Molecular classification and outcome of children with rare CNS embryonal tumors: results from St. Jude Children's Research Hospital including the multi-center SJYC07 and SJMB03 clinical trials.

Methylation profiling has radically transformed our understanding of tumors previously called central nervous system primitive neuro-ectodermal tumors (CNS-PNET). While this marks a momentous step toward defining key differences, reclassification has thrown treatment into disarray. To shed light on response to therapy and guide clinical decision-making, we report outcomes and molecular features of children with CNS-PNETs from two multi-center risk-adapted studies (SJMB03 for patients ≥ 3 years; SJYC07 for patients < 3 years) complemented by a non-protocol institutional cohort. Seventy patients who had a histological diagnosis of CNS-PNET or CNS embryonal tumor from one of the new categories that has supplanted CNS-PNET were included. This cohort was molecularly characterized by DNA methylation profiling (n = 70), whole-exome sequencing (n = 53), RNA sequencing (n = 20), and germline sequencing (n = 28). Clinical characteristics were detailed, and treatment was divided into craniospinal irradiation (CSI)-containing (SJMB03 and SJMB03-like) and CSI-sparing therapy (SJYC07 and SJYC07-like). When the cohort was analyzed in its entirety, no differences were observed in the 5-year survival rates even when CSI-containing therapy was compared to CSI-sparing therapy. However, when analyzed by DNA methylation molecular grouping, significant survival differences were observed, and treatment particulars provided suggestions of therapeutic response. Patients with CNS neuroblastoma with FOXR2 activation (CNS-NB-FOXR2) had a 5-year event-free survival (EFS)/overall survival (OS) of 66.7% ± 19.2%/83.3% ± 15.2%, and CIC rearranged sarcoma (CNS-SARC-CIC) had a 5-year EFS/OS both of 57.1% ± 18.7% with most receiving regimens that contained radiation (focal or CSI) and multidrug chemotherapy. Patients with high-grade neuroepithelial tumor with BCOR alteration (HGNET-BCOR) had abysmal responses to upfront chemotherapy-only regimens (5-year EFS = 0%), but survival extended with salvage radiation after progression [5-year OS = 53.6% ± 20.1%]. Patients with embryonal tumor with multilayered rosettes (ETMR) or high-grade glioma/glioblastoma multiforme (HGG/GBM) did not respond favorably to any modality (5-year EFS/OS = 10.7 ± 5.8%/17.9 ± 7.2%, and 10% ± 9.0%/10% ± 9.0%, respectively). As an accompaniment, we have assembled this data onto an interactive website to allow users to probe and query the cases. By reporting on a carefully matched clinical and molecular cohort, we provide the needed insight for future clinical management.

A phase 1 trial of everolimus and bevacizumab in children with recurrent solid tumors.

BACKGROUND: The prognosis for children with recurrent solid tumors generally is poor. Targeting mammalian target of rapamycin (mTOR) and vascular endothelial growth factor A with everolimus and bevacizumab, respectively, synergistically improves progression-free survival and is well tolerated in adults with solid tumors. METHODS: In the current phase 1 study, a total of 15 children with recurrent or refractory solid tumors were treated with bevacizumab and everolimus to establish the maximum tolerated dose, toxicity, and preliminary antitumor response (ClinicalTrials.gov identifier NCT00756340). The authors also evaluated everolimus-mediated inhibition of the mTOR pathway in the peripheral blood mononuclear cells of treated patients. RESULTS: Tumors predominantly were soft tissue and/or bone sarcomas (8 cases) and brain tumors (5 cases). The first 2 patients enrolled at dose level 1 (10 mg/kg of bevacizumab and 4 mg/m2 of everolimus) experienced dose-limiting toxicities (DLTs). The next 5 patients were enrolled at dose level 0 (8 mg/kg of bevacizumab and 4 mg/m2 of everolimus), and DLTs occurred in 2 patients. The authors then modified the protocol to permit expansion of dose 0, and 8 additional patients were added, with no DLTs reported. Of all the patients, stable disease occurred in 4 patients (30.8%; median, 2 courses), and progressive disease occurred in 9 patients (69.2%). Overall survival was 0.59 years (95% CI, 0.24-1.05 years). The mTOR biomarker phospho-4EBP1 Thr/37/46 significantly decreased from baseline to day 27 in peripheral blood mononuclear cells (P = .045). Phospho-AKT levels also decreased from those at baseline. CONCLUSIONS: The maximum tolerated dose of cotreatment with bevacizumab and everolimus was 8 mg/kg of bevacizumab and 4 mg/m2 of everolimus in a 4-week cycle for children with recurrent solid tumors.

Molecular heterogeneity and CXorf67 alterations in posterior fossa group A (PFA) ependymomas.

Of nine ependymoma molecular groups detected by DNA methylation profiling, the posterior fossa type A (PFA) is most prevalent. We used DNA methylation profiling to look for further molecular heterogeneity among 675 PFA ependymomas. Two major subgroups, PFA-1 and PFA-2, and nine minor subtypes were discovered. Transcriptome profiling suggested a distinct histogenesis for PFA-1 and PFA-2, but their clinical parameters were similar. In contrast, PFA subtypes differed with respect to age at diagnosis, gender ratio, outcome, and frequencies of genetic alterations. One subtype, PFA-1c, was enriched for 1q gain and had a relatively poor outcome, while patients with PFA-2c ependymomas showed an overall survival at 5 years of > 90%. Unlike other ependymomas, PFA-2c tumors express high levels of OTX2, a potential biomarker for this ependymoma subtype with a good prognosis. We also discovered recurrent mutations among PFA ependymomas. H3 K27M mutations were present in 4.2%, occurring only in PFA-1 tumors, and missense mutations in an uncharacterized gene, CXorf67, were found in 9.4% of PFA ependymomas, but not in other groups. We detected high levels of wildtype or mutant CXorf67 expression in all PFA subtypes except PFA-1f, which is enriched for H3 K27M mutations. PFA ependymomas are characterized by lack of H3 K27 trimethylation (H3 K27-me3), and we tested the hypothesis that CXorf67 binds to PRC2 and can modulate levels of H3 K27-me3. Immunoprecipitation/mass spectrometry detected EZH2, SUZ12, and EED, core components of the PRC2 complex, bound to CXorf67 in the Daoy cell line, which shows high levels of CXorf67 and no expression of H3 K27-me3. Enforced reduction of CXorf67 in Daoy cells restored H3 K27-me3 levels, while enforced expression of CXorf67 in HEK293T and neural stem cells reduced H3 K27-me3 levels. Our data suggest that heterogeneity among PFA ependymomas could have clinicopathologic utility and that CXorf67 may have a functional role in these tumors.

Phase 1 trial, pharmacokinetics, and pharmacodynamics of dasatinib combined with crizotinib in children with recurrent or progressive high-grade and diffuse intrinsic pontine glioma.

BACKGROUND: Progressive/recurrent high-grade and diffuse intrinsic pontine gliomas (DIPGs) are fatal. Treatments targeting molecular pathways critical for these cancers are needed. METHODS: We conducted a phase 1 study (rolling-six design) to establish the safety and maximum tolerated dose (MTD) of dasatinib, an oral platelet-derived growth factor receptor A (PDGFRA) inhibitor, and crizotinib, an oral c-Met inhibitor, in such patients. Pharmacokinetics of both agents were performed. Biomarkers of cellular pathway activation in peripheral-blood mononuclear cells (PBMC) were evaluated before and after administration of dasatinib. PDGFRA and MET amplification, and PDGFRA mutations were studied in tumor samples. RESULTS: Twenty-five patients were enrolled in this study (median age: 11.9 years). Eleven patients had DIPG. Glioblastoma accounted for 40% of cases. Dasatinib at 50 mg/m2 and crizotinib at 130 mg/m2 or 100 mg/m2 were poorly tolerated when administered twice daily. Drug administration was then switched to once daily. Dasatinib administered at 50 mg/m2 and crizotinib at 215 mg/m2 once daily was the MTD. Dose-limiting toxicities consisted of diarrhea, fatigue, proteinuria, hyponatremia, rash, and grade 4 neutropenia. Only two patients received therapy for at least 6 months. No objective radiologic responses were observed. Pharmacokinetics of dasatinib and crizotinib were comparable to previous studies. A statistically significant decrease in the ratio of p-AKT/total AKT in PBMC occurred after dasatinib administration. PDGFRA and MET amplification were found in four and two cases, respectively. Only one of 10 tumors harbored a PDGFRA mutation. CONCLUSIONS: This drug combination was poorly tolerated and its activity was minimal. We do not recommend further testing of this combination in children.

Zebrafish Cacna1fa is required for cone photoreceptor function and synaptic ribbon formation.

Mutations in the human CACNA1F gene cause incomplete congenital stationary night blindness type 2 (CSNB2), a non-progressive, clinically heterogeneous retinal disorder. However, the molecular mechanisms underlying CSNB2 have not been fully explored. Here, we describe the positional cloning of a blind zebrafish mutant, wait until dark (wud), which encodes a zebrafish homolog of human CACNA1F. We identified two zebrafish cacna1f paralogs and showed that the cacna1fa transcript (the gene mutated in wud) is expressed exclusively in the photoreceptor layer. We demonstrated that Cacna1fa localizes at the photoreceptor synapse and is absent from wud mutants. Electroretinograms revealed abnormal cone photoreceptor responses from wud mutants, indicating a defect in synaptic transmission. Although there are no obvious morphological differences, we found that wud mutants lacked synaptic ribbons and that wud is essential for the development of synaptic ribbons. We found that Ribeye, the most prominent synaptic ribbon protein, was less abundant and mislocalized in adult wud mutants. In addition to cloning wud, we identified synaptojanin 1 (synj1) as the defective gene in slacker (slak), a blind mutant with floating synaptic ribbons. We determined that Cacna1fa was expressed in slak photoreceptors and that Synj1 was initially expressed wud photoreceptors, but was absent by 5 days postfertilization. Collectively, our data demonstrate that Cacna1fa is essential for cone photoreceptor function and synaptic ribbon formation and reveal a previously unknown yet critical role of L-type voltage-dependent calcium channels in the expression and/or distribution of synaptic ribbon proteins, providing a new model to study the clinical variability in human CSNB2 patients.

Oncogenic KRAS promotes malignant brain tumors in zebrafish.

BACKGROUND: Zebrafish have been used as a vertebrate model to study human cancers such as melanoma, rhabdomyosarcoma, liver cancer, and leukemia as well as for high-throughput screening of small molecules of therapeutic value. However, they are just emerging as a model for human brain tumors, which are among the most devastating and difficult to treat. In this study, we evaluated zebrafish as a brain tumor model by overexpressing a human version of oncogenic KRAS (KRAS(G12V)). METHODS: Using zebrafish cytokeratin 5 (krt5) and glial fibrillary acidic protein (gfap) gene promoters, we activated Ras signaling in the zebrafish central nervous system (CNS) through transient and stable transgenic overexpression. Immunohistochemical analyses were performed to identify activated pathways in the resulting brain tumors. The effects of the MEK inhibitor U0126 on oncogenic KRAS were evaluated. RESULTS: We demonstrated that transient transgenic expression of KRAS(G12V) in putative neural stem and/or progenitor cells induced brain tumorigenesis. When expressed under the control of the krt5 gene promoter, KRAS(G12V) induced brain tumors in ventricular zones (VZ) at low frequency. The majority of other tumors were composed mostly of spindle and epithelioid cells, reminiscent of malignant peripheral nerve sheath tumors (MPNSTs). In contrast, when expressed under the control of the gfap gene promoter, KRAS(G12V) induced brain tumors in both VZs and brain parenchyma at higher frequency. Immunohistochemical analyses indicated prominent activation of the canonical RAS-RAF-ERK pathway, variable activation of the mTOR pathway, but no activation of the PI3K-AKT pathway. In a krt5-derived stable and inducible transgenic line, expression of oncogenic KRAS resulted in skin hyperplasia, and the MEK inhibitor U0126 effectively suppressed this pro-proliferative effects. In a gfap-derived stable and inducible line, expression of oncogenic KRAS led to significantly increased mitotic index in the spinal cord. CONCLUSIONS: Our studies demonstrate that zebrafish could be explored to study cellular origins and molecular mechanisms of brain tumorigenesis and could also be used as a platform for studying human oncogene function and for discovering oncogenic RAS inhibitors.

High-grade glioma in infants and young children is histologically, molecularly, and clinically diverse: Results from the SJYC07 trial and institutional experience.

BACKGROUND: High-grade gliomas (HGG) in young children pose a challenge due to favorable but unpredictable outcomes. While retrospective studies broadened our understanding of tumor biology, prospective data is lacking. METHODS: A cohort of children with histologically diagnosed HGG from the SJYC07 trial was augmented with nonprotocol patients with HGG treated at St. Jude Children's Research Hospital from November 2007 to December 2020. DNA methylome profiling and whole genome, whole exome, and RNA sequencing were performed. These data were integrated with histopathology to yield an integrated diagnosis. Clinical characteristics and preoperative imaging were analyzed. RESULTS: Fifty-six children (0.0-4.4 years) were identified. Integrated analysis split the cohort into four categories: infant-type hemispheric glioma (IHG), HGG, low-grade glioma (LGG), and other-central nervous system (CNS) tumors. IHG was the most prevalent (n = 22), occurred in the youngest patients (median age = 0.4 years), and commonly harbored receptor tyrosine kinase gene fusions (7 ALK, 2 ROS1, 3 NTRK1/2/3, 4 MET). The 5-year event-free (EFS) and overall survival (OS) for IHG was 53.13% (95%CI: 35.52-79.47) and 90.91% (95%CI: 79.66-100.00) vs. 0.0% and 16.67% (95%CI: 2.78-99.74%) for HGG (p = 0.0043, p = 0.00013). EFS and OS were not different between IHG and LGG (p = 0.95, p = 0.43). Imaging review showed IHGs are associated with circumscribed margins (p = 0.0047), hemispheric location (p = 0.0010), and intratumoral hemorrhage (p = 0.0149). CONCLUSIONS: HGG in young children is heterogeneous and best defined by integrating histopathological and molecular features. Patients with IHG have relatively good outcomes, yet they endure significant deficits, making them good candidates for therapy de-escalation and trials of molecular targeted therapy.

Differential expression patterns and developmental roles of duplicated scinderin-like genes in zebrafish.

Scinderin, the closest homologue of the actin-severing protein, gelsolin, has two similar paralogs (Scinla and Scinlb) in zebrafish. Scinla is abundant in the adult cornea; Scinlb comprises considerably less corneal protein. Here, we show that scinla is expressed in the nose, lens, brain, cornea and annular ligament of the iridocorneal angle; by contrast, scinlb is expressed in the hatching gland, floor plate, notochord, otic vesicle, brain, pharynx, cartilage, swim bladder and cornea. Activity of scinla and scinlb promoter fragments driving the EGFP reporter gene in transgenic zebrafish resembled scinla or scinlb expression. Previously, we showed that reduction of scinla by injection of antisense morpholino oligonucleotides ventralized embryos; here, specific reduction of scinlb expression led to subtle brain abnormalities associated with increased cell death, decreased shhb expression in the floor plate, and slightly reduced eye distance. Thus, scinla and scinlb have different expression patterns and developmental roles during zebrafish development.

Duplicated gelsolin family genes in zebrafish: a novel scinderin-like gene (scinla) encodes the major corneal crystallin.

We have previously identified a gelsolin-like protein (C/L-gelsolin) as a corneal crystallin in zebrafish. Here we show by phylogenetic analysis that there are at least six genes encoding gelsolin-like proteins based on their gelsolin domains in zebrafish: gsna and gsnb group with the vertebrate gelsolin gene, scina and scinb group with the scinderin (adseverin) gene, and scinla (C/L-gelsolin) and scinlb are novel scinderin-like genes. RT-PCR showed that scinla, scinlb, and gsnb are preferentially expressed in the adult cornea whereas gsna is expressed to a similar extent in cornea, lens, brain, and heart; scina and scinb expression were detectable only in whole zebrafish and not in these adult tissues. Quantitative RT-PCR and 2-dimensional polyacrylamide gel electrophoresis followed by MALDI/TOF mass spectroscopy confirmed high expression of beta-actin and scinla, moderate expression of scinlb, and very low expression of gsna and gsnb in the cornea. Finally, transgenic zebrafish carrying a green fluorescent protein reporter transgene driven by a 4 kb scinla promoter fragment showed expression in the cornea, snout, dorsal fin, and tail fin of 3-day-old zebrafish larvae. Our data suggest that scinla and scinlb are diverged paralogs of the vertebrate scinderin gene and show that scinla encodes the zebrafish corneal crystallin previously called C/L-gelsolin.

MicroRNA-mediated cancer metastasis regulation via heterotypic signals in the microenvironment.

MicroRNAs (miRNAs) are thought to regulate tumor progression and metastasis via direct interaction with target genes within cells. Emerging evidence has demonstrated the secretion of miRNAs into environment via cancer cell exosomes, called "exosomal shuttle small RNA". Microenvironmental miRNAs are important mediators of cell-to-cell communication, and they play important roles in regulating cancer metastasis. RNA analysis indicates enrichment of the miRNA population in cell-culturing medium. miRNA-conditioned medium is able to mediate the function of miRNAs in regulating cancer cell migration and invasion. Here we combine our recent work with literature discussing multiple mechanisms through which exosomal miRNAs regulate cancer cell migration, invasion and metastasis. We summarize a heterotypic signaling pathway by which miRNA regulates the cellular secretion and tumor microenvironment in control of breast cancer cell migration and invasion. In conclusion, exosomal miRNAs are able to regulate cancer metastasis via heterotypic signals in the microenvironment.