Manish K. Aghi

Hypoxia-Induced Autophagy Promotes Tumor Cell Survival and Adaptation to Antiangiogenic Treatment in Glioblastoma

Antiangiogenic therapy leads to devascularization that limits tumor growth. However, the benefits of angiogenesis inhibitors are typically transient and resistance often develops. In this study, we explored the hypothesis that hypoxia caused by antiangiogenic therapy induces tumor cell autophagy as a cytoprotective adaptive response, thereby promoting treatment resistance. Hypoxia-induced autophagy was dependent on signaling through the hypoxia-inducible factor-1α (HIF-1α)/AMPK pathway, and treatment of hypoxic cells with autophagy inhibitors caused a shift from autophagic to apoptotic cell death in vitro. In glioblastomas, clinically resistant to the VEGF-neutralizing antibody bevacizumab, increased regions of hypoxia and higher levels of autophagy-mediating BNIP3 were found when compared with pretreatment specimens from the same patients. When treated with bevacizumab alone, human glioblastoma xenografts showed increased BNIP3 expression and hypoxia-associated growth, which could be prevented by addition of the autophagy inhibitor chloroquine. In vivo targeting of the essential autophagy gene ATG7 also disrupted tumor growth when combined with bevacizumab treatment. Together, our findings elucidate a novel mechanism of resistance to antiangiogenic therapy in which hypoxia-mediated autophagy promotes tumor cell survival. One strong implication of our findings is that autophagy inhibitors may help prevent resistance to antiangiogenic therapy used in the clinic.

Oncolytic viral therapies – the clinical experience

It has been 9 years since the beginning of the first clinical trial in which an oncolytic virus was administered to cancer patients. Since then, oncolytic viruses from five different species have been taken to phase I and II clinical trials in over 300 cancer patients. While additional studies will be required to ascertain if the efficacy of any of these agents is high enough to warrant adding them to the existing therapeutic regimen, it has been reassuring that DNA viruses engineered to achieve tumor selectivity and RNA viruses with relative inherent natural tumor selectivity have proven reasonably safe at the wide range of doses that were tested. Here, we review the biology and clinical results of these five species of viruses and discuss lessons learned and challenges for the future.

Long-term recurrence rates of atypical meningiomas after gross total resection with or without postoperative adjuvant radiation.

OBJECTIVEAtypical meningioma (AM) patients often undergo gross total resection (GTR) at the time of presentation, but subsequent prognosis and optimal management remain unclear. We sought to define the long-term recurrence rate of AMs after GTR, along with factors predicting recurrence, including postoperative radiation. METHODSWe performed a retrospective review of 108 AMs after GTR at our institution from 1993 to 2004. Recurrence risk factors were analyzed using multivariate Cox regression. RESULTSThere were 600 patient-years of imaging follow-up on 48 men and 60 women. Of 108 tumors, 30 (28%) recurred 3 to 144 months after GTR (mean, 36 months). Actuarial tumor recurrence rates were 7% (1 year), 41% (5 years), and 48% (10 years). Of 108 patients, 8 received postoperative radiation without recurrence (P = 0.1). Multivariate analysis including age, sex, postoperative radiation, tumor location, MIB-1 labeling index, and 6 atypical-defining histological features identified recurrence-predicting factors: older age (hazard ratio, 1.6/decade; P = 0.01), sheeting (hazard ratio, 2.2; P = 0.025), and prominent nucleoli (hazard ratio, 2.1; P = 0.034). Recursive partitioning identified a subset, men with mitoses and prominent nucleoli, with 70% recurrence (n = 14). All patients with recurrences received radiation, and 22 of 30 patients underwent craniotomies (average, 2.7 craniotomies per patient with recurrence; range, 1–7 craniotomies). Only 1 of 22 re-resected meningiomas underwent malignant transformation. Of 30 patients with recurrence, 10 experienced tumor-induced mortality an average of 7 years after recurrence (range, 1–14 years). CONCLUSIONAfter GTR without postoperative radiation, AMs have a high recurrence rate. Most recurrences occurred within 5 years after resection. Recurrences caused numerous reoperations per patient and shortened survival. Our finding suggesting lower recurrence rates in patients undergoing immediate postoperative radiation should be investigated in larger, prospective series.

Human Glioblastoma–Derived Cancer Stem Cells: Establishment of Invasive Glioma Models and Treatment with Oncolytic Herpes Simplex Virus Vectors

Glioblastoma, the most malignant type of primary brain tumor, is one of the solid cancers where cancer stem cells have been isolated, and studies have suggested resistance of those cells to chemotherapy and radiotherapy. Here, we report the establishment of CSC-enriched cultures derived from human glioblastoma specimens. They grew as neurospheres in serum-free medium with epidermal growth factor and fibroblast growth factor 2, varied in the level of CD133 expression and very efficiently formed highly invasive and/or vascular tumors upon intracerebral implantation into immunodeficient mice. As a novel therapeutic strategy for glioblastoma-derived cancer stem-like cells (GBM-SC), we have tested oncolytic herpes simplex virus (oHSV) vectors. We show that although ICP6 (UL39)-deleted mutants kill GBM-SCs as efficiently as wild-type HSV, the deletion of gamma34.5 significantly attenuated the vectors due to poor replication. However, this was significantly reversed by the additional deletion of alpha47. Infection with oHSV G47Delta (ICP6(-), gamma34.5(-), alpha47(-)) not only killed GBM-SCs but also inhibited their self-renewal as evidenced by the inability of viable cells to form secondary tumor spheres. Importantly, despite the highly invasive nature of the intracerebral tumors generated by GBM-SCs, intratumoral injection of G47Delta significantly prolonged survival. These results for the first time show the efficacy of oHSV against human GBM-SCs, and correlate this cytotoxic property with specific oHSV mutations. This is important for designing new oHSV vectors and clinical trials. Moreover, the new glioma models described in this study provide powerful tools for testing experimental therapeutics and studying invasion and angiogenesis.

Impact of extent of resection for recurrent glioblastoma on overall survival: clinical article.

OBJECT Extent of resection (EOR) has been shown to be an important prognostic factor for survival in patients undergoing initial resection of glioblastoma (GBM), but the significance of EOR at repeat craniotomy for recurrence remains unclear. In this study the authors investigate the impact of EOR at initial and repeat resection of GBM on overall survival. METHODS Medical records were reviewed for all patients undergoing craniotomy for GBM at the University of California San Francisco Medical Center from January 1, 2005, through August 15, 2009. Patients who had a second craniotomy for pathologically confirmed recurrence following radiation and chemotherapy were evaluated. Volumetric EOR was measured and classified as gross-total resection (GTR, > 95% by volume) or subtotal resection (STR, ≤ 95% by volume) after independent radiological review. Overall survival was compared between groups using univariate and multivariate analysis accounting for known prognostic factors, including age, eloquent location, Karnofsky Performance Status (KPS), and adjuvant therapies. RESULTS Multiple resections were performed in 107 patients. Fifty-two patients had initial GTR, of whom 31 (60%) had GTR at recurrence, with a median survival of 20.4 months (standard error [SE] 1.0 months), and 21 (40%) had STR at recurrence, with a median survival of 18.4 months (SE 0.5 months) (difference not statistically significant). Initial STR was performed in 55 patients, of whom 26 (47%) had GTR at recurrence, with a median survival of 19.0 months (SE 1.2 months), and 29 (53%) had STR, with a median survival of 15.9 months (SE 1.2 months) (p = 0.004). A Cox proportional hazards model was constructed demonstrating that age (HR 1.03, p = 0.004), KPS score at recurrence (HR 2.4, p = 0.02), and EOR at repeat resection (HR 0.62, p = 0.02) were independent predictors of survival. Extent of initial resection was not a statistically significant factor (p = 0.13) when repeat EOR was included in the model, suggesting that GTR at second craniotomy could overcome the effect of an initial STR. CONCLUSIONS Extent of resection at recurrence is an important predictor of overall survival. If GTR is achieved at recurrence, overall survival is maximized regardless of initial EOR, suggesting that patients with initial STR may benefit from surgery with a GTR at recurrence.

Prodrug activation enzymes in cancer gene therapy

Among the broad array of genes that have been evaluated for tumor therapy, those encoding prodrug activation enzymes are especially appealing as they directly complement ongoing clinical chemotherapeutic regimes. These enzymes can activate prodrugs that have low inherent toxicity using both bacterial and yeast enzymes, or enhance prodrug activation by mammalian enzymes. The general advantage of the former is the large therapeutic index that can be achieved, and of the latter, the non‐immunogenicity (supporting longer periods of prodrug activation) and the fact that the prodrugs will continue to have some efficacy after transgene expression is extinguished. This review article describes 13 different prodrug activation schemes developed over the last 15 years, two of which – activation of ganciclovir by viral thymidine kinase and activation of 5‐fluorocytosine to 5‐fluorouracil – are currently being evaluated in clinical trials. Essentially all of these prodrug activation enzymes mediate toxicity through disruption of DNA replication, which occurs at differentially high rates in tumor cells compared with most normal cells. In cancer gene therapy, vectors target delivery of therapeutic genes to tumor cells, in contrast to the use of antibodies in antibody‐directed prodrug therapy. Vector targeting is usually effected by direct injection into the tumor mass or surrounding tissues, but the efficiency of gene delivery is usually low. Thus it is important that the activated drug is able to act on non‐transduced tumor cells. This bystander effect may require cell‐to‐cell contact or be mediated by facilitated diffusion or extracellular activation to target neighboring tumor cells. Effects at distant sites are believed to be mediated by the immune system, which can be mobilized to recognize tumor antigens by prodrug‐activated gene therapy. Prodrug activation schemes can be combined with each other and with other treatments, such as radiation, in a synergistic manner. Use of prodrug wafers for intratumoral drug activation and selective permeabilization of the tumor vasculature to prodrugs and vectors should further increase the value of this new therapeutic modality. Copyright

Tumor Stromal-Derived Factor-1 Recruits Vascular Progenitors to Mitotic Neovasculature, where Microenvironment Influences Their Differentiated Phenotypes

Mechanisms underlying tumor vasculogenesis, the homing and engraftment of bone marrow-derived vascular progenitors, remain undefined. We hypothesized that tumor cell-secreted factors regulate vasculogenesis. We studied vasculogenic and nonvasculogenic intracranial murine gliomas. A PCR screen identified stromal-derived factor-1 (SDF-1/CXCL12) and vascular endothelial growth factor (VEGF) expression by vasculogenic glioma cells and spontaneously arising vasculogenic tumors in NF1+/-:Trp53+/- mice, but not by nonvasculogenic glioma cells. Enforced SDF-1, not VEGF, expression in nonvasculogenic cells caused vasculogenesis. Combined SDF-1 and VEGF expression augmented vasculogenesis over SDF-1 expression alone. Blocking SDF-1 receptor CXCR4 reduced short-term homing and long-term engraftment of vascular progenitors. Implanting tumor cells secreting SDF-1 was therefore necessary and sufficient to incorporate marrow-derived precursors into tumor endothelium. SDF-1 seemed to exert these effects by acting locally intratumorally and did not cause an efflux of marrow-derived progenitors into circulation. Tumor microenvironment determined additional fates of marrow-derived cells. Hypoxia, observed with ectopic s.c. murine tumors at levels approximating that of intracranial human glioblastoma, interacted with tumor-secreted SDF-1 to expand engrafted vascular progenitor differentiated phenotypes to include pericytes as well as endothelium. In contrast, less hypoxic orthotopic intracranial murine gliomas contained only marrow-derived endothelium without marrow-derived pericytes. Furthermore, we found that vasculogenesis is significant for tumors because it generates endothelium with a higher mitotic index than endothelium derived from local sources. Although CXCR4 blockade selectively targeted endothelium generated by vasculogenesis, completely inhibiting vessel formation may require combination therapy targeting locally derived and marrow-derived endothelium.

Biology of angiogenesis and invasion in glioma

SummaryTreatment of adult brain tumors, in particular glioblastoma, remains a significant clinical challenge, despite modest advances in surgical technique, radiation, and chemotherapeutics. The formation of abnormal, dysfunctional tumor vasculature and glioma cell invasion along white matter tracts are believed to be major components of the inability to treat these tumors effectively. Recent insight into the fundamental processes governing glioma angiogenesis and invasion provide a renewed hope for development of novel strategies aimed at reducing the morbidity of this uniformly fatal disease. In this review, we discuss background biology of the blood brain barrier and its pertinence to blood vessel formation and tumor invasion. We will then focus our attention on the biology of glioma angiogenesis and invasion, and the key mediators of these processes. Last, we will briefly discuss recent and ongoing clinical trials targeting mediators of angiogenesis or invasion in glioma patients. The findings provide a renewed hope for those endeavoring to improve treatment of patients with glioma by providing a novel set of rational targets for translational drug discovery.

Application of novel response/progression measures for surgically delivered therapies for gliomas: Response Assessment in Neuro-Oncology (RANO) working group

BACKGROUND The Response Assessment in Neuro-Oncology (RANO) Working Group is an international, multidisciplinary effort to develop new standardized response criteria for clinical trials in brain tumors. The RANO group identified knowledge gaps relating to the definitions of tumor response and progression after the use of surgical or surgically based treatments. OBJECTIVE To outline a proposal for new response and progression criteria for the assessment of the effects of surgery and surgically delivered therapies for patients with gliomas. METHODS The Surgery Working Group of RANO identified surgically related end-point evaluation problems that were not addressed in the original Macdonald criteria, performed an extensive literature review, and used a consensus-building process to develop recommendations for how to address these issues in the setting of clinical trials. RESULTS Recommendations were formulated for surgically related issues, including imaging changes associated with surgical resection or surgically mediated adjuvant local therapies, the determination of progression in the setting where all enhancing tumor has been removed, and how new enhancement should be interpreted in the setting where local therapies that are known to produce nonspecific enhancement have been used. Additionally, the terminology used to describe the completeness of surgical resections has been recognized to be inconsistently applied to enhancing vs nonenhancing tumors, and a new set of descriptors is proposed. CONCLUSION The RANO process is intended to produce end-point criteria for clinical trials that take into account the effects of prior and ongoing therapies. The RANO criteria will continue to evolve as new therapies and technologies are introduced into clinical trial and/or practice.

Gene Expression Profile Identifies Tyrosine Kinase c-Met as a Targetable Mediator of Antiangiogenic Therapy Resistance

Purpose: To identify mediators of glioblastoma antiangiogenic therapy resistance and target these mediators in xenografts. Experimental Design: We conducted microarray analysis comparing bevacizumab-resistant glioblastomas (BRG) with pretreatment tumors from the same patients. We established novel xenograft models of antiangiogenic therapy resistance to target candidate resistance mediator(s). Results: BRG microarray analysis revealed upregulation versus pretreatment of receptor tyrosine kinase c-Met, which underwent further investigation because of its prior biologic plausibility as a bevacizumab resistance mediator. BRGs exhibited increased hypoxia versus pretreatment in a manner correlating with their c-Met upregulation, increased c-Met phosphorylation, and increased phosphorylation of c-Met–activated focal adhesion kinase and STAT3. We developed 2 novel xenograft models of antiangiogenic therapy resistance. In the first model, serial bevacizumab treatment of an initially responsive xenograft generated a xenograft with acquired bevacizumab resistance, which exhibited upregulated c-Met expression versus pretreatment. In the second model, a BRG-derived xenograft maintained refractoriness to the MRI tumor vasculature alterations and survival-promoting effects of bevacizumab. Growth of this BRG-derived xenograft was inhibited by a c-Met inhibitor. Transducing these xenograft cells with c-Met short hairpin RNA inhibited their invasion and survival in hypoxia, disrupted their mesenchymal morphology, and converted them from bevacizumab-resistant to bevacizumab-responsive. Engineering bevacizumab-responsive cells to express constitutively active c-Met caused these cells to form bevacizumab-resistant xenografts. Conclusion: These findings support the role of c-Met in survival in hypoxia and invasion, features associated with antiangiogenic therapy resistance, and growth and therapeutic resistance of xenografts resistant to antiangiogenic therapy. Therapeutically targeting c-Met could prevent or overcome antiangiogenic therapy resistance. Clin Cancer Res; 19(7); 1773–83. ©2012 AACR.