Jonathan W. Uhr - US grants

This program project is aimed at understanding the molecular basis of signalling B and T lymphocytes to become activated and differentiate. A multi-disciplinary effort utilizing molecular genetics, cell cloning technology, flow cytometry and protein chemistry will be utilized for the experiments. Thus, a number of T cell-derived lymphokines and their receptors on B lymphocytes are currently being isolated and characterized using monoclonal antibodies, protein chemistry and/or molecular genetics. Using these reagents, the mechanisms underlying activation of B lymphocytes and cytotoxic T lymphocyte precursors are being elucidated. The mechanisms underlying isotype switch in B lymphocytes are being studied by isolating cell populations that preferentially change isotype synthesis from IgM to particular subclasses of IgG. For the study of the generation of cytotoxic T cells (CTL), we will utilize recombinant inbred strains of mice in order to determine the genetic requirements for T help in the induction of CTL. Exon shuffling experiments using MHC products as well as MHC mutants will aid in the determination of structure-function relationships. We will investigate the role of the C2 domain of class I MHC molecules in delivering an activating signal to cytotoxic T lymphocytes (CTL). In order to determine the role of the C2 domain, we will analyze L cells transfected with mouse-human (hybrid) class I genes. We will determine the role of immune response genes in secondary CTL responses in vitro by assaying the ability of (B6 x bm12)F1 animals to respond in vitro to the H-Y alloantigen.

Ligand-ricin A chain conjugates (immunotoxins) will be prepared and utilized to modulate the immune response in a highly selective and predictable fashion. Hapten-protein carriers will be used to form immunotoxins and the effect of these immunotoxins on specifically reactive B cells will be studied in adoptive transfer systems and in vivo. The deletion of specific clones by this technique will be evaluated for specificity, duration of unresponsiveness, proof that B cells are the target cells and other parameters. A good deal of attention will be given to developing immunotoxins that are stable in vivo and that have reduced toxicity. Toxicity will be investigated with particular reference to the reticuloendothelial system (RES) with emphasis on the development of immunotoxins that will give minimal and possibly reversible damage to the RES. Immunotoxins will be prepared using major transplantatin antigens and their ability to delete appropriate subsets of specifically reactive T lymphocytes will be investigated in cytotoxic in vitro systems, after intravenous administration with in vitro analysis of lymphoid tissues from such animals and finally, in vivo, with the objective of obliterating specific homograft reactivity. Finally, in the latter phase of this study, regulation of the immune response will be attempted using immunotoxins directed to particular subsets of T lymphocytes that are known to suppress immune responses in specific experimental situations. This information should provide a board base for determining the potential effectiveness of these immunotoxins in clinical medicine.

BCL1 leukemia is the first B cell leukemia to be described in mice having arisen spontaneously in an elderly BALB/c mouse. One cell transferred to a syngeneic recipient causes progressive leukemia that is always detectable by 12 weeks and is followed by progressive tumor growth and death. Mice rendered chimeric at the H-2 locus by lethal X-irradiation followed by injection of T cell depleted allogeneic bone marrow allow progressive growth of subsequently injected BCL1 cells. However, after 6 weeks, the tumor recedes and the mice appear tumor-free as judged by idiotype analysis of blood cells, white blood count, and spleen size. Disease does not occur for as long as the mice have been followed (8 months). Nevertheless, cell transfer studies indicate a relatively large tumor load in the spleen (10-6 - 10-7 cells). We will study the biology of tumor dormancy in attempts to answer the following questions: 1) What is the cell cycle status of the dormant cells? 2) Are they different from the starting population as judged by immunophenotype, response to lymphokines and karyotype? 3) What are the host defense mechanisms that are required to induce dormancy? 4) What is the natural history of dormancy, e.g. do dormant BCL1 cells eventually die or do they begin to grow when immune responsiveness decreases with age? 5) What perturbations can initiate tumor growth? 6) Can immunotoxins specific to BCL1 eliminate this dormant population in vivo? With regard to a syngeneic model of tumor dormancy, immunity to the BCL1 tumor has been generated in both BALB/c mice previously immunized with purified BCL1-IgMlambda and in non-immunized congenic mice (B.A. 20) that differ at the IgH locus. We will further develop these 2 model systems to study tumor dormancy.

BCL1 is a high grade transplantable murine B cell lymphoma that is normally lethal for mice in 6-8 weeks. We have developed two models of tumor dormancy in BCL1 mice: prior immunization of BALB/c mice with BCL1-immunoglobulin or passive administration of murine polyclonal anti- idiotype in SCID mice. In both models, the majority of mice harbor 1-3 x 106 dormant BCL1 cells in their spleens for many months. There is a progressive but slow loss of dormancy during the 1 1/2 years of observation. By utilizing high resolution-multiparameter flow cytometry for cell analysis and sorting, we have succeeded in isolating and analyzing the population of dormant lymphoma cells. Preliminary studies using DNA dye (Hoechst) and also administration of BrdU indicate that a high proportion of the dormant cells are non-cycling. In the proposed studies, we will determine differences among dormant lymphoma cells, growing lymphoma cells in non-immune mice and growing lymphoma cells in previously dormant mice in an effort to elucidate mechanisms underlying dormancy. Thus, we will extend our studies on cell cycle analysis to determine the history of the dormant cells beginning with their induction from growing BCL1 to their loss of dormancy later in life. We will determine if there are differences in the above three populations with regard to immunophenotype including cytokine receptors, pattern of cytokine production, and the expression of messenger RNA levels of candidate oncogenes that appear to play a role in B cell growth, e.g., myc, Bcl-2, RB and p53. We will determine the in vivo distribution of the dormant cells with regard to organ site and histologic distribution within the spleen. We will determine if the regrowing population which is Id+ is no longer susceptible to the dormancy-inducing effect of anti- Id. Based on our preliminary results, our working hypothesis is: Dormancy is induced by cell cycle arrest of most but not all BCL1 cells due to an Id immune response; this results in down-regulation of growth- promoting oncogenes and up-regulation of suppressor genes; escape from dormancy is due in part to mutations in the subset of dormant BCL1 that is still cycling. The studies to be performed will test these hypotheses and should generate new ones about the mechanisms underlying tumor dormancy.

BCL1 is a high grade transplantable murine B cell lymphoma that is normally lethal for mice in 6-8 weeks. We have developed two models of tumor dormancy in BCL1 mice: prior immunization of BALB/c mice with BCL1-immunoglobulin or passive administration of murine polyclonal anti- idiotype in SCID mice. In both models, the majority of mice harbor 1-3 x 106 dormant BCL1 cells in their spleens for many months. There is a progressive but slow loss of dormancy during the 1 1/2 years of observation. By utilizing high resolution-multiparameter flow cytometry for cell analysis and sorting, we have succeeded in isolating and analyzing the population of dormant lymphoma cells. Preliminary studies using DNA dye (Hoechst) and also administration of BrdU indicate that a high proportion of the dormant cells are non-cycling. In the proposed studies, we will determine differences among dormant lymphoma cells, growing lymphoma cells in non-immune mice and growing lymphoma cells in previously dormant mice in an effort to elucidate mechanisms underlying dormancy. Thus, we will extend our studies on cell cycle analysis to determine the history of the dormant cells beginning with their induction from growing BCL1 to their loss of dormancy later in life. We will determine if there are differences in the above three populations with regard to immunophenotype including cytokine receptors, pattern of cytokine production, and the expression of messenger RNA levels of candidate oncogenes that appear to play a role in B cell growth, e.g., myc, Bcl-2, RB and p53. We will determine the in vivo distribution of the dormant cells with regard to organ site and histologic distribution within the spleen. We will determine if the regrowing population which is Id+ is no longer susceptible to the dormancy-inducing effect of anti- Id. Based on our preliminary results, our working hypothesis is: Dormancy is induced by cell cycle arrest of most but not all BCL1 cells due to an Id immune response; this results in down-regulation of growth- promoting oncogenes and up-regulation of suppressor genes; escape from dormancy is due in part to mutations in the subset of dormant BCL1 that is still cycling. The studies to be performed will test these hypotheses and should generate new ones about the mechanisms underlying tumor dormancy.

The long goal of this Program Project Grant is to improve therapy for B cell lymphomas by optimizing regimens of immunotoxin (IT) therapy and, eventually, combining this regimen with conventional chemotherapy in an effort to reduce mortality and morbidity from these diseases. Principles that emanate from such studies should be applicable to other types of cancer. The Program Project involves a core facility in which preparation, purification and pre-clinical testing of the immunotoxins is accomplished. Two immunotoxins that differ in antigenic specificity will be prepared for this project. Both consist of intact murine monoclonal IgG antibodies but one is directed to CD22 and the other to CD19. Each of these intact antibodies is covalently conjugated by a heterobifunctional crosslinker containing a hindered disulfide bond (SMPT) to the deglycosylated form of ricin A chain (dgA). A Phase I clinical trial is close to completion for the IgG-CD22 dgA. We have ben able to administer up to 50% of the calculated human LD50 dose with predictable and modest side effects and have not yet reached MTD. We believe that MTD may be reached with the next two dose escalations. Significant efficacy has been demonstrated. It is now planned to obtain FDA approval for a Phase II clinical trial. A Phase I clinical trial is planned for the IgG-CD19-SMPT-dgA. Approval has already been obtained from the FDA for this trial. Finally, a level covered under the aegis of another grant will help us evaluate strategies for designing optimal clinical trials and in particular, the potential combined use of both immunotoxins with conventional therapy.

DESCRIPTION: (adapted verbatim from the investigator's abstract) We have developed a highly sensitive test for detecting, enumerating and characterizing epithelial cells in the blood. The test can detect one such cell in 2 x 108 leukocytes. The cells can be obtained viable or fixed. The method allows characterization of the cells for their malignant nature, genetic alterations, organ of origin and aggressiveness. We have used the method to study the blood of 34 patients with breast cancer, 7 with prostate cancer and 23 controls (normal or non-neoplastic disease). Tumor cells were found in the blood of virtually all the cancer patients, including those that had a tumor clinically confined to the primary site. There was no overlap in the number of blood epithelial cells in the controls vs 21 of 23 cancer patients with clinically organ confined tumor. The latter have cells with the morphology of tumor cells and those with breast cancer, express Mucin-1, cytokeratin, 8, 18, and frequently Her-2 and steroid receptors. We plan to optimize the assay including confirming the cut-off value for distinguishing blood from controls vs patients with cancer, more assays to determine the breast origin of the tumor cells, analysis of DNA alterations by multi-color FISH (MCF), the presence of mutations in oncogenes, cytology and characterization of surface antigens associated with replication and aggressiveness. Our hypothesis is that the assay will detect carcinomas at a very early stage and can aid prognostication. Thus, 1) Can circulating cancer cells be detected in high risk individuals before detection of the primary tumor? 2) Can the test be used to monitor therapy? 3) Can the test predict whether metastatic disease will develop? We also attempt to do short-term culture of the tumor cells from individual patients to study their reactivity to conventional and novel therapeutic agents.

DESCRIPTION (provided by applicant): This proposal will test the hypotheses that: 1.There is concordance between the HER-2 gene status of metastases and circulating tumor cells (CTCs) in patients with [sic] whose primary tumor was HER-2 gene nonamplified and who acquired HER-2 gene amplification of their CTCs. 2. Such patients can respond significantly better to trastuzumab-containing therapy compared to a control group. At present, patients who acquire HER-2 gene amplification in their CTCs are rarely diagnosed and therefore do not receive needed targeted treatment. This is because the primary tumor is considered the "gold standard" for making the diagnosis of HER-2 gene amplification. This assumption should be questioned because the genetically unstable clone of tumor cells is rapidly mutating giving rise to genetic changes. Clinical experience shows that variants appear which are resistant to the particular chemotherapeutic regimen that was employed. Hence, it would be predicted that amplification of genes that make tumor cells more aggressive and resistant to chemotherapy would emerge with tumor progression. In preliminary experiments involving patients with primary breast carcinoma, concordance between the pathologists' evaluation of the primary tumor and FISH evaluation of CTCs was at the 97% level (29 of 30 patients). Of the breast cancer patients whose primary tumor was HER-2 gene non-amplified, HER-2 gene amplification was present in the CTCs of 37% of such patients (9 of 24) after a recurrence. Four of these patients were treated with trastuzumab-containing therapy. Three patients responded with a PR or CR. Testing of concordance between primary and metastatic tumor with concomitantly obtained CTCs will be extended to 275 patients to obtain statistically significant results. Our oncologists will treat patients who have acquired HER-2 gene amplification in their CTCs with targeted treatment. Finally, a new immunofluorescence method for quantifying HER-2 expression will be studied. The long-term goal is to validate the use of CTCs for determination of changes in immunophenotype and genotype of progressive cancer such as acquisition of HER-2 gene amplification. This would ensure that patients who acquire treatable changes in their tumor will receive optimal targeted therapy.

DESCRIPTION (provided by applicant): Breast cancer diagnosed at an early stage is usually cured, but a significant percentage of patients will experience a metastatic recurrence that is not curable despite advances in therapy. The very long interval of time to recurrence seen in some cases suggests the phenomenon of cancer dormancy, but very little is known about the physiology of metastasis and the determinants that govern the occurrence and timing of clinical recurrence. We have demonstrated the presence of circulating tumor cells (CTCs) in patients with prior breast cancer who are 7-22 years post-mastectomy and who have no evidence of disease (NED), thus very likely to be in a tumor dormancy state. Our preliminary data from 40 of these patients indicate that 18 have CTCs, using very stringent criteria including cytomorphology, immunophenotyping and aneusomy. Nineteen normal women had no cells in the blood that met the criteria for CTCs. All personnel involved in the procedures /interpretation were blinded. The procedure involved immunomagnetic purification followed by immunofluorescence analysis for CTCs on slides using the above criteria. From preliminary observations, the half-life of CTCs was estimated to be only several hours. Since 13 of the patients with CTCs are 13- 22 years post mastectomy with NED and the half-life of the CTCs is so short, we postulate that dormancy in breast cancer can represent a precise balance between cell replication and cell death. We plan to extend our studies to a large number (150) of patients 8 or more years after removal of a breast carcinoma with NED, a control group (50) of age-matched normal women, and 50 patients with metastatic breast carcinoma. The CTCs will be studied in-depth by counting, immunophenotyping and genotyping to determine if the CTCs from the dormant group differ from CTCs in metastatic breast carcinomas and whether a relapse is developing. The initial tumors and their treatments in the dormant vs. the metastatic tumor group will also be analyzed for differences. Exploratory experiments to determine the mechanisms responsible for this precise balance will be done. Providing concrete evidence of a dormancy state and being able to characterize the kinetics, genotype and phenotype of CTCs and the mechanisms by which they are regulated have enormous implications in cancer control and treatment.

DESCRIPTION (provided by applicant): This proposal will test the hypotheses that: 1.There is concordance between the HER-2 gene status of metastases and circulating tumor cells (CTCs) in patients with [sic] whose primary tumor was HER-2 gene nonamplified and who acquired HER-2 gene amplification of their CTCs. 2. Such patients can respond significantly better to trastuzumab-containing therapy compared to a control group. At present, patients who acquire HER-2 gene amplification in their CTCs are rarely diagnosed and therefore do not receive needed targeted treatment. This is because the primary tumor is considered the "gold standard" for making the diagnosis of HER-2 gene amplification. This assumption should be questioned because the genetically unstable clone of tumor cells is rapidly mutating giving rise to genetic changes. Clinical experience shows that variants appear which are resistant to the particular chemotherapeutic regimen that was employed. Hence, it would be predicted that amplification of genes that make tumor cells more aggressive and resistant to chemotherapy would emerge with tumor progression. In preliminary experiments involving patients with primary breast carcinoma, concordance between the pathologists' evaluation of the primary tumor and FISH evaluation of CTCs was at the 97% level (29 of 30 patients). Of the breast cancer patients whose primary tumor was HER-2 gene non-amplified, HER-2 gene amplification was present in the CTCs of 37% of such patients (9 of 24) after a recurrence. Four of these patients were treated with trastuzumab-containing therapy. Three patients responded with a PR or CR. Testing of concordance between primary and metastatic tumor with concomitantly obtained CTCs will be extended to 275 patients to obtain statistically significant results. Our oncologists will treat patients who have acquired HER-2 gene amplification in their CTCs with targeted treatment. Finally, a new immunofluorescence method for quantifying HER-2 expression will be studied. The long-term goal is to validate the use of CTCs for determination of changes in immunophenotype and genotype of progressive cancer such as acquisition of HER-2 gene amplification. This would ensure that patients who acquire treatable changes in their tumor will receive optimal targeted therapy.