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Abstracts of Oak Ridge Posters |
1 Johnson & Johnson Research Pty Limited, Australian Technology Park, Eveleigh NSW 1430, Australia
2 Kanematsu Laboratories, Royal Prince Alfred Hospital, Camperdown, Sydney NSW 2050, Australia
aaddress correspondence to this author at: Biomedical Bldg., Level 4, 1 Central Ave., Australian Technology Park, Eveleigh NSW 1430, Australia; fax 61-2-83965811, e-mail tapplega{at}medau.jnj.com
Acute promyelocytic leukemia (APL) is caused by inhibition of apoptosis and a block in myeloid differentiation resulting from a translocation invariably involving the retinoic acid 
(RAR) gene (1)(2). Ninety percent of breakpoints lie within intron 3 or 6 of the PML gene, producing fusion transcripts of the S-type (exon 3 PML/exon 3 RAR) or L-type (exon 6 PML/exon 3 RAR) isoform. The remaining patients have V-type transcripts, which vary in length depending on where the breakpoint lies within exon 6 of the PML gene. Although a combination of all-trans-retinoic acid (ATRA) differentiation therapy and chemotherapy successfully induces remission in the majority of patients harboring the PML/RAR transcript, approximately one-third of these patients relapse (3). Patient survival rates are improved by initiating salvage therapy at molecular relapse rather than waiting for overt hematologic relapse (4)(5). Recent preliminary results suggest that the predictive power of molecular monitoring may be improved by the application of new quantitative methods to patient management (6)(7). This report provides the most recent patient results obtained with the single-tube DzyNA reverse transcription-PCR (RT-PCR) assay recently developed in our laboratory for diagnosing and monitoring APL (8).
DzyNA RT-PCR assays for quantification of PML/RAR transcripts and endogenous BCR control transcripts were used to analyze total RNA from patients with APL. Specimens were analyzed with the investigator blinded to the patients’ clinical histories. Primers, substrate, and reaction conditions for DzyNA RT-PCR and confirmatory two-step nested qualitative RT-PCR were as described previously (8). The PML/RAR primers amplify all L-type, and most V-type, transcripts. PML/RAR concentrations were normalized by use of BCR concentrations, thus controlling for variations in RNA integrity from clinical specimens. Quantitative data were expressed as ng-equivalents of the total NB4 RNA. Relative transcript amounts were expressed as a percentage ratio of disease transcripts to control transcripts (RDC %) calculated by the following equation:
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RNA was analyzed from a cohort of 39 patients whose bone marrow was collected at the time of diagnosis of APL. PML/RAR transcripts were detected in all patients’ RNA, confirming the clinical diagnosis of APL, and there was complete concordance between DzyNA RT-PCR and nested RT-PCR. The overall average RDC was 185% (range, 16–2342%). This wide range provides an explanation, in addition to technical reasons, for the disparity in reports of detection limits determined by dilutions of patients’ RNA. There appeared to be higher amounts of transcripts in patients with V-type (n = 4; range, 258-2342%; mean, 998%) than in patients with L-type (n = 35 patients; range, 16–269%; mean, 94%). This is the first report of quantification of V-type transcripts, and although the number of data points is too small for statistical significance, these results are interesting in light of studies suggesting that V-form patients exhibit a worse prognosis than patients with other isoforms (9). Ongoing studies aim to determine whether transcript concentrations at diagnosis are of prognostic significance.
DzyNA RT-PCR was used to analyze serial samples from three APL patients. These patients are enrolled in the APML3 Clinical Trial currently being conducted by the Australasian Leukaemia and Lymphoma Group. This international multicenter trial is studying patients receiving ATRA combined with idarubicin. Bone marrow specimens were analyzed prospectively at the treating hospitals by standard nested RT-PCR (data not shown) and retrospectively in our laboratory by DzyNA RT-PCR and nested RT-PCR. Fig. 1
shows the results of molecular monitoring of patients A, B, and C, quantifying the expression of PML/RAR relative to BCR in panels A, B and C respectively.
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Trial patient A had above-average amounts of the L-form PML/RAR mRNA (RDC = 196%) at diagnosis (Fig. 1A
, points 1 and 2) and was treated with ATRA and idarubicin. Although normal bone marrow morphology and cytogenetics indicated hematologic remission (point 3), prospective nested RT-PCR detected fusion transcripts, and the patient was given a second course of idarubicin. Fusion transcripts were still detectable by nested RT-PCR after idarubicin therapy (point 4), and intermittent ATRA therapy was initiated shortly thereafter. Retrospective DzyNA RT-PCR and nested RT-PCR confirmed the presence of PML/RAR mRNA during this period and showed that the amounts had decreased at a constant rate over 37 weeks (points 2–5). Transcripts were still detectable by DzyNA RT-PCR at the conclusion of consolidation treatment (point 5), but were undetectable 3 months later (point 6). All subsequent samples (points 7–9) were negative for PML/RAR transcripts by all protocols.
Trial patient B presented with low amounts of L-type PML/RAR mRNA (RDC = 25%; Fig. 1B
, point 1). The patient was treated with ATRA (point 2), and fusion transcripts were undetectable by all protocols within 11 weeks (point 4). The patient remained in clinical remission for 8 months, during which fusion transcript values were either undetectable or low by DzyNA RT-PCR (points 4–7). The patient eventually relapsed (point 8) with both hematologic and molecular symptoms. ATRA treatment was offered but refused for 2.5 months (points 8 and 9). The patient then agreed to 1 month of ATRA treatment, and PML/RAR transcripts became undetectable (point 10). Consolidation therapy was refused, and the next samples showed a reemergence of PML/RAR transcripts (points 11–12), which was followed by frank hematologic relapse (point 13). Offers of ATRA treatment were refused during this period (points 11–13), but the patient again consented to 1 month of ATRA therapy, which led to clearance of the PML/RAR mRNA within 5 weeks (point 14). Stem cells were harvested (point 15), shown to be negative for disease by nested RT-PCR, and then transplanted back into the patient (point 16).
Trial patient C presented with very high amounts of V-type PML/RAR mRNA (RDC = 2342%; Fig. 1C
, point 1). The patient received ATRA therapy and achieved hematologic remission in 12 weeks (point 5). At this time, PML/RAR transcripts were still detectable by nested RT-PCR, but subsequent analysis by DzyNA RT-PCR showed that expression was very low. The fusion transcript became undetectable by both protocols 12 weeks later (point 6) and remained undetectable for 6 months (points 7 and 8). DzyNA analysis detected very low amounts of fusion transcripts (point 9), which increased in the subsequent sample (point 10). These fusion transcripts had been detected prospectively by nested RT-PCR in this sample (point 10), and the patient was offered ATRA despite the absence of evidence of hematologic relapse. Despite ATRA therapy, fusion transcripts remained high for the next 17 weeks (points 11–14). This patient then received an allogeneic bone marrow transplant, which was successful in inducing hematologic and molecular remission (point 15).
In the three cases above, the relative PML/RAR amounts determined by DzyNA RT-PCR correlated well with the clinical history. The assay detected increases in transcript concentrations 3–6 months before morphologic or cytologic symptoms of relapse. Although the literature reports that most patients achieve clinical remission within 1–2 months of ATRA therapy (10), the rates of clearance of fusion transcripts appear more variable. Patient A took 6 weeks to achieve clinical remission, but it was >9 months before fusion transcripts were undetectable. In contrast, patient B demonstrated rapid clearance of PML/RAR transcripts in as little as 4.5 weeks after treatment. Patient C initially achieved hematologic remission after initial ATRA therapy despite the very high leukemic burden. However, despite prompt re-initiation of therapy on detection of fusion transcripts, the amounts of fusion transcript remained high, indicating resistance to ATRA. Persistence of fusion transcripts should aid the identification of "nonresponders" who would benefit from alternative therapy. In patient B, fusion transcripts were detected at very low amounts in a sample (point 5) between two remission samples, which were negative for fusion transcripts. Ongoing studies will determine whether these "interruptions" of molecular remission are of prognostic significance and/or identify patients who have not completely cleared the malignant clone and may be at greater risk of relapse.
Studies monitoring APL patients by DzyNA RT-PCR will continue to assess the prognostic significance of several factors highlighted by the examples in this study. Potential factors influencing outcome may include the fusion transcript isoform, the relative amounts at diagnosis, and rates of clearance and accumulation, as well as intermittent detection of transcripts during clinical remission. Continuing studies aim to further demonstrate the potential for use of quantitative molecular monitoring to facilitate early prediction of imminent relapse and to investigate the impact on disease-free survival of efforts to optimize the timing of administration of salvage therapy based on molecular information.
Acknowledgments
We acknowledge Li Chong, Shane Supple, Juliet Ayling, Francisca Springall, and Albert Catalano from the Kanematsu Laboratories for assistance in clinical specimen collection and collation. We are very grateful to the Australasian Leukaemia and Lymphoma Group clinicians for allowing access to clinical specimens and patient histories.
References
transcripts with a sensitive competitive RT-PCR method. Leukemia 2001;15:1060-1065.[ISI][Medline]
[Order article via Infotrieve]
mRNA using real-time reverse-transcription polymerase chain reaction. J Mol Diagn 2001;3:141-149. fusion transcripts. Clin Chem 2002;48:1338-1343. isoform: results from intergroup protocol 0129. Blood 2000;95:398-403.
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