Pediatr Blood Cancer 2014;61:1761–1766
Monitoring the AML1/ETO Fusion Transcript to Predict Outcome in Childhood Acute Myeloid Leukemia Li Zhang, MD,1 Zeng Cao, MD,1 Min Ruan, MD,1 Qiang Zeng, MD,2 Liang Zhao, MD,2 Qinghua Li, MD,1 Yao Zou, MD,1 Jianxiang Wang, MD,3 and Xiaofan Zhu, MD1* Background. To determine the prognostic significance of the detection of the minimal residual disease (MRD) in children with AML1/ETO AML, we compared the results of reverse-transcription polymerase chain reaction (RT-PCR) and quantitative reversetranscription polymerase chain reaction (RQ-PCR). Procedure. Between January 2006 and February 2013, 70 patients (16 years of age) with AML1/ETO AML were included in our study. Bone marrow samples were evaluated using by both RT-PCR and RQ-PCR assays. AML1/ETO transcripts were normalized to 105 ABL copies. Results. When treated with fewer than four courses of therapy, no
association was found between positive RT-PCR results and relapse. After four courses of therapy, a positive RT-PCR result was correlated with a probability of relapse. After induction chemotherapy, a >1.8 log reduction in AML1/ETO transcripts in BM determined by RQ-PCR may represent a subgroup of patients at low risk for relapse. MRD levels after consolidation (Courses 2 and 3) were also informative. Conclusion. Both RT-PCR and RQ-PCR can be used to detect MRD in childhood AML1/ETO AML. RQ-PCR can identify patients who are at high risk of relapse earlier than can RT-PCR. Pediatr Blood Cancer 2014;61:1761–1766. # 2014 Wiley Periodicals, Inc.
Key words: acute; AML1/ETO; child; leukemia; myeloid; polymerase chain reaction
INTRODUCTION The t(8;21)(q22;q22) translocation, observed in approximately 12–14% of childhood acute myeloid leukemia (AML) cases, fuses the AML1 (RUNX1) gene located on chromosome 21 to the ETO (MTG8) gene located on Chromosome 8 and is generally associated with favorable prognosis [1–3]. However, relapse remains the most common reason for treatment failure in AML [1–5]. Therefore, the challenge is to identify patients who are at high risk of relapse early and deliver intensification treatment to improve the outcome. Monitoring of minimal residual disease (MRD) in these patients plays an important role in treatment evaluation and detection of early signs of relapse. Conventional qualitative reverse-transcription polymerase chain reaction (RT-PCR) assays for AML1/ETO have been used in the diagnosis and monitoring of MRD [6]. However, this method is very time-consuming, labor intensive, and limited in its dynamic range of quantification. In addition, some studies have shown that AML1/ ETO transcripts could be detected after chemotherapy as well as autologous and allogeneic bone marrow transplantation in many patients who are in long-term remission [7,8]. Recently, the quantification of the AML1/ETO copy number based on quantitative reverse-transcription polymerase chain reaction (RQ-PCR) has become a new alternative for monitoring disease progress [9–11]. This method is very fast, accurate, and reproducible, and it facilitates the assessment of kinetics. A comparison of RT-PCR and RQ-PCR in AML1/ETO AML showed a good correlation with respect to sensitivity and reproducibility [12]. There is now cumulative evidence supporting the notion that monitoring of MRD using RQ-PCR can predict relapse in adult AML1/ETO AML [9– 11]. At present, only a few RQ-PCR studies with a small number of patients have been performed to examine childhood AML1/ETO AML [13,14]. In addition, little is known about the kinetics of relapse in children. The present work analyzes the value of monitoring MRD for predicting relapse in pediatric AML1/ETO AML and compares the quantitative results with those of the conventional RT-PCR approach according to treatment phase. The aim of our study was to analyze associations between the risk of relapse and MRD C
2014 Wiley Periodicals, Inc. DOI 10.1002/pbc.25109 Published online 11 June 2014 in Wiley Online Library (wileyonlinelibrary.com).
results in different phases of treatment in children, with the goal being to identify patients who are at high risk of relapse early and deliver intensification treatment to improve the outcome.
METHODS Patients Between January 2006 and February 2013, 70 patients (16 years of age) with de novo AML1/ETO AML were admitted to the Department of Pediatrics, Institute of Hematology and Blood Disease Hospital. At diagnosis, all patients underwent routine cytogenetic investigations and were also screened for the presence of AML1/ETO transcripts. The diagnosis of AML1/ETO AML was based on a positive cytogenetic and/or molecular result. Sixty-two of the patients were included in the MRD study. BM samples were requested at diagnosis, after each course of chemotherapy, and serially every 3 months in the first year of 1
Department of Pediatrics, State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, P.R. China; 2Tianjin Centers for Disease Control and Prevention, Tianjin, P.R. China; 3State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, P.R. China Grant sponsor: Tianjin Science and Technology Support Plan; Grant number: 12ZCDZSY18100; Grant sponsor: Ministry of Science and Technology Major Projects; Grant number: 2011ZX09302-007; Grant sponsor: Youth Scientific Research Funds of Peking Union Medical College; Grant number: 2012J17; Grant sponsor: Natural Science Fund Foundation Project; Grant number: 81200396 Conflict of interest: The authors declare no conflict of interest. Correspondence to: Xiaofan Zhu, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 288 Nanjing Road, Tianjin 300020, P.R. China. E-mail: [email protected]
Received 19 December 2013; Accepted 23 April 2014
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Before November 2009, the induction therapy was homoharringtonine, (4 mg/m2/day, Days 1–7), cytarabine (150 mg/m2/day, Days 1–7), and daunorubicin (45 mg/m2/day, Days 1–3). After achieving CR, four to five courses of consolidation chemotherapy including high-dose cytarabine were given sequentially. The consolidation chemotherapy was daunorubicin þ cytarabine or mitoxantrone þ cytarabine or homoharringtonine þ cytarabine (daunorubicin, 45 mg/m2/day for 3 days; mitoxantrone, 5 mg/m2/ day for 3 days; homoharringtonine, 4 mg/m2/day for 7 days; cytarabine, 2 g/m2/q12 hr for 3 days). After November 2009, treatment was administered according to the AML-99 protocol, which was described previously [15]. These trials were approved by the Institutional Committee for Medical Care and Safety at Institute of Hematology and Blood Disease Hospital.
two, 36.5 copies after Course three, 1.8 log reduction after induction, 2.7 log reduction after Course two, and 2.6 log reduction after Course three. High WBC, PLT, copy numbers, and log reduction were defined as above the threshold. The dependent variables were disease-free survival (DFS) and overall survival (OS). DFS was calculated from the date of hematological complete remission achievement to the date of either last follow-up or an event (relapse or death); OS duration was calculated from the date of diagnosis to the date of either last follow-up or death. Proportions were compared using the chi-squared test. Differences between continuous variables were analyzed with Student’s t-test. The probability of relapse was calculated using cumulative incidence estimates (CIR). Cumulative incidence functions for competing events were constructed by the method of Kalbfleisch and Prentice and compared with Gray’s test. Gray’s test was used to compare CIR between two groups. The Kaplan–Meier method was used to estimate DFS and OS, and the log-rank test was used for comparisons. Relationships of clinical features and outcome were analyzed by Cox regression proportional hazard model. The statistical analyses were carried out using SPSS software (version 17.0) and R software (version 3.0).
RNA Extraction and cDNA Synthesis
RESULTS
Total RNA was isolated from BM samples using the acid guanidinium thiocyanate–phenol–chloroform extraction method [16]. Reverse transcription was performed using 1 mg of total RNA according to a previously described protocol [17].
Descriptive Analysis
follow-up, every 6 months in the second and third years, and at relapse. Written informed consent was obtained from the patients’ parents or guardians. Samples were collected as part of the treatment protocol.
Treatment Schedule
Qualitative RT-PCR and RQ-PCR (Real-Time) Assays To amplify the AML/ETO fusion gene, a two-step qualitative RT-PCR analysis was performed, as described previously [17]. The RQ-PCR method is based on cDNA and was established in our laboratory; using this method, a sensitivity of 1 105 was achieved for detection of AML/ETO in the Kasumi-1 cell line. The Abelson house-keeping gene (ABL) was selected as a control for RNA expression, as previously reported [18]. The AML1/ETO transcripts were quantified using the ABI PRISM 7500 DNA Sequence Detection System. The absolute copy numbers of fusion gene transcripts were normalized to ABL and expressed per 104 copies of ABL. All the results are reported as the normalized copy number (NCN). A result of less than 1 NCN was considered as RQ-PCR-negative. All RQ-PCR experiments were performed in duplicate [19]. Diagnostic and follow-up samples with 20 109/L PLT count (109/L) 30 109/L >30 109/L Copy number at Dx1 Few copies Many copies MRD 1 NCN 1 Few copies Many copies Log reduction 1 Low High MRD 2 NCN2 Few copies Many copies Log reduction 2 Low High MRD 3 NCN3 Few copies Many copies Log reduction 3 Low High
Range
N
%
70 9
14.3
28.5
30,464
230
23
12
3–16 3–9 10–16
41 29 35 35
50.0 50.0
33 37
47.1 52.9
44 26
62.9 37.1
36 34
51.4 48.6
32 38
45.7 54.3
33 25
56.9 43.1
20 38
34.5 65.5
30 25
54.5 45.5
21 34
38.2 61.8
37 16
69.8 30.2
13 40
24.5 75.5
0.8–83.8
4–116
516–184,138
0–6,796
0–2,466
0–1,581
5-year DFS (%)
5-year OS (%)
64.4 6.7% P ¼ 0.170 71.8 8.1% 53.8 11.0% P ¼ 0.170 55.2 9.5% 76.1 8.5% P ¼ 0.287 58.7 10.1% 69.5 8.7% P ¼ 0.129 72.4 7.9% 50.8 11.9% P ¼ 0.132 52.9 10.3% 75.5 8.1% P ¼ 0.034 50.0 10.2% 74.5 8.8%
65.6 6.5% P ¼ 0.302 68.1 8.6% 60.8 10.2% P ¼ 0.916 64.7 9.2% 66.1 9.4% P ¼ 0.160 57.5 9.8% 71.9 8.8% P ¼ 0.258 69.5 8.2% 60.1 10.4% P ¼ 0.118 56.6 9.7% 73.8 8.6% P ¼ 0.011 48.4 10.0% 79.6 7.6%
P < 0.001 86.7 6.2% 31.5 11.8% P < 0.001 23.3 11.9% 85.4 6.1%
P < 0.001 89.5 5.8% 38.5 11.5% P < 0.001 32.0 11.8% 87.8 5.8%
P ¼ 0.011 81.4 7.6% 44.6 11.0% P ¼ 0.007 39.7 11.6% 80.6 7.2%
P ¼ 0.065 80.9 7.8% 54.2 11.1% P ¼ 0.060 50.6 12.0% 79.9 7.5%
P ¼ 0.001 75.9 7.6% 37.5 12.1% P ¼ 0.001 30.8 12.8% 74.9 7.4%
P ¼ 0.008 78.8 7.3% 43.8 13.6% P ¼ 0.001 43.3 14.3% 77.2 7.2%
WBC, white blood cell; PLT, platelet; Dx1, diagnosis; MRD, minimal residual disease; NCN, normalized copy number.
was compared between the two groups. There was no significant difference between 5-year DFS and OS in the AML1/ETO-positive group and those in the AML1/ETO-negative group within four courses of chemotherapy. Five-year DFS and OS in the AML1/ETOnegative group were significantly higher than those in the AML1/ ETO-positive group after treatment with four courses of chemotherapy. The patients were divided into three groups according to the RTPCR results. Group I (continuous negative patients) included the patients with consistently disappearance of AML1/ETO fusion transcript. The median number of negative RT-PCR results in Group I was four (range, 3–7). Group II (continuous positive patients) included the patients with persistence of AML1/ETO fusion transcript. The median number of positive results in Group II was four (range, 3–8). Group III (recurrent positive patients) included the patients with at least one positive RT-PCR results after disappearance of AML1/ETO fusion transcript. The median number of checkpoint for Group III was 6.5 (range, 3–9). In our study, 28 Pediatr Blood Cancer DOI 10.1002/pbc
patients (Group I) were consistently negative, 21 patients (Group II) were continuous positive, and 10 patients (Group III) were recurrent positive. The patients in Group I had remarkably better prognosis than those in Group II or III (P < 0.001) (Fig. 1).
RQ-PCR in Different Phases of Treatment and Influence on Survival The median AML1/ETO copy number before treatment in all 70 patients was 30,464.9 (516.8–184,138.1). Pretreatment AML1/ ETO copy numbers did not correlate with age, WBC, or gender. However, copy number at diagnosis was significantly correlated with relapse. The impact of BM AML1/ETO copy numbers and log reduction on relapse risk is shown in Table I. After induction chemotherapy, patients with a >1.8 log reduction had a CIR at 5 years of 14.6 0.8% compared with 67.7 7.1% in those with 3 log reduction relapsed after introduction. We suggest that the AML1/ETO copy number after treatment may reflect the sensitivity of leukemic clones to treatment; thus, MRD monitoring by RQ-PCR might allow us to identify subgroups of pediatric patients who are at low risk of relapse sooner after induction. For patients with an AML1/ETO copy number reduction of less than 1.8 log after induction, MRD should be monitored sequentially so that treatment can be modified early to improve the outcome. It reported that an increased transcript level in patients with morphological CR predicted relapse [10]. We found that a 0.6 log increase in AML1/ETO transcripts during follow-up strongly predicted subsequent morphological relapse. Zhang et al. [26] found that a 0.5 log increase in AML1/ETO transcript during
TABLE III. Comparison of RQ-PCR and RT-PCR Results in Samples According to Therapeutic Phase
10 NCN Total
Post-induction (n ¼ 58)
Post-course 2 (n ¼ 55)
Post-course 3 (n ¼ 53)
Post-courses 4–6 (n ¼ 118)
Out of treatment (n ¼ 48)
POS
NEG
POS
NEG
POS
NEG
POS
NEG
POS
NEG
0 3 52 55
2 1 0 3
0 12 35 47
4 3 1 8
0 5 30 35
11 7 0 18
0 11 46 57
44 15 2 61
0 0 15 15
26 7 0 33
POS, positive; NEG, negative. Pediatr Blood Cancer DOI 10.1002/pbc
Discrepancies (%); total 0/87 33/64 3/18 36/332
(0) (51.6) (16.7) (10.8)
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follow-up strongly predicted morphological relapse, which was similar to our findings. Notably, three out of 14 patients developed to hematology relapse within 1 month in our study. Therefore, the optimal schedule for pre-emptive therapy in pediatric AML1/ETO AML should be the subject of further research. In conclusion, both RT-PCR and RQ-PCR can be used to detect MRD in pediatric AML1/ETO AML. RQ-PCR can indicate patients who are at a high risk of relapse earlier than can RT-PCR. Moreover, for patients with CR but continuous positive RT-PCR results, RQPCR should be performed more frequently. Although the small number of the patients and this retrospective analysis may not have sufficient power to lead to a conclusion, further clinical research with larger numbers of pediatric patients should be conducted. Also of note, the threshold values identified using ROC analysis may not be optimal and should be validated in subsequent studies.
ACKNOWLEDGMENTS This study was supported in parts by grant from the Tianjin Science and Technology Support Plan (12ZCDZSY18100), the Ministry of Science and Technology Major Projects (2011ZX09302-007), the Youth Scientific Research Funds of Peking Union Medical College (2012J17), and the Natural Science Fund Foundation Project (81200396).
REFERENCES 1. Harrison CJ, Hills RK, Moorman AV, et al. Cytogenetics of childhood acute myeloid leukemia: United Kingdom Medical Research Council Treatment trials AML 10 and 12. J Clin Oncol 2010;28:2674–2681. 2. von Neuhoff C, Reinhardt D, Sander A, et al. Prognostic impact of specific chromosomal aberrations in a large group of pediatric patients with acute myeloid leukemia treated uniformly according to trial AML-BFM 98. J Clin Oncol 2010;28:2682–2689. 3. Raimondi SC, Chang MN, Ravindranath Y, et al. Chromosomal abnormalities in 478 children with acute myeloid leukemia: Clinical characteristics and treatment outcome in a cooperative pediatric oncology group study-POG 882. Blood 1999;94:3707–3716. 4. Byrd JC, Dodge RK, Carroll A, et al. Patients with t(8;21)(q22;q22) and acute myeloid leukemia have superior failure-free and overall survival when repetitive cycles of high-dose cytarabine are administered. J Clin Oncol 1999;17:3767–3775. 5. Marcucci G, Mr ozek K, Ruppert AS, et al. Prognostic factors and outcome of core binding factor acute myeloid leukemia patients with t(8;21) differ from those of patients with inv(16): A Cancer and Leukemia Group B study. J Clin Oncol 2005;23:5705–5717.
Pediatr Blood Cancer DOI 10.1002/pbc
6. Satake N, Maseki N, Kozu T, et al. Disappearance of AML1-MTG8(ETO) fusion transcript in acute myeloid leukaemia patients with t(8;21) in long-term remission. Br J Haematol 1995;91:892–898. 7. Jurlander J, Caligiuri MA, Ruutu T, et al. Persistence of the AML1/ETO fusion transcript in patients treated with allogeneic bone marrow transplantation for t(8;21) leukemia. Blood 1996;88:2183–2191. 8. Miyamoto T, Nagafuji K, Akashi K, et al. Persistence of multipotent progenitors expressing AML1/ETO transcripts in long-term remission patients with t(8;21) acute myelogenous leukemia. Blood 1996; 87:4789–4796. 9. Yin JA, O’Brien MA, Hills RK, et al. Minimal residual disease monitoring by quantitative RT-PCR in core binding factor AML allows risk stratification and predicts relapse: Results of the United Kingdom MRC AML-15 trial. Blood 2012;120:2826–2835. 10. Lane S, Saal R, Mollee P, et al. A > or ¼1 log rise in RQ-PCR transcript levels defines molecular relapse in core binding factor acute myeloid leukemia and predicts subsequent morphologic relapse. Leuk Lymphoma 2008;49:517–523. 11. Jourdan E, Boissel N, Chevret S, et al. Prospective evaluation of gene mutations and minimal residual disease in patients with core binding factor acute myeloid leukemia. Blood 2013;121:2213–2223. 12. Wattjes MP, Krauter J, Nagel S, et al. Comparison of nested competitive RT-PCR and real-time RT-PCR for the detection and quantification of AML1/MTG8 fusion transcripts in t(8;21) positive acute myelogenous leukemia. Leukemia 2000;14:329–335. 13. Viehmann S, Teigler-Schlegel A, Bruch J, et al. Monitoring of minimal residual disease (MRD) by realtime quantitative reverse transcription PCR (RQ-RT-PCR) in childhood acute myeloid leukemia with AML1/ETO rearrangement. Leukemia 2003;17:1130–1136. 14. Kondo M, Kudo K, Kimura H, et al. Real-time quantitative reverse transcription-polymerase chain reaction for the detection of AML1-MTG8 fusion transcripts in t(8;21)-positive acute myelogenous leukemia. Leuk Res 2000;24:951–956. 15. Tsukimoto I, Tawa A, Horibe K, et al. Risk-stratified therapy and the intensive use of cytarabine improves the outcome in childhood acute myeloid leukemia: The AML99 trial from the Japanese Childhood AML Cooperative Study Group. J Clin Oncol 2009;27:4007–4013. 16. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate– phenol–chloroform extraction. Anal Biochem 1987;162:156–159. 17. Van Dongen JJM, Macintyre EA, Gabert JA, et al. Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease. Report of the BIOMED-1 Concerted Action: Investigation of minimal residual disease in acute leukemia. Leukemia 1999;13:1901–1928. 18. Gabert J, Beillard E, van der Velden VH, et al. Standardization and quality control studies of “realtime” quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia: A Europe Against Cancer program. Leukemia 2003;17:2318–2357. 19. Beillard E, Pallisgaard N, van der Velden VH, et al. Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patients using “real-time” quantitative reverse-transcriptase polymerase chain reaction (RQ-PCR). A Europe Against Cancer Program. Leukemia 2003;17:2474– 2486. 20. Arellano M, Bernal-Mizrachi L, Pan L, et al. Prognostic significance of leukopenia at the time of diagnosis in acute myeloid leukemia. Clin Lymphoma Myeloma Leuk 2011;11:427–432. 21. Mallett S, Timmer A, Sauerbrei W, et al. Reporting of prognostic studies of tumour markers: A review of published articles in relation to REMARK guidelines. Br J Cancer 2010;102:173–180. 22. Leroy H, de Botton S, Grardel-Duflos N, et al. Prognostic value of real-time quantitative PCR (RQ-PCR) in AML with t(8;21). Leukemia 2005;19:367–372. 23. Hoyos M, Nomdedeu JF, Esteve J, et al. Core binding factor acute myeloid leukemia: The impact of age, leukocyte count, molecular findings, and minimal residual disease. Eur J Haematol 2013;91:209–218. 24. Pollard JA, Alonzo TA, Gerbing RB, et al. Prevalence and prognostic significance of KIT mutations in pediatric patients with core binding factor AML enrolled on serial pediatric cooperative trials for de novo AML. Blood 2010;115:2372–2379. 25. Miyamoto T, Weissman IL, Akashi K. AML1/ETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 8;21 chromosomal translocation. Proc Natl Acad Sci USA 2000;97:7521– 7526. 26. Zhang L, Li Q, Li W, et al. Monitoring of minimal residual disease in acute myeloid leukemia with t(8;21) (q22;q22). Int J Hematol 2013;97:786–792.
Monitoring the AML1/ETO Fusion Transcript to Predict Outcome in Childhood Acute Myeloid Leukemia Li Zhang, MD,1 Zeng Cao, MD,1 Min Ruan, MD,1 Qiang Zeng, MD,2 Liang Zhao, MD,2 Qinghua Li, MD,1 Yao Zou, MD,1 Jianxiang Wang, MD,3 and Xiaofan Zhu, MD1* Background. To determine the prognostic significance of the detection of the minimal residual disease (MRD) in children with AML1/ETO AML, we compared the results of reverse-transcription polymerase chain reaction (RT-PCR) and quantitative reversetranscription polymerase chain reaction (RQ-PCR). Procedure. Between January 2006 and February 2013, 70 patients (16 years of age) with AML1/ETO AML were included in our study. Bone marrow samples were evaluated using by both RT-PCR and RQ-PCR assays. AML1/ETO transcripts were normalized to 105 ABL copies. Results. When treated with fewer than four courses of therapy, no
association was found between positive RT-PCR results and relapse. After four courses of therapy, a positive RT-PCR result was correlated with a probability of relapse. After induction chemotherapy, a >1.8 log reduction in AML1/ETO transcripts in BM determined by RQ-PCR may represent a subgroup of patients at low risk for relapse. MRD levels after consolidation (Courses 2 and 3) were also informative. Conclusion. Both RT-PCR and RQ-PCR can be used to detect MRD in childhood AML1/ETO AML. RQ-PCR can identify patients who are at high risk of relapse earlier than can RT-PCR. Pediatr Blood Cancer 2014;61:1761–1766. # 2014 Wiley Periodicals, Inc.
Key words: acute; AML1/ETO; child; leukemia; myeloid; polymerase chain reaction
INTRODUCTION The t(8;21)(q22;q22) translocation, observed in approximately 12–14% of childhood acute myeloid leukemia (AML) cases, fuses the AML1 (RUNX1) gene located on chromosome 21 to the ETO (MTG8) gene located on Chromosome 8 and is generally associated with favorable prognosis [1–3]. However, relapse remains the most common reason for treatment failure in AML [1–5]. Therefore, the challenge is to identify patients who are at high risk of relapse early and deliver intensification treatment to improve the outcome. Monitoring of minimal residual disease (MRD) in these patients plays an important role in treatment evaluation and detection of early signs of relapse. Conventional qualitative reverse-transcription polymerase chain reaction (RT-PCR) assays for AML1/ETO have been used in the diagnosis and monitoring of MRD [6]. However, this method is very time-consuming, labor intensive, and limited in its dynamic range of quantification. In addition, some studies have shown that AML1/ ETO transcripts could be detected after chemotherapy as well as autologous and allogeneic bone marrow transplantation in many patients who are in long-term remission [7,8]. Recently, the quantification of the AML1/ETO copy number based on quantitative reverse-transcription polymerase chain reaction (RQ-PCR) has become a new alternative for monitoring disease progress [9–11]. This method is very fast, accurate, and reproducible, and it facilitates the assessment of kinetics. A comparison of RT-PCR and RQ-PCR in AML1/ETO AML showed a good correlation with respect to sensitivity and reproducibility [12]. There is now cumulative evidence supporting the notion that monitoring of MRD using RQ-PCR can predict relapse in adult AML1/ETO AML [9– 11]. At present, only a few RQ-PCR studies with a small number of patients have been performed to examine childhood AML1/ETO AML [13,14]. In addition, little is known about the kinetics of relapse in children. The present work analyzes the value of monitoring MRD for predicting relapse in pediatric AML1/ETO AML and compares the quantitative results with those of the conventional RT-PCR approach according to treatment phase. The aim of our study was to analyze associations between the risk of relapse and MRD C
2014 Wiley Periodicals, Inc. DOI 10.1002/pbc.25109 Published online 11 June 2014 in Wiley Online Library (wileyonlinelibrary.com).
results in different phases of treatment in children, with the goal being to identify patients who are at high risk of relapse early and deliver intensification treatment to improve the outcome.
METHODS Patients Between January 2006 and February 2013, 70 patients (16 years of age) with de novo AML1/ETO AML were admitted to the Department of Pediatrics, Institute of Hematology and Blood Disease Hospital. At diagnosis, all patients underwent routine cytogenetic investigations and were also screened for the presence of AML1/ETO transcripts. The diagnosis of AML1/ETO AML was based on a positive cytogenetic and/or molecular result. Sixty-two of the patients were included in the MRD study. BM samples were requested at diagnosis, after each course of chemotherapy, and serially every 3 months in the first year of 1
Department of Pediatrics, State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, P.R. China; 2Tianjin Centers for Disease Control and Prevention, Tianjin, P.R. China; 3State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, P.R. China Grant sponsor: Tianjin Science and Technology Support Plan; Grant number: 12ZCDZSY18100; Grant sponsor: Ministry of Science and Technology Major Projects; Grant number: 2011ZX09302-007; Grant sponsor: Youth Scientific Research Funds of Peking Union Medical College; Grant number: 2012J17; Grant sponsor: Natural Science Fund Foundation Project; Grant number: 81200396 Conflict of interest: The authors declare no conflict of interest. Correspondence to: Xiaofan Zhu, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 288 Nanjing Road, Tianjin 300020, P.R. China. E-mail: [email protected]
Received 19 December 2013; Accepted 23 April 2014
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Before November 2009, the induction therapy was homoharringtonine, (4 mg/m2/day, Days 1–7), cytarabine (150 mg/m2/day, Days 1–7), and daunorubicin (45 mg/m2/day, Days 1–3). After achieving CR, four to five courses of consolidation chemotherapy including high-dose cytarabine were given sequentially. The consolidation chemotherapy was daunorubicin þ cytarabine or mitoxantrone þ cytarabine or homoharringtonine þ cytarabine (daunorubicin, 45 mg/m2/day for 3 days; mitoxantrone, 5 mg/m2/ day for 3 days; homoharringtonine, 4 mg/m2/day for 7 days; cytarabine, 2 g/m2/q12 hr for 3 days). After November 2009, treatment was administered according to the AML-99 protocol, which was described previously [15]. These trials were approved by the Institutional Committee for Medical Care and Safety at Institute of Hematology and Blood Disease Hospital.
two, 36.5 copies after Course three, 1.8 log reduction after induction, 2.7 log reduction after Course two, and 2.6 log reduction after Course three. High WBC, PLT, copy numbers, and log reduction were defined as above the threshold. The dependent variables were disease-free survival (DFS) and overall survival (OS). DFS was calculated from the date of hematological complete remission achievement to the date of either last follow-up or an event (relapse or death); OS duration was calculated from the date of diagnosis to the date of either last follow-up or death. Proportions were compared using the chi-squared test. Differences between continuous variables were analyzed with Student’s t-test. The probability of relapse was calculated using cumulative incidence estimates (CIR). Cumulative incidence functions for competing events were constructed by the method of Kalbfleisch and Prentice and compared with Gray’s test. Gray’s test was used to compare CIR between two groups. The Kaplan–Meier method was used to estimate DFS and OS, and the log-rank test was used for comparisons. Relationships of clinical features and outcome were analyzed by Cox regression proportional hazard model. The statistical analyses were carried out using SPSS software (version 17.0) and R software (version 3.0).
RNA Extraction and cDNA Synthesis
RESULTS
Total RNA was isolated from BM samples using the acid guanidinium thiocyanate–phenol–chloroform extraction method [16]. Reverse transcription was performed using 1 mg of total RNA according to a previously described protocol [17].
Descriptive Analysis
follow-up, every 6 months in the second and third years, and at relapse. Written informed consent was obtained from the patients’ parents or guardians. Samples were collected as part of the treatment protocol.
Treatment Schedule
Qualitative RT-PCR and RQ-PCR (Real-Time) Assays To amplify the AML/ETO fusion gene, a two-step qualitative RT-PCR analysis was performed, as described previously [17]. The RQ-PCR method is based on cDNA and was established in our laboratory; using this method, a sensitivity of 1 105 was achieved for detection of AML/ETO in the Kasumi-1 cell line. The Abelson house-keeping gene (ABL) was selected as a control for RNA expression, as previously reported [18]. The AML1/ETO transcripts were quantified using the ABI PRISM 7500 DNA Sequence Detection System. The absolute copy numbers of fusion gene transcripts were normalized to ABL and expressed per 104 copies of ABL. All the results are reported as the normalized copy number (NCN). A result of less than 1 NCN was considered as RQ-PCR-negative. All RQ-PCR experiments were performed in duplicate [19]. Diagnostic and follow-up samples with 20 109/L PLT count (109/L) 30 109/L >30 109/L Copy number at Dx1 Few copies Many copies MRD 1 NCN 1 Few copies Many copies Log reduction 1 Low High MRD 2 NCN2 Few copies Many copies Log reduction 2 Low High MRD 3 NCN3 Few copies Many copies Log reduction 3 Low High
Range
N
%
70 9
14.3
28.5
30,464
230
23
12
3–16 3–9 10–16
41 29 35 35
50.0 50.0
33 37
47.1 52.9
44 26
62.9 37.1
36 34
51.4 48.6
32 38
45.7 54.3
33 25
56.9 43.1
20 38
34.5 65.5
30 25
54.5 45.5
21 34
38.2 61.8
37 16
69.8 30.2
13 40
24.5 75.5
0.8–83.8
4–116
516–184,138
0–6,796
0–2,466
0–1,581
5-year DFS (%)
5-year OS (%)
64.4 6.7% P ¼ 0.170 71.8 8.1% 53.8 11.0% P ¼ 0.170 55.2 9.5% 76.1 8.5% P ¼ 0.287 58.7 10.1% 69.5 8.7% P ¼ 0.129 72.4 7.9% 50.8 11.9% P ¼ 0.132 52.9 10.3% 75.5 8.1% P ¼ 0.034 50.0 10.2% 74.5 8.8%
65.6 6.5% P ¼ 0.302 68.1 8.6% 60.8 10.2% P ¼ 0.916 64.7 9.2% 66.1 9.4% P ¼ 0.160 57.5 9.8% 71.9 8.8% P ¼ 0.258 69.5 8.2% 60.1 10.4% P ¼ 0.118 56.6 9.7% 73.8 8.6% P ¼ 0.011 48.4 10.0% 79.6 7.6%
P < 0.001 86.7 6.2% 31.5 11.8% P < 0.001 23.3 11.9% 85.4 6.1%
P < 0.001 89.5 5.8% 38.5 11.5% P < 0.001 32.0 11.8% 87.8 5.8%
P ¼ 0.011 81.4 7.6% 44.6 11.0% P ¼ 0.007 39.7 11.6% 80.6 7.2%
P ¼ 0.065 80.9 7.8% 54.2 11.1% P ¼ 0.060 50.6 12.0% 79.9 7.5%
P ¼ 0.001 75.9 7.6% 37.5 12.1% P ¼ 0.001 30.8 12.8% 74.9 7.4%
P ¼ 0.008 78.8 7.3% 43.8 13.6% P ¼ 0.001 43.3 14.3% 77.2 7.2%
WBC, white blood cell; PLT, platelet; Dx1, diagnosis; MRD, minimal residual disease; NCN, normalized copy number.
was compared between the two groups. There was no significant difference between 5-year DFS and OS in the AML1/ETO-positive group and those in the AML1/ETO-negative group within four courses of chemotherapy. Five-year DFS and OS in the AML1/ETOnegative group were significantly higher than those in the AML1/ ETO-positive group after treatment with four courses of chemotherapy. The patients were divided into three groups according to the RTPCR results. Group I (continuous negative patients) included the patients with consistently disappearance of AML1/ETO fusion transcript. The median number of negative RT-PCR results in Group I was four (range, 3–7). Group II (continuous positive patients) included the patients with persistence of AML1/ETO fusion transcript. The median number of positive results in Group II was four (range, 3–8). Group III (recurrent positive patients) included the patients with at least one positive RT-PCR results after disappearance of AML1/ETO fusion transcript. The median number of checkpoint for Group III was 6.5 (range, 3–9). In our study, 28 Pediatr Blood Cancer DOI 10.1002/pbc
patients (Group I) were consistently negative, 21 patients (Group II) were continuous positive, and 10 patients (Group III) were recurrent positive. The patients in Group I had remarkably better prognosis than those in Group II or III (P < 0.001) (Fig. 1).
RQ-PCR in Different Phases of Treatment and Influence on Survival The median AML1/ETO copy number before treatment in all 70 patients was 30,464.9 (516.8–184,138.1). Pretreatment AML1/ ETO copy numbers did not correlate with age, WBC, or gender. However, copy number at diagnosis was significantly correlated with relapse. The impact of BM AML1/ETO copy numbers and log reduction on relapse risk is shown in Table I. After induction chemotherapy, patients with a >1.8 log reduction had a CIR at 5 years of 14.6 0.8% compared with 67.7 7.1% in those with 3 log reduction relapsed after introduction. We suggest that the AML1/ETO copy number after treatment may reflect the sensitivity of leukemic clones to treatment; thus, MRD monitoring by RQ-PCR might allow us to identify subgroups of pediatric patients who are at low risk of relapse sooner after induction. For patients with an AML1/ETO copy number reduction of less than 1.8 log after induction, MRD should be monitored sequentially so that treatment can be modified early to improve the outcome. It reported that an increased transcript level in patients with morphological CR predicted relapse [10]. We found that a 0.6 log increase in AML1/ETO transcripts during follow-up strongly predicted subsequent morphological relapse. Zhang et al. [26] found that a 0.5 log increase in AML1/ETO transcript during
TABLE III. Comparison of RQ-PCR and RT-PCR Results in Samples According to Therapeutic Phase
10 NCN Total
Post-induction (n ¼ 58)
Post-course 2 (n ¼ 55)
Post-course 3 (n ¼ 53)
Post-courses 4–6 (n ¼ 118)
Out of treatment (n ¼ 48)
POS
NEG
POS
NEG
POS
NEG
POS
NEG
POS
NEG
0 3 52 55
2 1 0 3
0 12 35 47
4 3 1 8
0 5 30 35
11 7 0 18
0 11 46 57
44 15 2 61
0 0 15 15
26 7 0 33
POS, positive; NEG, negative. Pediatr Blood Cancer DOI 10.1002/pbc
Discrepancies (%); total 0/87 33/64 3/18 36/332
(0) (51.6) (16.7) (10.8)
1766
Zhang et al.
follow-up strongly predicted morphological relapse, which was similar to our findings. Notably, three out of 14 patients developed to hematology relapse within 1 month in our study. Therefore, the optimal schedule for pre-emptive therapy in pediatric AML1/ETO AML should be the subject of further research. In conclusion, both RT-PCR and RQ-PCR can be used to detect MRD in pediatric AML1/ETO AML. RQ-PCR can indicate patients who are at a high risk of relapse earlier than can RT-PCR. Moreover, for patients with CR but continuous positive RT-PCR results, RQPCR should be performed more frequently. Although the small number of the patients and this retrospective analysis may not have sufficient power to lead to a conclusion, further clinical research with larger numbers of pediatric patients should be conducted. Also of note, the threshold values identified using ROC analysis may not be optimal and should be validated in subsequent studies.
ACKNOWLEDGMENTS This study was supported in parts by grant from the Tianjin Science and Technology Support Plan (12ZCDZSY18100), the Ministry of Science and Technology Major Projects (2011ZX09302-007), the Youth Scientific Research Funds of Peking Union Medical College (2012J17), and the Natural Science Fund Foundation Project (81200396).
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