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Journal of Clinical Oncology, Vol 24, No 10 (April 1), 2006: pp. 1507-1515 © 2006 American Society of Clinical Oncology. DOI: 10.1200/JCO.2005.03.5303 High WT1 Expression After Induction Therapy Predicts High Risk of Relapse and Death in Pediatric Acute Myeloid Leukemia
From the Laboratoire d'hématologie, Service d'hématologie et d'oncologie pédiatrique, Service de réanimation pédiatrique, and Laboratoire de microbiologie, hôpital Trousseau; Laboratoire de cytogénétique, hôpital Saint-Antoine, Paris; Département d'hématologie et INSERM U524, hôpital Claude Huriez, Lille, France Address reprint requests to Judith Landman-Parker, MD, Service d'hématologie et d'oncologie pédiatrique, hôpital Armand Trousseau, 26 avenue Arnold Netter, 75012 Paris, France; e-mail: judith.landman-parker{at}trs.ap-hop-paris.fr
PURPOSE: To determine whether minimal residual disease (MRD) measured by Wilms' tumor gene 1 (WT1) expression is a prognostic marker in pediatric acute myeloid leukemia (AML), we quantified WT1 transcript by real-time quantitative-polymerase chain reaction in 92 AML at diagnosis and during follow-up. PATIENTS AND METHODS: Patients (median age, 6 years; cytogenetics, favorable 27%, intermediate 59%, poor 13%) were treated between 1995 and 2002 and enrolled in Leucémie aiguë Myéloblastique Enfant (LAME) 89/91, LAME 99 pilot study and Acute Promyelocytic Leukemia French collaborative protocols. With a median follow-up of 26 months, event-free survival was 56% with a standard deviation (SD) of 5% and overall survival of 62.5% with an SD of 6%. WT1 copy number was normalized by TATA box binding protein gene transcripts and expressed as WT1/TBP x 1,000 ratio. Median WT1 ratio in normal patient controls was 12 (range, 0 to 57). A level over two SD than normal bone marrow controls (ie, WT1 ratio > 50), was considered as significant overexpression.
RESULTS: At diagnosis, WT1 overexpression was detected in 78% of patients (72 of 92 patients; median copy ratio, 2231). The WT1 values were significantly higher (P = .01) in favorable cytogenetics and lower (P < .0001) in M5-FAB subtype, 11q23 rearrangements (P < .001), and infants (P = .003) and demonstrate a strong correlation with fusion transcript AML1-ETO, PML-RAR CONCLUSION: WT1 quantification is an informative molecular marker for MRD in pediatric AML and is now performed as prospective analysis in ELAM02 protocol.
Acute myeloid leukemia (AML) is a relatively rare malignancy in the pediatric population, comprising only 15% to 20% of the acute leukemia diagnosed in this age group.1 It remains a challenging disease with an inferior treatment outcome compared with pediatric acute lymphoblastic leukemia. Despite high-dose cytarabine consolidation phase and allogenic bone marrow (BM) transplantation, about 40% of the children with AML still died of their disease.2-4 In chronic myeloid leukemia and acute lymphoblastic leukemia, quantification of minimal residual disease (MRD) have provided useful data in predicting risk outcome and, therefore, is now an important parameter for treatment strategies.5-6 In AML, limited MRD data have been reported in adults, and few of them concerning pediatric cohorts of patients.7-9 Therefore, detection of MRD has become an essential tool for molecular monitoring of pediatric AML to improve patient decisions and care.
Currently used prognostic indicators include age, cytogenetic findings, WBC count, and the presence or absence of an antecedent hematologic disorder. Of these, cytogenetic findings represent the most important prognostic factor. According to the Medical Research Council's trial, AML can be classified into three prognostic groups: favorable, including t(15;17; PML-RAR At a molecular level, fusion transcript genes in pediatric AML represent 31% to 45% of all cases. Among them, MLL/11q23 abnormalities represent the major part with more than 30 different partners' genes; therefore, in most cases, fusion transcript expression data are not available. Fusion genes associated with CBF leukemia's are rare in young patients (15% to 18%).11-12 By contrast, the Wilms' tumor gene WT1, known as a tumor repressor gene, is found to be overexpressed at diagnosis in 70% to 80% of AML cases in adults13-17 and children.7-18 Several studies on WT1 as a molecular marker for MRD in AML have been published and results analyzed at different time points during postremission phase, after allogeneic BM transplantation, or autologous graft have been conflicting.14,16,19-20 However, the frequent overexpression of WT1 gene expression in AML creates an attractive tool for monitoring MRD quantification. In this study, we decided to assess WT1 expression by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) at diagnosis and after induction therapy in a cohort of 92 children with de novo AML to precise the impact of MRD value on survival and relapse. We first determined the positive range of WT1 transcript levels in normal BM samples. We investigated the prognostic value of WT1 expression level at diagnosis and its correlation with cytogenetic, clinical, and hematologic characteristics. Furthermore, we correlated WT1 expression during follow-up with fusion transcript expression, when possible.
Patients and Samples Patients (n = 92) were children with de novo AML diagnosed at Trousseau Hospital (Paris, France) and Claude Huriez Hospital (Lille, France) from March 1993 to January 2002. Control BM samples (n = 20) and peripheral blood (PB) samples (n = 10) were healthy volunteer donors (age range, 4 to 20 years). Informed consent was obtained from parents, guardians, or patients. Mononuclear cells from samples were prospectively isolated by density gradient centrifugation and cryopreserved in liquid nitrogen at the time of diagnosis (n = 92), at the end of induction therapy (35 to 50 days after diagnosis; n = 36), at 3 to 5 months (n = 25), at 6 to 8 months (n = 6), and at relapse (n = 11). Patients were enrolled in the molecular study on the basis of available cryopreserved samples at diagnosis. The diagnosis and classification of AML were based on morphologic, cytochemical, and immunophenotypic criteria according to the revised French-American-British classification. The patients were treated according to the French multicenter protocols Leucémie aiguë Myéloblastique Enfant (LAME) 89/91, LAME 99/01 pilot study and patient cases with AML3 according to acute promyelocytic leukemia’s protocols.3,21 A common induction treatment by cytarabine 200 mg/m2/d in continuous infusion for 7 days and mitoxantrone 12 mg/m2/d for 5 days has been done in the two LAME protocols. In these strategies, allogenic geno-identical BM transplant was indicated in first complete remission (CR1) or if no related donor was available. Patients received consolidation by high-doses cytarabine after induction and consolidation phases with a cumulative dose of 8 and 24 g/m2 in the LAME 89/91 and LAME 99/01, respectively. Infants were treated according to the same induction modalities with autologous BM transplant proposed in CR1 with busulfan, etoposide, and melphalan conditioning. Since 1996, maintenance treatment was no longer indicated as a result of LAME 89/91 randomized maintenance study. The clinical and hematologic characteristics of the 92 children enrolled in this analysis are presented in Table 1.
RNA Isolation, cDNA Synthesis, and RT-PCR Total RNA was extracted from the samples using RNAble (Eurobio, Les Ulis, France), according to the manufacturer's instructions. Then 1 µg of RNA was transcribed into cDNA using Superscript II (Invitrogen, Life Technologies, Cergy Pontoise, France) and random hexamers in a 20 µL reaction under standard conditions. One aliquot of one-tenth of the resulting reaction was used for quantitative PCR amplification. RT-PCR reaction was performed with the Light Cycler System (Hoffman-Roche, Penzberg, Germany), using 20 µL mix containing 100 ng cDNA sample; 3 mmol/L MgCl2; 400 nmol/L forward and reverse primer; 250 nmol/L probe, labeled at the 5' end with the reporter dye molecule 6-carboxy-fluorescein phosphoramidite (FAM) and at the 3' end with the quencher dye molecule TAMRA (6-carboxy-tetramethyl-rhodamine); 2 µL 1x LC FastStart master mix (Roche Diagnostics, Meylan, France); QSP water. The thermal cycling conditions included 10 minutes at 95°C followed by 50 cycles of denaturation for 15 seconds at 95°C and annealing/extension at 60°C for 60 seconds. Experiments were performed in duplicate for each data point. Each PCR run included the standard curve, a control without RT and a control without template. All the quality assessments were adapted from the BIOMED 1 protocol and the European SANCO concerted action.22
Primers and Probes
Quantitative Assessment of WT1
Experiments were performed in duplicate for each data point. The WT1 transcripts values obtained by RT-PCR were normalized with respect to the number of TBP transcripts and expressed as WT1 copy numbers every 103 copies of TBP (WT1/TBP x1,000). WT1 quantification by RT-PCR was validated by analysis on leukemia's cell lines, a pool of normal PB (n = 10) and BM from healthy donors (n = 20)
Quantitative Assessment of Fusion-Gene Transcripts in AML
Cytogenetics and Fluorescent In Situ Hybridization Studies
Statistical Methods
WT1 Expression in Normal Controls As also shown in previous study, the WT1 levels are detectable at low levels in normal samples.14 We tested a set of PB samples (n = 10) and normal BM samples (n = 20) from healthy donors. The median WT1 copy number was, respectively, four (range, five to 15) and 12 (range, zero to 57; Fig 2), corresponding to a value  10–3 dilution of the control cell lines REH and K562. A significant overexpression of WT1 gene was considered when WT1 copy number was over two standard deviation (SD) WT1 median copy number obtained in normal BM controls. This cutoff value was determined at 50 copies.
WT1 Expression at Diagnosis The expression levels of WT1 were investigated by real-time quantitative polymerase chain reaction (RQ-PCR) in BM of 92 children newly diagnosed with AML. Of these 92 patients, 72 patients (78.3%) expressed WT1 with a ratio level above 50 and a median value of 2231 (range, 53 to 429920; Fig 2). The remaining 20 patients (21.7%) had a low level of transcript between one and 49 (median, three), corresponding to levels less than 10-3 in the cell lines REH and K562 and to the baseline transcription of WT1 in normal marrow controls. Clinical data of the 72 patients with high WT1 expression (WT1+) at diagnosis are described in Table 1 and were compared to patients with low WT1 expression (WT1-). The major significant associations were overexpression of WT1 in all CBF leukemia (P = .01) and on the opposite low level of WT1 expression (n = 20) in the FAB M5 patient cases (P < .0001), and significant association with infant patient cases (P = .003) and strong correlation with the presence of MLL gene arrangements (P < .001), suggesting downregulation of WT1 activating pathway in this leukemia subset. There were no significant associations between WT1 level, WBC, intermediate or unfavorable cytogenetic groups, induction failure, relapse with 5 years EFS at 54% with an SD of 11%, 56.4% with an SD of 6%, 56% with an SD of 5%, and death with 5 years OS at 62.5% with an SD of 6%, 64% with an SD of 10%, 62 with an SD of 7% in WT1-, WT1+, and all patients, respectively (Table 1). At diagnosis, there was no evidence that WT1 expression could be used as a prognosis factor.
WT1 Expression After Induction Treatment
At D35-50 detection of WT1 transcript above 50 normalized copies (cutoff value) was found in nine of 36 assessed patient cases (25%), including three patients with residual blast cells in BM. The other six patients reached a morphologic CR1 but relapsed, respectively, at 9, 9, 13, 14, 15, and 28 months compared with seven of 27 patients who relapsed in CR1 with low levels of WT1 expression. Clinical characteristics of these six patients were as follows: three patients (age, 10 years, 4 years, 6 years) AML7 with, respectively, 46XY, 46XX,-7,+8, and 46 XY t(7;16;14) (p11;q12;q 23) karyotype, one patient (age, 10 years) AML4 with 46XX+8, one patient (age, 3 years) AML3 with PML-RAR  transcript, and one patient AML5 without available cytogenetic analysis. Of note three of six patients had no remarkable adverse cytogenetic prognostic factor, initially. At D35-50, after induction therapy, the risk of relapse in patients with high expression of WT1 was significant (P < .001). At 3 to 5 months, high WT1 levels were detected in five of 25 patients: (1) three patients were in CR1 and one of them relapsed, (2) two other patients had refractory disease and died without achieving CR, and (3) the remaining 20 assessed patients presented low levels of WT1 transcript and five of 20 relapsed compared with 16 of 44 in the CR1 patients without material valuable for analysis. At 3 to 5 months, after induction therapy, the risk of relapse in patients with high expression of WT1 showed only a tendency (P = .06).
WT1 Expression at Relapse
Relationship Between Expression Levels of WT1 Gene and Fusion Genes As Determined by Quantitative RT-PCR
Statistical Analysis Prognostic factors of events and deaths were analyzed in the cohort (n = 92) and reported in Table 4. In univariate analysis, MRC3 and M7-FAB were associated with adverse outcome. Overexpression of WT1 at the end of induction treatment demonstrated a high risk of relapse or failure in univariate analysis (P < .0001; Table 5). As shown on Figure 4, OS was 0% in those patients compared with 74% 5 years OS, if WT1 ratio was below 50 at the end of induction therapy. In multivariate analysis, taking into account cytogenetic, FAB subtype, and WBC count, WT1 overexpression, at the end of induction therapy, was the only independent factor of adverse outcome in our cohort (Table 5).
Low proportion of available data in patients analyzed at 3 to 5 months made no informative statistical analysis after initial point of follow-up.
Among children with AML, relapses occur in approximately 40% to 50% of patients who achieve remission, and only a minority are long-term survivors after further therapy.2,3,26 Established prognosis factors that affect outcome are: French-American-British classification, cytogenetic findings, early response to induction phase, dose-intensity level of induction and consolidation therapy, allogenic transplant from identical sibling in CR1, and length of CR1 before relapse. However, these different prognostic factors are not always observed in earlier reports in children.5,11-12,27-32 In AML, molecular-based techniques for MRD assessment have recently identified patients at high-risk of relapse in few reports.33-34 In this study, we investigated by RT-PCR WT1 gene expression as a marker for monitoring MRD in pediatric AML. Low levels of WT1 expression have been demonstrated in normal BM and progenitor cells CD34+, CD34+, CD38–.17,35-36 In our study, we defined significant WT1 overexpression when sample value is over two SD WT1 median copy number obtained in normal BM patient controls. This cutoff value corresponds to 50 normalized copies of the gene expression. In our cohort, the gene WT1 is highly expressed in 72% of AML at diagnosis. This value is comparable with the results of three other studies9,14-15 with, respectively, overexpression in 72%, 74%, and 73% of AML. This result confirms that taking together baseline value of WT1 detected in normal BM is comparable in these different studies and seems to be around 10–3 dilution of the standard K562 cDNA.
Our study confirms data by Ostergaard et al9 and demonstrates that leukemia AML1-ETO and PML-RAR
Our analysis in AML1-ETO and PML-RAR Our MRD study is based on an assessment after a first course of identical induction treatment for all patients. To date, this is the largest study in children with AML, regarding WT1 gene expression. Over the 72 WT1 positive patients at diagnosis, only 36 and 25 have been studied at D35-50 and months 3 to 5, respectively, on the basis of available cryopreserved samples at diagnosis with no major statistical difference between analyzed and nonanalyzed groups. However, at this time point WT1 overexpression is associated with a high risk of failure relapse and death with 74% OS in WT1- patients versus 0% in WT1+ patients. This is completely consistent with the initial study of Inoue et al13 in 54 adults with leukemia, including 40 with AML. Nine patients with WT1 levels never returned to normal BM levels and relapsed in all cases. Nevertheless, in this study, timing of assessment was heterogeneous and, for the majority, performed after 6 months of follow-up. These results were extended by the Garg study,16 five of 35 patients with persistent levels of WT1 > 10–3 relapsed within 6 months of follow-up although methodology of assessment was slightly different with PCR competitive assay. Few publications showed a good correlation for MRD detection between WT1 gene expression and flow cytometry analysis7 and between tricolor flow cytometry and risk of relapse and death.8 Nonetheless, as blast cells are heterogenous in antigen expression, flow cytometry sensitivity is still lower than molecular gene expression to date. The future may be different with the development of five- or six-color cytometers. Taking account, these results confirm that WT1 expression is a valuable and important tool for MRD in pediatric AML. The presence of MRD at the end of induction therapy is an extremely powerful adverse prognostic factor, supporting the importance of the induction treatment for long-term survival. Overall, WT1 overexpression during follow-up is highly predictive of relapse and, therefore, may reflect a specific functional state of leukemia residual cells. Moreover, during submission of this report, Weisser et al39 demonstrated, in a cohort of 116 adult patients, significant adverse prognostic value of WT1 overexpression at 2 to 3 and 3 to 6 months of follow-up. A prospective study is actually ongoing in the new French AML collaborative study ELAM02 to define a future adapted treatment strategy on the basis of WT1-expression results.
The authors indicated no potential conflicts of interest.
We thank David Young for editing.
Supported by the Association Pour La Recherche Dans Les Maladies Hématologiques De L'Enfant (ARMHE) and the Ligue Nationale Contre le Cancer (comité de Paris). Presented in part at the Annual Meeting of the French Society of Hematology (SFH) Paris, March 7-9, 2004 and at the American Society of Hematology 46th Annual Meeting, San Diego, California, December 4-7, 2004. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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Copyright © 2006 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
2 test and the Mann-Whitney U test. Event-free survival (EFS) and overall survival (OS) were calculated from the date of up-front randomization until the date of complete remission (CR) induction failure, first relapse, or death in CR. Patients were censored at the time of relapse or at the time of the last follow-up. EFS and OS were estimated by the product-limit method. The significance of differences between curves was estimated by the log-rank test (Cox-Mantel). Factors associated in univariate analysis with a P value under .10 were included in the final models. Multivariate analyses were performed using Cox proportional hazards regression for EFS and survival. Two different analyses were performed according to the selected population
