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Purpose: To investigate the role and significance of TP53 mutation in diffusely anaplastic Wilms tumors (DAWTs).Experimental Design: All DAWTs registered on National Wilms Tumor Study-5 (n = 118) with available samples were analyzed for TP53 mutations and copy loss. Integrative genomic analysis was performed on 39 selected DAWTs.Results: Following analysis of a single random sample, 57 DAWTs (48%) demonstrated TP53 mutations, 13 (11%) copy loss without mutation, and 48 (41%) lacked both [defined as TP53-wild-type (wt)]. Patients with stage III/IV TP53-wt DAWTs (but not those with stage I/II disease) had significantly lower relapse and death rates than those with TP53 abnormalities. In-depth analysis of a subset of 39 DAWTs showed seven (18%) to be TP53-wt: These demonstrated gene expression evidence of an active p53 pathway. Retrospective pathology review of TP53-wt DAWT revealed no or very low volume of anaplasia in six of seven tumors. When samples from TP53-wt tumors known to contain anaplasia histologically were available, abnormal p53 protein accumulation was observed by immunohistochemistry.Conclusions: These data support the key role of TP53 loss in the development of anaplasia in WT, and support its significant clinical impact in patients with residual anaplastic tumor following surgery. These data also suggest that most DAWTs will show evidence of TP53 mutation when samples selected for the presence of anaplasia are analyzed. This suggests that modifications of the current criteria to also consider volume of anaplasia and documentation of TP53 aberrations may better reflect the risk of relapse and death and enable optimization of therapeutic stratification. Clin Cancer Res; 22(22); 5582–91. ©2016 AACR.

Supplementary Table S2 from 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: Recent studies suggest that children Experimental Design: To define biologically distinct subsets, global gene expression analysis was done on 39 VLRWT that passed all quality-control parameters and the clusters identified were validated in an independent set of 11 VLRWT. Validation of select differentially expressed genes was done with immunohistochemistry on a tissue microarray from 20 of 39 tumors. Loss of heterozygosity (LOH) for 11p15, 1p, and 16q was analyzed in 52 tumors using PCR.Results: Two distinctive clusters were identified. One cluster included 9 tumors with epithelial differentiated tubular histology, paucity of nephrogenic rests, lack of LOH for 1p, 16q, and 11p, absence of relapse, and a unique gene expression profile consistent with arrest following mesenchymal-to-epithelial transition. The second cluster included 13 tumors with mixed histology, intralobar nephrogenic rests, and decreased expression of WT1. Three of 6 relapses occurred in this cluster. Of 43 informative tumors, 11p LOH was present in 5 of 5 relapses and 11 of 38 nonrelapses.Conclusions: Two subsets comprising a total of 56 of VLRWT are identified that have pathogenetic and molecular differences and apparent differences in risk for relapse. If these predictors can be prospectively validated, this would enable the refinement of clinical stratification and less arbitrary definition of VLRWT. (Clin Cancer Res 2009;15(22):68009)

This table shows the recurrent mutations that were identified as well as the gains/losses at previously reported chromosomal segments in the 39 DAWTs that were selected for in-depth analysis

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

Supplementary Data from Subsets of Very Low Risk Wilms Tumor Show Distinctive Gene Expression, Histologic, and Clinical Features

This table shows all TP53 variant that were identified by Whole Genome or Exome Sequencing in all DAWTs included in this study

This table shows the sequences of the probes used for MLPA

This table shows the clinical and pathological characteristics of all 118 DAWTs

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.

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.

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.

BACKGROUND Pediatric high-grade gliomas (pHGGs) are an aggressive CNS tumor which are often characterized by mutations in H3F3A, the gene that encodes Histone H3.3 (H3.3). A substitution of the Glycine at position 34 of H3.3 with either Arginine or Valine (H3.3G34R/V), was recently described in a large cohort of pHGG samples and has been characterized as occurring in anywhere between 5-20% of pHGGs. Attempts to study the mechanisms of H3.3G34R have proven difficult due to the developmental nature of the disease and the requirement of co-occurring mutations for model development. METHODS We utilized the RCAS/tv-a system to develop a genetically engineered mouse model (GEMM) that incorporates PDGF-A activation, TP53 loss and the H3.3G34R mutation both in the context of ATRX loss and ATRX presence in nestin expressing progenitor cells. RESULTS Transcriptomic analysis revealed that ATRX loss in the context of H3.3G34R upregulates the Hoxa cluster genes Hoxa2, Hoxa3, Hoxa5, and Hoxa7 (p < 0.05, unpaired t-test). H&E staining and EMA immune-staining indicate that ATRX loss is correlated with a higher incidence of ependymal differentiation in H3.3G34R expressing samples (p < 0.05, unpaired t-test). GSEA and RT-qPCR analysis demonstrate that H3.3G34R expression in the context of ATRX loss promotes increased expression in genes associated with neuronal lineage (FWER pval < 1.0) and exhibits upregulation of the neurofilament polypeptides Nefl, Nefm (p < 0.05, unpaired t-test) as well as the neuronal differentiation marker Stmn2 (p < 0.001, unpaired t-test). CONCLUSION Our study proposes a model in which cooperation between ATRX loss and H3.3G34R expression mediate major transcriptomic and histopathological changes in H3.3G34R pHGGs. Broadly, our work highlights the importance to study mechanisms of co-occurring genetic events separately and in combination.

BACKGROUND: Pediatric high-grade gliomas (pHGGs) are an aggressive CNS tumor which are often characterized by mutations in H3F3A, the gene that encodes Histone H3.3 (H3.3). A substitution of the Glycine at position 34 of H3.3 with either Arginine or Valine (H3.3G34R/V), was recently described in a large cohort of pHGG samples and has been characterized as occurring in anywhere between 5-20% of pHGGs. Attempts to study the mechanisms of H3.3G34R have proven difficult due to the developmental nature of the disease and the requirement of co-occurring mutations for model development. METHODS: We utilized the RCAS system to develop a genetically engineered mouse model (GEMM) that incorporates PDGF-A activation, TP53 loss and the H3.3G34R mutation both in the context of ATRX loss and ATRX presence in nestin expressing progenitors. RESULTS: We show that in H3.3G34R expressing mice, ATRX loss significantly increased tumor latency from 90 days to 143 days (p < 0.01, Log rank test) and decreased tumor incidence from 81% to 57% (p < 0.01, Fisher’s exact test). By contrast, H3.3G34R did not significantly impact tumor latency in either our ATRX loss (163 days to 143 days, p = 0.178, Log-rank test) or our ATRX expressing (95 days to 90 days, p = 0.415, Log-rank test) models. Transcriptomic analysis revealed that ATRX loss in the context of H3.3G34R upregulates the PRC2 associated genes Hoxa2, Hoxa3, Hoxa5, and Hoxa7 (p < 0.05, unpaired t-test). GSEA analysis and RT-qPCR data suggest that ATRX loss works synergistically with H3.3G34R to promote NOTCH pathway activation through upregulation of the NOTCH ligand Dll3 (p < 0.01, unpaired t-test). CONCLUSIONS: Our study proposes a model in which ATRX loss is the major contributor to transcriptomic changes in the majority of H3.3G34R pHGGs. Broadly, our work highlights the importance of studying mechanisms of co-occurring genetic events separately and in combination.

Long non-coding RNAs (lncRNAs) 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 curate RNA sequencing data for 1,044 pediatric leukemia and solid tumors and integrate paired tumor whole genome sequencing and epigenetic data in relevant cell line models to explore lncRNA expression, regulation, and association with cancer. We report a total of 2,657 robustly expressed lncRNAs across six pediatric cancers, including 1,142 exhibiting histotype-specific expression. DNA copy number alterations contributed to lncRNA dysregulation at a proportion comparable to protein coding genes. Application of a multi-dimensional 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-specific and developmental lncRNAs. We integrated our analyses to prioritize lncRNAs for experimental validation and showed that silencing of TBX2-AS1, our top-prioritized neuroblastoma-specific lncRNA, resulted in significant growth inhibition of neuroblastoma cells, confirming our 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.

Background: Long non-coding RNAs (lncRNAs) have emerged as key components of transcriptional and post-transcriptional gene regulation. Dysregulation of lncRNA expression has been widely observed in cancer and several lncRNAs are known to influence tumor initiation and progression. Despite this, the lncRNA landscape and regulatory networks across pediatric cancers remain relatively uncharted. Methods: To characterize the lncRNA landscape of pediatric cancers, we first curated RNA sequencing data for 1,044 pediatric leukemia and solid tumors from the Therapeutically Applicable Research To Generate Effective Treatments (TARGET) project to identify known and novel expressed lncRNAs. This data set included: 280 acute myeloid leukemia (AML), 190 acute B-cell leukemia (B-ALL), 244 acute T-cell leukemia (T-ALL), 121 Wilm's tumor (WT), 48 rhabdoid tumors (RT), and 161 neuroblastoma (NBL). Histotype-specific expression was assessed using the tau score. Whole genome sequencing (WGS) from 826 matched normal-tumor pairs was integrated to identify somatic copy number alterations (SCNAs) disrupting lncRNA expression. To further implicate cancer-relevant drivers of lncRNA expression, we used a unique combination of epigenetic data in pediatric cell lines, including ChIP-sequencing for cancer-specific transcription factors and genome-scale chromatin capture data. A global analysis of lncRNA function was performed using the lncMod method, in which expression data is modelled to identify lncRNA modulators that perturb transcription factor regulation of target genes. Functional prioritization of lncRNAs was obtained through integration of analyses per cancer. Biochemical assays in human-derived cell line models were utilized to validate the function of the top prioritized lncRNA in NBL. Results: We report a total of 2,657 robustly expressed lncRNAs across six pediatric cancers, including 1,142 lncRNAs exhibiting histotype-specific expression. SCNAs contributed to lncRNA dysregulation at a proportion comparable to protein coding genes. There were 207 (28%) lncRNAs in regions with SCNA that had significant expression dysregulation. LncMod analysis across the cancers revealed context-specific transcriptional gene networks per dysregulated lncRNA and enrichment for proliferation, metabolic, and DNA damage hallmarks. We further identified 547 cancer-associated lncRNAs in NBL based on upstream regulation via oncogenic transcription factors. The top-prioritized lncRNA, TBX2-AS1, was predicted to impact proliferation in NBL. Silencing of TBX2-AS1 using siRNAs achieved >90% knockdown in NBL cells and resulted in 46.6% decreased cell growth (p = 8.1 x 10-4). Conclusion: This study defines the lncRNA landscape across six pediatric cancers and provides a detailed catalog of how lncRNAs impact regulatory gene networks. These data serve as a robust resource for future hypothesis-driven mechanistic studies. Citation Format: Apexa Modi, Gonzalo Lopez, Karina L. Conkrite, Tsz Ching Leung, Sathvik Ramanan, Daphne Cheung, Chun Su, Matthew E. Johnson, Elisabetta Manduchi, Samantha Gadd, Jinghui Zhang, Malcolm A. Smith, Jaime M. Guidry Auvil, Daniela S. Gerhard, Soheil Meshinchi, Elizabeth J. Perlman, Stephen P. Hunger, John M. Maris, Andrew D. Wells, Struan F. Grant, Sharon J. Diskin. Integrative genomics reveals lncRNAs associated with pediatric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3028.

We performed genome-wide sequencing and analyzed mRNA and miRNA expression, DNA copy number, and DNA methylation in 117 Wilms tumors, followed by targeted sequencing of 651 Wilms tumors. In addition to genes previously implicated in Wilms tumors (WT1, CTNNB1, AMER1, DROSHA, DGCR8, XPO5, DICER1, SIX1, SIX2, MLLT1, MYCN, and TP53), we identified mutations in genes not previously recognized as recurrently involved in Wilms tumors, the most frequent being BCOR, BCORL1, NONO, MAX, COL6A3, ASXL1, MAP3K4, and ARID1A. DNA copy number changes resulted in recurrent 1q gain, MYCN amplification, LIN28B gain, and MIRLET7A loss. Unexpected germline variants involved PALB2 and CHEK2. Integrated analyses support two major classes of genetic changes that preserve the progenitor state and/or interrupt normal development.

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.

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., ACVR1 and H3.1K27M mutations co-occur in diffuse intrinsic pontine glioma. Here, the authors generate a mouse model that recapitulates these genetic lesions and show, using genetic and pharmacological approaches, that the bone morphogenetic protein pathway may be a therapeutic target in these tumours.

BACKGROUND Diffuse Intrinsic Pontine Glioma (DIPG) remains an incurable pediatric brain cancer. The oncohistone H3K27M implicated in 80% of the cases, is also predicted to target Enhancer of Zeste Homolog 2 (Ezh2), the catalytic component of the Polycomb Repressor Complex 2 (PRC2). There are no reported mutations of Ezh2 and its function in DIPG is not fully determined. This work aims to address the role of Ezh2 in DIPG. METHODS Brainstem tumors were established by intracranial injections of Nestin;Tv-a; Ezh2Y641F/+ (NTv-a; Ezh2Y641F/+) neonatal pups using Replication Competent Avian Sarcoma leucosis virus long terminal repeat with splice acceptor (RCAS) viruses, expressing PDGF-B, p53 shRNA, and RCAS-CRE/Y. Immunohistochemical staining for Ki-67 and H3K27me3 were performed on the Discovery ULTRA (Ventana). RESULTS Ezh2 overexpression (Ezh2Y641F/+, RCAS CRE) conferred a survival advantage of approximately 10 days (n=20 mice/group, p

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.

The X-linked BCL-6 co-repressor (BCOR) gene encodes a key constituent of a variant polycomb repressive complex (PRC) that is mutated or translocated in human cancers. Here we report on the identification of somatic internal tandem duplications (ITDs) clustering in the C terminus of BCOR in 23 of 27 (85%) pediatric clear cell sarcomas of the kidney (CCSK) from two independent cohorts. We profile CCSK tumours using a combination of whole-exome, transcriptome and targeted sequencing. Identical ITD mutations are found in primary and relapsed tumour pairs but not in adjacent normal kidney or blood. Mutant BCOR transcripts and proteins are markedly upregulated in ITD-positive tumours. Transcriptome analysis of ITD-positive CCSKs reveals enrichment for PRC2-regulated genes and similarity to undifferentiated sarcomas harbouring BCOR–CCNB3 fusions. The discovery of recurrent BCOR ITDs defines a major oncogenic event in this childhood sarcoma with significant implications for diagnostic and therapeutic approaches to this tumour., The genetic basis of clear cell sarcomas of the kidney is not well understood. In this study, Roy et al. perform whole-exome and RNA sequencing of these tumours and identify recurrent internal tandem duplications in BCOR, a key constituent of a variant polycomb repressive complex.

Wilms tumors (WT) have provided broad insights into the interface between development and tumorigenesis. Further understanding is confounded by their genetic, histologic, and clinical heterogeneity, the basis of which remains largely unknown. We evaluated 224 WT for global gene expression patterns; WT1, CTNNB1, and WTX mutation; and 11p15 copy number and methylation patterns. Five subsets were identified showing distinct differences in their pathologic and clinical features: these findings were validated in 100 additional WT. The gene expression pattern of each subset was compared with published gene expression profiles during normal renal development. A novel subset of epithelial WT in infants lacked WT1, CTNNB1, and WTX mutations and nephrogenic rests and displayed a gene expression pattern of the postinduction nephron, and none recurred. Three subsets were characterized by a low expression of WT1 and intralobar nephrogenic rests. These differed in their frequency of WT1 and CTNNB1 mutations, in their age, in their relapse rate, and in their expression similarities with the intermediate mesoderm versus the metanephric mesenchyme. The largest subset was characterized by biallelic methylation of the imprint control region 1, a gene expression profile of the metanephric mesenchyme, and both interlunar and perilobar nephrogenic rests. These data provide a biologic explanation for the clinical and pathologic heterogeneity seen within WT and enable the future development of subset-specific therapeutic strategies. Further, these data support a revision of the current model of WT ontogeny, which allows for an interplay between the type of initiating event and the developmental stage in which it occurs.

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.

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. 114 top genes were differentially expressed in RT (p2 or

Wilms tumour is an embryonal tumour of childhood that closely resembles the developing kidney. Genomic changes responsible for the development of the majority of Wilms tumours remain largely unknown. Here we identify recurrent mutations within Wilms tumours that involve the highly conserved YEATS domain of MLLT1 (ENL), a gene known to be involved in transcriptional elongation during early development. The mutant MLLT1 protein shows altered binding to acetylated histone tails. Moreover, MLLT1-mutant tumours show an increase in MYC gene expression and HOX dysregulation. Patients with MLLT1-mutant tumours present at a younger age and have a high prevalence of precursor intralobar nephrogenic rests. These data support a model whereby activating MLLT1 mutations early in renal development result in the development of Wilms tumour., Wilms tumour is a rare renal neoplasm that primarily affects children but the genomic changes responsible for its development are currently largely unknown. In this study, the authors identify somatic mutations of the MLLT1 gene that are potentially involved in the aetiology of a subset of Wilms tumours.

SummaryWe report the most common single-nucleotide substitution/deletion mutations in favorable histology Wilms tumors (FHWTs) to occur within SIX1/2 (7% of 534 tumors) and microRNA processing genes (miRNAPGs) DGCR8 and DROSHA (15% of 534 tumors). Comprehensive analysis of 77 FHWTs indicates that tumors with SIX1/2 and/or miRNAPG mutations show a pre-induction metanephric mesenchyme gene expression pattern and are significantly associated with both perilobar nephrogenic rests and 11p15 imprinting aberrations. Significantly decreased expression of mature Let-7a and the miR-200 family (responsible for mesenchymal-to-epithelial transition) in miRNAPG mutant tumors is associated with an undifferentiated blastemal histology. The combination of SIX and miRNAPG mutations in the same tumor is associated with evidence of RAS activation and a higher rate of relapse and death.

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.

Studies reported here tested the hypothesis that acetaminophen stimulates proliferation of E2-responsive cells by inducing expression of E2-regulated genes. Ribonuclease protection assays compared the effects of acetaminophen and E2 on expression of selected genes (c-myc, c-fos, cyclin D1, bcl-2, bax, gadd45, mcl-1, p53, p21(CIP1/WAF1), and bcl-xL) in E2-responsive breast cancer (MCF-7) and endometrial adenocarcinoma (Ishikawa) cells as well as in E2-nonresponsive (MDA-MB-231) breast cancer cells. Acetaminophen and E2 increased c-myc RNA levels in MCF-7 cells, consistent with a mitogenic activity of these compounds in MCF-7 cells. However, the magnitude and time course of acetaminophen and E2 induction of c-myc differed. Neither acetaminophen nor E2 induced c-myc in MDA-MB-231 cells, whereas E2, but not acetaminophen, weakly induced c-myc expression in Ishikawa cells. Furthermore, in these 3 cell types, the expression patterns of the other genes differed dramatically in response to acetaminophen and to E2, indicating that acetaminophen does not activate ER as a transcription factor in the same manner as does E2. Additionally, gel shift assays demonstrated that in MCF-7 cells, acetaminophen increased NF-kappaB activity approximately 40% and did not alter AP-1 activity, whereas E2 increased AP-1 activity approximately 50% and did not increase NF-B activity. These studies indicate that acetaminophen effects on gene expression and cell proliferation depend more on cell type/context than on the presence of ER.

Wilms tumor (WT) is the most common kidney tumor of children. Over the last three decades, clinical trials employing multi-modality therapies have resulted in overall survival of greater than 90% for patients with low-risk disease. In spite of these advances, treatment of patients with advanced, anaplastic, and relapsed Wilms tumors remains challenging, with substantial rates of treatment failure and death. To improve risk stratification and identify novel therapeutic targets, we used high-accuracy mass spectrometry urine proteomics to identify urine tumors markers associated with relapsed WT and non-WT renal tumors. We measured urine proteomes at diagnosis of 54 patients with renal WT, clear cell sarcoma, rhabdoid tumor, and age-matched controls, leading to the quantitation of 6,519 urine proteins. In particular, we identified specific urine WT markers, including those that were enriched in patients with relapsed WT, such as mitochondrial regulators prohibitin and DAD1, β-catenin antagonist DACT2, and DNA repair factor SUN1. Using a specific enzyme-linked immunosorbent assay (ELISA) developed to measure urine prohibitin in an independent cohort of 139 WT and control samples, we found that urine prohibitin concentrations over 1000 ng/mL were significantly associated with the risk of disease relapse, with an odds ratio of relapse of 153 and receiver operating characteristic area under the curve of 0.77 (95% confidence interval of 0.64-0.99). Immunohistochemical tumor analysis revealed that prohibitin was highly expressed in primary Wilms tumor specimens. Importantly, using loss- and gain-of-function genetic experiments, we found that prohibitin was required for the growth and survival of Wilms tumor cells, and its overexpression conferred concomitant resistance to vincristine, doxorubicin and actinomycin D. Consistent with prohibitin’s functions in mitochondria, we are using BH3 profiling to elucidate specific intrinsic apoptotic dependencies in distinct subsets of refractory Wilms tumors, as a prelude to rational combination blockade of chemotherapy resistance, such as blockade of BCL2 dependence using venetoclax. In all, the use of urine prohibitin measurements may improve initial therapy stratification, and enable monitoring of response to therapy and early detection of relapse. In addition, therapeutic targeting of chemotherapy resistance induced by prohibitin overexpression may offer improved therapies for patients with relapsed or refractory Wilms tumors. Citation Format: Michael Vincent Ortiz, Melissa Burns, Amy Eisenberg, Saima Ahmed, Lyvia Gaewsky, Gary Bradwin, Paolo Cifani, Anton Henssen, Ian Macarthur, Michael LaQuaglia, Anthony Letai, Arlene Naranjo, Samantha Gadd, Yueh-Yun Chi, Jeffrey Dome, Elizabeth Perlman, Elizabeth Mullen, Hanno Steen, Alex Kentsis. Prohibitin is a prognostic marker of treatment failure and therapeutic target to block chemotherapy resistance in Wilms tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 708. doi:10.1158/1538-7445.AM2017-708

Purpose: To investigate the role and significance of TP53 mutation in diffusely anaplastic Wilms tumors (DAWTs). Experimental Design: All DAWTs registered on National Wilms Tumor Study-5 (n = 118) with available samples were analyzed for TP53 mutations and copy loss. Integrative genomic analysis was performed on 39 selected DAWTs. Results: Following analysis of a single random sample, 57 DAWTs (48%) demonstrated TP53 mutations, 13 (11%) copy loss without mutation, and 48 (41%) lacked both [defined as TP53-wild-type (wt)]. Patients with stage III/IV TP53-wt DAWTs (but not those with stage I/II disease) had significantly lower relapse and death rates than those with TP53 abnormalities. In-depth analysis of a subset of 39 DAWTs showed seven (18%) to be TP53-wt: These demonstrated gene expression evidence of an active p53 pathway. Retrospective pathology review of TP53-wt DAWT revealed no or very low volume of anaplasia in six of seven tumors. When samples from TP53-wt tumors known to contain anaplasia histologically were available, abnormal p53 protein accumulation was observed by immunohistochemistry. Conclusions: These data support the key role of TP53 loss in the development of anaplasia in WT, and support its significant clinical impact in patients with residual anaplastic tumor following surgery. These data also suggest that most DAWTs will show evidence of TP53 mutation when samples selected for the presence of anaplasia are analyzed. This suggests that modifications of the current criteria to also consider volume of anaplasia and documentation of TP53 aberrations may better reflect the risk of relapse and death and enable optimization of therapeutic stratification. Clin Cancer Res; 22(22); 5582–91. ©2016 AACR.

Introduction: The National Cancer Institute's Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative seeks to characterize the genomes of high-risk pediatric tumors to identify therapeutic targets. The High-Risk Renal Tumor TARGET initiative includes the analysis of pre-therapy favorable histology Wilms Tumors (FHWT) that relapsed and tumors with diffuse anaplasia (unfavorable histology; DAWT). These two tumor subsets have survival rates of approximately 50% and 60%, respectively. Experimental procedures: Genomic sequencing (whole genome [WGS] or exome [WXS]), global copy number analysis, and global gene expression analysis were performed on a discovery set of 117 (78 FHWT, 39 DAWT) pre-therapy high-risk WTs treated on National Wilms Tumor Study-5 (NWTS-5). To determine the frequency of recurrent variants, targeted sequencing (Illumina HiSeq2500) was performed on a validation set of pre-therapy tumor DNA from a case-cohort of all FHWT treated on NWTS-5 (531 FHWT) and all available 118 DAWT treated on NWTS-5 (these groups include tumors from the discovery set). Results: WGS and WXS revealed an average of 21.74 ± 22.6 high-quality variants per DAWT (range, 3-131) and 13.8 ± 10.9 per FHWT (range 2-58). Genes previously reported to be recurrently mutated in WT were mutated at the following frequencies in the validation set: WTX (6%), CTNNB1 (15%), WT1 (7.5%), DROSHA (11%), DGCR8 (4.5%), XPO5 (2%), SIX1/2 (7%), and MLLT1 (3%). In addition, mutations were identified in three genes that impact the NMYC pathway, which is known to be involved in renal development. These include MYCN P44L/H (4%), MAX R60Q (2%), and novel mutations in NONO (2%); these mutations were mutually exclusive. Novel mutations in BCOR, a transcriptional corepressor that regulates both gene expression during development and chromatin modification, were found in 3% of validation set tumors. Analysis of global gene expression revealed significant up-regulation of genes associated with kidney development, extracellular matrix organization, and epithelial tube development in BCOR-mutant tumors compared with precursor lesions (5 hyperplastic perilobar nephrogenic rests). TP53 mutations were identified in 48% of DAWTs and 1% of FHWTs. The above data do not include copy number changes, which were recurrently detected in WT1, WTX, NMYC, and TP53. Conclusions: Through the TARGET initiative, we have identified several novel, potential driver mutations that occur in WT and have not been reported in other pediatric tumors. The majority of these genes are known to function in processes critical to early development and/or specifically in renal development. Many of these mutations are accompanied by Wnt activating mutations or 11p15 biallelic expression. However, approximately 50% of WTs lack clear driver mutations. Future studies will need to focus on elucidating epigenetic alterations in these tumors as well as genetic changes outside of protein-coding regions. Citation Format: Samantha L. Gadd, Amy L. Walz, Ariadne HAG Ooms, Vicki Huff, Daniela S. Gerhard, Malcolm A. Smith, Jaime M. Guidry Auvil, Leandro Hermida, Tanja Davidsen, Patee Gesuwan, Daoud Meerzaman, Yussanne Ma, Marco A. Marra, Jeffrey S. Dome, Charles G. Mullighan, David A. Wheeler, Oliver A. Hampton, Julie M. Gastier-Foster, Nicole Ross, Elizabeth J. Perlman. The genetic landscape of Wilms tumor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-180.

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.

Suppression of human immunodeficiency virus (HIV) replication by CD8+ T-cells (CD8 suppression) contributes to survival in adults and children

Purpose: Recent studies suggest that children Experimental Design: To define biologically distinct subsets, global gene expression analysis was done on 39 VLRWT that passed all quality-control parameters and the clusters identified were validated in an independent set of 11 VLRWT. Validation of select differentially expressed genes was done with immunohistochemistry on a tissue microarray from 20 of 39 tumors. Loss of heterozygosity (LOH) for 11p15, 1p, and 16q was analyzed in 52 tumors using PCR. Results: Two distinctive clusters were identified. One cluster included 9 tumors with epithelial differentiated tubular histology, paucity of nephrogenic rests, lack of LOH for 1p, 16q, and 11p, absence of relapse, and a unique gene expression profile consistent with arrest following mesenchymal-to-epithelial transition. The second cluster included 13 tumors with mixed histology, intralobar nephrogenic rests, and decreased expression of WT1. Three of 6 relapses occurred in this cluster. Of 43 informative tumors, 11p LOH was present in 5 of 5 relapses and 11 of 38 nonrelapses. Conclusions: Two subsets comprising a total of 56 of VLRWT are identified that have pathogenetic and molecular differences and apparent differences in risk for relapse. If these predictors can be prospectively validated, this would enable the refinement of clinical stratification and less arbitrary definition of VLRWT. (Clin Cancer Res 2009;15(22):68009)

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

Prolactin (PRL) is a pleiotrophic hormone that contributes to the growth of normal and malignant breast tissues. PRL signals through its receptor (PRLr), a transmembrane receptor that belongs to the cytokine receptor family. The mechanism of how the PRL:PRLr interaction triggers activation of signaling networks remains enigmatic. This review examines the effect of ligand binding on PRLr and the processes that initiate receptor-associated signaling. Evidence for PRLr predimerization in the absence of ligand and the actions of the prolyl isomerase cyclophilin A in ligand-induced activation of PRLr-associated Jak2 kinase are discussed. These studies reveal that ligand-induced conformational change of the PRLr complex is necessary for its function and open avenues for therapies to inhibit PRLr action in breast cancer.

Introduction: The TARGET initiative seeks to identify therapeutic targets through genomic sequencing supported by copy number and gene expression analysis. The High Risk Wilms Tumor TARGET project, currently in progress, includes the analysis of favorable histology (FH) tumors that relapsed and tumors with anaplasia (unfavorable histology). We report the identification of a group of Wilms tumors (WT) identified by a unique gene expression pattern. Methods and Results: 102 high risk WT (43 anaplastic, 59 relapsed FH) were analyzed using Affymetrix U133+2 chips. Unsupervised hierarchical analysis consistently selected 9 tumors (5 anaplastic, 4 FH) that shared a striking gene expression pattern. These patients presented at 15-96 months (median 51); 7 had a triphasic histology and 2 were blastemal predominate; 4 died of their disease. When the top differentially expressed genes (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1439. doi:1538-7445.AM2012-1439

Background: Rhabdoid tumors (RT) are aggressive tumors of infancy for which no effective treatment is currently available. These tumors are characterized by genetic loss of the SMARCB1 (SNF5, INI-1) component of the SWI/SNF chromatin remodeling complex. We previously reported an overall pattern of repression of genes associated with bivalent histone modification in embryonic stem cells within RT. We hypothesize that loss of SMARCB1 in RT results in an overall failure to release the repressive (H3K27) histone methylation in genes responsible for early differentiation. To test this hypothesis, we performed chromatin immunoprecipitation (ChIP) using antibodies specific for permissive (H3K4) and H3K27 trimethylation, comparing an RT cell line (G-410) with a human embryonic kidney cell line (HEK293). In addition, we report the global miRNA analysis of RT using the Illumina 96-sample Universal Matrix Array. Results: Those genes previously shown by others to be direct targets of SMARCB1 (PTN, CDK2NA, ATP1B1, HESR1, and FZD7) and genes previously shown to be down-regulated in RT (CDH2, SPON1, LEF1, SOX11, CDKN1B, SERPINE2, SPOCK1, DOCK4) all showed a significantly higher ratio of H3K27 methylation to H3K4 methylation (1.5-fold to 130-fold) in G410 cells compared with HEK293. In contrast, genes shown to have increased expression in RT (TFRC and DDX21) showed increased H3K4 methylation. Global miRNA analysis demonstrated striking downregulation of 156/158 (p Summary: Gene and miRNA expression strongly correlate with histone methylation patterns in the RT cell line. This suggests that loss of SMARCB1 in RTs results in striking repression of gene and miRNA expression which may be due to aberrant repressive (H3K27) histone methylation. The use of histone methylation inhibitors may therefore merit investigation for this pediatric tumor for which there is no effective therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 68. doi:10.1158/1538-7445.AM2011-68

Background: Wilms tumors (WT) commonly arise in the setting of precursor lesions known as nephrogenic rests. DHPLN is characterized by a continuous rind-like involvement of all or part of the kidney by nephrogenic rest. Patients with DHPLN are often difficult to manage clinically. This study investigates DHPLN in order to better understand the pathogenesis and to determine potential therapeutic targets. Methods: Five DHPLN were compared with 5 WT that developed from perilobar nephrogenic rests and with 3 fetal kidney samples. The following platforms were used: Illumina Human 610-Quad Beadchip, Affymetrix U133 plus 2 arrays, Illumina Infinium Human Methylation 27 DNA Analysis BeadChip, Illumina 96-sample Universal Matrix Array for miRNA analysis. In addition, key signaling pathway component analysis was performed using Reverse Phase Protein Array (RPPA) analysis. Lastly, we compared the gene expression of the above samples with 242 WT samples previously reported. Results: The five DHPLN samples showed 1, 1, 2, 3, and 10 small regions of low-level gain or loss. In contrast, the WT samples all showed larger and more numerous areas of gain or loss. The normal copy number for loci on chromosome 11p was present in all DHPLNs, although 2/5 showed allelic imbalance of distal 11p, consistent with uniparental isodisomy, confirmed by methylation analysis of H19DMR and KvDMR1. The remaining 3 DHPLN showed loss of imprinting with 100% methylation of H19DMR. The gene expression of several key inhibitors of the Wnt pathway were upregulated in DHPLN (Axin-10 GSK-3, WISP3, and APC). TCF21 (POD1), a potential tumor suppressor gene, was down-regulated in DHPLN compared with fetal kidney. Lastly, PLAG1, a transcription factor that positively regulates IGF2, was highly elevated in fetal kidney, down-regulated in DHPLN, and upregulated in most WT. Several significant genes were shown to be both highly methylated and down-regulated, including TCF21, and the Wnt-related genes ITGA6, SFRP1, and PLAGL2. Significantly increased expression of miRNAs overall was identified within DHPLN compared with WT, including the Let-7 miRNA cluster. Upregulation of miR135 may be the mechanism for down-regulation of PLAG1 in DHPLN. Finally, RPPA revealed up-regulation of multiple members of the mTOR pathway in both DHPLN and WT compared with fetal kidney. Conclusions: This data suggests the following hypotheses: DHPLN may arise due to biallelic expression of IGF2. Down-regulation of TCF21 may contribute to the formation of DHPLN. Clonal increased expression of PLAG1 may contribute to the development of WT within DHPLN. Potential therapeutic targets for both DHPLN and Wilms tumors developing with DHPLN include mTOR inhibitors and related cyclin-D1 inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5353. doi:10.1158/1538-7445.AM2011-5353

// Saskia L. Gooskens 1,2 , Samantha Gadd 3 , Jaime M. Guidry Auvil 4 , Daniela S. Gerhard 4 , Javed Khan 5 , Rajesh Patidar 5 , Daoud Meerzaman 6 , Qing-Rong Chen 6 , Chih Hao Hsu 6 , Chunhua Yan 6 , Cu Nguyen 6 , Ying Hu 6 , Charles G. Mullighan 7 , Jing Ma 7 , Lawrence J. Jennings 3 , Ronald R. de Krijger 8,9 , Marry M. van den Heuvel-Eibrink 2 , Malcolm A. Smith 10 , Nicole Ross 11 , Julie M. Gastier-Foster 11 and Elizabeth J. Perlman 3 1 Department of Pediatric Hematology and Oncology, Erasmus MC - Sophia Children’s Hospital, Rotterdam, The Netherlands 2 Department of Pediatric Oncology, Princess Maxima Center for Pediatric Oncology, Utrecht, The Netherlands 3 Department of Pathology, Ann and Robert H. Lurie Children’s Hospital of Chicago, Northwestern University’s Feinberg School of Medicine and Robert H. Lurie Cancer Center, Chicago, IL, USA 4 Office of Cancer Genomics, National Cancer Institute, Bethesda, MD, USA 5 Genetics Branch, Oncogenomics section, National Cancer Institute, Bethesda, MD, USA 6 Computational Genomics Research Group, Center for Biomedical Informatics and Information Technology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA 7 Department of Pathology, St. Jude Children’s Research Hospital, Memphis, TN, USA 8 Department of Pathology, Josephine Nefkens Institute, Erasmus MC, Rotterdam, The Netherlands 9 Department of Pathology, Reinier de Graaf Hospital, Delft, The Netherlands 10 Cancer Therapy Evaluation Program, National Cancer Institute, Bethesda, MD, USA 11 Department of Pathology and Laboratory Medicine, Nationwide Children’s Hospital, Ohio State University College of Medicine, Columbus, OH, USA Correspondence to: Elizabeth J. Perlman, email: // Keywords : clear cell sarcoma of the kidney, whole genome sequencing, methylation, TCF21, TARID Received : April 10, 2015 Accepted : June 07, 2015 Published : June 28, 2015 Abstract Clear Cell Sarcoma of the Kidney (CCSK) is a rare childhood tumor whose molecular pathogenesis remains poorly understood. We analyzed a discovery set of 13 CCSKs for changes in chromosome copy number, mutations, rearrangements, global gene expression and global DNA methylation. No recurrent segmental chromosomal copy number changes or somatic variants (single nucleotide or small insertion/deletion) were identified. One tumor with t(10;17)(q22;p13) involving fusion of YHWAE with NUTM2B was identified. Integrated analysis of expression and methylation data identified promoter hypermethylation and low expression of the tumor suppressor gene TCF21 ( Pod-1/capsulin/epicardin) in all CCSKs except the case with t(10;17)(q22;p13). TARID , the long noncoding RNA responsible for demethylating TCF21 , was virtually undetectable in most CCSKs. TCF21 hypermethylation and decreased TARID expression were validated in an independent set of CCSK tumor samples. The presence of significant hypermethylation of TCF21, a transcription factor known to be active early in renal development, supports the hypothesis that hypermethylation of TCF21 and/or decreased TARID expression lies within the pathogenic pathway of most CCSKs. Future studies are needed to functionally verify a tumorigenic role of TCF21 down-regulation and to tie this to the unique gene expression pattern of CCSK.