Samantha Gadd

Recurrent DGCR8, DROSHA, and SIX Homeodomain Mutations in Favorable Histology Wilms Tumors

We 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.

New mechanisms for PRLr action in breast cancer

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.

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.

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)

A Children's Oncology Group and TARGET initiative exploring the genetic landscape of Wilms tumor

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.

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

MLLT1 YEATS domain mutations in clinically distinctive Favourable Histology Wilms tumours.

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.

Significance of TP53 mutation in Wilms tumors with diffuse anaplasia : A report from the Children's Oncology Group

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.

A Parallel Study of mRNA and microRNA Profiling of Peripheral Blood in Young Adult Women

Background: Aging is a complex process that involves the interplay of genetic, epigenetic, and environmental factors. Identifying aging-related biomarkers holds great potential for improving our understanding of complex physiological changes, thereby providing a means to investigate the mechanism by which aging influences various diseases. Method and Results: We performed a parallel study of microRNA and gene expression profiling of peripheral blood in a group of healthy young adult women, among which 13 were aged 22–25 and 9 were aged 36–39 years old. We identified a significantly distinct pattern of microRNA, but not gene expression profiling, between these two young adult women groups. We also performed correlation analysis of expression levels between all pairs of age-associated microRNAs and genes and identified a weak global correlation between these two types of expression levels. A significant involvement of estrogen regulation was observed by pathway analysis of the most differentially expressed microRNAs that included miR-155, -18a, -142, -340, -363, -195, and -24. Conclusion: Our results suggest that the change in global microRNA expression in the peripheral blood is associated with normal aging in young adult women. This change may precede global gene expression changes. Future studies are needed to investigate the regulatory mechanism of the estrogen-related microRNAs and associated diseases.