Faculty Bibliography

PURPOSE/OBJECTIVE:Radiomics is the extraction of multidimensional imaging-features which when correlated with genomics is termed radiogenomics. However, radiogenomic biological validation is not sufficiently described in the literature. We seek to establish causality between differential gene expression status and MRI-extracted radiomic-features in glioblastoma. METHODS:Radiogenomic predictions and validation were done using the Cancer Genome Atlas and Repository of Molecular Brain Neoplasia Data glioblastoma patients (N=93) and orthotopic xenografts (OX)(N=40). Tumor phenotypes were segmented, and radiomic-features extracted using the developed radiome-sequencing pipeline. Patients and animals were dichotomized based on Periostin (POSTN) expression levels. RNA and protein levels confirmed RNAi-mediated POSTN knockdown in OX. Total RNA of tumor cells isolated from mouse brains (knockdown and control) was used for microarray-based expression profiling. Radiomic-features were utilized to predict POSTN expression status in patient, mouse, and inter-species. RESULTS:Our robust pipeline consists of segmentation, radiomic-feature extraction, feature normalization/selection, and predictive-modeling. The combination of skull stripping, brain-tissue focused normalization and patient-specific normalization are unique to this study, providing comparable cross-platform, cross-institution radiomic-features. POSTN expression status was not associated with qualitative or volumetric MRI parameters. Radiomic-features significantly predicted POSTN expression status in patients (AUC 76.56%, sensitivity/specificity: 73.91/78.26%) and OX (AUC 92.26%, sensitivity/specificity: 92.86%/91.67%). Furthermore, radiomic-features in OX were significantly associated with patients with similar POSTN expression levels (AUC 93.36%, sensitivity/specificity: 82.61%/95.74%; p-value 02.021E-15). CONCLUSION/CONCLUSIONS:We determined causality between radiomic texture features and POSTN expression levels in a pre-clinical model with clinical validation. Our biologically validated radiomic pipeline also showed the potential application for human-mouse matched co-clinical trials.

Purpose Both temozolomide (TMZ) and poly (ADP-ribose) polymerase (PARP) inhibitors are active in small-cell lung cancer (SCLC). This phase II, randomized, double-blind study evaluated whether addition of the PARP inhibitor veliparib to TMZ improves 4-month progression-free survival (PFS). Patients and Methods A total of 104 patients with recurrent SCLC were randomly assigned 1:1 to oral veliparib or placebo 40 mg twice daily, days 1 to 7, and oral TMZ 150 to 200 mg/m²/day, days 1 to 5, of a 28-day cycle until disease progression, unacceptable toxicity, or withdrawal of consent. Response was determined by imaging at weeks 4 and 8, and every 8 weeks thereafter. Improvement in PFS at 4 months was the primary end point. Secondary objectives included overall response rate (ORR), overall survival (OS), and safety and tolerability of veliparib with TMZ. Exploratory objectives included PARP-1 and SLFN11 immunohistochemical expression, MGMT promoter methylation, and circulating tumor cell quantification. Results No significant difference in 4-month PFS was noted between TMZ/veliparib (36%) and TMZ/placebo (27%; P = .19); median OS was also not improved significantly with TMZ/veliparib (8.2 months; 95% CI, 6.4 to 12.2 months; v 7.0 months; 95% CI, 5.3 to 9.5 months; P = .50). However, ORR was significantly higher in patients receiving TMZ/veliparib compared with TMZ/placebo (39% v 14%; P = .016). Grade 3/4 thrombocytopenia and neutropenia more commonly occurred with TMZ/veliparib: 50% versus 9% and 31% versus 7%, respectively. Significantly prolonged PFS (5.7 v 3.6 months; P = .009) and OS (12.2 v 7.5 months; P = .014) were observed in patients with SLFN11-positive tumors treated with TMZ/veliparib. Conclusion Four-month PFS and median OS did not differ between the two arms, whereas a significant improvement in ORR was observed with TMZ/veliparib. SLFN11 expression was associated with improved PFS and OS in patients receiving TMZ/veliparib, suggesting a promising biomarker of PARP-inhibitor sensitivity in SCLC.

OBJECTIVE Dexamethasone, a known regulator of mesenchymal programming in glioblastoma (GBM), is routinely used to manage edema in GBM patients. Dexamethasone also activates the expression of genes, such as CEBPB, in GBM stem cells (GSCs). However, the drug's impact on invasion, proliferation, and angiogenesis in GBM remains unclear. To determine whether dexamethasone induces invasion, proliferation, and angiogenesis in GBM, the authors investigated the drug's impact in vitro, in vivo, and in clinical information derived from The Cancer Genome Atlas (TCGA) cohort. METHODS Expression profiles of patients from the TCGA cohort with mesenchymal GBM (n = 155) were compared with patients with proneural GBM by comparative marker selection. To obtain robust data, GSCs with IDH1 wild-type (GSC3) and with IDH1 mutant (GSC6) status were exposed to dexamethasone in vitro and in vivo and analyzed for invasion (Boyden chamber, human-specific nucleolin), proliferation (Ki-67), and angiogenesis (CD31). Ex vivo tumor cells from dexamethasone-treated and control mice were isolated by fluorescence activated cell sorting and profiled using Affymetrix chips for mRNA (HTA 2.0) and microRNAs (miRNA 4.0). A pathway analysis was performed to identify a dexamethasone-regulated gene signature, and its relationship with overall survival (OS) was assessed using Kaplan-Meier analysis in the entire GBM TCGA cohort (n = 520). RESULTS The mesenchymal subgroup, when compared with the proneural subgroup, had significant upregulation of a dexamethasone-regulated gene network, as well as canonical pathways of proliferation, invasion, and angiogenesis. Dexamethasone-treated GSC3 demonstrated a significant increase in invasion, both in vitro and in vivo, whereas GSC6 demonstrated a modest increase. Furthermore, dexamethasone treatment of both GSC3 and GSC6 lines resulted in significantly elevated cell proliferation and angiogenesis in vivo. Patients with mesenchymal GBM had significant upregulation of dexamethasone-regulated pathways when compared with patients with proneural GBM. A prognostic (p = 0.0007) 33-gene signature was derived from the ex vivo expression profile analyses and used to dichotomize the entire TCGA cohort by high (median OS 12.65 months) or low (median OS 14.91 months) dexamethasone signature. CONCLUSIONS The authors present evidence that furthers the understanding of the complex effects of dexamethasone on biological characteristics of GBM. The results suggest that the drug increases invasion, proliferation, and angiogenesis in human GSC-derived orthotopic tumors, potentially worsening GBM patients' prognoses. The authors believe that careful investigation is needed to determine how to minimize these deleterious dexamethasone-associated side effects in GBM.

BACKGROUND:Meningioma is the most common primary brain tumor and has a variable risk of local recurrence. While World Health Organization (WHO) grade generally correlates with recurrence, there is substantial within-grade variation of recurrence risk. Current risk stratification does not accurately predict which patients are likely to benefit from adjuvant radiation therapy (RT). We hypothesized that tumors at risk for recurrence have unique gene expression profiles (GEP) that could better select patients for adjuvant RT. METHODS:We developed a recurrence predictor by machine learning modeling using a training/validation approach. RESULTS:Three publicly available AffymetrixU133 gene expression datasets (GSE9438, GSE16581, GSE43290) combining 127 primary, non-treated meningiomas of all grades served as the training set. Unsupervised variable selection was used to identify an 18-gene GEP model (18-GEP) that separated recurrences. This model was validated on 62 primary, non-treated cases with similar grade and clinical variable distribution as the training set. When applied to the validation set, 18-GEP separated recurrences with a misclassification error rate of 0.25 (log-rank p=0.0003). 18-GEP was predictive for tumor recurrence [p=0.0008, HR=4.61, 95%CI=1.89-11.23)] and was predictive after adjustment for WHO grade, mitotic index, sex, tumor location, and Simpson grade [p=0.0311, HR=9.28, 95%CI=(1.22-70.29)]. The expression signature included genes encoding proteins involved in normal embryonic development, cell proliferation, tumor growth and invasion (FGF9, SEMA3C, EDNRA), angiogenesis (angiopoietin-2), cell cycle regulation (CDKN1A), membrane signaling (tetraspanin-7, caveolin-2), WNT-pathway inhibitors (DKK3), complement system (C1QA) and neurotransmitter regulation (SLC1A3, Secretogranin-II). CONCLUSIONS:18-GEP accurately stratifies patients with meningioma by recurrence risk having the potential to guide the use of adjuvant RT.

Exosomes can mediate a dynamic method of communication between malignancies, including those sequestered in the central nervous system and the immune system. We sought to determine whether exosomes from glioblastoma (GBM)-derived stem cells (GSCs) can induce immunosuppression. We report that GSC-derived exosomes (GDEs) have a predilection for monocytes, the precursor to macrophages. The GDEs traverse the monocyte cytoplasm, cause a reorganization of the actin cytoskeleton, and skew monocytes toward the immune suppresive M2 phenotype, including programmed death-ligand 1 (PD-L1) expression. Mass spectrometry analysis demonstrated that the GDEs contain a variety of components, including members of the signal transducer and activator of transcription 3 (STAT3) pathway that functionally mediate this immune suppressive switch. Western blot analysis revealed that upregulation of PD-L1 in GSC exosome-treated monocytes and GBM-patient-infiltrating CD14⁺ cells predominantly correlates with increased phosphorylation of STAT3, and in some cases, with phosphorylated p70S6 kinase and Erk1/2. Cumulatively, these data indicate that GDEs are secreted GBM-released factors that are potent modulators of the GBM-associated immunosuppressive microenvironment.

Background/UNASSIGNED:The mesenchymal phenotype in glioblastoma (GBM) and other cancers drives aggressiveness and treatment resistance, leading to therapeutic failure and recurrence of disease. Currently, there is no successful treatment option available against the mesenchymal phenotype. Methods/UNASSIGNED:We classified patient-derived GBM stem cell lines into 3 subtypes: proneural, mesenchymal, and other/classical. Each subtype's response to the inhibition of diacylglycerol kinase alpha (DGKα) was compared both in vitro and in vivo. RhoA activation, liposome binding, immunoblot, and kinase assays were utilized to elucidate the novel link between DGKα and geranylgeranyltransferase I (GGTase I). Results/UNASSIGNED:Here we show that inhibition of DGKα with a small-molecule inhibitor, ritanserin, or RNA interference preferentially targets the mesenchymal subtype of GBM. We show that the mesenchymal phenotype creates the sensitivity to DGKα inhibition; shifting GBM cells from the proneural to the mesenchymal subtype increases ritanserin activity, with similar effects in epithelial-mesenchymal transition models of lung and pancreatic carcinoma. This enhanced sensitivity of mesenchymal cancer cells to ritanserin is through inhibition of GGTase I and downstream mediators previously associated with the mesenchymal cancer phenotype, including RhoA and nuclear factor-kappaB. DGKα inhibition is synergistic with both radiation and imatinib, a drug preferentially affecting proneural GBM. Conclusions/UNASSIGNED:Our findings demonstrate that a DGKα-GGTase I pathway can be targeted to combat the treatment-resistant mesenchymal cancer phenotype. Combining therapies with greater activity against each GBM subtype may represent a viable therapeutic option against GBM.

Background/UNASSIGNED:Glioma-associated macrophages and microglia (GAMs) are components of the glioblastoma (GBM) microenvironment that express MerTK, a receptor tyrosine kinase that triggers efferocytosis and can suppress innate immune responses. The aim of the study was to define MerTK as a therapeutic target using an orally bioavailable inhibitor, UNC2025. Methods/UNASSIGNED:We examined MerTK expression in tumor cells and macrophages in matched patient GBM samples by double-label immunohistochemistry. UNC2025-induced MerTK inhibition was studied in vitro and in vivo. Results/UNASSIGNED:MerTK/CD68+ macrophages increased in recurrent tumors while MerTK/glial fibrillary acidic protein-positive tumor cells did not. Pharmacokinetic studies showed high tumor exposures of UNC2025 in a syngeneic orthotopic allograft mouse GBM model. The same model mice were randomized to receive vehicle, daily UNC2025, fractionated external beam radiotherapy (XRT), or UNC2025/XRT. Although median survival (21, 22, 35, and 35 days, respectively) was equivalent with or without UNC2025, bioluminescence imaging (BLI) showed significant growth delay with XRT/UNC2025 treatment and complete responses in 19%. The responders remained alive for 60 days and showed regression to 1%-10% of pretreatment BLI tumor burden; 5 of 6 were tumor free by histology. In contrast, only 2% of 98 GBM mice of the same model treated with XRT survived 50 days and none survived 60 days. UNC2025 also reduced CD206+ macrophages in mouse tumor samples. Conclusions/UNASSIGNED:These results suggest that MerTK inhibition combined with XRT has a therapeutic effect in a subset of GBM. Further mechanistic studies are warranted.

Studies on melanoma brain metastases (MBM) with regard to mutational status are lacking. We investigated the outcomes of MBM in molecularly characterized patients for BRAF and NRAS mutations receiving conventional treatment. We investigated associations between outcomes [competing risk of local and distant brain failure (LF, DF) and overall survival (OS)] and clinical/pathological features of patients with known mutation status following initial treatment of MBM. Competing risk analysis was performed using the methods of Fine and Gray. We identified 235 patients with MBM diagnosed from 2005 to 2011. Mutation prevalence was BRAF ^non-V600K 98 (42%), BRAF ^V600K 34 (14%), NRAS 43 (18%), and wild-type for both genes (WT) 60 (26%) patients. Six month cumulative incidence LF rates were 3% for combined SRS or surgery with adjuvant radiation, 18% for surgery, 18% for SRS, 60% for WBRT, and 67% for systemic therapy. On multivariate analysis, only mutation status and initial treatment type were found to be independent predictors of local control. As compared to WT, NRAS (HR 2.58, 95% CI 1.18-5.67, p = 0.02) and BRAF ^V600K (HR 2.83, 95% CI 1.23-6.47, p = 0.01) mutational status were statistically significant while BRAF ^non-V600K status was not statistically significant (p = 0.23). Mutation status was not associated with DF or OS. BRAF ^V600K and NRAS mutation status predict increased LF following conventional treatments for MBM. These data can inform the design and interpretation of future MBM trials.

OBJECTIVE Mesenchymal stem cells (MSCs) have been shown to localize to gliomas after intravascular delivery. Because these cells home to areas of tissue injury, the authors hypothesized that the administration of ionizing radiation (IR) to tumor would enhance the tropism of MSCs to gliomas. Additionally, they sought to identify which radiation-induced factors might attract MSCs. METHODS To assess the effect of IR on MSC migration in vitro, transwell assays using conditioned medium (CM) from an irradiated commercially available glioma cell line (U87) and from irradiated patient-derived glioma stem-like cells (GSCs; GSC7-2 and GSC11) were employed. For in vivo testing, green fluorescent protein (GFP)-labeled MSCs were injected into the carotid artery of nude mice harboring orthotopic U87, GSC7-2, or GSC17 xenografts that were treated with either 0 or 10 Gy of IR, and brain sections were quantitatively analyzed by immunofluorescence for GFP-positive cells. These GSCs were used because GSC7-2 is a weak attractor of MSCs at baseline, whereas GSC17 is a strong attractor. To determine the factors implicated in IR-induced tropism, CM from irradiated GSC7-2 and from GSC11 was assayed with a cytokine array and quantitative ELISA. RESULTS Transwell migration assays revealed statistically significant enhanced MSC migration to CM from irradiated U87, GSC7-2, and GSC11 compared with nonirradiated controls and in a dose-dependent manner. After their intravascular delivery into nude mice harboring orthotopic gliomas, MSCs engrafted more successfully in irradiated U87 (p = 0.036), compared with nonirradiated controls. IR also significantly increased the tropism of MSCs to GSC7-2 xenografts (p = 0.043), which are known to attract MSCs only poorly at baseline (weak-attractor GSCs). Ionizing radiation also increased the engraftment of MSCs in strong-attractor GSC17 xenografts, but these increases did not reach statistical significance. The chemokine CCL2 was released by GSC7-2 and GSC11 after irradiation in a dose-dependent manner and mediated in vitro transwell migration of MSCs. Immunohistochemistry revealed increased CCL2 in irradiated GSC7-2 gliomas near the site of MSC engraftment. CONCLUSIONS Administering IR to gliomas enhances MSC localization, particularly in GSCs that attract MSCs poorly at baseline. The chemokine CCL2 appears to play a crucial role in the IR-induced tropism of MSCs to gliomas.