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Technical Briefs |
1 Molecular Biology, Department of Medical Biopathology, Hospital Universitario La Fe, Avda Campanar 21, 46009 Valencia, Spain;2 Hematopathology Unit, Hospital Clínic, Barcelona, Spain;3 Molecular Biology, Hematology, Hospital Gran Canaria Dr. Negrin, Las Paslmas de GC, Spain;4 Molecular Biology, Hematology, Hospital 12 de Octubre, Madrid, Spain;5 Immunopathology and Molecular Biology, Hematology, Hospital Clínico Universitario, Salamanca, Spain;6 Laboratory of Hematology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain;7 Laboratory of Cytogenetics and Molecular Biology, Service of Pathology, Hospital del Mar, Barcelona, Spain;8 Laboratory of Integrated Diagnosis of Oncohematologic Diseases, University Tor-Vergata, Rome, Italy;9 Laboratorio di Diagnostica Molecolare Oncoematologica, Dipartimento di Biotecnologie Cellulari ed Ematologia, Universitá degli Studi "La Sapienza", Rome, Italy;10 Biology, Hematology, Hospital de Jerez, Jerez de la Frontera, Cádiz, Spain;11 Molecular Cytogenetic Unit, Servicio de Hematologia, Hospital Universitario Puerta de Hierro, Madrid, Spain;12 Unified Laboratory, Immunology, Hospital Donostia, San Sebastian, Gupuzcoa, Spain;13 Molecular Medicine Unit-INGO (Sergas), University of Santiago de Compostela, Hospital Clínico Universitario de Santiago, Santiago de Compostela, Spain;14 Departamento de Estadistica e Investigación Operativa, Universidad Politécnica de Valencia, Valencia, Spain;15 Clinical Hematology, Service of Hematology, Hospital Universitario La Fe, Hospital Universitario La Fe, Valencia, Spain
aauthor for correspondence: fax 34961973030, e-mail bolufer_pas{at}gva.es
The recent introduction on the market of instruments for real-time PCR has prompted the development of quantitative assays for the most common fusion transcripts detectable in hematologic malignancies. However, because the ABI PRISM apparatus (ABI; Applied Biosystems) was the first available instrument for real-time PCR, most of the methods developed for the ABI PRISM use TaqMan probe chemistry (1)(2)(3). With the introduction of other real-time PCR instruments, such as the LightCycler (LC; Roche), other methods have been described (4)(5)(6)(7). The instruments differ in several respects, including the light sources and the approach to acquisition of fluorescence data. Few reports have compared the results obtained with different types of real-time PCR instruments (8). To the best of our knowledge, no such multicenter studies with common calibrators and common methods have been reported.
In the present study we analyzed the results obtained with two of the more widely used instruments for real time PCR, i.e., the ABI and LC, for amplifying two rearrangements frequently detectable in human leukemia, the BCR-ABL and PML-RARa fusion genes. For BCR-ABL several quantitative methods have been established for both instruments (3)(4)(5)(6)(7), whereas for PML-RARa most of the quantitative methods have been developed for the ABI PRISM (1).
The quantification of BCR-ABL transcripts is clinically relevant for monitoring patients with chronic myeloid leukemia undergoing allogeneic hematopoietic stem cell transplantation (4)(9) or treatment with interferon-a or imatinib mesylate (9)(10)(11). For example, low numbers of BCR-ABL transcripts after 2 weeks of imatinib treatment predict a good response to imatinib after 4 weeks (9). With respect to PML-RARa, recent reports have shown that quantitative assessment of PML-RARa transcripts allowed efficient monitoring of minimal residual disease (1) and assessment of the effects of the treatment given (12). Furthermore, patients who had transcription values above an empirical checkpoint after consolidation therapy had an increased risk of relapse (13).
Ten laboratories participated in the trial for PML-RARa analysis (6 using LC and 4 ABI), and 11 laboratories were involved in the BCR-ABL assay (7 using LC and 4 ABI; see the list of participating laboratories in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol50/issue6/). In the trial for PML-RARa, each laboratory received 20 samples: 2 reagent blanks, 4 BCR1-positive, and 4 BCR3-positive cDNA samples in two replicates. In the trial for BCR-ABL, the laboratories received 10 samples: 1 blank and 9 patient cDNA samples (5 B3A2-positive and 4 B2A2-positive).
The laboratories also received calibrators prepared by cloning PCR products of samples from positive patients (B3A2 isoform for BCR-ABL, and BCR1 or BCR3 isoforms for PML-RARa) into CR® II-TOPO® vector (TOPOTM Cloning® Kit). For PML-RARa, two sets of calibrators were prepared: one for BCR1 and the other for the BCR3 isoform. Calibrators were provided at the following concentrations: 2 x 105, 2 x 104, 2 x 102, 2 x 101, and 2 copies/µL. Samples and calibrators were shipped on dry ice by overnight courier and stored refrigerated until used. The calibrators were analyzed in duplicate and the samples in triplicate.
For PML-RARa quantification, the laboratories equipped with ABI used the reagents and protocol established by Gabert et al. (14)(15) for this instrument in the Europe Against Cancer Program (EAC Protocol April 2002). The method designed for the ABI PRISM was optimized in the LC by use of a final volume of 10 µL, including 2 µL of cDNA samples (unknowns) or 2 µL of calibrators (for the calibration curve; see the Methods file in the online Data Supplement).
For the BCR-ABL rearrangement the laboratories equipped with ABI used the reagents and protocol designed by Gabert et al. (14)(15). The ABI method was also optimized for the LC as described (see the Methods file in the online Data Supplement).
The quantification of transcripts was carried out automatically with the software provided in each type of equipment (see the section on quantification in the Methods file in the online Data Supplement).
To compare the results among the laboratories or samples for each real-time instrument, we performed a multivariate ANOVA for laboratories and samples as described in the Statistics file in the online Data Supplement.
The efficiencies estimated from the slopes of the calibration curves for the PML-RARa BCR1 and BCR3 isoforms for the ABI and LC were very similar (Table 1
in the online Data Supplement). One false-positive result was detected in a BCR1 blank sample (Table 1
).
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All laboratories detected transcripts in the replicate of the BCR1-positive sample with the lowest number of copies, and three of the four ABI and two of the seven LC laboratories detected the replicate sample BCR3 with the lowest number of copies (Table 1
). These results suggest that both instruments are capable of detecting 1–5 copies/µL of the PML-RARa isoforms.
We observed significant differences among global means of the PML-RARa results of the laboratories with LC or ABI (P = 0.000). However, the global means of all samples assessed with LC or ABI showed no statistical difference (Fig. 1A
), as reflected in the profiles of the means of the samples processed by each real-time instrument (Fig. 1B
).
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We found a difference in the interaction reproducibility between the two instruments at the limits of statistical significance (P 
0.05; Table 2 in the online Data Supplement), which was attributable to the larger variance of the LC for this component.
The efficiencies estimated from the slope of the calibration curves for BCR-ABL were similar for the two instruments [mean (SD) 1.79 (0.04) for the LC and 1.84 (0.02) for the ABI; Table 1 in the online Data Supplement]. No false-positive results were reported for the blank controls (Table 1
).
All participating laboratories except one using the LC were able to detect transcripts in the sample with the lowest amount of BCR-ABL transcripts (1–5 copies/µL; Table 1
).
We observed significant differences among the global means of the laboratories using LC or ABI (P <0.001). Conversely, we found no statistically significant differences between the ABI and LC instruments for the global means of pooled samples of the laboratories (Fig. 1C
). In addition, the profiles of the means of log(BCR-ABL) obtained for each sample within the laboratories using an ABI or LC instrument were nearly identical (Fig. 1D
).
None of the precision components differed between the ABI and LC (Table 2 in the online Data Supplement).
In summary, this multicenter study showed that the ABI and LC instruments performed similarly. As a multilaboratory trial, the results obtained can be expected to better transfer to the data reported in clinical trials than if the comparison were performed by a single laboratory. The study confirms the ability of the LC to use the TaqMan technology (6)(16) as an alternative to the HybProbes technology originally developed for this system (5)(17).
In this study, the main statistical differences were among the pooled means of the data. These individual differences among the laboratories with the same instrument could be attributable to variability in the stability of control samples or reagents, methodologic proficiency, or instrument maintenance.
The small difference in the interaction reproducibility (P 0.05) for PML-RARa could reflect a difference in sensitivity of LC to the influence of noncontrolled effects.
In conclusion, despite differences in reagents in our study, the results for the LC and ABI instruments were equivalent with respect to the means and precision, suggesting that the choice of instrument has little to do with results when laboratories use the same methods and calibrators. Standardization of quantitative real-time PCR studies in a multiinstitutional context will require adoption of common methods and calibrators. The harmonization of the results should in turn allow better comparison of data obtained in different therapeutic trials.
Acknowledgments
This study was supported financially by Roche Applied Science, Spain.
References
-positive acute promyelocytic leukemia patients: real time quantification of minimal residual disease. A pilot study. Leukemia 2001;15:1696-1700.[Web of Science][Medline]
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