Pre-Clinical Validation of a Novel, Highly Sensitive Assay to Detect PML-RARα mRNA Using Real-Time Reverse-Transcription Polymerase Chain Reaction

Abstract

We have developed a sensitive and quantitative reverse-transcription polymerase chain reaction (RT-PCR) assay for detection of PML-RARalpha, the fusion oncogene present as a specific marker in >99% of cases of acute promyelocytic leukemia (APL). The assay is linear over at least 5 orders of magnitude of input DNA or RNA, and detects as few as 4 copies of PML-RARalpha plasmid DNA. PML-RARalpha transcripts could be detected in mixtures containing 2 to 5 pg of RNA from fusion-containing cells in a background of 1 microg of RNA from PML-RARalpha-negative cells. Using 1.0 to 2.5 microg of input RNA, the sensitivity of the assay was between 10(-5) and 10(-6). Furthermore, determination of GAPDH copy number in each reaction allowed an accurate assessment of sample-to-sample variation in RNA quality and reaction efficiency, with consequent definition of a detection limit for each sample assayed. Using an internal calibrator, assay precision was high, with coefficients of variation between 10 and 20%. An interlaboratory study using coded samples demonstrated excellent reproducibility and high concordance between laboratories. This assay will be used to test the hypothesis that sensitive and quantitative measurement of leukemic burden, during or after therapy of APL, can stratify patients into discrete risk groups, and thereby serve as a basis for risk-adapted therapy in APL.

Standardization and quality control studies of ‘real-time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia – A Europe Against Cancer Program

“…However, there have been relatively few studies reporting the use of RQ-PCR in APL patients. 41,[159][160][161] Although the molecular diagnosis and monitoring of APL patients represents one of the most relevant examples of the impact of molecular genetics in clinical hematology, further investigations are still needed.…”

“…Most publications show data from a single laboratory, but, to our knowledge, just one study involves the standardization between three laboratories for one target. 159 The majority of publications used a copy number ratio between the FG and the CG with a standard curve, this ratio being expressed as a decimal value or a percentage. To obtain the standard curve, laboratories used either cell line cDNA 37,39,128,190 or plasmid DNA.…”

Relapse of acute promyelocytic leukemia with PML-RARα mutant subclones independent of proximate all-trans retinoic acid selection pressure

“…5,19 Also, methods for PML-RARa type determination, 20 monitoring of MRD by real-time quantitative RT-PCR (Q-PCR), 18 measurement of ATRA binding to PMLRARa, 6 and measurement of ATRA transactivation of mutant and wild-type (wt) recombinant PML-RARa constructs in a retinoic acid response-element(RARE)-dependent reporter vector transient transfection assay 6 have been previously reported.…”

PCR-based Calibration Curves for Studies of Quantitative Gene Expression in Human Monocytes: Development and Evaluation

“…Measurements of specific mRNAs through quantitative RT-PCR poses numerous problems; in fact, it requires the isolation of intact mRNA species, quantitative reverse transcription, and quantitative PCR amplification (4,32,33 ). In the present study, the first step focused on the development and evaluation of PCR amplicon-based calibrators to generate calibration curves that could be used to quantify specific target mRNAs.…”