Shot of a lifetime: How pharmacists stay ahead of the season.

OBJECTIVES: The profession of pharmacy has long advocated for the advancement of practice through increased clinical responsibility. Provision of immunization related services has been one service pharmacists have been able to provide to add to their existing responsibilities. A universal influenza vaccination has been under investigation and is nearing success. While other clinical services should be considered, now more than ever, development of the universal vaccine should provide a pause for the profession and consideration of not only the impact on student learning opportunities but also pharmacy revenue. SUMMARY: The development of the universal influenza vaccination poses a potential challenge to existing service-related revenue models for community pharmacies. There are many other opportunities pharmacists can capitalize on including, but not limited to, travel and other vaccinations, point-of-care testing, and transitions-of-care. In addition, through initiatives such as "Flip the Pharmacy" and Community Pharmacy Enhanced Service Network, pharmacists are in a great position to be innovative with clinical services while continuing to provide learners with training opportunities. CONCLUSION: Many opportunities exist for pharmacists to expand services that lean into their clinical training and add other vaccination opportunities. These opportunities can augment revenue streams and still provide learners with training.

ONCOGENE PANEL SEQUENCING ANALYSIS IDENTIFIES CANDIDATE ACTIONABLE GENES IN ADVANCED WELL-DIFFERENTIATED GASTROENTEROPANCREATIC NEUROENDOCRINE TUMORS.

Objective: To report the rate of candidate actionable somatic mutations in patients with locally advanced and metastatic gastro-enteropancreatic (GEP) neuroendocrine tumors (NET) and of other genetic alterations that may be associated with tumorigenesis. Methods: A phase II mutation targeted therapy trial was conducted in patients with advanced well-differentiated G1/G2 GEP-NET. Mutations found in the mTOR pathway-associated genes led to treatment with the mTOR inhibitor everolimus, and were defined as actionable. Tumor deoxyribonucleic acid (DNA) from GEP-NET were sequenced and compared with germline DNA, using the OncoVAR-NET assay, designed for hybrid capture sequencing of 500 tumor suppressor genes and oncogenes. Somatic variants were called and copy-number (CN) variant analysis was performed. Results: Thirty patients (14 small-intestine, 8 pancreatic, 3 unknown primary NET, and 5 of other primary sites) harbored 37 lesions (4 patients had DNA of multiple lesions sequenced). Only 2 patients with sporadic NET (n = 26) had an actionable mutation leading to treatment with everolimus. Driver somatic mutations were detected in 18 of 30 patients (21/37 lesions sequenced). In the remaining samples without a driver mutation, CN alterations were found in 11/16 tumors (10/12 patients), including CN loss of chromosome (Chr) 18 (P<.05), CN gain of Chr 5, and loss of Chr 13. CN losses in Chr 18 were more common in patients without driver mutations detected. Pronounced genetic heterogeneity was detected in patients with multiple lesions sequenced. Conclusion: Genome-wide DNA sequencing may identify candidate actionable genes and lead to the identification of novel target genes for advanced well-differentiated GEP-NET. Abbreviations: Chr = chromosome; CN = copy number; DNA = deoxyribonucleic acid; FDA = Food and Drug Administration; GEP = gastro-enteropancreatic; MEN-1 = multiple endocrine neoplasia syndrome type 1; mTOR = mammalian target of rapamycin; NET = neuroendocrine tumor; PFS = progression-free survival; PNET = pancreatic neuroendocrine tumors; SINET = small-intestine neuroendocrine tumor.

Detailed longitudinal sampling of glioma stem cells in situ reveals Chr7 gain and Chr10 loss as repeated events in primary tumor formation and recurrence.

Intratumoral heterogeneity at the genetic, epigenetic, transcriptomic, and morphologic levels is a commonly observed phenomenon in many aggressive cancer types. Clonal evolution during tumor formation and in response to therapeutic intervention can be predicted utilizing reverse engineering approaches on detailed genomic snapshots of heterogeneous patient tumor samples. In this study, we developed an extensive dataset for a GBM case via the generation of polyclonal and monoclonal glioma stem cell lines from initial diagnosis, and from multiple sections of distant tumor locations of the deceased patient's brain following tumor recurrence. Our analyses revealed the tissue-wide expansion of a new clone in the recurrent tumor and chromosome 7 gain and chromosome 10 loss as repeated genomic events in primary and recurrent disease. Moreover, chromosome 7 gain and chromosome 10 loss produced similar alterations in mRNA expression profiles in primary and recurrent tumors despite possessing other highly heterogeneous and divergent genomic alterations between the tumors. We identified ETV1 and CDK6 as putative candidate genes, and NFKB (complex), IL1B, IL6, Akt and VEGF as potential signaling regulators, as potentially central downstream effectors of chr7 gain and chr10 loss. Finally, the differences caused by the transcriptomic shift following gain of chromosome 7 and loss of chromosome 10 were consistent with those generally seen in GBM samples compared to normal brain in large-scale patient-tumor data sets.

Epigenetic-mediated dysfunction of the bone morphogenetic protein pathway inhibits differentiation of glioblastoma-initiating cells.

Despite similarities between tumor-initiating cells with stem-like properties (TICs) and normal neural stem cells, we hypothesized that there may be differences in their differentiation potentials. We now demonstrate that both bone morphogenetic protein (BMP)-mediated and ciliary neurotrophic factor (CNTF)-mediated Jak/STAT-dependent astroglial differentiation is impaired due to EZH2-dependent epigenetic silencing of BMP receptor 1B (BMPR1B) in a subset of glioblastoma TICs. Forced expression of BMPR1B either by transgene expression or demethylation of the promoter restores their differentiation capabilities and induces loss of their tumorigenicity. We propose that deregulation of the BMP developmental pathway in a subset of glioblastoma TICs contributes to their tumorigenicity both by desensitizing TICs to normal differentiation cues and by converting otherwise cytostatic signals to proproliferative signals.

Neuronal and glioma-derived stem cell factor induces angiogenesis within the brain.

Stem cell factor (SCF) is overexpressed by neurons following brain injury as well as by glioma cells; however, its role in gliomagenesis remains unclear. Here, we demonstrate that SCF directly activates brain microvascular endothelial cells (ECs) in vitro and induces a potent angiogenic response in vivo. Primary human gliomas express SCF in a grade-dependent manner and induce normal neurons to express SCF in brain regions infiltrated by glioma cells, areas that colocalize with prominent angiogenesis. Downregulation of SCF inhibits tumor-mediated angiogenesis and glioma growth in vivo, whereas overexpression of SCF is associated with shorter survival in patients with malignant gliomas. Thus, the SCF/c-Kit pathway plays an important role in tumor- and normal host cell-induced angiogenesis within the brain.

Predicting in vitro drug sensitivity using Random Forests.

MOTIVATION: Panels of cell lines such as the NCI-60 have long been used to test drug candidates for their ability to inhibit proliferation. Predictive models of in vitro drug sensitivity have previously been constructed using gene expression signatures generated from gene expression microarrays. These statistical models allow the prediction of drug response for cell lines not in the original NCI-60. We improve on existing techniques by developing a novel multistep algorithm that builds regression models of drug response using Random Forest, an ensemble approach based on classification and regression trees (CART). RESULTS: This method proved successful in predicting drug response for both a panel of 19 Breast Cancer and 7 Glioma cell lines, outperformed other methods based on differential gene expression, and has general utility for any application that seeks to relate gene expression data to a continuous output variable. IMPLEMENTATION: Software was written in the R language and will be available together with associated gene expression and drug response data as the package ivDrug at http://r-forge.r-project.org.

Publication Rates of Pediatric-Focused Resident Research Projects Presented at The Pediatric Pharmacy Association Bruce Parks Memorial Residency Showcase.

OBJECTIVES: The primary objective was to identify the number of residency projects presented at the Pediatric Pharmacy Association (PPA) Bruce Parks Memorial Residency Showcase that were subsequently published. Secondary objectives included a comparison of subsequent publications after residency completion between those who did and did not publish their residency project and an analysis of factors associated with subsequent publications. METHODS: This was a descriptive study including all pediatric-focused resident projects presented at the PPA Bruce Parks Memorial Residency Showcase from 2006 to 2015. Literature searches for all the pediatric-focused residency projects and any subsequent publications were performed. Data collection included residency type (i.e., postgraduate year 1 [PGY1], postgraduate year 2 [PGY2]), project category, and initial position after residency. A zero-inflated Poisson regression was used to analyze subsequent publication status while controlling for other factors. Statistical analyses were performed using SAS/STAT, with a priori p value < 0.05. RESULTS: There were 434 projects presented by 401 residents. Seventy-four (17.1%) were published, with the majority being PGY2s (74.3%). Subsequent publications were identified for 162 residents (40.4%), with a higher percentage in those who published their pediatric-focused residency project versus those who did not, 59.5% versus 32.8%, p < 0.001. Factors associated with subsequent publications were those who published their residency project, initial position in academia, and PGY2s. CONCLUSIONS: Of the residency projects presented at the showcase <20% were subsequently published. Those who published their residency research project were more likely to have subsequent publications. Future efforts should be taken to ensure that residents have the tools/confidence to independently publish their research/scholarship.

A phase I trial of enzastaurin in patients with recurrent gliomas.

PURPOSE: Enzastaurin is a selective inhibitor of protein kinase C beta. Prior phase I studies did not show increased drug exposures with escalating once daily administration. Limits from gastrointestinal absorption may be overcome by twice daily dosing, potentially improving antitumor effects. EXPERIMENTAL DESIGN: We conducted a phase I dose escalation study in 26 patients with recurrent malignant glioma, stratified by use of enzyme-inducing antiepileptic drugs, to investigate whether divided twice daily dosing results in higher exposures compared with once daily dosing. Phosphorylated glycogen synthase 3 beta was analyzed as a potential biomarker of enzastaurin activity. RESULTS: Enzastaurin was poorly tolerated at all dose levels evaluated (500, 800, and 1,000 mg total daily), with thrombocytopenia and prolonged QTc as dose-limiting toxicities. The average drug concentration of enzastaurin under steady-state conditions was doubled by twice daily dosing compared with daily dosing [1.990; 90% confidence interval (CI), 1.450-2.730]. Additionally, geometric mean ratios doubled with 800 versus 500 mg dosing for both daily (2.687; 90% CI, 1.232-5.860) and twice daily regimens (1.852; 90% CI, 0.799-4.292). Two patients achieved long-term benefit (over 150 weeks progression free). CONCLUSIONS: Higher and more frequent dosing of enzastaurin resulted in improved drug exposure but with unacceptable toxicity at the doses tested. Phosphorylated glycogen synthase 3 beta may be a useful biomarker of the biological activity of enzastaurin. Enzastaurin has activity in a subset of malignant glioma patients and warrants continued study in combination with other agents using a maximal once daily dose of 500 mg.

Gene pathways and subnetworks distinguish between major glioma subtypes and elucidate potential underlying biology.

Molecular diagnostic tools are increasingly being used in an attempt to classify primary human brain tumors more accurately. While methods that are based on the analysis of individual gene expression prove to be useful for diagnostic purposes, they are devoid of biological significance since tumorgenesis is a concerted deregulation of multiple pathways rather than single genes. In a proof of concept, we utilize two large clinical data sets and show that the elucidation of enriched pathways and small differentially expressed sub-networks of protein interactions allow a reliable classification of glioblastomas and oligodendrogliomas. Applying a feature selection method, we observe that an optimized subset of pathways and subnetworks significantly improves the prediction accuracy. By determining the enrichment of altered genes in pathways and subnetworks we show that optimized subsets of genes rarely seem to be a target of genomic alteration. Our results suggest that groups of genes play a decisive role for the phenotype of the underlying tumor samples that can be utilized to reliably distinguish tumor types. In the absence of enrichment of genes that are genomically altered we assume that genetic changes largely exert an indirect rather than direct regulatory influence on a number of tumor-defining regulatory networks.

Author Correction: Recurrent PTPRT/JAK2 mutations in lung adenocarcinoma among African Americans.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genomic changes and gene expression profiles reveal that established glioma cell lines are poorly representative of primary human gliomas.

Genetic aberrations, such as gene amplification, deletions, and loss of heterozygosity, are hallmarks of cancer and are thought to be major contributors to the neoplastic process. Established cancer cell lines have been the primary in vitro and in vivo models for cancer for more than 2 decades; however, few such cell lines have been extensively characterized at the genomic level. Here, we present a high-resolution genome-wide chromosomal alteration and gene expression analyses of five of the most commonly used glioma cell lines and compare the findings with those observed in 83 primary human gliomas. Although genomic alterations known to occur in primary tumors were identified in the cell lines, we also observed several novel recurrent aberrations in the glioma cell lines that are not frequently represented in primary tumors. Additionally, a global gene expression cluster distinct from primary tumors was identified in the glioma cell lines. Our results indicate that established cell lines are generally a poor representation of primary tumor biology, presenting a host of genomic and gene expression changes not observed in primary tissues, although some discrete features of glioma biology were conserved in the established cell lines. Refined maps of genetic alterations and transcriptional divergence from the original tumor type, such as the one presented here, may help serve as a guideline for a more biologically rational and clinically relevant selection of the most appropriate glioma model for a given experiment.

Recurrent PTPRT/JAK2 mutations in lung adenocarcinoma among African Americans.

Reducing or eliminating persistent disparities in lung cancer incidence and survival has been challenging because our current understanding of lung cancer biology is derived primarily from populations of European descent. Here we show results from a targeted sequencing panel using NCI-MD Case Control Study patient samples and reveal a significantly higher prevalence of PTPRT and JAK2 mutations in lung adenocarcinomas among African Americans compared with European Americans. This increase in mutation frequency was validated with independent WES data from the NCI-MD Case Control Study and TCGA. We find that patients carrying these mutations have a concomitant increase in IL-6/STAT3 signaling and miR-21 expression. Together, these findings suggest the identification of these potentially actionable mutations could have clinical significance for targeted therapy and the enrollment of minority populations in clinical trials.

A Core Regulatory Circuit in Glioblastoma Stem Cells Links MAPK Activation to a Transcriptional Program of Neural Stem Cell Identity.

Glioblastoma, the most common primary malignant brain tumor, harbors a small population of tumor initiating cells (glioblastoma stem cells) that have many properties similar to neural stem cells. To investigate common regulatory networks in both neural and glioblastoma stem cells, we subjected both cell types to in-vitro differentiation conditions and measured global gene-expression changes using gene expression microarrays. Analysis of enriched transcription factor DNA-binding sites in the promoters of differentially expressed genes was used to reconstruct regulatory networks involved in differentiation. Computational predictions, which were biochemically validated, show an extensive overlap of regulatory circuitry between cell types including a network centered on the transcription factor KLF4. We further demonstrate that EGR1, a transcription factor previously shown to be downstream of the MAPK pathway, regulates KLF4 expression and that KLF4 in turn transcriptionally activates NOTCH as well as SOX2. These results demonstrate how known genomic alterations in glioma that induce constitutive activation of MAPK are transcriptionally linked to master regulators essential for neural stem cell identify.

Common Molecular Subtypes Among Asian Hepatocellular Carcinoma and Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) and hepatocellular carcinoma (HCC) are clinically disparate primary liver cancers with etiological and biological heterogeneity. We identified common molecular subtypes linked to similar prognosis among 199 Thai ICC and HCC patients through systems integration of genomics, transcriptomics, and metabolomics. While ICC and HCC share recurrently mutated genes, including TP53, ARID1A, and ARID2, mitotic checkpoint anomalies distinguish the C1 subtype with key drivers PLK1 and ECT2, whereas the C2 subtype is linked to obesity, T cell infiltration, and bile acid metabolism. These molecular subtypes are found in 582 Asian, but less so in 265 Caucasian patients. Thus, Asian ICC and HCC, while clinically treated as separate entities, share common molecular subtypes with similar actionable drivers to improve precision therapy.

Micro-environment causes reversible changes in DNA methylation and mRNA expression profiles in patient-derived glioma stem cells.

In vitro and in vivo models are widely used in cancer research. Characterizing the similarities and differences between a patient's tumor and corresponding in vitro and in vivo models is important for understanding the potential clinical relevance of experimental data generated with these models. Towards this aim, we analyzed the genomic aberrations, DNA methylation and transcriptome profiles of five parental tumors and their matched in vitro isolated glioma stem cell (GSC) lines and xenografts generated from these same GSCs using high-resolution platforms. We observed that the methylation and transcriptome profiles of in vitro GSCs were significantly different from their corresponding xenografts, which were actually more similar to their original parental tumors. This points to the potentially critical role of the brain microenvironment in influencing methylation and transcriptional patterns of GSCs. Consistent with this possibility, ex vivo cultured GSCs isolated from xenografts showed a tendency to return to their initial in vitro states even after a short time in culture, supporting a rapid dynamic adaptation to the in vitro microenvironment. These results show that methylation and transcriptome profiles are highly dependent on the microenvironment and growth in orthotopic sites partially reverse the changes caused by in vitro culturing.

Identification of molecular pathways facilitating glioma cell invasion in situ.

Gliomas are mostly incurable secondary to their diffuse infiltrative nature. Thus, specific therapeutic targeting of invasive glioma cells is an attractive concept. As cells exit the tumor mass and infiltrate brain parenchyma, they closely interact with a changing micro-environmental landscape that sustains tumor cell invasion. In this study, we used a unique microarray profiling approach on a human glioma stem cell (GSC) xenograft model to explore gene expression changes in situ in Invading Glioma Cells (IGCs) compared to tumor core, as well as changes in host cells residing within the infiltrated microenvironment relative to the unaffected cortex. IGCs were found to have reduced expression of genes within the extracellular matrix compartment, and genes involved in cell adhesion, cell polarity and epithelial to mesenchymal transition (EMT) processes. The infiltrated microenvironment showed activation of wound repair and tissue remodeling networks. We confirmed by protein analysis the downregulation of EMT and polarity related genes such as CD44 and PARD3 in IGCs, and EFNB3, a tissue-remodeling agent enriched at the infiltrated microenvironment. OLIG2, a proliferation regulator and glioma progenitor cell marker upregulated in IGCs was found to function in enhancing migration and stemness of GSCs. Overall, our results unveiled a more comprehensive picture of the complex and dynamic cell autonomous and tumor-host interactive pathways of glioma invasion than has been previously demonstrated. This suggests targeting of multiple pathways at the junction of invading tumor and microenvironment as a viable option for glioma therapy.

Recurrent epimutation of SDHC in gastrointestinal stromal tumors.

Succinate dehydrogenase (SDH) is a conserved effector of cellular metabolism and energy production, and loss of SDH function is a driver mechanism in several cancers. SDH-deficient gastrointestinal stromal tumors (dSDH GISTs) collectively manifest similar phenotypes, including hypermethylated epigenomic signatures, tendency to occur in pediatric patients, and lack of KIT/PDGFRA mutations. dSDH GISTs often harbor deleterious mutations in SDH subunit genes (SDHA, SDHB, SDHC, and SDHD, termed SDHx), but some are SDHx wild type (WT). To further elucidate mechanisms of SDH deactivation in SDHx-WT GIST, we performed targeted exome sequencing on 59 dSDH GISTs to identify 43 SDHx-mutant and 16 SDHx-WT cases. Genome-wide DNA methylation and expression profiling exposed SDHC promoter-specific CpG island hypermethylation and gene silencing in SDHx-WT dSDH GISTs [15 of 16 cases (94%)]. Six of 15 SDHC-epimutant GISTs occurred in the setting of the multitumor syndrome Carney triad. We observed neither SDHB promoter hypermethylation nor large deletions on chromosome 1q in any SDHx-WT cases. Deep genome sequencing of a 130-kbp (kilo-base pair) window around SDHC revealed no recognizable sequence anomalies in SDHC-epimutant tumors. More than 2000 benign and tumor reference tissues, including stem cells and malignancies with a hypermethylator epigenotype, exhibit solely a non-epimutant SDHC promoter. Mosaic constitutional SDHC promoter hypermethylation in blood and saliva from patients with SDHC-epimutant GIST implicates a postzygotic mechanism in the establishment and maintenance of SDHC epimutation. The discovery of SDHC epimutation provides a unifying explanation for the pathogenesis of dSDH GIST, whereby loss of SDH function stems from either SDHx mutation or SDHC epimutation.

G-cimp status prediction of glioblastoma samples using mRNA expression data.

Glioblastoma Multiforme (GBM) is a tumor with high mortality and no known cure. The dramatic molecular and clinical heterogeneity seen in this tumor has led to attempts to define genetically similar subgroups of GBM with the hope of developing tumor specific therapies targeted to the unique biology within each of these subgroups. Recently, a subset of relatively favorable prognosis GBMs has been identified. These glioma CpG island methylator phenotype, or G-CIMP tumors, have distinct genomic copy number aberrations, DNA methylation patterns, and (mRNA) expression profiles compared to other GBMs. While the standard method for identifying G-CIMP tumors is based on genome-wide DNA methylation data, such data is often not available compared to the more widely available gene expression data. In this study, we have developed and evaluated a method to predict the G-CIMP status of GBM samples based solely on gene expression data.

Histone demethylase Jumonji D3 (JMJD3) as a tumor suppressor by regulating p53 protein nuclear stabilization.

Histone methylation regulates normal stem cell fate decisions through a coordinated interplay between histone methyltransferases and demethylases at lineage specific genes. Malignant transformation is associated with aberrant accumulation of repressive histone modifications, such as polycomb mediated histone 3 lysine 27 (H3K27me3) resulting in a histone methylation mediated block to differentiation. The relevance, however, of histone demethylases in cancer remains less clear. We report that JMJD3, a H3K27me3 demethylase, is induced during differentiation of glioblastoma stem cells (GSCs), where it promotes a differentiation-like phenotype via chromatin dependent (INK4A/ARF locus activation) and chromatin independent (nuclear p53 protein stabilization) mechanisms. Our findings indicate that deregulation of JMJD3 may contribute to gliomagenesis via inhibition of the p53 pathway resulting in a block to terminal differentiation.