Christopher J. Farrell

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.

Lytic replication-associated protein (RAP) encoded by Kaposi sarcoma-associated herpesvirus causes p21CIP-1-mediated G1 cell cycle arrest through CCAAT/enhancer-binding protein-α

Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic DNA virus that causes Kaposi sarcoma and AIDS-related primary effusion lymphoma (PEL). Here we show that KSHV lytic cycle replication in PEL cells induces G1 cell cycle arrest, presumably to facilitate the progression of viral DNA replication. Expression of a KSHV-encoded early lytic protein referred to as RAP or K8 is induced within 12–24 h after the onset of lytic cycle induction in host PEL cells, and coincides with increased levels of both the endogenous C/EBPα and p21CIP-1 proteins in the nucleus of the same cells. The KSHV RAP protein binds to C/EBPα in vitro and stimulates C/EBPα-induced expression from both the C/EBPα and p21 promoters in cotransfected cells. A recombinant adenovirus expressing the RAP protein induced the expression of both the C/EBPα and p21 proteins in primary human fibroblasts, and flow cytometric analysis revealed a dramatic inhibition of G1 to S cell cycle progression in the same cells. All of these effects were abolished in cells that lack C/EBPα or by deletion of the basic/leucine zipper region in RAP that interacts with C/EBPα. Therefore, C/EBPα is essential for the p21-mediated inhibition of G1 to S-phase progression by RAP in KSHV-infected host cells.

CCAAT/Enhancer Binding Protein α Interacts with ZTA and Mediates ZTA-Induced p21CIP-1 Accumulation and G1 Cell Cycle Arrest during the Epstein-Barr Virus Lytic Cycle

ABSTRACT Cellular CCAAT/enhancer binding protein α (C/EBPα) promotes cellular differentiation and has antimitotic activities involving cell cycle arrest at G1/S through stabilization of p21CIP-1/WAF1 and through transcriptional activation of the p21 promoter. The Epstein-Barr virus lytic-cycle transactivator protein ZTA is known to arrest the host cell cycle at G1/S via a p53-independent p21 pathway, but the detailed molecular mechanisms involved have not been defined. To further evaluate the role of ZTA in cell cycle arrest, we constructed a recombinant adenovirus vector expressing ZTA (Ad-ZTA), whose level of expression at a low multiplicity of infection in normal human diploid fibroblast (HF) cells was lower than or equal to the physiological level seen in Akata cells lytically induced by EBV (EBV-Akata cells). Fluorescence-activated cell sorting analysis of HF cells infected with Ad-ZTA confirmed that G1/S cell cycle arrest occurred in the majority of ZTA-positive cells, but not with an adenovirus vector expressing green fluorescent protein. Double-label immunofluorescence assays (IFA) performed with Ad-ZTA-infected HF cells revealed that only ZTA-positive cells induced the expression of both endogenous C/EBPα and p21 and blocked the progression into S phase, as detected by a lack of incorporation of bromodeoxyuridine. The stimulation of endogenous ZTA protein expression either through treatment with tetradecanoyl phorbol acetate in D98/HR1 cells or through B-cell receptor cross-linking with anti-immunoglobulin G antibody in EBV-Akata cells also coincided with the induction of both C/EBPα and p21 and their mRNAs, as assayed by Northern blot, Western blot, and IFA experiments. Mechanistically, the ZTA protein proved to directly interact with C/EBPα by coimmunoprecipitation in EBV-Akata cells and with DNA-bound C/EBPα in electrophoretic mobility shift assay experiments, and the in vitro interaction domain encompassed the basic leucine zipper domain of ZTA. ZTA also specifically protected C/EBPα from degradation in a protein stability assay with a non-EBV-induced Akata cell proteasome extract. Furthermore, both C/EBPα and ZTA were found to specifically associate with the C/EBPα promoter in chromatin immunoprecipitation assays, but the interaction with ZTA appeared to be mediated by C/EBPα because it was abolished by clearing with anti-C/EBPα antibody. ZTA did not bind to or activate the C/EBPα promoter directly but cooperatively enhanced the positive autoregulation of the C/EBPα promoter by cotransfected C/EBPα in transient luciferase reporter gene assays with Vero and HeLa cells as well as with DG75 B lymphocytes. Similarly, ZTA alone had little effect on the p21 promoter in transient reporter gene assays, but in the presence of cotransfected C/EBPα, ZTA enhanced the level of C/EBPα activation. This effect proved to require a previously unrecognized region in the proximal p21 promoter that contains three high-affinity C/EBPα binding sites. Finally, in C/EBPα-deficient mouse embryonic fibroblasts (MEF), Ad-ZTA was unable to induce either p21 or G1 arrest, whereas it was able to induce both in wild-type MEF. Overall, we conclude that C/EBPα is essential for at least one pathway of ZTA-induced G1 arrest during EBV lytic-cycle DNA replication and that this process involves a physical piggyback interaction between ZTA and C/EBPα leading to greatly enhanced C/EBPα and p21 levels through both transcriptional and posttranslational mechanisms.

EBNA2 Is Required for Protection of Latently Epstein-Barr Virus-Infected B Cells against Specific Apoptotic Stimuli

ABSTRACT In addition to functioning as a transcriptional transactivator, Epstein-Barr virus EBNA2 interacts with Nur77 to protect against Nur77-mediated apoptosis. Estrogen-regulated EBNA2 in EREB2-5 cells was replaced by either EBNA2 or EBNA2 with a deletion of conserved region 4 (EBNA2ΔCR4). Both EBNA2-converted and EBNA2ΔCR4-converted EREB2-5 cells grew in the absence of estrogen and expressed LMP1. Treatment with tumor necrosis factor alpha did not induce apoptosis of EBNA2- or EBNA2ΔCR4-expressing cells, but EBNA2ΔCR4 cells were susceptible to etoposide and 5-fluorouracil, Nur77-mediated inducers of apoptosis. Thus, EBNA2 protects B cells against specific apoptotic agents against which LMP1 is not effective.

Somatic mutations to CSMD1 in colorectal adenocarcinomas

The short arm of chromosome 8 is frequently deleted in advanced human colorectal cancers, suggesting the presence of one or more tumor suppressor genes having a major role in tumor progression and metastasis. Comprehensive sequencing of over 18,000 genes in colon and breast cancers identified somatic mutations in CUB and Sushi Domains 1 (CSMD1) which is located on the p arm of chromosome 8. In this report, we describe a novel, robust, high-throughput gene mutation profiling strategy based on massively parallel picotiter plate pyrosequencing and have used this approach to identify additional somatic mutations to CSMD1 in early and late stage colorectal cancers. Using this strategy, we identified five nonsynonymous somatic mutations in CSMD1 among 28 colorectal cancers. Interestingly, these mutations occurred predominantly in advanced colorectal tumors, suggesting a role for CSMD1 in the development of late-stage metastatic disease.

Surgical management of temporal meningoencephaloceles, cerebrospinal fluid leaks, and intracranial hypertension: treatment paradigm and outcomes.

OBJECT Thinning of the tegmen tympani and mastoideum components of the temporal bone may predispose to the development of meningoencephaloceles and spontaneous CSF leaks. Surgical repair of these bony defects and associated meningoencephaloceles aids in the prevention of progression and meningitis. Intracranial hypertension may be a contributing factor to this disorder and must be fully evaluated and treated when present. The purpose of this study was to establish a treatment paradigm for tegmen defects and elucidate causative factors. METHODS The authors conducted a retrospective review of 23 patients undergoing a combined mastoidectomy and middle cranial fossa craniotomy for the treatment of a tegmen defect. RESULTS The average body mass index (BMI) among all patients was 33.2 ± 7.2 kg/m(2). Sixty-five percent of the patients (15 of 23) were obese (BMI > 30 kg/m(2)). Preoperative intracranial pressures (ICPs) averaged 21.8 ± 6.0 cm H(2)O, with 10 patients (43%) demonstrating an ICP > 20 cm H(2)O. Twenty-two patients (96%) had associated encephaloceles. Five patients underwent postoperative ventriculoperitoneal shunting. Twenty-two CSF leaks (96%) were successfully repaired at the first attempt (average follow-up 10.4 months). CONCLUSIONS Among all etiologies for CSF leaks, those occurring spontaneously have the highest rate of recurrence. The surgical treatment of temporal bone defects, as well as the recognition and treatment of accompanying intracranial hypertension, provides the greatest success rate in preventing recurrence. After tegmen dehiscence repair, ventriculoperitoneal shunting should be considered for patients with any combination of the following high-risk factors for recurrence: spontaneous CSF leak not caused by another predisposing condition (that is, trauma, chronic infections, or prior surgery), high-volume leaks, CSF opening pressure > 20 cm H(2)O, BMI > 30 kg/m(2), preoperative imaging demonstrating additional cranial base cortical defects (that is, contralateral tegmen or anterior cranial base) and/or an empty sella turcica, and any history of an event that leads to inflammation of the arachnoid granulations and impairment of CSF absorption (that is, meningitis, intracranial hemorrhage, significant closed head injury, and so forth).

Combination Immunotherapy for Tumors via Sequential Intratumoral Injections of Oncolytic Herpes Simplex Virus 1 and Immature Dendritic Cells

Purpose: Oncolytic herpes simplex virus 1 (oHSV) vectors treat tumors in preclinical models and have been used safely in phase I clinical trials for patients with cancer. Infection of tumors with oHSV also induces specific antitumor immunity. We investigated whether this immunotherapeutic effect is enhanced by combining oHSV infection with intratumoral administration of immature myeloid dendritic cells (iDC). Experimental Design: Subcutaneous neuroblastoma tumors were established in syngeneic immunocompetent mice and sequentially treated with oHSV(G47Δ) and intratumoral iDCs. Tumor volumes and survival were monitored. Antitumor immune responses were evaluated by immunohistochemistry, IFN-γ ELISPOT, and CTL assay. Treatment was also evaluated in immunodeficient NOD-SCID mice. Results: We observed significant reductions in tumor volumes in mice receiving G47Δ + iDCs compared with those treated with G47Δ or iDC monotherapy. Survival was prolonged, with ∼90% of tumors eradicated in the combination group. Combination therapy led to enhancement of antitumor immune responses, confirmed by increases in IFN-γ expression by splenocytes harvested from G47Δ + iDC-treated mice. Splenocytes harvested from G47Δ + iDC-treated mice were effective against neuroblastoma tumor cells in a CTL assay. Immunohistochemistry of combination-treated tumors revealed robust lymphocytic infiltrates. Adding iDCs to G47Δ infection in tumors in NOD-SCID mice did not reduce the rate of growth. Substitution of lipopolysaccharide-matured dendritic cells abrogated the enhanced tumor volume reduction seen with combination therapy with iDCs. Conclusions: Combination treatment of murine tumors with oHSV and iDCs reduces the volume of established tumors and prolongs survival via enhancement of antitumor immunity.

Limitations of diffusion-weighted imaging in the diagnosis of postoperative infections.

OBJECTIVEDiffusion-weighted imaging (DWI) has assumed a rapidly emerging role in the diagnosis of intracranial infection; however, its usefulness in the recognition of postoperative infection has been largely unexplored. We sought to determine the ability of DWI to accurately detect a broad range of postneurosurgical infections as well as identify individual factors that may limit its applicability. METHODSWe retrospectively identified 65 patients who had undergone surgery for the confirmed diagnosis of infection between August 2001 and February 2005 and had received preoperative magnetic resonance imaging evaluation, including DWI. Fifty patients developed infections after a primary neurosurgical procedure (“postoperative” infections), whereas 15 infections occurred without antecedent intervention (“spontaneous” infections). Logistic regression analysis was used to identify factors associated with false-negative DWI findings. Additionally, we investigated the false-positive rate of DWI by retrospectively reviewing a series of 30 consecutive patients who underwent craniotomy and received postoperative DWI. RESULTSSpontaneously developing cranial infections exhibited evidence of restricted diffusion in 14 out of 15 (93%) patients; however, infections that occurred postoperatively were associated with a significant false-negative rate using DWI (36%; P < 0.01). Within the subset of patients with postoperative infection, location of infection significantly correlated with the DWI false-negative rate. Infections located extradurally were less likely to demonstrate restricted diffusion compared with those located primarily within the subdural or intraparenchymal spaces. Additionally, false-positive DWI findings were observed in 11 of the 30 patients (37%) who had DWI obtained postoperatively in the absence of infection. CONCLUSIONUtilization of DWI for the diagnosis of infection after primary neurosurgical intervention is associated with an elevated false-negative rate. The absence of restricted diffusion is not sufficient to exclude the presence of pyogenic postcraniotomy infection and should not be used as the principle determinant of patient management in this clinical setting.

The effects of anatomic variations on stereotactic laser amygdalohippocampectomy and a proposed protocol for trajectory planning.

BACKGROUND: Stereotactic laser amygdalohippocampectomy (SLAH) is a promising minimally invasive alternative for mesial temporal lobe epilepsy. As seizure outcome has been associated with the extent of amygdalar and hippocampal ablation, it is important to select a safe trajectory optimizing involvement of both structures; however, variations in temporal anatomy significantly affect the overall complexity of planning. OBJECTIVE: To quantify anatomic variables of SLAH and facilitate stereotactic planning by developing a protocol for optimally targeting the amygdalohippocampal complex (AHC). METHODS: We performed a retrospective analysis of 19 SLAHs. Anatomic measurements from preoperative magnetic resonance imaging and laser trajectory measurements from coregistered postoperative magnetic resonance imaging were taken in 11 patients. Simple linear regression analysis was performed to identify significant predictor variables determining ablation extent. Based on these data, a protocol for optimal trajectory planning was developed and subsequently implemented in 8 patients. RESULTS: The medial angle of the laser trajectory correlated with the medial angle of the AHC. The length of amygdalar cannulation was predictive of its ablation volume. All trajectories passed through a posteroinferior corridor formed by the lateral ventricle superiorly and collateral sulcus inferiorly. Our protocol facilitated planning and increased the volume of AHC ablation. CONCLUSION: The medial AHC angle dictates the medial trajectory angle and a path from the posteroinferior corridor through the hippocampus and the center of the amygdala dictates the caudal angle. These observations led to a protocol for long-axis AHC cannulation that maintains an extraventricular trajectory to minimize hemorrhage risk and targets the center of the amygdala to optimize ablation volumes. ABBREVIATIONS: AHC, amygdalohippocampal complex MTLE, mesial temporal lobe epilepsy SLAH, stereotactic laser amygdalohippocampectomy

Airway Management and Perioperative Concerns in Acromegaly Patients Undergoing Endoscopic Transsphenoidal Surgery for Pituitary Tumors

Objectives Patients with acromegaly present unique challenges to cranial base surgery and anesthesia teams in the perioperative period, especially with regard to airway management. Abnormal airway anatomy may result from soft tissue hypertrophy and bony alterations. Additional perioperative challenges relate to the management of medical comorbidities. We aim to review perioperative airway concerns in acromegalic patients for the skull base surgeon in order to reduce preventable perioperative complications. Study Design Case series with chart review. Setting Tertiary care academic institution. Subjects Thirty-two acromegaly patients undergoing endoscopic transsphenoidal pituitary surgery. Results Videoscopic direct laryngoscopy intubation was required in 7 of 32 patients (21.9%) and fiberoptic intubation in 4 of 32 patients (12.5%). Overall failure rate for first intubation technique used was 12.5% (4/32). Cardiovascular comorbidities (hypertension and conduction abnormalities predominated) were present in 16 of 32 patients (50%), and obstructive sleep apnea, or other respiratory conditions, existed in 12 of 32 patients (37.5%). Conclusions Acromegaly patients present a particular challenge to the endoscopic skull base surgeon. Despite preoperative anesthesia and otolaryngology evaluation, many of these patients will experience an unanticipated airway challenge during intubation. Preoperative preparation and perioperative awareness of anatomic and physiologic abnormalities of acromegalic patients is essential for successful endoscopic surgery in this unique population.