Andrew Sloan, Ph.D. - US grants

Deficits in fine-grained auditory pitch perception have been implicated in language learning disorders, certain forms of dyslexia, and more severe neurological conditions. Fine-grained frequency discrimination is believed to be a function of the primary auditory cortex (A1), but the mechanism responsible is still unclear. In the time-course of individual A1 neural responses to iso-frequency tones, relatively non-frequency- selective phasic excitation gives way to more narrowly selective tonic excitation that is sustained throughout the duration of the tone. Tonic responses in A1 may code for fine-grained spectral perception. This study will investigate the role and functionality of tonic responses with respect to pitch perception of pure tones in awake rats using a combination of neural recording, microstimulation, and behavioral techniques. Neural recordings from multichannel electrode arrays will be used to fully characterize tonic neural responses in A1 to iso-frequency tones. A simultaneous neural recording and microstimulation paradigm will be used to examine the contribution of intracortical modulation to processing of iso-frequency stimuli. Finally, microstimulation will be paired with a behavioral paradigm to investigate the perceived pitch of sensation elicited by artificial activation of A1 in a pattern mimicking natural iso-frequency responses. The combination of these techniques will provide a uniquely detailed view of the mechanisms and functionality of tonic responses in A1 with respect to fine-grained pitch perception. This study will help in understanding how the brain breaks down sound information into sound frequency. The knowledge gained from this study could be useful in developing better treatments for childhood language learning disorders or for language disorders stemming from strokes and other brain damage.

Project Summary Glioblastoma (GBM), the most common primary brain tumor, has median patient survival of little more than one year. Surgical resection of all enhancing tumor prolongs survival, yet successful resection is challenging because GBM tumors diffusely infiltrate the brain, which results in >90% local recurrence. Development of a next-generation fluorescent molecular imaging agent to help surgeons differentiate cancerous from normal tissue intraoperatively will help reduce the rate of local recurrence and improve patient survival outcomes. No such agent for guided surgical resection of GBM currently has US FDA approval. An extracellular fragment of the cell surface adhesion molecule PTPµ is a unique imaging biomarker of the tumor microenvironment. The PTPµ fragment arises from proteolytic cleavage of the receptor protein tyrosine phosphatase (PTPµ), a proteolysis seen in multiple tumor types including GBM. An agent that binds to this PTPµ fragment, SBK2, recognizes human GBM tumors. Systemic delivery of the SBK2 agent results in binding to tumor cells within minutes in orthotopic xenografts in rodents, and can label virtually all the dispersing brain tumor cells several millimeters away from the main tumor mass in mouse brains in vivo. This proposal aims to translate this highly-selective fluorescent SBK2 agent for pre-surgical systemic delivery to cancerous GBM tissue and render it visible in real-time by current surgical microscopy adapted with standard fluorescent filters. The fluorescent SBK2 molecular imaging agent would enhance the efficacy and efficiency of GBM surgery by allowing real-time decisions regarding tumor borders. In this Academic-Industrial Partnership, a team with expertise in molecular biology, neurosurgery and molecular imaging regulatory approval, will work together to translate SBK2 from a preclinical to a clinical agent. To achieve this goal, the team will need to perform cGMP synthesis and toxicology studies before obtaining FDA regulatory approval for investigational use. Upon FDA approval, the cGMP SBK2 agents will be tested in a Phase 0 eIND imaging trial during surgical resection of GBM for determination of safety profile and imaging efficacy. We expect that this translational plan for the SBK2 imaging agent will yield a new clinical imaging capability for surgeon end users to reduce tumor burden and prolong patient survival.

ABSTRACT Glioblastoma (GBM) remains a devastating diagnosis with a median survival of 12-14 months. Although radio-chemotherapy improves outcomes, benefit is primarily in the subset of patients bearing tumors with low expression of the MGMT gene, which comprises less than 45% of all patients. Initial studies demonstrated that the MGMT inhibitor, O-6benzylguanine (BG) depletes AGT and sensitized GBM to TMZ. However, toxicity was high and this was abandoned. To reduce the hematopoietic toxicity of adding BG to chemoradiation, hematopoietic progenitor cells (HPCs) were genetically engineered to express an MGMT mutant (P140K) first identified by PI Gerson, which has low affinity to BG, but removes O-6 methyl adducts with similar efficacy as AGT. This innovative approach protects the hematopoietic progenitor cells (HPC) from BG/TMZ treatment in both in vitro and in vivo preclinical studies and early clinical trials suggest clinical efficacy. Although our original hypothesis was that this treatment strategy would improve survival by improving tolerance to dose-escalated chemotherapy, the emerging recognition of the importance of the immune system in controlling cancer has raised additional important questions not anticipated during initial trial design. Is the observed improved survival due simply to chemoprotecton of the hematopoietic compartment resulting in increased tolerance to cytotoxic chemotherapy as originally hypothesized? Alternatively, does this strategy alter the balance between anti-tumor immune immunosuppressive subpopulations? Which immune subpopulations might be rendered chemo-resistant by transduced P140K-MGMT, and does this contribute to treatment response or resistance? To address these questions, we propose a collaborative project between the Case Comprehensive Cancer Center (CCCC) and the NIH Clinical Center (NIHCC) to expand an ongoing active IND phase I trial and incorporate additional analysis of biospecimens from patients with newly diagnosed GBM. The improved patient access, clinical expertise, and unique, cutting edge immunological monitoring capabilities at the NCICC will speed up accrual, help assess the feasibility of this complex trial, and help quantify the changes in quantity and function of various immune subpopulations, which will markedly enhance the impact of this innovative treatment approach. The overall goal of this proposal is to define the longitudinal changes in both the quantity and function of immune cell subtypes in blood, marrow and the tumor microenvironment, while documenting treatment tolerance to chemotherapy, the persistence of the transduced gene, integration site safety, and changes in tumor mutation profile after treatment. Our hypothesis is that GBM patients treated with MGMT-P140K transduced HPC followed by BG/TMZ, will: A). tolerate treatment better, maintain improved hematopoietic and immune function, and demonstrate increased progression-free and overall survival; and B). demonstrate altered proportions of effector and suppressor immune subpopulations before and after treatment. The collaboration with NIHCC and intramural investigators is essential to these to these ongoing studies given the complementary expertise in genetically engineered HPCs at CCCC; the increased access for patient accrual and the unique immunological monitoring and clinical expertise at NIHCC. This project also synergizes with the ongoing collaboration between the PIs.

One major clinical challenge for diagnosis of recurrent glioblastoma (GBM) is assessment of response to treatment. While standard chemo-radiotherapy improves survival, it also complicates assessment of recurrence. Indeed, radiation effects which present as enhancing masses indistinguishable from recurrent tumor occur in nearly 30% of GBM patients. Myeloid-derived suppressor cells (MDSC) are important immunosuppressive cells that appear in and around solid tumors, including GBM, as well as in the peripheral blood of many cancer patients. Recruitment to the local tumor microenvironment is thought to mediate active suppression of the host immune response by the tumor. These observations make MDSCs potentially useful for detecting recurrence of GBM and monitoring response to therapy in a noninvasive manner, while avoiding the inconvenience, cost, and risk of more expensive Magnetic Resonance Imaging (MRI) and/or invasive biopsy. Given the invasiveness, risk and cost of surgical intervention and the radiological challenges involved, a minimally invasive ?liquid biopsy?, with high sensitivity and specificity represents a transformative technology. Our preliminary data suggests that a MDSC based biomarker known as DVI can differentiate patients with recurrent GBM from other etiologies of enhancing masses including radiation necrosis, scar, and pseudoprogression using only peripheral blood. To further assess the sensitivity and specificity of this test, we propose the following aims: 1.) Validate the sensitivity and specificity of DVI for distinguishing true recurrence of GBM (rGBM) from other etiologies of MRI imaging enhancement; 2.) Determine the performance characteristics of DVI relative to conventional imaging at differentiating true recurrence (rGBM) from treatment effect in patients under treatment; and 3) Identify potential mechanism(s) hereby VNN2 levels are modulated by GBM. The ability to perform a clinically safe and easy test to quantify the DVI will advance the current diagnostic criteria for distinguishing RN from GBM tumor recurrence and could be easily adapted and implemented by clinical flow cytometry laboratories nationwide. The ability to objectively assess response to treatment using a liquid biopsy will be transformative and lead to both better treatment and improving the value of care by avoiding risky and expensive surgical procedures.