Expression of Wilms’ Tumor Gene 1 and Its Clinical Significance in Children with Acute Lymphocytic Leukemia

Expression of Wilms’ Tumor Gene 1 and Its Clinical Significance in Children with Acute Lymphocytic Leukemia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research article Expression of Wilms’ Tumor Gene 1 and Its Clinical Significance in Children with Acute Lymphocytic Leukemia Mengmeng Yin, Aiguo Liu, Ai Zhang, Yaqin Wang, Qun Hu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-105168/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Wilms’ Tumor Gene 1 (WT1) is a potential valuable parameter in prognosis of childhood acute lymphoblastic leukemia (ALL). However, studies on prevalence of WT1 and its correlation to clinical features and prognosis in pediatric patients were not well done. In this study we attempted to identify the correlation between WT1 and childhood ALL. Methods: The expression levels of WT1 in bone marrow cells of 188 children diagnosed with ALL from 2015 to 2018 were detected using real-time quantitative polymerase chain reaction (RQ-PCR). The relationship between expression levels of WT1 and patients’ characteristics, remission status (complete remission/relapse), fusion genes and prognosis of childhood ALL were analyzed and revealed. Results: 1. 147 (78.2%) cases had positive WT1 expression, and the average level was 1.76 (0.3, 6.03) %. 2. The CR and relapse rates of ALL children with positive WT1 were not significantly different from those of WT1 negative group, respectively (87.76% vs 82.93%, P =0.42 and 14.29% vs 17.1%, P =0.658). 3. The WT1 expression level in patients at CR was significantly lower than when at diagnosis ( P <0.001) and the expression of WT1 increased obviously after induction therapy in 21 patients who relapsed ( P =0.003) .4. The WT1 expression was related to lymphadenectasis ( P =0.004) and immunophenotyping ( P =0.009), but not to fusion genes ( P =0.912). Conclusion: The WT1 i n ALL children can be employed as an independent tool to evaluate the prognosis and curative effect of the disease. Pediatrics Acute Lymphoblastic Leukemia Children Wilms’ tumor 1 gene Clinical significance Figures Figure 1 Figure 2 1. Background Wilms’ tumor gene 1 ( WT1 ) located on human chromosome 11p13 was isolated from nephroblastoma in children in 1990 [1]. As a transcription factor encoded by tumor suppressor genes that can regulate cell growth, it is closely related to the occurrence and development of Wilms’ tumors. The WT1 encodes a zinc finger transcription factor. Posttranscriptional mRNA modification and the existence of several possible transcriptional initiation sites produce many different WT1 protein isomers (at least 32), which are located in specific subcellular and subnuclear regions and have different functions, some of which are partially overlapping. WT1 modulates the transcription of various target genes and participates in the processing of posttranscriptional mRNA. It has bidirectional transcriptional regulation and participates in the regulation of hematopoietic transcription [2]. WT1 plays a major carcinogenic role in malignant hematological tumors, inhibiting the function of transcription factors and hindering the normal proliferation and differentiation of hematopoietic stem cells. At the same time, many factors can lead to mutation of WT1 , rendering the WT1 protein unable to regulate specific DNA and thus promoting the growth of tumor cells [3]. There is a significant correlation between the expression of WT1 and the poor prognosis of malignant tumors in the human hematopoietic system. Although WT1 is not specific to acute malignant hematological diseases, its continuous expression can be detected in almost all leukemia cells. Several studies have demonstrated that WT1 was over-expressed in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML), and it was highly expressed in more than 80% of ALL patients in bone marrow (BM) cells [4]. Therefore, WT1 was considered to be a new "pan-leukemia" gene that involved in the proliferation and differentiation of leukemia cells. The quantitative assessment of WT1 in acute leukemia could represent a universal molecular marker of malignant hematopoiesis and was suggested to monitor minimal residual disease (MRD) and to predict the progression and prognosis of disease [5]. The biological functions of WT1 in childhood ALL are not understood clearly and completely, but it has been suggested that WT1 can impact the pathogenesis of leukemia during cellular proliferation and differentiation. In this paper, we analyzed WT1 expression level in ALL children to reveal the predictive role of WT1 as an MRD marker in this group of patients. 2. Methods 2.1 Patients, treatment and follow-up A total of 188 children newly diagnosed with ALL between 2015 and 2018 in our hospital were enrolled. We collected all the patients’ characteristics and measured the expression levels of WT1 at diagnosis and during treatment. All patients were followed up as of October 2020. A cohort of the 188 patients with ALL were consisted of 118 males (62.8%) and 70 females (37.2%), with a median age of 3.2 years. In accordance with WHO leukemia diagnostic criteria，all the patients were diagnosed by morphology combined with immunology, cytogenetics and molecular biology (MICM) of bone marrow cells. Patients’ treatment was given based on CCCG (Chinese Children Cancer Group) –ALL 2015 regimen. MRD testing is used to detect the proportion of remaining leukemia cells in bone marrow at the end of induction chemotherapy (IC). A cut-off point for MRD testing by flow cytometry (FCM) is defined at the level of 0.01%. Thus, MRD lower than 0.01% means complete remission (CR). 2.2 Morpholog y Morphologic criteria were established based on French-American-British (FAB), WHO 2004 or WHO 2008 classification. 2.3 Immunophenotyping Immunophenotyping was conducted by FCM, using monoclonal antibodies to determine lineage-specific antigens, and the panel of tests included at least the following: CD10, CD19, CD20, CD23, cCD79a, SmIg for B-ALL or CD1, CD2, CD3, CD4, CD5, CD7, CD8, TdT for T-ALL. 2.4 Cytogenetics Chromosome banding of BM samples was performed using R-banding or G-banding technique with short-term cultures. The reverse-transcription polymerase chain reaction (RT-PCR) or fluorescence in situ hybridization (FISH) was performed to detect common fusion genes such as TEL-AML1 , E 2A (also called TCF3 )- PBX1 , BCR-ABL , and MLL (also called KMT2A )-related rearrangements. The real-time quantitative PCR (RQ-PCR) was applied to measure the WT1 expression level in BM samples from the 188 ALL children. We cloned both WT1 and ABL genes for creating a standard curve and calculated copy numbers of WT1 gene in these patients. RNA extraction: 4ml bone marrow was collected from the patients and put into 2% EDTA for anticoagulation. Mononuclear cells were separated by Ficoll lymphocyte isolate solution. After washing the cells with PBS, 1 mL Trizol (invitrogen) was added, blown evenly and stored in -80℃ until the RNA was extracted. The extraction of total RNA was carried out according to the instructions of Trizol reagent. The A 260nm /A 280nm ratio was determined by ultraviolet spectrophotometer to identify the purity and quantity of RNA and then frozen under -80℃.The extracted RNA were mixed with RNase-free water and dissolved, and the 15 mL treatment fluid was used as the PCR reaction template. Real-time quantitative PCR: PCR method was used to detect WT1 gene mRNA. The instrument was a ABI7500 fluorescence detector, the reagent was derived from the tumor-related WT1 gene detection kit produced by Shanghai Yuanqi Biotechnology Co. Ltd. The steps were operated according to the operating instructions of the kit. The reaction conditions were 42℃ for 30 min, 94℃ for 5 min, 94℃ denaturation for 15 s, 60℃ annealing for 30 cycles. The reaction system was 25μL. The fluorescence signal was collected at 60℃ in the second step of PCR cycle. Calculation of WT1 gene expression level: Four successful serial dilutions (1×10 6 , 1×10 5 , 1×10 4 and 1×10 3 copies) of each gene were prepared. The copy number of Abelson proto-oncogene ( ABL ) and WT1 gene were calculated according to the standard curve by ABL expression as the internal reference. When 1×10 2 copies≤ WT1 RNA detection concentration≤1×10 7 copies and 1×10 2 copies≤ ABL RNA detection concentration≤1×10 7 copies, the WT1 expression level = ( WT1 copy numbers/ ABL copy numbers)× 100%. 2.5 Statistical analyses Statistical analysis was carried out using SPSS software version 23.0 (IBM Corporation, Armonk, USA), and the count data were expressed as percentage or constituent ratios. Parametric and nonparametric tests including Mann–Whitney, Chi-square test，Student's t test and ANOVA were applied according to data distribution. Experiment data were described in the form of mean ± standard deviation or median (25th, 75th) in accordance with data distribution in our study and P < 0.05 was considered statistically significant. 3. Results 3.1 WT1 expression in ALL children Among the 188 children with ALL, 147 were tested positive for WT1 , 41 were negative, as the positive rate was 78.2%. The average expression level was 1.76 (0.3，6.03) % in WT1 positive group. We compared WT1 expression level in different patients’ characteristics and found that it was only related to lymphadenectasis ( P =0.004) (Table 1). 3.2 Association between WT1 and immunophenotyping In our study cohort, there were 22 children with T-ALL, of whom 17 (77.27%) with WT1 positive, and 78.31% (130/166) of B-ALL children were WT1 positive. There was no significant difference in the positive rate of WT1 between the two groups (77.27% vs 78.31%, P =0.912), but expression level of WT1 in T-ALL children [7.4(1.03, 18.91) %] was obviously higher than that in B-ALL children [1.61(0.23, 4.84) %] ( P =0.009). 3.3 Association between WT1 and fusion gen es In our group, a total of 62 cases had fusion genes including TEL-AML1 , BCR-ABL1 , E2A-PBX1 , MLL rearrangements and SIL-TAL1 . 88.9% (32/36) of children with TEL-AML1 were positive for WT1 gene. The incidence of WT1in BCR-ABL1 , E2A-PBX1 and MLL rearrangements group was 87.5% (7/8), 88.9% (8/9) and 85.7% (6/7), respectively. The WT1 was positive in both of the children with SIL-TAL1 . There were 55 cases carried WT1 and other fusion genes simultaneously, the other 7 cases with single fusion gene. There was no significant difference in expression of WT1 among ALL children carried the above five fusion genes when at diagnosis and at CR, respectively ( P = 0.415, P = 0.861) (Table 2). 3.4 Comparison of CR and relapse rate s in different groups There were 129 children achieved CR in the WT1 positive group and 34 children achieved CR in the WT1 negative group. The CR rates of WT1 gene-positive group and gene-negative group after induction chemotherapy were 87.76% and 82.93%, respectively ( P =0.42). By October 2020, there were 21 children relapsed in the positive group and 7 children relapsed in the negative group, with relapse rates of 14.29% and 17.1%, respectively ( P = 0.658). The CR and relapse rates of two groups both had no significant difference. 3.5 E xpression of WT1 during the course of disease The expression of WT1 in the 147 positively expressed ALL children was monitored after chemotherapy. Among the 129 children with CR after IC, the expression level of WT1 decreased in 109 cases (84.5%, 109/129) and increased in 20 cases (15.5%, 20/129). The expression of WT1 between at diagnosis and CR had a significant difference ( P < 0.001). The patients at CR showed a significantly lower expression of WT1 than at diagnosis. However, WT1 expression of 18 children who didn’t get CR had no significant difference between at diagnosis and after IC (Table 3, Figure 1). Finally, 14 children (12.84%, 14/109) relapsed in the 109 children with decreased WT1 expression and 4 relapsed in 20 ones with increased WT1 expression (20%, 4/20).There was no significant difference (12.84% vs 20%, P =0.47), meaning risk of relapse is not correlated on initial WT expression levels. 3.6 Changes of WT1 expression in ALL children after relapse Up to the follow-up time, there were 21 children relapsed in WT1 gene-positive group finally, and 18 of whom achieved CR after IC (Table 4). Among the 21 relapsed patients with positive WT1 , their WT1 expression after relapse [2.28（0.25,1.69）%] showed higher than that after IC [0.33（0.045,0.66）%] ( P =0.003) (Figure 2). 4. Discussion The innovation of chemotherapeutic drugs and allogeneic hematopoietic stem cell transplantation (allo-HSCT) can significantly improve the remission and survival rates of children with ALL, but the relapse after tumor remission is still a difficult problem [6]. The results of MRD at the end of induction phase of chemotherapy in BM testing are used to reclassify risk levels of ALL patients and guide changes in treatment according to their future risk of relapse. The timely monitoring of MRD in ALL children can provide them individualized and intensified treatment [7]. An enormous amount of literature suggested that high expression of WT1 before and after allo-HSCT is a poor prognosis factor. It is of clinical practical value to use WT1 as a transplant recommendation indicator for ALL patients and as a marker to monitor MRD dynamically. Specific marker genes of ALL, such as TEL/AML1 , BCR/ABL1 and E2A/PBX1 can be monitored. Although the altered WT1 expression seems inherent in leukemia cells, the molecular mechanisms by which WT1 contribute to leukemogenesis have not yet been fully elucidated. The role of WT1 dosage in steady-state and pathologic hematopoiesis needs to be investigated by further studies [8]. The literature reported the positive rate of WT1 in ALL children was more than 80%, and in our study the positive rate was 78.2% which was consistent with that reported in the literature. By FCM, BM samples can give a sufficient sensitivity in the vast majority of patients. WT1 expression can be used as a panleukemic marker and is also a good marker for ALL patients with no other molecular or cytogenetic abnormalities [9]. Since the majority of ALL children expressed very high values of WT1 at diagnosis, the detection of WT1 can be considered a tool of value to monitor the persistence of the disease after chemotherapy or BM transplantation or during the treatment [10]. We collected all patient characteristics including age, sex, chromosome and so on to analyze whether there was an association between WT1 and them. Among all characteristics, we only found lymphadenectasis was related to WT1 in childhood ALL. The phenomenon of lymphadenectasis happened in ALL children without WT1 expression was more common than those with WT1 (53.66% vs 29.25%, P =0.004). T-ALL is biologically distinct from B-ALL and differences in response to chemotherapy have been observed. The prognosis of children with T-ALL is worse than those with B-ALL, which is caused by different fusion transcripts and prognostic factors had been investigated extensively. To determine whether immunophenotyping of ALL can impact WT1 expression, we compared expression of WT1 in T-ALL and B-ALL children. Later findings demonstrated that WT1 expression upregulated in T-ALL due to the continuous activation of NF-KB pathway, which meanwhile has recognized as a vital role in the mechanism of refractory relapse in T-ALL children [11]. In our present study, incidence of WT1 in T-ALL and B-ALL children did not differ, but expression level of WT1 in T-ALL children was significantly higher than that in B-ALL children. Considering this result, we can put forward a hypothesis that WT1 involved in the progression and relapse of T-ALL and contributed to diverse prognosis in different immunophenotyping of ALL. Bordin et al. reported that loss-of-function mutations and deletions in WT1 emerged in approximately 10% of T-ALL children. Clinically, WT1 mutations are enriched in relapsed series and play a critical role in the response to DNA damage in T-cell leukemia. Some studies based on WT1 positively affecting the expression of the X-linked inhibitor of apoptosis protein (XIAP) to restore sensitivity to γ-radiation therapy supported the view that anti-XIAP targeted therapies might be beneficial to treatment of T-ALL patients with WT1 [12]. We carried out a study which compared the WT1 expression between individuals positive for fusion genes including TEL-AML1 , BCR-ABL1 , E2A-PBX1 , MLL rearrangements and SIL-TAL1 . Through the statistical data, we found WT1 and other fusion genes coexisting in ALL children were common. This was also in accordance with the theory that WT1 presented in majority of ALL children. The various fusion transcripts have been confirmed to be highly predictive of clinical outcomes and play different roles in the prognosis of ALL. For example, the Philadelphia chromosome-positive (Ph+) ALL is defined by the t (9; 22) (q34; q11) translocation that forms the fusion gene BCR-ABL1 , which produces an abnormal protein, contributing to the sustaining activation of tyrosine kinase. It predicts poor clinical outcomes due to its resistance to chemotherapy and high relapse risk. Several researches suggested that BCR-ABL1 fusion protein may prompt WT1 expression by activating some signaling pathways, and decrease the sensitivity to chemotherapeutic drugs [13]. Svensson et al. presented that BCR-ABL1 fusion protein could increase the expression of WT1 mRNA and protein via the phosphatidylinositol-3 kinase (PI3K)-Akt pathway. Their experiment results indicated that WT1 expression was induced by oncogenic signaling from BCR-ABL1 and that WT1 contributed to resistance against apoptosis induced by imatinib[14]. The t (12; 21) (p13;q22) producing TEL-AML1 is the most common reciprocal translocation in childhood B-ALL. It is associated with favorable prognosis following conventional therapeutic strategies. But the interaction between TEL-AML1 fusion protein and WT1 is not certain. Similarly, the role of WT1 in other fusion transcripts is still unclear. Qin et al. reported that WT1 expression was obviously different among diverse cytogenetics groups ( TEL-AML1 , E2A-PBX1 and MLL rearrangements) [15]. However, there was no difference in expression level of WT1 among these five different fusion transcripts groups when at diagnosis and CR in our study. The discrepancy might be caused by the small number of patients and different detection methods. Among the 129 ALL children having achieved CR, 109 (84.5%, 109/129) got their WT1 expression descended or negative, 20 (15.5%, 20/129) got elevated in WT1 expression conversely. Among the 18 relapsed children with WT1 , the expression of WT1 decreased in 14 cases and increased in 4 cases after CR. Moreover, the expression levels of WT1 after relapse were all higher than those after IC. These results were in agreement with literature data [16]. The clinical value of altered WT1 expression in response to chemotherapy, guiding therapeutic intervention and predicting relapse is still not determined, and the conclusions of different reports are inconsistent. Some researches have found that WT1 was over-expressed in both peripheral blood (PB) and BM cells of the majority of ALL children, at a level that enabled a significantly more sensitive evaluation of residual disease than standard immunophenotypic and morphological analyses. Meantime, WT1 could have abnormally high expression in malignant cells compared with normal controls, which might be a candidate for MRD monitoring. Several studies confirmed that the expression of WT1 in ALL could cause resistance of cells to differentiation and apoptosis, and it might lead to poor clinical outcomes. For example, it has been reported that expression level of WT1 after T-ALL transplantation was significantly negatively correlated with relapse risk [17]. Based on the information contained in other studies, they thought the expression of WT1 in ALL children is so variable that cannot be accurately calculated as MRD during treatment. Because of the inaccuracy of the assessment, WT1 cannot evaluate the prognosis of ALL [2]. While Inoue et al compared CR rate of ALL children in WT1 gene-positive group and gene-negative group, it revealed a significant difference [18]. However, Chiusa et al. reported that no correlation was observed between WT1 and therapeutic effect of leukemia [19]. In our group, the CR rate of WT1 gene-positive patients (87.76%) and gene-negative patients (82.93%) did not statistically differ. The interaction between therapy response and WT1 is still required more studies to be established. The presence of MRD following therapy for ALL has been demonstrated to be a crucial predictor of relapse in many current studies. MRD typically detected by FCM is on the base of leukemic cells expressing combinations of antigens that are different from those exist in normal BM cells. Particularly in ALL children, the value of MRD must be balanced against other well-established prognostic indexes, but its correlation with other prognostic indexes has not been fully assessed [20]. In this analysis, we found 14.29% (21/147) in patients with WT1 and 17.1% (7/41) without WT1 relapsed after therapy. The relapse rate in these two groups was not significantly altered. The small number of cases and short follow-up time may lead to the lack of significant difference between the two groups. Some studies have found that there was a horizontal relationship between expression level of WT1 and progression of ALL, which would increase, decrease and then increase corresponding to the stage of disease diagnosis, remission and relapse, respectively. A similar trend was observed in our study. Significant decrease in WT1 expression was observed in 129 patients after CR compared with those at diagnosis. That means it can indict the patients’ therapeutic effect. Meanwhile, a significant increase in WT1 expression was witnessed in ALL patients after relapse. 21 relapsed children got their WT1 over-expressed which accorded with some literature documents [5]. From the results obtained so far, it seems that prognosis in ALL children is inversely associated with the expression of WT1 . There are several limitations in our study. First, as there were not enough patients, especially for some fusion genes group, patients are so few that could not be compared comprehensively. Second, a little shorter follow-up time may leave out some relapsed patients. 5. Conclusion From the above discussion, the conclusion can be reached that WT1 can be used as a suitable marker to monitor MRD, assess response to chemotherapy and predict relapse in the majority of ALL children. Adding to the patients carrying a fusion gene, we can also use RQ‐PCR technology to detect WT1 to assess disease risk. By employing such methods, we can determine the effect of induction chemotherapy and state in disease after therapy. Collectively, our study results support the use of WT1 as a marker to monitor childhood ALL progression dynamically. Abbreviations WT1 : Wilms’ Tumor Gene 1 ALL: acute lymphoblastic leukemia RT-PCR: reverse-transcription polymerase chain reaction RQ-PCR: real-time quantitative polymerase chain reaction MRD: minimal residual disease AML: acute myeloid leukemia CML: chronic myeloid leukemia BM: bone marrow MICM: morphology, immunology, cytogenetics and molecular biology CCCG: Chinese Children Cancer Group FAB: French-American-British IC: induction chemotherapy FISH: fluorescence in situ hybridization FCM: flow cytometry ABL : Abelson proto-oncogene PB: peripheral blood Declarations Acknowledgements Not applicable Authors’ contributions Conception and design: QH and MM. Data analysis and interpretation: MM. Writing, review, and revision of the manuscript: QH and MM. Collection and assembly of data: all authors. The authors read and approved the final manuscript. Funding No funding. Availability of data and materials The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate This study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice. This study was approved by the Ethical review committee of Tongji Medical College, Huazhong University of Science and Technology. The reference number: S207. All written informed consent was obtained from a parent or guardian for participants under 16 years old. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References Lee, S.B. and D.A. Haber, Wilms tumor and the WT1 gene. Exp Cell Res, 2001. 264(1): p. 74-99. Boublikova, L., et al., Wilms' tumor gene 1 (WT1) expression in childhood acute lymphoblastic leukemia: a wide range of WT1 expression levels, its impact on prognosis and minimal residual disease monitoring. Leukemia,2006. 20(2): p. 254-63. Toska, E. and S.G. Roberts, Mechanisms of transcriptional regulation by WT1 (Wilms' tumour 1). Biochem J, 2014. 461(1): p. 15-32. Wang, X.R., et al., Overexpressed WT1 exhibits a specific immunophenotype in intermediate and poor cytogenetic risk acute myeloid leukemia. Ann Hematol, 2020. 99(2): p. 215-221. Dou, C.Y., et al., [Relationship between Quantitative Monitoring of WT1 Gene and Prognosis in Patients with Acute Leukemia]. Zhongguo Shi Yan Xue Ye Xue Za Zhi, 2018. 26(6): p. 1632-1636. Jiang, B.Q., et al., [The effect of WT1 expression on the prognosis of allogeneic hematopoietic stem cell transplantation in acute leukemia]. Zhonghua Xue Ye Xue Za Zhi, 2018. 39(12): p. 989-993. 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Bordin, F., et al., WT1 loss attenuates the TP53-induced DNA damage response in T-cell acute lymphoblastic leukemia. Haematologica, 2018. 103(2): p. 266-277. Qin, Y.Z., et al., The prognostic significance of Wilms' tumor gene 1 (WT1) expression at diagnosis in adults with Ph-negative B cell precursor acute lymphoblastic leukemia. Ann Hematol, 2019. 98(11): p. 2551-2559. Svensson, E., et al., Deregulation of the Wilms' tumour gene 1 protein (WT1) by BCR/ABL1 mediates resistance to imatinib in human leukaemia cells. Leukemia, 2007. 21(12): p. 2485-94. Qin, Y.Z., et al., [An interlaboratory comparison study on the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels]. Zhonghua Xue Ye Xue Za Zhi, 2019. 40(11): p. 889-894. Owen, C., J. Fitzgibbon and P. Paschka, The clinical relevance of Wilms Tumour 1 (WT1) gene mutations in acute leukaemia. Hematol Oncol, 2010. 28(1): p. 13-9. 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Patient characteristics Characteristic WT1 (+)（ n =147） WT1 (-)（ n =41） P Sex Male Female Age, years Immunophenotyping T-ALL B-ALL Karyotype Hyperdiploid(46 chromosomes) Liver/splenomegaly (subcostal >5 cm) lymphadenectasis CR after IC Relapse Leukocyte count，×10 9 /L Hemoglobin count, g/L Platelet count, ×10 9 /L Myeloid juvenile cell，% Serum LDH level, IU/L 93 54 5.9（3.3,8） 17 130 37 103 7 21 43 129 21 31.07（4.05,27.94） 77.52±23.6 93.24(31,135) 41.64（10,71） 572.3±133.6 23 18 5.1（3,6.2） 5 36 10 29 2 126 22 34 7 18.62（2.8,18.4） 74.69±24.89 110.7(36,129) 40.02（8.5,69.5） 472.93±124.9 0.404 0.191 0.912 0.995 0.732 0.004* 0.42 0.658 0.242 0.503 0.652 0.765 0.23 Counting data were expressed by number of people, and measurement data were described in the form of mean ± standard deviation or median (25th, 75th) were in accordance with data distribution. ALL: acute lymphoblastic leukemia; CR: complete remission; IC: induction chemotherapy; LDH, lactate dehydrogenase. * P<0.05: statistically significant. Table 2. Changes in the expression level of WT1 in fusion genes CR: complete remission Table 3. WT1 expression level after IC CR: complete remission; IC: induction chemotherapy. * P<0.05: statistically significant. Table 4. Dynamic changes in expression of WT1 in relapsed patients Case Diagnosis (%) After IC (%) Relapse (%) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 2.4 0.91 1.2 0.01 0.14 0.46 2.08 0.17 0.81 0.99 0.75 0.86 0.05 0.9 1.76 0.38 0.06 1.39 1.55 4.7 0.3 0.02 0.19 0.56 0 0.29 0.15 0.18 0.02 0.67 0 0.3 0.15 0.68 1.12 0.15 0.02 0.64 0.07 0.16 0.9 0.67 0.14 0.39 1.94 0.2 0.19 22.82 0.91 0.3 1.43 0.06 0.45 2.7 0.5 4.41 0.3 1.04 7.89 0.31 1.37 0.4 0.06 ALL: acute lymphoblastic leukemia; IC: induction chemotherapy Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-105168","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":4513303,"identity":"78741549-ec77-4afb-a49d-0a27422bf5a8","order_by":0,"name":"Mengmeng Yin","email":"","orcid":"","institution":"Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mengmeng","middleName":"","lastName":"Yin","suffix":""},{"id":4513304,"identity":"ad402946-3a07-4f8e-a599-5808bb1d5afc","order_by":1,"name":"Aiguo Liu","email":"","orcid":"","institution":"Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Aiguo","middleName":"","lastName":"Liu","suffix":""},{"id":4513305,"identity":"cea0a13a-088d-445d-9985-d00e23a28e47","order_by":2,"name":"Ai Zhang","email":"","orcid":"","institution":"Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ai","middleName":"","lastName":"Zhang","suffix":""},{"id":4513306,"identity":"ece82f95-a7a4-49e9-9016-0079e29674ea","order_by":3,"name":"Yaqin Wang","email":"","orcid":"","institution":"Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yaqin","middleName":"","lastName":"Wang","suffix":""},{"id":4513307,"identity":"463184e9-cd76-4a08-95c6-6bbe562fbbf0","order_by":4,"name":"Qun Hu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAx0lEQVRIiWNgGAWjYHACNgaGCgkeefbGxocfiNdyxkbGsOdws7EE0VoY29JsGG6ktwnwEKOe70buscc8bId5GGc+bGOQYLCT020goEXyRl66MQ/PYR526cS2BwUMycZmBwhoMbiRYybNIwG0ZXZiu4EEw4HEbcRpMTjMw3DzYJsED/FaEtJ4GG4wEqlF8swbM8k5B2x4DHsSgYFsQIRf+I7nmEm8/SdhL89+/OHDDxV2cgS1MKAqMCCkHFPLKBgFo2AUjAIsAAAjYT/BKeYOoAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-1508-2796","institution":"Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Qun","middleName":"","lastName":"Hu","suffix":""}],"badges":[],"createdAt":"2020-11-09 14:25:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-105168/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-105168/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":3580977,"identity":"9d63a2df-f4dc-4e8a-8bfb-4d2c87488da1","added_by":"auto","created_at":"2020-11-13 22:51:03","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":433077,"visible":true,"origin":"","legend":"Changes of WT1 expression during course of ALL disease\nThe red and green graphics show the expression level of WT1 at diagnosis and CR in ALL children, respectively. There was a significant difference between WT1 expression at diagnosis and when at CR (P \u003c 0.001). The patients achieved CR showed a significantly lower expression of WT1 than at diagnosis.","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-105168/v1/7194c9bdb22325a750bcfedd.jpg"},{"id":3580978,"identity":"020779fe-c5f4-419a-a930-56abe24d37ae","added_by":"auto","created_at":"2020-11-13 22:51:03","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":415690,"visible":true,"origin":"","legend":"Changes in WT1 expression in ALL children after relapse\nThe red, green and blue graphics show the WT1 expression level of relapsed patients at diagnosis, CR and after relapse to be compared, respectively. A change trend can be observed from this figure. There was a significant difference between WT1 expression at CR and when after relapse (P =0.003). For 21 relapsed patients with WT1, their expression of WT1 after relapse showed higher than that after induction chemotherapy.","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-105168/v1/33c02bd2f367e155fd62b9be.jpg"},{"id":13616043,"identity":"18f82c5c-6a9a-4d75-9fac-acd7ccb759f0","added_by":"auto","created_at":"2021-09-17 06:47:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":458768,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-105168/v1/dc03b80b-ff10-4414-bbe1-b1316553a3cd.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eExpression of Wilms’ Tumor Gene 1 and Its Clinical Significance in Children with Acute Lymphocytic Leukemia\u003c/p\u003e","fulltext":[{"header":"1. Background","content":"\u003cp\u003eWilms\u0026rsquo; tumor gene 1 (\u003cem\u003eWT1\u003c/em\u003e) located on human chromosome 11p13 was isolated from nephroblastoma in children in 1990 [1]. As a transcription factor encoded by tumor suppressor genes that can regulate cell growth, it is closely related to the occurrence and development of Wilms\u0026rsquo; tumors. The \u003cem\u003eWT1\u003c/em\u003e encodes a zinc finger transcription factor. Posttranscriptional mRNA modification and the existence of several possible transcriptional initiation sites produce many different \u003cem\u003eWT1\u003c/em\u003e protein isomers (at least 32), which are located in specific subcellular and subnuclear regions and have different functions, some of which are partially overlapping. \u003cem\u003eWT1\u003c/em\u003e modulates the transcription of various target genes and participates in the processing of posttranscriptional mRNA. It has bidirectional transcriptional regulation and participates in the regulation of hematopoietic transcription [2]. \u003cem\u003eWT1\u003c/em\u003e plays a major carcinogenic role in malignant hematological tumors, inhibiting the function of transcription factors and hindering the normal proliferation and differentiation of hematopoietic stem cells. At the same time, many factors can lead to mutation of \u003cem\u003eWT1\u003c/em\u003e, rendering the \u003cem\u003eWT1\u003c/em\u003e protein unable to regulate specific DNA and thus promoting the growth of tumor cells [3].\u003c/p\u003e\n\u003cp\u003eThere is a significant correlation between the expression of \u003cem\u003eWT1\u003c/em\u003e and the poor prognosis of malignant tumors in the human hematopoietic system. Although \u003cem\u003eWT1\u003c/em\u003e is not specific to acute malignant hematological diseases, its continuous expression can be detected in almost all leukemia cells. Several studies have demonstrated that \u003cem\u003eWT1 \u003c/em\u003ewas over-expressed in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML),\u0026nbsp;and chronic myeloid leukemia (CML), and it was highly expressed in more than 80% of ALL patients in bone marrow (BM) cells [4]. Therefore, \u003cem\u003eWT1\u003c/em\u003e was considered to be a new \"pan-leukemia\" gene that involved in the proliferation and differentiation of leukemia cells. The quantitative assessment of \u003cem\u003eWT1 in acute leukemia could represent a universal molecular marker of malignant hematopoiesis and was suggested to \u003c/em\u003emonitor minimal residual disease (MRD) and to predict the progression and prognosis of disease [5].\u003c/p\u003e\n\u003cp\u003eThe biological functions of \u003cem\u003eWT1 in childhood ALL are not understood clearly and completely, but it has been suggested that WT1\u003c/em\u003e can impact the pathogenesis of leukemia during cellular proliferation and differentiation. In this paper, we analyzed \u003cem\u003eWT1\u003c/em\u003e expression level in ALL children to reveal the predictive role of \u003cem\u003eWT1\u003c/em\u003e as an MRD marker in this group of patients.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Patients, treatment and follow-up\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 188 children newly diagnosed with ALL between 2015 and 2018 in our hospital were enrolled. We collected all the patients\u0026rsquo; characteristics and measured the expression levels of \u003cem\u003eWT1\u003c/em\u003e at diagnosis and during treatment. All patients were followed up as of October 2020. A cohort of the 188 patients with ALL were consisted of 118 males (62.8%) and 70 females (37.2%), with a median age of 3.2 years. In accordance with WHO leukemia diagnostic criteria，all the patients were diagnosed by morphology combined with immunology, cytogenetics and molecular biology (MICM) of bone marrow cells. Patients\u0026rsquo; treatment was given based on CCCG (Chinese Children Cancer Group) \u0026ndash;ALL 2015 regimen.\u003c/p\u003e\n\u003cp\u003eMRD testing is used to detect the proportion of remaining leukemia cells in bone marrow at the end of induction chemotherapy (IC). A cut-off point for MRD testing by flow cytometry (FCM) is defined at the level of 0.01%. Thus, MRD lower than 0.01% means complete remission (CR).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eMorpholog\u003c/strong\u003e\u003cstrong\u003ey\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMorphologic criteria were established based on French-American-British (FAB), WHO 2004 or WHO 2008 classification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Immunophenotyping\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunophenotyping was conducted by FCM, using monoclonal antibodies to determine lineage-specific antigens, and the panel of tests included at least the following: CD10, CD19, CD20, CD23, cCD79a, SmIg for B-ALL or CD1, CD2, CD3, CD4, CD5, CD7, CD8, TdT for T-ALL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eCytogenetics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChromosome banding of BM samples was performed using R-banding or G-banding technique with short-term cultures. The reverse-transcription polymerase chain reaction (RT-PCR) or fluorescence \u003cem\u003ein situ \u003c/em\u003ehybridization (FISH) was performed to detect common fusion genes such as \u003cem\u003eTEL-AML1\u003c/em\u003e, \u003cem\u003eE\u003c/em\u003e\u003cem\u003e2A\u003c/em\u003e (also called \u003cem\u003eTCF3\u003c/em\u003e)-\u003cem\u003ePBX1\u003c/em\u003e, \u003cem\u003eBCR-ABL\u003c/em\u003e, and \u003cem\u003eMLL \u003c/em\u003e(also called\u003cem\u003e KMT2A\u003c/em\u003e)-related rearrangements.\u003c/p\u003e\n\u003cp\u003eThe real-time quantitative PCR (RQ-PCR) was applied to measure the \u003cem\u003eWT1\u003c/em\u003e expression level in BM samples from the 188 ALL children. We cloned both \u003cem\u003eWT1\u003c/em\u003e and \u003cem\u003eABL\u003c/em\u003e genes for creating a standard curve and calculated copy numbers of \u003cem\u003eWT1\u003c/em\u003e gene in these patients.\u003c/p\u003e\n\u003col\u003e\n\u003cli\u003e\n\u003cp\u003eRNA extraction: 4ml bone marrow was collected from the patients and put into 2% EDTA for anticoagulation. Mononuclear cells were separated by Ficoll lymphocyte isolate solution. After washing the cells with PBS, 1 mL Trizol (invitrogen) was added, blown evenly and stored in -80℃ until the RNA was extracted. The extraction of total RNA was carried out according to the instructions of Trizol reagent. The A 260nm /A 280nm ratio was determined by ultraviolet spectrophotometer to identify the purity and quantity of RNA and then frozen under -80℃.The extracted RNA were mixed with RNase-free water and dissolved, and the 15 mL treatment fluid was used as the PCR reaction template.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eReal-time quantitative PCR: PCR method was used to detect \u003cem\u003eWT1\u003c/em\u003e gene mRNA. The instrument was a ABI7500 fluorescence detector, the reagent was derived from the tumor-related \u003cem\u003eWT1\u003c/em\u003e gene detection kit produced by Shanghai Yuanqi Biotechnology Co. Ltd. The steps were operated according to the operating instructions of the kit. The reaction conditions were 42℃ for 30 min, 94℃ for 5 min, 94℃ denaturation for 15 s, 60℃ annealing for 30 cycles. The reaction system was 25\u0026mu;L. The fluorescence signal was collected at 60℃ in the second step of PCR cycle.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eCalculation of \u003cem\u003eWT1\u003c/em\u003e gene expression level: Four successful serial dilutions (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e, 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e, 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e and 1\u0026times;10\u003csup\u003e 3\u003c/sup\u003ecopies) of each gene were prepared. The copy number of Abelson proto-oncogene (\u003cem\u003eABL\u003c/em\u003e) and \u003cem\u003eWT1\u003c/em\u003e gene were calculated according to the standard curve by \u003cem\u003eABL\u003c/em\u003e expression as the internal reference.\u0026nbsp;When 1\u0026times;10\u003csup\u003e2\u003c/sup\u003e copies\u0026le; \u003cem\u003eWT1\u003c/em\u003e RNA detection concentration\u0026le;1\u0026times;10\u003csup\u003e7\u003c/sup\u003e copies and 1\u0026times;10\u003csup\u003e2\u003c/sup\u003e copies\u0026le; \u003cem\u003eABL\u003c/em\u003e RNA detection concentration\u0026le;1\u0026times;10\u003csup\u003e7\u003c/sup\u003e copies, the \u003cem\u003eWT1\u003c/em\u003e expression level = (\u003cem\u003eWT1\u003c/em\u003e copy numbers/ \u003cem\u003eABL\u003c/em\u003e copy numbers)\u0026times; 100%.\u003c/p\u003e\n\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 \u003c/strong\u003e\u003cstrong\u003eStatistical\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eanalyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis was carried out using SPSS software version 23.0 (IBM Corporation, Armonk, USA), and the count data were expressed as percentage or constituent ratios. Parametric and nonparametric tests including Mann\u0026ndash;Whitney,\u0026nbsp;Chi-square test，Student's t test and ANOVA were applied according to data distribution. Experiment data were described in the form of mean \u0026plusmn; standard deviation or median (25th, 75th) in accordance with data distribution in our study and \u003cem\u003eP\u003c/em\u003e\u0026nbsp;\u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 \u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eWT1\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e expression in\u003c/strong\u003e\u003cstrong\u003e ALL \u003c/strong\u003e\u003cstrong\u003echildren\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmong the 188 children with ALL, 147 were tested positive for \u003cem\u003eWT1\u003c/em\u003e, 41 were negative, as the positive rate was 78.2%. The average expression level was 1.76 (0.3，6.03) % in \u003cem\u003eWT1\u003c/em\u003e positive group. We compared \u003cem\u003eWT1\u003c/em\u003e expression level in different patients\u0026rsquo; characteristics and found that it was only related to lymphadenectasis (\u003cem\u003eP\u003c/em\u003e=0.004) (Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Association\u003c/strong\u003e \u003cstrong\u003ebetween \u003cem\u003eWT1\u003c/em\u003e and immunophenotyping\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn our study cohort, there were 22 children with T-ALL, of whom 17 (77.27%) with \u003cem\u003eWT1\u003c/em\u003e positive, and 78.31% (130/166) of B-ALL children were \u003cem\u003eWT1 \u003c/em\u003epositive. There was no significant difference in the positive rate of \u003cem\u003eWT1\u003c/em\u003e between the two groups (77.27% \u003cem\u003evs\u003c/em\u003e 78.31%, \u003cem\u003eP \u003c/em\u003e=0.912), but expression level of \u003cem\u003eWT1 in\u003c/em\u003e T-ALL children [7.4(1.03, 18.91) %] was obviously higher than that in B-ALL children [1.61(0.23, 4.84) %] (\u003cem\u003eP\u003c/em\u003e=0.009).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Association\u003c/strong\u003e \u003cstrong\u003ebetween\u003cem\u003e WT1\u003c/em\u003e and fusion gen\u003c/strong\u003e\u003cstrong\u003ees\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn our group, a total of 62 cases had fusion genes including \u003cem\u003eTEL-AML1\u003c/em\u003e\u003cem\u003e,\u003c/em\u003e\u003cem\u003e BCR-ABL1\u003c/em\u003e\u003cem\u003e,\u003c/em\u003e\u003cem\u003e E2A-PBX1\u003c/em\u003e\u003cem\u003e,\u003c/em\u003e\u003cem\u003e MLL\u003c/em\u003e rearrangements and \u003cem\u003eSIL-TAL1\u003c/em\u003e. 88.9% (32/36) of children with \u003cem\u003eTEL-AML1\u003c/em\u003e were positive for \u003cem\u003eWT1\u003c/em\u003e gene. The incidence of \u003cem\u003eWT1in\u003c/em\u003e\u003cem\u003e BCR-ABL1\u003c/em\u003e\u003cem\u003e,\u003c/em\u003e\u003cem\u003e E2A-PBX1\u003c/em\u003e and \u003cem\u003eMLL\u003c/em\u003e rearrangements group was 87.5% (7/8), 88.9% (8/9) and 85.7% (6/7), respectively. The \u003cem\u003eWT1\u003c/em\u003e was positive in both of the children with \u003cem\u003eSIL-TAL1\u003c/em\u003e. There were 55 cases carried \u003cem\u003eWT1\u003c/em\u003e and other fusion genes simultaneously, the other 7 cases with single fusion gene. There was no significant difference in expression of \u003cem\u003eWT1\u003c/em\u003e among ALL children carried the above five fusion genes when at diagnosis and at CR, respectively (\u003cem\u003eP \u003c/em\u003e= 0.415, \u003cem\u003eP \u003c/em\u003e= 0.861) (Table 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 \u003c/strong\u003e\u003cstrong\u003eComparison of CR and relapse rate\u003c/strong\u003e\u003cstrong\u003es\u003c/strong\u003e \u003cstrong\u003ein different groups\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere were 129 children achieved CR in the \u003cem\u003eWT1\u003c/em\u003e positive group and 34 children achieved CR in the \u003cem\u003eWT1\u003c/em\u003e negative group. The CR rates of \u003cem\u003eWT1\u003c/em\u003e gene-positive group and gene-negative group after induction chemotherapy were 87.76% and 82.93%, respectively (\u003cem\u003eP\u003c/em\u003e=0.42). By October 2020, there were 21 children relapsed in the positive group and 7 children relapsed in the negative group, with relapse rates of 14.29% and 17.1%, respectively (\u003cem\u003eP \u003c/em\u003e= 0.658). The CR and relapse rates of two groups both had no significant difference.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 E\u003c/strong\u003e\u003cstrong\u003expression\u003c/strong\u003e\u003cstrong\u003e of \u003cem\u003eWT1\u003c/em\u003e \u003c/strong\u003e\u003cstrong\u003eduring\u003c/strong\u003e \u003cstrong\u003ethe course of \u003c/strong\u003e\u003cstrong\u003edisease\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expression of \u003cem\u003eWT1 in \u003c/em\u003ethe 147 positively expressed ALL children was monitored after chemotherapy. Among the 129 children with CR after IC, the expression level of \u003cem\u003eWT1\u003c/em\u003e decreased in 109 cases (84.5%, 109/129) and increased in 20 cases (15.5%, 20/129). The expression of \u003cem\u003eWT1\u003c/em\u003e between at diagnosis and CR had a significant difference (\u003cem\u003eP \u003c/em\u003e\u003cem\u003e\u0026lt;\u003c/em\u003e0.001). The patients at CR showed a significantly lower expression of \u003cem\u003eWT1\u003c/em\u003e than at diagnosis. However, \u003cem\u003eWT1\u003c/em\u003e expression of 18 children who didn\u0026rsquo;t get CR had no significant difference between at diagnosis and after IC (Table 3, Figure 1). Finally, 14 children (12.84%, 14/109) relapsed in the 109 children with decreased \u003cem\u003eWT1\u003c/em\u003e expression and 4 relapsed in 20 ones with increased \u003cem\u003eWT1 \u003c/em\u003eexpression (20%, 4/20).There was no significant difference (12.84% \u003cem\u003evs \u003c/em\u003e20%, \u003cem\u003eP\u003c/em\u003e=0.47), meaning risk of relapse is not correlated on initial WT expression levels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 \u003c/strong\u003e\u003cstrong\u003eChanges \u003c/strong\u003e\u003cstrong\u003eof\u003c/strong\u003e \u003cstrong\u003e\u003cem\u003eWT1\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e expression in\u003c/strong\u003e\u003cstrong\u003e ALL\u003c/strong\u003e \u003cstrong\u003echildren\u003c/strong\u003e \u003cstrong\u003eafter\u003c/strong\u003e \u003cstrong\u003erelapse\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUp to the follow-up time, there were 21 children relapsed in \u003cem\u003eWT1\u003c/em\u003e gene-positive group finally, and 18 of whom achieved CR after IC (Table 4). Among the 21 relapsed patients with positive \u003cem\u003eWT1\u003c/em\u003e, their \u003cem\u003eWT1\u003c/em\u003e expression after relapse [2.28（0.25,1.69）%] showed higher than that after IC [0.33（0.045,0.66）%] (\u003cem\u003eP \u003c/em\u003e=0.003) (Figure 2).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe innovation of chemotherapeutic drugs and allogeneic hematopoietic stem cell transplantation (allo-HSCT) can significantly improve the remission and survival rates of children with ALL, but the relapse after tumor remission is still a difficult problem [6]. The results of MRD at the end of induction phase of chemotherapy in BM testing are used to reclassify risk levels of ALL patients and guide changes in treatment according to their future risk of relapse. The timely monitoring of MRD in ALL children can provide them individualized and intensified treatment [7]. An enormous amount of literature suggested that high expression of \u003cem\u003eWT1\u003c/em\u003e before and after allo-HSCT is a poor prognosis factor. It is of clinical practical value to use \u003cem\u003eWT1\u003c/em\u003e as a transplant recommendation indicator for ALL patients and as a marker to monitor MRD dynamically. Specific marker genes of ALL, such as \u003cem\u003eTEL/AML1\u003c/em\u003e, \u003cem\u003eBCR/ABL1\u003c/em\u003e and \u003cem\u003eE2A/PBX1\u003c/em\u003e can be monitored. Although the altered \u003cem\u003eWT1\u003c/em\u003e expression seems inherent in leukemia cells, the molecular mechanisms by which \u003cem\u003eWT1\u003c/em\u003e contribute to leukemogenesis have not yet been fully elucidated. The role of \u003cem\u003eWT1\u003c/em\u003e dosage in steady-state and pathologic hematopoiesis needs to be investigated by further studies [8]. The literature reported the positive rate of \u003cem\u003eWT1 in\u003c/em\u003e ALL children was more than 80%, and in our study the positive rate was 78.2% which was consistent with that reported in the literature. By FCM, BM samples can give a sufficient sensitivity in the vast majority of patients. \u003cem\u003eWT1\u003c/em\u003e expression can be used as a panleukemic marker and is also a good marker for ALL patients with no other molecular or cytogenetic abnormalities [9]. Since the majority of ALL children expressed very high values of \u003cem\u003eWT1\u003c/em\u003e at diagnosis, the detection of \u003cem\u003eWT1\u003c/em\u003e can be considered a tool of value to monitor the persistence of the disease after chemotherapy or BM transplantation or during the treatment [10].\u003c/p\u003e\n\u003cp\u003eWe collected all patient characteristics including age, sex, chromosome and so on to analyze whether there was an association between \u003cem\u003eWT1\u003c/em\u003e and them. Among all characteristics, we only found lymphadenectasis was related to \u003cem\u003eWT1 in childhood ALL. The phenomenon of lymphadenectasis happened in ALL children without WT1 \u003c/em\u003eexpression was more common than those with \u003cem\u003eWT1\u003c/em\u003e (53.66% \u003cem\u003evs \u003c/em\u003e29.25%, \u003cem\u003eP\u003c/em\u003e=0.004).\u003c/p\u003e\n\u003cp\u003eT-ALL is biologically distinct from B-ALL and differences in response to chemotherapy have been observed. The prognosis of children with T-ALL is worse than those with B-ALL, which is caused by different fusion transcripts and prognostic factors had been investigated extensively. To determine whether immunophenotyping of ALL can impact \u003cem\u003eWT1\u003c/em\u003e expression, we compared expression of \u003cem\u003eWT1 in T-ALL and B-ALL children. Later findings demonstrated that WT1\u003c/em\u003e expression upregulated in T-ALL due to the continuous activation of NF-KB pathway, which meanwhile has recognized as a vital role in the mechanism of refractory relapse in T-ALL children [11]. In our present study, incidence of \u003cem\u003eWT1 in \u003c/em\u003eT-ALL and B-ALL children did not differ, but expression level of \u003cem\u003eWT1 in\u003c/em\u003e T-ALL children was significantly higher than that in B-ALL children. Considering this result, we can put forward a hypothesis that \u003cem\u003eWT1\u003c/em\u003e involved in the progression and relapse of T-ALL and contributed to diverse prognosis in different immunophenotyping of ALL. Bordin et al. reported that loss-of-function mutations and deletions in \u003cem\u003eWT1\u003c/em\u003e emerged in approximately 10% of T-ALL children. Clinically, \u003cem\u003eWT1\u003c/em\u003e mutations are enriched in relapsed series and play a critical role in the response to DNA damage in T-cell leukemia. Some studies based on \u003cem\u003eWT1\u003c/em\u003e positively affecting the expression of the X-linked inhibitor of apoptosis protein (XIAP) to restore sensitivity to \u0026gamma;-radiation therapy supported the view that anti-XIAP targeted therapies might be beneficial to treatment of T-ALL patients with \u003cem\u003eWT1 \u003c/em\u003e[12].\u003c/p\u003e\n\u003cp\u003eWe carried out a study which compared the \u003cem\u003eWT1\u003c/em\u003e expression between individuals positive for fusion genes including \u003cem\u003eTEL-AML1\u003c/em\u003e\u003cem\u003e, \u003c/em\u003e\u003cem\u003eBCR-ABL1\u003c/em\u003e\u003cem\u003e,\u003c/em\u003e\u003cem\u003e E2A-PBX1\u003c/em\u003e\u003cem\u003e,\u003c/em\u003e\u003cem\u003e MLL\u003c/em\u003e rearrangements and \u003cem\u003eSIL-TAL1\u003c/em\u003e. Through the statistical data, we found \u003cem\u003eWT1\u003c/em\u003e and other fusion genes coexisting in ALL children were common. This was also in accordance with the theory that \u003cem\u003eWT1\u003c/em\u003e presented in majority of ALL children. The various fusion transcripts have been confirmed to be highly predictive of clinical outcomes and play different roles in the prognosis of ALL. For example, the Philadelphia chromosome-positive (Ph+) ALL is defined by the t (9; 22) (q34; q11) translocation that forms the fusion gene \u003cem\u003eBCR-ABL1\u003c/em\u003e, which produces an abnormal protein, contributing to the sustaining activation of tyrosine kinase. It predicts poor clinical outcomes due to its resistance to chemotherapy and high relapse risk. Several researches suggested that \u003cem\u003eBCR-ABL1\u003c/em\u003e fusion protein may prompt \u003cem\u003eWT1\u003c/em\u003e expression by activating some signaling pathways, and decrease the sensitivity to chemotherapeutic drugs [13]. Svensson et al. presented that \u003cem\u003eBCR-ABL1\u003c/em\u003e fusion protein could increase the expression of \u003cem\u003eWT1\u003c/em\u003e mRNA and protein via the phosphatidylinositol-3 kinase (PI3K)-Akt pathway. Their experiment results indicated that \u003cem\u003eWT1\u003c/em\u003e expression was induced by oncogenic signaling from \u003cem\u003eBCR-ABL1\u003c/em\u003e and that \u003cem\u003eWT1\u003c/em\u003e contributed to resistance against apoptosis induced by imatinib[14]. The t (12; 21) (p13;q22) producing \u003cem\u003eTEL-AML1\u003c/em\u003e is the most common reciprocal translocation in childhood B-ALL. It is associated with favorable prognosis following conventional therapeutic strategies.\u0026nbsp;But the interaction between \u003cem\u003eTEL-AML1\u003c/em\u003e fusion protein and \u003cem\u003eWT1\u003c/em\u003e is not certain. Similarly, the role of \u003cem\u003eWT1 in other \u003c/em\u003efusion transcripts is still unclear. Qin et al. reported that \u003cem\u003eWT1\u003c/em\u003e expression was obviously different among diverse cytogenetics groups (\u003cem\u003eTEL-AML1\u003c/em\u003e\u003cem\u003e, \u003c/em\u003e\u003cem\u003eE2A-PBX1\u003c/em\u003e and \u003cem\u003eMLL\u003c/em\u003e rearrangements) [15]. However, there was no difference in expression level of \u003cem\u003eWT1\u003c/em\u003e among these five different fusion transcripts groups when at diagnosis and CR in our study. The discrepancy might be caused by the small number of patients and different detection methods.\u003c/p\u003e\n\u003cp\u003eAmong the 129 ALL children having achieved CR, 109 (84.5%, 109/129) got their \u003cem\u003eWT1\u003c/em\u003e expression descended or negative, 20 (15.5%, 20/129) got elevated in \u003cem\u003eWT1\u003c/em\u003e expression conversely. Among the 18 relapsed children with \u003cem\u003eWT1\u003c/em\u003e, the expression of \u003cem\u003eWT1\u003c/em\u003e decreased in 14 cases and increased in 4 cases after CR. Moreover, the expression levels of \u003cem\u003eWT1\u003c/em\u003e after relapse were all higher than those after IC. These results were in agreement with literature data [16].\u003c/p\u003e\n\u003cp\u003eThe clinical value of altered \u003cem\u003eWT1\u003c/em\u003e expression in response to chemotherapy, guiding therapeutic intervention and predicting relapse is still not determined, and the conclusions of different reports are inconsistent. Some researches have found that \u003cem\u003eWT1\u003c/em\u003e was over-expressed in both peripheral blood (PB) and BM cells of the majority of ALL children, at a level that enabled a significantly more sensitive evaluation of residual disease than standard immunophenotypic and morphological analyses. Meantime, \u003cem\u003eWT1\u003c/em\u003e could have abnormally high expression in malignant cells compared with normal controls, which might be a candidate for MRD monitoring. Several studies confirmed that the expression of \u003cem\u003eWT1 in ALL could cause resistance of cells to \u003c/em\u003edifferentiation and apoptosis, and it might lead to poor clinical outcomes. For example, it has been reported that expression level of WT1 after T-ALL transplantation was significantly negatively correlated with relapse risk [17]. Based on the information contained in other studies, they thought the expression of \u003cem\u003eWT1 in\u003c/em\u003e ALL children is so variable that cannot be accurately calculated as MRD during treatment. Because of the inaccuracy of the assessment,\u003cem\u003e WT1\u003c/em\u003e cannot evaluate the prognosis of ALL [2]. While Inoue et al compared CR rate of ALL children in \u003cem\u003eWT1\u003c/em\u003e gene-positive group and gene-negative group, it revealed a significant difference [18]. However, Chiusa et al. reported that no correlation was observed between \u003cem\u003eWT1\u003c/em\u003e and therapeutic effect of leukemia [19]. In our group, the CR rate of \u003cem\u003eWT1\u003c/em\u003e gene-positive patients (87.76%) and gene-negative patients (82.93%) did not statistically differ. The interaction between therapy response and \u003cem\u003eWT1\u003c/em\u003e is still required more studies to be established.\u003c/p\u003e\n\u003cp\u003eThe presence of MRD following therapy for ALL has been demonstrated to be a crucial predictor of relapse in many current studies. MRD typically detected by FCM is on the base of leukemic cells expressing combinations of antigens that are different from those exist in normal BM cells. Particularly in ALL children, the value of MRD must be balanced against other well-established prognostic indexes, but its correlation with other prognostic indexes has not been fully assessed [20]. In this analysis, we found 14.29% (21/147) in patients with \u003cem\u003eWT1\u003c/em\u003e and 17.1% (7/41) without\u003cem\u003e WT1\u003c/em\u003e relapsed after therapy. The relapse rate in these two groups was not significantly altered. The small number of cases and short follow-up time may lead to the lack of significant difference between the two groups. Some studies have found that there was a horizontal relationship between expression level of \u003cem\u003eWT1\u003c/em\u003e and progression of ALL, which would increase, decrease and then increase corresponding to the stage of disease diagnosis, remission and relapse, respectively. A similar trend was observed in our study. Significant decrease in \u003cem\u003eWT1\u003c/em\u003e expression was observed in 129 patients after CR compared with those at diagnosis. That means it can indict the patients\u0026rsquo; therapeutic effect. Meanwhile, a significant increase in \u003cem\u003eWT1 \u003c/em\u003eexpression was witnessed in ALL patients after relapse. 21 relapsed children got their \u003cem\u003eWT1\u003c/em\u003e over-expressed which accorded with some literature documents [5]. From the results obtained so far, it seems that prognosis in ALL children is inversely associated with the expression of \u003cem\u003eWT1\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eThere are several limitations in our study. First, as there were not enough patients, especially for some fusion genes group, patients are so few that could not be compared comprehensively. Second,\u0026nbsp;a little shorter follow-up time may leave out some relapsed patients.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eFrom the above discussion, the conclusion can be reached that \u003cem\u003eWT1\u003c/em\u003e can be used as a suitable marker to monitor MRD, assess response to chemotherapy and predict relapse in the majority of ALL children. Adding to the patients carrying a fusion gene, we can also use RQ‐PCR technology to detect \u003cem\u003eWT1 \u003c/em\u003eto assess disease risk. By employing such methods, we can determine the effect of induction chemotherapy and state in disease after therapy. Collectively, our study results support the use of \u003cem\u003eWT1\u003c/em\u003e as a marker to monitor childhood ALL progression dynamically.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cem\u003eWT1\u003c/em\u003e: Wilms\u0026rsquo; Tumor Gene 1\u003c/p\u003e\n\u003cp\u003eALL: acute lymphoblastic leukemia\u003c/p\u003e\n\u003cp\u003eRT-PCR: reverse-transcription polymerase chain reaction\u003c/p\u003e\n\u003cp\u003eRQ-PCR: real-time quantitative polymerase chain reaction\u003c/p\u003e\n\u003cp\u003eMRD: minimal residual disease\u003c/p\u003e\n\u003cp\u003eAML: acute myeloid\u0026nbsp;leukemia\u003c/p\u003e\n\u003cp\u003eCML: chronic myeloid leukemia\u003c/p\u003e\n\u003cp\u003eBM: bone marrow\u003c/p\u003e\n\u003cp\u003eMICM: morphology, immunology, cytogenetics and molecular biology\u003c/p\u003e\n\u003cp\u003eCCCG: Chinese Children Cancer Group\u003c/p\u003e\n\u003cp\u003eFAB: French-American-British\u003c/p\u003e\n\u003cp\u003eIC: induction chemotherapy\u003c/p\u003e\n\u003cp\u003eFISH: fluorescence \u003cem\u003ein situ \u003c/em\u003ehybridization\u003c/p\u003e\n\u003cp\u003eFCM: flow cytometry\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eABL\u003c/em\u003e: Abelson proto-oncogene\u003c/p\u003e\n\u003cp\u003ePB: peripheral blood\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConception and design: QH and MM. Data analysis and interpretation: MM. Writing, review, and revision of the manuscript: QH and MM. Collection and assembly of data: all authors. The authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice. This study was approved by the Ethical review committee of Tongji Medical College, Huazhong University of Science and Technology. The reference number: S207. All written informed consent was obtained from a parent or guardian for participants under 16 years old.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLee, S.B. and D.A. Haber, Wilms tumor and the WT1 gene. Exp Cell Res, 2001. 264(1): p. 74-99.\u003c/li\u003e\n\u003cli\u003eBoublikova, L., et al., Wilms' tumor gene 1 (WT1) expression in childhood acute lymphoblastic leukemia: a wide range of WT1 expression levels, its impact on prognosis and minimal residual disease monitoring. Leukemia,2006. 20(2): p. 254-63.\u003c/li\u003e\n\u003cli\u003eToska, E. and S.G. Roberts, Mechanisms of transcriptional regulation by WT1 (Wilms' tumour 1). Biochem J, 2014. 461(1): p. 15-32.\u003c/li\u003e\n\u003cli\u003eWang, X.R., et al., Overexpressed WT1 exhibits a specific immunophenotype in intermediate and poor cytogenetic risk acute myeloid leukemia. Ann Hematol, 2020. 99(2): p. 215-221.\u003c/li\u003e\n\u003cli\u003eDou, C.Y., et al., [Relationship between Quantitative Monitoring of WT1 Gene and Prognosis in Patients with Acute Leukemia]. Zhongguo Shi Yan Xue Ye Xue Za Zhi, 2018. 26(6): p. 1632-1636.\u003c/li\u003e\n\u003cli\u003eJiang, B.Q., et al., [The effect of WT1 expression on the prognosis of allogeneic hematopoietic stem cell transplantation in acute leukemia]. Zhonghua Xue Ye Xue Za Zhi, 2018. 39(12): p. 989-993.\u003c/li\u003e\n\u003cli\u003eMinimal Residual Disease Evaluation in Childhood Acute Lymphoblastic Leukemia: A Clinical Evidence Review. Ont Health Technol Assess Ser, 2016. 16(7): p. 1-52.\u003c/li\u003e\n\u003cli\u003ePronier, E., et al., Genetic and epigenetic evolution as a contributor to WT1-mutant leukemogenesis. Blood, 2018. 132(12): p. 1265-1278.\u003c/li\u003e\n\u003cli\u003eGupta, S., et al., Flow-cytometric vs. -morphologic assessment of remission in childhood acute lymphoblastic leukemia: a report from the Children's Oncology Group (COG). Leukemia, 2018. 32(6): p. 1370-1379.\u003c/li\u003e\n\u003cli\u003eZhang, R., et al., Comparison of minimal residual disease (MRD) monitoring by WT1 quantification between childhood acute myeloid leukemia and acute lymphoblastic leukemia. Eur Rev Med Pharmacol Sci, 2015. 19(14): p. 2679-88.\u003c/li\u003e\n\u003cli\u003eGao, H.L., et al., [Expression Levels of HES1, C-MYC and NF-kB in Peripheral Blood of Patients with T Cell Acute Lymphoblastic Leukemia and Their Significance]. Zhongguo Shi Yan Xue Ye Xue Za Zhi, 2019. 27(5): p. 1449-1454.\u003c/li\u003e\n\u003cli\u003eBordin, F., et al., WT1 loss attenuates the TP53-induced DNA damage response in T-cell acute lymphoblastic leukemia. Haematologica, 2018. 103(2): p. 266-277.\u003c/li\u003e\n\u003cli\u003eQin, Y.Z., et al., The prognostic significance of Wilms' tumor gene 1 (WT1) expression at diagnosis in adults with Ph-negative B cell precursor acute lymphoblastic leukemia. Ann Hematol, 2019. 98(11): p. 2551-2559.\u003c/li\u003e\n\u003cli\u003eSvensson, E., et al., Deregulation of the Wilms' tumour gene 1 protein (WT1) by BCR/ABL1 mediates resistance to imatinib in human leukaemia cells. Leukemia, 2007. 21(12): p. 2485-94.\u003c/li\u003e\n\u003cli\u003eQin, Y.Z., et al., [An interlaboratory comparison study on the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels]. Zhonghua Xue Ye Xue Za Zhi, 2019. 40(11): p. 889-894.\u003c/li\u003e\n\u003cli\u003eOwen, C., J. Fitzgibbon and P. Paschka, The clinical relevance of Wilms Tumour 1 (WT1) gene mutations in acute leukaemia. Hematol Oncol, 2010. 28(1): p. 13-9.\u003c/li\u003e\n\u003cli\u003eWoehlecke, C., et al., Prognostic impact of WT1 expression prior to hematopoietic stem cell transplantation in children with malignant hematological diseases. J Cancer Res Clin Oncol, 2015. 141(3): p. 523-9.\u003c/li\u003e\n\u003cli\u003eInoue, K., et al., WT1 as a new prognostic factor and a new marker for the detection of minimal residual disease in acute leukemia. Blood,1994. 84(9): p. 3071-9.\u003c/li\u003e\n\u003cli\u003eChiusa, L., et al., Prognostic value of quantitative analysis of WT1 gene transcripts in adult acute lymphoblastic leukemia. Haematologica, 2006. 91(2): p. 270-1.\u003c/li\u003e\n\u003cli\u003eHagag, A.A., et al., Prognostic Impact of WT-1 Gene Expression in Egyptian Children with Acute Lymphoblastic Leukemia. Mediterr J Hematol Infect Dis, 2016. 8(1): p. e2016008.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1. Patient characteristics\u003c/p\u003e\n\u003ctable style=\"width: 634px;\" border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 255px;\"\u003e\n\u003cp\u003eCharacteristic\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 147px;\"\u003e\n\u003cp\u003e\u003cem\u003eWT1 \u003c/em\u003e(+)（\u003cem\u003en\u003c/em\u003e=147）\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 134px;\"\u003e\n\u003cp\u003e\u003cem\u003eWT1 \u003c/em\u003e(-)（\u003cem\u003en\u003c/em\u003e=41）\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 70px;\"\u003e\n\u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 255px;\"\u003e\n\u003cp\u003eSex\u003c/p\u003e\n\u003cp\u003eMale\u003c/p\u003e\n\u003cp\u003eFemale\u003c/p\u003e\n\u003cp\u003eAge, years\u003c/p\u003e\n\u003cp\u003eImmunophenotyping\u003c/p\u003e\n\u003cp\u003eT-ALL\u003c/p\u003e\n\u003cp\u003eB-ALL\u003c/p\u003e\n\u003cp\u003eKaryotype\u003c/p\u003e\n\u003cp\u003eHyperdiploid(\u0026lt;46 chromosomes)\u003c/p\u003e\n\u003cp\u003eDiploid(=46 chromosomes)\u003c/p\u003e\n\u003cp\u003eHypodiploid(\u0026gt;46 chromosomes)\u003c/p\u003e\n\u003cp\u003eLiver/splenomegaly (subcostal \u0026gt;5 cm)\u003c/p\u003e\n\u003cp\u003elymphadenectasis\u003c/p\u003e\n\u003cp\u003eCR after IC\u003c/p\u003e\n\u003cp\u003eRelapse\u003c/p\u003e\n\u003cp\u003eLeukocyte count，\u0026times;10\u003csup\u003e9\u003c/sup\u003e/L\u003c/p\u003e\n\u003cp\u003eHemoglobin count, g/L\u003c/p\u003e\n\u003cp\u003ePlatelet count, \u0026times;10\u003csup\u003e9\u003c/sup\u003e/L\u003c/p\u003e\n\u003cp\u003eMyeloid juvenile cell，%\u003c/p\u003e\n\u003cp\u003eSerum LDH level, IU/L\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 147px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e93\u003c/p\u003e\n\u003cp\u003e54\u003c/p\u003e\n\u003cp\u003e5.9（3.3,8）\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e17\u003c/p\u003e\n\u003cp\u003e130\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e37\u003c/p\u003e\n\u003cp\u003e103\u003c/p\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003cp\u003e21\u003c/p\u003e\n\u003cp\u003e43\u003c/p\u003e\n\u003cp\u003e129\u003c/p\u003e\n\u003cp\u003e21\u003c/p\u003e\n\u003cp\u003e31.07（4.05,27.94）\u003c/p\u003e\n\u003cp\u003e77.52\u0026plusmn;23.6\u003c/p\u003e\n\u003cp\u003e93.24(31,135)\u003c/p\u003e\n\u003cp\u003e41.64（10,71）\u003c/p\u003e\n\u003cp\u003e572.3\u0026plusmn;133.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 134px;\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e23\u003c/p\u003e\n\u003cp\u003e18\u003c/p\u003e\n\u003cp\u003e5.1（3,6.2）\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003cp\u003e36\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003cp\u003e29\u003c/p\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003cp\u003e126\u003c/p\u003e\n\u003cp\u003e22\u003c/p\u003e\n\u003cp\u003e34\u003c/p\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003cp\u003e18.62（2.8,18.4）\u003c/p\u003e\n\u003cp\u003e74.69\u0026plusmn;24.89\u003c/p\u003e\n\u003cp\u003e110.7(36,129)\u003c/p\u003e\n\u003cp\u003e40.02（8.5,69.5）\u003c/p\u003e\n\u003cp\u003e472.93\u0026plusmn;124.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 70px;\"\u003e\n\u003cp\u003e0.404\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e0.191\u003c/p\u003e\n\u003cp\u003e0.912\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e0.995\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e0.732\u003c/p\u003e\n\u003cp\u003e0.004*\u003c/p\u003e\n\u003cp\u003e0.42\u003c/p\u003e\n\u003cp\u003e0.658\u003c/p\u003e\n\u003cp\u003e0.242\u003c/p\u003e\n\u003cp\u003e0.503\u003c/p\u003e\n\u003cp\u003e0.652\u003c/p\u003e\n\u003cp\u003e0.765\u003c/p\u003e\n\u003cp\u003e0.23\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eCounting data were expressed by number of people, and measurement data were described in the form of mean \u0026plusmn; standard deviation or median (25th, 75th) were in accordance with data distribution.\u003c/p\u003e\n\u003cp\u003eALL: acute lymphoblastic leukemia; CR: complete remission; IC: induction chemotherapy; LDH, lactate dehydrogenase.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05: statistically significant.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 2. Changes in the expression level of \u003cem\u003eWT1 in fusion genes\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/58893_b39df98f09c4a4bb/58893_custom_files/img1605274827.png\" alt=\"\" /\u003e\u003c/p\u003e\n\u003cp\u003eCR: complete remission\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 3. \u003cem\u003eWT1\u003c/em\u003e expression level after IC\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/58893_b39df98f09c4a4bb/58893_custom_files/img1605274884.png\" alt=\"\" /\u003e\u003c/p\u003e\n\u003cp\u003eCR: complete remission; IC: induction chemotherapy.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05: statistically significant.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 4. Dynamic changes in expression of \u003cem\u003eWT1 in relapsed patients\u003c/em\u003e\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003eCase\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"169\"\u003e\n\u003cp\u003e\u0026nbsp;Diagnosis (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003eAfter IC (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003eRelapse (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"115\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003cp\u003e11\u003c/p\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003cp\u003e13\u003c/p\u003e\n\u003cp\u003e14\u003c/p\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003cp\u003e16\u003c/p\u003e\n\u003cp\u003e17\u003c/p\u003e\n\u003cp\u003e18\u003c/p\u003e\n\u003cp\u003e19\u003c/p\u003e\n\u003cp\u003e20\u003c/p\u003e\n\u003cp\u003e21\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"169\"\u003e\n\u003cp\u003e2.4\u003c/p\u003e\n\u003cp\u003e0.91\u003c/p\u003e\n\u003cp\u003e1.2\u003c/p\u003e\n\u003cp\u003e0.01\u003c/p\u003e\n\u003cp\u003e0.14\u003c/p\u003e\n\u003cp\u003e0.46\u003c/p\u003e\n\u003cp\u003e2.08\u003c/p\u003e\n\u003cp\u003e0.17\u003c/p\u003e\n\u003cp\u003e0.81\u003c/p\u003e\n\u003cp\u003e0.99\u003c/p\u003e\n\u003cp\u003e0.75\u003c/p\u003e\n\u003cp\u003e0.86\u003c/p\u003e\n\u003cp\u003e0.05\u003c/p\u003e\n\u003cp\u003e0.9\u003c/p\u003e\n\u003cp\u003e1.76\u003c/p\u003e\n\u003cp\u003e0.38\u003c/p\u003e\n\u003cp\u003e0.06\u003c/p\u003e\n\u003cp\u003e1.39\u003c/p\u003e\n\u003cp\u003e1.55\u003c/p\u003e\n\u003cp\u003e4.7\u003c/p\u003e\n\u003cp\u003e0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003e0.02\u003c/p\u003e\n\u003cp\u003e0.19\u003c/p\u003e\n\u003cp\u003e0.56\u003c/p\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003cp\u003e0.29\u003c/p\u003e\n\u003cp\u003e0.15\u003c/p\u003e\n\u003cp\u003e0.18\u003c/p\u003e\n\u003cp\u003e0.02\u003c/p\u003e\n\u003cp\u003e0.67\u003c/p\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003cp\u003e0.3\u003c/p\u003e\n\u003cp\u003e0.15\u003c/p\u003e\n\u003cp\u003e0.68\u003c/p\u003e\n\u003cp\u003e1.12\u003c/p\u003e\n\u003cp\u003e0.15\u003c/p\u003e\n\u003cp\u003e0.02\u003c/p\u003e\n\u003cp\u003e0.64\u003c/p\u003e\n\u003cp\u003e0.07\u003c/p\u003e\n\u003cp\u003e0.16\u003c/p\u003e\n\u003cp\u003e0.9\u003c/p\u003e\n\u003cp\u003e0.67\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"142\"\u003e\n\u003cp\u003e0.14\u003c/p\u003e\n\u003cp\u003e0.39\u003c/p\u003e\n\u003cp\u003e1.94\u003c/p\u003e\n\u003cp\u003e0.2\u003c/p\u003e\n\u003cp\u003e0.19\u003c/p\u003e\n\u003cp\u003e22.82\u003c/p\u003e\n\u003cp\u003e0.91\u003c/p\u003e\n\u003cp\u003e0.3\u003c/p\u003e\n\u003cp\u003e1.43\u003c/p\u003e\n\u003cp\u003e0.06\u003c/p\u003e\n\u003cp\u003e0.45\u003c/p\u003e\n\u003cp\u003e2.7\u003c/p\u003e\n\u003cp\u003e0.5\u003c/p\u003e\n\u003cp\u003e4.41\u003c/p\u003e\n\u003cp\u003e0.3\u003c/p\u003e\n\u003cp\u003e1.04\u003c/p\u003e\n\u003cp\u003e7.89\u003c/p\u003e\n\u003cp\u003e0.31\u003c/p\u003e\n\u003cp\u003e1.37\u003c/p\u003e\n\u003cp\u003e0.4\u003c/p\u003e\n\u003cp\u003e0.06\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eALL: acute lymphoblastic leukemia; IC: induction chemotherapy\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Acute Lymphoblastic Leukemia, Children, Wilms’ tumor 1 gene, Clinical significance","lastPublishedDoi":"10.21203/rs.3.rs-105168/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-105168/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Wilms’ Tumor Gene 1 \u003cem\u003e(WT1) \u003c/em\u003eis a potential valuable parameter in prognosis of childhood acute lymphoblastic leukemia (ALL). However, studies on prevalence of \u003cem\u003eWT1\u003c/em\u003e and its correlation to clinical features and prognosis in pediatric patients were not well done. In this study we attempted to identify the correlation between \u003cem\u003eWT1 \u003c/em\u003eand childhood ALL.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eThe expression levels of \u003cem\u003eWT1 in bone marrow cells of 188 \u003c/em\u003echildren diagnosed with ALL from 2015 to 2018 were detected using real-time quantitative polymerase chain reaction (RQ-PCR). The relationship between expression levels of \u003cem\u003eWT1\u003c/em\u003e and patients’ characteristics, remission status (complete remission/relapse), fusion genes and prognosis of childhood ALL were analyzed and revealed. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e 1. 147 (78.2%) cases had positive \u003cem\u003eWT1\u003c/em\u003e expression, and the average level was 1.76 (0.3, 6.03) %. 2. The CR and relapse rates of ALL children with positive \u003cem\u003eWT1\u003c/em\u003e were not significantly different from those of \u003cem\u003eWT1\u003c/em\u003e negative group, respectively (87.76% \u003cem\u003evs \u003c/em\u003e82.93%, \u003cem\u003eP\u003c/em\u003e=0.42 and 14.29% \u003cem\u003evs\u003c/em\u003e 17.1%, \u003cem\u003eP\u003c/em\u003e=0.658). 3. The \u003cem\u003eWT1\u003c/em\u003e expression level in patients at CR was significantly lower than when at diagnosis (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001) and the expression of \u003cem\u003eWT1 \u003c/em\u003eincreased obviously after induction therapy in 21 patients who relapsed (\u003cem\u003eP\u003c/em\u003e=0.003) .4. The \u003cem\u003eWT1\u003c/em\u003e expression was related to lymphadenectasis (\u003cem\u003eP\u003c/em\u003e=0.004) and immunophenotyping (\u003cem\u003eP\u003c/em\u003e=0.009), but not to fusion genes (\u003cem\u003eP\u003c/em\u003e=0.912). \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eThe \u003cem\u003eWT1 i\u003c/em\u003en ALL children can be employed as an independent tool to evaluate the prognosis and curative effect of the disease.\u003c/p\u003e","manuscriptTitle":"Expression of Wilms’ Tumor Gene 1 and Its Clinical Significance in Children with Acute Lymphocytic Leukemia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-11-13 22:51:02","doi":"10.21203/rs.3.rs-105168/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3a69ce76-3279-4067-a076-5760154f13b4","owner":[],"postedDate":"November 13th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":1069429,"name":"Pediatrics"}],"tags":[],"updatedAt":"2020-11-19T13:12:37+00:00","versionOfRecord":[],"versionCreatedAt":"2020-11-13 22:51:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-105168","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-105168","identity":"rs-105168","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}