David S. Baskin

Combinational targeting offsets antigen escape and enhances effector functions of adoptively transferred T cells in glioblastoma.

Preclinical and early clinical studies have demonstrated that chimeric antigen receptor (CAR)-redirected T cells are highly promising in cancer therapy. We observed that targeting HER2 in a glioblastoma (GBM) cell line results in the emergence of HER2-null tumor cells that maintain the expression of nontargeted tumor-associated antigens. Combinational targeting of these tumor-associated antigens could therefore offset this escape mechanism. We studied the single-cell coexpression patterns of HER2, IL-13Rα2, and EphA2 in primary GBM samples using multicolor flow cytometry and immunofluorescence, and applied a binomial routine to the permutations of antigen expression and the related odds of complete tumor elimination. This mathematical model demonstrated that cotargeting HER2 and IL-13Rα2 could maximally expand the therapeutic reach of the T cell product in all primary tumors studied. Targeting a third antigen did not predict an added advantage in the tumor cohort studied. We therefore generated bispecific T cell products from healthy donors and from GBM patients by pooling T cells individually expressing HER2 and IL-13Rα2-specific CARs and by making individual T cells to coexpress both molecules. Both HER2/IL-13Rα2-bispecific T cell products offset antigen escape, producing enhanced effector activity in vitro immunoassays (against autologous glioma cells in the case of GBM patient products) and in an orthotopic xenogeneic murine model. Further, T cells coexpressing HER2 and IL-13Rα2-CARs exhibited accentuated yet antigen-dependent downstream signaling and a particularly enhanced antitumor activity.Preclinical and early clinical studies have demonstrated that chimeric antigen receptor (CAR)-redirected T cells are highly promising in cancer therapy. We observed that targeting HER2 in a glioblastoma (GBM) cell line results in the emergence of HER2-null tumor cells that maintain the expression of nontargeted tumor-associated antigens. Combinational targeting of these tumor-associated antigens could therefore offset this escape mechanism. We studied the single-cell coexpression patterns of HER2, IL-13Rα2, and EphA2 in primary GBM samples using multicolor flow cytometry and immunofluorescence, and applied a binomial routine to the permutations of antigen expression and the related odds of complete tumor elimination. This mathematical model demonstrated that cotargeting HER2 and IL-13Rα2 could maximally expand the therapeutic reach of the T cell product in all primary tumors studied. Targeting a third antigen did not predict an added advantage in the tumor cohort studied. We therefore generated bispecific T cell products from healthy donors and from GBM patients by pooling T cells individually expressing HER2 and IL-13Rα2-specific CARs and by making individual T cells to coexpress both molecules. Both HER2/IL-13Rα2-bispecific T cell products offset antigen escape, producing enhanced effector activity in vitro immunoassays (against autologous glioma cells in the case of GBM patient products) and in an orthotopic xenogeneic murine model. Further, T cells coexpressing HER2 and IL-13Rα2-CARs exhibited accentuated yet antigen-dependent downstream signaling and a particularly enhanced antitumor activity.

HER2-Specific Chimeric Antigen Receptor–Modified Virus-Specific T Cells for Progressive Glioblastoma: A Phase 1 Dose-Escalation Trial

Importance Glioblastoma is an incurable tumor, and the therapeutic options for patients are limited. Objective To determine whether the systemic administration of HER2-specific chimeric antigen receptor (CAR)–modified virus-specific T cells (VSTs) is safe and whether these cells have antiglioblastoma activity. Design, Setting, and Participants In this open-label phase 1 dose-escalation study conducted at Baylor College of Medicine, Houston Methodist Hospital, and Texas Children’s Hospital, patients with progressive HER2-positive glioblastoma were enrolled between July 25, 2011, and April 21, 2014. The duration of follow-up was 10 weeks to 29 months (median, 8 months). Interventions Monotherapy with autologous VSTs specific for cytomegalovirus, Epstein-Barr virus, or adenovirus and genetically modified to express HER2-CARs with a CD28.&zgr;-signaling endodomain (HER2-CAR VSTs). Main Outcomes and Measures Primary end points were feasibility and safety. The key secondary end points were T-cell persistence and their antiglioblastoma activity. Results A total of 17 patients (8 females and 9 males; 10 patients ≥18 years [median age, 60 years; range, 30-69 years] and 7 patients <18 years [median age, 14 years; range, 10-17 years]) with progressive HER2-positive glioblastoma received 1 or more infusions of autologous HER2-CAR VSTs (1u2009×u2009106/m2 to 1u2009×u2009108/m2) without prior lymphodepletion. Infusions were well tolerated, with no dose-limiting toxic effects. HER2-CAR VSTs were detected in the peripheral blood for up to 12 months after the infusion by quantitative real-time polymerase chain reaction. Of 16 evaluable patients (9 adults and 7 children), 1 had a partial response for more than 9 months, 7 had stable disease for 8 weeks to 29 months, and 8 progressed after T-cell infusion. Three patients with stable disease are alive without any evidence of progression during 24 to 29 months of follow-up. For the entire study cohort, median overall survival was 11.1 months (95% CI, 4.1-27.2 months) from the first T-cell infusion and 24.5 months (95% CI, 17.2-34.6 months) from diagnosis. Conclusions and Relevance Infusion of autologous HER2-CAR VSTs is safe and can be associated with clinical benefit for patients with progressive glioblastoma. Further evaluation of HER2-CAR VSTs in a phase 2b study is warranted as a single agent or in combination with other immunomodulatory approaches for glioblastoma.

Autologous HER2 CMV bispecific CAR T cells are safe and demonstrate clinical benefit for glioblastoma in a Phase I trial.

Glioblastoma (GBM) remains incurable with current standard-of-care therapies. Adoptive T cell transfer holds the promise to improve outcomes for GBM patients. We report on the results of the Phase I clinical study, NCT01109095, administering autologous CMV.pp65 T cells grafted with a second generation HER2 chimeric antigen receptor (CAR) with a CD28.zeta signaling domain to patients with progressive GBM. n nSeventeen CMV-seropositive patients with radiologically progressive HER2+ GBM were enrolled. The median age was 49 years (range 11 to 71; 6 children; 11 adults). Children enrolled had significantly larger tumor volumes at infusion. A cell product was successfully generated for all patients from a peripheral blood draw (maximum 90mL). A median of 67% (range: 46-82) of T cells expressed the HER2 CAR, and exhibited a median 985.5 (range 390 to 1292) CMV.pp65 reactivity in an IFN-γ Elispot assay (SFC/105 T cells). Infusions of 1x106/m2-1x108/m2 were well tolerated without severe adverse events or cytokine release syndrome. HER2 CMV T cells were detected in the peripheral blood for up to 12 weeks post infusion, as judged by rtPCR of a CAR-specific amplicon. Out of 16 evaluable patients, 8 had progressive disease, 8/16 patients had objective responses: 1 patient had a partial response with a ~62% reduction in tumor volume lasting 8 months, 7 patients had stable disease for more than 6 weeks (of these 5 were durable >10 weeks) and 3 subjects are currently with a follow up 24 to >30 months, after T cell infusion. The median survival was 11.6 months from infusion and 24.8 months from diagnosis. The median survival for adults was 30 months from diagnosis. n nWe conclude that systemically administered HER2 CAR CMV bispecific T cells are safe. A durable clinical benefit was observed in ~38% of patients.

mRNA stability alterations mediated by HuR are necessary to sustain the fast growth of glioma cells

Regulation of mRNA decay is an important mechanism controlling gene expression. Steady state levels of mRNAs can be markedly altered by changes in the decay rate. The control of mRNA stability depends on sequences in the transcript itself and on RNA-binding proteins that dynamically bind to these sequences. A well characterized sequence motif, which has been shown to be present in many short-lived mRNAs, is the de-stabilizing adenylate/uridylate-rich element (ARE) located at the 3′ untranslated region (3′UTR) of mRNAs. HuR is an RNA-binding protein, which binds to AREs and in doing so, increases the half-life and steady state levels of the corresponding mRNA. Using tissue microarray technology, we found that HuR is over-expressed in human gliomas. We also found that there is a change in HuR localization from being solely in the nucleus to being expressed at high levels in the cytosol. Moreover, a positive correlation was found between total HuR levels, cytosolic localization and tumor grade. We also studied the decay rate of several HuR target mRNAs and found that these mRNAs have a slower rate of decay in glioma cell lines than in astrocytes. Finally, we have been able to decrease both the stability and steady state level of these transcripts in glioma cells using an RNA decoy. More importantly, the decoy transfected cells and cells exposed to a HuR inhibitor have reduced cell growth. In addition, pharmacological inhibition of HuR also resulted in glioma cell growth inhibition. In conclusion, our data suggest that post-transcriptional control abnormalities mediated by HuR are necessary to sustain the rapid growth of this devastating type of cancer.

Neuroendoscopic Resection of Intraventricular Tumors: A Systematic Outcomes Analysis

Introduction. Though traditional microsurgical techniques are the gold standard for intraventricular tumor resection, the morbidity and invasiveness of microsurgical approaches to the ventricular system have galvanized interest in neuroendoscopic resection. We present a systematic review of the literature to provide a better understanding of the virtues and limitations of endoscopic tumor resection. Materials and Methods. 40 articles describing 668 endoscopic tumor resections were selected from the Pubmed database and reviewed. Results. Complete or near-complete resection was achieved in 75.0% of the patients. 9.9% of resected tumors recurred during the follow-up period, and procedure-related complications occurred in 20.8% of the procedures. Tumor size ≤ 2cm (P = 0.00146), the presence of a cystic tumor component (P < 0.0001), and the use of navigation or stereotactic tools during the procedure (P = 0.0003) were each independently associated with a greater likelihood of complete or near-complete tumor resection. Additionally, the complication rate was significantly higher for noncystic masses than for cystic ones (P < 0.0001). Discussion. Neuroendoscopic outcomes for intraventricular tumor resection are significantly better when performed on small, cystic tumors and when neural navigation or stereotaxy is used. Conclusion. Neuroendoscopic resection appears to be a safe and reliable treatment option for patients with intraventricular tumors of a particular morphology.

Stereotactic radiosurgery for trigeminal neuralgia utilizing the BrainLAB Novalis system.

Stereotactic radiosurgery (SRS) is one of the least invasive treatments for trigeminal neuralgia (TN). To date, most reports have been about Cobalt-based treatments (i.e., Gamma Knife) with limited data on image-guided stereotactic linear accelerator treatments. We describe our initial experience of using BrainLAB Novalis stereotactic system for the radiosurgical treatment of TN. A total of 20 patients were treated between July 2004 and February 2007. Each SRS procedure was performed using the BrainLAB Novalis System. Thin cuts MRI images of 1.5 mm thickness were acquired and fused with the simulation CT of each patient. Majority of the patients received a maximum dose of 90 Gy. The median brainstem dose to 1.0 cc and 0.1 cc was 2.3 Gy and 13.5 Gy, respectively. In addition, specially acquired three-dimensional fast imaging sequence employing steady-state acquisition (FIESTA) MRI was utilized to improve target delineation of the trigeminal proximal nerve root entry zone. Barrow Neurological Index (BNI) pain scale for TN was used for assessing treatment outcome. At a median follow-up time of 14.2 months, 19 patients (95%) reported at least some improvement in pain. Eight (40%) patients were completely pain-free and stopped all medications (BNI Grade I) while another 2 (10%) patients also stopped medications but reported occasional pain (BNI Grade II). Another 2 (10%) patients reported no pain and 7 (35%) patients only occasional pain while continuing medications, BNI Grade IIIA and IIIB, respectively. Median time to pain control was 8.5 days (range: 1–70 days). No patient reported severe pain, worsening pain or any pain not controlled on their previously taken medication. Intermittent or persistent facial numbness following treatments occurred in 35% of patients. No other complications were reported. Stereotactic radiosurgery using the BrainLAB Novalis system is a safe and effective treatment for TN. This information is important as more centers are obtaining image-guided stereotactic-based linear accelerators capable of performing radiosurgery.

First results on survival from a large Phase 3 clinical trial of an autologous dendritic cell vaccine in newly diagnosed glioblastoma

BackgroundStandard therapy for glioblastoma includes surgery, radiotherapy, and temozolomide. This Phase 3 trial evaluates the addition of an autologous tumor lysate-pulsed dendritic cell vaccine (DCVax®-L) to standard therapy for newly diagnosed glioblastoma.MethodsAfter surgery and chemoradiotherapy, patients were randomized (2:1) to receive temozolomide plus DCVax-L (nu2009=u2009232) or temozolomide and placebo (nu2009=u200999). Following recurrence, all patients were allowed to receive DCVax-L, without unblinding. The primary endpoint was progression free survival (PFS); thexa0secondary endpoint was overall survival (OS).ResultsFor the intent-to-treat (ITT) population (nu2009=u2009331), median OS (mOS) was 23.1xa0months from surgery. Because of the cross-over trial design, nearly 90% of the ITT population received DCVax-L. For patients with methylated MGMT (nu2009=u2009131), mOS was 34.7xa0months from surgery, with a 3-year survival of 46.4%. As of this analysis, 223 patients areu2009≥u200930xa0months past their surgery date; 67 of these (30.0%) have livedu2009≥u200930xa0months and have a Kaplan-Meier (KM)-derivedxa0mOS of 46.5xa0months. 182 patients areu2009≥u200936xa0months past surgery; 44 of these (24.2%) have livedu2009≥u200936xa0months and have a KM-derived mOS of 88.2xa0months. A population of extended survivors (nu2009=u2009100) with mOS of 40.5xa0months, not explained by known prognostic factors, will be analyzed further. Only 2.1% of ITT patients (nu2009=u20097) had a grade 3 or 4 adverse event that was deemed at least possibly related to the vaccine. Overall adverse events with DCVax were comparable to standard therapy alone.ConclusionsAddition of DCVax-L to standard therapy is feasible and safe in glioblastoma patients, and may extend survival.Trial registration Funded by Northwest Biotherapeutics; Clinicaltrials.gov number: NCT00045968; https://clinicaltrials.gov/ct2/show/NCT00045968?term=NCT00045968&rank=1; initially registered 19 September 2002

Quantification and calibration of images in fluorescence microscopy.

Fluorescence microscopy is a method widely used in life sciences to image biological processes in living and fixed cells or in fixed tissues. Quantification and calibration of images in fluorescence microscopy is notoriously difficult. We have developed a new methodology to prepare tissue phantoms that contain known amounts of (i) fluorophore, (ii) DNA, (iii) proteins, and (iv) DNA oligonucleotide standards. The basis of the phantoms is the ability of gelatin to act as a matrix for the conjugation of fluorophores as either a free-flowing liquid or a gelatinous solid depending on temperature (> or = 40 and < or = 4 degrees C).

Sellar Floor Reconstruction with the Medpor Implant Versus Autologous Bone After Transnasal Transsphenoidal Surgery: Outcome in 200 Consecutive Patients.

OBJECTIVEnThe Medpor porous polyethylene implant provides benefits to perform sellar floor reconstruction when indicated. This material has been used for cranioplasty and reconstruction of skull base defects and facial fractures. We present the most extensive use of this implant for sellar floor reconstruction and document the safety and benefits provided by this unique implant.nnnMETHODSnThe medical charts for 200 consecutive patients undergoing endonasal transsphenoidal surgery from April 2008 through December 2011 were reviewed. Material used for sellar floor reconstruction, pathologic diagnosis, immediate inpatient complications, and long-term complications were documented and analyzed. Outpatient follow-up was documented for a minimum of 1-year duration, extending in some patients up to 5 years.nnnRESULTSnOf the 200 consecutive patients, 136 received sellar floor cranioplasty using the Medpor implant. Postoperative complications included 6 complaints of sinus irritation or drainage, 1 postoperative cerebrospinal fluid leak requiring operative re-exploration, 1 event of tension pneumocephalus requiring operative decompression, 1 case of aseptic meningitis, 1 subdural hematoma, and 1 case of epistaxis. The incidence of these complications did not differ from the autologous nasal bone group in a statistically significant manner.nnnCONCLUSIONSnSellar floor reconstruction remains an important part of transsphenoidal surgery to prevent postoperative complications. Various autologous and synthetic options are available to reconstruct the sellar floor, and the Medpor implant is a safe and effective option. The complication rate after surgery is equivalent to or less frequent than other methods of reconstruction and the implant is readily incorporated into host tissue after implantation, minimizing infectious risk.

Successful Treatment of Intracranial Glioblastoma Xenografts With a Monoamine Oxidase B-Activated Pro-Drug

The last major advance in the treatment of glioblastoma multiforme (GBM) was the introduction of temozolomide in 1999. Treatment with temozolomide following surgical debulking extends survival rate compared to radiotherapy and debulking alone. However, virtually all glioblastoma patients experience disease progression within 7 to 10 months. Although many salvage treatments, including bevacizumab, rechallenge with temozolomide, and other alkylating agents, have been evaluated, none of these clearly improves survival. Monoamine oxidase B (MAOB) is highly expressed in glioblastoma cell mitochondria, and mitochondrial function is intimately tied to treatment-resistant glioblastoma progression. These glioblastoma properties provide a strong rationale for pursuing a MAOB-selective pro-drug treatment approach that, upon drug activation, targets glioblastoma mitochondria, especially mitochondrial DNA. MP-MUS is the lead compound in a family of pro-drugs designed to treat GBM that is converted into the mature, mitochondria-targeting drug, P+-MUS, by MAOB. We show that MP-MUS can successfully kill primary gliomas in vitro and in vivo mouse xenograft models.