Thomas E. Carey, MD - US grants

The long-range goals of our study are to identify cell surface antigens of human squamous-cell carcinoma using monoclonal antibodies. Particular emphasis will be placed on identifying those antigens that are expressed solely or in greatly increased amounts by tumor cells. To achieve these goals, we will make use of our large panel of human squamous carcinoma cell (SCC) lines and our capacity to culture the corresponding normal cell, the epidermal keratinocyte, as in vitro test systems. We will immunize mice with selected SCC cell lines and select monoclonal antibodies that bind specifically to membrane antigens of this cancer type using an extensive panel of representative cell lines as test cells and hemadsorption assays to assess binding. Antibodies that define antigens that appear to be restricted to squamoua cancers will be further tested by immunoperoxidase tests on tissue sections from a range of normal and tumor tissue. We will study selected antigen-antibody systems further by labeling antigen-bearing target cells with metabolic precursor molecules and solubilizing membrsne antigens. Individual antigens will be selectively precipitated with the corresponding monoclonal antibody and studied for molecular size using polyacrylamide gel electrophoresis and autoradiography enhanced for fluorographic techniques. From this study, we hope to identify antibodies that will bind in a specific or enhanced manner to tumor cells and which may find use as diagnostic or even therapeutic reagents. Initial studies suggest that increased expression on cancer cells of the antigen detected by monoclonal antibody A9 is associated with more aggressive growth in vitro and in vivo (in nude mice). Furthermore, this antibody when labeled with radioactive iodine localizes human squamous cancers in nude mice with a tumor to normal tissue ration of 18:1. (AG)

Sensorineural hearing loss (SNHL) of unexplained etiology is a common entity with an incidence rate of 1:10,000 for which there are few effective treatments. McCabe proposed that a subset of patients presenting with sudden or rapidly progressive onset of hearing loss, vestibular involvement, or systemic immune disease, such as vasculitis, might be of autoimmune origin. Some of these patients have antibodies to inner ear antigens and show improvement of symptoms on immunosuppressive therapy but such therapy is dangerous and should be used only when clearly indicated. Thus it is important to develop animal model systems for autoimmune sensorineural hearing loss (AISNHL) which can be used to determine if antibodies to inner ear antigens do cause hearing loss, and to determine the mechanism(s) by which the hearing loss occurs. We previously developed monoclonal antibodies (mabs) to inner ear antigens and demonstrated that mice carrying high titers of the KHRI-3 mab to a prominent cochlear antigen, develop high frequency hearing loss and corresponding outer hair cell loss in the basal turn of the cochlea. Furthermore, intracochlear infusion of this antibody in Guinea pigs results in in vivo antibody binding to the supporting cells, hearing loss, and pathologic changes that include loss of outer hair cells. and supporting cells. These tests indicate that exogenously administered antibody to a cochlear antigen can induce hearing loss. We will use this system to: test the hypothesis that antibodies to inner ear antigens mediate SNHL, and we will determine the pathogenic mechanism of antibody induced hearing loss. We will affinity purify mabs raised to inner ear proteins (such as KHRI-3, -4 and 5) as well as control mabs and prepare Fab and Fab' antibody fragments. These preparations will be infused in mice and Guinea pigs in which hearing, inner ear pathology and antibody binding will be tested. Proteins identified by mabs that induce hearing loss, will be affinity purified on mab columns and used as antigen to induce AISNHL. Preliminary studies using highly purified KHRI-3 Mab has replicated earlier results obtained with crude antibody preparations supporting the hypothesis that this Mab, and not some other component of the hybridoma, is responsible for the induced hearing loss. Mabs to inner ear proteins that produce antibody- mediated auditory damage provide a useful small animal model of AISNHL. Eventually these studies should lead to better diagnosis test and therapy of AISNHL.

nucleic acid sequence; gene expression; ear hair cell; human genetic material tag; molecular cloning; computer assisted sequence analysis;

Squamous cell carcinoma (SCC) of oral cavity is a devastating and deadly disease. At present, there are few definitive indicators for predicting outcome and determining the clinical management other than tumor state and lymph node involvement. Studies over the past two decades have shown that gene defects underlie cancer development and progression, and play a critical role in defining the natural history and biological behavior of cancers. The Principal Investigator has shown that losses affecting the long arm of chromosome 18 (18q) occur in 55-60 percent of oral SCC. The preliminary data indicate that 18q loss is associated with poor prognosis and death from cancer in head and neck SCCs. Moreover, loss of heterozygosity (LOH) on 18q is associated with progression in individual patients. If the Principal Investigator can identify the basis for these findings, then genetic markers could be used in selecting high-risk patients for more aggressive therapy, and spare low risk patients from unnecessary treatment morbidity. Chromosomal regions frequently affected by LOH are thought to indicate the presence of a tumor suppressor gene (TSG) within the affected region. Three candidate TSGs have been identified on 18q; DCC (deleted in colon cancer), DPC4 (deleted in pancreatic cancer), and MADR2 (mad related gene 2). The goals of the application are: to establish the smallest region of loss on 18q in oral SCC, to test the hypothesis that one or more TSGs within the smallest region of loss on 18q is associated with tumor progression; to test the hypothesis that restoration of the affected TSG will affect tumor growth or invasive behavior; and using markers identified in Aim 1 and the tissue specimens from a large, controlled, randomized treatment trial for SCC, test the hypothesis that 18q LOH is associated with survival and/or response to therapy. From these studies, the Principal Investigator expects to further define chromosome 18q alterations as an important feature of biologically advanced disease and to develop the knowledge for designing new strategies to counter the effect of tumor suppressor gene inactivation.

DESCRIPTION: (Adapted from applicant's description) Autoimmune sensorineural hearing loss (AISNHL) in man may be the result of antibodies to inner ear antigens. A monoclonal antibody KHRI-3 that binds to supporting cells in the organ of Corti and a 68 kD protein in guinea pig inner ear extracts causes hearing loss and loss of outer hair cells in the guinea pig. In sera from 50-60% of patients with AISNHL we found antibodies with similarities to KHRI-3. We will test the hypothesis that patients with AISNHL have antibody to a 68 kD inner ear antigen, that this antigen is expressed on supporting cells in the organ of Corti, and that the same antigen is also expressed in human inner ear. We will test a second hypothesis that human autoantibodies and KHRI-3 bind to the same inner ear protein, that this protein is distinct from heat shock protein 70 (HSP-70), that the inner protein can be affinity purified on a KHRI-3 column, sequenced, and that the gene for this protein can be cloned, sequenced, expressed in vitro and used as an antigenic substrate for detecting antibodies in patients with AISNHL. Preliminary results show that human sera with antibodies to a 68 kD guinea pig inner ear antigen also bind to supporting cells in the guinea pig inner ear. Human inner ear tissue can specifically absorb this antibody reactivity. A 68 kD protein immunoprecipitated from guinea pig inner ear extracts by KHRI-3 is stained by AISNHL sera, but not by normal donors' sera and not by antibodies to HSP-70. These preliminary data support our hypotheses and suggest that an immunoaffinity column prepared with KHRI-3 antibodies will isolate a protein of sufficient purity for amino acid sequencing. Based on the sequence we will screen inner ear libraries, isolate and clone the gene for the inner protein, express the full length cDNA in an in vitro translation system, and test whether this material can be used as a substrate for detecting AISNHL antibodies. If we are successful this could lead to a rational, accurate test for AISNHL and a better understanding of the mechanism of autoimmune damage to the inner ear.

A critical research goal in head and neck oncology is to define the use of chemotherapy plus radiation regimens that allow organ preservation and preserve quality of life. Progress has been limited by the failure of some tumors to respond to chemoradiation, and the lack of predictive markers for identifying those likely to respond. Furthermore, persistent or recurrent disease is often detected late when the effectiveness of salvage treatment options is limited. Oral cancer is a devastating disease for which better therapies are needed. Surgery is effective in some patients but the surgical defects can be functionally and cosmetically unacceptable and many patients recur after surgery. Thus, the development of predictive markers to select patients for appropriate therapy is needed. Our preliminary results suggest that p53 expression or mutation and expression of the apoptosis-blocking proteins, Bcl-2 and Bcl-x, interact to determine responsiveness to chemoradiation. We propose to determine in patients with oral cancers whether chemotherapy response, organ preservation and/or survival is predicted by p53 overexpression and gene mutation, and we will determine how this is influenced by the expression of Bcl-2 and Bcl-x. Our preliminary in vivo and in vitro data support the concept that tumors with mutant p53 are susceptible to cisplatin-based chemotherapy regimens but that those with wild type p53 are resistant. We will test our hypotheses in tumor samples obtained from patients before and after organ sparing therapy and we will test these hypotheses with in vitro experiments using cell lines with known p53 and Bcl-2/Bcl-x status. Specimens from patients undergoing conventional treatment will also be tested to determine how expression and mutation of these genes may influence outcome in the absence of organ sparing procedures. Furthermore, our preliminary data indicate that the presence and persistence of serum antibodies to p53 in patients with head and neck cancer is associated with poor outcome. However, we have also observed that following successful treatment the antibodies disappear and may reappear with relapse. Thus, we plan to investigate the value of antibodies to p53 as a predictive marker for persistence, recurrence, and successful therapy as well as to correlate p53 antibody data with p53 mutation data. The information developed in this work should result in new trials that employ predictive markers to select the most appropriate and effective treatment for oral cancer. This together with close monitoring for early recurrence should in turn lead to improved overall survival.