Introduction

The prognostic impact of residual BCR-ABL transcripts has been established using sensitive quantitative reverse transcription–PCR methodologies in chronic myeloid leukemia (CML) patients after allogeneic stem cell transplantation,¹ interferon-² and imatinib^{3, 4} therapy. International recommendations on the generation and interpretation of molecular data in the era of tyrosine kinase inhibitors have been published.^{5, 6} However, diversity of molecular approaches lead to incomparable results between different laboratories and studies.^{7, 8, 9} Thus, there is an unmet need for harmonization of both procedures and expression of results.¹⁰ In a series of consensus meetings on the initiative of the European LeukemiaNet, a catalogue of prerequisites to achieve optimal sensitivity and standardization has been elaborated: (a) use of at least 10 ml peripheral blood (PB); (b) processing within 36 h;⁸ (c) bedside RNA stabilization for multicenter trials;¹¹ (d) standardized PCR protocols;^{12, 13} (e) use of a plasmid containing target and housekeeping genes to avoid dilution errors; (f) use of total ABL, BCR and/or glucuronidase- (GUS) as internal controls.¹⁴ To substantiate this catalogue, an international multicenter trial involving 39 laboratories in 14 countries was initiated. The aim of the study was to assess the variability of results obtained by different laboratories using the PAXgene Blood RNA Kit (PreAnalytiX, Hombrechtikon, Switzerland) for RNA extraction, individual protocols for cDNA synthesis, three different PCR platforms (TaqMan (TM), n=25; LightCycler (LC), n=13; Rotorgene (RG) n=1) and optimized quantitative reverse transcription–PCR conditions.

Patients, materials and methods

Construction of a plasmid containing BCR-ABL and GUS (pME-2)

The pCR 2.1 TOPO Vector (Invitrogen, Carlsbad, CA, USA) was used to clone a sequence of b3a2 (e14a2) BCR-ABL derived from cDNA of the K562 cell line. An 855 bp PCR product was generated (forward primer: 5'-AGAGTTACACGTTCCTGATCTCCTCT-3', reverse primer: 5'-AGACAGTGGGCTTGTTGCGCTTTGGG-3') using a proofreading polymerase under the following conditions: 32 cycles of 30 s at 97 °C, 50 s at 60 °C, 50 s at 73 °C. Ligation of the PCR product was performed following the recommendations of the TOPO TA Cloning kit. Escherichia coli (E. coli) (One Shot Top 10 F') (Invitrogen) were transformed with an aliquot of the ligation reaction mix. After selection of one BCR-ABL-containing clone, the plasmid DNA was eluted. The insertion of a GUS sequence was performed as follows: after amplification of a 487 bp PCR product using the K562 cell line (forward primer: 5'-CAACTCTAGAAACGTTCTGGTCTGCCGTGAACA-3', reverse primer: 5'-TGGTTCTAGAACTGGTATAAGAAGTATCAGAAG-3'), using the above-mentioned conditions, both the BCR-ABL-containing plasmid and the GUS product were digested in separate tubes applying the restriction enzyme XbaI. The ligation step was carried out using the T4 ligase followed by transformation of E. coli. After selective growth of one clone containing both designated sequences, the plasmid DNA was eluted and linearized using the restriction enzyme SpeI. The total number of plasmids was estimated and nine serial dilutions (2 to 2 10⁸ copies per l) were centrally established in TER buffer (Tris, EDTA, rRNA). BCR-ABL and GUS inserts were chosen in accordance with primers and probes of the Europe Against Cancer (EAC) protocol^{12, 14} for TM laboratories and for laboratories using LC technology.¹³ Sequence analysis revealed a single base mismatch in exon 11 of the GUS sequence affecting the binding of the EAC primer ENF1102 (Figure 1). However, ENF1102 maps to a single-nucleotide polymorphism (SNP) (rs1061361, dbSNP126). Sequencing of GUS exon 11 in 19 randomly selected CML patients revealed the plasmid sequence in 13 (68%) patients, SNP heterozygosity in 5 (26%) patients and SNP homozygosity in 1 (5%) patient, which is in line with population-based allele frequencies of the SNP (http://www.ncbi.nlm.nih.gov/SNP). Crossover experiments evaluating GUS quantifications (10-fold) of pME-2 plasmid (4 10⁴ and 4 10⁶ copies) showed no statistical difference using ENF1102 (SNP) or ENF1102-wt (wild type) as forward primers (4 10⁴ copies: median CP 24.65 vs 24.65, P=0.88; 4 10⁶ copies: median CP 17.79 vs 17.82, P=0.81). Furthermore, no difference was detected comparing 10-fold repetitions of a CML patient's cDNA with wild-type GUS (median CP 25.71 vs 25.77, P=0.38) and a CML patient's cDNA with homozygous SNP (median CP 25.10 vs 25.14, P=1.0). All BCR-ABL dilution samples contained varying proportions of polymorphic GUS, since each dilution was prepared using one heterozygous individual (b3a2 dilutions: CML patient heterozygous, healthy control wild type; b2a2 dilutions: CML patient wild type, healthy control heterozygous).

Figure 1.

Map of new plasmid (pME-2) consisting of PCR 2.1-TOPO vector and sequences of b3a2 BCR-ABL and glucuronidase- (GUS). The GUS sequence was highlighted, indicating exon boundaries (: exon 11/exon 12; : exon 10/exon 11). Primers for amplification with TaqMan (TM) and LightCycler technology are highlighted and transcript lengths described. The forward primer ENF1102 shows a single base mismatch due to a single-nucleotide polymorphism (rs1061361), which affects TM laboratories only.

Full figure and legend (123K)

Establishment of an RQ–PCR for GUS quantification with LightCycler technology

For TM users, the EAC protocol for GUS transcript quantification was suggested.¹⁴ For LC users, a new PCR protocol for GUS transcript quantification was established: As reverse primer, the EAC (ENR1162: 5'-CCGAGTGAAGATCCCCTTTTTA-3', GUS exon 12) was used and combined with a forward primer on GUS exon 10 (GUS10-LC: 5'-AGAAACGATTGCAGGGTTTCAC-3'). Hybridization probes were placed on exon 11: 5'-TGATCCAGACCCAGATGGTACTGCT-F3'; 5'LC Red640-TAGCAGACTTTTCTGGTACTCTTCAGTGAACA-P3' (F=fluorescein, P=phosphate). PCR conditions were adjusted to 45 cycles of 1 s at 95 °C, 10 s at 58 °C and 26 s at 72 °C.

Patients and healthy controls

After informed consent, PB samples of two CML patients (patient A: 68-year-old female, b2a2=e13a2 transcript positive, ratio BCR-ABL/ABL 47%; patient B: 71-year-old male, b3a2=e14a2 transcript positive, ratio BCR-ABL/ABL 66%) with relapsing or refractory disease after therapy were used for spiking PB samples from healthy donors (female donor, age 30 years; male donor, age 33 years).

Sample preparation

Red blood cell lysis of PB samples of BCR-ABL-positive patients was performed by adding four volumes of NH₄Cl solution (0.15 M) to one volume of PB, followed by 10 min on ice and a 10 min spin at 400 g. After decanting and resuspension of the pellet in phosphate-buffered saline, white blood cells (WBC) were counted in a Neubauer chamber. A stock solution was prepared for each BCR-ABL transcript type adapted to the WBC count of the respective healthy control blood. A total of 1200 ml of PB from healthy donors was used for preparation of dilutions 1:10 (10%), 1:50 (2%), 1:100 (1%) and 1:1000 (0.1%) of both b3a2 and b2a2 dilutions as well as negative controls. Four hundred aliquots of 2.5 ml were injected manually into PAXgene tubes containing stabilizing agent, blinded and frozen at -20 °C until shipment.

RNA extraction

Participants (n=39) were asked to follow the manufacturers' protocol of the PAXgene Blood RNA Kit for RNA extraction after thawing and incubation at room temperature for 2 h of the 10 blinded samples. Five of the resulting 40 l of the eluate were used to determine RNA concentration by ultraviolet spectrophotometry. Laboratories without experience in PAXgene extraction (23 of 39 laboratories, 59%) had the opportunity to practice with 20 test kits.

cDNA synthesis

RNA was transcribed to cDNA following individually established protocols at each site. All laboratories used either random hexamer or nonamer priming, and most of them used the M-MLV reverse transcriptase (n=17; AMV n=6; Superscript n=5; others n=11).

Quantitative real-time RT–PCR on LightCycler platforms

Quantitative real-time RT–PCR (RQ-PCR) for BCR-ABL, total ABL and GUS transcripts was performed in triplicate from cDNA of all samples (n=10). In most of the LC laboratories (11 of 13, 85%), a common protocol¹³ was already established that was extended by providing GUS-specific primers and hybridization probes. Two of 13 LC laboratories used the EAC protocol. Seven serial dilutions of 4 to 4 10⁶ copies of plasmid pME-2 per reaction were used to provide an external control within each LC run.

Quantitative real-time RT–PCR on TM and RG platforms

RQ-PCR for BCR-ABL, total ABL and GUS transcripts was also performed in triplicate on the TM platforms; all of them (23 of 23, 100%) used the primers and probes published in the EAC protocol. In addition to the plasmid dilutions, GUS-specific primers and probes were provided if not pre-existing in the respective laboratories. One laboratory using the RG platform performed RQ-PCR following a commercially available kit (FusionQuant, Ipsogen, Marseille, France).

Calculation of the results

Absolute transcript counts were enlisted at each site, transferred via e-mail and centrally decoded. After calculation of the mean for each triple measure, ratios BCR-ABL/total ABL and ratios BCR-ABL/GUS were calculated and expressed in percent. Single outliers were excluded for calculation of the mean. At least 2 of 3 measures were required to be positive to judge the sample as BCR-ABL positive.

Statistical methods

Correlation coefficients and P-values were calculated according to Spearman's rank test. Comparison of slopes of the regression curves was performed using the non-parametric Mann–Whitney test. The non-parametric Wilcoxon matched pairs test was applied for evaluation of PCR efficiencies using GUS primers with or without the SNP rs1061361. All tests, including regression analyses, were calculated using the GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA).

Results

Data were received from 37 of 39 participating laboratories. Reasons for not providing data were loss of samples in one laboratory and methodological problems in the second.

RNA yield

The RNA concentrations measured by 35 laboratories are depicted according to LC, TM or RG platform in Figure 2. The median RNA yield per laboratory differed between 8 and 260 ng l^-1 (overall median 133 ng l^-1) corresponding to a median absolute RNA yield of 5.3 g per 2.5 ml PAXgene tube (expectation according to the manufacturers' protocol: >3 g in 95% of cases). Three laboratories did not achieve a proper RNA concentration (13, 8 and 9 ng l^-1 resulting in RNA yield of 0.52, 0.32 and 0.36 g, respectively). These laboratories were provided with cDNA from two laboratories having achieved adequate RNA yield.

Figure 2.

RNA concentrations as determined by ultraviolet spectrophotometry. Box-whisker plots of 35 individual laboratories combining 10 samples each. Boxes are marked by the first and third quartiles, and the whiskers extend to the range. The dotted circles indicate the three laboratories failing to achieve sufficient RNA yield. The dashed line marks the median RNA yield of 133 ng l^-1.

Full figure and legend (74K)

Correlation of ratios BCR-ABL/ABL and BCR-ABL/GUS

Ratios BCR-ABL/ABL and BCR-ABL/GUS from the same samples correlated well (r=0.90, P<0.0001, 95% confidence interval (CI), 0.87–0.92).

Ratios BCR-ABL/ABL and BCR-ABL/GUS: individual results

The data are summarized in Figure 3. Median BCR-ABL/ABL ratios (95% CI) of 37 laboratories were 9.1% (7.8–11.9), 1.8% (1.6–2.2), 0.85% (0.76–1.2) and 0.11% (0.10–0.16) in b3a2 samples and 9.5% (7.9–12.5), 1.6% (1.4–2.0), 0.84% (0.76–1.1) and 0.11% (0.07–0.26) in b2a2 samples, respectively (dilutions 10, 2, 1 and 0.1% patient WBC in healthy control WBC; Figure 3a). Median ratios BCR-ABL/GUS (95% CI) turned out to be 3.4% (2.8–4.6), 0.77% (0.60–1.1), 0.37% (0.29–0.55) and 0.042% (0.031–0.082) in b3a2 samples and 2.8% (2.3–3.8), 0.48% (0.36–0.68), 0.29% (0.22–0.37) and 0.031% (0.017–0.074) in b2a2 samples (Figure 3b). The coefficients of variation (CV) for all participants were 0.62 for ratios BCR-ABL/ABL and 1.03 for ratios BCR-ABL/GUS. Five of 37 evaluable laboratories (13%; TM laboratories, n=5) detected low BCR-ABL copy numbers in negative control samples (positive in one of two negative control samples, n=4; positive in both negative control samples, n=1). One laboratory (3%) failed to detect BCR-ABL in the 1:1,000 dilution of b2a2 while achieving a low RNA yield in that particular sample (21 ng l^-1).

Figure 3.

Individual results of ratios BCR-ABL/ABL (a) and ratios BCR-ABL/GUS (b) from 37 participating laboratories examining serial dilutions of b3a2 and b2a2 BCR-ABL transcripts. Six low-level BCR-ABL measures in negative control samples are derived from five TaqMan laboratories. One LightCycler laboratory showed a false-negative result in the 0.1% b2a2 dilution.

Full figure and legend (105K)

Regression analysis revealed, for most of the laboratories, parallel regression curves for BCR-ABL/ABL ratios (median slope 0.93, R² 0.9974, standard error 0.21) and BCR-ABL/GUS ratios (median slope 0.33, R² 0.9978, standard error 0.08; Figure 4). Eight laboratories (22%; TM, n=6; LC, n=2) showed problems concerning linearity of the results (R²<0.97 and/or standard error >0.40). Overall, mean BCR-ABL/ABL ratios from TM laboratories were 1.7 times (95% CI, 1.5–2.0; Figure 5) higher than in LC laboratories, and mean BCR-ABL/GUS ratios were 2.2 times (95% CI, 1.8–2.6; data not shown) higher, which indicates a difference in the amplification efficiency. This is reflected in differences of the median slopes of the regression curves resulting from plotting dilutions (10, 2, 1, 0.1%) toward BCR-ABL/ABL ratios (TM 1.08 vs LC 0.58, P=0.0002) and BCR-ABL/GUS ratios (TM 0.38 vs LC 0.18, P=0.0046).

Figure 4.

Regression analysis of resulting ratios BCR-ABL/ABL and BCR-ABL/GUS compared to b3a2 dilutions.

Full figure and legend (72K)

Figure 5.

BCR-ABL/ABL ratios from 37 participating laboratories divided in LightCycler (LC) laboratories (L, circles) and TaqMan (TM) laboratories (T, squares). TM results were 1.7 times (95% CI, 1.5–2.0) higher than LC results.

Full figure and legend (58K)

Discussion

Considering the increasing number of laboratories adopting methods for molecular monitoring in CML and Philadelphia chromosome-positive acute lymphoblastic leukemia, we sought to harmonize quantitative PCR results for BCR-ABL transcripts by introducing a newly designed plasmid containing BCR-ABL and GUS sequences for external calibration. By this approach, two of the housekeeping genes commonly used for minimal residual disease monitoring (ABL and GUS) were implemented.¹⁴ Dilution series of WBC were established, blinded and distributed¹⁰ from untreated CML patients in PB of healthy donors. Ultraviolet spectrophotometry showed a wide range of RNA yields. Only three laboratories (8%) failed to achieve sufficient RNA amounts although 23 of the 39 (59%) participating laboratories had no previous experience in performing PAXgene RNA extraction. The good correlation of BCR-ABL/ABL with BCR-ABL/GUS ratios validated the usability of both housekeeping genes for CML monitoring as proposed previously.¹⁴ BCR-ABL ratios of each dilution revealed a good concordance within the 37 evaluable laboratories, which was favorable using ABL (CV=0.62) compared to GUS (CV=1.03) as the housekeeping gene. Hitherto insufficient experience with GUS quantification and the need for most of the laboratories to newly establish a protocol in the context of this study might be the main reason for the increased variance. Most laboratories provided data with a linear correlation of BCR-ABL/housekeeping gene ratios and the cell dilutions, whereas eight laboratories (22%) revealed problems of linearity. Five TM laboratories (13%) detected low levels of BCR-ABL in the negative control samples (one of two measures positive, n=4; two of two measures positive, n=1), whereas one LC laboratory (3%) was false-negative in one of the 1:1000 dilutions. This agrees with systematic differences between LC and TM platforms, which have been recognized previously and will be overcome by the introduction of external calibrators and regular control rounds.^{10, 15} Control rounds like this turn out to be efficient tools for detection of PCR assay deficits in individual laboratories, providing an opportunity for correction. Since the SNP in the GUS gene has not been considered in the EAC protocol, we suggest changing the TM protocol by using a chimeric primer taking into account both SNP and wild-type sequences or by avoiding the polymorphic sequence.

The study revealed an overall beneficial applicability of the new plasmid (pME-2) for quantification of BCR-ABL, total ABL and GUS independent of the PCR platform. It enables the harmonization of results irrespective of the use of different protocols for cDNA synthesis or PCR and provides a solid base for the establishment of an international standard.

Notes

Participating laboratories

Division of Hematology and Internal Medicine, Department of Clinical and Biological Sciences, University of Turin, Turin, Italy (Giuseppe Saglio, Enrico Gottardi); Klinisches Institut für Medizinische und Chemische Labordiagnostik, Ehemalige I. Medizinische Universitätsklinik, Vienna, Austria (Harald Esterbauer); Molecular Biology, Children's Cancer Research Institute, Vienna, Austria (Thomas Lion); Department of Molecular Genetics, Institute of Hematology and Blood Transfusion, Prague, Czech Republic (Jana Rulcova, Cedric Haskovec); Department of Hematology, Aarhus University Hospital, Aarhus, Denmark (Charlotte Guldborg Nyvold); Department of Pathology, Odense University Hospital, Odense, Denmark (Niels Pallisgaard); Laboratoire Central d'Hématologie, Hôpital Saint-Louis, Paris, France (Jean-Michel Cayuela); Luminy Biotech Entreprises, Marseille, France (Fabienne Hermitte); INSERM U 817, Institut de Recherche contre le Cancer, Lille, France (Claude Preudhomme); INSERM E 217, Université Victor Segalen, Bordeaux, France (Francois-Xavier Mahon); Laboratoire de cytogénétique et biologie moléculaire, Centre hospitalier Lyon sud, Pierre Bénite, France (Sandrine Hayette); Klinik für Hämatologie, Onkologie und klinische Immunologie, Universität Düsseldorf, Düsseldorf, Germany (Ralf Kronenwett); Zell- und Molekularpathologie, Medizinische Hochschule, Hannover, Germany (Nils von Neuhoff); MLL-Munich Leukemia Lab, München, Germany (Susanne Schnittger); II. Medizinische Klinik, Universität Leipzig, Leipzig, Germany (Thoralf Lange); III. Medizinische Klinik, Universität Ulm, Ulm, Germany (Konstanze Döhner); Medizinische Klinik und Poliklinik I, Universität Dresden, Dresden, Germany (Christian Thiede); III. Medizinische Klinik, Universität Mainz, Mainz, Germany (Georg He); Klinik für Innere Medizin C, Universtität Greifswald, Greifswald, Germany (Frank Schüler); Institute of Hematology and Medical Oncology, University of Bologna, Bologna, Italy (Angela Poerio, Giovanni Martinelli); Department of Hematology, University of Naples, Naples, Italy (Fabrizio Pane); Department of Hematology, Jagiellonian University, Cracow, Poland (Tomasz Sacha); Department of Hematology at Hospital de la Santa Creu i Sant Pau, Universitat Autonoma de Barcelona, Barcelona, Spain (Josep Nomdedeu); Department of Hematopathology, University of Barcelona, Hospital Clinic, Barcelona, Spain (Dolors Colomer); Hematology Department, Hospital Universitario de Salamanca, Salamanca, Spain (Marcos Gonzalez Diaz); Laboratorio de Biologia Molecular, Hospital Universitario La Fe, Valencia, Spain (Pascual Bolufer); Molecular Medicine and Surgery, Karolinska Hospital, Stockholm, Sweden (Gisela Barbany); Molekulare Diagnostik, Inselspital, Bern, Switzerland (Elisabeth Oppliger-Leibundgut); Department of Hematology, VU University Medical Center, Amsterdam, The Netherlands (Quinten Waisfisz); Department of Genetics, Istanbul University , Istanbul, Turkey (Ugur Ozbek); Department of Clinical Haematology, Manchester Royal Infirmary, Manchester, United Kingdom (Abida Awan); Molecular Haematology, Glasgow Royal Infirmary, Glasgow, United Kingdom (Fiona Reid, Jan Baird); Department of Haematology, University of Liverpool, Liverpool, United Kingdom (Richard E Clark); II. Medizinische Klinik, Universität Frankfurt, Germany (Heike Pfeifer); Department of Pathology, Oregon Health and Science University, Portland OR, USA (Richard D Press); Department of Clinical Pathology, Cleveland Clinic Lerner College of Medicine, Cleveland, OH, USA (Raymond R Tubbs).

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Acknowledgements

The study was supported by the Competence Network 'Acute and chronic leukemias,' sponsored by the German Bundesministerium für Bildung und Forschung (Projektträger Gesundheitsforschung; DLR e.V.- 01 GI9980/6), the European LeukemiaNet (WP 4+12) within the 6th European Framework Programme for Research and Technological Development, the German José Carreras-Foundation, Qiagen, Hilden, Germany, and Novartis Pharma, Nürnberg, Germany.