David N. Louis, MD - US grants

DESCRIPTION: (Adapted from the investigator's abstract) Malignant gliomas are the most common human brain tumors. He has clarified some of the genetic events that underlie the formation of these tumors, but a number of genes involved in glioma tumorigenesis have not yet been cloned. Of the various glioma-related tumor suppressor loci, the chromosome 19q locus is the only one that is deleted in all three major subtypes of malignant diffuse glioma, suggesting that this gene encodes a critical glial growth regulatory protein. Chromosome 19q deletions are common, with allelic loss estimated to occur in approximately 6000 new gliomas each year in the United States. His mapping of the chromosome 19q tumor suppressor gene over the past few years suggests that the gene lies in band 19q 13.3 telomeric to the marker D19S412 and centromeric to the marker STD. He has cloned this candidate region and has further localized the gene to an approximately 200 kb BAC-PAC contig just centromeric to STD. Gene identification approaches to this region have yielded a number of partial coding sequences. To identify and characterize the chromosome 19q glioma tumor suppressor gene, therefore, he proposes: 1) to complete identification of candidate genes, using approaches such as exon trapping, cDNA selection; 2) to identify glioma-specific alterations, using mutation detection techniques such as single strand conformation polymorphism (SSCP) analysis; 3) to define the mutational spectrum of the gene in different types of primary gliomas using SSCO. If the gene is cloned within the proposed funding period, he then proposes: 1) to begin characterization of expression of the 19q gene, including evaluation of the protein encoded by the 10q gene, by generating specific antibodies for Western blotting and immunohistochemistry; and 2) to identify other proteins that interact with the product of the chromosome 19q gene, using immunoprecipitation and yeast two-hybridy screening The identification and characterization of the chromosome 19q glioma tumor suppressor gene will contribute towards understanding a critical step in human glial tumorigenesis and may eventually lead to improved treatment for these malignant tumors.

DESCRIPTION (provided by applicant): Problems in the pathological classification of the common malignant gliomas complicate predicting patient prognosis and response to therapy. The long-term goal of this research is to provide a comprehensive understanding of the genetic events that characterize glioma tumorigenesis and to introduce genetic analyses into routine classification. During the two prior funding periods of this grant, we have clarified genetic events that underlie glioma formation, have correlated genotype with clinicopathological parameters, and have introduced genetic analyses into clinical practice. This work now raises three hypotheses that can be tested using proven translational research strategies. 1) To test the hypothesis that molecular profiles can divide histologically classic and non-classic malignant gliomas into improved prognostic and predictive subgroups in a practical manner, we propose to define markers capable of robust distinction of chemosensitive and better prognosis malignant gliomas from chemoresistant and poor prognosis malignant gliomas. 2) To test the hypothesis that increased cellular invasion correlates with prognosis independent of histological appearance and that such a molecular phenotype can be readily detected, we propose to define an "invasion genotype/phenotype" that correlates with clinicopathological endpoints in patients with malignant gliomas. 3) Finally, to test the hypothesis that ongoing selection pressures operate to determine tumor genotype, and that molecular diagnostic approaches should take such heterogeneity into account, we propose to characterize intratumoral selection in glioblastoma perinecrotic pseudopalisades to define a molecular signature that correlates with clinicopathological endpoints in patients with anaplastic astrocytomas. Each aim thus tests a related hypothesis that a particular molecular analysis can augment current approaches to glioma classification, and each aim also follows a similar experimental design. The further clarification of the genetic basis of human gliomas will continue to contribute to a glioma classification system that will more accurately reflect tumor behavior and response to therapy than current histopathological schemes.

DESCRIPTION: The core has two specific aims: 1) To provide a neuropathology core for use by investigators interested in neurogenetics research. The resources include neuropathological expertise as well as a nervous system tumor and tissue bank; the bank features frozen and fixed tissues, tumor cell lines, DNA and RNA extracts from tumors, and constitutional tissue and DNA from patients with inherited nervous system tumor syndromes (e.g., NF1, NF2, TS); and 2) To maintain a data base of human nervous system tumors which have been thoroughly and uniformly evaluated by a number of laboratory parameters, including histopathology, proliferation markers, and genetic analyses. This contributes to studies of molecular correlates of brain tumor prognosis in the inherited CNS tumor syndromes, NF1, NF2 and TS, as well as the same tumors occurring sporadically; 3) To characterize the cellular and subcellular localization of the NF2, TSC1 and TSC2 gene products, as well as their respective novel interacting proteins, in the normal adult and developing nervous system and in nervous system tumors; 4) To analyze human tissues from patients with NF2 and TS, as well as the corresponding sporadic tumors (e.g. meningiomas, schwannomas), to elucidate the molecular pathology underlying these conditions; and 5) to provide neuropathological expertise in the histological evaluation and image analysis of experimental gene therapy/gene delivery models as well as mouse models of tuberous sclerosis.

DESCRIPTION (provided by applicant): The purpose of this postdoctoral research program continues to be to train a select group of young physicians and scientists in the fundamentals of basic research in cancer and immunology, with emphasis on basic mechanisms of tumor growth, invasion and metastasis, the corresponding immune response and the use and development of immunologic techniques to detect, diagnose and treat cancer. The goal is to provide young scientists with the possibility to develop into outstanding investigators capable of addressing a broad range of fundamental questions related to the immune response and the biology of cancer. Trainees will be selected primarily from outstanding residents in our pathology program, who have completed 2-3 years of postdoctoral training and who demonstrate a strong interest in pursuing a basic research career. The research training program includes selected outstanding M.D. or Ph.D. candidates from outside the pathology program. Criteria for selection include a commitment to a career in research and teaching and the consensus by the faculty that the candidate has outstanding potential for success in such a career. Continued support for 5 trainees/year is requested, distributed approximately equally among the first, second and third year of training. Training will require 2-3 years. Our program is modeled on the highly successful Research Associate Program at the NIH in which research training is primarily accomplished by intense daily exposure of trainees to one or a small group of outstanding preceptors who are themselves bench scientists and who are committed to teaching. Such training will be supplemented by extensive reading of the literature, departmental and interdepartmental seminars, formal course work and participation in national scientific meetings. During the 2-3 year training program, trainees will be exposed to a wide variety of techniques and strategies designed to study the immunology, and cellular and molecular biology of cancer. This approach is designed to provide them with the knowledge required to address a broad range of biological problems, related to immunology and cancer in subsequent years. Trainees will acquire expertise in a selected number of these fields by choosing specific topics on which they focus their research interest. Following the completion of the program, trainees will be expected to have acquired the experience and skills necessary to engage in an independent career in cancer or basic immunology research.

DESCRIPTION (provided by applicant): Malignant gliomas are common primary human brain tumors, but problems in their pathological classification compromise patient management. These difficulties have sparked considerable interest in molecular genetic approaches. Clinically relevant genetic associations have been discovered, but practical problems have prevented widespread diagnostic application of this knowledge. We hypothesize that custom array comparative genomic hybridization (aCGH) could provide a sensitive, specific, cost-effective and rapid method to assess human malignant gliomas for a variety of clinicopathologically relevant genetic changes and that such first-generation custom CGH arrays will provide the basis for improving diagnostic correlations for future assays. To evaluate this possibility, we propose a two-stage plan that capitalizes on our existing strengths in molecular genetics, pathology, biostatistics and clinical databases. For the R21 component, we will: 1) evaluate custom aCGH sensitivity and specificity in comparison to standard assays;and 2) generate custom BAG arrays for CGH that include targets providing broad genomic coverage as well as focused coverage of chromosomes 1, 7, 9, 10, 19 and X, and other select loci. Once we have met the Milestones from the above R21 Aims, we will proceed in the R33 component to: 1) evaluate whether alterations of particular regions on the assayed chromosomes, as revealed by aCGH, offer improved and/or novel correlations with chemoresponse and survival in two carefully annotated cohorts of malignant glioma patients: a) a retrospective series of anaplastic oligodendroglioma patients;and b) a prospective series of malignant glioma patients. We will then: 2) develop an aCGH-based classification of malignant gliomas using the data from Aim 1 of the R33. The long-term goal of this project is the implementation of a practical molecular assay to detect a variety of clinicopathologically relevant genetic alterations in malignant gliomas. We also anticipate that such information will contribute to identification of key glioma genes as well as to construction of next-generation diagnostic approaches. Given these endpoints, the application is highly responsive to PA-04-102, "Phased application awards in cancer prognosis and prediction."

To provide tissues for research purposes, it is therefore necessary to process tumors in a[unreadable] uniform manner, with tissue saved in a variety of standard ways. The direct support and/or[unreadable] guidance from a Neuropathology Core provides a means of ensuring:[unreadable] 1.) that all tissues are processed in the same, technically adequate way;[unreadable] 2.) that all specimens are analyzed by an experienced pathologist;[unreadable] 3.) that all human tumors are classified, analyzed and graded in a uniform manner;[unreadable] 4.) that appropriately processed experimental tissues are available for all researchers.

DESCRIPTION (provided by applicant): Glioblastoma is the most common and most malignant of human brain tumors. Recent therapeutic advances have improved control of primary tumors, with a significant fraction of tumors responding to initial therapies. Unfortunately, all glioblastomas recur and lead to patient death. For this reason, the present proposal focuses on the crucial issue of recurrent glioblastoma-with studies extending from biomarker generation and evaluation, to understanding mechanisms of therapeutic resistance, to exploration of novel therapeutic compounds. Over the past 15 years, studies supported by this grant have clarified the molecular genetic basis of glioblastomas and other malignant gliomas and have shown that the treatment of such tumors can be guided by molecular markers;in particular, the now widespread clinical use of 1p/19q testing in oligodendrogliomas emerged from studies performed under this grant. Preliminary data gathered during the last funding period have shown: glioblastomas become resistant to the effects of the alkylating agent used to treat all glioblastomas, temozolomide (TMZ), through a mechanism associated with inactivation of MSH6;MSH6-deficient glioblastomas grow more rapidly during TMZ therapy;and in vitro resources exist to evaluate defects in the TMZ-induced DNA damage response and therapeutic resistance. To address this critical issue, we propose three Specific Aims: 1) To define the spectrum of DNA repair defects that arise after TMZ therapy in glioblastoma, and to generate relevant biomarkers relating these events to clinical outcome;2) To define the mechanism whereby deficiencies in DNA damage responses enable therapeutic resistance;and 3) To define pathways that could be targeted to overcome therapeutic resistance due to defects in DNA damage response. The proposed approach will therefore extend our prior clinically relevant biomarker work to the particularly vexing problem of recurrent glioblastoma, a tumor that confounds current treatment attempts. Importantly, through continuing the work supported by this grant over the past three funding cycles, we aim to generate biomarkers and elucidate mechanisms of therapeutic resistance that could translate in the short term to changes in current diagnostic and therapeutic approaches and, in the long term, to the development of novel targeted therapies. PUBLIC HEALTH RELEVANCE: The purpose of the work outlined in this proposal is to investigate defects in MSH6 and related genes/gene products that are selected for during current glioblastoma therapy and how these defects lead to therapeutic resistance and eventual patient death. We aim to generate biomarkers and elucidate mechanisms of therapeutic resistance that could translate in the short term to changes in current diagnostic and therapeutic approaches and, in the long term, to the development of novel targeted therapies.