Upregulation of HOXA3 by isoform-specific Wilms tumour 1 drives chemotherapy resistance in acute myeloid leukaemia.

Upregulation of the Wilms' tumour 1 (WT1) gene is common in acute myeloid leukaemia (AML) and is associated with poor prognosis. WT1 generates 12 primary transcripts through different translation initiation sites and alternative splicing. The short WT1 transcripts express abundantly in primary leukaemia samples. We observed that overexpression of short WT1 transcripts lacking exon 5 with and without the KTS motif (sWT1+/- and sWT1-/-) led to reduced cell growth. However, only sWT1+/- overexpression resulted in decreased CD71 expression, G1 arrest, and cytarabine resistance. Primary AML patient cells with low CD71 expression exhibit resistance to cytarabine, suggesting that CD71 may serve as a potential biomarker for chemotherapy. RNAseq differential expressed gene analysis identified two transcription factors, HOXA3 and GATA2, that are specifically upregulated in sWT1+/- cells, whereas CDKN1A is upregulated in sWT1-/- cells. Overexpression of either HOXA3 or GATA2 reproduced the effects of sWT1+/-, including decreased cell growth, G1 arrest, reduced CD71 expression and cytarabine resistance. HOXA3 expression correlates with chemotherapy response and overall survival in NPM1 mutation-negative leukaemia specimens. Overexpression of HOXA3 leads to drug resistance against a broad spectrum of chemotherapeutic agents. Our results suggest that WT1 regulates cell proliferation and drug sensitivity in an isoform-specific manner.

Interplay of cell-cell contacts and RhoA/MRTF-A signaling regulates cardiomyocyte identity.

Cell-cell and cell-matrix interactions guide organ development and homeostasis by controlling lineage specification and maintenance, but the underlying molecular principles are largely unknown. Here, we show that in human developing cardiomyocytes cell-cell contacts at the intercalated disk connect to remodeling of the actin cytoskeleton by regulating the RhoA-ROCK signaling to maintain an active MRTF/SRF transcriptional program essential for cardiomyocyte identity. Genetic perturbation of this mechanosensory pathway activates an ectopic fat gene program during cardiomyocyte differentiation, which ultimately primes the cells to switch to the brown/beige adipocyte lineage in response to adipogenesis-inducing signals. We also demonstrate by in vivo fate mapping and clonal analysis of cardiac progenitors that cardiac fat and a subset of cardiac muscle arise from a common precursor expressing Isl1 and Wt1 during heart development, suggesting related mechanisms of determination between the two lineages.

Maintenance of WT1 expression in tumor cells is associated with a good prognosis in malignant glioma patients treated with WT1 peptide vaccine immunotherapy.

We have previously revealed the overexpression of Wilms' tumor gene 1 (WT1) in malignant glioma and developed WT1 peptide vaccine cancer immunotherapy. A phase II clinical trial indicated the clinical efficacy of the WT1 peptide vaccine for recurrent malignant glioma. Here, we aimed to investigate the immunological microenvironment in glioma tissues before and after WT1 peptide vaccine treatment. Paired tissue samples were obtained from 20 malignant glioma patients who had received the WT1 peptide vaccine for > 3 months and experienced tumor progression, confirmed radiographically and/or clinically, during vaccination. We discovered that the expression of WT1 and HLA class I antigens in the tumor cells significantly decreased after vaccination. Maintenance of WT1 expression, which is the target molecule of immunotherapy, in tumor cells during the vaccination period was significantly associated with a longer progression-free and overall survival. A high expression of HLA class I antigens and low CD4+/CD8+ tumor-infiltrating lymphocytes (TIL) ratio in pre-vaccination specimens, were also associated with a good prognosis. No statistically significant difference existed in the number of infiltrating CD3+ or CD8+ T cells between the pre- and post-vaccination specimens, whereas the number of infiltrating CD4+ T cells significantly decreased in the post-vaccination specimens. This study provides insight into the mechanisms of intra-tumoral immune reaction/escape during WT1 peptide vaccine treatment and suggests potential clinical strategies for cancer immunotherapy.

Giant benign lymphangioendothelioma with positive expression of Wilms tumor 1: A case report.

Benign lymphangioendothelioma (BL, acquired progressive lymphangioma) is a rare, slow-growing lymphatic tumor, first described 40 years ago, with fewer than 50 published cases. Clinically, it presents as a skin-colored or erythematous patch. Definitive diagnosis requires histopathological examination. The immunohistochemical staining profile is still controversial regarding Wilms tumor 1 (WT1) expression, a marker of proliferative and neoplastic, rather than malformative nature. Here, we report a case of a 60-cm-long BL on the breast of an adult female. Biopsy revealed irregular vascular spaces dissecting the collagen bundles lined by swollen endothelial cells but without cellular atypia. Positivity for podoplanin (D2-40), CD31, and WT1 was observed, supporting the neoplastic nature of this lesion. Dermatologists and pathologists must be aware of this entity for early diagnosis and treatment.

MicroRNA-216a targets WT1 expression and regulates KRT7 transcription to mediate the progression of pancreatic cancer-A transcriptome analysis.

Gene expression profiling has been broadly performed in the field of cancer research. This study aims to explore the key gene regulatory network and focuses on the functions of microRNA (miR)-216a in pancreatic cancer (PC). PC datasets GSE15471, GSE16515, and GSE32676 were used to screen the differentially expressed genes (DEGs) in PC. A miRNA microarray analysis and gene oncology analysis suggested miR-216a as an important differentially expressed miRNA in PC. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis suggested that miR-216a and the DEGs are largely enriched on the phosphatidyl inositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathway. miR-216a targeted Wilms Tumor 1 (WT1), while WT1 promoted transcription activity of keratin 7 (KRT7). Upregulation of miR-216a reduced proliferation and invasiveness of PC cells, while further upregulation of WT1 blocked the functions of miR-216a. Silencing of KRT7 diminished the oncogenic role of WT1. The in vitro results were reproduced in vivo. High expression of miR-216a while poor expression of WT1 indicated better prognosis of PC patients. The miR-216a/WT1/KRT7 axis influenced the activity of the PI3K/AKT pathway. To conclude, this study evidenced that miR-216a suppressed WT1 expression and blocked KRT7 transcription, which inactivated the PI3K/AKT signaling and reduced PC progression.

RUNX1 mutation in a patient with myelodysplastic syndrome and decreased erythrocyte expression of blood group A antigen.

BACKGROUND: Loss of blood group ABO antigens on red blood cells (RBCs) is well known in patients with leukemias, and such decreased ABO expression has been reported to be strongly associated with hypermethylation of the ABO promoter. We investigated the underlying mechanism responsible for A-antigen reduction on RBCs in a patient with myelodysplastic syndrome. STUDY DESIGN AND METHODS: Genetic analysis of ABO was performed by PCR and sequencing using peripheral blood. RT-PCR were carried out using cDNA prepared from total bone marrow (BM) cells. Bisulfite genomic sequencing was performed using genomic DNA from BM cells. Screening of somatic mutations was carried out using a targeted sequencing panel with genomic DNA from BM cells, followed by transient transfection assays. RESULTS: Genetic analysis of ABO did not reveal any mutation in coding regions, splice sites, or regulatory regions. RT-PCR demonstrated reduction of A-transcripts when the patient's RBCs were not agglutinated by anti-A antibody and did not indicate any significant increase of alternative splicing products in the patient relative to the control. DNA methylation of the ABO promoter was not obvious in erythroid cells. Targeted sequencing identified somatic mutations in ASXL1, EZH2, RUNX1, and WT1. Experiments involving transient transfection into K562 cells showed that the expression of ABO was decreased by expression of the mutated RUNX1. CONCLUSION: Because the RUNX1 mutation encoded an abnormally elongated protein without a transactivation domain which could act as dominant negative inhibitor, this frame-shift mutation in RUNX1 may be a genetic candidate contributing to A-antigen loss on RBCs.

Overexpressed WT1 exhibits a specific immunophenotype in intermediate and poor cytogenetic risk acute myeloid leukemia.

Many studies have confirmed that overexpressed WT1 exists in leukemic cells, especially in AML. However, the immunophenotypic features of this sort of leukemic cells remain to be unclarified. We retrospectively analyzed the immunophenotype of 283 newly diagnosed AML patients with intermediated and poor cytogenetic risk to evaluate the correlation between phenotype and WT1 overexpression. EVI1 transcripts, KMT2A-PTD, FLT3-ITD, and NPM1 mutations were simultaneously assessed. Our results revealed that overexpressed WT1 was significantly associated with the expression of CD117, CD13, and CD123. Besides, leukemic cells with WT1 overexpression also lacked lymphoid and myeloid differentiation-related markers. FAB subtype M2 patients had higher WT1 levels, compared with other FAB subtype. Multivariate analysis was proved that NPM1 mutation, M2 subtype, and the expression of CD123 were independently associated with WT1 overexpression. These indicated that AML with overexpressed WT1 was proliferated and blocked in the early stage of AML development. It presumably provided some clues to detect overexpressed WT1 cells via multiparameter flow cytometry. CD123-targeted drugs might become one of the alternative treatments for patients with WT1 overexpression.

Histone demethylase KDM6B regulates human podocyte differentiation in vitro.

Podocytes are terminally differentiated and highly specialized glomerular cells, which have an essential role as a filtration barrier against proteinuria. Histone methylation has been shown to influence cell development, but its role in podocyte differentiation is less understood. In this study, we first examined the expression pattern of histone demethylase KDM6B at different times of cultured human podocytes in vitro We found that the expression of KDM6B and podocyte differentiation markers WT1 and Nephrin are increased in the podocyte differentiation process. In cultured podocytes, KDM6B knockdown with siRNA impaired podocyte differentiation and led to expression down-regulation of WT1 and Nephrin. The treatment of podocytes with GSK-J4, a specific KDM6B inhibitor, can also obtain similar results. Overexpression of WT1 can rescue differentiated phenotype impaired by disruption of KDM6B ChIP (chromatin immunoprecipitation) assay further indicated that KDM6B can bind the promoter region of WT1 and reduce the histone H3K27 methylation. Podocytes in glomeruli from nephrotic patients exhibited increased KDM6B contents and reduced H3K27me3 levels. These data suggest a role for KDM6B as a regulator of podocyte differentiation, which is important for the understanding of podocyte function in kidney development and related diseases.

Evidence that downregulation of Wilms' tumor 1 (WT1) is involved in cortical stromal cell differentiation into theca cells in adult bovine ovaries.

Bovine theca cells are thought to differentiate from cortical stromal cells, and ovary-derived Wilms' tumor 1+ (WT1+ ) cells are the primary source of mouse theca cells. However, it is not known whether the differentiation of cortical stromal cells is regulated by WT1. Here, we identified WT1 in the cortical stroma and theca layer of the bovine ovary and analyzed the theca cell functional markers in cortical stromal cells and theca cells; in addition, we determined the effects of this gene on the secretion of androstenedione and progesterone by cortical stromal cells and the responsiveness of cortical stromal cells to luteinizing hormone (LH) in vitro. We used quantitative reverse-transcription polymerase chain reaction (RT-qPCR), western blot analysis, and immunohistochemistry to discover that the cortical stroma had higher WT1 expression than the theca layer. We used RT-qPCR and ELISA analyses to determine that the cortical stromal cells had lower levels of androstenedione and progesterone secretion and LHR messenger RNA expression than the levels of the theca cells. In cultured bovine cortical stromal cells, we found that WT1 downregulation increased androstenedione and progesterone secretion but had no effect on the LH responsiveness. Notably, the increase in androstenedione and progesterone secretion was associated with an increase in 3-ß-hydroxysteroid dehydrogenase expression. In conclusion, the results suggest that WT1 is involved in the differentiation of cortical stromal cells into cells with characteristics similar to theca cells of antral follicles in adult bovine ovaries.

The prognostic significance of Wilms' tumor gene 1 (WT1) expression at diagnosis in adults with Ph-negative B cell precursor acute lymphoblastic leukemia.

The prognostic significance of Wilms' tumor gene 1 (WT1) expression at diagnosis in adults with B cell precursor acute lymphoblastic leukemia (BCP-ALL) remains poorly understood. A total of 257 adults with Ph-negative BCP-ALL who were consecutively diagnosed and received at least 1 course of induction therapy at our institute were retrospectively analyzed. The WT1 expression patterns were significantly different among the molecularly and cytogenetically defined groups (E2A-PBX1, TEL-AML1, and MLL rearrangements; high hyperdiploidy and B-other). By considering the WT1 expression pattern and the relapse status, 2 cutoff values, 1.8% and 7.2%, were arbitrarily selected to place patients into WT1-low, WT1-inter, and WT1-high groups. In the B-other patients who achieved complete remission (CR), WT1-low and WT1-high patients had similar 3-year relapse-free survival (RFS), disease-free survival (DFS), and overall survival (OS) rates, which were all significantly lower than those of WT1-inter patients. The combined WT1-low/high expression group (n = 132) had significantly lower 3-year RFS, DFS, and OS rates compared with the WT1-inter group (n = 63) of B-other patients (RFS and DFS all P < 0.0001; OS P = 0.0018 and 0.0008). WT1 low/high expression as well as treating with chemotherapy only was independent poor prognostic factors for RFS, DFS, and OS in the B-other patients who achieved CR. Therefore, the molecularly and cytogenetically defined adult Ph-negative BCP-ALL groups have characteristic WT1 expression patterns, and WT1 low/high expression at diagnosis predicts poor outcome in B-other patients.

Investigation of Insulin-Like Growth Factor-1 (IGF-1), P53, and Wilms' Tumor 1 (WT1) Expression Levels in the Colon Polyp Subtypes in Colon Cancer.

BACKGROUND There is no study in the literature investigating the expression levels of WT1, p53, and IGF-1 in colon polyp subtypes. In this study, we aimed to investigate the expression levels of IGF-1, p53, and WT1 in colon polyp subtypes and to determine whether expression levels are correlated with each other. MATERIAL AND METHODS Tissue specimens were obtained from 105 patients (80 men, 25 women; age range, 30-91 years) who underwent surgical resection for colorectal cancer (CRC) at Ordu University School of Medicine, Department of Pathology between January 2015 and 2017. Parameters such as age, sex, region of origin, and pathological diagnosis type were determined. The preparations were immunohistochemically stained with corresponding markers. RESULTS The results of the study showed that there was a statistically significant relationship between WT1 expression (negative - positive) in polyps and the place where the sample was taken (P=0.011). There is a positive relationship between P53 staining score (0-3) and positive frequency of IGF-1 (60.9-85.7%). There was a statistically significant change in P53 scores and location (P=0.006, p=0.015, respectively). As the P53 score of the polyps increased (0 to 3), the rate of adenomatous (34.8-78.4%) increased, so a positive relationship was found. WT1 and IGF-1 gene expression was associated with tumor location, p53 staining score, and sex. CONCLUSIONS WT1 and IGF-1 are appropriate markers for CRC, and WT1 expression in CRC primary tumors especially could be a novel independent marker for prognosis and tumor progression.

Alteration of Connexin43 expression in a rat model of obesity-related glomerulopathy.

It is accepted that alteration of connexin43 (Cx43) expression in glomeruli is a common pathological response in several forms of kidney diseases. To date, however the change of the Cx43 expression in obesity-related glomerulopathy (ORG) has not been reported. In this study, the alteration of Cx43 expression in the glomeruli of rat with ORG was defined. Five-week-old rats were fed with high-fat diet for 18weeks to establish the ORG model, then the histological change of glomeruli, the foot process effacement of podocyte, the markers for podocyte injury (nephrin,podocin and WT1) and Cx43 expression in glomeruli were examined respectively. The results demonstrated metabolic disorder, hyperinsulinemia, systemic inflammation and microalbuminuria in ORG rats. There was significant hypertrophy, glomerular expansion and inflammatory cell infiltration in the kidney of ORG rats compared to the control group. Significant foot process effacement of the podocyte in the glomeruli, nephrin loss and density reduction were shown in the ORG rats, and Cx43 expression was significant upregulated in glomeruli of ORG rats compared to the control group. The results indicate the correlation of overexpressed Cx43 with the obesity related renal inflammation and suggest that Cx43 might be a potential target in the development of obesity related glomerulopathy.

A Proposed Kinetic Model for the Diagnostic and Prognostic Value of WT1 and p53 in Acute Myeloid Leukemia.

BACKGROUND: Wilms tumor (WT1) and p53 proteins were identified in the pathogenesis of several malignancies, including hematological malignancies. As a result of their interaction and diverse context-specific functions, this study aimed to emphasize the diagnostic and prognostic impacts of WT1 and p53 expression in acute myeloid leukemia (AML). METHODS: Twelve bone marrow (BM) biopsies were obtained from AML patients who were diagnosed in accordance with the French-American-British diagnostic criteria. For comparative purposes, nine normal BM biopsies were included. The expression rate of WT1 and p53 were determined by an immunohistochemistry assay. RESULTS: A significantly higher (p < 0.005) and strongly correlated ( = 0.855, p = 0.001) expression rates of WT1 and p53 were observed in the BM of AML patients in comparison to control BM. Furthermore, relapsed AML patients had significantly higher expression of WT1, but not p53, when compared to newly diagnosed patients. In regard of patient's responsiveness to chemotherapy, no significant difference was reported between good and poor responders. However, the relative ratio of p53 to WT1 expression was evidently correlated to the responsiveness groups (p < 0.05), where the ratio was observed to be significantly higher among poor responders. Poor responders were characterized by a statistically significant and dominant p53 expression (p53/WT1 > 1.0) while both good responding patients and control subjects had a dominant WT1 expression (p53/WT1 < 1.0). CONCLUSIONS: The enhanced expression levels of WT1 and p53 proteins in the BM of AML patients is supportive of their intermediate role in the pathogenesis of the disease. WT1 expression rate may encompass a negative prognostic value of the disease. Furthermore, the ratio of p53/WT expression may serve as a hallmark of the patient's responsiveness to chemotherapy, where a dominant WT1 expression may reveal good responsiveness to chemotherapy. Herein, we are proposing a kinetic model where the p53/WT1 ratio might be useful as a laboratory approach to evaluate the prognostic value of AML including the patient's responsiveness to chemotherapeutic regimen.

Focal and segmental glomerulosclerosis in murine models: a histological and ultrastructural characterization with immunohistochemistry correlation of glomerular CD44 and WT1 expression.

AIM: Focal segmental glomerulosclerosis (FSGS) is a common progressive chronic renal disease. Podocyte injury and loss are the postulated pivotal events that trigger FSGS. In this study, the authors aim to examine the evolution of FSGS in murine models histologically, ultrastructurally and immunohistochemically with special emphasis on podocytes and parietal epithelial cells (PECs). MATERIAL AND METHODS: FSGS resembling primary FSGS in humans was initiated in Wistar rats using intravenous Adriamycin injections. Blood and urine analysis were performed at 0, 8, and 12 weeks. Both the control kidneys and the test kidneys were harvested at 8 and 12 weeks, examined histologically and ultrastructurally and the findings correlated with the glomerular expression of immunostains specific for podocytes (WT-1) and for activated PECs (CD44). RESULTS: FSGS developed in both 8 and 12 weeks test groups showing progressive proteinuria, podocytopathy and segmental glomerular scarring. There was a decrease in the glomerular expression of WT-1 with a concurrent increase in the glomerular expression of CD44, indicating podocyte loss with synchronous increase in activated PECs. The evolving FSGS correlated negatively with podocytes and positively with activated PECs. CONCLUSION: Our study shows that with podocyte injury there is podocyte effacement and loss, proteinuria, glomerular segmental adhesion and scarring, all culminating in FSGS. In addition, there is activation, hyperplasia and hypertrophy of PECs. This demonstrates that both podocyte loss and PEC activation promote FSGS. Our findings are consistent with recent investigations. More studies are required to further understand the role of these cells in the evolution of FSGS and subsequently introduce new targeted treatment modalities.

Extremely High Expression of Antisense RNA for Wilms' Tumor 1 in Active Osteoclasts: Suppression of Wilms' Tumor 1 Protein Expression during Osteoclastogenesis.

Wilms' tumor 1 (WT1), a zinc-finger transcription regulator of the early growth response family, identified as the product of a tumor suppressor gene of Wilms' tumors, bears potential ability to induce macrophage differentiation in blood cell differentiation. Herein, we examined the involvement of WT1 in the regulation of osteoclastogenesis. We detected a high level of WT1 protein expression in osteoclast precursors; however, WT1 expression was markedly suppressed during osteoclastogenesis. We examined expression of WT1 transcripts in bone tissue by RNA in situ hybridization. We found a high level of antisense transcripts in osteoclasts actively resorbing bone in mandible of newborn rats. Expression of antisense WT1 RNA in mandible was also confirmed by Northern blot analysis and strand-specific RT-PCR. Overexpression of antisense WT1 RNA in RAW-D cells, an osteoclast precursor cell line, resulted in a marked enhancement of osteoclastogenesis, suggesting that antisense WT1 RNA functions to suppress expression of WT1 protein in osteoclastogenesis. High level expression of antisense WT1 RNA may contribute to commitment to osteoclastogenesis, and may allow osteoclasts to maintain or stabilize their differentiation state.

A comparison of p53 and WT1 immunohistochemical expression patterns in tubo-ovarian high-grade serous carcinoma before and after neoadjuvant chemotherapy.

AIMS: The treatment of patients with tubo-ovarian high-grade serous carcinoma (HGSC) is increasingly based on diagnosis on small biopsy samples, and the first surgical sample is often taken post-chemotherapy. p53 and WT1 are important diagnostic markers for HGSC. The effect of neoadjuvant chemotherapy on p53 and WT1 expression has not been widely studied. We aimed to compare p53 and WT1 expression in paired pre-chemotherapy and post-chemotherapy samples of HGSC. METHODS AND RESULTS: Immunohistochemistry (IHC) was carried out for p53 and WT1 on paired omental HGSC samples pre-chemotherapy and post-chemotherapy. p53 IHC was recorded as normal (wild-type) or abnormal (mutation-type), and was further classified as overexpression, complete absence, or cytoplasmic. WT1 IHC was classified as positive or negative. A subset of cases were further assessed for the extent of nuclear immunoreactivity of WT1 by use of the H-score. Fifty-seven paired samples were stained with p53. Fifty-six of 57 (98%) cases showed mutation-type p53 staining. Pre-chemotherapy and post-chemotherapy IHC results were concordant in 55 of 57 (96%) cases. For WT1, pre-chemotherapy and post-chemotherapy IHC results were concordant in 56 of 58 (97%) cases. In 23 paired WT1 cases, the mean post-treatment H-score decreased from 227 [range 20-298, standard deviation (SD) 64] to 151 (range 0-288, SD 78) (P = 0.0008). CONCLUSIONS: Immunohistochemical expression of p53 (abnormal/mutation-type pattern) and WT1 in HGSC is almost universal and is largely concordant before and after chemotherapy. This finding underscores the reliability of these diagnostic markers in small samples and in surgical samples following neoadjuvant chemotherapy, with very few exceptions. A novel finding was the significant diminution in intensity of WT1 staining following chemotherapy.

Evaluation of ETV4 and WT1 expression in CIC-rearranged sarcomas and histologic mimics.

A distinct subset of round cell sarcomas harbors capicua transcriptional repressor (CIC) rearrangement. Diagnosing these sarcomas can be difficult owing to their resemblance to Ewing sarcoma and other 'small round blue cell tumors'; molecular techniques are generally required. Recent gene expression studies of CIC-rearranged sarcomas identified the upregulation of ETV4. We assessed the sensitivity and specificity of ETV4 and WT1 immunohistochemistry for CIC-rearranged sarcoma. We evaluated whole-tissue sections from 40 CIC-rearranged sarcomas, 40 Ewing sarcomas, 4 BCOR-CCNB3 sarcomas, 6 unclassified round cell sarcomas, and 150 histologic mimics. Moderate-to-strong nuclear immunoreactivity for ETV4 in at least 50% of cells was observed in 36 (90%) CIC-rearranged sarcomas and 10 (5%) other tumors, including 5 unclassified round cell sarcomas, 2 Wilms tumors, and 1 each desmoplastic small round cell tumor, melanoma, and small cell carcinoma. Thirty-eight (95%) CIC-rearranged sarcomas showed nuclear staining for WT1, and 34 (85%) were positive for both ETV4 and WT1. Of 182 other tumors evaluated, 34 (19%) showed nuclear WT1 positivity, including all Wilms tumors and desmoplastic small round cell tumors, 5 unclassified round cell sarcomas, and a subset of lymphoblastic lymphomas, rhabdomyosarcomas, mesenchymal chondrosarcomas, carcinomas, and melanomas. In summary, diffuse moderate-to-strong ETV4 expression is present in most CIC-rearranged sarcomas and unclassified round cell sarcomas. More limited expression is seen in small subsets of various other round cell neoplasms. Nuclear WT1 expression is also present in most CIC-rearranged sarcomas and unclassified round cell sarcomas, along with Wilms tumors and desmoplastic small round cell tumors, and subsets of various histologic mimics. The sensitivity and specificity of diffuse ETV4 expression for CIC-rearranged sarcomas are 90% and 95%, respectively, whereas the sensitivity and specificity of WT1 are 95% and 81%, respectively. Diffuse ETV4 along with at least focal WT1 expression is helpful to distinguish CIC-rearranged sarcoma from Ewing sarcoma and other histologic mimics.

Diagnostic utility of Wilms' tumour-1 protein (WT-1) immunostaining in paediatric renal tumours.

BACKGROUND & OBJECTIVES: Renal tumours constitute about 7 per cent of all neoplasms in children. It is important to differentiate Wilms' tumour (commonest tumour) from non-Wilms' tumours. The aim of this study was to evaluate the immunoexpression and diagnostic role of Wilms' tumour-1 protein (WT1) in paediatric renal tumours. METHODS: A total of 53 cases of renal tumours in children (below 18 yr) who underwent total nephrectomy were included in this retrospective study. WT1 immunostaining was done using mouse monoclonal WT1 antibody (clone: 6F-H2). RESULTS: Of the 53 cases, 38 (72%) were of Wilms' tumour. Non-Wilms' group (15) included six cases of mesoblastic nephroma (MN), two each of clear cell sarcoma (CCSK), renal cell carcinoma (RCC) and peripheral neuroectodermal tumour (PNET) and one each of angiomyolipoma (AML), rhabdomyosarcoma (RMS) and malignant rhabdoid tumour (MRT). Proportion of WT1 positivity in Wilms' tumour was 100 per cent in contrast to 26.7 per cent in non-Wilms' tumours ( P<0.001). Epithelial and blastemal components of Wilms' tumour showed moderate (2+) nuclear and cytoplasmic staining in 80 (24/30) and 75 per cent (24/32) cases, respectively. MN, PNET, CCSK and AML were negative for WT1. RMS, RCC and MRT showed cytoplasmic staining, strongest in RMS. No significant association was seen between WT1 expression and NWTSG (National Wilms' Tumor Study Group) stage. INTERPRETATION & CONCLUSIONS: WT1 helps to differentiate Wilms' tumour from other paediatric renal tumours. It may help in differentiating the two subgroups of Wilms' tumour which have distinct molecular pathogenesis and biological behaviour, however, further prospective studies are required for validation of this hypothesis.

Molecular and morphological differentiation of testes and ovaries in relation to the thermosensitive period of gonad development in the snapping turtle, Chelydra serpentina.

Ambient temperatures during embryonic development determine gonadal sex in many reptiles. The temperature sensitive period for sex determination has been defined by shifting eggs between female- and male-producing temperatures in a few species. This phase spans 20-35% of embryogenesis in most species, which makes it difficult to define the mechanisms that transduce temperature into a signal for ovarian versus testicular development. We present an extensive set of studies that define a brief period when high temperature specifies, and then determines, ovarian fate in a northern population of snapping turtles, Chelydra serpentina. We shifted embryos from male to female temperatures, or vice versa, at various stages of development. Gonads in embryos incubated at female temperatures commit to ovarian fate earlier (by stage 18) than gonads in embryos incubated at male temperatures commit to testicular fate (by stages 19-21). In double shift studies, embryos were incubated at a female temperature, exposed to a male temperature for set times, and shifted back to the original temperature, or vice versa. The time required to induce ovarian development (≤6 days at female temperatures) was much shorter than the time required to induce testicular formation (>20 days at male temperatures). Differentiation of the gonads at the histological level occurred after the sex-determining period. Nevertheless, we found that a change in temperature rapidly (within 24h) influenced expression and splicing of WT1 mRNA: the absolute abundance of WT1 mRNA, the relative abundance of +KTS versus -KTS isoforms, as well as the ratio of +KTS:-KTS isoforms was higher in gonads at a male versus a female temperature. In conclusion, ovarian fate is more readily determined than testicular fate in snapping turtle embryos. The short sex-determining period in this species (6-8% of embryogenesis) will facilitate studies of molecular mechanisms for specification and determination of gonad fate by temperature.

Down-regulatory mechanism of mammea E/BB from Mammea siamensis seed extract on Wilms' Tumor 1 expression in K562 cells.

BACKGROUND: Wilms' tumor 1 (WT1) is a biological marker for predicting leukemia progression. In this study, mammea E/BB, an active compound from Saraphi (Mammea siamensis) seed extract was examined for its effect on down-regulatory mechanism of WT1 gene expression, WT1 protein and mRNA stability, and cell proliferation in K562 cell line. METHODS: M. siamensis seeds were obtained from the region of Chiang Mai (North of Thailand). Mammea E/BB was extracted from seeds of M. siamensis. WT1 protein expression and stability were evaluated by Western blot analysis. WT1 mRNA stability was assessed by qRT-PCR. WT1-DNA binding and WT1 promoter activity were assayed by ChIP assay and luciferase-reporter assay, respectively. Cell cycle arrest was studied by flow cytometry. RESULTS: Treatment with mammea E/BB led to down-regulation of WT1 expression. The suppression of WT1 expression did not involve protein and mRNA degradation. Rather, WT1 protein was down-regulated through disruption of transcriptional auto-regulation of the WT1 gene. Mammea E/BB inhibited WT1-DNA binding at the WT1 promoter and decreased luciferase activity. It also disrupted c-Fos/AP-1 binding to the WT1 promoter via ERK1/2 signaling pathway and induced S phase cell cycle arrest in K562 cells. CONCLUSION: Mammea E/BB had pleotropic effects on kinase signaling pathways, resulting in inhibition of leukemia cell proliferation.