Pediatric Nephroblastoma Research Articles

Prognostic Correlation of Basic Fibroblast Growth Factor and Vascular Endothelial Growth Factor with Radiological Tumor Size in Pediatric Nephroblastoma and Neuroblastoma: A Prospective Study

ABSTRACT Introduction: Angiogenesis plays an important role in the growth, progression, and metastasis of solid tumors. Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF); both are potent angiogenic factors implicated with the growth and metastasis of solid tumors mostly in adult studies. Rapidly growing and large-size tumors have a positive correlation with these factors which are measured in serum and urine samples of patients. There is little literature available for pediatric patients that compare the computed tomography findings (size) of pediatric solid tumors with serum VEGF and serum/urinary bFGF. This prospective study aims to determine the correlation of serum VEGF and serum/urinary bFGF with the size of common pediatric solid tumors (nephroblastoma and neuroblastoma [NB]) to determine its diagnostic and prognostic significance. Materials and Methods: A prospective case–control study was done for 3 years (December 2020 to November 2023) at our institute. All children aged 1 day to 18 years admitted with the diagnosis of NB and nephroblastoma (Wilms’ tumor [WT]) were included after parental consent. The control group includes children of varying ages without any malignancy. Blood and urinary samples were collected at admission before or a week after the biopsy and the level of bFGF and VEGF was analyzed with the ELISA method. Triple-phase computed tomographies along with recommended evaluation were performed according to the tumor as per international standard protocols. Appropriate statistical analyses were done. Results: A total of 30 patients were included in the cohort, of which 13 patients were with the diagnosis of WT. Only 7 patients had metastatic disease. All the patients were treated with neoadjuvant chemotherapy, surgery, and radiotherapy according to their presentation and tumor stage. Both the growth factors, i.e., VEGF (median value - 314.38 pg/mL) and bFGF (median value - 18.96 pg/mL), are elevated in all the tumor patients in comparison with the controls (median VEGF - 100.51 pg/mL and bFGF - 15.6 pg/mL). When compared with the tumor size, no significant associations are found (with VEGF P - 0.40 and with bFGF P - 0.44). Conclusion: Although no satistically significant correlations were found between serum/urinary levels of bFGF and VEGF with tumor size, this study showed the raised median serum value of bFGF and VEGF in patients with NB and WT in comparison to controls and provided the rationale to explore this issue on a larger group of patients.

The high levels of fibrinogen and platelets are associated with poor survival in nephroblastoma in children.

Pediatric nephroblastoma is the most common abdominal malignancy in children. Hyperfibrinogenemia and thrombocytosis are often associated with malignancy and poor prognosis. This study aimed to investigate the relationship between high fibrinogen and platelet levels and the clinicopathologic features as well as overall survival in pediatric nephroblastoma. We recruited a total of 129 nephroblastoma patients in this research. The patients were evaluated retrospectively using the Kaplan-Meier method and Log rank test to investigate the correlation between patient survival and fibrinogen and platelet levels. A multivariate Cox regression model and a predictive nomogram have also been constructed. Our findings indicated that fibrinogen levels were associated with Children's Oncology Group (COG) stage (P<0.001), pathological typing (P<0.001), hematuresis (P=0.002), hypertension (P=0.02), and venous thrombosis (P<0.001). Platelet levels were associated with COG stage (P<0.001), pathological typing (P<0.001), hematuresis (P=0.001), and venous thrombosis (P<0.001). Elevated fibrinogen and platelet levels are associated with poor overall survival (P<0.001). Multivariate analysis also indicated that elevated fibrinogen and platelet were independent risk factors (P=0.02, and P<0.001). The nomogram model demonstrates its application value in predicting the prognosis of pediatric nephroblastoma. The calibration curve validates that the predictions made by the nomogram model are in agreement with the actual observed survival outcomes. High levels of platelets and fibrinogen may have negative effects on the prognosis of children with nephroblastoma.

CO187 On Track for WHO Global Initiatives for Childhood Cancer: A Comparison of Survival Rates in Pediatric Nephroblastoma (Wilms Tumor) in Sub-Saharan Africa

CO187 On Track for WHO Global Initiatives for Childhood Cancer: A Comparison of Survival Rates in Pediatric Nephroblastoma (Wilms Tumor) in Sub-Saharan Africa

A novel model incorporating chromatin regulatory factors for risk stratification, prognosis prediction, and characterization of the microenvironment in Wilms tumor.

Wilms tumor, also known as nephroblastoma, a pediatric most-frequent malignant-kidney tumor, may be regulated and influenced by transcriptional and epigenetic mechanisms. Chromatin regulatory factors (CRs) play key roles in epigenetic regulation. The present study aimed to explore the involvement of CRs in the development of nephroblastoma. RNA-sequencing and clinical information of nephroblastoma samples were obtained by downloading data from the TARGET database. The Limma package was utilized to perform differential expression analysis of genes (DEGs) between the tumor group and the control group. A Venn map was used for intersection of differential genes and CRs and to perform Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis of DEGs using the clusterProfiler package. LASSO and Cox analyses were used to construct CR-related risk models and were evaluated based on clinical parameters. A receiver operating characteristic curve was employed to assess the diagnostic performance of risk model. Furthermore, we used a single-sample gene set enrichment analysis algorithm for immune cell infiltration analysis. Finally, to confirm the transcriptome expression of pivotal genes in human nephroblastoma cell lines, a quantitative real-time PCR was employed. Fifteen key CRs were obtained through analysis in nephroblastoma and then the risk model based on 13 important CRs was constructed using the transcriptome data of nephroblastoma. Using the risk model, pediatric nephroblastoma patients were stratified into high- and low-risk groups based on their individual risk scores. The risk score of CRs can predict adverse outcomes in pediatric nephroblastoma, and this gene cluster is closely related to various immunity characteristics of nephroblastoma. Moreover, the nephroblastoma cell line exhibited higher expression levels of prognostic genes (VRK1, ARNTL, RIT1, PRDM6, and TSPY1) compared to the HEK293 T cell line. The risk characteristics derived from CRs have tremendous significance in predicting prognosis and guiding clinical classification and intervention strategies for pediatric nephroblastoma.

The relationship between vascular endothelial growth factor expression and the risk of childhood nephroblastoma: systematic review and meta-analysis.

BackgroundThis study explores the correlation between vascular endothelial growth factor expression and the risk of childhood nephroblastoma.MethodsPubMed, Embase, Cochrane Library, China National Knowledge Infrastructure (CNKI), Wanfang database were searched to collect independent study data published in China between 2010 and 2021 on the expression and significance of VEGF in childhood nephroblastoma, and literature heterogeneity was tested. The odds ratio (OR) value was used as the effect indicator. Meta-analysis software RevMan 4.2.2 was used, and the original data of each study were statistically processed to calculate the combined OR value and 95% confidence interval (CI).ResultsTwelve studies involving 1,226 cases of pediatric nephroblastoma were included for systematic evaluation. The 12 randomized controlled studies reported the expression of VEGF in childhood nephroblastoma (OR =9.06, 95% CI: 6.97–11.78, P<0.00001). There was a statistically significant difference in expression of VEGF between the unfavorable histology (UH) group and the favorable histology (FH) group (OR =1.17, 95% CI: 1.07–1.27, P=0.0006) and expression of VEGF in different clinical stages of nephroblastoma, including stage I–II and III–IV (OR =0.49, 95% CI: 0.42–0.58, P<0.00001). Positive expression of VEGF showed no significant statistical difference between cases with and without tumor metastasis (OR =1.08, 95% CI: 0.86–1.36, P=0.50).ConclusionsThe expression of VEGF may play an important role in the occurrence and development of childhood nephroblastoma and could help guide clinicians to judge disease and treatment.

Expressions of hypoxia inducible factor-1α and angiopoietin-2 in pediatric nephroblastoma

Objective To explore the expressions of hypoxia-inducible factor-1α(HIF-1α) and angiopoietin-2(Ang-2) in children with nephroblastoma(NB) and its correlation with clinicopathology, prognosis and microvessel density(MVD). Methods Forty-five cases of NB treated by Pediatric Surgery Department of the First Affiliated Hospital of Guangxi Medical University, with NB resected from Jan.2008 to Jan.2012, were divided into groups according to histopathological type, clinical stage and National Wilm's Tumor Study-5 risk factors.The expressions of HIF-1α, Ang-2 and CD34/MVD in NB tissues and adjacent tissues were tested by immunohistochemical method, and the findings were analyzed with the combination of the clinical data and results of the follow-up. Results The ratio of HIF-1α and Ang-2 expressions in NB group was higher than that in the normal adjacent tissues(95.6% vs 40% and 86% vs31.0%, respectively, P<0.05). HIF-1α and Ang-2 expressions were positively correlated with clinic stage and risk factors, and the postoperative cumulated survival rate was higher in low expression group of HIF-1α, Ang-2 compared with the higher expression group of HIF-1α, Ang-2(P<0.05). MVD was higher in NB group than in normal adjacent tissue groups(58.22±10.009 vs 49.43±8.114, P<0.05) and had a positive correlation with HIF-1α(r=0.442, P<0.05) and Ang-2(r=0.590, P<0.05). HIF-1α had a positive correlation with Ang-2(r=0.442, P<0.05). Conclusion HIF-1α and Ang-2 could promote angiogenesis and proliferation in NB, which affects the prognosis. Key words: Hypoxia-inducible factor-1α; Angiopoietin-2; Microvessel density; Nephroblastoma; Prognosis

Wilms’ tumor 1 gene (WT1) is overexpressed and provides an oncogenic function in pediatric nephroblastomas harboring the wild-type WT1

Wilms' tumor 1 gene (WT1) is known to be a tumor suppressor gene in the subset of nephroblastomas that harbors WT1 mutations. However, its role in nephroblastomas without mutations remains unclear. This study aimed to evaluate the expression of WT1 and its potential oncogenic role in pediatric nephroblastoma with wild-type WT1. A total of 24 nephroblastomas were studied for WT1 mRNA expression by quantitative reverse-transcription polymerase chain reaction. The expression levels were compared between nephro-blastomas with and without WT1 mutations, as well as to normal kidney tissue, other pediatric renal tumors and neuroblastomas. Immunohistochemistry was used to evaluate expression patterns at the tissue level. Post-transcriptional inhibition of WT1 was performed in primary cultures of wild-type nephroblastoma using WT1 siRNA. The average WT1 expression level in nephroblastoma tissue was significantly higher than that in normal kidney tissue and neuroblastomas. Expression at the mRNA level was not different between nephroblastomas with WT1 mutations (4 cases) and those with wild-type WT1 (20 cases). However, while WT1 immunoreactivity was positive in all of the nephroblastoma components in the tumors with wild-type WT1, the protein expression was weaker and limited to stromal components in the tumors with mutated WT1, where it co-localized with β-catenin nuclear accumulation. The post-transcriptional inhibition of WT1 resulted in growth retardation and a significantly increased apoptotic fraction. Our study found overexpression of the WT1 gene in pediatric nephroblastomas with wild-type WT1. Moreover, the study suggests an oncogenic role of WT1 in this tumor subset.

Wilms-Tumor bei Erwachsenen

Wilms' tumor (nephroblastoma) is the most frequent renal tumor in childhood. In contrast nephroblastoma in adults is rare, and the disease used to have a poor prognosis. Of 1,300 registered patients, a total of 41 patients older than 16 years were enrolled in the pediatric nephroblastoma trial from 1994 to 2005. Median age at diagnosis was 25.4 years (range: 16-62 years). Treatment was given according to the pediatric protocol. The adults had higher local stages, more frequent metastasis, and developed more toxicity due to therapy. Vincristine caused severe neurotoxicity in many cases. The distribution of histological subtypes was similar to the children's. The outcome is better than previously described with an overall survival of 71%. Patients with local stage I and II have an event-free survival of 84%. This is comparable to children's survival rates. Adults with nephroblastoma have a very good prognosis if treated according to a pediatric protocol.

Mice lacking the 68-amino-acid, mammal-specific N-terminal extension of WT1 develop normally and are fertile.

Mutations in the Wilms' tumor 1 gene, WT1, cause pediatric nephroblastoma and the severe genitourinary disorders of Frasier and Denys-Drash syndromes. High levels of WT1 expression are found in the developing kidney, uterus, and testis--consistent with this finding, the WT1 knockout mouse demonstrates that WT1 is essential for normal genitourinary development. The WT1 gene encodes multiple isoforms of a zinc finger-containing protein by a combination of alternative splicing and alternative translation initiation. The use of an upstream, alternative CUG translation initiation codon specific to mammals results in the production of WT1 protein isoforms with a 68-amino-acid N-terminal extension. To determine the function in vivo of mammal-specific WT1 isoforms containing this extension, gene targeting was employed to introduce a subtle mutation into the WT1 gene. Homozygous mutant mice show a specific absence of the CUG-initiated WT1 isoforms yet develop normally to adulthood and are fertile. Detailed histological analysis revealed normal development of the genitourinary system.

Regulation of gene expression by WT1 in development and tumorigenesis.

WT1 encodes a zinc finger transcription factor implicated in normal development and tumorigenesis. Germline mutation or deletion of WT1 results in a spectrum of abnormal kidney development, male-to-female intersex disorders, and predisposition to pediatric nephroblastoma, Wilms tumor. Initially thought to encode a transcriptional repressor, WT1-dependent functions are now more clearly linked to its property as a transcriptional activator of genes involved in renal development and sex determination. WT1 is expressed in 4 isoforms as a result of 2 alternative messenger RNA splicing events, the more significant of which encodes the 3 amino acids lysine, threonine, and serine (KTS) between zinc fingers 3 and 4. Although WT1 isoforms lacking KTS act as sequence-specific DNA binding factors, a large body of evidence now implicates the KTS-containing isoforms in RNA processing. In keeping with distinct biochemical mechanisms for these isoforms, genetic data from humans and mice point to separate but partially overlapping roles for WT1 (+KTS) and (-KTS) during genitourinary development. Recently, a hematopoietic model system has been used to study functional properties of WT1 in vitro. WT1 expression in primary hematopoietic cells leads to stage-specific effects that may be relevant to WT1-mediated tumor suppression.

Differential Effects of Wilms Tumor WT1 Splice Variants on the Insulin Receptor Promoter

The Wilms tumor gene WT1 has been implicated in the early development of the kidney. Mutations in WT1 are found in a small fraction of Wilms tumor, a pediatric nephroblastoma, and Denys–Drash syndrome, characterized by genitourinary abnormalities. The WT1 gene product functions as a transcriptional repressor of growth factor-related genes. The kidney is one of the major sites of insulin actionin vivoand expresses high levels of insulin receptors (IR). IR expression has been detected during early embryogenesis, suggesting that it may play a role in development. We investigated whether two WT1 splice variants lacking or including a three-amino-acid (KTS) insertion between the third and fourth zinc finger in the DNA-binding domain could repress the IR promoterin vitro.We show that the +KTS variant effectively represses promoter activity under all conditions tested but the −KTS variant was only able to repress in the presence of cotransfected C/EBPβ or a dominant-negative p53 mutation. Deletional mapping indicated that distinct regions of the IR promoter mediated the effects of the two isoforms and DNaseI footprint analysis identified potential WT1 binding sites within these regions.

The WT1 Wilms tumor gene product: a developmentally regulated transcription factor in the kidney that functions as a tumor suppressor

Alteration of transcription factor function is becoming a common theme in molecular mechanisms of oncogenesis. A recent example of this trend is the isolation and characterization of the chromosome 11p13 Wilms tumor suppressor gene, WT1. The WT1 protein contains a DNA binding domain consisting of four zinc fingers of the Cys2-His2 class and a proline-glutamine rich region capable of regulating transcription. Deletions of the WT1 gene or point mutations which destroy the DNA binding activity of the protein are associated with the development of the pediatric nephroblastoma Wilms tumor and Denys-Drash syndrome. This article reviews the role of WT1 in normal kidney development processes, the known biochemical functions of the protein and the status of identifying target genes regulated by this potentially oncogenic transcription factor.

Correlation of the inability to sustain growth in defined serum-free medium with the suppression of tumorigenicity in Wilms' nephroblastoma

We report the investigation of the growth properties of tumorigenic and reverted nontumorigenic Wilms' nephroblastoma cells when cultured in serum-free medium. Wilms' tumor, a pediatric nephroblastoma, has been associated with deletions encompassing the p13 band of chromosome 11 and an independent loss of heterozygosity at 11p15. Weissman et al. (Science 236:175-180, 1987) transferred a human der(11) chromosome into the G401.6TG.6 Wilms' tumor cell line via the microcell-mediated chromosome transfer technique. The resulting microcell hybrids were nontumorigenic when assayed in nude mice; however these cells retained all of the in vitro growth and morphological characteristics of the tumorigenic parental cells in 10% fetal calf serum (FCS). Segregation of the der(11) chromosome from the nontumorigenic microcell hybrid cells resulted in the reappearance of the tumorigenic phenotype in vivo. In vitro culture of these cell lines in serum-free medium supplemented with 0.1% bovine serum albumin (BSA) and 10 ng/ml Na2O3Se resulted in sustained growth of both the tumorigenic parent and the tumorigenic segregant while the nontumorigenic microcell hybrids were unable to divide. The separate addition of either 10 ng/ml of epidermal growth factor (EGF) or 5 micrograms/ml of insulin did not alter this effect. However, the addition of 5 micrograms/ml of transferrin stimulated the nontumorigenic microcell hybrid cells to grow at a rate comparable to the tumorigenic cells. In addition, conditioned serum-free medium from the tumorigenic parental or tumorigenic segregant cell lines was able to stimulate the growth of the nontumorigenic microcell hybrid cells, whereas the reciprocal experiment had no effect on the growth of the tumorigenic cells. These data suggest that the inability of the microcell hybrid cells to grow in serum-free conditions is correlated with their genetic nontumorigenic phenotype and that a specific growth factor, transferrin, can bypass or alter this negative growth regulatory pathway(s) in vitro.

The isolation and characterization of a novel cDNA demonstrating an altered mRNA level in nontumorigenic Wilms' microcell hybrid cells.

Wilms' tumor, a pediatric nephroblastoma, has been associated with genetic alterations of the 11p13 and 11p15 regions. The introduction of a der(11) chromosome into the G401 Wilms' tumor cell line has been shown previously to revert the tumorigenic phenotype. A subtractive cDNA/RNA hybridization performed between the tumorigenic parent (G401) and a nontumorigenic microcell hybrid of G401 (110.1/G401.1) containing the der(11) chromosome resulted in the identification of a single novel cDNA clone, designated QM. The cDNA is 745 nucleotides in length and encodes a predicted hydrophilic 25 kd basic protein, primarily consisting of alpha helices. The QM transcript is expressed in a wide variety of embryonic and adult tissues and demonstrates a down regulation of expression in adult kidney and heart. QM is also a member of a multigene family members of which map to chromosomes 6 and 14. The QM mRNA level is modulated between the tumorigenic and nontumorigenic cell lines and therefore may be involved in the maintenance of the nontumorigenic phenotype.

Role of the retinoblastoma gene in the oncogenesis of human breast carcinoma.

The notion that genetic alterations are involved in cancer has gained support from many studies. Certain genetic alterations in tumors have been precisely defined. First, the activated proto-oncogenes were isolated from tumors by their abilities to transform nonneoplastic cells in culture [1]. To date many genes potentially involved in cancer have been isolated based on the sequence homology to oncogenes of retroviruses [for review see 2]. In contrast to the activated oncogenes in tumor cells, a different class of cancer genes has been suggested by somatic cell hybrid studies. Fusion cells made between tumor cells and normal fibroblasts, lymphocytes, or kerati- nocytes are often nontumorigenic, an effect contributed by chromosomes [1–5]. A correlation exists between suppression of tumorigenicity and the retention of certain chromosomes in the fused cells [6]. For example, the introduction of chromosome 11, but not chromosome X nor 13, suppresses the tumorigenic phenotype of a Wilm’s tumor cell line, indicating the tumor suppressor gene for pediatric nephroblastoma is located on chromosome 11 [7]. These genes are actively expressed in normal cells and are mutationally inactivated in tumor cells. They are termed ’tumor suppressor genes’, since the presence of one or more normal alleles is thought to prevent the tumor phenotype [for review see 8, 9]. A complementary line of evidence comes from cytogenetic observations in which a specific chromosomal deletion occurs in somatic or tumor cells from patients with retinoblastoma, Wilm’s tumor, and bilateral acoustic neurofibromatosis [10–12]. This suggests the existence of a tumor suppressor gene in normal cells in specific chromosomal regions. Additional support for tumor suppressor genes is derived from the isolation of DNA fragments capable of reverting the transformed phenotypes of cultured cells [13]. A recent study led to the identification of the Krev—1 gene that converts the H-ras transformed cells into flat revertants upon transfection. Sequence analysis indicates that significant amino acid homology exists between Krev-1 and H-ras [14].

Introduction of a normal human chromosome 11 into a Wilms' tumor cell line controls its tumorigenic expression.

The development of Wilms' tumor, a pediatric nephroblastoma, has been associated with a deletion in the p13 region of chromosome 11. The structure and function or functions of this deleted genetic material are unknown. The role of this deletion in the process of malignant transformation was investigated by introducing a normal human chromosome 11 into a Wilms' tumor cell line by means of the microcell transfer technique. These variant cells, derived by microcell hybridization, expressed similar transformed traits in culture as the parental cell line. Furthermore, expression of several proto-oncogenes by the parental cells was unaffected by the introduction of this chromosome. However, the ability of these cells to form tumors in nude mice was completely suppressed. Transfer of other chromosomes, namely X and 13, had no effect on the tumorigenicity of the Wilms' tumor cells. These studies provide support for the existence of genetic information on chromosome 11 which can control the malignant expression of Wilms' tumor cells.