Pre-clinical validation of a novel, highly sensitive assay to detect PML-RARalpha 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.

Figure 1.

Primers and probe used in real-time PML-RARα PCR. The primer and probe names, locations, and sequences are shown, along with the final concentrations used in PCR, and the S- and L-form amplicon sizes. PML exons 1–6 are shown as shaded rectangles, while RARα exon 3 is represented by an open rectangle. The S and L genomic breakpoints are designated by double slanted lines.

Figure 2.

GAPDH copy number related to sample processing technique, type of primer used to initiate cDNA synthesis, and tissue source. A: GAPDH standard curve generated with 10-fold serial dilutions of GAPDH plasmid standards. B: Effects of sample processing and RNA extraction. Solid columns: bone marrow was collected in EDTA or heparin and RNA was isolated using STAT-60. Cross-hatched columns: bone marrow was collected in heparin and cryopreserved. Vials were thawed, divided into two aliquots, and RNA was extracted using either STAT-60 or Qiagen RNeasy columns. In all cases, equivalent amounts of RNA (measured by spectrophotometry) were reverse-transcribed into cDNA and equal amounts of cDNA were amplified using GAPDH primers and probe. The number of GAPDH copies was determined by reference to the GAPDH standard curve shown in A. C: Comparison of random hexamers to gene-specific primers in cDNA synthesis. Equal amounts of the RNA from the cells shown were reverse-transcribed into cDNA using either gene-specific primers or random hexamers, and GAPDH copy number was calculated from the same amount of cDNA equivalent using real-time PCR. D: GAPDH copy number/μg of RNA from different tissue sources. The cell lines were a mixture of hematopoietic and epithelial cells. RNA from normal tissue (spleen, placenta, colon, and lung, all pooled) was purchased from Clontech. All RNAs, except those purchased from Clontech, were isolated using Qiagen RNeasy columns.

Figure 3.

A: Detection limit and dynamic range of detection of PML-RARα using plasmid DNA standards. Ten-fold serial dilutions of PML-RARα standards (L and S isoform) were amplified in TaqMan PCR using conditions outlined in Materials and Methods. The threshold cycle C_T was computed using the 7700 Sequence Detector software and is plotted against the logarithm of the input copy number. The linear regression equations and correlation coefficients are shown. B: Detection limit and dynamic range of detection of PML-RARα using RNA from APL cell lines. NB4 or UF1 total RNA was mixed with HL-60 total RNA at dilutions ranging from 10⁻¹ to 10⁻⁶. Five μg of RNA from each dilution was reverse-transcribed into cDNA and 20% (1 μg RNA equivalent) was amplified in TaqMan PCR reactions using the PR-L or -S primer/probe set. The amount of NB4 or UF1 RNA equivalent in each TaqMan reaction ranged from 1 pg (10⁻⁶ dilution) to 100 ng (10⁻¹ dilution).

Figure 4.

Application of real-time PML-RARα RT-PCR to analysis of clinical specimens. Total RNA was isolated from bone marrow (A) or blood (B) mononuclear cells from a single patient with APL (L-isoform) who was treated with all-trans retinoic acid (RA) until complete remission (CR), and then with 2 cycles of daunorubicin and cytosine arabinoside consolidation chemotherapy. Real-time RT-PCR was performed as described in Materials and Methods using both L-form and GAPDH primers and probes. Dx, diagnosis; Consol1, sample obtained after one cycle of consolidation; Pre-M, sample obtained after two cycles of consolidation and before randomization to maintenance therapy. Additional samples were taken at 6-month intervals during follow-up. Pathological analysis of the sample taken at 2 years revealed relapsed APL. The definitions of NQ value and detection limit are given in the text.