Christian A. Graves

Opposing effects of pigment epithelium-derived factor on breast cancer cell versus neuronal survival: implication for brain metastasis and metastasis-induced brain damage.

Brain metastases are a significant cause of morbidity and mortality for patients with cancer, yet preventative and therapeutic options remain an unmet need. The cytokine pigment epithelium-derived factor (PEDF) is downregulated in resected human brain metastases of breast cancer compared with primary breast tumors, suggesting that restoring its expression might limit metastatic spread. Here, we show that outgrowth of large experimental brain metastases from human 231-BR or murine 4T1-BR breast cancer cells was suppressed by PEDF expression, as supported by in vitro analyses as well as direct intracranial implantation. Notably, the suppressive effects of PEDF were not only rapid but independent of the effects of this factor on angiogenesis. Paralleling its cytotoxic effects on breast cancer cells, PEDF also exerted a prosurvival effect on neurons that shielded the brain from tumor-induced damage, as indicated by a relative 3.5-fold reduction in the number of dying neurons adjacent to tumors expressing PEDF. Our findings establish PEDF as both a metastatic suppressor and a neuroprotectant in the brain, highlighting its role as a double agent in limiting brain metastasis and its local consequences.

Imaging Biomarker Dynamics in an Intracranial Murine Glioma Study of Radiation and Antiangiogenic Therapy

PURPOSE There is a growing need for noninvasive biomarkers to guide individualized spatiotemporal delivery of radiation therapy (RT) and antiangiogenic (AA) therapy for brain tumors. This study explored early biomarkers of response to RT and the AA agent sunitinib (SU), in a murine intracranial glioma model, using serial magnetic resonance imaging (MRI). METHODS AND MATERIALS Mice with MRI-visible tumors were stratified by tumor size into 4 therapy arms: control, RT, SU, and SU plus RT (SURT). Single-fraction conformal RT was delivered using MRI and on-line cone beam computed tomography (CT) guidance. Serial MR images (T2-weighted, diffusion, dynamic contrast-enhanced and gadolinium-enhanced T1-weighted scans) were acquired biweekly to evaluate tumor volume, apparent diffusion coefficient (ADC), and tumor perfusion and permeability responses (K(trans), K(ep)). RESULTS Mice in all treatment arms survived longer than those in control, with a median survival of 35 days for SURT (P<.0001) and 30 days for RT (P=.009) and SU (P=.01) mice vs 26 days for control mice. At Day 3, ADC rise was greater with RT than without (P=.002). Sunitinib treatment reduced tumor perfusion/permeability values with mean K(trans) reduction of 27.6% for SU (P=.04) and 26.3% for SURT (P=.04) mice and mean K(ep) reduction of 38.1% for SU (P=.01) and 27.3% for SURT (P=.02) mice. The magnitude of individual mouse ADC responses at Days 3 and 7 correlated with subsequent tumor growth rate R values of -0.878 (P=.002) and -0.80 (P=.01), respectively. CONCLUSIONS Early quantitative changes in diffusion and perfusion MRI measures reflect treatment responses soon after starting therapy and thereby raise the potential for these imaging biomarkers to guide adaptive and potentially individualized therapy approaches in the future.

The translational significance of epithelial-mesenchymal transition in head and neck cancer

Positive markers of epithelial-mesenchymal transition (EMT) in head and neck cancers complicate clinical management and are associated with reduced survival. We discuss recent translational discoveries in EMT and suggest additional actionable molecular pathways, biomarkers, and clinical agents.

Prognostic significance of serum levels of vascular endothelial growth factor and insulin-like growth factor-1 in advanced gastric cancer patients treated with FOLFOX chemotherapy.

Background: Tumor vascular endothelial growth factor (VEGF) is a key angiogenic factor and may have an impact on tumor progression and response to chemotherapy. The insulin-like growth factor (IGF) system is related to cell proliferation and tumor growth. However, there is limited available data regarding the clinical and prognostic significance of VEGF or IGF-1 in advanced gastric cancer. The aim of this study was to evaluate the prognostic significance of serum VEGF and IGF-1 levels in advanced gastric cancer patients who were treated with oxaliplatin/5-fluorouracil (FOLFOX). Methods: The study population consisted of 100 advanced gastric cancer patients (median age 56 years). Patients were treated with oxaliplatin 85 mg/m2 as a 2-hour infusion on day 1 plus leucovorin 20 mg/m2 over 10 min, followed by a 5-fluorouracil (5-FU) bolus 400 mg/m2 and 22 h of continuous infusion of 600 mg/m2 on days 1–2. Treatment was repeated in 2-week intervals. The levels of serum VEGF and IGF-1 were measured using enzyme-linked immunoassays. Results: There was a significant correlation between the serum level of VEGF and Lauren’s classification (p = 0.030) and previous operations (p = 0.010). IGF-1 was associated with the number of metastases (p = 0.012). The median level of serum VEGF was decreased after FOLFOX chemotherapy (p = 0.034). However, none of the measured serum markers were significantly correlated with response. In univariate analysis, overall survival (p < 0.001) was significantly shorter in patients with high serum levels of VEGF. Multivariate analysis revealed that VEGF was an independent factor for overall survival (HR 2.221; 95% CI 1.377–3.583, p = 0.001). Furthermore, IGF-1 had no significant influence on the clinical outcome. Conclusion: A high level of serum VEGF is an independent prognostic factor in patients with advanced gastric cancer treated with chemotherapy. This may help to identify the patients who are more sensitive to the FOLFOX regimen.

Human papillomavirus status and gene expression profiles of oropharyngeal and oral cancers from European American and African American patients.

Disparities in prevalence, human papillomavirus (HPV) status, and mortality rates for head and neck cancer have been described between African American and European American patients.

Neuroendocrine Merkel Cell Carcinoma Is Associated with Mutations in Key DNA Repair, Epigenetic and Apoptosis Pathways: A Case-Based Study Using Targeted Massively Parallel Sequencing

Introduction: Merkel cell carcinoma (MCC) is a rare neuroendocrine carcinoma with a poorly understood molecular etiology. We implemented a comprehensive deep sequencing approach to identify mutations in the tumor DNA from a cohort of patients treated at our institution over the past 15 years. Our results indicate mutations that may constitute therapeutic targets in MCC. Methods: Five patients were treated for MCC within the study interval. Patients with adequate tissue (n = 4), positive neuroendocrine differentiation (chromogranin, synaptophysin, and cytokeratin 20), and histopathological confirmation of MCC were included in the study. DNA was extracted from archival tumor tissue samples and analyzed by massively parallel sequencing using a targeted, multiplex PCR approach followed by semiconductor sequencing. Results: We demonstrate high-penetrance nonsense mutations in PDE4DIP (n = 4) as well as various missense mutations in the DNA damage response (PRKDC, AURKB, ERCC5, ATR, and ATRX) and epigenetic modulating enzymes (MLL3). Conclusion: We describe several mutations in potential disease-relevant genes and pathways. These targets should be evaluated in a larger cohort to determine their role in the molecular pathogenesis of MCC.

Radiosensitization by the novel DNA intercalating agent vosaroxin

PurposeVosaroxin is a first in class naphthyridine analog structurally related to quinolone antibacterials, that intercalates DNA and inhibits topoisomerase II. Vosaroxin is not a P-glycoprotein receptor substrate and its activity is independent of p53, thus evading common drug resistance mechanisms. To evaluate vosaroxin as a clinically applicable radiation sensitizer, we investigated its effects on tumor cell radiosensitivity in vitro and in vivo.MethodsVosaroxins effect on post-irradiation sensitivity of U251, DU145, and MiaPaca-2 cells was assessed by clonogenic assay. Subsequent mechanistic and in vivo studies were performed with U251 cells. Cell cycle distribution and G2 checkpoint integrity was analyzed by flow cytometry. DNA damage and repair was evaluated by a high throughput gamma-H2AX assay. Apoptosis was assessed by flow cytometry. Mitotic catastrophe was assessed by microscopic evidence of fragmented nuclei by immunofluorescence. In vivo radiosensitization was measured by subcutaneous tumor growth delay.Results50-100 nmol/L treatment with vosaroxin resulted in radiosensitization of all 3 cell lines tested with a dose enhancement factor of 1.20 to 1.51 measured at a surviving fraction of 0.1. The maximal dose enhancement was seen in U251 cells treated with 75 nmol/L vosaroxin (DEF 1.51). Vosaroxin exposure did not change cell cycle distribution prior to irradiation nor alter G2 checkpoint integrity after irradiation. No difference was seen in the apoptotic fraction regardless of drug or radiation treatment. The number of cells in mitotic catastrophe was significantly greater in irradiated cells treated with vosaroxin than cells receiving radiation only at 72 hr (p = 0.009). Vosaroxin alone did not significantly increase mitotic catastrophe over control (p = 0.53). Cells treated with vosaroxin and radiation maintained significantly higher gamma-H2AX levels than cells treated with vehicle control (p = 0.014), vosaroxin (p = 0.042), or radiation alone (p = 0.039) after 24 hr. In vivo tumor growth delay was 1.5 days for vosaroxin alone (IV 10 mg/kg), 1.0 days for radiation (3 Gy) alone, and 8.6 days for the group treated with vosaroxin 4 hours prior to radiation.ConclusionsVosaroxin enhanced tumor cell radiosensitivity in vitro and in vivo. The mechanism appears to be related to inhibition of DNA repair and increased mitotic catastrophe.

Abstract 450: Head and neck cancer and HPV infection: A potential role for peritumoral neurogenesis in HPV-associated malignancy

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Head and neck cancers account for 5% of all cancer related deaths and present with complex molecular pathology and clinical features owing to their functional anatomical location. The molecular etiology of head and neck squamous cancer (HNSC) has gained extensive attention recently revealing key mutations, viral infection, and an emerging landscape of varying disease sub-types. While perineural invasion has long been associated with poor outcome in HNSC, the association of neurogenesis-related genes with cancer progression has only recently come into focus in HNSC. Using prospectively acquired patient samples and TCGA HNSC mutational analyses we further characterized the sequelae of molecular changes in neurogenesis-related genes and propose that these genes play a significant role in HNSC development. Methods: All clinical specimens were collected under approval by Institutional Review Board. Prospectively collected samples were analyzed for HPV DNA by linear arrays (InnoLipa) and q-PCR for E7-RNA. Gene expression arrays were conducted to determine differences in HPV activity and neuronal associated gene ontologies (Agilent). Gene expression profiles were compared in oral HPV+ and HPV- cultured oral keratinocytes as well as the TCGA Kandoth & Tang datasets. Gene targets were validated by Immunohistochemistry and qPCR. Results: Initial unsupervised hierarchical clustering analysis of our HNSC cohort revealed a separation of HPV+ HPV+E7-(HPV-inactive: HPVin), and HPV- tumors with changes in the expression of key neurogenesis-related genes. For example, POU4F1, NGFR, and GRIN2C were significantly up-regulated (P<0.05) in HPV+ vs HPVin cancers. Additional analysis of TCGA data revealed divergence of the mutational spectra in HNSC with and without perineural invasion. Conclusion: Our preliminary findings indicate the emerging importance of neurogenesis-related genes and perineural involvement in HNSC and support a divergence of HPV+, HPVin, and HPV- HNSC sub-types. Citation Format: Christian A. Graves, Swati Tomar, Diego Altomare, James R. Wells, Kim E. Creek, Lucia Pirisi. Head and neck cancer and HPV infection: A potential role for peritumoral neurogenesis in HPV-associated malignancy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 450. doi:10.1158/1538-7445.AM2014-450

Abstract 4026: HPV16-positive and HPV16-negative head and neck squamous cell carcinomas display different patterns of expression of genes involved in the control of growth and EMT.

Up to 60% of oropharyngeal cancers and 25% of head and neck squamous cell carcinomas (HNSCC) are high-risk human papillomavirus (HPV) positive, primarily HPV16. Differences in risk factors, age of presentation and clinical behavior of these tumors indicate that HPV+ and HPV- tumors develop with different molecular mechanisms and are biologically distinct. Based upon a gene expression profile comparison of HPV- and HPV+ HNSCC specimens, obtained from the Medical University of South Carolina ENT/Head and Neck Surgery clinic, we identified 384 candidate genes differentially expressed (125 genes up-regulated and 223 down-regulated) in HPV+ in comparison with HPV- tumors. GO analysis of the microarray data indicated that gene expression changes in HPV+ tumors affected primarily proliferation and cell cycle control, while HPV- tumors showed changes in genes involved in EMT, cell motility, and angiogenesis. We selected five genes (c-MET, TP53, TGF-beta2, BRCA1 and SIX1) for further analysis in a set of 44 tumor and four normal (tonsil and uvula) samples. Seventeen tumor samples were from African American (AA) patients, and 27 from European Americans (EA). The prevalence of HPV infection was 30% in samples from AA patients, and 65% in those from EA patients. Most HPV+ tumor samples (20 of 24) were positive for HPV16 and a subset of the HPV16+ tumor samples (55%) expressed HPV16 E7, as documented by real-time RT/PCR using tumor-derived RNA. The prevalence of active HPV infections (where E7 was expressed) was 17% in AA and 50% in EA patients. These tumor samples were utilized to explore the expression of the selected genes by real-time RT/PCR. Data were normalized to normal human keratinocytes based on three reference genes derived from a panel of seven candidate reference genes, using Normfinder, BestKeeper and GeNorm. Normalized relative quantities for each gene of interest were then calculated using qbasePLUS (Biogazelle.) c-MET was overexpressed, when compared to control tissue, in all tumor samples but levels of c-MET were the highest in HPV- EA samples, and comparatively lower in HPV+ samples. TP53 was overexpressed in HPV+ samples; TGF-beta2 and Six1 were clearly overexpressed in all tumor samples, compared to control tissue samples; and BRCA1 levels were higher in HPV+ samples, compared to HPV- samples and controls. These data are consistent with our microarray results that indicate that HPV- tumors exhibit gene expression profiles indicative of activation of EMT. We are investigating the molecular interactions that lead to overexpression of SIX1, c-MET and BRCA1 in these tumors, and the functional consequences of this overexpression in terms of cell growth and EMT. Citation Format: Swati Tomar, Sangeeta Kowli, Diego Altomare, Christian Graves, Susannah Kassler, Natalie A. Sutkowski, Marion Boyd Gillespie, Saundra H. Glover, Kim E. Creek, Lucia A. Pirisi-Creek. HPV16-positive and HPV16-negative head and neck squamous cell carcinomas display different patterns of expression of genes involved in the control of growth and EMT. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4026. doi:10.1158/1538-7445.AM2013-4026

Abstract 4539: Vascular endothelial growth factor gene polymorphisms and clinical outcome in advanced gastric cancer treated with FOLFOX

Background: Single-nucleotide polymorphisms (SNPs) of the vascular endothelial growth factor (VEGF) gene may have an impact on tumor progression, and response to chemotherapy. The aim of this study is to evaluate the associations between VEGF SNPs and clinical outcome in advanced gastric cancer patients treated with oxaliplatin, 5-fluorouracil, and leucovorin (FOLFOX). Methods: One hundred ninety recurrent or metastatic gastric cancer patients were enrolled in this study and treated with FOLFOX regimen. Genomic DNA was isolated from whole blood, and six VEGF (-2578C/A, –2489C/T, –1498T/C, –634G/C, 936C/T, and 1612G/A) gene polymorphisms were analyzed by PCR. Results: Patients genotyped G/G for –634G/C gene polymorphism had lower response rate (22.2%) than those G/C or C/C (32.3%, 51.1%; p = 0.034). The median serum levels of VEGF was higher in G/G genotypes than G/C + C/C (724.1 pg/ml vs. 462.4 pg/ml, p = 0.041). Patients with the VEGF –634CG/C polymorphism G/C + C/C genotype had a longer time to progression (TTP) of 4.9 months, compared with the TTP of 3.5 months for those with the G/G (p = 0.012, log-rank test). By multivariate analysis, this G/G genotype of –634G/C polymorphism was identified as an independent prognostic factor (Hazard ratio 1.497, p = 0.017). No significant influence on overall survival (OS) was observed by the –634 G/C. However, other SNPs were not related to response rate, TTP, or OS. Conclusion: Our data suggest that G/G genotype of –634G/C polymorphism is related to the higher serum levels of VEGF, and poor clinical outcome in advanced gastric cancer patients. It may help to identify patients who are more sensitive to FOLFOX regimen. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4539. doi:1538-7445.AM2012-4539