Chiang Ching Huang

Clear Cell Sarcoma of the Kidney: Up-regulation of Neural Markers with Activation of the Sonic Hedgehog and Akt Pathways

Purpose and Experimental Design: Clear cell sarcoma of the kidney (CCSK), the second most common renal tumor in children, poses significant diagnostic challenges. No positive diagnostic markers are available, and the pathogenesis of CCSK remains an enigma. To address these challenges, the gene expression patterns of 14 CCSKs were compared with 15 Wilms tumors and 3 fetal kidney samples using oligonucleotide arrays. Results: Using unsupervised methods, the gene expression profile of CCSK was distinctive: differentially expressed genes could largely be grouped into four categories: (a) a wide variety of neural markers, (b) members of the Sonic hedgehog pathway, (c) members of the phosphoinositide-3-kinase/Akt cell proliferation pathway, and (d) known therapeutic targets. Corresponding changes in critical proteins using Western blot and/or immunohistochemistry confirmed the up-regulation of these pathways and proteins. In particular, CD117 and epidermal growth factor receptor are up-regulated at the protein level in many CCSKs, providing potential therapeutic targets. One of the neural markers, nerve growth factor receptor, represents a promising diagnostic tool for CCSK. Conclusions: This study suggests that CCSKs arise within a renal mesenchymal cell that shows a wide variety of neural markers. As such, it seems to be susceptible to genetic changes also seen in a variety of other neuroectodermal and neuronal tumors, including activation of Sonic hedgehog and phosphoinositide-3-kinase/Akt pathways. Involvement of these pathways in CCSKs implicates their widening role in tumorigenesis.

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.

Pilot study of etanercept in patients with refractory juvenile dermatomyositis

To evaluate the efficacy of etanercept in patients with juvenile dermatomyositis (DM) refractory to standard treatment.

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.

Classification of malignant pediatric renal tumors by gene expression

The most common malignant renal tumors of childhood are Wilms tumor (WT), clear cell sarcoma of the kidney (CCSK), cellular mesoblastic nephroma (CMN), and rhabdoid tumor of the kidney (RTK). Because these tumors present significant diagnostic difficulties, the goal was to define diagnostically useful signatures based on gene expression.

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

Purpose: Recent studies suggest that children <24 months with stage I favorable histology Wilms tumors <550 g [very low risk Wilms tumors (VLRWT)] have an excellent prognosis when treated with nephrectomy only, without adjuvant chemotherapy. The identification of risk categories within VLRWT may enable refinement of their definition and optimization of their therapy. 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)

Upregulation of mir-221 and mir-222 in atypical teratoid/rhabdoid tumors: potential therapeutic targets

PurposeThe aim of this study is to search for new therapeutic targets for atypical teratoid–rhabdoid tumors (ATRT).MethodsTo achieve this, we compared the expression of 365 microRNAs among ATRT, medulloblastomas, and normal brain.ResultsMiR-221 and miR-222 were within the top differentially expressed microRNAs. The deregulated expression of miR221/222 was demonstrated to inhibit the expression of the tumor suppressor and inhibitor of cell cycle p27Kip1. Here, we demonstrated the negative regulation of p27Kip1 by miR-221/222 in ATRT using microarray, real-time reverse transcriptase polymerase chain reaction, and immunohistochemistry.ConclusionAs anti-miR therapy was recently proposed as an alternative treatment for cancer, these findings suggest that anti-miR-221/222 therapy might have therapeutic potential in ATRT.

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. Copyright

MicroRNA expression profiling for molecular classification of pediatric brain tumors.

To the Editor: Our group performed a pilot study with 18 primary fresh-frozen pediatric brain tumors and we would like to share our experience of an approach similar to Birks et al. [1] using a different platform. Our study included six medulloblastomas (MB), four atypical teratoid/rhabdoid tumors (AT/RT), four juvenile pilocytic astrocytomas (JPA), and four high-grade gliomas/glioblastomas (HGG/GBM). RNA was extracted using Trizol Reagent (Invitrogen, Carlsbad, CA). Patients underwent surgical resection at the Children’s Memorial Hospital (Chicago, IL). MicroRNA expression profiles were determined by the Taq Man Real-Time PCR (TLDAs) v1.o (Applied Biosystems, Carlsbad, CA) that quantifies 365 mature microRNAs as described previously [2]. The Ct values of microRNA probes were normalized using Real-Time StatMiner softwareversion 3.0 (Integromics, Philadelphia, PA). Unsupervised hierarchical clustering of the microRNA data was performed using R packages (www.bioconductor.org) (Fig. 1A). We performed microarray gene expression profiling for the same samples using Illumina HT-12 BeadChip whole-genome expression arrays (Illumina, Inc., San Diego, CA). Gene expression data were normalized using the quantile normalization procedure from the bioconductor package (www.bioconductor.org). Over 48,000 transcripts were used for

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, χ2 test), and 38 such genes (P < 0.0001, χ2 test) in a second adult study. In addition, two exosome components and some RNAs 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. J. Med. Virol. 83:24–32, 2011.