Improving vitamin D testing and supplementation in children with newly diagnosed cancer: A quality improvement initiative at Rady Children's Hospital San Diego.

BACKGROUND: Vitamin D deficiency and insufficiency have been associated with poorer health outcomes. Children with cancer are at high risk for vitamin D deficiency and insufficiency. At our institution, we identified high variability in vitamin D testing and supplementation in this population. Of those tested, 65% were vitamin D deficient/insufficient. We conducted a quality improvement (QI) initiative with aim to improve vitamin D testing and supplementation among children aged 2-18 years with newly diagnosed cancer to ≥80% over 6 months. METHODS: An inter-professional team reviewed baseline data, then developed and implemented interventions using Plan-Do-Study-Act (PDSA) cycles. Barriers were identified using QI tools, including lack of automated triggers for testing and inconsistent supplementation criteria and follow-up testing post supplementation. Interventions included an institutional vitamin D guideline, clinical decision-making tree for vitamin D deficiency, insufficiency and sufficiency, electronic medical record triggers, and automated testing options. RESULTS: Baseline: N = 26 patients, four (15%) had baseline vitamin D testing; two (8%) received appropriate supplementation. Postintervention: N = 33 patients; 32 (97%) had baseline vitamin D testing; 33 (100%) received appropriate supplementation and completed follow-up testing timely (6-8 weeks post supplementation). Change was sustained over 24 months. CONCLUSIONS: We achieved and sustained our aim for vitamin D testing and supplementation in children with newly diagnosed cancer through inter-professional collaboration of hematology/oncology, endocrinology, hospital medicine, pharmacy, nursing, and information technology. Future PDSA cycles will address patient compliance with vitamin D supplementation and impact on patients' vitamin D levels.

Reciprocal H3.3 gene editing identifies K27M and G34R mechanisms in pediatric glioma including NOTCH signaling.

Histone H3.3 mutations are a hallmark of pediatric gliomas, but their core oncogenic mechanisms are not well-defined. To identify major effectors, we used CRISPR-Cas9 to introduce H3.3K27M and G34R mutations into previously H3.3-wildtype brain cells, while in parallel reverting the mutations in glioma cells back to wildtype. ChIP-seq analysis broadly linked K27M to altered H3K27me3 activity including within super-enhancers, which exhibited perturbed transcriptional function. This was largely independent of H3.3 DNA binding. The K27M and G34R mutations induced several of the same pathways suggesting key shared oncogenic mechanisms including activation of neurogenesis and NOTCH pathway genes. H3.3 mutant gliomas are also particularly sensitive to NOTCH pathway gene knockdown and drug inhibition, reducing their viability in culture. Reciprocal editing of cells generally produced reciprocal effects on tumorgenicity in xenograft assays. Overall, our findings define common and distinct K27M and G34R oncogenic mechanisms, including potentially targetable pathways.

Histone H3.3 regulates dynamic chromatin states during spermatogenesis.

The histone variant H3.3 is involved in diverse biological processes, including development, transcriptional memory and transcriptional reprogramming, as well as diseases, including most notably malignant brain tumors. Recently, we developed a knockout mouse model for the H3f3b gene, one of two genes encoding H3.3. Here, we show that targeted disruption of H3f3b results in a number of phenotypic abnormalities, including a reduction in H3.3 histone levels, leading to male infertility, as well as abnormal sperm and testes morphology. Additionally, null germ cell populations at specific stages in spermatogenesis, in particular spermatocytes and spermatogonia, exhibited increased rates of apoptosis. Disruption of H3f3b also altered histone post-translational modifications and gene expression in the testes, with the most prominent changes occurring at genes involved in spermatogenesis. Finally, H3f3b null testes also exhibited abnormal germ cell chromatin reorganization and reduced protamine incorporation. Taken together, our studies indicate a major role for H3.3 in spermatogenesis through regulation of chromatin dynamics.

Induced pluripotency and oncogenic transformation are related processes.

Induced pluripotent stem cells (iPSCs) have the potential for creating patient-specific regenerative medicine therapies, but the links between pluripotency and tumorigenicity raise important safety concerns. More specifically, the methods employed for the production of iPSCs and oncogenic foci (OF), a form of in vitro produced tumor cells, are surprisingly similar, raising potential concerns about iPSCs. To test the hypotheses that iPSCs and OF are related cell types and, more broadly, that the induction of pluripotency and tumorigenicity are related processes, we produced iPSCs and OF in parallel from common parental fibroblasts. When we compared the transcriptomes of these iPSCs and OF to their parental fibroblasts, similar transcriptional changes were observed in both iPSCs and OF. A significant number of genes repressed during the iPSC formation were also repressed in OF, including a large cohort of differentiation-associated genes. iPSCs and OF shared a limited number of genes that were upregulated relative to parental fibroblasts, but gene ontology analysis pointed toward monosaccharide metabolism as upregulated in both iPSCs and OF. iPSCs and OF were distinct in that only iPSCs activated a host of pluripotency-related genes, while OF activated cellular damage and specific metabolic pathways. We reprogrammed oncogenic foci (ROF) to produce iPSC-like cells, a process dependent on Nanog. However, the ROF had reduced differentiation potential compared to iPSC, suggesting that oncogenic transformation leads to cellular changes that impair complete reprogramming. Taken together, these findings support a model in which OF and iPSCs are related, yet distinct cell types, and in which induced pluripotency and induced tumorigenesis are similar processes.