The expression of WT1 gene and its clinical characteristics in children with acute lymphoblastic leukemia / 中国医师进修杂志

Objective:To investigate the expression of WT1 gene in children with acute lymphoblastic leukemia (ALL), and explore its clinical characteristics and correlation with the prognosis of ALL.Methods:The clinical data of 183 children with newly diagnosed ALL in Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology from January 2015 to May 2019 were retrospectively analyzed. The expression level of WT1 gene in bone marrow samples was detected by real-time fluorescence quantitative polymerase chain reaction. The children were followed up to June 2021 with a median follow-up time of 46 months (0 to 63 months).Results:Among 183 children with ALL, the WT1 gene positive was in 130 cases (71.04%), and the expression level was 1.41% (0.26%, 6.73%); WT1 gene negative was in 53 cases (28.96%). The expression levels of WT1 gene in children with T-cell lymphoblastic leukemia (T-ALL), non-hyperdiploid and middle/high-risk were significantly increased, and there were statistical differences ( P<0.05 or <0.01); however, there were no statistical differences in the expression levels of WT1 gene between children with different gender, chromosome karyotype, hepatosplenomegaly and the first diagnosis white blood cell count ( P>0.05). There were no statistical differences in complete remission rate and recurrence rate after induction chemotherapy between WT1 gene positive children and WT1 gene negative children: 87.69% (114/130) vs. 86.79% (46/53) and 16.15% (21/130) vs. 18.87% (10/53), P>0.05. By the end of follow-up, 179 children were followed up, and there was no statistical difference in survival rate between WT1 gene positive children and WT1 gene negative children: 89.68% (113/126) vs. 86.79% (46/53), P>0.05. Among the children with WT1 gene positive, relapse was in 21 cases, and there was no statistical difference in the expression level of WT1 gene after complete remission or after relapse, compared with that while the first diagnosis ( P>0.05); among non-relapse children, 96 completed the detection, the expression level of WT1 gene after complete remission was significantly lower than the first diagnosis: 0.17% (0.04%, 0.49%) vs. 2.01% (0.41%, 8.82%), and there was statistical difference ( P<0.01). Kaplan-Meier survival curve analysis result showed there was no statistical difference in survival time between WT1 gene positive children and WT1 gene negative children ( P>0.05). According to the median expression level of WT1 gene (1.41%), the children with WT1 gene positive were divided into high expression (66 cases) and low expression (64 cases), there was no statistical difference in survival time between high expression children and low expression children ( P>0.05). Conclusions:WT1 gene is commonly expressed in children with ALL and is associated with some clinical features and prognosis of the children. Decreased WT1 gene expression may result in better prognosis.

The Distribution and Significance of Activated T Cells and Lymphocyte Subsets in Myelodysplastic Syndrome / 中国实验血液学杂志

OBJECTIVE@#To investigate the distribution of bone marrow lymphocyte subsets in patients with myelodysplastic syndrome(MDS),the proportion of activated T cells with immunophenotype CD3+HLA-DR+ in the lymphocytes and its clinical significance, and to understand the effects of different types of MDS, different immunophenotypes, and different expression levels of WT1 on the proportion of lymphocyte subsets and activated T cells.@*METHODS@#The immunophenotypes of 96 MDS patients, the subsets of bone marrow lymphocytes and activated T cells were detected by flow cytometry. The relative expression of WT1 was detected by real-time fluorescent quantitative PCR, and the first induced remission rate (CR1) was calculated, the differences of lymphocyte subsets and activated T cells in MDS patients with different immunophenotype, different WT1 expression, and different course of disease were analyzed.@*RESULTS@#The percentage of CD4+T lymphocyte in MDS-EB-2, IPSS high-risk, CD34+ cells >10%, and patients with CD34+CD7+ cell population and WT1 gene overexpression at intial diagnosis decreased significantly (P<0.05), and the percentage of NK cells and activated T cells increased significantly (P<0.05), but there was no significant difference in the ratio of B lymphocytes. Compared with the normal control group, the percentage of NK cells and activated T cells in IPSS-intermediate-2 group was significantly higher(P<0.05), but there was no significant difference in the percentage of CD3+T, CD4+T lymphocytes. The percentage of CD4+T cells in patients with complete remission after the first chemotherapy was significantly higher than in patients with incomplete remission(P<0.05), and the percentage of NK cells and activated T cells was significantly lower than that in patients with incomplete remission (P<0.05).@*CONCLUSION@#In MDS patients, the proportion of CD3+T and CD4+T lymphocytes decreased, and the proportion of activated T cells increased, indicating that the differentiation type of MDS is more primitive and the prognosis is worse.

Síndrome de Frasier: ¿Cómo diagnosticar una enfermedad rara en la consulta de Ginecología? Reporte de caso / Frasier Syndrome: How to diagnose a rare disease in a Gynecology consultation? A case report

Resumen INTRODUCCIÓN: El síndrome de Frasier es una enfermedad de herencia autosómica dominante con una prevalencia inferior a 1 caso por cada millón de recién nacidos vivos. Hasta la fecha se han descrito unos 150 casos. Este síndrome se caracteriza por pseudohermafroditismo masculino, disgenesia gonadal 46, XY y enfermedad glomerular, todo ello producido por una mutación del gen WT1. CASO CLÍNICO: Paciente de 16 años que consultó por amenorrea primaria y ausencia de caracteres sexuales secundarios. Antecedentes: glomerulonefritis focal segmentaria corticorresistente desde la infancia. En la exploración física se objetivó un estadio Tanner 1. Las pruebas complementarias pusieron de manifiesto la ausencia de útero y anejos y un hipogonadismo hipergonadotrópico con cariotipo 46, XY. Ante los hallazgos se decidió la laparoscopia exploradora y salpingooforectomía bilateral. El informe anatomopatológico fue de disgerminoma de ovario derecho. La sospecha clínica se confirmó en el estudio genético, que reportó una mutación del gen WT1, diagnóstica de síndrome de Frasier. En la actualidad, la paciente recibe tratamiento inmunosupresor y hormonal sustitutivo, con una evolución favorable. CONCLUSIÓN: El diagnóstico temprano del síndrome de Frasier es fundamental en virtud del riesgo asociado de malignidad. La baja frecuencia de la enfermedad y la asociación común con retraso puberal en pacientes con enfermedades crónicas puede favorecer el retraso del diagnóstico. El reporte de los casos diagnosticados de este síndrome, y el tratamiento multidisciplinario son decisivos para mejorar el conocimiento de esta rara enfermedad.

Abstract INTRODUCTION: Frasier Syndrome is an autosomal dominant inherited disease with a prevalence of less than 1 per million live births. To date, about 150 cases have been described. This syndrome is characterized by male pseudohermaphroditism, 46, XY gonadal dysgenesis, and glomerular disease, all caused by a mutation of the WT1 gene. It is essential to learn more about this disease, not only because of the high risk of ovarian neoplasia, but also because its early diagnosis will improve the prognosis. CLINICAL CASES: We report the case of a 16-year-old woman who consulted for primary amenorrhea and absence of secondary sexual characteristics. As medical history, she highlighted steroid-resistant focal segmental glomerulonephritis since childhood. The examination revealed Tanner stage 1. Complementary tests revealed the absence of the uterus and adnexa and hypergonadotropic hypogonadism with a 46, XY karyotype. Given the findings, it was decided to perform an exploratory laparoscopy and bilateral salpingo-oophorectomy. The anatomopathological result reported dysgerminoma of the right ovary. The clinical suspicion was confirmed by genetic study, which reported a mutation of the WT1 gene, diagnostic of Frasier Syndrome. Currently, the patient undergoes, along with immunosuppressive treatment, hormone replacement therapy, with a favorable evolution. CONCLUSION: Early diagnosis of Frasier Syndrome is essential given the associated risk of malignancy. The low frequency of the disease and the usual association of delayed puberty in patients with chronic diseases may lead to a diagnostic delay. Therefore, reporting the diagnosed cases of this syndrome, as well as its multidisciplinary management, is essential to improve knowledge about this rare disease.

Expression Levels of the CA9, WT1, and PRAME genes and genotyping-associated antigens for the diagnosis and prognosis of colorectal cancer

Background: Colorectal cancer (CRC) is the third most prevalent type of cancer worldwide, and is one of the major health problems in Asia, Africa, Europe, and America. The tumor antigens recently are of interesting indicators as diagnostic and prognostic tools, The aim of the present study is to detect the expression levels of carbonic anhydrase IX (CA9), the Wilms tumor gene (WT1), and the preferentially expressed antigen in melanoma (PRAME) in the peripheral blood of CRC patients in comparison with healthy controls. Methods: A prospective case-control study of CRC patients was conducted. We included 25 newly-diagnosed CRC eligible patients and obtained peripheral blood samples of them as well as 10 blood samples from the control group. All samples were then submitted to deoxyribonucleic acid (DNA) extraction and a molecular study through real-time polymerase chain reaction (PCR). Results: The CRC group consisted of 15 (60%) female and 10 (40%) male patients with a mean age of 50.52 ± 9.8 years, while the control group included 4 (40%) female and 6 (60%) male patients with a mean age of 47.7 ± 7.9 years. The CRC group, 24 (96%) of patient samples were CA9-positive with strong statistically significant differences (p < 0.00001; sensitivity: 96%; specificity: 90%). Regarding the WT1 gene, there were 11 (44%) positive samples in the CRC group, with no statistically significant differences (p = 0.055; sensitivity: 44%; specificity: 90%). The PRAME gene was positive in 9 (36%) samples in the CRC group, with no statistically significant differences (p = 0.357; sensitivity: 36%; specificity: 80%. Among CA9 (24 patients; 96%) of patients with CRC expressed positive results, in WT1 11(91.6%) CRC patients expressed gene, and in PRAME gene, 9 patients with CRC (81.8%) expressed positive results. Conclusion: Overexpression of the CA9 gene in CRC of high sensitivity and specificity to be used as a tool to discriminate CRC from benign associate with high accuracy compare to WT1 and PRAME genes. (AU)

Correlation of peripheral blood lymphocyte-to-monocyte ratio after induction chemotherapy with treatment efficacy and prognosis in acute myeloid leukemia / 白血病·淋巴瘤

Objective:To explore the value of peripheral blood lymphocyte-to-monocyte ratio (LMR) after induction chemotherapy in patients with acute myeloid leukemia (AML) for the judgment of curative effect and prognosis.Methods:The clinical data of 63 newly-treated AML patients (except for acute promyelocytic leukemia) in Heze Municipal Hospital of Shandong Province from January 2015 to January 2020 were retrospectively analyzed. No blasts were seen on the blood films of all patients at one week after induction chemotherapy. The receiver operating characteristic (ROC) curve was used to determine the best cut-off value of LMR at one week after the completion of all induction chemotherapy for predicting complete remission (CR) of patients, and based on this value, the patients were divided into the low LMR group (LMR <the best cut-off value) and the high LMR group (LMR ≥ the best cut-off value). The differences in clinical characteristics, laboratory test indicators, treatment efficacy, recurrence and survival between the two groups of patients were compared.Results:Sixty-three patients were enrolled in the study. The median LMR of 63 patients was 3.64 (0.13-88.01) at one week after the completion of all induction chemotherapy. Fifty-one patients (81.0%) achieved CR after one course of induction chemotherapy, 54 patients (85.7%) achieved CR after two courses, and there were finally 56 patients (88.9%) with CR. The ROC curve determined that the best cut-off value of LMR was 1.515, and there were 20 cases and 43 cases in the low LMR group and the high LMR group, respectively. There were no significant differences in age, gender, hemoglobin, bone marrow blast cell ratio, white blood cell count, platelet count, and lactate dehydrogenase levels between the two groups (all P > 0.05). The CR rates after 1 course of treatment in the low LMR group and the high LMR group were 65.0% (13/20) and 88.4% (38/43), respectively, and the difference was statistically significant ( χ2=4.836, P=0.028). In the low LMR group, 3 of the 13 patients who achieved CR within 1 course of treatment relapsed; in the high LMR group, 2 of the 38 patients who achieved CR within 1 course of treatment relapsed. The 3-year RFS rates of the low LMR group and the high LMR group were 64% and 80%, respectively, and the difference was not statistically significant ( χ2=2.897, P=0.089); the 3-year OS rates were 84% and 80%, respectively, and the difference was not statistically significant ( χ2=0.136, P=0.712). Conclusion:For newly-treated AML patients with no nucleated cells in blood smear microscopy at one week after the completion of induction chemotherapy, LMR may be used to evaluate the treatment efficacy and recurrence.

Therapeutic targets of desmoplastic small round cell tumor and future direction / 中国肿瘤临床

Desmoplastic small round cell tumor (DSRCT) is a rare and highly malignant soft tissue sarcoma. Most patients are diagnosed at advanced stage unless DSRCT happens to be discovered accidentally. DSRCT mainly occurs in the abdomen and pelvis, spreading along the peritoneal surface. At the time of diagnosis, most patients have passed the operable stage. The diagnosis of DSRCT is based on the histological analysis of a biopsy. It typically manifests as small round blue cells in the nest, separated by a large number of fibroproliferative stroma. A stable cytogenetic feature of DSRCT is that the characteristic t(11; 22) (P13; Q12) chromosome produces the EWSWT1 fusion gene. The prognosis of patients with DSRCT is very poor, and the 5-year survival rate is about 15%. Despite the use of active treatment methods, such as chemotherapy, surgery, and total abdominal radiotherapy, about 60% 70% of patients with DSRCT die within 2- 3 years. Targeted therapy, immunotherapy, and other methods have been trialed recently in the treatment of DSRCT due to the development of DSRCT molecular genetics.

LncRNA WT1-AS Knockdown Promotes Invasion and Migration of Triple Negative Breast Cancer MDA-MB-231 Cells / 肿瘤防治研究

Objective To investigate the effect of LncRNA WT1-AS on the invasion and migration of triple-negative breast cancer MDA-MB-231 cells. Methods qRT-PCR was used to detect the expression of WT1-AS and the efficiency of gene silencing in MDA-MB-231 cells. Transwell invasion test and scratch test were used to detect the invasion and migration of MDA-MB-231 cells; Western blot was used to detect the expression of E-cadherin, N-cadherin, Vimentin and Snail in MDA-MB-231 cells. Results Compared with normal human mammary epithelial cells, the expression of WT1-AS in MDA-MB-231, MDA-MB-453 and MDA-MB-468 cells were significantly up-regulated. Knockdown of WT1-AS significantly reduced the invasion and migration abilities of MDA-MB-231 cells, the expression of E-cadherin was increased but N-cadherin, Vimentin and Snail expression were decreased. Conclusion WT1-AS could promote the invasion and migration of triple-negative breast cancer cells MDA-MB-231 via regulating epithelial-mesenchymal transition.

Síndrome WAGR por deleción en heterocigosis del gen WT1: Caso clínico pediátrico / WAGR syndrome by heterozygous deletion of the WT1 gene: Pediatric case report

El síndrome WAGR (tumor de Wilms, aniridia, anomalías genitourinarias y retraso mental) es un trastorno genético infrecuente debido a la deleción de la región 11p13, que contiene los genes WT1 y PAX6. Comprende una combinación distintiva de afecciones clínicas; la aniridia y el tumor de Wilms son las más notables. Se presenta a un lactante de 17 meses con microcefalia, alteraciones oculares (buftalmos, leucocoria, aniridia bilateral), hipoplasia escrotal, testículos en la región inguinal y retraso en el neurodesarrollo, a quien se le realizó el estudio de amplificación de sondas dependiente de ligandos múltiples para WT1, que mostró haploinsuficiencia en las sondas que hibridaban la región 11p13, compatible con una deleción en heterocigosis del gen. Posteriormente, se diagnosticó tumor de Wilms. Dada su baja prevalencia, es importante difundir sus características clínicas y hacer énfasis en un manejo interdisciplinario centrado en la identificación precoz del síndrome y de sus posibles complicaciones. .

WAGR syndrome (Wilms tumor, aniridia, genitourinary anomalies and mental retardation) is an uncommon genetic disorder due to the deletion of the 11p13 region that contains the WT1 and PAX6 genes. It involves a distinctive combination of clinical conditions, with aniridia and Wilms tumor being the most notable. We present a 17-month-old infant with microcephaly, ocular alterations (buphthalmos, leukocoria, bilateral aniridia), scrotal hypoplasia, undescended testes and neurodevelopmental delay who underwent multiplex ligation-dependent probe amplification study for WT1, showing haploinsufficiency in the probes that hybridize to the 11p13 region, compatible with an heterozygous deletion of the gene. Wilms tumor was later diagnosed. WAGR syndrome is infrequent; its report in Latin America is low. It is important to disseminate its clinical characteristics, emphasizing an interdisciplinary management focused on the early identification of both the syndrome and its possible complications.

A Rare Case of Sinonasal Ameloblastoma with Immunohistochemistry.

Introduction: Ameloblastoma is benign locally aggressivedentine epithelial tumor. Mandible and maxilla are commonsites of involvement. Calretinin is specific marker and usefulto differentiate ameloblastoma from Keratocystic odontogenictumor. The rarity of site and presentation of lesion makes usreport this case.Case report: 46 year old female had recurrent nasalblockage. Polyps in right nostrils were seen on Rhinoscopy.Polypectomy was done for suspected antrochoanal polyps.Ameloblastoma was diagnosed on histopathology andconfirmed by Immunohistochemistry with Cytokeratin,Calretinin and WT1.Conclusion: Preoperative diagnosis by biopsy in unilateralnasal masses should be carried out to expect the unexpected.Immunohistohemistry should be performed in cases havingrare site to confirm the diagnosis.

G-quadruplex forming region within WT1 promoter is selectively targeted by daunorubicin and mitoxantrone: A possible mechanism for anti-leukemic effect of drugs

Wilms tumor 1 (WT1) has long been overexpressed in acute myeloid leukemia and has a prognostic value in its diagnosis.Lately, the formation of G-quadruplexes in oncogenic promoters like WT1 has been widely investigated since stabilizationof these structures leads to transcriptional inhibition of the oncogene. Daunorubicin and mitoxantrone considered as crucialcomponents of almost all standard acute myeloid leukemia induction regimens. Herein we have proposed a probablemolecular mechanism of action through which the drugs may stabilize WT1 promoter G-quadruplexes. Differential pulsevoltammetry, circular dichroism, and polyacrylamide gel electrophoresis, electrophoretic mobility shifts assay, polymerasechain reaction (PCR) stop assays, and quantitative RT-PCR were performed in order to better understanding the nature ofinteractions between the drugs and G-quadruplexes. Data revealed that both drugs had potential to stabilize G-quadruplexesand down-regulate WT1 transcription but daunorubicin exposed more silencing impact. The results illustrated the therapeutic association of these two commercial FDA-approved drugs in WT1 transcriptional down-regulation. Since WT1 hasknown as a transcriptional regulator of at least 137 target genes, so the new data are significant for the development of newapproaches to regulating WT1 and other target genes by employing special drugs in cancer treatment.

Correlation between WT1 gene polymorphism and multiple myeloma susceptibility / 中国肿瘤生物治疗杂志

@#Objective: To analyze the correlation between WT1 gene polymorphism and multiple myeloma (MM) susceptibility in 168 patients. Methods: One hundred and sixty eight MM patients, who were hospitalized in our hospital and Hebei Provincial People's Hospital from January 2013 to December 2017, were researched in this study. There were 121 males (72%) and 47 females(28%) with a median age of 62.4 years old (36~83 years old). Polymorphism of WT1 gene of the samples was detected and analyzed by SSP-PCR and SBT-PCR. Results: Eleven WT1 alleles were detected in MM patients, WT1*010 and WT1*012 alleles occupied a higher frequency in MM group (WT1*010: OR=6.13, 95%CI:3.5~10.75, PC<0.000; WT1*012: OR=2.06, 95%CI:1.23~1.44, PC<0.051). STR genotype frequency of WT1*A5 markedly increased (OR=1.62, 95%CI:1.18~2.23, PC<0.05). Genotype frequency of WT1*010/010 also obviously increased (OR=6.28, 95%CI:1.81～21.76, PC<0.05). Conclusion: WT1 allele is highly polymorphic in MM patients and homozygote WT1*010/010 is a susceptible genotype of MM, indicating that the occurrence and development of MM are related to the polymorphism of WT1 gene.

Relationship between WT1 gene mutation and prognosis of acute myeloid leukemia: a Meta鄄analysis / 白血病·淋巴瘤

Objective To summarize the relationship between WT1 gene mutation and prognosis of acute myeloid leukemia (AML). Methods The related literatures were searched in PubMed, Google Scholar and Cochrane Library databases, and the deadline was April 2, 2018. The Meta_analysis was performed by using Review Manager 5.2 software. Results A total of 9 literatures were included. Meta analysis showed that for the pediatric AML patients, the overall survival (OS) time in the WT1 gene mutated group was shorter than that in the wild_type group ( HR＝1.41, 95% CI 1.07-1.87, P＝0.01). For the total AML patients, the relapse_free survival (RFS) time in the WT1 gene mutated group was shorter than that in the wild_type group ( HR＝2.21, 95% CI 1.15-3.93, P＝0.02), but there was no significant difference in OS and disease_free survival (DFS) time between the two groups ( HR＝1.65, 95% CI 0.97-2.80, P＝0.06; HR＝0.46, 95% CI 0.08-2.57, P＝0.38). For the AML patients with normal karyotype and adult AML patients, there was no difference in OS time between the WT1 gene mutated group and wild_type group ( HR＝ 2.66, 95% CI 0.57-12.31, P＝ 0.21;HR＝ 2.10, 95% CI 0.70-6.30, P＝ 0.18). Conclusion WT1 gene mutation is a risk factor affecting OS of pediatric AML patients and RFS of general AML patients.

Significance of WT1 gene detection in the prognosis of acute myeloid leukemia patients with normal karyotype after hematopoietic stem cell transplantation / 白血病·淋巴瘤

Objective To investigate the monitoring significance of WT1 gene level in the prognosis of acute myeloid leukemia (AML) patients with normal karyotype after hematopoietic stem cell transplantation (HSCT). Methods The clinical data of 115 AML patients with normal karyotype who were treated with HSCT from July 2009 to March 2017 in the First Affiliated Hospital of Soochow University were retrospectively analyzed. The dynamic detection of bone marrow WT1 gene was carried out by using reverse transcription_polymerase chain reaction (RT_PCR). According to the relative expression level median of WT1 gene before transplantation, the whole patients were divided into the two groups (<median group and ≥median group) for survival analysis. Results There were 52 males and 63 females in 115 patients. The average age was (39± 10) years old. The median white blood cell count at initial diagnosis was 20.45×109/L [(0.5-355.9)×109/L], the ratio of blast cells in the bone marrow was 0.60±0.28, and the relative expression level median of WT1 gene was 87×104, while the median time of the follow_up was 24 (3-79) months. Among 115 patients, 19 cases relapsed. Remission group (96 cases) and relapse group (19 cases) were followed up. The WT1 gene level was monitored by using bone marrow puncture in 1 month, 3 months, 6 months, 12 months after transplantation. It was found that the WT1 gene relative expression level of relapse group was higher than that of remission group, and the differences between the two groups at 6 month_point [remission group (187±50)×104, relapse group (871±211)×104, t ＝ 2.519, P＝ 0.014] and 12 month_point [remission group (51±9)×104, relapse group (1 797±312)×104, t ＝ 4.000, P< 0.05] were statistically different. The overall survival (OS) rate of 2_year, progression_free survival rate in WT1 gene relative expression level < 87×104 group were higher than those in WT1 gene relative expression level ≥87×104 group, the relapse rate in WT1 gene relative expression level <87×104 group was lower than that in WT1 gene relative expression level ≥87×104 group, and the differences were statistically different (all P<0.05). Multivariate analysis showed that the level of WT1 gene at 12 months after transplantation was an independent factor affecting OS ( HR＝4.12, P＝0.046) and PFS ( HR＝5.95, P＝0.001). There were 19 cases of recurrence (16.5%), with a median relapsed time of 11 (1-60) months. When WT1 gene relative expression level was significantly increased in 19 patients, firstly immunosuppressive agents were reduced, of which 6 patients were not influenced by this intervention; in other 13 cases, only 5 cases were influenced by intervention. Conclusions For CN_AML patients, the expression level of WT1 gene before transplantation has a negative correlation with the prognosis. The expression level of WT1 gene at 12 months after transplantation is an independent factor for affecting the survival. The relapsed patients have a higher WT1 expression level, and clinical interventions for relapsed patients have a favorable effect.

Changes of WT1 mRNA expression level in patients with myelodysplastic syndromes after hypomethylating agents and its prognostic significance / 中华血液学杂志

Objective@#To monitor the WT1 mRNA level and its dynamic changes in patients with myelodysplastic syndromes (MDS) after hypomethylating agents (HMA) , as well as to assess the significance of WT1 mRNA levels and its dynamic changes in evaluating the efficacy of HMA and distinguishing the disease status of heterogeneous patients with stable disease (SD) .@*Methods@#Bone marrow or peripheral blood samples of 56 patients with MDS who underwent hypomethylating agents (≥4 cycles) from November 2009 to March 2018 were tested by real-time quantitative polymerase chain reaction (PCR) to detect the expression of WT1 mRNA, and to observe the correlation between the dynamic changes of WT1 mRNA expression and clinical efficacy and prognosis of patients.@*Results@#WT1 mRNA expression levels of MDS patients decreased significantly after 3 cycles of hypomethylating agent treatment. Besides, the WT1 mRNA expression levels of patients increased significantly after diseases progression. According to the dynamic changes of WT1 mRNA expression levels during SD, 45 cases could be further divided into increased group and non-increased group. In those SD patients with increased WT1 mRNA expression level, the ratio of suffering disease progression or transformation to AML was 95.65% (22/23) , whereas the ratio turned to be 9.09% (2/22) for the non-increased group (χ2=33.852, P<0.001) . Compared with those SD patients reporting no increase in WT1 mRNA expression level, the overall survival[17 (95%CI 11-23) months vs not reached, P<0.001] and progression-free survival [13 (95%CI 8-18) months vs not reached, P<0.001] of those SD patients reporting increase in WT1 mRNA expression level were significantly shorter.@*Conclusion@#WT1 mRNA expression level is a useful indicator to assess the efficacy of hypomethylating agents in MDS patients. Especially in patients with SD, detection of the changes in WT1 mRNA expression level is able to predict disease progression and help to make clinical decision.

An interlaboratory comparison study on the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels / 中华血液学杂志

Objective@#To investigate the current status and real performance of the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels in China through interlaboratory comparison.@*Methods@#Peking University People’s Hospital (PKUPH) prepared the samples for comparison. That is, the fresh RUNX1-RUNX1T1 positive (+) bone morrow nucleated cells were serially diluted with RUNX1-RUNX1T1 negative (-) nucleated cells from different patients. Totally 23 sets with 14 different samples per set were prepared. TRIzol reagent was added in each tube and thoroughly mixed with cells for homogenization. Each laboratory simultaneously tested RUNX1-RUNX1T1 and WT1 transcript levels of one set of samples by real-time quantitative PCR method. All transcript levels were reported as the percentage of RUNX1-RUNX1T1 or WT1 transcript copies/ABL copies. Spearman correlation coefficient between the reported transcript levels of each participated laboratory and those of PKUPH was calculated.@*Results@#①RUNX1-RUNX1T1 comparison: 9 samples were (+) and 5 were (-) , the false negative and positive rates of the 20 participated laboratories were 0 (0/180) and 5% (5/100) , respectively. The reported transcript levels of all 9 positive samples were different among laboratories. The median reported transcript levels of 9 positive samples were from 0.060% to 176.7%, which covered 3.5-log. The ratios of each sample’s highest to the lowest reported transcript levels were from 5.5 to 12.3 (one result which obviously deviated from other laboratories’ results was not included) , 85% (17/20) of the laboratories had correlation coefficient ≥0.98. ②WT1 comparison: The median reported transcript levels of all 14 samples were from 0.17% to 67.6%, which covered 2.6-log. The ratios of each sample’s highest to the lowest reported transcript levels were from 5.3-13.7, 62% (13/21) of the laboratories had correlation coefficient ≥0.98. ③ The relative relationship of the reported RUNX1-RUNX1T1 transcript levels between the participants and PKUPH was not always consistent with that of WT1 transcript levels. Both RUNX1-RUNX1T1 and WT1 transcript levels from 2 and 7 laboratories were individually lower than and higher than those of PKUPH, whereas for the rest 11 laboratories, one transcript level was higher than and the other was lower than that of PKUPH.@*Conclusion@#The reported RUNX1-RUNX1T1 and WT1 transcript levels were different among laboratories for the same sample. Most of the participated laboratories reported highly consistent result with that of PKUPH. The relationship between laboratories of the different transcript levels may not be the same.

Anti-tumorimmune effects of WT1 polypeptide vaccine on SCID mice with xenografted human monocytic leukemia / 西安交通大学学报(医学版)

Objective: To study the anti-tumor immune effects of WT1 peptide vaccine in SCID mice with xenografted human monocytic leukemia. Methods: Twenty-four hours after intraperitoneal injection of human peripheral blood lymphocytes, the xenograft human monocytic leukemia model in SCID mice was established by subcutaneous inoculation of THP1 cells. The mice were randomly divided into three groups with eight mice in each. Blank control group was vaccinated with incomplete Freund's adjuvant (IAF). Helper T cell epitope group was vaccinated with helper T cell epitopes and IAF. WT1 group was vaccinated with WT1 peptide, helper T cell epitopes and IAF. When the tumor volume grew to 100 mm3, intraperitoneal injection of vaccine components started. The SCID mice were killed 14 days after vaccination. LDH release method was adopted to detect the specific CTL killing activity of spleen cells. Histological characteristics of tumor tissue were observed under microscope after HE staining. Flow cytometry was used to test the levels of peripheral blood CD3+/CD4+T cells, CD3+/CD8+T cells and CD4+CD25+ Treg cells. ELISA method was applied to detect the levels of serum immunoglobulin, IL-2, γ-interferon, TGF-β and IL-10. Results: The xenograft human monocytic leukemia model was successfully established in SCID mice and tumor developed in all the SCID mice. In WT1 group, the activity of mouse spleen cells on THP1 cells was significantly higher than that in helper T cell epitope group and control group (P<0.05). The mean weight and volume of tumor were significantly lower in WT1 group than in helper T cell epitope group and control group (P<0.05). A large amount of tumor cell degeneration and necrosis was observed under the microscope in WT1 group mice and few tumor cells survived. Peripheral blood levels of CD3+/CD4+T cells, CD3+/CD8+T cells, IgG, IFN-γ and IL-2 were all higher in WT1 group than in helper T cell epitope group and control group (P<0.05). However, peripheral blood levels of CD4+/CD25+Treg cells, TGF-β and IL-10 were all lower in WT1 group than in helper T cell epitope group and control group (P<0.05). Conclusion: WT1 polypeptide vaccine can effectively produce anti-tumor immunity and kill leukemia cells in SCID mice with exnografted human monocytic leukemia.

WT1 expression as a potential biomarker of malignancy in canine breast tumor / La expresión de WT1 como un biomarcador potencial maligno en un tumor de mama canino

SUMMARY: Veterinary oncology is very important nowadays to get a better understanding of human carcinogenesis. Estrogen receptor, progesterone receptor and Human Epidermal Growth Factor receptor 2 are frequently evaluated by immunohistochemistry (HIC) in human breast tumor. WT1 is an oncogene, its overexpression has been detected in leukemia and diverse solid tumors like breast cancer, lung cancer and mesothelioma in humans. WT1 expression was evaluated in 15 canine breast tumors (CBT) diagnosed by histopathological analysis to find its relationship with neoplasia and malignancy. IHC and RT-PCR were performed in CBT tissues. Fisher´s test was used to analyze WT1 relationship with malignancy. Of the 15 tumors, 9 (60 %) were diagnosed as benign and 6 (40 %) were malignant. With IHC, WT1 expression was positive only in biopsies diagnosed as malignant. Expression of WT1 by RT-PCR was detected in 14 of the 15 tumors (93.33 %) as well as in control healthy mammary gland. Clinical significance: This study describes for the first time a close correlation between CBT and a positive result for WT1 expression with IHC; hence, it can be used as a biomarker for this neoplasia and as an indicator of malignancy. RT-PCR analysis also showed to be good option to detect WT1 expression. These results will be useful to further investigations to elucidate WT1-related signaling pathways in CBT. Also to know molecules that regulate the translation of this protein as a marker for tumor progression.

RESUMEN: La oncología veterinaria es muy importante hoy en día para comprender mejor la carcinogénesis humana. El receptor de estrógeno, el receptor de progesterona y el receptor 2 del factor de crecimiento epidérmico humano se evalúan con frecuencia mediante inmunohistoquímica (HIC) en tumores de mama humanos. WT1 es un oncogén, su sobreexpresión se ha detectado en leucemia y en diversos tumores sólidos como el cáncer de mama, cáncer de pulmón y mesotelioma en humanos. La expresión de WT1 se evaluó en 15 tumores de mama caninos (TCC) diagnosticados mediante análisis histopatológico para encontrar su relación con la neoplasia y la malignidad. IHC y RT-PCR se realizaron en tejidos CBT. La prueba de Fisher se utilizó para analizar la relación de WT1 con la malignidad. De los 15 tumores, 9 (60 %) fueron diagnosticados como benignos y 6 (40 %) fueron malignos. Con IHC, la expresión de WT1 fue positiva solo en biopsias diagnosticadas como malignas. La expresión de WT1 por RT-PCR se detectó en 14 de los 15 tumores (93,33 %), así como en el control de la glándula mamaria sana. Importancia clínica: este estudio describe por primera vez una estrecha correlación entre la TCC y un resultado positivo para la expresión de WT1 con IHC; por lo tanto, se puede utilizar como un biomarcador para esta neoplasia y como un indicador de malignidad. El análisis por RT-PCR también demostró ser una buena opción para detectar la expresión de WT1. Estos resultados serán útiles para futuras investigaciones para dilucidar las vías de señalización relacionadas con WT1 en la TCC. También para conocer moléculas que regulan la traducción de esta proteína como marcador de progresión tumoral.

Expression of WT1 gene in bone marrow mononuclear cells of patients with myelodysplastic syndromes / 白血病·淋巴瘤

Objective To analyze the relationship between the expression of WT1 gene in bone marrow mononuclear cells and the cytogenetic characteristics, curative effect and prognosis in patients with myelodysplastic syndromes (MDS). Methods The quantitative expression of WT1 gene was detected by real-time quantitative polymerase chain reaction (RQ-PCR) in bone marrow mononuclear cells of 115 MDS patients who were admitted to Taixing People's Hospital from August 2010 to March 2018, and the relationship between the expression of WT1 gene and the cytogenetic characteristics, curative effect and prognosis were evaluated. Results WT1 gene was highly expressed in 80 cases (69.6 %), lowly expressed in 15 cases (13.0 %), and unexpressed in 20 cases (17.4 %). Of the 80 patients with high WT1 expression, 56 cases (70.0 %) were refractory anemia with excess blasts (RAEB), and 22 cases (27.7 %) were refractory cytopenia with multilineage dysplasia (RCMD). In 55 patients with very poor cytogenetic prognosis, 44 patients had high expression of WT1 gene, and in 28 patients with poor prognosis, 19 patients had high expression of WT1 gene. The complete remission rate of WT1 gene high expression group [12.5 % (10/80)] was lower than that of low expression group [26.7 % (4/15)] and unexpressed group [40.0 % (8/20)], and the difference of complete remission rate between the three groups was statistically significant (χ2＝ 8.96, P< 0.05). Conclusion MDS patients with high expression of WT1 gene have low remission rate and poor prognosis.

Clinical and pathological features and mutational types of WT1 mutation-associated nephropathy / 中华儿科杂志

Objective@#To explore the clinical and pathological features and mutational types and their relations with WT1 mutation-associated nephropathy (WT1MAN).@*Methods@#The clinical and pathological data and the results of WT1 mutation analysis of the cases from Nanfang Hospital of Southern Medical University, Sun Yat-sen Memorial Hospital and The First Affiliated Hospital of Sun Yat-sen University whom we recruited recently and reported during the last ten years were analyzed.@*Results@#Totally, 20 cases (6 males and 14 females), included 5 newly diagnosed cases, were recruited. (1) Ten children were diagnosed with Denys-Drash syndrome (DDS): The median onset age of proteinuria was 1 year and 7 months. Diffuse mesangial sclerosis (DMS) were revealed in 3 cases, minimal lesions (MCD) in 4 cases, and focal segmental glomerulosclerosis (FSGS) in 1 case; renal pathology was not available in the other 2 cases. Glomerular basement membrane (GBM) thickening was observed in 2 cases. Calcineurin inhibitors (CNIs) were administered in 5 cases, complete remission of proteinuria was observed in 3 cases, partial remission in the other 2 cases. Genetic analysis revealed that six cases had WT1 missense mutation, 3 had nonsense mutation, and 1 had frameshift mutation. (2) Two cases were diagnosed with Frasier syndrome (FS): proteinuria was observed at 1 year and 1 month of age and 1 year and 9 months of age, respectively. FSGS with GBM layering were observed in both cases. They progressed to ESRD at 1 year and 6 months of age and 6 years and 6 months of age, respectively. CNI was tried in 1 case with partial proteinuria remission. Both patients were detected to have WT1 splice mutation. (3) Isolated nephropathy (IN) was observed in 8 cases: three had splice mutation, 5 had missense mutation. Of the 3 patients with splice mutation, one was found to have nephropathy and renal failure at the age of 5 months. The other two cases (1 was FSGS and another MCD), both had GBM layering. CNIs were tried on both of them, one got partial remission with normal renal function at the age of fourteen years, the other one had no response and entered ESRD at the age of 6 years and 9 months. Of the 5 cases with missense mutation, 3 had DMS, 2 of them entered ESRD within 6 months of age, another case had DMS entered ESRD at 9 years of age. One case with FSGS, was treated with CNIs and got complete remission.@*Conclusions@#Slow progression (7/10) nephropathy was observed in DDS patients. Missense mutation (11/20) was the most common type of WT1 variants, followed by splice mutation (5/20) in this group of patients. Early onset nephropathy (4/5), rapid progression (4/5) and GBM layering (4/4) wereobserved in patients with splice mutation. CNI was effective in reducing or even eliminating proteinuria in WT1 MAN patients (8/9).

Clinical implication of minimal residue disease monitoring by WT1 gene detection and flow cytometry in myelodysplastic syndrome with allogeneic stem cell transplantation / 中华血液学杂志

Objective@#To investigate the clinical significance of minimal residual disease (MRD) monitoring by using WT1 gene and flow cytometry (FCM) in patients with myelodysplastic syndrome (MDS) who receiving allogeneic stem cell transplantation (allo-HSCT).@*Methods@#WT1 gene and MDS-related abnormal immunophenotype were examined by real-time quantitative polymerase chain reaction (RQ-PCR) and FCM, respectively. The bone marrow samples were collected from patients with MDS who received allo-HSCT from Feb, 2011 to Oct, 2015 in Peking University People’s Hospital before and after transplantation.@*Results@#Among 92 MDS patients, 40 (48.2%) patients were positive for WT1 (WT1+) and 9 (10.8%) patients were positive for flow cytometry (FCM+). 27 patients (29.3%) met the criteria of our combinative standard, MRDco (MRDco+). Only FCM+ post-transplant (P<0.001) and MRDco+ (P=0.017) were associated with relapse. The cumulative incidence of relapse (CIR) at 2 years were 66.7% and 1.2% (P<0.001) in FCM+ and FCM- groups. MRDco+ group had a 2-year CIR of 23.0% while MRDco- group had a 2-year CIR of 1.6% (P=0.004). The specificity of post-transplant WT1, FCM and MRDco to predict relapse was 59.0%, 96.4% and 74.7%, respectively. The sensitivity of these three MRD parameters to predict relapse was 66.7%.@*Conclusion@#Post-transplant FCM and MRDco are useful tools to monitor MRD for MDS after transplantation. The preemptive intervention based on MRDco is able to reduce the relapse rate.