Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patients using ‘real-time’ quantitative reverse-transcriptase polymerase chain reaction (RQ-PCR) – a Europe against cancer program

Abstract

Real-time quantitative RT-PCR (RQ-PCR) is a sensitive tool to monitor minimal residual disease (MRD) in leukemic patients through the amplification of a fusion gene (FG) transcript. In order to correct variations in RNA quality and quantity and to calculate the sensitivity of each measurement, a control gene (CG) transcript should be amplified in parallel to the FG transcript. To identify suitable CGs, a study group within the Europe Against Cancer (EAC) program initially focused on 14 potential CGs using a standardized RQ-PCR protocol. Based on the absence of pseudogenes and the level and stability of the CG expression, three genes were finally selected: Abelson (ABL), beta-2-microglobulin (B2M), and beta-glucuronidase (GUS). A multicenter prospective study on normal (n ¼ 126) and diagnostic leukemic (n ¼ 184) samples processed the same day has established reference values for the CG expression. A multicenter retrospective study on over 250 acute and chronic leukemia samples obtained at diagnosis and with an identified FG transcript confirmed that the three CGs had a stable expression in the different types of samples. However, only ABL gene transcript expression did not differ significantly between normal and leukemic samples at diagnosis. We therefore propose to use the ABL gene as CG for RQ-PCRbased diagnosis and MRD detection in leukemic patients. Overall, these data are not only eligible for quantification of fusion gene transcripts, but also for the quantification of aberrantly expressed genes.

Imatinib as the first‐line treatment of patients with chronic myeloid leukemia diagnosed in the chronic phase: Can we compare real life data to the results from clinical trials?

“…An optimized multiplex reverse transcription-polymerase chain reaction (RT-PCR) was adopted from Cross et al [15] to determine the type of BCR-ABL transcript. Real-time quantitative PCR (RQ RT-PCR), standardized within the international program EUTOS for CML [16], was performed according to Europe Against Cancer recommendations [17,18]. B2M or ABL were applied as housekeeping genes in the Prague and Brno laboratories, respectively.…”

Standardized definitions of molecular response in chronic myeloid leukemia

“…36 With regard to definitions of MR, however, a new problem arises in how to define assay sensitivity in a standardized manner when BCR-ABL mRNA is undetectable. Assay sensitivity was initially considered by the Europe Against Cancer Group, 37 but the criteria they established only works for assays using an internal control gene that is independent of the fusion being tested. For CML, by far the most widely used internal control gene is normal ABL, which is not independent.…”

Real-time PCR quantification of major Wilms’ tumor gene 1 (WT1) isoforms in acute myeloid leukemia, their characteristic expression patterns and possible functional consequences

“…The reactions characteristics were compared with those of qPCR for total WT1 and for the control gene ABL. [43][44][45] For further reference and correlation, qPCR reactions detecting only one of the two splicing regions-variants…”