahashi T, Ueda R . Two distinct portions of LTG19/ENL at 19p13 are involved in t(11;19) leukemia Oncogene 1993 8: 2617–2625
Yamamoto K, Seto M, Iida S, Komatsu H, Kamada N, Kojima S, Kodera Y, Nakazawa S, Saito H, Takahashi T, Ueda R . A reverse transcriptase-polymerase chain reaction detects heterogeneous chimeric mRNAs in leukemias with 11q23 abnormalities Blood 1994 83: 2912–2921
Lozzio CB, Lozzio BB . Human chronic myelogenous leukemia cell line with positive Philadelphia chromosome Blood 1975 45: 321–334
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: 1080–1086
Fioretos T, Nilsson PG, Aman P, Heim S, Kristoffersson U, Malm C, Simonsson B, Turesson I, Mitelman F . Clinical impact of breakpoint position within M-bcr in chronic myeloid leukemia Leukemia 1993 7: 1225–1231
Nilson I, Reichel M, Ennas MG, Greim R, Knorr C, Siegler G, Greil J, Fey GH, Marschalek R . Exon/intron structure of the human AF-4 gene, a member of the AF-4/LAF-4/FMR-2 gene family coding for a nuclear protein with structural alterations in acute leukaemia Br J Haematol 1997 98: 157–169
Hunger SP, Cleary ML . What significance should we attribute to the detection of MLL fusion transcripts? Blood 1998 92: 709–711
Nam DK, Honoki K, Yu M, Yunis JJ . Alternative RNA splicing of the MLL gene in normal and malignant cells Gene 1996 178: 169–175
Caldas C, So CW, MacGregor A, Ford AM, McDonald B, Chan LC, Wiedemann LM . Exon scrambling of MLL transcripts occur commonly and mimic partial genomic duplication of the gene Gene 1998 208: 167–176
Cimino G, Rapanotti MC, Sprovieri T, Elia L . ALL1 gene alterations in acute leukemia: biological and clinical aspects Haematologica 1998 83: 350–357
Nakamura T, Alder H, Gu Y, Prasad R, Canaani O, Kamada N, Gale RP, Lange B, Crist WM, Nowell PC, Croce CM, Canaani E . Genes on chromosomes 4, 9, and 19 involved in 11q23 abnormalities in acute leukemia share sequence homology and/or common motifs Proc Natl Acad Sci USA 1993 90: 4631–4635
Hilden JM, Chen CS, Moore R, Frestedt J, Kersey JH . Heterogeneity in MLL/AF-4 fusion messenger RNA detected by the polymerase chain reaction in t(4;11) acute leukemia Cancer Res 1993 53: 3853–3856
Domer PH, Fakharzadeh SS, Chen CS, Jockel J, Johansen L, Silverman GA, Kersey JH, Korsmeyer SJ . Acute mixed-lineage leukemia t(4;11)(q21;q23) generates an MLL-AF4 fusion product Proc Natl Acad Sci USA 1993 90: 7884–7888
Uckun FM, Herman-Hatten K, Crotty ML, Sensel MG, Sather HN, Tuel-Ahlgren L, Sarquis MB, Bostrom B, Nachman JB, Steinherz PG, Gaynon PS, Heerema N . Clinical significance of MLL-AF4 fusion transcript expression in the absence of a cytogenetically detectable t(4;11)(q21;q23) chromosomal translocation Blood 1998 92: 810–821
Biondi A, Rambaldi A, Rossi V, Elia L, Caslini C, Basso G, Battista R, Barbui T, Mandelli F, Masera G, Croce C, Canaani E, Cimino G . Detection of ALL-1/AF4 fusion transcript by reverse transcription-polymerase chain reaction for diagnosis and monitoring of acute leukemias with the t(4;11) translocation Blood 1993 82: 2943–2947
van Dongen JJ, Macintyre EA, Gabert JA, Delabesse E, Rossi V, Saglio G, Gottardi E, Rambaldi A, Dotti G, Griesinger F, Parreira A, Gameiro P, Diaz MG, Malec M, Langerak AW, San Miguel JF, Biondi A . 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: 1901–1928
Prasad R, Gu Y, Alder H, Nakamura T, Canaani O, Saito H, Huebner K, Gale RP, Nowell PC, Kuriyama K, Miyazaki Y, Croce CM, Canaani E . Cloning of the ALL-1 fusion partner, the AF-6 gene, involved in acute myeloid leukemias with the t(6;11) chromosome translocation Cancer Res 1993 53: 5624–5628
Poirel H, Rack K, Delabesse E, Radford-Weiss I, Troussard X, Debert C, Leboeuf D, Bastard C, Picard F, Veil-Buzyn A, Flandrin G, Bernard O, Macintyre E . Incidence and characterization of MLL gene (11q23) rearrangements in acute myeloid leukemia M1 and M5 Blood 1996 87: 2496–2505
Super HG, Strissel PL, Sobulo OM, Burian D, Reshmi SC, Roe B, Zeleznik-Le NJ, Diaz MO, Rowley JD . Identification of complex genomic breakpoint junctions in the t(9;11) MLL-AF9 fusion gene in acute leukemia Genes Chromosomes Cancer 1997 20: 185–195
Mitterbauer G, Zimmer C, Fonatsch C, Haas O, Thalhammer-Scherrer R, Schwarzinger I, Kalhs P, Jaeger U, Lechner K, Mannhalter C . Monitoring of minimal residual leukemia in patients with MLL-AF9 positive acute myeloid leukemia by RT-PCR Leukemia 1999 13: 1519–1524
Iida S, Seto M, Yamamoto K, Komatsu H, Tojo A, Asano S, Kamada N, Ariyoshi Y, Takahashi T, Ueda R . MLLT3 gene on 9p22 involved in t(9;11) leukemia encodes a serine/proline rich protein homologous to MLLT1 on 19p13 Oncogene 1993 8: 3085–3092
Borkhardt A, Haas OA, Strobl W, Repp R, Mann G, Gadner H, Lampert F . A novel type of MLL/AF10 fusion transcript in a child with acute megakaryocytic leukemia (AML-M7) Leukemia 1995 9: 1796–1797
Hjorth-Sorensen B, Pallisgaard N, Gronholm M, Hokland P, Clausen N, Jorgensen P . A novel MLL-AF10 fusion mRNA variant in a patient with acute myeloid leukemia detected by a new asymmetric reverse transcription PCR method Leukemia 1997 11: 1588–1593
Chaplin T, Bernard O, Beverloo HB, Saha V, Hagemeijer A, Berger R, Young BD . The t(10;11) translocation in acute myeloid leukemia (M5) consistently fuses the leucine zipper motif of AF10 onto the HRX gene Blood 1995 86: 2073–2076
Chaplin T, Ayton P, Bernard OA, Saha V, Della Valle V, Hillion J, Gregorini A, Lillington D, Berger R, Young BD . A novel class of zinc finger/leucine zipper genes identified from the molecular cloning of the t(10;11) translocation in acute leukemia Blood 1995 85: 1435–1441
Rubnitz JE, Morrissey J, Savage PA, Cleary ML . ENL, the gene fused with HRX in t(11;19) leukemias, encodes a nuclear protein with transcriptional activation potential in lymphoid and myeloid cells Blood 1994 84: 1747–1752
Rubnitz JE, Behm FG, Curcio-Brint AM, Pinheiro RP, Carroll AJ, Raimondi SC, Shurtleff SA, Downing JR . Molecular analysis of t(11;19) breakpoints in childhood acute leukemias Blood 1996 87: 4804–4808
Mitani K, Kanda Y, Ogawa S, Tanaka T, Inazawa J, Yazaki Y, Hirai H . Cloning of several species of MLL/MEN chimeric cDNAs in myeloid leukemia with t(11;19)(q23;p13.1) translocation Blood 1995 85: 2017–2024
Thirman MJ, Levitan DA, Kobayashi H, Simon MC, Rowley JD . Cloning of ELL, a gene that fuses to MLL in a t(11;19)(q23;p13.1) in acute myeloid leukemia Proc Natl Acad Sci USA 1994 91: 12110–12114
Harrison CJ, Cuneo A, Clark R, Johansson B, Lafage-Pochitaloff M, Mugneret F, Moorman AV, Secker-Walker LM, on behalf of the European 11q23 Workshop participants . Ten novel 11q23 chromosomal partner sites Leukemia 1998 12: 811–822
Acknowledgements
This study was supported by grants from the Swedish Cancer Society, the Swedish Child Cancer Fund, and the Lund University Medical Faculty.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Andersson, A., Höglund, M., Johansson, B. et al. Paired multiplex reverse-transcriptase polymerase chain reaction (PMRT-PCR) analysis as a rapid and accurate diagnostic tool for the detection of MLL fusion genes in hematologic malignancies. Leukemia 15, 1293–1300 (2001). https://doi.org/10.1038/sj.leu.2402189
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/sj.leu.2402189
Keywords
This article is cited by
-
Rare MLL-ELL fusion transcripts in childhood acute myeloid leukemia—association with young age and myeloid sarcomas?
Experimental Hematology & Oncology (2015)
-
Single Fluorescence Channel-based Multiplex Detection of Avian Influenza Virus by Quantitative PCR with Intercalating Dye
Scientific Reports (2015)
-
Risk-based classification of leukemia by cytogenetic and multiplex molecular methods: results from a multicenter validation study
Blood Cancer Journal (2012)
-
Complex MLL rearrangements in t(4;11) leukemia patients with absent AF4 · MLL fusion allele
Leukemia (2007)
-
Monitoring minimal residual disease by quantification of genomic chromosomal breakpoint sequences in acute leukemias with MLL aberrations
Leukemia (2006)