Summary
In acute myeloid leukemia (AML), molecular diagnosis for the optimal management of patients and for minimal residual disease (MRD) monitoring is of extreme importance. Cumulative data suggest that quantitative monitoring or MRD in AML with fusion transcripts corresponding to 5(I;21), inv(16), and t(15;17) is useful in distinguishing patients at high risk of relapse from those in durable remission. Real-time quantitative polymerase chain reaction (RQPCR) is by far the most sensitive assay in the context of MRD detection. We present herein an overview of the principles of RQ-PCR encompassing both the chemistries (double-stranded DNA detection or specific fragment detection) and the instruments. The absolute and relative quantification and the most commonly used methods for calculation of MRD results in absolute quantification are also described.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Laczika K., Novak M., Hilgarth B., et al. (1998) Competitive CBFbeta/MYH11 reverse-transcriptase polymerase chain reaction for quantitative assessment of minimal residual disease during postremission therapy in acute myeloid leukemia with inversion(16): a pilot study. J. Clin. Oncol. 16, 1519ā1525.
Tobal K. and Liu Yin J. A. (1998) Molecular monitoring of minimal residual disease in acute myeloblastic leukemia with t(8;21) by RT-PCR. Leuk. Lymphoma 31, 115ā120.
Barragan E., Bolufer P., Moreno I., et al. (2001) Quantitative detection of AML1-ETO rearrangement by real-time RT-PCR using fluorescently labeled probes. Leuk. Lymphoma 42, 747ā756.
Higuchi R., Fockler C., Dollinger G., and Watson R. (1993) Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology 11, 1026ā1030.
Wittwer C. T., Herrmann M. G., Moss A. A., and Rasmussen R. P. (1997a) Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques 22, 130ā131, 134-138.
Vandesompele J., De Paepe A., and Speleman F. (2002) Elimination of primerdimer artifacts and genomic coamplification using a two-step SYBR green I realtime RT-PCR. Anal. Biochem. 303, 95ā98.
Ririe K. M., Rasmussen R. P., and Wittwer C. T. (1997) Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal. Biochem. 245, 154ā160.
Nygren J., Svanvik N., and Kubista M. (1998) The interactions between the fluorescent dye thiazole orange and DNA. Biopolymers 46, 39ā51.
Bengtsson M., Karlsson H. J., Westman G., and Kubista M. (2003) A new minor groove binding asymmetric cyanine reporter dye for real-time PCR. Nucleic Acids Res. 31, e45.
Svanvik N., Westman G., Wang D., and Kubista M. (2000) Light-up probes: thiazole orange-conjugated peptide nucleic acid for detection of target nucleic acid in homogeneous solution. Anal. Biochem. 281, 26ā35.
Stahlberg A., Aman P., Ridell B., Mostad P., and Kubista M. (2003) Quantitative real-time PCR method for detection of B-lymphocyte monoclonality by comparison of kappa and lambda immunoglobulin light chain expression. Clin. Chem. 49, 51ā59.
Nazarenko I. A., Bhatnagar S. K., and Hohman R. J. (1997) A closed tube format for amplification and detection of DNA based on energy transfer. Nucleic Acids Res. 25, 2516ā2521.
Nazarenko I., Pires R., Lowe B., Obaidy M., and Rashtchian A. (2002) Effect of primary and secondary structure of oligodeoxyribonucleotides on the fluorescent properties of conjugated dyes. Nucleic Acids Res. 30, 2089ā2195.
Applegate T. L., Iland H. J., Mokany E., and Todd A. V. (2002) Diagnosis and molecular monitoring of acute promyelocytic leukemia using DzyNA reverse transcription-PCR to quantify PML/RARalpha fusion transcripts. Clin. Chem. 48, 1338ā1343.
Holland P. M., Abramson R. D., Watson R., and Gelfand D. H. (1991) Detection of specific polymerase chain reaction product by utilizing the 5,Ćā¤-3,Ćā¤ exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. USA 88, 7276ā80.
Fƶrster V. (1948) Zwischenmolekular Energiewanderung und Fluoreszenz. Ann. Phy. 2, 55ā57.
Clegg R. M., Murchie A. I., Zechel A., Carlberg C., Diekmann S., and Lilley D. M. (1992) Fluorescence resonance energy transfer analysis of the structure of the four-way DNA junction. Biochemistry 31, 4846ā4856.
Lyamichev V., Brow M. A., and Dahlberg J. E. (1993) Structure-specific endonucleolytic cleavage of nucleic acids by eubacterial DNA polymerases. Science 260, 778ā783.
de Kok J. B., Wiegerinck E. T., Giesendorf B. A., and Swinkels D. W. (2002) Rapid genotyping of single nucleotide polymorphisms using novel minor groove binding DNA oligonucleotides (MGB probes). Hum. Mutat. 19, 554ā559.
Latorra D., Arar K., and Hurley J. M. (2003) Design considerations and effects of LNA in PCR primers. Mol. Cell. Probes 17, 253ā259.
Letertre C., Perelle S., Dilasser F., Arar K., and Fach P.. (2003) Evaluation of the performance of LNA and MGB probes in 5ā²-nuclease PCR assays. Mol. Cell. Probes 17, 307ā311.
Wittwer C. T., Ririe K. M., Andrew R. V., David D. A., Gundry R. A., and Balis U. J. (1997b) The LightCycler: a microvolume multisample fluorimeter with rapid temperature control. Biotechniques 22, 176ā181.
Meuer S. Wittwer C., and Nakagawara K. (eds) (2001) Rapid Cycle Real-Time PCR: Methods and Applications. Springer, Heidelberg.
Tyagi S. and Kramer F. R. (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14, 303ā308.
Leone G., van Schijndel H., van Gemen B., Kramer F. R., and Schoen C. D. (1998) Molecular beacon probes combined with amplification by NASBA enable homogeneous, real-time detection of RNA. Nucleic Acids Res. 26, 2150ā2155.
Whitcombe D., Theaker J., Guy S. P., Brown T., and Little S. (1999) Detection of PCR products using self-probing amplicons and fluorescence. Nat. Biotechnol. 17, 804ā807.
Thelwell N., Millington S., Solinas A., Booth J., and Brown T. (2000) Mode of action and application of Scorpion primers to mutation detection. Nucleic Acids Res. 28, 3752ā3761.
Solinas A., Brown L. J., McKeen C., et al. (2001) Duplex Scorpion primers in SNP analysis and FRET applications. Nucleic Acids Res. 29, E96.
Silvy M., Mancini J., Thirion X., Sigaux F., and Gabert J. (2005) Evaluation of real-time quantitative PCR machines for the monitoring of fusion gene transcripts using the Europe Against Cancer protocol. Leukemia 19, 305ā307.
Pattyn F., Speleman F., De Paepe A., and Vandesompele J. (2003) RTPrimerDB: the real-time PCR primer and probe database. Nucleic Acids Res. 31, 122ā123.
Gabert J., Beillard E., van der Velden V. H., et al. (2003) Standardization and quality control studies of "e;real-time"e; quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia-a Europe Against Cancer program. Leukemia 17, 2318ā2357.
Beillard E., Pallisgaard N. van der Velden V. H. et al. (2003) Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patients using "e;real-time"e; quantitative reverse-transcriptase polymerase chain reaction (RQ-PCR)-a Europe against cancer program. Leukemia 17, 2474ā2486.
Freeman W. M., Walker S. J., and Vrana K. E. (1999) Quantitative RT-PCR: pitfalls and potential. Biotechniques 26, 112ā122, 124-125.
Keilholz U., Willhauck M., Rimoldi D., et al. (1998) Reliability of reverse transcription-polymerase chain reaction (RT-PCR)-based assays for the detection of circulating tumour cells: a quality-assurance initiative of the EORTC Melanoma Cooperative Group. Eur. J. Cancer 34, 750ā753.
Bolufer P., Lo Coco F., Grimwade D., et al. (2001) Variability in the levels of PML-RAR alpha fusion transcripts detected by the laboratories participating in an external quality control program using several reverse transcription polymerase chain reaction protocols. Haematologica 86, 570ā576.
Saldanha J. (2001) Validation and standardisation of nucleic acid amplification technology (NAT) assays for the detection of viral contamination of blood and blood products. J. Clin. Virol. 20, 7ā13.
Saldanha J., Lelie N., Yu M. W., and Heath A. (2002) Establishment of the first World Health Organization International Standard for human parvovirus B19 DNA nucleic acid amplification techniques. Vox Sang. 82, 24ā31.
Saldanha J. and Heath A. (2003) Collaborative study to calibrate hepatitis C virus genotypes 2ā6 against the HCV International Standard, 96/790 (genotype 1). Vox Sang. 84, 20ā27.
Kƶhler T., Schill C., Deininger M. W., et al. (2002) High Bad and Bax mRNA expression correlate with negative outcome in acute myeloid leukemia (AML). Leukemia 16, 22ā29.
Dobbs L. J., Madigan M. N., Carter A. B., and Earls L. (2002) Use of FTA gene guard filter paper for the storage and transportation of tumor cells for molecular testing. Arch. Pathol. Lab. Med. 126, 56ā63.
Kline M. C., Duewer D. L., Redman J. W., Butler J. M., and Boyer D. A. (2002) Polymerase chain reaction amplification of DNA from aged blood stains: quantitative evaluation of the "e;suitability for purpose"e; of four filter papers as archival media. Anal. Chem. 74, 1863ā1869.
Vandesompele J., De Preter K., Pattyn F., et al. (2003) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, RESEARCH0034.
Pfaffl M. W. (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, e45.
Liu W. and Saint D. A. (2002) A new quantitative method of real time reverse transcription polymerase chain reaction assay based on simulation of polymerase chain reaction kinetics. Anal. Biochem. 302, 52ā59.
Ramakers C., Ruijter J. M., Deprez R. H., and Moorman A. F. (2003) Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci. Lett. 339, 62ā66.
Tichopad A., Dilger M., Schwarz G., and Pfaffl M. W. (2003) Standardized determination of real-time PCR efficiency from a single reaction set-up. Nucleic Acids Res. 31, e122.
Gerard C. J., Olsson K., Ramanathan R., Reading C., and Hanania E. G. (1998) Improved quantitation of minimal residual disease in multiple myeloma using realtime polymerase chain reaction and plasmid-DNA complementarity determining region III standards. Cancer Res. 58, 3957ā3964.
Silvy M., Pic G, Gabert J., and Picard C. (2004) Improvement of gene expression analysis by RQ-PCR technology: addition of BSA. Leukemia 18, 1022ā1025.
Quina A. S., Gameiro P., Sa da Costa M., Telhada M., and Parreira L. (2000) PML-RARA fusion transcripts in irradiated and normal hematopoietic cells. Genes Chromosomes Cancer 29, 266ā275.
Lossos I. S, Czerwinski D. K., Alizadeh A. A., et al. (2004) Prediction of survival in diffuse large-B-cell lymphoma based on the expression of six genes. N. Engl. J. Med. 350, 1828ā1837.
Marcucci G., Livak K. J., Bi W., Strout M. P., Bloomfield C. D., and Caligiuri M. A. (1998) Detection of minimal residual disease in patients with AML1/ETOassociated acute myeloid leukemia using a novel quantitative reverse transcription polymerase chain reaction assay. Leukemia 12, 1482ā1489.
Cassinat B., Zassadowski F., Balitrand N., et al. (2000) Quantitation of minimal residual disease in acute promyelocytic leukemia patients with t(15;17) translocation using real-time RT-PCR. Leukemia 14, 324ā328.
Fujimaki S., Funato T., Harigae H., et al. (2000) A quantitative reverse transcriptase polymerase chain reaction method for the detection of leukaemic cells with t(8;21) in peripheral blood. Eur. J. Haematol. 64, 252ā258.
Sugimoto T., Das H., Imoto S., et al. (2000) Quantitation of minimal residual disease in t(8;21)-positive acute myelogenous leukemia patients using real-time quantitative RT-PCR. Am. J. Hematol. 64, 101ā106.
Krauter J., Hoellge W., Wattjes M. P., et al. (2001) Detection and quantification of CBFB/MYH11 fusion transcripts in patients with inv(16)-positive acute myeloblastic leukemia by real-time RT-PCR. Genes Chromosomes Cancer 30, 342ā348.
Kwong Y. L., Au W. Y., Chim C. S., Pang A., Suen C., and Liang R. (2001) Arsenic trioxide-and idarubicin-induced remissions in relapsed acute promyelocytic leukaemia: clinicopathological and molecular features of a pilot study. Am. J. Hematol. 66, 274ā279.
Marcucci G., Caligiuri M. A., Dohner H., et al. (2001) Quantification of CBFbeta/MYH11 fusion transcript by real time RT-PCR in patients with INV(16) acute myeloid leukemia. Leukemia 15, 1072ā1080.
Slack J. L., Bi W., Livak K. J., et al. (2001) Pre-clinical validation of a novel, highly sensitive assay to detect PML-RARalpha mRNA using real-time reversetranscription polymerase chain reaction. J. Mol. Diagn. 3, 141ā149.
Visani G., Buonamici S., Malagola M., et al. (2001) Pulsed ATRA as single therapy restores long-term remission in PML-RARalpha-positive acute promyelocytic leukemia patients: real time quantification of minimal residual disease. A pilot study. Leukemia 15, 1696ā1700.
Buonamici S., Ottaviani E., Testoni N., et al. (2002) Real-time quantitation of minimal residual disease in inv(16)-positive acute myeloid leukemia may indicate risk for clinical relapse and may identify patients in a curable state. Blood 99, 443ā449.
Cilloni D., Gottardi E., De Micheli D., et al. (2002) Quantitative assessment of WT1 expression by real time quantitative PCR may be a useful tool for monitoring minimal residual disease in acute leukemia patients. Leukemia 16, 2115ā2121.
Guerrasio A., Pilatrino C., De Micheli D., et al. (2002) Assessment of minimal residual disease (MRD) in CBFbeta/MYH11-positive acute myeloid leukemias by qualitative and quantitative RT-PCR amplification of fusion transcripts. Leukemia 16, 1176ā1181.
Gallagher R. E., Yeap B. Y., Bi W., et al. (2003) Quantitative real-time RTPCR analysis of PML-RAR alpha mRNA in acute promyelocytic leukemia: assessment of prognostic significance in adult patients from intergroup protocol 0129. Blood 101, 2521ā2528.
Martinelli G., Buonamici S., Visani G., et al. (2003) Molecular monitoring of acute myeloid leukemia associated with inv(16): threshold of CBFbeta/MYH11 transcript copy number above which relapse occurs and below which continuous complete remission is likely. Leukemia 17, 650ā651.
Scholl C., Breitinger H., Schlenk R. F., Dohner H., Frohling S., and Dohner K. (2003) AML Study Group Ulm. Development of a real-time RT-PCR assay for the quantification of the most frequent MLL/AF9 fusion types resulting from translocation t(9;11)(p22;q23) in acute myeloid leukemia. Genes Chromosomes Cancer 38, 274ā280.
Viehmann S., Teigler-Schlegel A., Bruch J., Langebrake C., Reinhardt D., and Harbott J. (2003) Monitoring of minimal residual disease (MRD) by real-time quantitative reverse transcription PCR (RQ-RT-PCR) in childhood acute myeloid leukemia with AML1/ETO rearrangement. Leukemia 17, 1130ā1136.
Satake N., Maseki N., Kozu T., et al. (1995) Disappearance of AML1-MTG8(ETO) fusion transcript in acute myeloid leukaemia patients with t(8;21) in long-term remission. Br. J. Haematol. 91, 892ā898.
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
Ā© 2006 Humana Press Inc.
About this protocol
Cite this protocol
Picard, C., Silvy, M., Gabert, J. (2006). Overview of Real-Time RT-PCR Strategies for Quantification of Gene Rearrangements in the Myeloid Malignancies. In: Iland, H., Hertzberg, M., Marlton, P. (eds) Myeloid Leukemia. Methods In Molecular Medicineā¢, vol 125. Humana Press. https://doi.org/10.1385/1-59745-017-0:27
Download citation
DOI: https://doi.org/10.1385/1-59745-017-0:27
Publisher Name: Humana Press
Print ISBN: 978-1-58829-485-2
Online ISBN: 978-1-59745-017-1
eBook Packages: Springer Protocols