A novel mouse model of diffuse midline glioma initiated in neonatal oligodendrocyte progenitor cells highlights cell-of-origin dependent effects of H3K27M.

Diffuse midline glioma (DMG) is a type of lethal brain tumor that develops mainly in children. The majority of DMG harbor the K27M mutation in histone H3. Oligodendrocyte progenitor cells (OPCs) in the brainstem are candidate cells-of-origin for DMG, yet there is no genetically engineered mouse model of DMG initiated in OPCs. Here, we used the RCAS/Tv-a avian retroviral system to generate DMG in Olig2-expressing progenitors and Nestin-expressing progenitors in the neonatal mouse brainstem. PDGF-A or PDGF-B overexpression, along with p53 deletion, resulted in gliomas in both models. Exogenous overexpression of H3.3K27M had a significant effect on tumor latency and tumor cell proliferation when compared with H3.3WT in Nestin+ cells but not in Olig2+ cells. Further, the fraction of H3.3K27M-positive cells was significantly lower in DMGs initiated in Olig2+ cells relative to Nestin+ cells, both in PDGF-A and PDGF-B-driven models, suggesting that the requirement for H3.3K27M is reduced when tumorigenesis is initiated in Olig2+ cells. RNA-sequencing analysis revealed that the differentially expressed genes in H3.3K27M tumors were non-overlapping between Olig2;PDGF-B, Olig2;PDGF-A, and Nestin;PDGF-A models. GSEA analysis of PDGFA tumors confirmed that the transcriptomal effects of H3.3K27M are cell-of-origin dependent with H3.3K27M promoting epithelial-to-mesenchymal transition (EMT) and angiogenesis when Olig2 marks the cell-of-origin and inhibiting EMT and angiogenesis when Nestin marks the cell-of-origin. We did observe some overlap with H3.3K27M promoting negative enrichment of TNFA_Signaling_Via_NFKB in both models. Our study suggests that the tumorigenic effects of H3.3K27M are cell-of-origin dependent, with H3.3K27M being more oncogenic in Nestin+ cells than Olig2+ cells.

BET bromodomain inhibition potentiates radiosensitivity in models of H3K27-altered diffuse midline glioma.

Diffuse midline glioma (DMG) H3K27-altered is one of the most malignant childhood cancers. Radiation therapy remains the only effective treatment yet provides a 5-year survival rate of only 1%. Several clinical trials have attempted to enhance radiation antitumor activity using radiosensitizing agents, although none have been successful. Given this, there is a critical need for identifying effective therapeutics to enhance radiation sensitivity for the treatment of DMG. Using high-throughput radiosensitivity screening, we identified bromo- and extraterminal domain (BET) protein inhibitors as potent radiosensitizers in DMG cells. Genetic and pharmacologic inhibition of BET bromodomain activity reduced DMG cell proliferation and enhanced radiation-induced DNA damage by inhibiting DNA repair pathways. RNA-Seq and the CUT&RUN (cleavage under targets and release using nuclease) analysis showed that BET bromodomain inhibitors regulated the expression of DNA repair genes mediated by H3K27 acetylation at enhancers. BET bromodomain inhibitors enhanced DMG radiation response in patient-derived xenografts as well as genetically engineered mouse models. Together, our results highlight BET bromodomain inhibitors as potential radiosensitizer and provide a rationale for developing combination therapy with radiation for the treatment of DMG.

Hallmark discoveries in the biology of Wilms tumour.

The modern study of Wilms tumour was prompted nearly 50 years ago, when Alfred Knudson proposed the 'two-hit' model of tumour development. Since then, the efforts of researchers worldwide have substantially expanded our knowledge of Wilms tumour biology, including major advances in genetics - from cloning the first Wilms tumour gene to high-throughput studies that have revealed the genetic landscape of this tumour. These discoveries improve understanding of the embryonal origin of Wilms tumour, familial occurrences and associated syndromic conditions. Many efforts have been made to find and clinically apply prognostic biomarkers to Wilms tumour, for which outcomes are generally favourable, but treatment of some affected individuals remains challenging. Challenges are also posed by the intratumoural heterogeneity of biomarkers. Furthermore, preclinical models of Wilms tumour, from cell lines to organoid cultures, have evolved. Despite these many achievements, much still remains to be discovered: further molecular understanding of relapse in Wilms tumour and of the multiple origins of bilateral Wilms tumour are two examples of areas under active investigation. International collaboration, especially when large tumour series are required to obtain robust data, will help to answer some of the remaining unresolved questions.

Mediators of receptor tyrosine kinase activation in infantile fibrosarcoma: a Children's Oncology Group study.

Infantile fibrosarcoma (IFS; also known as cellular congenital mesoblastic nephroma, CMN, when in the kidney) is a rare, undifferentiated tumour often characterized by the ETV6-NTRK3 fusion transcript. Our goal was to identify downstream pathways, diagnostic markers and potential therapeutic targets for IFS/CMN. Global gene expression, reverse-phase protein array and ETV6-NTRK3 fusion analyses were performed on 14 IFS/CMN and compared with 41 other paediatric renal tumours. These analyses confirm significant receptor tyrosine kinase (RTK) activation, with evidence of PI3-Akt, MAPK and SRC activation. In particular, GAB2 docking protein, STAT5-pTyr-694, STAT3-pSer-729 and YAP-pSer-127 were elevated, and TAZ-pSer-89 was decreased. This provides mRNA and proteomic evidence that GAB2, STAT activation and phosphorylation of the Hippo pathway transcription co-activators YAP and TAZ contribute to the RTK signal transduction in IFS/CMN. All IFS/CMN tumours displayed a distinctive gene expression pattern that may be diagnostically useful. Unexpectedly, abundant ETV6-NTRK3 transcript copies were present in only 7/14 IFS, with very low copy number in 3/14. An additional 4/14 were negative by RT-PCR and absence of ETV6-NTRK3 was confirmed by FISH for both ETV6 and NTRK3. Therefore, molecular mechanisms other than ETV6-NTRK3 fusion are responsible for the development of some IFS/CMNs and the absence of ETV6-NTRK3 fusion products should not exclude IFS/CMN as a diagnosis.

Novel genetically engineered H3.3G34R model reveals cooperation with ATRX loss in upregulation of Hoxa cluster genes and promotion of neuronal lineage.

Background: Pediatric high-grade gliomas (pHGGs) are aggressive pediatric CNS tumors and an important subset are characterized by mutations in H3F3A, the gene that encodes Histone H3.3 (H3.3). Substitution of Glycine at position 34 of H3.3 with either Arginine or Valine (H3.3G34R/V), was recently described and characterized in a large cohort of pHGG samples as occurring in 5-20% of pHGGs. Attempts to study the mechanism of H3.3G34R have proven difficult due to the lack of knowledge regarding the cell-of-origin and the requirement for co-occurring mutations for model development. We sought to develop a biologically relevant animal model of pHGG to probe the downstream effects of the H3.3G34R mutation in the context of vital co-occurring mutations. Methods: We developed a genetically engineered mouse model (GEMM) that incorporates PDGF-A activation, TP53 loss and the H3.3G34R mutation both in the presence and loss of Alpha thalassemia/mental retardation syndrome X-linked (ATRX), which is commonly mutated in H3.3G34 mutant pHGGs. Results: We demonstrated that ATRX loss significantly increases tumor latency in the absence of H3.3G34R and inhibits ependymal differentiation in the presence of H3.3G34R. Transcriptomic analysis revealed that ATRX loss in the context of H3.3G34R upregulates Hoxa cluster genes. We also found that the H3.3G34R overexpression leads to enrichment of neuronal markers but only in the context of ATRX loss. Conclusions: This study proposes a mechanism in which ATRX loss is the major contributor to many key transcriptomic changes in H3.3G34R pHGGs. Accession number: GSE197988.

Integrative Genomic Analyses Identify LncRNA Regulatory Networks across Pediatric Leukemias and Solid Tumors.

Long noncoding RNAs (lncRNA) play an important role in gene regulation and contribute to tumorigenesis. While pan-cancer studies of lncRNA expression have been performed for adult malignancies, the lncRNA landscape across pediatric cancers remains largely uncharted. Here, we curated RNA sequencing data for 1,044 pediatric leukemia and extracranial solid tumors and integrated paired tumor whole genome sequencing and epigenetic data in relevant cell line models to explore lncRNA expression, regulation, and association with cancer. A total of 2,657 lncRNAs were robustly expressed across six pediatric cancers, including 1,142 exhibiting histotype-elevated expression. DNA copy number alterations contributed to lncRNA dysregulation at a proportion comparable to protein coding genes. Application of a multidimensional framework to identify and prioritize lncRNAs impacting gene networks revealed that lncRNAs dysregulated in pediatric cancer are associated with proliferation, metabolism, and DNA damage hallmarks. Analysis of upstream regulation via cell type-specific transcription factors further implicated distinct histotype-elevated and developmental lncRNAs. Integration of these analyses prioritized lncRNAs for experimental validation, and silencing of TBX2-AS1, the top-prioritized neuroblastoma-specific lncRNA, resulted in significant growth inhibition of neuroblastoma cells, confirming the computational predictions. Taken together, these data provide a comprehensive characterization of lncRNA regulation and function in pediatric cancers and pave the way for future mechanistic studies. SIGNIFICANCE: Comprehensive characterization of lncRNAs in pediatric cancer leads to the identification of highly expressed lncRNAs across childhood cancers, annotation of lncRNAs showing histotype-specific elevated expression, and prediction of lncRNA gene regulatory networks.

A tumor suppressor role for EZH2 in diffuse midline glioma pathogenesis.

Pediatric high-grade gliomas, specifically diffuse midline gliomas, account for only 20% of clinical cases but are 100% fatal. A majority of the DMG cases are characterized by the signature K27M mutation in histone H3. The H3K27M mutation opposes the function of enhancer of zeste homolog 2 (EZH2), the methyltransferase enzyme of the polycomb repressor complex 2. However, the role of EZH2 in DMG pathogenesis is unclear. In this study, we demonstrate a tumor suppressor function for EZH2 using Ezh2 loss- and gain-of-function studies in H3WT DMG mouse models. Genetic ablation of Ezh2 increased cell proliferation and tumor grade while expression of an Ezh2 gain-of-function mutation significantly reduced tumor incidence and increased tumor latency. Transcriptomic analysis revealed that Ezh2 deletion upregulates an inflammatory response with upregulation of immunoproteasome genes such as Psmb8, Psmb9, and Psmb10. Ezh2 gain-of-function resulted in enrichment of the oxidative phosphorylation/mitochondrial metabolic pathway namely the isocitrate dehydrogenase Idh1/2/3 genes. Pharmacological inhibition of EZH2 augmented neural progenitor cell proliferation, supporting the tumor suppressive role of EZH2. In vivo 7-day treatment of H3K27M DMG tumor bearing mice with an EZH2 inhibitor, Tazemetostat, did not alter proliferation or significantly impact survival. Together our results suggest that EZH2 has a tumor suppressor function in DMG and warrants caution in clinical translation of EZH2 inhibitors to treat patients with DMG.

Genetic changes associated with relapse in favorable histology Wilms tumor: A Children's Oncology Group AREN03B2 study.

Over the last decade, sequencing of primary tumors has clarified the genetic underpinnings of Wilms tumor but has not affected therapy, outcome, or toxicity. We now sharpen our focus on relapse samples from the umbrella AREN03B2 study. We show that over 40% of relapse samples contain mutations in SIX1 or genes of the MYCN network, drivers of progenitor proliferation. Not previously seen in large studies of primary Wilms tumors, DIS3 and TERT are now identified as recurrently mutated. The analysis of primary-relapse tumor pairs suggests that 11p15 loss of heterozygosity (and other copy number changes) and mutations in WT1 and MLLT1 typically occur early, but mutations in SIX1, MYCN, and WTX are late developments in some individuals. Most strikingly, 75% of relapse samples had gain of 1q, providing strong conceptual support for studying circulating tumor DNA in clinical trials to better detect 1q gain earlier and monitor response.

Differential gene expression of soluble CD8+ T-cell mediated suppression of HIV replication in three older children.

Suppression of human immunodeficiency virus (HIV) replication by CD8+ T-cells (CD8 suppression) contributes to survival in adults and children <1 year. Soluble CD8 suppression can also be seen in some older children with AIDS. The factor responsible, CD8-derived antiviral factor (CAF), acts at the level of HIV RNA transcription. Differential gene expression techniques have been used to define the gene(s) mediating this phenomenon in adults. Recently, CAF has been linked to exosomes secreted by CD8+ T-cells. To compare the gene expression profiles from pediatric patients with each other, with those reported in 2 previous studies in adults and in those reportedly related to exosomes, we used differential gene expression to study three older children with HIV infection, one who did demonstrate soluble CD-8 suppression and two who did not. Eighteen differentially expressed genes were also seen in one adult study (P = 0.002, χ(2) test), and 38 such genes (P < 0.0001, χ(2) test) in a second adult study. In addition, two exosome components and some RNA's related to exosomal proteins were also differentially expressed. In children with HIV infection, we found significant differentially expressed genes that correlated to those previously reported in two studies in adults. Our data also lends some support to the recent identification of CAF with exosomes secreted by CD8+ T-cells.

Prenatal overexpression of platelet-derived growth factor receptor A results in central nervous system hypomyelination.

BACKGROUND: Platelet-derived growth factor (PDGF) signaling, through the ligand PDGF-A and its receptor PDGFRA, is important for the growth and maintenance of oligodendrocyte progenitor cells (OPCs) in the central nervous system (CNS). PDGFRA signaling is downregulated prior to OPC differentiation into mature myelinating oligodendrocytes. By contrast, PDGFRA is often genetically amplified or mutated in many types of gliomas, including diffuse midline glioma (DMG) where OPCs are considered the most likely cell-of-origin. The cellular and molecular changes that occur in OPCs in response to unregulated PDGFRA expression, however, are not known. METHODS: Here, we created a conditional knock-in (KI) mouse that overexpresses wild type (WT) human PDGFRA (hPDGFRA) in prenatal Olig2-expressing progenitors, and examined in vivo cellular and molecular consequences. RESULTS: The KI mice exhibited stunted growth, ataxia, and a severe loss of myelination in the brain and spinal cord. When combined with the loss of p53, a tumor suppressor gene whose activity is decreased in DMG, the KI mice failed to develop tumors but still exhibited hypomyelination. RNA-sequencing analysis revealed decreased myelination gene signatures, indicating a defect in oligodendroglial development. Mice overexpressing PDGFRA in prenatal GFAP-expressing progenitors, which give rise to a broader lineage of cells than Olig2-progenitors, also developed myelination defects. CONCLUSION: Our results suggest that embryonic overexpression of hPDGFRA in Olig2- or GFAP-progenitors is deleterious to OPC development and leads to CNS hypomyelination.

Rhabdoid tumor: gene expression clues to pathogenesis and potential therapeutic targets.

Rhabdoid tumors (RT) are aggressive tumors characterized by genetic loss of SMARCB1 (SNF5, INI-1), a component of the SWI/SNF chromatin remodeling complex. No effective treatment is currently available. This study seeks to shed light on the SMARCB1-mediated pathogenesis of RT and to discover potential therapeutic targets. Global gene expression of 10 RT was compared with 12 cellular mesoblastic nephromas, 16 clear cell sarcomas of the kidney, and 15 Wilms tumors. In all, 114 top genes were differentially expressed in RT (P<0.001, fold change >2 or <0.5). Among these were downregulation of SMARCB1 and genes previously associated with SMARCB1 (ATP1B1, PTN, DOCK4, NQO1, PLOD1, PTP4A2, PTPRK); 28/114 top differentially expressed genes were involved with neural or neural crest development and were all sharply downregulated. This was confirmed by Gene Set Enrichment Analysis (GSEA). Neural and neural crest stem cell marker proteins SOX10, ID3, CD133, and Musashi were negative by immunohistochemistry, whereas Nestin was positive. Decreased expression of CDKN1A, CDKN1B, CDKN1C, CDKN2A, and CCND1 was identified, while MYC-C was upregulated. GSEA of independent gene sets associated with bivalent histone modification and polycomb group targets in embryonic stem cells showed significant negative enrichment in RT. Several differentially expressed genes were associated with tumor suppression, invasion, and metastasis, including SPP1 (osteopontin), COL18A1 (endostatin), PTPRK, and DOCK4. We conclude that RTs arise within early progenitor cells during a critical developmental window in which loss of SMARCB1 directly results in repression of neural development, loss of cyclin-dependent kinase inhibition, and trithorax/polycomb dysregulation.

Predicting relapse in favorable histology Wilms tumor using gene expression analysis: a report from the Renal Tumor Committee of the Children's Oncology Group.

PURPOSE: The past two decades has seen significant improvement in the overall survival of patients with favorable histology Wilms tumor (FHWT); however, this progress has reached a plateau. Further improvements may rely on the ability to better stratify patients by risk of relapse. This study determines the feasibility and potential clinical utility of classifiers of relapse based on global gene expression analysis. EXPERIMENTAL DESIGN: Two hundred fifty FHWT of all stages enriched for relapses treated on National Wilms Tumor Study-5 passed quality variables and were suitable for analysis using oligonucleotide arrays. Relapse risk stratification used support vector machine; 2- and 10-fold cross-validations were applied. RESULTS: The number of genes associated with relapse was less than that predicted by chance alone for 106 patients (32 relapses) with stages I and II FHWT treated with chemotherapy, and no further analyses were done. This number was greater than expected by chance for 76 local stage III patients. Cross-validation including an additional 68 local stage III patients (total 144 patients, 53 relapses) showed that classifiers for relapse composed of 50 genes were associated with a median sensitivity of 47% and specificity of 70%. CONCLUSIONS: This study shows the feasibility and modest accuracy of stratifying local stage III FHWT using a classifier of <50 genes. Validation using an independent patient population is needed. Analysis of genes differentially expressed in relapse patients revealed apoptosis, Wnt signaling, insulin-like growth factor pathway, and epigenetic modification to be mechanisms important in relapse. Potential therapeutic targets include FRAP/MTOR and CD40.

ACVR1 R206H cooperates with H3.1K27M in promoting diffuse intrinsic pontine glioma pathogenesis.

Diffuse intrinsic pontine glioma (DIPG) is an incurable pediatric brain tumor, with approximately 25% of DIPGs harboring activating ACVR1 mutations that commonly co-associate with H3.1K27M mutations. Here we show that in vitro expression of ACVR1 R206H with and without H3.1K27M upregulates mesenchymal markers and activates Stat3 signaling. In vivo expression of ACVR1 R206H or G328V with H3.1K27M and p53 deletion induces glioma-like lesions but is not sufficient for full gliomagenesis. However, in combination with PDGFA signaling, ACVR1 R206H and H3.1K27M significantly decrease survival and increase tumor incidence. Treatment of ACVR1 R206H mutant DIPGs with exogenous Noggin or the ACVR1 inhibitor LDN212854 significantly prolongs survival, with human ACVR1 mutant DIPG cell lines also being sensitive to LDN212854 treatment. Together, our results demonstrate that ACVR1 R206H and H3.1K27M promote tumor initiation, accelerate gliomagenesis, promote a mesenchymal profile partly due to Stat3 activation, and identify LDN212854 as a promising compound to treat DIPG.

Prohibitin is a prognostic marker and therapeutic target to block chemotherapy resistance in Wilms' tumor.

Wilms' tumor is the most common type of childhood kidney cancer. To improve risk stratification and identify novel therapeutic targets for patients with Wilms' tumor, we used high-resolution mass spectrometry proteomics to identify urine tumor markers associated with Wilms' tumor relapse. We determined the urine proteomes at diagnosis of 49 patients with Wilms' tumor, non-Wilms' tumor renal tumors, and age-matched controls, leading to the quantitation of 6520 urine proteins. Supervised analysis revealed specific urine markers of renal rhabdoid tumors, kidney clear cell sarcomas, renal cell carcinomas as well as those detected in patients with cured and relapsed Wilms' tumor. In particular, urine prohibitin was significantly elevated at diagnosis in patients with relapsed as compared with cured Wilms' tumor. In a validation cohort of 139 patients, a specific urine prohibitin ELISA demonstrated that prohibitin concentrations greater than 998 ng/mL at diagnosis were significantly associated with ultimate Wilms' tumor relapse. Immunohistochemical analysis revealed that prohibitin was highly expressed in primary Wilms' tumor specimens and associated with disease stage. Using functional genetic experiments, we found that prohibitin was required for the growth and survival of Wilms' tumor cells. Overexpression of prohibitin was sufficient to block intrinsic mitochondrial apoptosis and to cause resistance to diverse chemotherapy drugs, at least in part by dysregulating factors that control apoptotic cytochrome c release from mitochondrial cristae. Thus, urine prohibitin may improve therapy stratification, noninvasive monitoring of treatment response, and early disease detection. In addition, therapeutic targeting of chemotherapy resistance induced by prohibitin dysregulation may offer improved therapies for patients with Wilms' and other relapsed or refractory tumors.

A unique subset of low-risk Wilms tumors is characterized by loss of function of TRIM28 (KAP1), a gene critical in early renal development: A Children's Oncology Group study.

This study explores the genomic alterations that contribute to the formation of a unique subset of low-risk, epithelial differentiated, favorable histology Wilms tumors (WT), tumors that have been characterized by their expression of post-induction renal developmental genes (Subset 1 WT). We demonstrate copy neutral loss of heterozygosity involving 19q13.32-q13.43, unaccompanied by evidence for imprinting by DNA methylation. We further identified loss-of-function somatic mutations in TRIM28 (also known as KAP1), located at 19q13, in 8/9 Subset 1 tumors analyzed. An additional germline TRIM28 mutation was identified in one patient. Retrospective evaluation of previously analyzed WT outside of Subset 1 identified an additional tumor with anaplasia and both TRIM28 and TP53 mutations. A major function of TRIM28 is the repression of endogenous retroviruses early in development. We depleted TRIM28 in HEK293 cells, which resulted in increased expression of endogenous retroviruses, a finding also demonstrated in TRIM28-mutant WT. TRIM28 has been shown by others to be active during early renal development, and to interact with WTX, another gene recurrently mutated in WT. Our findings suggest that inactivation of TRIM28 early in renal development contributes to the formation of this unique subset of FHWTs, although the precise manner in which TRIM28 impacts both normal renal development and oncogenesis remains elusive.

Polo-Like Kinase 4 (PLK4) Is Overexpressed in Central Nervous System Neuroblastoma (CNS-NB).

Neuroblastoma (NB) is the most common extracranial solid tumor in pediatrics, with rare occurrences of primary and metastatic tumors in the central nervous system (CNS). We previously reported the overexpression of the polo-like kinase 4 (PLK4) in embryonal brain tumors. PLK4 has also been found to be overexpressed in a variety of peripheral adult tumors and recently in peripheral NB. Here, we investigated PLK4 expression in NBs of the CNS (CNS-NB) and validated our findings by performing a multi-platform transcriptomic meta-analysis using publicly available data. We evaluated the PLK4 expression by quantitative real-time PCR (qRT-PCR) on the CNS-NB samples and compared the relative expression levels among other embryonal and non-embryonal brain tumors. The relative PLK4 expression levels of the NB samples were found to be significantly higher than the non-embryonal brain tumors (p-value < 0.0001 in both our samples and in public databases). Here, we expand upon our previous work that detected PLK4 overexpression in pediatric embryonal tumors to include CNS-NB. As we previously reported, inhibiting PLK4 in embryonal tumors led to decreased tumor cell proliferation, survival, invasion and migration in vitro and tumor growth in vivo, and therefore PLK4 may be a potential new therapeutic approach to CNS-NB.

Ligand-independent dimerization of the human prolactin receptor isoforms: functional implications.

Prolactin (PRL) contributes to the growth of normal and malignant breast tissues. PRL initiates signaling by engaging the PRL receptor (PRLr), a transmembrane (TM) receptor belonging to the cytokine receptor family. The accepted view has been that PRL activates the PRLr by inducing dimerization of the receptor, but recent reports show ligand-independent dimerization of other cytokine receptors. Using coimmunoprecipitation assays, we have confirmed ligand-independent dimerization of the PRLr in T47D breast cancer and HepG2 liver carcinoma cells. In addition, mammalian cells transfected with differentially epitope-tagged isoforms of the PRLr indicated that long, intermediate, and DeltaS1 PRLrs dimerized in a ligand-independent manner. To determine the domain(s) involved in PRLr ligand-independent dimerization, we generated PRLr constructs as follows: (1) the TM-ICD, which consisted of the TM domain and the intracellular domain (ICD) but lacked the extracellular domain (ECD), and (2) the ECD-TM, which consisted of the TM domain and the ECD but lacked the ICD. These constructs dimerized in a ligand-independent manner in mammalian cells, implicating a significant role for the TM domain in this process. These truncated PRLrs were functionally inert alone or in combination in cells lacking the PRLr. However, when introduced into cells containing endogenous PRLr, the ECD-TM inhibited human PRLr signaling, whereas the TM-ICD potentiated human PRLr signaling. These studies indicate that the ECD-TM and the TM-ICD are capable of modulating PRLr function. We also demonstrated an endogenous TM-ICD in T47D cells, suggesting that these findings are relevant to PRL-signaling pathways in breast cancer.

BRAF exon 15 mutations in pediatric renal stromal tumors: prevalence in metanephric stromal tumors.

Metanephric stromal tumors (MSTs) are rare renal stromal tumors that predominantly affect children. They belong to the metanephric family of tumors, along with metanephric adenofibroma and metanephric adenoma. The previous documentation of BRAF exon 15 mutations in 88% of metanephric adenomas and in isolated cases of metanephric adenofibroma prompted us to investigate the prevalence of these mutations in MSTs and in other pediatric renal stromal tumors. In this study, 17 MSTs, 22 congenital mesoblastic nephromas, and 6 ossifying renal tumors of infancy were selected for BRAF exon 15 testing. Tumor genomic DNA was extracted from formalin-fixed paraffin-embedded tissue, followed by polymerase chain reaction amplification and Sanger dideoxy sequencing with primers flanking the BRAF exon 15 gene. BRAF exon 15 mutations were found in 11 (65%) of the 17 cases of MST, all corresponding to a thymidine-to-adenine substitution at codon 600 (BRAF V600E). All other renal stromal tumors tested were negative for BRAF exon 15 mutations. In conclusion, BRAF V600E mutations are encountered in most MSTs, supporting a link with other metanephric tumors and suggesting a clonal event possibly affecting primordial renal cells. In addition, BRAF V600E mutations have been associated with oncogene-induced senescence in other benign tumors, providing clues to the pathogenesis of metanephric neoplasms in keeping with their overall benign behavior. Our results also suggest a potential diagnostic use for BRAF exon 15 mutations in differentiating MSTs from other pediatric renal stromal tumors, particularly in limited samples.