Molecular Pathology Research for Cancer Diagnostics and Biomarkers

Division Of Basic Sciences - Nci

Investigators

Abstract

In this project research is conducted to characterize and develop new animal models of human disease and to develop the means to better characterize a models relevance for a human condition. The project aims include the evaluation of research technologies and markers. Progress was made in developing cancer diagnostics and in research resources useful in developing and characterizing new models of human cancer. Methods and reagents were developed and technologies were applied for use in experimental molecular imaging. Research resulted in development of a novel cancer cell line with a molecular tag that permits the cancer cell line to be tracked during the process of an experimental cancer study. In this way, this technology was used to assess cancer staging, diagnosis, and vaccine efficacy in testing host immune response to the vaccine and of immunotherapy against experimental cancers. A benefit included reduction in the number of research animals required and refinement of the animal welfare while on study due to the use of noninvasive imaging. More specifically, the research evaluated the fact that many strategies have been used to enhance the peptide vaccine immune response and to establish therapeutic benefits. This includes the utilization of cytokines to improve antigen presentation or enhance T cell response. In this research the combination of granulocyte-monocyte-colony stimulating factor (GM-CSF) and interleukin-2 (IL-2) was tested as locally administered adjuvants to enhance the immune response to the human papilloma virus type-16 (HPV16) E7 peptide. Female C57BL/6 mice were immunized intradermally with a 9-mer HPV16 E7 peptide (aa: 4957) alone, or in combination with GM-CSF, IL-2, or both cytokines. Specific immune responses were measured by enzyme-linked immunosorbant assay (ELISA) and Chromium-Release Assays. Furthermore, therapeutic effects of these vaccines and long term tumor protection were assessed in mice bearing established tumors. We showed that GM-CSF and IL-2, when co-administered locally in an emulsion with peptide, exert a synergistic effect in enhancing the immune response to the antigen. This combination induced higher cytotoxic T-lymphocyte (CTL) and cytokine release responses and did not increase the regulatory T-cell population. Therapeutic intervention with this synergistic combination led to a complete response of established tumors. Furthermore, this combination induced a memory response which protected mice against subsequent additional tumor challenge. We identified a new vaccine adjuvant, a local combination of GM-CSF and IL-2, which is synergistic in enhancing peptide specific immune response through local effect without increasing regulatory T-cells. This immune response was found to be long lasting and protective in tumor bearing mice. The scientific benefits will permit better design and delivery strategies for cancer vaccines. A second area of research involved developing methods for the research community that can be used to estimate the amount of body fat burden in the abdominal cavity using a noninvasive nuclear magnetic resonance imaging method to detect molecular signature of fat as present in specific depots of fat in the body. The physiology of fat is unique in different depots of body fat. The application of the research applies to studying obesity as a cancer related risk factor. Research in this project assessed proton magnetic resonance spectroscopy (1H-MRS) as a molecular diagnostic means to distinguish among mice with disparate intra-abdominal body fat compositions, and to measure changes in intra-abdominal fat burden during weight loss and regain. Intra-abdominal fat burden was analyzed as a ratio of integrated areas under the curves of fat to water 1H-MRS signals collected from a region of interest standardized across B6.V-Lepob, C57BL/6, and A-ZIP/F mice that exhibited various genotypically related body fat compositions, ranging from obese (B6.V-Lepob) to minimal body fat (A-ZIP/F). 1H-MRS analysis of fat burden was compared with intra-abdominal fat volume and with a single cross-sectional intra-abdominal fat area calculated from segmented magnetic resonance images. Similar measurements were made from obese B6.VLepob mice before, during, and after they were induced to lose weight by leptin administration. Relative amounts of intra-abdominal fat analyzed by 1H-MRS differed significantly according to body composition and genotype of the three strains of mice (p less than 0.05). Intra-abdominal fat assessed by 1H-MRS correlated with both intra-abdominal fat volume (r=0.88, p 0.001) and body weight (r = 0.82, p less than 0.001) among, but not within, all three genotypes. During weight loss and regain, there was a significant overall pattern of changes in intraabdominal fat quantity that occurred, which was reflected by 1H-MRS (p less than 0.006). Results support the use of localized 1H-MRS for assessing differences in intra-abdominal fat. This research project included developing capabilities in molecular diagnostics for cancer on studies utilizing repository biospecimens from animal models of human cancer disease. Historical archival specimens are being evaluated and used in research aimed at identifying environmental risk factors for development and progression of human cancer. Chemical exposures are important risks for development of hepatocellular carcinoma (HCC). One such chemical, diethylnitrosamine (DENA), is present in food products as well as in industrial and research settings. Further examination of tumors induced by DENA may yield clues to human risk. HCC from animals exposed to DENA were selected from a tissue archive to examine for evidence of Wnt/beta catenin signaling events, which are frequently associated with HCC. DENA exposure durations ranged from 8 to 207 months and total accumulated dose ranged from 0.7 to 4.08 mg. Unexposed colony breeder served as controls. Previously unrecognized HCC metastases were discovered in lungs of three animals. Primary HCC, and all metastatic HCC, are being evaluated for overexpression of beta catenin and glutamine synthetase to assess the possible role of Wnt/beta-catenin signaling in the evolution of HCC due to exposure of DENA. The significant materials, equipment or methods in this project include use of recombinant DNA technology, in vitro cell culture, DNA sequence analysis, immunodiagnostics, molecular imaging, magnetic resonance spectroscopy, magnetic resonance imaging, morphometrics, computer assisted image analysis, optical imaging, and veterinary medical diagnosis.