Bob Carter, MD - US grants

Core B- Bob S. Carter PI Project Summary/ Abstract The non-invasive diagnosis of human glioma could improve the quality of life of patients with this disease by allowing patients to be directly diagnosed or stratified for treatment without the need of an invasive brain biopsy. In Core B, the investigators propose to develop a novel clinical sample core to study extracellular RNA biomarkers for the diagnosis of human glioma tumors. In collaboration with the P01 project investigators, we will study EV proteins, nucleic acids, and physical characteristics as potential biomarkers for glioma diagnosis or progression. We will work with a consortium of institutions to provide rapid prospective collection of tumor tissue, CSF, and blood from glioma patients. In additon, the Core will provide biostatistical support to all Projects.

Project? ?Summary Extracellular vesicles (EVs) and cell free DNA have emerged as a promising surrogate for the tissue biopsy, potentially enabling non-invasive, real-time cancer monitoring. Most cancer cells release high levels of EVs and cell-free DNA into circulation that carry molecular constituents of the parent tumor. Gliomas are among the most lethal of cancers and despite improved survival associated with surgery, utilizing intraoperative MR imaging guidance, as well as the postoperative use of the alkylating agent temozolomide, in combination with conformal radiation therapy and the angiogenesis inhibitor bevacizumab, glial tumors remain uniquely morbid, costly, and incurable. Brain tumor biopsy, necessary for clinical information, is itself associated with substantial morbidity and mortality. Consequently, there is a crucial need for more effective ways to determine the mutational landscape of early-stage tumors, via minimally invasive platforms, allowing for effective follow-up and biomarker-based care. The main goal of this proposal is to optimize liquid biopsy assays for the IDH1.R132H and EGFRvIII mutations in partnership with industry. This alliance includes Exosome Diagnostics, an industry leader in EV-based cancer diagnostics, offering ready capacity to develop and manufacture in-vitro diagnostic assays, and the Department of Neurosurgery at Massachusetts General Hospital, a pioneer in developing liquid biopsy signatures for brain cancer. These teams bring in their multidisciplinary expertise, innovative technologies and complementary resources to carry? ?out? ?the? ?proposed? ?work.

Extracellular vesicles (EVs) have emerged as a promising surrogate for tissue biopsy, potentially enabling non-invasive, real-time cancer monitoring. Most cancer cells release large numbers of EVs into circulation that carry molecular constituents reflective of the heterogeneity of the parent tumor. This project is designed to optimize a liquid biopsy to diagnose malignant glioma tumors. Currently, such tumors are diagnosed through a brain tissue biopsy which involves considerable risk for patients and doesn?t allow for longitudinal follow up of clinical care. Current EV isolation and characterization methods yield inconsistent results and render data reproducibility challenging, often leading to unpredictable conclusions. The ?goals? of this project are to i) address variability among the different EV isolation methods and platforms currently available, and to ii) pinpoint to the ?best? method to validate candidate biomarkers for glioma diagnosis. Our exceptional investigative team brings together experts in malignant glioma treatment, the field of nano-engineering, vesicular research, assay development and droplet digital PCR technology to optimize the necessary elements for the development of a blood-based assay capable of moving towards clinical settings. Through a simple blood test, clinicians will be able to diagnose, stratify and monitor a tumor without the need for tissue biopsy. Our strategic partnership with ?Exosome Diagnostics?, an industry leader in EV-based cancer diagnostics, offers us venues allowing for the translation of our findings, coupled with access to clinical grade kits, platforms and study design. The D? epartment of Neurosurgery? and the ?Center for Systems Biology? at the Massachusetts General Hospital comprise multidisciplinary clinical expertise, innovative technologies and complementary resources to carry out the following translational projects: ?First,? based on our prior kit comparison work, we have picked two top EV isolation kits and enrichment platforms to test in a series of well controlled, reference standards to determine an optimal EV isolation method. ?Second,? we will test whether EV gene signatures can be used as biomarkers for cancer detection as well as tracking recurrence. By following quality control on device design and sample processing, accruing well-annotated patient and control samples, and performing multi site testing, we will ?ensure assay reliability and reproducibility? to deliver clinically translatable EV diagnostics. Fourteen genes were selected through literature data mining based on the putative evidence that they can distinguish gliomas from controls. ?Finally,? a gene?s signature with the highest sensitivity and specificity will be validated in a large cohort of patient samples. The technical and scientific outcomes of this research could have a significant ?translational impact in gliomas?, establishing a robust, highly specific assay to guide treatment decision and assess tumor recurrence.

Malignant gliomas (MG) are the most aggressive malignancy of the central nervous system with the mean survival time of 15 months. Fast progression and high heterogeneity of MG mandates frequent imaging (MRI) to measure therapeutic responses. Imaging alone, however, is limited by false positives (?pseudoprogression?) and lacks molecular specificity. Tissue analyses could augment imaging results, but carries the risk of comorbidity and sampling errors. The goal of this project is to address such challenges and advance a minimally invasive, hence serially repeatable clinical assay specific to MG. We will exploit extracellular vesicles (EVs) as a new class of circulating cancer biomarker. Increasing number of studies evidence EVs' potential utility: these vesicles, are released from all cells, function as reliable cellular surrogates and reflect global tumor burden, overcoming limitations of tumor heterogeneity and sampling bias. We now seek to establish a translational EV assay for MG diagnosis and treatment monitoring, and compare its performance with gold standard imaging-based diagnostics. First, we will standardize EV assay protocols for clinical workflow. We will adopt our validated technologies: ExoLution, a clinical grade kit for EV isolation; Shahky, a high-throughput plasmonic instrument for EV protein analyses; and digital droplet PCR for highly sensitive EV mRNA detection. Leveraging the developmental and regulatory expertise of Exosome Diagnostics, we will make these platforms ready for translation into clinical diagnostic laboratories. Second, we will perform a targeted clinical study, critically assessing EVs' diagnostic and prognostic capacity for GBM. We will collect circulating EVs from GBM patients undergoing therapies and monitor serial changes of EV protein/mRNA profiles, particularly to detect the early sign of acquired resistance. We will compare results from EV assays and accompanying MRI, and build an integrative model for treatment monitoring. We formed a powerful multidisciplinary team to conduct these projects: Brain Tumor Center at Massachusetts General Hospital (MGH) which operates a vast biobank program of human clinical samples; Center for Systems Biology at MGH, a pioneer in developing new technologies for EV analyses; and Exosome Diagnostics, a de-facto industry leader in EV-based liquid biopsy with extensive experience in assay standardization. Collectively, the team has a track record of studying EVs' potential as a gliomas biomarker and has established optimal EV assays for molecular analyses. We will ensure assay reliability and reproducibility to deliver clinically translatable EV tests. We will also impose stringent quality controls on assay design and sample processing, accrue well-annotated patient and control samples, and perform statistically powered clinical studies. The technical and scientific outcomes of this research could have a significant translational impact in gliomas care by establishing a robust and highly specific clinical tool for gliomas diagnostics and treatment monitoring.