Joanna J. Phillips, MD PhD - US grants

DESCRIPTION (provided by applicant): Project Summary: This proposal describes a 5-year-long career development program whose goal is to prepare Dr. Phillips for the role of an independent academic investigator. The project sponsor, Dr. Zena Werb, will provide the principle guidance. Dr. Werb is an internationally recognized expert on the tumor microenvironment and has a distinguished record of training independent scientists. The project co-sponsor, Dr. David Rowitch, is an internationally recognized expert on brain tumor development with a demonstrated commitment to training clinician-scientists. In addition to Drs. Werb and Rowitch, the members of the advisory committee will provide additional scientific and career development related guidance. The research program will address the hypothesis that microglia and macrophages accelerate brain tumor progression and infiltration. Microglia and macrophages are a significant component of the brain tumor microenvironment; however, their activation phenotype and function are unclear. In this project, the role of microglia and macrophages in the development of infiltrating glial tumors will be assessed in three specific aims. First, I will examine the activation patterns of microglia and macrophages in infiltrating glial tumors over time. Studies will be performed using a robust murine model for infiltrating glioma and the results will be confirmed in human tumor tissue. Second, I will determine whether microglia and macrophages promote tumor cell infiltration. Using novel imaging technology I will monitor the dynamic interactions of immune cells and tumor cells. Third, I will determine the in vivo significance of the microglial and macrophage response to brain tumor by depleting it or modifying it. The research training will also include a formal didactic program in research ethics, tumor biology, advanced molecular techniques, and tumor immunology. The UCSF Pathology Department is fully committed to Dr. Phillips' career development and UCSF provides an ideal setting for the training of independent physician scientists. Relevance: In 2007, it is estimated that 20,500 individuals will be diagnosed with cancer of the brain and nervous system in the United States. Despite current therapies infiltrating gliomas, the most common type of primary brain tumor, continues to progress and is often fatal. The research proposed in this project will yield important insight into the factors in the microenvironment that contribute to brain tumor progression and infiltration and potentially may aid in the development of new therapies for glioma.

DESCRIPTION (provided by applicant): Among all cancers, non-small cell lung cancer (NSCLC) and glioblastoma (the most common and most malignant type of astrocytic brain cancer) are two of the most deadly cancers with survival measured in months. Earlier diagnosis using a robust biomarker would likely improve survival. However, currently no such biomarker exists due to the heterogeneity of the diseases and the lack of biomarkers with functional relevance in tumorigenesis. The extracellular heparan sulfate endosulfatases, SULF1 and SULF2, are overexpressed in a wide variety of human cancers, and SULF2, in particular, has been shown by the PIs of this proposal, to drive carcinogenesis in NSCLC and malignant astrocytoma through regulation of several cancer-promoting pathways. The overall goal of the proposed study is to evaluate plasma for the presence of the SULFs and consequences of SULF-bioactivity in order to develop novel biomarkers for the early detection of NSCLC and malignant astrocytoma. The SULFs are unique as they modulate the binding of a multiplicity of signaling molecules, including Wnts, VEGF, and FGF-1, by acting on the 6O-sulfation of glucosamine (6OS) on heparan sulfate proteoglycans (HSPGs). As extracellular enzymes that are both tethered to the cell membrane and secreted, the SULFs and their HSPG substrates are present in the extracellular environment and have great potential as novel biomarkers for early detection of cancer in body fluids. This innovative project assembles a diverse team of investigators to integrate mechanistic studies of SULF function in cancer with practical applications of this knowledge to validate a novel biomarker for the early detection of cancer. Our Aims are: Aim 1: Characterize the expression levels of SULFs and the expression and sulfation status of their HSPG substrates in tumor samples from NSCLC and astrocytoma patients. Aim 2: Determine SULF levels in plasma and correlate these with tumor presence and progression Aim 3: Determine the 6OS sulfation status of HSPGs in plasma and correlate these with tumor presence and progression. Aim 4: Characterize the plasma levels of heparin-binding factors (growth factors, morphogens, cytokines, chemokines) that are potentially mobilized by SULF action in the tumor and correlate with tumor SULF levels. Our investigation of novel SULF-based biomarkers could lead to ELISAs for the early diagnosis of NSCLC and malignant astrocytoma and could become a new basis for patient management. In addition, these studies will promote collaborations and stimulate Trans-Alliance projects aimed at identifying how changes in cellular carbohydrates promote cancer progression and how to use this information to identify biomarkers for the early cancer detection. PUBLIC HEALTH RELEVANCE: This novel project will confirm the clinical utility of plasma levels of the extracellular heparan sulfate endosulfatases, or SULFs, as robust biomarkers for the early detection of non-small lung cancer (NSCLC) and malignant astrocytoma, two of the most deadly cancers due to failure in early diagnosis. As the SULFs are overexpressed and help to drive carcinogenesis in a number of cancers including the two in this proposal, our results could have a major impact upon patient care.

DESCRIPTION (provided by applicant): Glioblastoma (GBM), a uniformly lethal brain cancer, is characterized by diffuse invasion and abnormal activation of multiple receptor tyrosine kinase (RTK) signaling pathways (1). Despite current therapies, the prognosis for GBM is poor and mean survival remains less than 2 years. An improved understanding of the mechanisms driving abnormal cell signaling is essential for improving treatment outcomes. The long-term goal of this innovative proposal is to define tumor-microenvironment interactions critical in brain cancer and identify clinically relevant, druggable therapeutic targets. Specifically, we focus on the role of extracellular heparan sulfate proteoglycans (HSPGs) as they regulate the activity of multiple ligand-mediated signaling pathways (2), are altered in malignant brain tumors (3, 4), and have the potential to influence both tumor cells and critical tumor-microenvironment interactions, including the tumor-associated microglia/macrophage response. HSPGs, present on the cell surface and in the extracellular matrix, regulate signaling via their ability to bind and alter the bioavailability of diverse ligands, including growth factors, morphogens, chemokines, and enzymes. SULF2, an extracellular heparan sulfate endosulfatase, actively regulates HSPG-dependent signaling by removing the sulfate from 6-O- of glucosamine (6OS) and liberating protein ligands from HSPG sequestration (5). Alterations in HSPG core protein expression and SULF2 expression are common in diverse cancers and the PI of this proposal has shown SULF2 can drive carcinogenesis in malignant astrocytoma through regulation of RTK signaling pathways. As extracellular enzymes that are both tethered to the cell membrane and secreted, the SULFs and their HSPG substrates are present in the extracellular environment and have great potential as novel therapeutic targets. Our Aims are: Aim 1: In human infiltrating astrocytomas, identify the alterations in HSPG expression and sulfation associated with tumor malignancy. Aim 2: Determine HSPG changes driving tumor biologic behavior, including microglia/macrophage response to tumor. Aim 3: Identify how HSPG alterations activate signaling pathways to promote GBM malignant behaviors. The proposed research will determine the mechanisms by which alterations in HSPGs drive oncogenic cell signaling pathways in malignant brain cancer and validate HSPGs as clinically relevant, novel therapeutic targets. Successful completion of these studies provides a preclinical basis to study agents that target HSPGs as a novel therapeutic option in malignant brain cancer. PUBLIC HEALTH RELEVANCE: The mortality rates for primary malignant brain cancer have remained stable despite substantial advances in our understanding of disease biology. In the present proposal we will use innovative approaches to define how alterations in the tumor microenvironment drive oncogenic signaling pathways critical in brain cancer and identify clinically relevant, druggable therapeutic targets.

The overarching goal of this Core is to provide the high degree of technical expertise required to perform and interpret advanced MR imaging studies; facilitate the collection of image guided surgical specimens; and relate the quantitative information derived from the imaging data to molecular markers of the biological properties of the tissue. It should be stressed that many of the procedures required to achieve this objective are nonstandard, requiring specialized knowledge, cutting-edge equipment, and sophisticated experimental or computer analysis in order to extract the information necessary for testing the hypotheses associated with each of the Scientific Projects. The decision to create a single Core that would integrate the imaging and tissue analysis reflects the high priority placed upon acquiring image-guided samples that can provide a direct link between the clinical imaging variables extracted from the noninvasive metabolic and physiologic imaging data and the tissue markers of tumor biology. The interaction of the Imaging and Tissue Procurement Core with the rest of the UCSF TMEN Brain Cancer Center is shown in the figure immediately below. Working closely with Co-Principal Investigators Gabriele Bergers and Valerie M. Weaver, Co-Project Leaders Joanna J. Phillips and Soonmee Cha will hold the primary responsibility for planning, directing, and executing the Imaging and Tissue Procurement activities ofthe TMEN U54 UCSF Brain Cancer Center. They will be assisted by the Administrative Core Manager Annette Bistrup and will exploit the Center's webpage for inquiries and protocols for tissue and imaging requests/requirements. The major purpose of the Core is to identify potential research subjects, schedule imaging acquisition, identify potential biopsy sites, procure tissue from the operating room, de-identify tissue samples, distribute fresh tumor tissue and paired non-tumor tissue, and properly handle and store tissue for future use. The Core will also provide expertise in neuroradiology, neuropathology, and morphologic analytical techniques to the UCSF TMEN Brain Cancer Center. In addition, the Core will provide training and information with respect to the analysis of imaging and pathology variables and the optimal use and handling of human tissue. Critical to the success of the TMEN Brain Cancer Center is access to primary brain tumor tissue and non-tumor tissue for analysis. The organizational structure for the Imaging and Tissue Procurement Core is depicted in the figure below. The progress of tissue procurement will be monitored through weekly teleconference meetings. Our goal is to obtain fresh tissue from 3 patients every 2 months for a total accrual of 90 patients over 5 years.

PROJECT SUMMARY The UCSF Brain Tumor SPORE Biospecimen/Pathology Core provides staff and technology dedicated to the procurement, processing, storing, distribution, and histopathologic analysis of high-quality brain tumor biospecimens for translational science research. Our mission is to enhance biospecimen quality and utility through use of optimized standard operating procedures, multi-modality preservation, integrated histopathologic and molecular annotation, and a computerized inventory, request and tracking system. All aspects of this Core are performed in accordance with the guiding principles set forth in the 2016 National Cancer Institute Best Practice for Biorepository Guidelines. In order to maximize sharing and integration of SPORE projects, the Biospecimen Core collects and makes available data derived from all distributed brain tumor biospecimens. Specific Aims of SPORE Biospecimen/Pathology Core: 1. To procure brain tumor patient biospecimens from the operating room and from animal models used by the Projects with optimized handling to maximize cell viability and/or minimize the cold-ischemia time so as to meet the tissue accrual requirements for all of the proposed Brain Tumor SPORE projects and clinical trials. 2. To perform quality control assays on archived biospecimens collected from the operating room and animal models, to ensure availability of adequate numbers of consistently handled specimens that will yield high- quality data for SPORE projects and clinical trials. High quality biospecimens are critical for all proposed SPORE projects. 3. To provide standardized routine and advanced tissue handling/processing and analytical techniques, including immunohistochemistry, fluorescence in situ hybridization, tissue microarray construction, DNA/RNA extraction, protein isolation, and preparation of viable cells that will allow each SPORE project to fulfill its goals. 4. To maintain a SPORE Biospecimen/Pathology Core database containing demographic data, integrated histopathologic and molecular annotation, results from molecular analyses, and tissue distributions (internal and external) that will be linked to relational clinical databases maintained by the SPORE Biostatistics and Clinical Core and used by all SPORE Projects.

The Biospecimen and Biomarker Core provides the leadership and expertise in biospecimen sciences and molecular analyses needed in this P01 to investigate the metabolic signatures of molecular subtypes of glioma to improve patient management. High-quality, well-annotated biospecimens and rigorous and standardized analysis of tumor tissue are essential for testing the hypotheses associated with the scientific Projects. The Core will acquire, annotate, distribute, track, and analyze brain tumor patient biospecimens from the operating room. This will include the acquisition and analysis of image-guided and multiple spatially mapped samples from patients with newly-diagnosed and recurrent glioma. In addition, the Core will provide histopathologic and molecular analysis of human glioma xenografts. All aspects of this Core are performed in accordance with the guiding principles set forth in the 2016 National Cancer Institute Best Practice for Biorepository Guidelines. The specific aims of the Biospecimen and Biomarker Core are as follows: 1. To acquire and preserve high-quality biospecimens, including image-guided and spatially mapped tissue samples, from the operating room to meet the tissue accrual requirements for the scientific projects. 2. To perform the clinically relevant molecular analyses required by the Projects. 3. To provide the neuropathologic expertise for interpretation of the processed tissue sections and extraction of appropriate quantitative and semi-quantitative parameters. 4. To maintain a tissue inventory and database of associated molecular parameters that is linked to the Brain Tumor Center Database, integrating imaging, clinical, molecular, and pathology data. 5. To provide consistent oversight of the Core activities that will ensure the stringent compliance with regulations governing informed consent and patient confidentiality, as well as management of the biospecimens and the information derived from the biospecimens.

PROJECT SUMMARY Diffuse gliomas, including oligodendroglioma, astrocytoma, and the highly malignant glioblastoma (GBM), are the most common type of primary malignant brain tumor. Increased activation of the phosphatidylinositol 3 (PI3)-kinase/AKT/mTOR signaling pathway is common in GBM and appears to be one mechanism of malignant transformation of tumors from lower grade diffuse glioma to GBM. Given the importance of this pathway in disease, several therapeutic strategies that target it are being tested in early phase clinical trials. To maximize benefit and minimize toxicity to these therapies, however, accurate and robust biomarkers for patient stratification and assessment of disease response to therapy are needed. Analysis of protein phosphorylation provides integrated, functional information about signaling pathway activity. The goal of this proposal is to use biospecimen sciences to identify and mitigate the preanalytic factors, including intratumoral heterogeneity, tissue acquisition, and confounding non-neoplastic cells, that can obscure accurate analysis of protein phosphorylation in diffuse glioma. We hypothesize that accurate assessment will require small, well-preserved and biologically targeted tumor biopsies. To investigate this hypothesis we propose a multidisciplinary team of investigators with expertise in the acquisition and analysis of multiple and image-guided biopsies, brain tumor biology, biobanking, neuropathology, imaging, biostatistics, and neuro-oncology. First, we investigate innovative approaches to target tumor biopsies to biologically aggressive tumor regions using pre-operative MR spectroscopic imaging and intra-operative tumor metabolism using 5-aminolevulinic acid (ALA)-based imaging. Second, we use clinically validated assays and state-of-the-art single cell mass cytometry to evaluate PI3K/AKT/mTOR signaling pathway activity and determine the contribution of non-neoplastic cells to overall phosphoprotein levels. Third, using the evidence-based methodologies and approaches we develop, we will investigate the prognostic significance of PI3K/AKT/mTOR signaling pathway activity as determined by protein phosphorylation in diffuse glioma and in patients treated with an mTOR inhibitor by integration with our Phase 2 clinical trial targeting the PI3K/AKT/mTOR signaling pathway (NCT02023905).

PROJECT SUMMARY/ABSTRACT The Immune Monitoring and Biospecimen (IMB) Core is a dedicated to provide the outstanding leadership and expertise in immune monitoring and biospecimen sciences needed in this U19 to develop novel effective agents that can cross the blood brain barrier (BBB) and test them in the clinic. Working collaboratively as a team the investigators at University of California San Francisco and Northwestern University will acquire, process, and analyze biospecimens from enrolled patients and from preclinical models for GBM. Many of the procedures required to achieve these objectives are non-standard, requiring specialized knowledge and sophisticated experimental analysis. In addition, the Core will provide histopathologic and molecular analyses of human glioma xenografts, central histopathologic review, and ensure strict quality control in all aspects of biospecimen handling. The Core will address these needs based on the following specific aims: Specific Aims 1. To provide the staff and expertise for the acquisition and preservation of high-quality brain tumor patient biospecimens and preclinical models to meet the tissue accrual requirements for the projects and clinical trials. 2. To provide routine and advanced tissue handling/processing and analytical techniques and expertise for human and murine tumors, including high-dimensional flow cytometry, multiplex immunoflourescence, Cytek, and single cell RNA sequencing, that will advance project hypothesis development and goal attainment. 3. To provide the neuropathologic expertise for interpretation of the tumor tissue and extraction of appropriate quantitative and semi-quantitative parameters. 4. To provide consistent oversight of the Core activities that will ensure the stringent compliance with regulations governing informed consent and patient confidentiality, as well as management of the biospecimens and the information derived from the biospecimens.