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Objectives The goals of this study were to: 1) evaluate the cardiac magnetic resonance (CMR) characteristics of pediatric cardiac masses from a large international cohort; 2) test the accuracy of previously developed CMR-based diagnostic criteria; and 3) expand diagnostic criteria using new information. Background After diagnosis of a cardiac mass, clinicians must weigh the benefits and risks of ascertaining a tissue diagnosis. Limited data are available on the accuracy of previously developed noninvasive pediatric CMR-based diagnostic criteria. Methods CMR studies (children 0-18 years of age) with confirmatory histological and/or genetic diagnosis were analyzed by 2 reviewers, without knowledge of prior diagnosis. Diagnostic accuracy was graded as: 1) single correct diagnosis; 2) correct diagnosis among a differential; or 3) incorrect diagnosis. Results Of 213 cases, 174 (82%) had diagnoses that were represented in the previously published diagnostic criteria. In 70% of 174 cases, both reviewers achieved a single correct diagnosis (94% of fibromas, 71% of rhabdomyomas, and 50% of myxomas). When ≤2 differential diagnoses were included, both reviewers reached a correct diagnosis in 86% of cases. Of 29 malignant tumors, both reviewers indicated malignancy as a single diagnosis in 52% of cases. Including ≤2 differential diagnoses, both reviewers indicated malignancy in 83% of cases. Of 6 CMR sequences examined, acquisition of first-pass perfusion and late gadolinium enhancement were independently associated with a higher likelihood of a single correct diagnosis. Conclusions CMR of cardiac masses in children leads to an accurate diagnosis in most cases. A comprehensive imaging protocol is associated with higher diagnostic accuracy.

Supplementary Data from EP300 Selectively Controls the Enhancer Landscape of MYCN-Amplified Neuroblastoma

Gene expression is regulated by promoters and enhancers marked by histone H3 lysine 27 acetylation (H3K27ac), which is established by the paralogous histone acetyltransferases (HAT) EP300 and CBP. These enzymes display overlapping regulatory roles in untransformed cells, but less characterized roles in cancer cells. We demonstrate that the majority of high-risk pediatric neuroblastoma (NB) depends on EP300, whereas CBP has a limited role. EP300 controls enhancer acetylation by interacting with TFAP2β, a transcription factor member of the lineage-defining transcriptional core regulatory circuitry (CRC) in NB. To disrupt EP300, we developed a proteolysis-targeting chimera (PROTAC) compound termed “JQAD1” that selectively targets EP300 for degradation. JQAD1 treatment causes loss of H3K27ac at CRC enhancers and rapid NB apoptosis, with limited toxicity to untransformed cells where CBP may compensate. Furthermore, JQAD1 activity is critically determined by cereblon (CRBN) expression across NB cells.Significance:EP300, but not CBP, controls oncogenic CRC-driven transcription in high-risk NB by binding TFAP2β. We developed JQAD1, a CRBN-dependent PROTAC degrader with preferential activity against EP300 and demonstrated its activity in NB. JQAD1 has limited toxicity to untransformed cells and is effective in vivo in a CRBN-dependent manner.This article is highlighted in the In This Issue feature, p. 587

PDF file 17K, Supplementary Figure 1: Staining of additional Ewing sarcoma cell lines. All Ewing sarcoma cell lines tested stain equivalently positive for DN16. In all panels, unstained and isotype matched IgG are controls. Supplementary Figure 2: MRI detection of experimental metastases. Experimental liver metastases were induced by IV injection of TC32 cells. Imaging with a 7T MRI demonstrates 1-2 mm metastatic lesions in the liver (white arrows)

Purpose: Ewing sarcoma is a tumor of the bone and soft tissue characterized by diffuse cell membrane expression of CD99 (MIC2). Single-site, surgically resectable disease is associated with an excellent 5-year event-free survival; conversely, patients with distant metastases have a poor prognosis. Noninvasive imaging is the standard approach to identifying sites of metastatic disease. We sought to develop a CD99-targeted imaging agent for staging Ewing sarcoma and other CD99-expressing tumors.Experimental Design: We identified a CD99 antibody with highly specific binding in vitro and labeled this antibody with 64Cu. Mice with either subcutaneous Ewing sarcoma xenograft tumors or micrometastases were imaged with the 64Cu-labeled anti-CD99 antibody and these results were compared with conventional MRI and 2[18F]fluoro-2-deoxy-d-glucose–positron emission tomography (FDG–PET) imaging.Results:64Cu-labeled anti-CD99 antibody demonstrated high avidity for the CD99-positive subcutaneous tumors, with a high tumor-to-background ratio, greater than that demonstrated with FDG–PET. Micrometastases, measuring 1 to 2 mm on MRI, were not detected with FDG–PET but were readily visualized with the 64Cu-labeled anti-CD99 antibody. Probe biodistribution studies demonstrated high specificity of the probe for CD99-positive tumors.Conclusions:64Cu-labeled anti-CD99 antibody can detect subcutaneous Ewing sarcoma tumors and metastatic sites with high sensitivity, outperforming FDG–PET in preclinical studies. This targeted radiotracer may have important implications for the diagnosis, surveillance, and treatment of Ewing sarcoma. Similarly, it may impact the management of other CD99 positive tumors. Clin Cancer Res; 20(3); 678–87. ©2013 AACR.

PDF file 373K, Supplementary Figure 1: Staining of additional Ewing sarcoma cell lines. Supplementary Figure 2: MRI detection of experimental metastases

Supplementary Figures 1-2 from Differentiation of NUT Midline Carcinoma by Epigenomic Reprogramming

Fewer than 15% of gastrointestinal stromal tumors (GIST) in pediatric patients harbor KIT or platelet-derived growth factor receptor α (PDGFRA) mutations in contrast to a mutation rate of 80% in adult GISTs. However, some therapeutic inhibitors of KIT have efficacy in pediatric GIST, suggesting that KIT may, nevertheless, play an important role in oncogenesis. In adult GIST, characteristic cytogenetic changes occur during progression to malignancy. A better understanding of mechanisms of genetic progression and KIT and PDGFRA transforming roles in pediatric GIST might facilitate treatment advances. KIT and PDGFRA mutation analysis was done in 27 pediatric GISTs. The activation status of KIT, PDGFRA, and downstream signaling intermediates was defined, and chromosomal aberrations were determined by single nucleotide polymorphism assays. Mutations in KIT or PDGFRA were identified in 11% of pediatric GISTs. KIT and the signaling intermediates AKT and mitogen-activated protein kinase were activated in pediatric GISTs. In particular, most pediatric KIT–wild-type GISTs displayed levels of KIT activation similar to levels in adult KIT-mutant GISTs. Pediatric KIT–wild-type GISTs lacked the typical cytogenetic deletions seen in adult KIT-mutant GISTs. Notably, most pediatric KIT–wild-type GISTs progress to malignancy without acquiring large-scale chromosomal aberrations, which is a phenomenon not reported previously in malignant solid tumors. KIT activation levels in pediatric KIT–wild-type GISTs are comparable with those in KIT-mutant GISTs. Therapies that inhibit KIT activation, or crucial KIT signaling intermediates, should be explored in pediatric KIT–wild-type GIST. [Cancer Res 2007;67(19):9084–8]

NUT midline carcinoma (NMC) is a lethal pediatric tumor defined by the presence of BRD-NUT fusion proteins that arrest differentiation. Here we explore the mechanisms underlying the ability of BRD4-NUT to prevent squamous differentiation. In both gain-of and loss-of-expression assays, we find that expression of BRD4-NUT is associated with globally decreased histone acetylation and transcriptional repression. Bulk chromatin acetylation can be restored by treatment of NMC cells with histone deacetylase inhibitors (HDACi), engaging a program of squamous differentiation and arrested growth in vitro that closely mimics the effects of siRNA-mediated attenuation of BRD4-NUT expression. The potential therapeutic utility of HDACi differentiation therapy was established in three different NMC xenograft models, where it produced significant growth inhibition and a survival benefit. Based on these results and translational studies performed with patient-derived primary tumor cells, a child with NMC was treated with the FDA-approved HDAC inhibitor, vorinostat. An objective response was obtained after five weeks of therapy, as determined by positron emission tomography. These findings provide preclinical support for trials of HDACi in patients with NMC. Cancer Res; 71(7); 2686–96. ©2011 AACR.

Supplementary Table 1 from Pediatric KIT–Wild-Type and Platelet-Derived Growth Factor Receptor α–Wild-Type Gastrointestinal Stromal Tumors Share KIT Activation but not Mechanisms of Genetic Progression with Adult Gastrointestinal Stromal Tumors