Faculty Bibliography

BACKGROUND:Repurposed memantine, mefloquine, and metformin have putative anticancer activity. The objective of this phase 1 study was to determine the maximum tolerated doses (MTDs) of combinations of these agents with temozolomide (TMZ). METHODS:Adults with newly diagnosed glioblastoma who completed chemoradiation were eligible. The patients were assigned to receive doublet, triplet, or quadruplet therapy with TMZ combined with mefloquine, memantine, and/or metformin. Dose-limiting toxicities (DLTs) were determined, using a 3 + 3 study design. RESULTS:Of 85 enrolled patients, 4 did not complete cycle 1 (the DLT observation period) for nontoxicity reasons, and 81 were evaluable for DLT. The MTDs for doublet therapy were memantine 20 mg twice daily, mefloquine 250 mg 3 times weekly, and metformin 850 mg twice daily. For triplet therapy, the MTDs were memantine 10 mg twice daily, mefloquine 250 mg 3 times weekly, and metformin 850 mg twice daily. For quadruplet therapy, the MTDs were memantine 10 mg twice daily, mefloquine 250 mg 3 times weekly, and metformin 500 mg twice daily. DLTs included dizziness (memantine) and gastrointestinal effects (metformin). Lymphopenia was the most common adverse event (66%). From study entry, the median survival was 21 months, and the 2-year survival rate was 43%. CONCLUSIONS:Memantine, mefloquine, and metformin can be combined safely with TMZ in patients with newly diagnosed glioblastoma.

Molecular markers are redefining classification of lower grade gliomas and ushering in a paradigm shift in their management. Our objective was to evaluate the differences in pattern of care and outcome by comparing grade II and grade III molecularly defined 1p19q co-deleted gliomas. We evaluated 1618 patients in the National Cancer Database diagnosed with 1p19q co-deleted gliomas from 2010 through 2014 and treated with surgery followed by radiation therapy (RT), chemotherapy (CT), or combined-modality therapy. Differences in patterns of care included that fifty-one percent of grade II tumors received surgery alone, whereas most patients with grade III tumors (86%) received surgery or biopsy followed by a form of post-operative therapy (p < 0.001). In a propensity score matched cohort, the Cox multivariable proportional hazards model with frailty testing identified significant covariates were age, comorbidity, histology and grade. Outcomes were different in overall survival even after adjusting for treatment received. The hazard for death for grade III 1p19q co-deleted gliomas was about 3.6 times higher ([HR] 3.69, 95% confidence interval [CI] 2.03-6.68, p < 0.001) than grade II 1p19q gliomas. Oligodendroglioma histology was associated with a lower likelihood of death (HR 0.40, 95% CI 0.23-0.70, p < 0.001). Our study is among the largest series to report on 1p19q co-deleted gliomas, which would otherwise require decades to acquire outside of large databases.

Macroautophagy/autophagy is a natural intracellular process that maintains cellular homeostasis and protects cells from death under stress conditions. Autophagy sustains tumor survival and growth when induced by common cancer treatments, including IR and cytotoxic chemotherapy, thereby contributing to therapeutic resistance of tumors. In this study, we report that the expression of MIR93, noted in two clinically relevant tumor subtypes of GBM, influenced GSC phenotype as well as tumor response to therapy through its effects on autophagy. Our mechanistic studies revealed that MIR93 regulated autophagic activities in GSCs through simultaneous inhibition of multiple autophagy regulators, including BECN1/Beclin 1, ATG5, ATG4B, and SQSTM1/p62. Moreover, two first-line treatments for GBM, IR and temozolomide (TMZ), as well as rapamycin (Rap), the prototypic MTOR inhibitor, decreased MIR93 expression that, in turn, stimulated autophagic processes in GSCs. Inhibition of autophagy by ectopic MIR93 expression, or via autophagy inhibitors NSC (an ATG4B inhibitor) and CQ, enhanced the activity of IR and TMZ against GSCs. Collectively, our findings reveal a key role for MIR93 in the regulation of autophagy and suggest a combination treatment strategy involving the inhibition of autophagy while administering cytotoxic therapy. Abbreviations: ACTB: actin beta; ATG4B: autophagy related 4B cysteine peptidase; ATG5: autophagy related 5; BECN1: beclin 1; CL: classical; CQ: chloroquine diphosphate; CSCs: cancer stem cells; GBM: glioblastoma; GSCs: glioma stem-like cells; HEK: human embryonic kidney; IB: immunoblotting; IF: immunofluorescent staining; IR: irradiation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MES: mesenchymal; MIR93: microRNA 93; MIRC: a control miRNA; miRNA/miR: microRNA; MTOR: mechanistic target of rapamycin kinase; NSC: NSC185085; PN: proneural; qRT-PCR: quantitative reverse transcription-polymerase chain reaction; Rap: rapamycin; SQSTM1/p62: sequestosome 1; TCGA: the cancer genome atlas; TMZ: temozolomide; WT: wild type; ZIP93: lentiviral miRZIP targeting MIR93; ZIPC: lentiviral miRZip targeting control miRNA.

Background: Treatment effectiveness and overall prognosis for glioma patients depend heavily on the genetic and epigenetic factors in each individual tumor. However, intra-tumoral genetic heterogeneity is known to exist and needs to be managed. Currently, evidence for genetic changes varying spatially within the tumor is qualitative, and quantitative data is lacking. We hypothesized that a greater genetic diversity or "genetic distance" would be observed for distinct tumor samples taken with larger physical distances between them. Methods: Stereotactic biopsies were obtained from untreated primary glioma patients as part of a clinical trial between 2011 and 2016, with at least one biopsy pair collected in each case. The physical (Euclidean) distance between biopsy sites was determined using coordinates from imaging studies. The tissue samples underwent whole exome DNA sequencing and epigenetic methylation profiling and genomic distances were defined in three separate ways derived from differences in number of genes, copy number variations (CNV), and methylation profiles. Results: Of the 31 patients recruited to the trial, 23 were included in DNA methylation analysis, for a total of 71 tissue samples (14 female, 9 male patients, age range 21-80). Samples from an 8 patient subset of the 23 evaluated patients were further included in whole exome and copy number variation analysis. Physical and genomic distances were found to be independently and positively correlated for each of the three genomic distance measures. The correlation coefficients were 0.63, 0.65, and 0.35, respectively for (a) gene level mutations, (b) copy number variation, and (c) methylation status. We also derived quantitative linear relationships between physical and genomic distances. Conclusion: Primary brain tumors are genetically heterogeneous, and the physical distance within a given glioma correlates to genomic distance using multiple orthogonal genomic assessments. These data should be helpful in the clinical diagnostic and therapeutic management of glioma, for example by: managing sampling error, and estimating genetic heterogeneity using simple imaging inputs.

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.

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.

Mutational inactivation of ATRX (a-thalassemia mental retardation X-linked) represents a defining molecular alteration in large subsets of malignant glioma. ATRX encodes a chromatin binding protein widely implicated in epigenetic regulation and remodeling. However, multiple studies have also associated its loss in cancer with replication stress, DNA damage, and copy number alterations (CNAs). The mechanisms by which ATRX deficiency drives this global genomic instability remain unclear. Here we report that ATRX inactivation in isogenic glioma model systems induces replication stress and DNA damage by promoting the formation of deleterious G-quadruplex (G4) secondary structure in DNA. Moreover, these effects are associated with the acquisition of disease-relevant CNAs over time. Prior work has linked G4s with genomic instability as well as CNAs in cancer. We then demonstrate, both in vitro and in vivo, that chemical G4 stabilization with CX-3543 (Quarfloxin) selectively enhances cell death in ATRX deficient isogenic cell lines as well as ATRX-mutant primary glioma stem cells derived from patients. Finally, we show that G4 stabilization synergizes with other DNA-damaging therapies, including ionizing radiation, in the ATRX-deficient context. Our findings clarify distinct mechanisms by which DNA secondary structure influences ATRX-deficient glioma pathogenesis and indicate that G4-stabilization may represent and attractive therapeutic strategy for the selective targeting of ATRX-mutant cancers. Opportunities for the development of radiosensitization approaches based on G4-stabilization are particularly intriguing, as ionizing radiation remains among the most effective non-surgical treatments for malignant glioma

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.