Evaluation of molecular response is important for the diagnosis and monitoring of minimal residual disease in patients with acute promyelocytic leukemia (APL). In this study, we analyzed the molecular response by regular reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR in 31 newly diagnosed patients. The real-time RT-PCR results are reported as normalized DoseN and log-reduction (3.0–4.9 log-reduction as minor and ⩾5.0 log-reduction as major molecular response). After induction therapy and completion of consolidation, minor molecular response was documented in 35.5 and 96.8% patients, respectively, which was equivalent to the regular RT-PCR (22.6 and 96.8%), whereas the major molecular response rate was significantly lower (12.9 and 90.3%, respectively). All patients achieved major molecular response during and after maintenance therapy. During the follow-up study, loss of major molecular response was observed in two patients, which was associated with subsequent loss of minor molecular response, positive RT-PCR and then documentation of central nervous system leukemia or clinical relapse in 3–6 months. For summary, we demonstrated that the real-time RT-PCR is potentially superior to regular RT-PCR in evaluation of molecular response in APL patients and that reporting real-time RT-PCR data by log-reduction is feasible and clinically relevant.
Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia characterized by t (15; 17) translocation, which fuses the promyelocytic leukemia (PML) gene on chromosome 15 to the retinoic acid receptor (RARα) gene on chromosome 17 resulting in the chimeric gene encoding PML-RARα fusion protein.1 The introduction of all-trans retinoic acid (ATRA) improved the complete remission (CR) rate in newly diagnosed patients to 85–90% and combination of ATRA with chemotherapy in induction or post-remission therapy improved long-term survival.2, 3 Since 1990s, arsenic compound was proved to be effective in the treatment of relapsed APL.4, 5 More recently, combination or sequential use of ATRA and As2O3 synergy to eradicate the leukemia in mice model, and primary data from clinical trial also demonstrate that the combination therapy with ATRA plus As2O3 and chemotherapy might be potential curative therapy for APL.6, 7, 8, 9
The PML-RARα mRNA can be detected by the reverse transcription-polymerase reaction (RT-PCR) assay, which provided an important tool in the monitoring of minimal residual disease (MRD).10 Achievement of undetectable level of PML-RARα mRNA by PCR assay was required for long-term survivors, whereas the persistence and/or re-emergence of PML-RARα fusion transcripts have been found to be predictive of hematological relapse.11, 12, 13, 14, 15 Thus, achievement of a negative RT-PCR is considered as important therapeutic goal and surrogate marker for the evaluation of treatment protocol.16, 17 In recent years, quantitative real-time RT-PCR was developed to monitor MRD, providing a more sensitive tool than regular RT-PCR. Although both regular and real-time RT-PCR assays were commonly used in the clinical setting, their exact clinical significance and/or superiority was still not fully determined.18, 19, 20 In this study, we systemically compare the clinical relevance, and kinetics of molecular response of qualitative and quantitative RT-PCR in newly diagnosed APL.
Materials and methods
A total of 31 newly diagnosed APL patients who have been admitted to our hospital since April 2001 and entered CR were included in this study. Diagnosis was determined based on clinical manifestations, bone marrow examination and presence of t (15; 17) chromosomal translocation with PML-RARα transcript by RT-PCR assay. All 31 patients included in this study completed the entire planned treatment and have been followed up for 27–46 months (median 38) since the beginning of treatment. The bone marrow samples at pre-treatment and post-treatment time points were obtained regularly for evaluation of molecular response in all patients.
All patients received the treatment protocol with ATRA at 25 mg/m2/day plus As2O3 at 0.16 mg/kg/day till CR. Chemotherapy (Daunorubicin (DA) 30 mg/m2/day plus cytosine arabinoside 100 mg/m2/day) was added in the case of white blood count (WBC) over 10 × 109/l. After achieving CR, all patients were given three courses of consolidation chemotherapy with three regimens, that is, DA (30–45 mg/m2/day for 3 days; cytosine arabinoside 100 mg/m2/day for 7 days), HA (homoharringtonine 2–3 mg/m2/day for 3 days; cytosine arabinoside 100 mg/m2/day for 7 days) and Ara-C pulse regimen (cytosine arabinoside 1.5–2.0 g/m2 for 3 days). The maintenance treatment include five cycles of sequential use of ATRA (25 mg/m2 daily for 15–30 days for the first month), As2O3 (0.16 mg/kg daily for 30 days for second month) and then methotrexate (MTX, 10 mg/m2 weekly) for third month.9
Assessment of molecular response
For assessment of molecular response, serial bone marrow samples were obtained at following time points: before and after the induction therapy; the end of consolidation chemotherapy; and then every 3–6 months during maintenance therapy and then every 6–12 months afterwards. For direct comparison of qualitative and quantitative RT-PCR assays, both regular and real-time RT-PCR assays were performed on the same total RNA extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA).
The conditions for PCR reactions were the same as previously described.19, 21 RT was performed on samples (200 ng) of total RNA. After 10 min at 25°C, RNA was incubated for 50 min at 45°C with 50 U of Superscript II Reverse transcriptase (GIBCO BRL, Gaithersburg, MD, USA), 20 U of RNase inhibitor (GIBCO), 2.5 mM random hexamers and dNTP at 0.5 mM in a final volume of 20 ml. Remaining reverse transcriptase was then inactivated by heating for 5 min at 95°C. cDNA (10 μl) synthesized above were used for regular RT-PCR and real-time quantitative RT-PCR. The real-time RT-PCR reactions were performed in triplicate with Taqman PCR core reagent kit on ABI PRISM7700 DNA sequence Detection System via 50 cycles, as previously described.21 Reported PML-RARα and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) copy numbers were the geometric average of these three determinations and then calculated by comparing the Ct value for each sample with the GAPDH and PML-RARα standard curve. Real-time RT-PCR amplification of the serially diluted Long (L) and Short (S) isoforms of PML-RARα and GAPDH standard RNA was run in quadruplicate to establish standard curves and the Ct values were averaged from the values obtained in each reaction. The standard curve was constructed by plotting the Ct vs the known copy number of each standard sample. For interassay analysis by testing four standard curves of L- and S-form PML-RARα and GAPDH resulted in a coefficient of variation (CV) less than 10%. Based on the standard curves, the lowest sensitivity limit of PML-RARα DoseN in our PCR system was at 0.0116 and 0.0124, respectively, for L- and S-type PML-RARα fusion transcripts, respectively. Besides, we also perform a direct comparison of the sensitivity of the RT-PCR and real-time RT-PCR assay by serial dilution of RNA from APL patient and RNA from NB4 cells serially diluted with RNA derived from HL-60 cells. The RT-PCR limit for L and S isoforms of PML-RARα was 1:1 × 103–4, whereas the real-time RT-PCR was 1:1 × 105–6. No detectable signal was found among HL-60 cells, showing that the real-time RT-PCR system is also highly specific to the determination of PML-RARα fusion transcripts.
To report the real-time RT-PCR data, two methods were used in our study: (1) normalized values of the PML-RARα fusion transcript dose (PML-RARα DoseN) were calculated as PML-RARα fusion transcript copy number/GAPDH transcript copy number × 1000; (2) log-reduction of DoseN was calculated based on the comparison of post-treatment DoseN vs pretreatment DoseN in individual patient. For the log-reduction calculation, all samples from same patients were run in the same experiments.
Analysis of the Flt3 internal tandem duplication
For the screening of internal tandem duplication of Flt3 gene (Flt3 ITD), we analyzed total RNA in all 31 patients. In selected cases, both DNA and RNA from the same patient were analyzed in parallel. ITDs were investigated by PCR with the primers described by Kiyoi et al.22 Primers 11F-11R and R5-R6, which amplify the juxtamembrane domain of the receptor, were used to amplify DNA and cDNA, respectively. The amplified products were finally electrophoresed on a 1.5% agarose gel stained with ethidium bromide.
The disease-free survival (DFS) and overall survival (OS) were calculated according to the Kaplan and Meier method with surviving patients censored at the point of last follow-up.23 Log-rank test and χ2 test were performed by JMPin (SAS Institute Inc.) and Excel software (Microsoft) accordingly.24
For all 31 patients, the median peripheral white blood cells (WBC) was 2.4 × 109/l (0.9–72 × 109/l), in which WBC was less than 10 × 109/l in 23 patients and other eight patients presented with initial hyperleukocytosis (⩾10 × 109/l). Chemotherapy was added for all patients with hyperleukocytosis simultaneously with ATRA and As2O3. All patients enter CR with median time to attain CR at 27 days (range: 15–32). At the last follow-up in February 2005, 29 patients remained in continuous clinical remission and two patients experienced central nervous system (CNS) leukemia and/or clinical relapse but remain alive. The median DFS and OS were not reached and estimated 3-year DFS and OS were 91.7% and 100%, respectively.
Evaluation of molecular response by regular RT-PCR
To regular RT-PCR, the complete molecular remission was defined as negative result by two repeated assays. The RT-PCR results were positive in all 31 patients before the treatment. When clinical remission was achieved, 11 (35.5%) patients achieved molecular remission by regular RT-PCR. At the end of consolidation chemotherapy, 30 of 31 patients (96.8%) achieved negative RT-PCR and finally all patients entered into complete molecular remission during maintenance therapy (Table 1).
Evaluation of molecular response by real-time RT-PCR
Reporting real-time RT-PCR data by normalized PML-RARα DoseN
For patient samples, the median level of PML-RARα DoseN before treatment in all 31 patients was 7816.1 (1119.4–25575.4). The initial PML-RARα DoseN in patients with L- and S-type PML-RARα transcript level was similar at 7763.5±6467.94 and 7921.2±6231.3, respectively. After induction therapy, DoseN decreased significantly to a median of 17.6 (<0.0116 or 0.0124 to 4080.9) and further decrease to a median DoseN at 0.1 (<0.0116 or 0.0124 to 23.6) was achieved after completion of consolidation chemotherapy.
Reporting real-time RT-PCR data by log-reduction
Based on the observation of significant DoseN variance (1119.4 vs 25575.4) in the pretreatment samples between individual patients, we considered that the calculation of DoseN might not be the ideal method to evaluate the molecular response during maintenance and follow-up. It is also impossible to determine a clear-cut criterion defining molecular response for all patients with such significant variation. Thus we introduced the fold-reduction analysis by comparing post-treatment and pretreatment samples and tried to assess its feasibility in the clinical evaluation of MDR.
To establish a more reliable log-reduction scheme, firstly we group the result of real-time RT-PCR in the evaluable 181 post-treatment samples from 31 patients according to the level of log-reduction: <3.0; 3.0–3.9; 4.0–4.9; 5.0–5.9 and ⩾6.0. The correlation of real-time RT-PCR data with regular RT-PCR was demonstrated as shown in Table 2. Briefly, regular RT-PCR test was positive in 80.5% (33/41), 25%(2/8) and 8.3%(1/12) samples when the log-reduction of real-time RT-PCR was <3.0 log, 3.0–3.9 log and 4.0–4.9 log, respectively, and all post-treatment samples with 5.0–5.9 and ⩾6.0 log-reduction by real-time RT-PCR were negative in regular RT-PCR. As there was no statistic difference between groups of 3.0–3.9 and 4.0–4.9 log-reduction as well as groups of 5.0–5.9 and ⩾6.0 log-reduction, we simply classified the 181 post-treatment samples into three groups by log-reduction: <3.0, 3.0–4.9 and ⩾5.0. Secondly, we propose the criteria of molecular evaluation by real-time RT-PCR: 3.0–4.9 log-reduction as minor molecular response and ⩾5.0 log-reduction as major molecular response. To verify its clinical relevance, we analyzed the molecular response rate in patients at different time points. For all 31 patients after induction therapy, 22.6% patients achieved at least minor molecular response (⩾3.0 log-reduction) and 96.8% after consolidation chemotherapy, which was similar to the molecular response rate assessed by regular RT-PCR (35.5 vs 96.8%, respectively, P=0.26 and P=1.0). For achievement of major molecular response (⩾5.0 log-reduction), the molecular response rate was 12.9% after induction therapy, which was significantly lower than regular RT-PCR (P=0.037). Of notice, the molecular response rate defined by regular RT-PCR or minor/major molecular response of real-time RT-PCR was similar after completion of consolidation therapy and maintenance therapy (Table 1). Thirdly, we further analyzed the time required to achieve the molecular response accordingly. Regular RT-PCR turned negative with a median of 2 months compared to a median of 3 months for achievement of minor molecular response by real-time RT-PCR (P=0.56; Figure 2a), whereas there was a significant delay in time to achieve major molecular response with a median of 4 months as compared to minor molecular response (P=0.045, Figure 1a) or regular RT-PCR (P=0.0036, Figure 2b).
Evaluation of the clinical samples with PML-RARα DoseN below detection limit
In 181 post-treatment samples, there are sizable proportions of samples presenting PML-RARα DoseN below the lowest sensitivity of real-time RT-PCR assay and thus was considered as complete molecular response. After induction and consolidation chemotherapy, the complete molecular response rate was 3.23 and 77.4%, respectively, and all patients achieved complete molecular response after maintenance therapy with a median of 4 months. These data were not significantly different from major molecular response (P=0.161 and 0.167, respectively, as shown in Table 1; P=0.84 in Figure 1c). Of notice, all these samples also had a ⩾5.0 log-reduction.
Molecular evaluation during the follow-up
During the follow-up, in 29 patients who remained in continuous clinical remission, the RT-PCR study remained negative, whereas the log-reduction was consistently ⩾5.0 and PML-RARα DoseN below detection level by real-time RT-PCR study. CNS leukemia and bone marrow relapse were documented in patients UPN19 and UPN20 (as shown in Table 3). The PML-RARα DoseN was significantly increased to 38.95–1484 together with the log-reduction less than 3.0 when CNS leukemia and relapse were documented. Except for the first episode of CNS disease in UPN19, other three CNS diseases or bone marrow relapse were/was preceded for about 3–6 months by an increase of PML-RARα DoseN over the detection limit (3.35–13.86) and decrease of log-reduction to less than 5.0.
Impact of clinical and biological features on molecular remission with real-time RT-PCR
Both the major or complete molecular remission rate and time to achieve at least major molecular remission by real-time RT-PCR were irrelevant to the initial hyperleukocytosis. (Figure 3a).
Rapidity of early molecular response by RT-PCR
To evaluate the impact of early molecular response, we analyzed the kinetics of major or complete molecular response by real-time RT-PCR in correlation with regular RT-PCR result early after induction. The time to molecular response by real-time RT-PCR was not associated with status of molecular response by regular RT-PCR at post-induction stage (Figure 3b).
PML-RARα transcript isoform
In our series, a total of 18 patients carried long-form PML-RARα transcript, whereas 13 patients carried the short-form. The molecular response rate and time to molecular response in two PML-RARα transcript types (L and S types) have no significant difference (Figure 3c).
In our series, a total of five patients harbored Flt-3 ITD, whereas no D835 mutation was identified in all 31 patients. Interestingly, all these 5 patients with Flt-3-ITD carried the short-form PML-RARα transcript. Upon real-time PCR, there is no significant difference in molecular remission rate and time to molecular response in patients with or without Flt3-ITD (Figure 3d).
Evaluation of molecular response is important for the diagnosis and monitoring of MRD in patients with APL.16 Elimination of residual disease with consistent negative RT-PCR is necessary for patients to achieve long-term remission and is considered as an independent prognostic factor for APL.11, 12, 13, 14, 15, 25, 26, 27, 28 Nevertheless, the achievement of PCR negativity cannot be equated with cure because clinical relapses are often preceded by negative PCR tests.14 The failure to detect residual disease highlights the limited sensitivity of standard RT-PCR in predicting clinical realpse.16, 29 Quantitative real-time RT-PCR with higher sensitivity has been developed and applied for MRD monitoring in various leukemias.30 Owing to lack of standard criteria for quantitative evaluation and significant variance between individual patients and different PCR systems, no superiority of real-time RT-PCR was established over regular RT-PCR in clinical setting by direct comparison data.
In this study, we analyzed systemically the molecular response defined by real-time RT-PCR: minor (⩾3.0 log-reduction); major (⩾5.0 log-reduction) and complete (PML-RARα DoseN below the sensitivity limit of real-time RT-PCR assay) and compared these data directly with regular RT-PCR. First of all, regular RT-PCR was negative in almost all post-treatment samples (137/140) with ⩾3.0 log-reduction compared with positive result in 33/41 sample with less than 3 log-reduction. We considered that the minor molecular response of real-time RT-PCR was equivalent to molecular response using regular RT-PCR. This assumption was further supported by the fact that minor molecular response rate and median time to achieve the response were equivalent to regular RT-PCR. More importantly, the loss of minor molecular response in patients during follow-up was associated with a positive regular RT-PCR test (as shown in Table 3).
Secondary, our data clearly demonstrated the clinical relevance of major and complete molecular responses by real-time RT-PCR. All samples with major or complete response by real-time RT-PCR were negative in RT-PCR. More importantly, 29 out of 31 patients who remained in continuous major and or complete molecular response were disease-free with at least 3-year follow-up, whereas the loss of major or complete molecular response was months before the regular PCR turned positive and the subsequent documentation of CNS leukemia or relapse in other two patients. This observation highly suggested a superior role of evaluating the major/complete molecular response by quantitative RT-PCR in predicting the disease progression than by regular RT-PCR.
Thirdly, a step-wise reduction of PML-RARα transcript level and increase of molecular response rate were documented throughout the treatment procedure and all patients achieved major and/or complete molecular response after maintenance therapy. The molecular response and kinetics is irrelevant to the molecular response assess early after induction therapy, thus suggested that early evaluation of molecular response has limited clinical value and might not be necessary. As to the complete molecular response, it may not be necessary to be true ‘complete’, simply because patients with complete molecular response can still harbor minimum amount of PML-RARα transcript just below the detection limit of real-time RT-PCR assay. Meanwhile the sensitivity of real-time RT-PCR may also vary significantly between different laboratories and studies, thus making it difficult to compare the result from different studies. Given the fact that complete molecular response rate, and time to achieve the molecular response were almost the same as major molecular response, we considered that it is more convenient to use only major molecular response in the clinical setting.
More importantly, our study also demonstrated the feasibility of reporting real-time RT-PCR data by log-reduction formula in case of substantial failure in obtaining pretreatment BM samples. We analyzed the fold reduction data by replacing the actual individual pretreatment DoseN with a common pretreatment DoseN based on the average of all 31 patients. The result showed that the correlation between regular and real-time RT-PCR maintained significance (data not shown) and more importantly with the same criteria defining minor and major molecular response (3.0 and 5.0 logfold-reduction), the loss of major molecular response was months before positive result by regular PCR and the subsequent documentation of active leukemia and relapse. Thus we suggest that the evaluation of real-time RT-PCR for patients in whom a pretreatment bone marrow is not available is still feasible by using an average pretreatment DoseN value.
Based on above observations with limited assay points, we may conclude as follows: early analysis of molecular response by either regular or real-time RT-PCR is of little relevance to clinical outcome; a close monitoring of MRD by molecular evaluation is critical and must be performed throughout the maintenance therapy and afterwards; the achievement and maintenance of major molecular response by real-time RT-PCR can be considered as required for the long-term disease control; real-time RT-PCR is potentially superior in monitoring of MDR during follow-up, especially the loss of major molecular response may predict a potential disease progression; molecular evaluation with real-time RT-PCR should be adopted as indicating marker for molecular relapse and the anticipation of salvage treatment is warranted.14, 16
As in various reports, including ours, several clinical and biological features of APL, such as initial WBC count, PML-RARα isoform and Flt3-ITD are reported to be associated with diagnostic characteristics and potentially clinical outcome.21, 29, 31, 32, 33, 34, 35, 36, 37, 38, 39 For example, patients with short-isoform APL have been associated with high initial WBC count and adverse prognosis, but the statistically significant correlation has not been established with clinical outcome in all studies, whereas patients carrying Flt3 ITD are associated with high initial WBC count and potential inferior DFS, although there are also substantial conflicting data about the relationship of Flt3 ITD to the clinical outcome.21, 32, 33, 34, 35, 36, 37, 38, 39 As to the initial WBC, it has been demonstrated to be an prognostic factor in our historical study and other reports;11, 25 it has been impossible to correlate all these features with the treatment outcome in this study because only two patients had CNS leukemia or relapsed. Both patients had short-form PML-RARα with initial WBC at 2.46 × 109/l and 45.6 × 109/l, respectively, and no Flt-3 ITD documented. We also observed no impact of all these three factors on the overall molecular response and kinetics by regular and real-time RT-PCR analysis based on our treatment protocol with upfront use of ATRA and As2O3.
In summary, we determined that molecular evaluation by real-time RT-PCR was sensitive and valuable in clinical settings. We established criteria of minor (⩾3.0 log-reduction of DoseN) and major (⩾5.0 log-reduction of DoseN) molecular response, which proved to be feasible and clinically relevant. The achievement and maintenance of major molecular response is associated with constant clinical remission and the loss of major molecular response indicates an increased risk of potential disease progression.
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Grants and financial support: This work was supported in part by Grants from Co-Pi Program of Samuel-Waxman Cancer Research Foundation, Chinese National High Tech Program (863), National Key Program for Basic Research (973), National Natural Science Foundation of China, Shanghai Municipal Commission for Education (Y0201) and the Shanghai Municipal Commission for Science and Technology.
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