We have developed a sensitive and quantitative reverse-transcription polymerase chain reaction (RT-PCR) assay for detection of PML-RARα, 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-RARα plasmid DNA. PML-RARα transcripts could be detected in mixtures containing 2 to 5 pg of RNA from fusion-containing cells in a background of 1 μg of RNA from PML-RARα-negative cells. Using 1.0 to 2.5 μg of input RNA, the sensitivity of the assay was between 10^{and 10}^{. 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.}

Abstract

Many cases of leukemia are caused by translocations that result in the formation and expression of chimeric fusion genes,¹ and it is now routinely possible to detect extremely small numbers of transcripts from these leukemia-associated fusion genes in the blood or bone marrow of affected patients. Clinical application of these exquisitely sensitive reverse-transcription polymerase chain reaction (RT-PCR) assays has been slow due to a variety of factors, including a lack of interlaboratory standardization and the non-quantitative nature of most such assays. Interpretation of negative results is complicated by issues of RNA integrity and assay sensitivity that can vary dramatically from one center to another and even from day to day in the same laboratory. Positive results are more readily accepted, but the mere detection of transcripts of leukemia-associated fusion genes has not proven to be a reliable marker for impending relapse.²³⁴ The accumulated evidence suggests that there exists a threshold of leukemic burden above which relapse is highly likely, but below which continued remission is the expected outcome. Definition of such a threshold is an important goal in APL and other leukemias, and will require a standardized, sensitive, and quantitative assay that can expeditiously analyze large numbers of samples from uniformly treated patients enrolled on collaborative clinical trials.

Themajority of APL patients harbor a translocation between chromosomes 15 and 17, with resultant fusion of the PML gene, at 15q12, with the retinoic acid receptor α (RARα) gene, at 17q22.⁵ This gene fusion results in production of a leukemia-specific chimeric mRNA, PML-RARα. In the present study, we present the technical details, pre-clinical validation, and initial clinical application of a sensitive, quantitative RT-PCR assay that can detect and measure the PML-RARα fusion transcript. The assay utilizes the 5′-nuclease technique, in which nuclease degradation of a dual-labeled fluorogenic probe results in a measurable fluorescence signal that is detected during the PCR process (“real-time” PCR, or TaqMan assay).⁶⁷ Our data indicate that the sensitivity of this quantitative method is at least equal to that of a published manual RT-PCR assay for detection of PML-RARα, and that it is 100% specific. Furthermore, the precision of this method is good, and it is amenable to interlaboratory standardization; these features suggest that it is ideally suited for high throughput analysis of large numbers of clinical specimens.

One laboratory (University of New Mexico) prepared coded samples of UF1 or NB4 RNA mixed with RNA from normal blood leukocytes at dilutions ranging from 10^{to 10}^{(or RNA from normal bone marrow and blood mononuclear cells). Samples were assayed independently in both laboratories using the same amount (1 μg) of RNA for RT, and the same amounts of cDNA for TaqMan PCR with PR-L, PR-S, and GAPDH probe and primer sets (225 ng cDNA equivalent for PR-L and PR-S, and 100 ng for GAPDH).}

NQ, normalized quantity (number of PML-RARα copies per 1 × 10^{GAPDH copies).}

MNCs, mononuclear cells. UNM, University of New Mexico.

Assays were performed four times on 4 successive days. Results shown are the means (ranges) of the four independent determinations, with standard deviation (SD) and coefficient of variation (CV) shown.

RNA from L-isoform blasts at diagnosis of APL. High, undiluted RNA; low, RNA diluted 1:100 in RNA from normal peripheral blood mononuclear cells.

Number of PML-RARα copies per μg of input RNA, calculated by reference to a standard curve.

NQ, normalized quantity: number of PML-RARα copies per 10^{GAPDH copies.}

NQ_C, normalized, calibrated quantity: number of PML-RARα copies per 10^{GAPDH copies divided by an internal calibrator.}

For the manual assay, 1 μg of RNA equivalent cDNA was used in each PCR reaction, and samples were interpreted as positive or negative based on the presence or absence of strong ethidium-bromide staining bands of appropriate size after double nested PCR and gel analysis. A corresponding reaction for GAPDH was performed for each sample in the presence of a fixed amount of competitor DNA using 0.1 μg RNA equivalent to ensure that negative reactions were not false negatives due to RNA degradation or failure of RNA transcription.¹⁴ For real-time RT-PCR, performed in a separate laboratory, the amount of input RNA equivalent cDNA varied for each dilution, as indicated (average 4.52 μg). For each real-time PCR assay, 1/20 of the RT reaction product was amplified using GAPDH primers and probe to confirm RNA integrity.

Acknowledgments

We thank Drs. M. Lanotte and M. Kizaki for NB4 and UF1 cells, respectively, and Dr. Srinivas Rao for assistance with the development of the high-sensitivity manual RT-PCR assay.

Acknowledgments

Footnotes

Supported by National Institutes of Health grants CA75049 (to J.L.S.) and CA56771 (to R.E.G.).

Address requests for reprints to James L. Slack, MD, Department of Medicine, Roswell Park Cancer Institute, Elm & Carlton Streets, Buffalo, NY 14263. E-mail: james.slack@roswellpark.org.

Footnotes

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