WT1 Gene Research Articles

Comprehensive genomic profiling of malignant peritoneal mesothelioma (MPeM) reveals key genomic alterations (GAs) distinct from malignant pleural mesothelioma (MPM).

8557 Background: MPeM is a rare and aggressive cancer with very limited treatment options. Lack of dedicated research has impeded improvements in outcomes. Defining prevalent GAs is a critical unmet need for use of targeted therapies in these patients. Although MPeM is notably distinct from MPM vis-à-vis epidemiologic and clinical attributes, the genomic underpinings of these differences have yet to be established. We aimed at describing a comprehensive genomic profile (CGP) of MPeM in comparison to MPM. Methods: We performed a retrospective comparative analysis between 89 patients with MPeM and 241 patients with MPM (N = 330) who underwent CGP using CLIA certified next-generation sequencing assays. The cohort was generated using mesothelioma patients at MD Anderson Cancer Center (N = 223) and supplemented by additional mesothelioma patients (N = 107) from a publicly available database from Memorial Sloan Kettering Cancer Center, the MSK-IMPACT database. Essential clinicopathological variables were collected. Descriptive statistics, Fisher’s exact and Mann-Whitney tests were used for comparison. Kaplan-meier method and log rank tests were used for overall survival (OS) estimates. Results: MPeM cohort (vs. MPM) had more women (54% vs. 31%, P < 0.001) and younger age at diagnosis (56 vs. 69 years, P < 0.001). Histology was epithelioid, biphasic and sarcomatoid in 86%, 7% and 7% cases, a distribution similar to MPM cohort. At least 1 GA was found in 64 (72% vs. 82% in MPM, P = 0.044) of MPeM patients with a median of 1 (range 1 – 12) (vs. a median of 2, range 1 – 24, P < 0.001) GA per patient. A significantly lower proportion of MPeM patients had ≥ 3 mutations (14% vs. 26%, OR 2.1, P = 0.028) per patient. The most frequent mutations were present in the following genes: TP53 (24%), BAP1 (16%), NF2 (15%), MET (9%) and TRAF7, KIT and PIK3CA (each 6%). MPeM patients harbored more mutations in MET (9% vs. < 1%, P < 0.001) and TRAF7 (6% vs. < 1%, P = 0.02) but fewer mutations in BAP1 (16% vs. 32%, P = 0.003) and CDKN2B (0% vs. 5%, P = 0.041). The most common copy number variations (CNVs: amplifications or deletions) were seen in BAP1, MCL1, SETD2, WT1 (each 2%) and AURKA (1%) genes. Among genes with CNVs, MPeM had a lower rate of deletions in CDKN2A (1% vs. 6%, P = 0.040). Among more common GAs, only BAP1 mutations appeared to be associated with poor OS (45.7 vs. 127.1 months, HR 2.5, 95%CI: 0.6 – 10.1, P = 0.050) in patients with MPeM. Conclusions: In this large cohort with CGP, we identified potential molecular drivers in MPeM and demonstrated key genomic differences between MPeM and MPM. MPeM is frequently driven by GAs involved in cell cycle control, a potentially targetable pathway. Despite this insight from CGP, a large subset of patients do not have actionable GAs and for these patients, further collaborative trans-“omic” research efforts are needed to advance potential therapeutic options.

14q32 rearrangements deregulating BCL11B mark a distinct subgroup of T-lymphoid and myeloid immature acute leukemia

AbstractAcute leukemias (ALs) of ambiguous lineage are a heterogeneous group of high-risk leukemias characterized by coexpression of myeloid and lymphoid markers. In this study, we identified a distinct subgroup of immature acute leukemias characterized by a broadly variable phenotype, covering acute myeloid leukemia (AML, M0 or M1), T/myeloid mixed-phenotype acute leukemia (T/M MPAL), and early T-cell precursor acute lymphoblastic leukemia (ETP-ALL). Rearrangements at 14q32/BCL11B are the cytogenetic hallmark of this entity. In our screening of 915 hematological malignancies, there were 202 AML and 333 T-cell acute lymphoblastic leukemias (T-ALL: 58, ETP; 178, non-ETP; 8, T/M MPAL; 89, not otherwise specified). We identified 20 cases of immature leukemias (4% of AML and 3.6% of T-ALL), harboring 4 types of 14q32/BCL11B translocations: t(2,14)(q22.3;q32) (n = 7), t(6;14)(q25.3;q32) (n = 9), t(7;14)(q21.2;q32) (n = 2), and t(8;14)(q24.2;q32) (n = 2). The t(2;14) produced a ZEB2-BCL11B fusion transcript, whereas the other 3 rearrangements displaced transcriptionally active enhancer sequences close to BCL11B without producing fusion genes. All translocations resulted in the activation of BCL11B, a regulator of T-cell differentiation associated with transcriptional corepressor complexes in mammalian cells. The expression of BCL11B behaved as a disease biomarker that was present at diagnosis, but not in remission. Deregulation of BCL11B co-occurred with variants at FLT3 and at epigenetic modulators, most frequently the DNMT3A, TET2, and/or WT1 genes. Transcriptome analysis identified a specific expression signature, with significant downregulation of BCL11B targets, and clearly separating BCL11B AL from AML, T-ALL, and ETP-ALL. Remarkably, an ex vivo drug-sensitivity profile identified a panel of compounds with effective antileukemic activity.

Generation of the induced pluripotent stem cell line (NCKDi002-A) from a 22-year-old patient with Focal Segmental Glomerular Sclerosis carrying a heterozygous mutation in WT1 gene

Focal Segmental Glomerular Sclerosis (FSGS) is a glomerular disease which can be classified into primary, secondary, genetic, and unknown forms. WT1 mutation has been shown to be associated with this disorder. Recently, we identified a mutation in the Zinc finger C2H2 domain of WT1 gene in a patient with FSGS who also carried a family history of end-stage renal disease (ESRD). The Peripheral Blood Mononuclear Cells (PBMCs) of the patient were obtained and a line of induced pluripotent stem cells (iPSCs) was successfully generated. The iPSC line will be useful for further study of the pathogenesis and drug screening for FSGS.

Molecular Study of Childhood Steroid-Resistant Nephrotic Syndrome: A Hospital-Based Study.

Steroid-resistant nephrotic syndrome (SRNS) patients with genetic mutations most commonly have histology of focal segmental glomerulosclerosis (FSGS) and do not respond to immunosuppressive drugs. We report the molecular screening results of 18 pediatric SRNS cases presented to our nephrology clinic. Three pathogenic variants have been detected, two previously reported and one novel variant. The reported pathogenic variants have been detected in NPHS1 and NPHS2 genes. A novel pathogenic variant has been detected in the inverted formin 2 gene ( INF2 ) gene. We did not detect any variant of the WT1 gene. There were 13 males. Mean age of study participants at enrollment was 69 months. There were 12 cases of primary SRNS. The mean duration from onset of symptoms to SRNS diagnosis was 13 months. FSGS and minimal change disease (MCD) were present in the same number of cases. The response rate (complete or partial) to immunosuppressive drugs was seen in only one patient in the genetic SRNS group ( n = 3), while the response rate in nongenetic cases ( n = 15) was 80%. Two nonresponders in the genetic SRNS group had FSGS for histopathology and pathogenic variants (NPHS2 and INF2). The other three nonresponders in the nongenetic SRNS group had both FSGS ( n = 1) and MCD ( n = 2) histopathology. There were two deaths in the study cohort of the nongenetic SRNS group. This study highlights the screening of the SRNS cohort by a panel of extended genes rather focussing on the three most common genes ( NPHS1 , NPHS2 , and WT1 ). This further confirms the molecular etiology of SRNS in three cases and extends the list of pathogenic variants of genetic SRNS in the North Indian population. This is the first study in the eastern part of Uttar Pradesh in India.

Desmoplastic Small Round Cell Tumor: A Review of Main Molecular Abnormalities and Emerging Therapy.

Simple SummaryDesmoplastic small round cell tumor is a rare neoplasm with extremely aggressive behavior. Despite the multimodal treatment for newly diagnosed patients with chemotherapy, cytoreductive surgery and radiation, the cure rate is still low. For relapsed or progressive disease, there is limited data regarding second and third-line therapies. Novel agents have shown only modest activity. Recent molecular changes have been identified in this disease and this opens opportunities to be explored in future clinical trials.Desmoplastic small round cell tumor (DSRCT) is an extremely rare, aggressive sarcoma affecting adolescents and young adults with male predominance. Generally, it originates from the serosal surface of the abdominal cavity. The hallmark characteristic of DSRCT is the EWSR1–WT1 gene fusion. This translocation up-regulates the expression of PDGFRα, VEGF and other proteins related to tumor and vascular cell proliferation. Current management of DSRCT includes a combination of chemotherapy, radiation and aggressive cytoreductive surgery plus intra-peritoneal hyperthermic chemotherapy (HIPEC). Despite advances in multimodal therapy, outcomes remain poor since the majority of patients present disease recurrence and die within three years. The dismal survival makes DSRCT an orphan disease with an urgent need for new drugs. The treatment of advanced and recurrent disease with tyrosine kinase inhibitors, such as pazopanib, sunitinib, and mTOR inhibitors was evaluated by small trials. Recent studies using comprehensive molecular profiling of DSRCT identified potential therapeutic targets. In this review, we aim to describe the current studies conducted to better understand DSRCT biology and to explore the new therapeutic strategies under investigation in preclinical models and in early phase clinical trials.

Surgical reconstruction of abnormally located penis in urorectal septum malformation sequence: report of a case

Urorectal septum malformation sequence (URSM) is a rare congenital anomaly of caudal end of mesoderm. There is usually a single or no opening in the perineum. An infant with dysmorphic facial features, ambiguous genitalia, meningomyelocele, pes equinovarus, left renal agenesis, severe hydronephrosis of the right kidney, and anuria requiring right ureterocutaneostomy during the neonatal period is reported. Initial physical examination revealed fusion of labioscrotal folds forming a single sac without a raphe, and a single gonad on the left side of the sac, less than 1 mm in size. There was no phallus, urethral opening, or any palpable cavernous body. Karyotype analysis from peripheral blood was 46XY. Chromosomal microarray analysis using Agilent 8X60K platform revealed arr [hg19](1-22)x2,(X,Y)x1. DNA sequence analysis for WT1 gene was normal. During inguinal hernia repair, cavernous bodies were detected in the retroperitoneal space under the hernia sac intraoperatively. Penis was reconstructed using two rotational flaps from the midline skin above the pubic bone to cover the cavernous bodies. This report aims to discuss the clinical features as well as surgical options for reconstruction of an abnormally located penis in an infant with partial URSM. Level of evidence: Level V, therapeutic study.

Moyamoya Syndrome in Post-Transplant Children with Type 1 Neurofibromatosis and Congenital Nephrotic Syndrome

Background: Neurofibromatosis (NF) is a genetic disorder of the nervous system that affects the development and growth of neuronal tissues. It is characterized by the development of malignant and benign tumors of the peripheral nerves and meningiomas, and the clinical manifestations can vary, including cerebral vascular compromise that precipitates symptoms characteristic of Moyamoya syndrome. The behavior of a posttransplant child with type 1 NF who presents with characteristics of Moyamoya syndrome and is a carrier of a WT1 genetic mutation is unknown, which is why the report was made. Case Report: A child with type I NF with a history of cryptorchidism, hypospadias, bilateral sensorineural hearing loss, and chronic renal failure was arising from a congenital nephrotic syndrome due to a WT1 gene mutation at 3 years of age began renal replacement therapy initially with chronic ambulatory peritoneal dialysis and hemodialysis for refractory peritonitis. He underwent two episodes of transient ischemic attack (TIA) characterized by right hemiparesis during hemodialysis therapy and during six months post-kidney transplantation present a similar episode. Studies for other causes were negative, and imaging studies show characteristics of NF-related vasculitis, cortical lesions, and focal lesions in the left frontal subcortical white matter with annular enhancement and subcortical edema in the parasagittal region of the left parietal lobe, as well as a decrease in flow in the cortical branches of the middle cerebral artery were described. After four weeks, the clinical manifestations of hemiparesis completely disappeared without leaving any sequels continuing with the regular immunosuppression of the everolimus, mycophenolate and low dose steroids. Conclusion: These findings are suggestive of small and mediumsized vessel vasculitis compatible with Moyamoya syndrome in child post-transplantation associated with arteriopathy by NF.

Comprehensive Biology and Genetics Compendium of Wilms Tumor Cell Lines with Different WT1 Mutations.

Simple SummaryWilms tumor is a childhood kidney tumor arising from embryonal cells. Wilms tumors are heterogeneous with several distinct subgroups that differ in their response to treatment. The genetic basis for these diverse forms of Wilms tumor is not fully understood. One subgroup of Wilms tumors is associated with mutations in the WT1 gene, encoding a transcription factor with a role in early kidney differentiation. Patients with WT1 mutant Wilms tumor may harbor germline mutations in this gene. Cell lines from Wilms tumors are notoriously difficult to establish and only few exist. We developed a method to cultivate cells from the WT1 mutant subtype of Wilms tumors and have established 11 cell lines with different mutations in WT1 to date. These cells will be instrumental to study the biology and genetics ultimately to develop precision treatmentsPurpose: WT1 mutant Wilms tumors represent a distinct subgroup, frequently associated with CTNNB1 mutations. The genetic basis for the development of this subtype is currently not fully understood. Methods: Live WT1 mutant Wilms tumors were collected during surgery of patients and cell cultures established in mesenchymal stem cell medium. They were studied for mutations in WT1 and CTNNB1, their differentiation capacity and protein activation status. Four cell lines were immortalized with a triple mutant ts SV40 largeT antigen and Telomerase. Results: 11 cell lines were established from Wilms tumors of nine patients, including a left and right tumor from the same patient and a primary and second tumor from another patient. Six patients had germ line and three were tumor specific mutations. All cell lines harbored only mutant or deleted WT1 genes. CTNNB1 was wild type in three, all others carried mutations affecting amino acid S45. They had variable and limited capacities for mesenchymal differentiation, a high migratory capacity and a low invasive potential. All cells showed an activation of multiple receptor tyrosine kinases and downstream signaling pathways. Conclusions: These cell lines represent an important new tool to study WT1 mutant Wilms tumors, potentially leading to new treatment approaches.

Differentially methylated CpG sites associated with the high-risk group of prostate cancer

Prostate cancer (PC) is one of the most common and socially significant oncological diseases among men. Bioinformatic analysis of omics data allows identifying molecular genetic changes associated with the disease development, as well as markers of prognosis and response to therapy. Alterations in DNA methylation and histone modification profiles widely occur in malignant tumors. In this study, we analyzed changes in DNA methylation in three groups of PC patients based on data from The Cancer Genome Atlas project (TCGA, https://portal.gdc.cancer.gov): (1) high- and intermediate-risk of the tumor progression, (2) favorable and unfavorable prognoses within the high-risk group, and (3) TMPRSS2-ERG-positive (tumors with TMPRSS2-ERG fusion transcript) and TMPRSS2-ERG-free cases within the high-risk group. We found eight CpG sites (cg07548607, cg13533340, cg16643088, cg18467168, cg23324953, cg23753247, cg25773620, and cg27148952) hypermethylated in the high-risk group compared with the intermediate-risk group of PC. Seven differentially methylated CpG sites (cg00063748, cg06834698, cg18607127, cg25273707, cg01704198, cg02067712, and cg02157224) were associated with unfavorable prognosis within the high-risk group. Six CpG sites (cg01138171, cg14060519, cg19570244, cg24492886, cg25605277, and cg26228280) were hypomethylated in TMPRSS2-ERG-positive PC compared to TMPRSS2-ERG-negative tumors within the high-risk group. The CpG sites were localized, predominantly, in regulatory genome regions belonging to promoters of the following genes: ARHGEF4, C6orf141, C8orf86, CLASP2, CSRNP1, GDA, GSX1, IQSEC1, MYOF, OR10A3, PLCD1, PLEC1, PRDM16, PTAFR, RP11-844P9.2, SCYL3, VPS13D, WT1, and ZSWIM2. For these genes, analysis of differential expression and its correlation with CpG site methylation (β-value level) was also performed. In addition, STK33 and PLCD1 had similar changes in colorectal cancer. As for the CSRNP1, the ARHGEF4, and the WT1 genes, misregulated expression levels were mentioned in lung, liver, pancreatic and androgen-independent prostate cancer. The potential impact of changed methylation on the mRNA level was determined for the CSRNP1, STK33, PLCD1, ARHGEF4, WT1, SCYL3, and VPS13D genes. The above CpG sites could be considered as potential prognostic markers of the high-risk group of PC.

Expression of WT1 in renal rhabdoid tumors: immunohistochemistry and molecular genetics

Renal rhabdoid tumor (RRT) is the most malignant and aggressive renal tumors in children. The results of immunohistochemistry show co-expression of epithelial and mesenchymal markers with total loss of INI1 expression in tumor cells. Aim. To determine the frequency of WT1 expression in RRT using three different clones and to investigate WT1 gene status. This study is supported by the Independent Ethics Committee and approved by the Academic Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. 28 patients with RRT were included in our study from a period 2006–2019. Immunohistochemical staining included Vimentin, panCK, CK19, EMA, INI1, CD34, WT1 (clones: polyclonal, WT49, 6FH2). Additionally, in order to evaluate genetic events known to be significant in the RRT pathogenesis, molecular genetic testing by multiplex ligase-dependent probe amplification (NGS) and next-generation sequencing (NGS) of customized gene panel were conducted in 15 cases with available material. All tumors had classical rhabdoid morphology with co-expression of epithelial and mesenchymal markers and total loss of INI1 expression. Two tumors were WT1-positive (WT49 and Polyclonal). 5 out of 15 tumors harbored different in length SMARCB1 gene deletions. 7 cases studied by NGS for nucleotide substitutions and small indels included 5 patients lacking SMARCB1 deletions based on the MLPA data and 2 cases of RRT with WT1 protein nuclear expression. All evaluated patients had pathogenic or likely-pathogenic genetic variants in genes encoding core subunits of SWI/ SNF chromatin remodeling complex (predominantly, SMARCB1 and SMARCA4 genes). No pathogenic variants were revealed in WT1 gene. The literature describes isolated cases of WT1 expression in RRT elements. Often, the cellular elements of a rhabdoid tumor after preoperative chemotherapy are regarded as elements of the stromal component of nephroblastoma, maturing into rhabdomyoblasts. In the presence of WT1 expression, these tumors are considered as nephroblastomas. In this study we showed that RRTs are molecularly heterogeneous tumors with aberrant immunophenotype. Thus, our experience proves the importance of an integrated approach to differential diagnosis and the need for routine use of the INI1 antibody in panels for the study of kidney tumors in children to avoid misinterpretation of morphological data.

Differential expression of microRNAs in the hippocampi of male and female rodents after chronic alcohol administration

BackgroundWomen are more vulnerable than men to the neurotoxicity and severe brain damage caused by chronic heavy alcohol use. In addition, brain damage due to chronic heavy alcohol use may be associated with sex-dependent epigenetic modifications. This study aimed to identify microRNAs (miRNAs) and their target genes that are differentially expressed in the hippocampi of male and female animal models in response to alcohol.MethodsAfter chronic alcohol administration (3~3.5 g/kg/day) in male (control, n = 10; alcohol, n = 12) or female (control, n = 10; alcohol, n = 12) Sprague-Dawley rats for 6 weeks, we measured body weights and doublecortin (DCX; a neurogenesis marker) concentrations and analyzed up- or downregulated miRNAs using GeneChip miRNA 4.0 arrays. The differentially expressed miRNAs and their putative target genes were validated by RT-qPCR.ResultsAlcohol attenuated body weight gain only in the male group. On the other hand, alcohol led to increased serum AST in female rats and decreased serum total cholesterol concentrations in male rats. The expression of DCX was significantly reduced in the hippocampi of male alcohol-treated rats. Nine miRNAs were significantly up- or downregulated in male alcohol-treated rats, including upregulation of miR-125a-3p, let-7a-5p, and miR-3541, and downregulation of their target genes (Prdm5, Suv39h1, Ptprz1, Mapk9, Ing4, Wt1, Nkx3-1, Dab2ip, Rnf152, Ripk1, Lin28a, Apbb3, Nras, and Acvr1c). On the other hand, 7 miRNAs were significantly up- or downregulated in alcohol-treated female rats, including downregulation of miR-881-3p and miR-504 and upregulation of their target genes (Naa50, Clock, Cbfb, Arih1, Ube2g1, and Gng7).ConclusionsThese results suggest that chronic heavy alcohol use produces sex-dependent effects on neurogenesis and miRNA expression in the hippocampus and that sex differences should be considered when developing miRNA biomarkers to diagnose or treat alcoholics.

WT-1, BAALC, and ERG Expressions in Iranian Patients with Acute Myeloid Leukemia Pre- and Post-chemotherapy.

Purpose: Acute myeloid leukemia (AML) is the most prevalent acute leukemia in adults. It possesses different cytogenetic and molecular features. The expression of Wilms tumor-1 (WT1), brain and acute leukemia, cytoplasmic (BAALC) and ETS-related gene (ERG) might be considered as prognostic factors in AML patients. The aim of this study was to determine the mRNA expressions of WT-1, BAALC and ERG genes in bone marrow of mononuclear cells and their effects on complete remission in the Iranian AML patients, pre- and post- chemotherapy. Methods: Forty AML patients with normal karyotype were evaluated. The mRNA gene expressions were measured with quantitative real-time PCR in bone marrow of mononuclear cells of AML patients at the baseline and after chemotherapy. The subtypes of AML and flow cytometry panel were also assessed. Complete remission (CR) after the treatment was addressed for all patients. Results: The mRNA expressions of WT-1, BAALC and ERG were significantly decreased after the treatment (p = 0.001, 0.017, 0.036). WT-1 mRNA expression was inversely correlated with CR after chemotherapy (P =0.024). There was also significant correlation between baseline expression of BAALC and CR (P =0.046). No significant correlation was observed between ERG and CR pre- and post- chemotherapy (P =0.464 and 0.781). There was also significant correlation between BAALC mRNA expression and CD34+ (P <0.001). Conclusion: The present study showed that WT-1 decreased significantly after standard chemotherapy which could have favorable effects on CR. Also, the high expression of BAALC could have a poor prognostic role in AML patients. The identification of these gene expressions can be an efficient approach in targeted therapy among AML patients.

The Prognostic Significance of Wilms' Tumor Gene 1 in Adult AML with Different Risk Stratification Following Allo-HSCT

INTRODUCTION: Wilms' tumor gene 1 is a "pan-leukemic marker" for evaluating measurable residual disease (MRD) in leukemia. The prognostic value in prediction of relapse following chemotherapy or allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been recognized. However, there lack relevant guidelines for WT1 gene quantification as a MRD assessment for patients with acute myeloid leukemia (AML) undergoing allo-HSCT. The optimal monitoring time and threshold of WT1 expression level for MRD assessment following allo-HSCT are still unclear. METHODS: We retrospectively investigated the dynamic change of WT1 expression level in bone marrow in 151 adult patients with AML following allo-HSCT. All patients were divided into two subgroup including complete remission (CR) and non-remission (NR), which was based on the disease status before HSCT. WT1 expression levels were compared between two subgroups. Meanwhile, Receiver operating characteristic (ROC) curve analysis was performed on WT1 expression levels at different time points and the highest value of WT1 before relapse-related intervention to find the cutoff values for relapse prediction. Furthermore, the cutoffs were tested by cumulative incidence of relapse (CIR), overall survival (OS) and disease-free survival (DFS), which were analyzed through the Kaplan-Meier method. RESULTS: Compared with complete remission (CR) subgroup, non-remission (NR) subgroup has a trend of a higher expression baseline and a rapidly increasing from 2 to 3 months after allo-HSCT. ROC curve analysis results showed that 0.6% was the optimal cutoff value for relapse prediction at 3 months after allo-HSCT. The cutoff level 0.6% was effective in identifying patients in CR and NR group. Among the patients with CR status (n=131), patients with WT1≥0.6% had a significantly higher 5-year cumulative incidence of relapse (CIR, 27.3% vs. 6.0%; p=0.006), than patients with WT1&amp;lt;0.6%. Meanwhile, among the patients with NR status before allo-HSCT (n=20), patients with WT1≥0.6% had a significantly higher 5-year cumulative incidence of relapse (CIR, 75.7% vs. 40.0%; p=0.021), than patients with WT1&amp;lt;0.6%.Although 0.6% was effective in both group, there was no significant difference in OS between the patients with WT1≥0.6% and &amp;lt;0.6% in CR group (p&amp;gt;0.05). Moreover, ROC analysis was performed using the highest value of WT1 before relapse-related intervention, which showed that 2.25% was the upper threshold for predicting relapse after transplantation. The upper cutoff 2.25% was only effective in CR group. In CR group, patients with WT1≥2.25% had a significantly higher 5-year cumulative incidence of relapse (CIR, 100% vs. 7.1%; p&amp;lt;0.001), than patients with WT1&amp;lt;2.25%. In NR group, there was no significant difference in the 5-year CIR between patients with WT1≥2.25% and patients with WT1&amp;lt;2.25% (CIR, 75.0% vs. 53.8%; p=0.215). Results showed that the cutoff 0.6% was the optimal threshold for NR patients, while the cutoff 2.5% or 1.2% might be the optimal threshold for CR group in relapse prediction. Because the intervention guided by higher cutoff value would decrease the transplant-related mortality which might be caused by more aggressive relapse intervention in CR group. CONCLUSION: Our results suggest that the relapse predictive WT1 level is different in patients with different risk stratification before HSCT and the 3th months after allo-HSCT is the most meaningful time point for WT1-based MRD monitoring and relapse intervention. Different intervention thresholds should be used according to the pre-transplant disease status. Patients in NR before transplant should strictly use 0.6% as the threshold, while for patients in CR can be appropriately widened. Disclosures No relevant conflicts of interest to declare.

Comparative Genomic Analysis of Adolescents and Young Adults Versus Elderly with Acute Myeloid Leukemia

Prognosis and survival outcomes of acute myeloid leukemia (AML) strongly depend on age at diagnosis. In fact, long-term survival rates range from 60% in children and adolescent/young adults (AYA-AML: aged 15-39 years) to 10-15% in elderly patients (E-AML: aged &amp;gt;70 years). The currently available classification and prognostication models incorporate genomic features that include cytogenetics and molecular mutations based on adult AML clinical trials that mainly enroll patients outlying these two age categories. Consequently, prognostication models might still not account for the invariant genomic background of each subgroup. In this study, we aimed to dissect the genomic landscape of AYA-AML and E-AML cases in order to uncover patterns of distinct genomic features of important pathogenetic information, knowing how fundamental it is for the development of novel treatment approaches and more importantly for providing opportunities of individualization therapies. We studied the clinical and genomic characteristics of fully annotated sets of 198 AYA-AML and 414 E-AML cases from the Cleveland Clinic and publicly available data. Median age at diagnosis was 31 years for AYA-AML (range: 16-39 years) and 76 years (range: 71-100 years) for E-AML patients with a significant male preponderance noted in the latter cohort (65% vs 46%, P&amp;lt;0.0001). Based on the 2016 WHO classification, AML subtypes were differently distributed within the two subgroups. Low-risk AML (PML-RARA AML, core-binding factor AML, and CEBPA biallelic AML) was more prevalent among AYA cases while a higher odd of secondary AML (sAML) was found in E-AML. Compared to AYA-AML, E-AML were more typically presenting with a less proliferative disease phenotype, as outlined by a lower bone marrow blast infiltration (median: 34% vs 51%, P=0.04) and a higher frequency of leukopenia at baseline (66% vs 27%, P=0.009). In terms of cytogenetic abnormalities, when compared with AYA-AML, E-AML cases harbored more complex karyotype (32% vs 13%, P=0.002) and other cytogenetic abnormalities e.g., -5/del(5q) (14% vs 2%, P&amp;lt;0.0001), -7/del(7q) (14% vs 5%, P=0.001), -17/del(17p) (5% vs 1%, P=0.02), del(20q) (4% vs &amp;lt;1%, P=0.01) and trisomy 8 (14% vs 5%, P=0.0007) while a significantly lower odd of -9/del(9q) (&amp;lt;1% vs 4%, P=0.006). A total of 1092 somatic mutations were identified by next-generation sequencing of common myeloid genes. The AYA-AML cohort had a total of 249 mutations (ratio 1.25) while 843 mutations were reported in E-AML patients (ratio 2.04). Older patients, as compared to AYA-AML, had more mutations in age-related myeloid genes linked to clonal hematopoiesis, including mutations in DNMT3A (15% vs 6%, P=0.001), ASXL1 (14% vs 4%, P=0.0005), and TET2 (19% vs 7%, P=0.0002). As inferred from the cytogenetic comparisons, TP53 mutations were more frequently detected in E-AML (10% vs 2%, P=0.002) compared to AYA-AML, as they carried more chromosomal 5 and 17 abnormalities which are commonly associated with this genomic lesion. On the other hand, AYA-AML had a higher frequency of mutated WT1 gene (9% vs 2%, P=.0009). Multivariate Cox-Proportional hazard was performed to identify group-specific genomic lesions associated with survival in each cohort. In AYA-AML, normal karyotype (HR:0.59, P=0.02), t(15;17) translocation (HR:0.24, P=0.0001), inv(16)/t(8;21) (HR:0.52, P=0.02) and CEBPA biallelic (HR:0.50, P=0.02) were favorable lesions while complex karyotype had an adverse impact on survival (HR:3.0, P=0.0003). Moreover, while normal karyotype and t(15;17) translocation had a favorable impact in E-AML (HR:0.73, P=0.0001 and HR:0.28, P&amp;lt;0.0001, respectively), mutations in RUNX1 (HR:1.2, P=0.4), TET2 (HR:1.2, P=0.01), and TP53 (HR:1.97, P&amp;lt;0.0001) lead to worse outcomes. In conclusion, we demonstrated that E-AML cases are typically characterized by mutations belonging to the genomic subgroups of DNA methylation and chromatin modifiers, which have been reported to occur in age-related hematopoiesis or CHIP. Moreover, the complexity of aberrations (including cytogenetics) emphasizes that in this group of patients AML arises from a stepwise mutation acquisition model. In contrast, AYA-AML are typically characterized by favorable cytogenetic translocations and less clonal instability, dominated by the enrichment in myeloid leukemia driver gene mutations such as WT1. Disclosures No relevant conflicts of interest to declare.

Effects of Toxicity Induced by Gentamicin on the Kidney of Killifish Aphaniops hormuzensis and the Role of Wt1 and MMP9 Genes in Response to This Toxicity

Background: Aminoglycoside antibiotics such as gentamicin are used to cure bacterial infections in humans and other animals, but they can cause nephritic damage, as well. Nephrotoxicity is one of the side effects of gentamicin. Objectives: The objective of this study was to investigate the effects of toxicity induced by gentamicin on the kidney of killifish Aphaniops hormuzensis. Also, we aimed to study the expression pattern of Wt1 and MMP9 genes by real-time PCR in response to this toxicity. Methods: First, 10 µg/g (sub-lethal dose) gentamicin was given to adult fish. The kidney tissues were dissected and preserved in 10% formalin for a 24-hour; then, they underwent standard histological procedures. The sections were prepared at 3 μm and stained with Haematoxylin &amp; Eosin (H&amp;E). The slide microphotography process was done by an Olympus CH2 microscope. The RNA was isolated, and cDNA was synthesized with a standard protocol, and the expression patterns of Wt1 and MMP9 genes were examined by real-time PCR. Results: Nephrotoxicity occurred 10 hours after the injection of gentamicin, and the injury was detected in the epithelium of kidney tubules. The kidney tubule regenerated itself within 10 days post-injection (dpi). On 7 dpi, the nephrogenic body formation occurred and was differentiated into renal nephrons. The Wt1 gene was upregulated (two-fold) on 5 dpi after kidney damage and then had a down-regulation on 7 dpi when the kidney began to regenerate. The MMP9 gene showed increased expression in comparison with the control sample in the study days, and this expression increased on 7 dpi by 6.6 folds. Conclusions: The results of this study, for the first time, highlighted that nephritic damage appears in the kidney of A. hormuzensis after toxicity induced by gentamicin and that changes in the expression of the examined genes are consistent with their roles in the process of renal regeneration in this species.

WT1 Gene Expression in Peripheral Blood Before and After Allogeneic Stem Cell Transplantation is a Clinically Relevant Prognostic Marker in AML – A Single-center 14-year Experience

BackgroundThis work summarizes our experience with WT1 monitoring before and after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Patients and MethodsThe expression of WT1 gene was measured by real-time polymerase chain reaction in peripheral blood according to the European Leukemia Net recommendations. Between May 2005 and August 2019, we analyzed 147 consecutive patients with acute myeloid leukemia with high WT1 expression at diagnosis, transplanted in first (CR1) or second (CR2) complete remission. ResultsAt the time of allo-HSCT, 107 patients had WT1-normal expression (WT1 ≤ 50 copies), and 40 patients had WT1-high expression. The median follow-up was 21 months. The estimated 5-year overall survival and event-free survival was significantly better in the WT1-normal cohort (65% and 57% vs. 37% and 25%; P = .0003 and P < .0001, respectively) and 5-year cumulative incidence of relapse was significantly lower in the WT1-normal group (19% vs. 53%; P < .0001). Five-year non-relapse mortality was not significantly different (20% and 23%). Multivariate analysis revealed WT1-high expression and acute graft-versus-host disease grade 3/4 as significantly negative prognostic factors for OS. Overall, 49 patients developed WT1 molecular relapse in the post-transplant period; in 14 cases, the therapeutic intervention was done. In all but 1 relapsed patient where WT1 minimal residual disease (MRD) was monitored (38 patients), we detected WT1-high levels (sensitivity of 97%). ConclusionThe results of the analysis confirmed our previous experience that WT1 status before allo-HSCT is a strong prognostic factor for both OS and relapse risk. In addition, we confirmed the usefulness of this marker for MRD monitoring after allo-HSCT. The main advantage is the possibility of frequent MRD monitoring in peripheral blood and early bone marrow examination based on WT1-high expression.

A sporadic case of congenital aniridia caused by pericentric inversion inv(11)(p13q14) associated with a 977\u2009kb deletion in the 11p13 region

BackgroundBecause of the significant occurrence of “WAGR-region” deletions among de novo mutations detected in congenital aniridia, DNA diagnosis is critical for all sporadic cases of aniridia due to its help in making an early diagnosis of WAGR syndrome. Standard cytogenetic karyotype study is a necessary step of molecular diagnostics in patients with deletions and in the patients’ parents as it reveals complex chromosomal rearrangements and the risk of having another affected child, as well as to provide prenatal and/or preimplantation diagnostics.Case presentationDNA samples were obtained from the proband (a 2-year-old boy) and his two healthy parents. Molecular analysis revealed a 977.065 kb deletion that removed loci of the ELP4, PAX6, and RCN1 genes but did not affect the coding sequence of the WT1 gene. The deletion occurred de novo on the paternal allele. The patient had normal karyotype 46,XY and a de novo pericentric inversion of chromosome 11, inv(11)(p13q14).ConclusionsWe confirmed the diagnosis of congenital aniridia at the molecular level. For the patient, the risk of developing Wilms’ tumor is similar to that in the general population. The recurrence risk for sibs in the family is low, but considering the possibility of gonadal mosaicism, it is higher than in the general population.

Penetrance of WT1 and WT2 Gene Mutation and Loss of Heterozygosity in Wilms tumors in Indian Population

Wilms tumor is a nephroblastoma of pediatric age group and heterogeneous in nature. WT1 and WT2 gene mutations are involved in onset of tumorigenesis in syndromic and non syndromic cases of Wilms tumor. Present study has been designed with aim to evaluate the frequency of WT1 and WT2 gene mutation, loss of heterozygosity (LOH) and DNA copy number variation (CNV) in clinically diagnosed cases of Wilms tumor using RT-PCR based analysis. Findings reveals that there was vast difference in the mutation frequency between WT1 (7.5%) and WT2 (17.5) gene, and statistical analysis shows significant difference (p &lt; 0.05) in WT2 mutation and calculated value of C.I. varies between 0.886-1.990 at 95% with odd ratio (7.52). Interestingly, the frequency of LOH (loss of heterozygosity and DNA copy number variations (CNVs) shows significant difference (p &lt; 0.01), suggesting increase of genetic susceptibility and penetrance of gene in proband of the family tumor resulting increase of risk factor after using three different microsatellite DNA markers (DS11S935, DS11S904 and DS11S1363). Genetic heterogenecity were also observed by calculating Tm values, between cases and controls (GAPDH), and C.I. varies between 0.292 - 2.270 at 95% with O.R = 0.54 in D11S1363 and maximum value of C.I. was 1.165-2.165 at 95% with O.R = 0.50 in D11S1905 and the p = 0.194 &amp; p = 0.522 respectively, between cases and controls..

WT1 Expression in Peripheral Blood at Diagnosis and During the Course of Early Consolidation Treatment Correlates With Survival in Patients With Intermediate and Poor-Risk Acute Myeloid Leukemia

BackgroundUp to 55% of non-APL acute myeloid leukemias (AML) lack a molecular target suitable for standardized disease monitoring. We aimed to evaluate the prognostic significance of WT1 gene expression at early stages of intensive treatment. Patients and MethodsA total of 106 consecutive patients with intermediate and high-risk AML who had WT1 expression at diagnosis >500 copies/104ABL and who achieved remission after 1 to 2 cycles of induction treatment were analyzed. WT1 expression was measured in peripheral blood using a standardized European LeukemiaNet method. Overexpression was defined as >50 copies/104ABL. The median follow-up was 30 months. ResultsPatients with normal versus high WT1 expression after 2 cycles of chemotherapy had overall survival (OS) at 3 years of 66% versus 41% (P = .01); event-free survival (EFS) 45% versus 22% (P = .01). Prognostic significance of WT1 expression after 2 cycles of treatment was maintained in the group of patients treated with chemotherapy alone without hematopoietic stem cell transplantation in first line treatment (OS 70% vs. 36%, P = .02; EFS 35% vs. 0%, P = .03). Significant prognostic factors for EFS on multivariate analysis were the achievement of molecular remission (<50 copies of WT1) at any time during treatment (hazard ratio [HR] 0.47, P = .04) and increased WT1 expression after 2 cycles of chemotherapy (HR 2.0, P = .03). ConclusionIncreased WT1 expression after 2 cycles of chemotherapy is a negative prognostic factor for survival. WT1 remains a valuable molecular marker in AML without any leukemia-specific mutation, especially if next generation sequencing and/or digital polymerase chain reaction are not routinely available.

A Unique Presentation of XY Gonadal Dysgenesis in Frasier Syndrome due to WT1 Mutation and a Literature Review.

Frasier syndrome (FS), a rare disease caused by inherited or de novo mutation in Wilm's Tumor suppressor gene 1 (WT1), is characterized by slow progressive nephropathy, XY gonadal dysgenesis (XY-DSD), and increased risk for gonadal tumors. Early childhood (1-6 years) nephropathy progresses with age to refractory nephrotic syndrome, and end-stage renal failure in late adolescence, when delayed puberty and/or primary amenorrhea are clinically evident. We report a unique case of FS presenting initially with primary amenorrhea at 16 years, without previous or concomitant renal damage. Only subsequently she developed an extremely late-onset nephropathy. Genetic analysis revealed the IVS9 + 5 G>A mutation in intron 9 of the WT1 gene. This clinical presentation and review of WT1 literature highlights the importance of considering FS in the differential diagnosis of patients with 46,XY disorders of Sexual development, even without nephropathy. Furthermore, the identification WT1 gene mutation prior to evident renal dysfunction indicates an immediate and close surveillance of renal function enabling an optimal and timely medical response.