Leukemia
SEARCH     advanced search my account e-alerts subscribe register
Journal home
Advance online publication
Current issue
Archive
Press releases
For authors
For referees
Contact editorial office
About the journal
For librarians
Subscribe
Advertising
naturereprints
Contact Springer Nature
Customer services
Site features
NPG Subject areas
Access material from all our publications in your subject area:
Biotechnology Biotechnology
Cancer Cancer
Chemistry Chemistry
Dentistry Dentistry
Development Development
Drug Discovery Drug Discovery
Earth Sciences Earth Sciences
Evolution & Ecology Evolution & Ecology
Genetics Genetics
Immunology Immunology
Materials Materials Science
Medical Research Medical Research
Microbiology Microbiology
Molecular Cell Biology Molecular Cell Biology
Neuroscience Neuroscience
Pharmacology Pharmacology
Physics Physics
Browse all publications
 
Journal home
Advance online publication
Current issue
Archive
Press releases
For authors
For referees
Contact editorial office
About the journal
For librarians
Subscribe
Advertising
naturereprints
Contact Springer Nature
Customer services
Site features
NPG Subject areas
Access material from all our publications in your subject area:
Biotechnology Biotechnology
Cancer Cancer
Chemistry Chemistry
Dentistry Dentistry
Development Development
Drug Discovery Drug Discovery
Earth Sciences Earth Sciences
Evolution & Ecology Evolution & Ecology
Genetics Genetics
Immunology Immunology
Materials Materials Science
Medical Research Medical Research
Microbiology Microbiology
Molecular Cell Biology Molecular Cell Biology
Neuroscience Neuroscience
Pharmacology Pharmacology
Physics Physics
Browse all publications
 
February 2000, Volume 14, Number 2, Pages 324-328
Table of contents    Previous  Article  Next   [PDF]
Bio-Technical Methods Section (BTS)
Quantitation of minimal residual disease in acute promyelocytic leukemia patients with t(15;17) translocation using real-time RT-PCR
B Cassinat1, F Zassadowski1, N Balitrand1, C Barbey1, J D Rain1, P Fenaux2, L Degos2, M Vidaud3 and C Chomienne1

1Laboratory of Cellular Biology, Nuclear Medicine Department, Paris V University, Paris, France

2Coordinators of APL 93 trial, Paris V University, Paris, France

3Laboratory of Molecular Genetic, Paris V University, Paris, France

Correspondence to: C Chomienne, LBCH, Service de Médecine Nucléaire, Hôpital Saint Louis, 1 rue Claude Vellefaux, 75010 Paris, France; Fax: 33 1 40 00 01 60

Abstract

We took advantage of a recently developed system allowing performance of real-time quantitation of polymerase chain reaction to develop a quantitative method of measurement of PML-RARalpha transcripts which are hallmarks of acute promyelocytic leukemia (APL) with t(15;17) translocation. Indeed, although quantitation of minimal residual disease has proved to be useful in predicting clinical outcome in other leukemias such as chronic myeloid leukemia or acute lymphoblastic leukemia, no quantitative data have been provided in the case of APL. We present here a method for quantitation of the most frequent subtypes of t(15;17) transcripts (namely bcr1 and bcr3). One specific forward primer is used for each subtype in order to keep amplicon length under 200 bp. The expression of PML-RARalpha transcripts is normalized using the housekeeping porphobilinogen deaminase (PBGD) gene. This technique allows detection of 10 copies of PML-RARalpha or PBGD plasmids, and quantitation was efficient up to 100 copies. One t(15;17)-positive NB4 cell could be detected among 106 HL60 cells, although quantitation was efficient up to one cell among 105. Repeatability and reproducibility of the method were satisfying as intra- and inter-assay variation coefficients were not higher than 15%. The efficiency of the method was finally tested in patient samples, showing a decrease of the PML-RARalpha copy number during therapy, and an increase at the time of relapse. Leukemia (2000) 14, 324-328.

Keywords

APL; PML-RAR; RT-PCR; real-time quantitation; TaqMan; minimal residual disease

Introduction

Acute promyelocytic leukemia (APL) is characterized by the t(15;17) translocation which fuses the PML gene to the retinoic acid receptor alpha (RARalpha) gene.1 The breakpoint in chromosome 17 is always located in the second intron of RARalpha gene, but in chromosome 15 different breakpoint cluster regions (bcr) may exist. Usually bcr1, bcr2 and bcr3 encompass intron 6, exon 6 and intron 3, respectively,2 resulting in three kind of transcripts, although some atypical localizations can occur.3 In the recent APL93 trial,4 combining ATRA and chemotherapy, although 90% of patients achieved complete remission (CR), and survival at 5 years is significantly improved, 5-15% still relapse and the level of mortality for patients in CR remains high, suggesting that individualization of treatment is needed. Thus, the identification of novel criteria which could allow prediction of the outcome of patients in CR is required. We postulated that a sensitive quantitative reverse transcription-polymerase chain reaction (RT-PCR) might be a valuable tool for assessing prognosis of patients, by providing quantitative kinetic data on the disappearance of leukemic cells. Indeed, the quantitation of minimal residual disease (MRD) after induction of remission has recently been shown to be a powerful prognostic factor for childhood acute lymphoblastic leukemia,5 and the kinetics of increasing numbers of Bcr-Abl transcripts predict the relapse of chronic myeloid leukemia patients after bone marrow transplantation.6 Although one paper reported the development of a quantitative method to detect PML-RARalpha transcripts by competitive PCR,7 to date, no kinetic data have yet been published for APL, probably because this analysis is laborious. To develop a new quantitative assay for t(15;17) transcripts, we took advantage of the development of an integrated system performing both thermal cycling, real-time detection of the amplification and subsequent analysis, that has contributed to the development of highly sensitive, accurate and really quantitative assays,8 particularly in the field of hematology in leukemias with fusion genes Bcr-Abl or AML1-ETO and ALL.9,10,11

This is the first report of a fast quantitative assay for bcr1 and bcr3 PML-RARalpha transcripts that does not necessitate post-PCR sample handling and which is characterized by good reproducibility and sensitivity, with a high dynamic range. Finally, we show that the technique is successful for the accurate quantification of MRD in APL patients.

Materials and methods

RNA isolation and cDNA synthesis

Total RNA was extracted from mononucleated cells prepared from the bone marrow of APL patients or the NB4 t(15;17) APL cell line, as previously described.12 Briefly, total RNA was prepared by ultracentrifugation of a guanidine-isothiocyanate lysate through a cesium chloride cushion. Pellet was washed with 70% ethanol and dissolved in diethylpyrocarbonate (DEPC)-treated water. RNA was reprecipitated by 10% sodium acetate 3M and 2.5 volume of ethanol, centrifuged, washed with 70% ethanol, and redissolved in DEPC-treated water, and then stored at -80°C. Integrity of RNA was assessed by migration on a formaldehyde-agarose gel.

Reverse transcription was performed on 1 mug of total RNA or less when the yield was low. After 3 min at 95°C, RNA was incubated for 1 h at 42°C with 50 units of M-MLV reverse transcriptase (Perkin Elmer), 20 units of RNase inhibitor (Perkin Elmer), 2.5 mM random hexamers and dNTP at 1 mM in a final volume of 20 mul. Finally, reverse transcriptase was denatured by heating for 5 min at 95°C.

Principle of quantitation using TaqMan probes

The TaqMan probe was double labeled by a fluorogenic reporter dye and a quencher dye. During the PCR reaction, upon hybridization to the template DNA, the probe was hydrolyzed by the 5' nuclease activity of the Taq DNA polymerase.13 Thus, the release of the reporter dye causes an increase in fluorescence intensity that is proportional to the accumulation of PCR products. The system generates an amplification plot based upon the fluorescence signal normalized with the passive reference ROX (a rhodamin derivative) contained in the buffer, in order to correct for PCR-independent fluorescence variation. Determination of the amplification plot is possible because a CCD camera measures the target-specific fluorescence emission spectrum from 500 to 650 nm in real time during each elongation step, and data are integrated by the Sequence Detector software V 1.6 (Perkin Elmer Applied Biosystems, Courtaboeuf, France). The software establishes the standard deviation of the baseline fluorescence usually between the 3rd and the 15th cycles. The threshold cycle (Ct) is defined as the fractional cycle where the fluorescence intensity of a sample reached 10 times the standard deviation of the baseline. The CT is invertionally proportional to the initial number of copies,14 thus if the starting copy number is important, the specific signal is detected early and the CT value is low. In order to correct variations linked to differences in the amount of RNA taken for the reaction or to different levels of inhibition during RT or PCR, we normalized the number of target gene copies using as a reference gene the porphobilinogene deaminase (PBGD) gene, a ubiquitously expressed housekeeping gene.15,16 The number of PML-RARalpha transcripts was normalized for 105 copies of PBGD transcripts.

Primers and probe

To achieve the aim of defining amplicons not longer than 200 bp, one set of primers was designed for each type of PML-RARalpha transcript, QP3 and QR1 for Bcr1, QP1 and QR1 for Bcr3 (Table 1). These primers were located in exons, thus each primer spanning an intron. Indeed, the breakpoint in chromosome 17 is always located in the intron 2 of the RARalpha gene, and in the majority of bcr1 and bcr3 transcripts the breakpoint is located, respectively, in introns 3 and 6 of the PML gene.2 The TaqMan probe is located in the RARalpha gene so as to be convenient for both bcr1 and bcr3 transcripts (Table 1). One set of primers was also defined for the reference PBGD gene (Table 1). Only the transcripts resulting from the housekeeping promoter in the PBGD gene were amplified using a forward primer in the first exon.15 Furthermore, to avoid a contaminating signal from PBGD genomic DNA, these primers were also located in different exons and the probe was located on the junction between two exons. Both primers and TaqMan probes were designed with Primer Express software (Perkin Elmer, Foster City, CA, USA). Probes labelled by a 5' FAM reporter and a 3' TAMRA quencher group were synthesized according to Lee et al17 by Perkin Elmer/Applied Biosystems.

PCR conditions

We used 1.25 U AmpliTaq Gold DNA polymerase, 0.5 U AmpErase uracil N-glycosylase (UNG), 200 muM dATP, dCTP, dGTP, 400 muM dUTP (Perkin Elmer/Applied Biosystems), forward and reverse primers at 200 muM, specific TaqMan probe at 100 nM, in TaqMan Buffer A with 6 mM MgCl2, in a final volume of 50 mul containing 5 mul of cDNA or plasmid. All the reagents were from Perkin Elmer/Applied Biosystems.

PCR reactions were set in a MicroAmp optical 96-well reaction plate (Perkin Elmer/Applied Biosystems) which were closed using MicroAmp optical caps (Perkin Elmer/Applied Biosystems). After 2 min at 50°C to allow the destruction by UNG of potential contaminant PCR products, and 10 min at 95°C to denature UNG and activate AmpliTaq Gold, the amplification was carried out by 50 cycles at 95°C for 15 s and 65°C for 1 min in the ABI/Prism 7700 Sequence Detector System (ABI/Perkin Elmer, Foster City, CA, USA).

Establishment of the standard curves for PML-RARalpha and PBGD

Quantitation utilized standard curves which have been established with plasmids containing specific sequences of each gene studied. For PML-RARalpha bcr1 subtype, we used a plasmid containing the complete cDNA. For PML-RARalpha bcr3 subtype, we cloned a PCR product (obtained from clinical samples) longer than the amplicon used for TaqMan assay, into the pCR II-TOPO vector using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA, USA). A plasmid containing the cDNA coding for the PBGD gene was a kind gift of Dr H Cave (Hôpital Robert Debre, Paris, France). Serial dilutions were prepared once for all tests presented in this study and conserved at -20°C. For each assay 10-fold dilutions starting at 108 copies of the specific plasmid were analyzed in triplicate, and the Sequence Detector system software V 1.6 (Perkin Elmer, Applied Biosystems) established a standard curve by plotting the CT vs the known copy number on a logarithm scale. The standard curve allowed us to interpolate the unknown copy number of specific cDNA in each sample.

Results and discussion

We present here a sensitive, specific and quantitative method to follow the minimal residual disease in APL patients, based on the TaqMan technology. Simultaneous amplification of serial dilutions of known quantities of a plasmid containing the bcr1 or bcr3 target sequence and a plasmid containing the PBGD reference gene sequence were performed on six plasmid dilutions and a standard curve was constructed by the Sequence Detector software for both bcr1 and bcr3 transcripts, and the PBGD reference gene. The standard curves obtained showed that quantitation of each target gene is linear on a scale of at least 6 orders of magnitude with excellent correlation factors of 0.99 and optimal PCR efficiencies for the three plasmids (bcr1, bcr3 and PBGD) between 93 and 96% corresponding to slope values18 ranging between 3.4 and 3.5 (Figure 1 and data not shown).

Monitoring of MRD in APL samples has been to date performed by nested RT-PCR.12,19,20,21 The sensitivity of our method proved identical for the three targets studied as we could detect as low as 10 copies of each plasmid; however, to ensure an accurate quantitation we set the threshold of detection at 100 copies for the three plasmids. Indeed, under this copy number, quantitation was not sufficiently efficient or reproducible (data not shown). We then assessed the sensitivity of the method on RNA preparations from myeloid cells and patient samples. Figure 2 shows that even on NB4 cell RNA extracts, the technique proved to be excellent with a very good linearity of the dilutions up to 1 bcr1 cell among 105 negative cells, although one cell among 106 could sometimes be detected in one of the duplicates. This experiment is equally representative of the specificity of the test described, as the presence of the negative control cells (HL-60) did not alter the amplification of the specific target sequence of the leukemic bcr1-positive NB4 cells. Thus, the sensitivity of the technique is extremely good and within the range of the sensitivity usually obtained with conventional RT-PCR of NB4 cells.12,19,20,21 This technique allowed us to quantify, for the first time, the copy number of PML-RARalpha and PBGD transcripts present in NB4 cells (1 mug of total RNA from NB4 cells contained 104 copies of PML-RARalpha and 105 copies of PBGD). Both on plasmids and cell extracts, the precision of the TaqMan technology in quantifying PML-RARalpha transcripts was tested. For intra-assay repeatability on RNA extracts, reverse transcription products of cells from the bcr1 cell line NB4 or of cells from a bcr3 patient were analyzed in five replicates in the same reaction plate. The variation coefficient (cv) of the number of copies calculated from these replicates was about 10% for each of the three transcripts studied (bcr1, bcr3 and PBGD). Likewise, a variation coefficient of the same magnitude was obtained when dilutions of bcr1, bcr3 or PBGD plasmids were analyzed in 10 replicates in the same experiment. The inter-assay analysis was performed by testing five reverse transcriptions obtained from the same preparation of total RNA, and tested in triplicates in different PCR reactions. We obtained a cv of about 5% in the copy number once normalized with PBGD. When performed with dilutions of bcr1, bcr3 or PBGD plasmids, the inter-assay analysis was characterized by cvs between 10 and 15%. Thus intra-assay repeatability and inter-assay reproducibility confirm the precision of this technique.

Finally, in order to test if this method was efficient in monitoring MRD, we analyzed bone marrow samples of five APL patients (three patients known to harbor a bcr1 transcript and two patients with a bcr3 transcript). The samples were obtained at diagnosis, and at different time intervals during follow-up (Figure 3). We found that the normalized number of PML-RARalpha transcripts at diagnosis was different from one patient to another and that a significant decrease in copy number was evident during therapy. However, the reduction of copy number was variable from one patient to another (Figure 3a). The patient presented in Figure 3b still had an important number of copies (800) after 3 months of consolidation therapy (APL93 trial), although he was in complete remission. The copy number increased by 30-fold (30 ´ 103) when he relapsed 5 months later. This level was efficiently reduced (2.8 ´ 103) after ATRA plus chemotherapy. Quantitative RT-PCR monitoring in this patient effectively reflects the clinical evolution. The preliminary results presented here suggest that differences in the kinetics of PML-RARalpha transcript decrease can be expected during therapy and could provide the basis for comparison of therapeutic response. In addition, the relapse of the patient presented in this study is well correlated to an increase in the number of PML-RARalpha copies.

In summary, we show here that real-time RT-PCR is efficient to perform quantitation of PML-RARalpha transcripts of bcr1 or bcr3 subtypes in patient samples. Because about 10% of APL patients have a transcript of intermediate length called bcr2 it will be necessary to quantitate such transcripts, however, bcr2 transcripts are more complex and no correct set of primers is yet available. This technique allowed to detect 104 PML-RARalpha copies in approximately 1 mug of RNA from patient samples at diagnosis. This level of detection is a little higher than the 103 copies previously reported,7 but further confirms that PML-RARalpha is weakly expressed as previously described.22,23 This quantitative method will in fact be a useful tool to optimize the upstream steps of the RT-PCR reaction such as patient cell handling, anticoagulant used, shipment, RNA extraction, and reverse transcription, in order to increase the yield in cDNA, an important limiting factor specifically noted in APL cells.

This approach has several advantages when compared to classical end-point quantitative methods. TaqMan technology utilizes hybridization probes, which confers to the method a high degree of specificity, without the need to analyze PCR products on a gel, with subsequent transfer and hybridization, a time-consuming step with a high risk of inter-assay contamination. In addition, compared to the standard end-point methods in which large variations can occur24,25 the method is already known for its very efficient reproducibility and repeatability, which we have confirmed for PML-RARalpha transcripts. Finally, the 96-well reaction plate we have tested, provides a convenient tool for simultaneous standard and patient sample testing. The automation allows the study of several patient samples within 2 h, significantly reducing the time taken for the RT-PCR analysis itself. Thus defined, this technology should allow ongoing studies to assess the potential role of this assay in predicting the clinical outcome of patients based on the kinetic quantification of their MRD during follow-up.

Acknowledgements

This work was supported by the Ligue Contre le Cancer des Hauts de Seine and the Association pour la Recherche contre le Cancer.

References

1 De The H, Chomienne C, Lanotte M, Degos L, Dejean A. The t(15;17) translocation of acute promyelocytic leukaemia fuses the retinoic acid receptor alpha gene to a novel transcribed locus. Nature 1990; 347: 558-561, MEDLINE

2 Pandolfi PP, Alcalay M, Fagioli M, Zangrilli D, Mencarelli A, Diverio D, Biondi A, Lo Coco F, Rambaldi A, Grignani F, Rochette-Egly C, Gaube MP, Chambon P, Pelicci PG. Genomic variability and alternative splicing generate multiple PML-RARalpha transcripts that encode aberrant PML proteins and PML-RARalpha isoforms in acute promyelocytic leukemia. EMBO J 1992; 11: 1397-1407, MEDLINE

3 Geng JP, Tong JH, Dong S, Wang ZY, Chen SJ, Chen Z, Zelent A, Berger R, Larsen CJ. Localization of the chromosome 15 breakpoints and expression of multiple PML-RARa transcripts in acute promyelocytic leukemia: a study of 28 Chinese patients. Leukemia 1996; 7: 20-26,

4 Fenaux P, Chastang C, Chevret S, Sanz M, Dombret H, Archimbaud E, Fey M, Rayon C, Huguet F, Sotto JJ, Cony Makhoul P, Travade P, Solary E, Fegueux N, Bordessoule D, San Miguel J, Link H, Desablens B, Stamatoulas A, Deconinck E, Maloisel F, Castaigne S, Preudhomme C, Degos L, European APL group. A randomized comparison of all trans retinoic acid followed by chemotherapy and ATRA plus chemotherapy, and the role of maintenance therapy in newly diagnosed acute promyelocytic leukemia. Blood 1999; 94: 1192-1200, MEDLINE

5 Cave H, Van der Werff ten Bosch J, Suciu S, Guidal C, Waterkeyn C, Otten J, Bakkus M, Thielemans K, Grandchamp B, Vilmer E. Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia. New Engl J Med 1998; 339: 591-598, MEDLINE

6 Lin F, van Rhee F, Goldman JM, Cross NCP. Kinetics of increasing Bcr-Abl transcripts numbers in chronic myeloid leukemia patients who relapse after bone marrow transplantation. Blood 1996; 87: 4473-4478, MEDLINE

7 Seale JRC, Varma S, Swirsky DM, Pandolfi PP, Goldman JM, Cross NCP. Quantification of PML-RARalpha transcripts in acute promyelocytic leukemia: explanation for the lack of sensitivity of RT-PCR for the detection of minimal residual disease and induction of the leukaemia-specific mRNA by alpha interferon. Br J Haematol 1996; 95: 95-101, MEDLINE

8 Gibson UEM, Heid CA, Williams PM. A novel method for real time quantitative RT-PCR. Genome Res 1996; 6: 995-1001, MEDLINE

9 Mensink E, van de Locht A, Schattenberg A, Linders E, Schaap N, Geurts van Kessel A, de Witte T. Quantitation of minimal residual disease in Philadelphia chromosome positive chronic myeloid leukaemia patients using real-time quantitative RT-PCR. Br J Haematol 1998; 102: 768-774, Article MEDLINE

10 Marcucci G, Livak KJ, Bi W, Strout MP, Bloomfield CD, Caliguri 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, MEDLINE

11 Pongers-Willemse MJ, Verhagen OJHM, Tibbe GJM, Wijkhuijs AJM, de Haas V, Roovers E, van der Schoot CE, van Dongen JJM. Real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia using junctional region specific TaqMan probes. Leukemia 1998; 12: 2006-2014, MEDLINE

12 Castaigne S, Balitrand N, de The H, Dejean A, Degos L, Chomienne C. A PML/retinoic acid receptor alpha fusion transcript is constantly detected by RNA-based polymerase chain reaction in acute promyelocytic leukemia. Blood 1992; 79: 3110-3115, MEDLINE

13 Holland PM, Abramson RD, Watson R, Gelfand GH. Detection of specific polymerase chain reaction products by utilizing the 5' to 3' exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci USA 1991; 88: 7276-7280, MEDLINE

14 Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res 1996; 6: 986-994, MEDLINE

15 Chretien S, Dubart A, Beaupain D, Raich N, Grandchamp B, Rosa J, Goosens M, Romeo PH. Alternative transcription and splicing of the human porphobilinogen deaminase gene result either in tissue-specific or in housekeeping expression. Proc Natl Acad Sci USA 1988; 85: 6-10, MEDLINE

16 Fincke J, Fritzen R, Ternes P, Lange W, Dolken G. An improved strategy and a useful housekeeping gene for RNA analysis from formalin-embedded tissues by PCR. Biotechniques 1993; 14: 448-453, MEDLINE

17 Lee LG, Connell CR, Bloch W. Allelic discrimination by nick-translation PCR with fluorogenic probes. Nucleic Acids Res 1993; 21: 3761-3766, MEDLINE

18 Fink L, Seeger W, Ermert L, Hanze J, Stahl U, Grimminger F, Kummer W, Bohle RM. Real-time quantitative RT-PCR after laser-assisted cell picking. Nature Med 1998; 4: 1329-1333, Article MEDLINE

19 Lo Coco F, Diverio D, Pandolfi PP, Biondi A, Rossi V, Avvisati G, Rambaldi A, Arcese W, Petti MC, Meloni G, Mandeli F, Grignani F, Macera G, Barbui T, Pelicci PG. Molecular evaluation of residual disease as a predictor of relapse in acute promyelocytic leukemia. Lancet 1992; 340: 1437-1438, MEDLINE

20 Miller WH, Kakizuka A, Frankel SR, Warrel RP, DeBlasio A, Levine K, Evans RM, Dmitrovsky E. Reverse transcription polymerase chain reaction for the rearranged retinoic acid receptor alpha clarifies diagnosis and detects minimal residual disease in acute promyelocytic leukemia. Proc Natl Acad Sci USA 1992; 89: 2694-2698, MEDLINE

21 Grimwade D, Howe K, Langabeer S, Burnett S, Goldstone A, Solomon E. Minimal residual disease detection in acute promyelocytic leukemia by reverse-transcriptase PCR: evaluation of PML-RAR alpha and RAR alpha-PML assessment in patients who ultimately relapse. Leukemia 1996; 10: 61-66, MEDLINE

22 Chomienne C. RT-PCR in acute promyelocytic leukemia: second workshop of the European Retinoic Group. Leukemia 1996; 10: 368-371, MEDLINE

23 Bolufer P, Barragan E, Sanz MA, Martin G, Bornstein R, Colomer D, Delgado MD, Gonzalez M, Marugan I, Roman J, Gomez MT, Anguita E, Diverio D, Chomienne C, Briz M. Preliminary experience in external quality control of RT-PCR PML-RAR alpha detection in promyelocytic leukemia. Leukemia 1998; 12: 2024-2028, MEDLINE

24 Gerard CJ, Olsson K, Ramanathan R, Reading C, Hanania EG. Improved quantitation of minimal residual disease in multiple myeloma using real-time polymerase chain reaction and plasmid-DNA complementarity determining in region III standards. Cancer Res 1998; 58: 3957-3964, MEDLINE

25 Henry JM, Sykes PJ, Brisco MJ, To LB, Juttner CA. Comparison of myeloma cell contamination of bone marrow and peripheral blood stem cell harvests. Br J Haematol 1996; 92: 614-619, MEDLINE

Figures

Figure 1  Standard curves with Bcr1 plasmid. (a) Real-time amplification plots of the 10-fold plasmid dilutions. (b) Standard curve showing linear correlation between Ct and the initial amount of plasmid copies.

Figure 2  Dilution of t(15;17)-positive NB4 cells in t(15;17)-negative HL60 cells. (a) Real-time amplification plots of the 10-fold dilution. (b) Linearity of the correlation between Ct and initial number of NB4 cells.

Figure 3  (a) Quantitation of PML-RARalpha transcripts before, during and after treatment, in four different patients harboring bcr1 or bcr2 transcripts. (b) Quantitation of PML/RARalpha transcripts in one bcr3 patient during the course of treatment and relapse.

Tables

Table 1  Primer and probe sequences

Received 19 May 1999; accepted 24 September 1999
February 2000, Volume 14, Number 2, Pages 324-328
Table of contents    Previous  Article  Next    [PDF]