Abstract
Background Accurate assessment of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) patients after initial chemotherapy is essential to evaluate the efficacy of therapeutic regimens. Wilms tumor 1 (WT1) is a pan-leukemic marker used for identification of the leukemic clone rather than the use of individual specific molecular aberration of ALL.
Methods Using a real-time quantitative polymerase chain reaction, bone marrow samples from 41 newly diagnosed Egyptian ALL patients; 22 adults and 19 children were examined for WT1 expression. After induction therapy, WT1 expression was reestimated in 20 ALL patients.
Results WT1 was overexpressed in adult and pediatric ALL patients (95.4% and 89.4%, respectively). WT1 expression at diagnosis had no statistically significant impact on disease-free survival of patients (P = 0.054). However, WT1 expression increased after induction chemotherapy in the 3 pediatric patients who had relapse.
Conclusions WT1 is a leukemia-associated molecular marker that may be used for the diagnosis and for monitoring clinical progress in ALL; it also can be used as a molecular target for adoptive immunotherapy.
The Wilms tumor 1 gene (WT1) encodes a transcription factor involved in normal and malignant hematopoiesis.1 WT1 gene was first identified in patients with Wilms tumor, located on chromosome 11p13.2Posttranscriptional messenger RNA modifications and the presence of possible several transcription initiation sites give rise to a number of different WT1 protein isoforms (at least 32) that are localized in specific subcellular and subnuclear regions and show different, partially overlapping, but distinct functions.3–5
WT1 regulates the transcription of a variety of target genes and is involved in posttranscriptional messenger RNA processing. It can act as a transcription activator or repressor, depending on the WT1 isoform, interaction with other isoforms, and interaction with other transcription regulators. In this way, WT1 can control proliferation, differentiation, cell cycle, and apoptosis; however, its regulatory properties have not yet been fully understood.6
WT1 is necessary for the normal development of mesothelial tissue including the hemopoietic system. Hemopoietic precursors show transient biphasic WT1 over expression, which has a stage- specific effect; WT1 maintains primitive stem cells in a quiescent state, whereas it promotes differentiation of more mature linage-committed progenitors.7,8
Unlike other tumor suppressor genes, such as Rb and p53, the expression of the WT1 gene is restricted to a limited set of tissues (fetal kidney, ovary, testis, and spleen). Highest expression levels were observed in the developing kidney, thus WT1 expression is strongly regulated in a time- and tissue-specific manner. In normal bone marrow (BM), WT1 is expressed at a very low level by normal primitive CD34 progenitor cells and its level of expression is downregulated with further differentiation.2
Accurate assessment of minimal residual disease (MRD) in ALL patients after initial chemotherapy is essential to evaluate the efficacy of treatment regimens, to establish early diagnosis of impending relapse, and to individualize treatment protocols. Detection of early relapse allow intervention at a more favorable stage than at overt relapse.9However, so far, the applicability of this strategy has been limited to those leukemia subsets characterized by genetic markers amenable to sensitive detection by polymerase chain reaction (PCR) like immunoglobulin and T-cell receptor rearrangement in acute lymphoblastic leukemia (ALL).10A “pan-leukemic” marker such as WT1 could therefore simplify leukemia diagnosis and MRD detection.11
The current study was designed to investigate the WT1 gene expression status in BM of 41 newly diagnosed Egyptian ALL patients. The study also explored the possible clinical application of WT1 as a molecular marker for monitoring the response to chemotherapy and for MRD detection in 20 of them after receiving standard induction chemotherapy protocols to identify the subset of patients who are at high risk of impeding relapse. Furthermore, according to its expression frequency among Egyptian ALL, it can be considered as a molecular marker for adoptive immunotherapy.
MATERIALS AND METHODS
This study included 41 consecutive patients (27 males and 14 females) with newly diagnosed ALL. Twenty of them were studied also after induction therapy. Eight PB and BM samples from nonleukemic patients (idiopathic thrombocytopenia and hypersplensim before splenctomy) were enrolled in the study as a control group. All patients were diagnosed and treated at Kasr El-Aini Teaching Hospital, Faculty of Medicine, Cairo University.
Sampling
Bone marrow samples were obtained for morphologic assessment, cytochemical staining, immunophenotyping, and cytogenetic studies. Formal written consents were obtained from the adult patients or from the caregivers of the participants who agreed to their child’s participation in the study. For each patient, 2 mL of BM aspirate was collected on an EDTA vacutainer for quantitative detection of WT1 transcript using real-time quantitative PCR (RQ-PCR).
For optimal results, BM samples were immediately transported to the laboratory at room temperature. The initial processing of the samples was performed within 4 hours after their collection. Total cellular RNA was extracted from BM samples using QIAamp RNA Blood Mini Kit, following the manufacturer’s instructions (Qiagen cat. no. 52304), and the concentration of extracted RNA was evaluated by spectrophotometry using Nanodrop ND-1000 (Thermo Scientific, Rockford, IL). All samples had an optical density 260/280 nm ratio higher than 1.8, indicating high purity. Complementary DNA (cDNA) synthesis reaction was performed with 2 μg of RNA by reverse transcription using random primers with a high-capacity cDNA archive kit (PN4322171 Applied Biosystem, Foster City, CA) according to the manufacturer’s instructions. The reaction was inactivated by heating at 90°C for 10 minutes. The formed cDNA was stored at −20°C until use.
Detection of WT1 Transcripts
Real-time quantitative PCR for detection and quantification of WT1 was developed according to European Against Cancer protocols, using Quant Kit (PQPP-01). Oligoneucleotide probe consisted of oligoneucleotide labeled with 5′ reporter dye (FAM) and dowenstream, 3′ quencher dye (TAMRA). As a control gene (CG), ABL was detected in samples without BCR-ABL translocation, the RQ-PCR reaction was performed on ABI Prism 7700 TaqMan instrument (Applied Biosystems, Carlsbad, CA). The WT1 (profile gene) and CG copy number and WT1 normalized copy number (NCN = WT1/ABL) were calculated according to the standard curve method. Standard curves were created using plasmid DNA calibrators (Ipsogen, Marseilles, France). In the TaqMan technology, the number of PCR cycles necessary to detect a signal above the threshold is called the cycle threshold (C t) and is directly proportional to the amount of target gene present at the beginning of the reaction. Ipsogen recommends a threshold set at 0.1 on a TaqMan machine to be in the exponential phase, and a baseline set between cycles 3 and 15.
All patients were investigated at the time of diagnosis. After induction chemotherapy, a good quality of cDNA was obtained from 20 patients, mostly due to marked BM hypoplasia secondary to chemotherapy.
Therapeutic Regimen of ALL Patients
All patients received Berlin Frankfurt Munster (BFM 90/95) protocol for treatment of ALL. This protocol depends on risk stratification of patients into standard (SRG), medium (MRG), and high-risk groups (HRG). The main criteria for stratification were the leukemic cell mass estimate, initial response to steroids, presence of T-cell immunophenotype, t(9;22), and CNS involvement.
Follow-up of leukemic patients was carried out for 50 months to study any possible association between the WT1 expression and the response to chemotherapy. The response to treatment was classified as follows:
− Death during induction: death during or after the first course of therapy with aplastic or hypocellular marrow.
− Complete remission (CR): cellular marrow with blast cells of 5% or less, peripheral blood picture shows a neutrophil count of 1.5 × 103/mL or higher, platelet count of 100 × 103/mL or higher, and no evidence of leukemia in other sites.
− Primary resistance: cellular marrow with more than 5% blasts or evidence of leukemia in other sites.
Achievement of CR was considered to be the mark of good prognosis in acute leukemia.12
Statistical Analysis
Data were coded and entered using the Statistical Package (SSPS) version 15. Data were summarized using mean, SD, median, and range for quantitative variables, whereas number and percent were used for qualitative variables. Receiver operator characteristic (ROC) analysis was performed to determine sensitivity and specificity of WT1-NCN at diagnosis in early detection of relapse. The impact of WT1 expression levels at diagnosis on disease-free survival (DFS) was determined by Cox proportional hazards regression model. P ⩽ 0.05 was considered statistically significant.
RESULTS
The current study included 41 ALL patients. Patients were diagnosed and treated at Kasr Al-Aini teaching Hospital, Faculty of Medicine, Cairo University. They were 28 B-ALL and 13 T-ALL cases. Patients were divided into 2 groups according to their age:
Group 1: Adult group (aged >15 years) including 22 patients (53.7%). They were 7 males and 15 females. Fourteen patients were B-ALL, whereas 8 patients were T-ALL.
Group 2: Pediatric group (aged ⩽15 years) including 19 patients (46.3%). They were 7 males and 12 females. Fourteen patients were B-ALL, whereas 5 patients were T-ALL.
Conventional cytogenetic analysis revealed that all the cases were Philadelphia chromosome negative.
WT1 Expression in Normal Controls
WT1 levels were detectable at low levels in normal samples: both PB and BM samples. The median WT1 copy number was 15 × 10−3 in PB (ranged from 0 to 73 × 10−3), and in BM, it was 22 × 10−3 (ranged from 2 to 89 × 10−3). A significant overexpression of WT1 gene was considered when WT1 copy number was more than 2 SD of WT1 median copy number obtained in normal BM controls.13This cutoff value was determined at 59 copies.
WT1 Expression in ALL Patients
WT1 expression level (WT1-NCN) in adult patients at diagnosis ranged from 0.7 to 269 × 10−3, with a median of 100 × 10−3. Only 1 patient had low WT1 expression (below the cutoff value), whereas 95.4% had an overexpression of WT1. WT1-NCN, after induction chemotherapy, ranged from 0.34 to 390 × 10−3, with a median of 239 × 10−3.
WT1-NCN in pediatric patients at diagnosis ranged from 0 to 6890 × 10−3, with a median of 3.3 × 10−3. One patient did not express WT1, whereas 1 patient showed a low expression of WT1. Although 89.4% had overexpression of WT1, WT1-NCN after induction chemotherapy ranged from 0 to 8000 × 10−3, with a median of 580 × 10−3 (Table 1).
Role of WT1-NCN at Diagnosis in Predicting Poor Outcome
Statistical analysis of the results revealed that WT1-NCN was significantly higher in male pediatric patients compared with female pediatric cases (P = 0.048). Otherwise, there was no statistically significant difference between WT1-NCN among ALL patients regarding clinical presentation (fever, bony pains, lymphadenopathy, pallor, weakness, petechiae, and ecchymosis), laboratory data (total leukocyte count, hemoglobin level, platelet count, peripheral blood, and BM blast percentages, FAB classification, whether L1 or L2, or immunophenotyping being B-ALL or T-ALL), or their response to induction chemotherapy.
Receiver operator characteristic analysis was done to determine sensitivity and specificity of WT1-NCN at diagnosis in the early detection of relapse.
In group 1, ROC analysis revealed that the calculated optimum cutoff limit was 210 × 10−3 with 83.3% sensitivity and 68.6% specificity. Thus, WT1-NCN level at diagnosis has a limited value in the detection of poor outcome. WT1 overexpression at diagnosis was not associated with increased incidence of relapse (P = 1).
In group 2, ROC analysis revealed that the calculated optimum cutoff limit was 210 × 10−3 with sensitivity 75% and specificity 46.7%. Thus, WT1-NCN levels above the 0.021 cutoff value at diagnosis raise the incidence of relapse to 46.7%. WT1 overexpression at diagnosis was not associated with an increased incidence of relapse (P = 0.068).
Impact of WT1 Gene Expression at Diagnosis on DFS in ALL Patients
Follow-up of patients was achieved via accessing their files. The duration of follow-up was 50 months. The impact of WT1 expression levels at diagnosis on the DFS was determined by Cox proportional hazards regression model. There was no significant impact on DFS in both adult and pediatric groups (Figs. 1 and 2).
Impact of WT1 Gene Expression (WT1-NCN) After Induction Chemotherapy on Prognosis
After induction chemotherapy, patients were classified into 2 groups: those who either achieved CR or had bad treatment outcome (had relapse or died).
In group 1, 2 adult patients (2/22, 9%) had relapse; both were from the HRG and postinduction WT1 expression analysis was not done because of their cDNA.
In group 2, 4 pediatric patients (4/19, 21%) had poor treatment outcome; 1 died during induction chemotherapy (she was 6-month-old neonate and she was from the HRG) and 3 patients had relapse (1 was from the HRG and 2 were from the MRG). All the 3 pediatric patients who had relapsed had a higher postinduction WT1 expression than that detected at the time of diagnosis (3.2 × 10−3, 12 × 10−3, and 1450 × 10−3 at diagnosis; 25 × 10−3, 104 × 10−3, and 8000 × 10−3 after induction, respectively).
DISCUSSION
A number of studies have been performed in an attempt to identify cytogenetic and molecular abnormalities associated with leukemic transformation to detect clonality markers suitable for MRD monitoring. The quantitative assessment of WT1 transcript may be used as a molecular marker for the detection of the leukemic clone useful for monitoring the presence of MRD in all the patients affected by acute and chronic leukemias.14
In this work, WT1 expression in ALL patients at diagnosis and after induction chemotherapy was evaluated to identify the subset of patients who are at high risk of early relapse. This could help to clarify the prognostic relevance of WT1 expression at diagnosis and early follow-up. Twenty-one adult ALL patients (95.4%) and 17 pediatric ALL patients (89.4%) enrolled in this study showed WT1 overexpression at diagnosis. In agreement with our findings, Miwa et al.15reported an overexpression of WT1 in 86% of newly diagnosed ALL patients, whereas Chiusa et al.16detected an overexpression of WT1 gene in all ALL examined samples (100%). Consistent with our results, the study of Chen et al.17reported overexpression of more than 90% of WT1 gene in several hematological malignancies including ALL. Moreover, Chen et al.18demonstrated overexpression of the WT1 transcript in 82.1% of childhood ALL at diagnosis. Regarding the strength of expression of WT1 gene among ALL patients, they proved not only that the WT1 transcript level varies widely in childhood ALL but also that the high expression of the WT1 transcript at presentation occurs in only a minority of patients.
On the contrary, Menssen et al.19detected WT1 expression in only 44% of newly diagnosed ALL patients by conventional reverse transcriptase-PCR. This controversy may be attributed to the higher sensitivity and specificity of the RQ-PCR technique that was used in our study. Also, Spanaki et al.2demonstrated that significant levels of WT1 were expressed in 35.7% of children with newly diagnosed acute leukemia. This may be attributed to ethnic difference or different sample sizes included in these studies.
Regarding the immunophenotypic subtypes of ALL and WT1 expression in our patients, no difference between T-ALL and B-ALL was found regarding WT1 expression both in adult and pediatric groups. Most of the studies did not distinguish between B-ALL and T-ALL; in those analyzing these subtypes separately, controversial results were found, with higher WT1 overexpression detected in B-ALL in some of them20and in T-ALL in others.21However, these studies investigated WT1 mostly in adult ALL patients or in heterogenous groups of children and adults, in PB samples, using potentially less sensitive PCR techniques for WT1 detection.22,23In contrast to our finding, a study carried out by Chen et al.18demonstrated that in children with T-ALL, WT1 level at diagnosis seems to be higher than that in children with B-ALL and was in no case lower than the reference limit.
In the present work, patients were followed up for 50 months, and the influence of WT1 expression levels at diagnosis on the DFS was determined. There was no statistically significant impact of the gene’s overexpression on DFS either in adult or in pediatric patients. This comes consistent with the studies of Magyarosy et al.,24Imashuku et al.,25and Boublikova et al.,6who reported that higher levels of WT1 gene expression at diagnosis were not associated with shorter DFS.
However, Inoue et al.26reported a significant correlation between low WT1 expression at diagnosis and higher CR rate and longer survival. Also, Chiusa et al.16clearly demonstrated the high prognostic value of the amount of WT1 expressed in adult ALL at presentation: patients with high WT1 expression had shorter DFS and overall survival.
In addition, Spanaki et al.2demonstrated that a high WT1 expression could correlate with an unfavorable outcome in acute leukemia at childhood, and WT1 expression was the most significant independent prognostic factor in the multivariate analysis.
In our study, MRD detection after induction therapy was done for 20 patients using WT1 detection. Among the 3 pediatric patients who had relapse, WT1 expression was higher after induction than at the time of diagnosis. So, quantitative determination of WT1 transcript levels after induction therapy may contribute to an effective monitoring of MRD and in predicting early relapse in pediatric ALL. In fact, the role of WT1 as a marker of MRD has been validated by many investigators. Our results are consistent with those of Imashuku et al.25who showed that WT1 detection after induction is an independent prognostic risk factor of relapse and death.
Cilloni and Saglio27studied a number of patients bearing a fusion gene transcript suitable for the quantitative assessment of the amount of MRD by RQ-PCR and performed a simultaneous analysis of the amount of WT1 at sequential time intervals during the follow-up. The WT1 levels were shown to be strictly parallel to the behavior of the other molecular markers (fusion gene transcripts) used for MRD monitoring.
In 2010, the study of Heesch et al.1reported that WT1 overexpression after induction and consolidation chemotherapy was associated with shorter overall survival, and hence, WT1 expression analysis could be a useful tool for MRD monitoring in acute leukemia. Moreover, Kang Lim et al.28demonstrated that WT1 gene might be used not only as a potential molecular marker for diagnosis and for monitoring the clinical progress and response to treatment but also as a target for the development of novel therapeutic approaches.
WT1 is now regarded as a molecular target for immunotherapy for many cancers. The oncogenic properties of the WT1 gene have recently been reported in various malignancies.29Peptides from WT1, upregulated in many hematopoietic and solid tumors, can be recognized by T cells, and numerous efforts are underway to engineer WT1-based cancer vaccines.30Clinical trials of WT1 peptide-based immunotherapy for cancer have already been started, and WT1 peptide vaccination has been shown to be safe and clearly effective against several types of adulthood leukemia and solid cancers.31
In conclusion, a single assessment of WT1 expression at diagnosis may be of limited prognostic value, and it has no obvious impact on DFS. But still, quantitative assessment of WT1 transcripts could be a useful molecular marker for MRD detection in leukemic patients being a pan-leukemic marker. Moreover, WT1 could be a molecular target for cancer immunotherapy.