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Prognostic Value of Minimal Residual Disease Quantification by Real-Time Reverse Transcriptase Polymerase Chain Reaction in Patients With Core Binding Factor Leukemias

Jürgen Krauter, Kerstin Görlich, Oliver Ottmann, Michael Lübbert, Hartmut Döhner, Wolfgang Heit, Lothar Kanz, Arnold Ganser, Gerhard Heil

From the Department of Hematology/Oncology, Hannover Medical School, Hannover; Department of Internal Medicine III, University of Frankfurt, Frankfurt; Department of Internal Medicine I, University of Freiburg, Freiburg; Department of Internal Medicine III, University of Ulm, Ulm; Department of Hematology/Oncology, Ev. Krankenhaus Essen-Werden, Essen; and Department of Internal Medicine II, University of Tübingen, Tübingen, Germany.

Address reprint requests to Jürgen Krauter, MD, Department of Hematology/Oncology, Hannover Medical School, Carl-Neuberg-Str 1, D-30625 Hannover, Germany; e-mail: krauter.juergen{at}mh-hannover.de.

	ABSTRACT

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Purpose: In patients with acute myeloblastic leukemia with t(8;21) or inv(16) aberrations (core binding factor [CBF] leukemias), minimal residual disease (MRD) can be sensitively detected during and after chemotherapy by use of molecular methods. However, the prognostic impact of qualitative MRD detection is still under debate. In this study, the prognostic value of MRD quantification in patients with CBF leukemias was assessed.

Patients and Methods: We quantified MRD at various time points during and after therapy by real-time reverse transcriptase polymerase chain reaction (RT-PCR) for AML1/MTG8 and CBFB/MYH11 in 37 patients with CBF leukemias treated within a multicenter trial.

Results: At initial diagnosis, the patients showed a heterogenous fusion gene expression relative to glyceraldehyde 3-phosphate dehydrogenase with a variation of more than two log steps. According to MRD status during/after therapy, two groups of patients were separated. Of the 26 patients who had MRD levels of less than 1% in relation to initial diagnosis at all time points tested after induction chemotherapy, only two experienced relapse after a median follow-up of 19 months. Of the 11 patients who had a sample with an MRD level 1% at least at one time point after induction therapy, 10 experienced relapse, with a median remission duration of 10 months (P < .001). The median interval between the informative MRD sample and clinical relapse in these patients was 3 months.

Conclusion: MRD quantification by real-time RT-PCR allows the identification of patients with a high risk of relapse among the CBF leukemias.

	INTRODUCTION

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THE DETECTION of minimal residual disease (MRD) in complete hematologic remission (CR) by sensitive molecular methods can provide important prognostic information in patients with acute leukemia.¹ In acute lymphoblastic leukemia, most patients can be analyzed for MRD by the use of immunoglobulin and T-cell receptor gene rearrangements as a molecular marker.^2,3 In contrast, in acute myeloblastic leukemia (AML), molecular MRD detection is mainly restricted to patients with defined chromosomal aberrations.

Among the most frequent of these genomic lesions in AML are the reciprocal translocation t(8;21)(q22;q22) and the pericentric inversion of chromosome 16, inv(16)(p13q22). The t(8;21) fuses the AML1 gene (also called CBFA or RUNX1) on chromosome 21 to the MTG8 gene (also called ETO or CBF2T1) on chromosome 8, resulting in a chimeric AML1/MTG8 mRNA.⁴ In the inv(16), a CBFB/MYH11 fusion mRNA is transcribed.⁵ Because both aberrations affect the genes for the subunits of the core binding factor (CBF) transcription factor complex, they are also referred to as CBF leukemias.⁶ Patients with CBF leukemias are usually younger than 60 years of age.⁷ The t(8;21) is often accompanied by a French-American-British M2 subtype, whereas patients with an inv(16) normally show a myelomonocytic leukemia with abnormal eosinophils (French-American-British M4eo). It is generally accepted that in patients with CBF leukemias, a high CR rate can be achieved after a standard induction chemotherapy with cytarabine and an anthracycline.⁸ Moreover, after an intensive consolidation therapy with high-dose cytarabine or an autologous stem-cell transplantation, long-term remission rates of 60% to 70% have been reported.^9,10 However, even after such aggressive therapies, a considerable proportion of the patients suffers from relapse. To identify these high-risk patients, several risk factors have been defined during the last years. Among others, these factors comprise extramedullary disease and CD56 expression for the t(8;21)^11,12 and a high leukocyte count or WBC index for both types of CBF leukemias.^13,14 These risk factors are assessed before initiation of treatment. Therefore, MRD analysis during and after treatment might provide additional information about response to a given therapy in the individual patient. In both types of CBF leukemias, the fusion mRNA can be sensitively detected by reverse transcriptase polymerase chain reaction (RT-PCR). However, studies using qualitative RT-PCR in CBF leukemias have produced conflicting results, because there was no clear correlation between qualitative MRD detection and clinical outcome.^15–17 Recent studies show that quantification of MRD by competitive RT-PCR might be more clinically relevant in this patient group.^18,19 The major drawback of this method, however, is that it is labor-intensive and carries a high risk of contamination.

Recently, real-time RT-PCR has been introduced. This method allows the reliable semi-automatic quantification of RNA and DNA targets by the use of fluorescence-labeled DNA probes included in the PCR reaction.²⁰ Pilot studies with small numbers of patients have shown that real-time RT-PCR can also be used to quantify MRD in patients with CBF leukemias.^21–23 In our study, we applied this technique to a homogenously treated patient cohort with t(8;21) or inv(16) aberrations and evaluated its prognostic value.

	PATIENTS AND METHODS

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Patients
Thirty-seven patients with t(8;21) (n = 22) or inv(16) (n = 15) were analyzed in this study. In all patients, the genetic aberration was detected at initial diagnosis by conventional cytogenetics and confirmed by qualitative PCR for AML1/MTG8 and CBFB/MYH11, respectively. All inv(16) patients had a type A transcript with breakpoint at base pair 1921 of the MYH11 gene.

Real-Time PCR for AML1/MTG8, CBFB/MYH11, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
Real-time RT-PCR for AML1/MTG8 and CBFB/MYH11 was performed as previously described.^21,22,24 The sequences of the primers and probes are outlined in Table 1. Briefly, the PCR reaction was carried out in 25 µL with 1 x Taqman PCR Mastermix (Applied Biosystems, Foster City, CA), 150nmol probe, 300nmol primers and 1 µL cDNA template (equal to 50 ng of reversely transcribed RNA). The reaction conditions were 95°C for 10 minutes followed by 40 cycles of 95°C for 15 seconds and 59°C for 60 seconds for AML1/MTG8. For CBFB/MYH11 the temperature for annealing and extension was 60°C. Both PCR reactions had a sensitivity of 1:10⁵ with RNA and cDNA dilutions of the Kasumi-1 or ME-1 cell line and reliably detected 10 copies of a plasmid with the target sequence of AML1/MTG8 or CBFB/MYH11 respectively.^21,22,24,25 As a control gene, GAPDH was used. GAPDH quantification was performed under the same conditions using the GAPDH control reagents kit (Applied Biosystems). All three PCR reactions showed efficiencies near 100% (Fig 1). All experiments were carried out in duplicate. Fluorescence spectra were continuously monitored and analyzed by an SDS7700 system (Applied Biosystems, software version 1.6.3).

View larger version (74K): [in this window] [in a new window]   Fig 1. Amplification plot and standard curve for AML1/MTG8 (A, B) and CBFB/MYH11 (C, D) real-time reverse transcriptase polymerase chain reaction (RT-PCR).

View larger version (50K): [in this window] [in a new window]   Fig 2. Scheme of the treatment protocol for induction and consolidation I (A) and consolidation II (B). FLAG-Ida-I, fludarabine, intermediate-dose AraC, idarubicin, and granulocyte colony-stimulating factor; IVA-I, cytarabine, etoposide, and idarubicin; DNR, daunorubicin; auto, autologous; allo, allogeneic, PBPCT, peripheral blood progenitor cell transplantation; BMT, bone marrow transplantation.

AML1/MTG8 and CBFB/MYH11 were quantified relative to GAPDH expression. For a given cDNA sample, the real-time PCR threshold cycle (C_T) for the fusion gene (FG) and for GAPDH was determined. The ratio FG:GAPDH was calculated as follows: FG:GAPDH = 2^{CT(GAPDH)-CT(FG)} [ C_T-method.²⁷]

Statistical Analysis
Relapse-free and overall survival were calculated according to the method of Kaplan and Meier. Relapse-free survival was calculated from the day entering complete remission to relapse or death in CR. Overall survival was calculated from the day of first diagnosis to the day of death or last follow-up. Comparisons regarding relapse-free and overall survival were performed with the log-rank test. Patient groups A and B were compared using the Wilcoxon and ²-test.

	RESULTS

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Patients and Samples
A total of 37 patients with CBF leukemias were included in the study. Twenty-two patients had a t(8;21) and 15 had an inv(16) aberration. The median age at initial diagnosis was 39 years (range, 16 to 61 years). The median leukocyte count at initial diagnosis was 14.0 x 10⁹/liter (range, 1.2 to 284 x 10⁹/liter). Thirty-two of 37 patients had a good response (< 5% blasts in the day 15 bone marrow) to the first induction cycle. All patients analyzed achieved a hematologic CR after two cycles of induction chemotherapy.

A total of 267 samples of the 37 patients were analyzed by real-time RT-PCR from initial diagnosis to the last follow-up in CR or first relapse, respectively. In all these samples the quality of the RNA/cDNA as assessed by GAPDH quantification allowed a theoretical sensitivity for the detection of leukemic cells of at least 1:10⁴. The median number of samples analyzed per patient was six (range, three to 18 samples). The median molecular follow-up in first CR/until first relapse for the whole patient group is 474 days (range, 94 to 2188 days).

Fusion Gene Expression at Initial Diagnosis
At initial diagnosis, patients with t(8;21) as well as with inv(16) showed a considerable heterogeneity in the expression of the fusion gene relative to GAPDH, although the material analyzed contained 80% blasts in all cases under study (Fig 3). In the t(8;21) patients, the median AML1/MTG8:GAPDH ratio was 0.2531 (range, 0.011 to 3.66). The corresponding figure for CBFB/MYH11:GAPDH in the inv(16) patients was 0.0252 (range, 0.0039 to 0.956).

View larger version (6K): [in this window] [in a new window]   Fig 3. Variation of AML1/MTG8 and CBFB/MYH11 expression at initial diagnosis.

View larger version (20K): [in this window] [in a new window]   Fig 4. Relapse-free survival of the whole patient group (A) and according to minimal residual disease status (B).

View larger version (25K): [in this window] [in a new window]   Fig 5. Minimal residual disease (MRD) analysis of patients in group A (A, B) and group B (C, D). PCR, polymerase chain reaction.

Concerning MRD kinetics, two patterns of relapse could be distinguished in group B: four patients (nos. 25, 28, 31, and 34; Table 2) had the MRD level 1% directly after induction or after consolidation I and received further therapy thereafter. Three of these patients experienced relapse despite subsequent aggressive chemotherapy (Fig 5C). Median time to relapse of these patients was 302 days. In the other seven patients, MRD levels were less than 1% from achievement of CR to the end of therapy. In these patients, a secondary increase in MRD levels was observed in CR after the end of therapy followed by clinical relapse (Fig 5D). The median time to relapse of these patients was 423 days. The median interval between MRD levels 1% in CR and hematologic relapse in the group B patients was 99 days (range, 22 to 315 days).

When groups A and B were compared with regard to overall survival, there was no significant difference (P = .12, log-rank test; Fig 6). The outcome of the relapsed patients of group B is outlined in Table 4.

View larger version (13K): [in this window] [in a new window]   Fig 6. Overall survival of patient groups A and B according to minimal residual disease status.

	DISCUSSION

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We investigated the prognostic impact of MRD quantification by real-time RT-PCR in CBF leukemias. We focused on this subgroup of AML because a major proportion of these patients are believed to be cured by standard induction and a consolidation with high-dose AraC or an autologous stem-cell transplantation.^9,10 Therefore, further intensification or modification of the therapy, eg, by an allogeneic stem-cell transplantation or the use of investigational drugs, is not generally recommended. The detection of MRD is an attractive approach to identify patients at high risk for relapse who might benefit from alternative treatment strategies. Several studies have shown, however, that the qualitative detection of MRD in this patient group does not necessarily indicate subsequent relapse.^17,28 Therefore, the quantification of the residual tumor burden is necessary. Real-time RT-PCR is generally accepted as a reliable method for the quantification of RNA and DNA targets in hematologic malignancies. Moreover, several studies with smaller numbers of patients have shown that it can be used for the quantification of the fusion mRNAs AML1/MTG8 and CBFB/MYH11 in the CBF leukemias.^21–23

We evaluated the prognostic value of these analyses in a group of 37 patients treated within a multicenter trial. As previously described, at initial diagnosis there was a considerable heterogeneity of the fusion gene expression with a more than two-log difference between individual patients.^21,22 To take these differences into account, we quantified the MRD levels in relation to the patients’ fusion gene:GAPDH ratio at initial diagnosis. In patients without subsequent relapse, these relative MRD levels in CR were significantly lower than in patients who later experienced relapse. Moreover, using a relative MRD level of 1% as a threshold, two groups of patients could be separated: group A included 26 patients with a rapid decrease in tumor burden and MRD levels that at any time after induction chemotherapy were below 1% of the levels at initial diagnosis. All of these patients also had a significant decline in terms of absolute fusion gene copy numbers, and most of them became PCR-negative. As expected, these patients had a good prognosis and only two relapses occurred in this group. These findings are in line with data from Buonamici et al.²⁹ In their study with inv(16)-positive patients, they found CBFB/MYH11:abl ratios of 19% to 60% at initial diagnosis and in relapse. In patients without subsequent relapse, the CBFB/MYH11:abl ratios in CR were always less than 0.25%. When the MRD levels of Buonamici’s study are expressed in relation to initial diagnosis (like in our analysis), this corresponds to relative MRD levels of less than 0.4 to 1.3% in CR. This supports our finding that a durable, at least two-log reduction of the leukemic burden is a prerequisite for long-term CR.

Despite the good prognosis of group A, two relapses occurred. This demonstrates that relapse kinetics can be quite fast. Thus MRD testing must initially be performed at least every 3 months to be informative.

In the second group of 11 patients (group B), an MRD level of 1% was observed at least at one point after the completion of two cycles of induction therapy. The patients in this group had a dismal prognosis, and 10 of 11 patients experienced relapse, with a median remission duration of approximately 10 months. It is noteworthy that there was no significant difference between the two groups with respect to other known risk factors.^13,14,30 Both groups had approximately the same age and the same median leukocyte count at diagnosis, and also the percentage of bad responders to the first induction cycle did not differ. In addition, a systematic error in the follow-up of the two groups can be largely ruled out, because the frequency of follow-up PCRs did not significantly differ between the groups. Thus MRD status represents an additional prognostic factor. By MRD quantification, two patterns of relapse kinetics could be identified: three relapsing patients had an MRD level 1% directly after induction therapy or during ongoing consolidation. Therefore, CBF patients with a tumor reduction of less than two logs during ongoing therapy should be considered as high risk. In contrast, in seven of the 10 patients who experienced relapse, a good initial reduction of fusion gene levels was observed, and some of them even became PCR-negative. However, after the end of therapy, an increase in MRD levels above the threshold was observed in CR, and the patients ultimately experienced relapse. This confirms a recent study by Marcucci et al.³¹ In their patients, the authors also found low CBFB/MYH11 copy numbers during intensive chemotherapy, and relapse was preceded by a secondary increase in fusion gene expression. All relapses in our patients occurred during the first 27 months after initiation of treatment. However, the time span, during which frequent serial MRD testing is necessary, is still unknown and must be defined in larger trials.

In conclusion, our data show that the quantification of MRD by real-time RT-PCR in CBF patients is of prognostic value. On one hand, patients with persistently low MRD levels have a good prognosis, and for this low-risk group, therapy should not be further intensified. On the other hand, patients with a high risk of relapse can be identified. The overall survival was not significantly different between the two risk groups. Although this finding needs to be confirmed in a larger number of patients, it implies that durable second remissions can be achieved in these patients after salvage therapy, especially after an allogeneic stem-cell transplantation (Table 4). It is therefore conceivable that an allogeneic stem-cell transplantation in first CR might be of value in high-risk patients identified by MRD quantification. The median interval between the MRD level 1% and clinical relapse in our patients was greater than 3 months. This time span offers the opportunity to initiate the search for an HLA-identical stem-cell donor and to prepare the patient for an allogeneic stem-cell transplantation. Another approach could be the modulation of MRD levels by additional chemotherapy or the use of investigational drugs in a similar way as described for acute promyelocytic leukemia.³² Of special interest in this context are histone deacetylase inhibitors, which in vitro have activity in leukemic blasts with CBF aberrations.^33–35 Thus molecular analysis of the remission status of AML patients allows the discrimination of risk groups who require specific postinduction therapy.

	AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

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The authors indicated no potential conflicts of interest.

	ACKNOWLEDGMENTS

 
We thank I. Schäfer, G. Samson, and Peter Krauter for their help in data acquisition and statistical analysis. We also thank the members of the AML 2/95 and AML 01/99 study groups for their continuous support of the treatment protocols.

	NOTES

 
Supported by a grant from the Deutsche José Carreras Leukämie-Stiftung e.V., Munich; (DJCLS-R01/08) and the Kompetenznetz Akute und chronische Leukämien, funded by the German Bundesministerium für Bildung und Forschung, Berlin, Germany.

	REFERENCES

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Submitted March 26, 2003; accepted September 3, 2003.

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