Kategorie: Maligní lymfomy a leukémie
Téma: Acute lymphoblastic leukemia - Biology 2
Číslo abstraktu: 0614
Autoři: MD Heike Pfeifer; Dr. ssa Orietta Spinelli, PhD, BiolSC; Jean-Michel Cayuela, PhD.; Dr. Helene Cavé; MD Peter Vandenberghe (Vandenberge), Ph.D.; Carmen Chillon; MD Tomasz Sacha; Sandrine Hayette, Ph.D.; Silja Roettgers, Ph.D.; Giovanni (Gianni) Cazzaniga, PhD; Prof. MD Thomas Lion, PhD; MD Letizia Foroni, PhD; Vincent H.J. Van der Velden, PhD; Doc. MUDr. Jan Zuna, Ph.D.; Monica Hermanson, PhD; Martin Christian Mueller, MD; MD Thoralf Lange; MD Miroslaw Majewski; MD Israel Bendit; MD Fabrizio Pane; Ilaria Iacobucci, PhD; MD Veli Kairisto, PhD; Christa Homburg; Smadar Avigad, PhD; M.D. Allen Eng Juh Yeoh; Beat Schäfer; MB BS Adele Fielding, PhD; Loredana Elia; Katarzyna Borg; Eric Delabesse; MD Jacques J.M. van Dongen, PhD; MD Oliver G. Ottmann, PhD; Sandra Markovic; Prof. MUDr. Dieter Hoelzer, Dr. h.c.
Background. Minimal residual disease is well established as an important prognostic parameter and is used for stratification and treatment decisions in acute lymphoblastic leukaemia (ALL). DNA- based PCR analysis of Ig-/TCRrearrangements have been well standardised during the last decade, resulting in robust systems for MRD analysis with an intra- and inter-assay variability of less than half a log. In Ph+ALL, MRD analysis relies on RNA-based RT-PCR techniques that are highly sensitive but as yet lack standardisation between laboratories. Since laboratories differ substantially in both their methodology and analysis strategy, interpretation and meaningful comparison of results between laboratories and from different studies is difficult or impossible. Moreover, and in contrast to CML, there are no generally accepted definitions of molecular response that could identify thresholds on which to base therapeutic decisions. Aims. To assess i) the variability of BCR-ABL quantification between laboratories with recognized expertise in bcr-abl analysis, ii) identify optimal laboratory methods and iii) to standardize analysis and interpretation of RT-PCR results for Ph+ALL, the EWALL and ESG-MRD-ALL consortia initiated a multinational project for quality assurance involving more than 30 laboratories from 14 countries worldwide. We here report the results of the first seven m-BCRABL laboratory control rounds, performed between march 2008 and march 2012, that focussed on inter-intraassay variability of rt-PR techniques, comparison of housekeeping genes, plasmids, platforms and reagents. Methods. Serial dilutions of the BCR-ABL positive cell line Sup B15 with the BCR-ABL negative cell line Nalm 6 covering 5 logs were produced. In the 7 lab rounds, 1355 aliquots, each generated with a total amount of 5 x 10E+06 cells in a final volume of 1 ml were produced, stabilized in TRIZOL or buffer RLT and frozen at -20°C until shipment or centralized isolation of RNA and reverse transcribed. Participants were asked to process the material using predefined procedures. Results. In the first QC round a major finding was the high variability in RNAyield between laboratories despite using the same extraction method, with an up to 3 log difference in ABL copy numbers. In addition, there was rare falsepositivity and false negativity (specifity 94.8%). With centrally produced cDNA the variability improved and there was a lower frequency of false-positivity reaching 100% specifity in QC 2. By using standardized assays, in 12 of 33 laboratories no conversion factor is needed, 20 of 33 laboratories are at the moment within one log difference when comparing the BCR-ABL/ABL ratios. Comparison of housekeeping genes (ABL/GUS) revealed non-linearity at high transcript levels resulting in an underestimation of intraindividual log reductions and may be relevant when assessing disease prognosis. The quantitative range was generally above 10E-04. Conclusions. Initiation of laboratory control rounds and implementation of standardized methodology improved the variability and reduced the frequency of false-positive results. Sensitivity of current techniques is still unsatisfactory for diseases with aggressive growth kinetics such as Ph*ALL. Further standardisation of technical and analytical methodology
Haematologica, 2012; 97(s1): 254
Datum přednesení příspěvku: 14. 6. 2012