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An international study to standardize the detection of BCR-ABL transcripts from stabilized peripheral blood preparations by quantitative RT-PCR


, : An international study to standardize the detection of BCR-ABL transcripts from stabilized peripheral blood preparations by quantitative RT-PCR. Blood 102(11): 64a, November 16

Quantitative determination of residual disease after treatment of patients with BCR-ABL positive leukemias may be achieved by various methods. However, there is a need to standardize methodologies and interpretation of results to reduce interlaboratory variation. The amount of BCR-ABL transcripts has been shown to be a reliable surrogate parameter for progression free survival after treatment with interferon alpha or imatinib and after allogeneic stem cell transplantation. The major aim of quantitative RT-PCR analyses is to identify patients who need alternative treatment strategies. Standardized molecular response data are required for new treatment protocols and to compare different treatment strategies. Hence, a multicenter study was performed to detect BCR-ABL transcripts from peripheral blood (PB) preparations utilizing RNA stabilization and extraction (PAXgene Blood RNA Kit, Preanalytix, Hombrechtikon, Switzerland) and real time RT-PCR technology (Lightcycler, LC, Roche Diagnostics, Mannheim, Germany, and TaqMan, TM, ABI Prism, Foster City, CA). Objectives were the definition of a clinically relevant detection limit for low tumor burden (optimal sample volume), the applicability and comparison of the two control genes glucose-6-phosphate dehydrogenase (G6PD) and total ABL, and comparison of the LC and TM RT-PCR methodologies to develop a standardized PCR strategy for the molecular monitoring of residual disease in CML patients after therapeutic intervention. A total of 186 PB samples (2.5, 5, and 10 ml) derived from healthy donors and spiked with serial dilutions (1:20 to 1:2X106) of b2a2 (n=66), b3a2 (n=36), and e1a2 (n=36) BCR-ABL+ cells from leukemic patients and negative controls (n=48) were stabilized using the PAXgene system, blinded, frozen and distributed to six laboratories. RNA extraction and cDNA synthesis were performed according to a common protocol, but PCR conditions for quantification of BCR-ABL, ABL and G6PD were optimized for the LC (three labs) and TM (three labs) platforms. A calibrator RNA was used as reference for both target and standard genes. The calculation of the final results was performed using the relative quantification software (Roche). BCR-ABL transcripts were detected in 99/138 positive samples and 0/48 negative controls. The coefficients of variation for samples tested positive were 0.70 and 0.75 for ratios BCR-ABL/ABL and BCR-ABL/G6PD, respectively (n.s.). There was no significant difference of the results achieved with LC and TM technologies. Up to a dilution of 1:1000, 27/30 2.5 ml samples were tested positive. For higher dilutions, a volume of 5 or 10 ml PB was required to achieve positive results. We conclude that (i) standardization of quantitative PCR is feasible independent of the PCR machine used. (ii) Sensitivity of the test depends on the volume of PB used. To detect a three-log reduction of the tumor load, 2.5 ml PB are sufficient in most cases. Higher sensitivities can be achieved using 10 ml PB. (iii) There is no difference in interlaboratory variation between ABL and G6PD as housekeeping genes.

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