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Organization of the Organ Systems Branch Current SPORE Programs Information for the Public PART Program Information for Applicants IntraSPORE Communications
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Georgetown University Medical Center
Project 3 We have initiated a series of pilot studies in human breast cancer cell lines and xenografts culture and in needle biopsies of breast lesions in patients to determine the optimal conditions for tissue collection and processing for gene and tissue microarray analyses. We also have developed a novel series of data analysis algorithms for the mining of complex data sets. The analysis of these data sets should allow us to identify molecular profiles that will predict the response to systemic cytotoxic and endocrine therapies in breast cancer patients. Furthermore, we anticipate that the more Ainformation-rich@ genes, identified within the molecular profiles, contribute to their associated phenotype. We will select these genes for further analysis in experimental models to determine their likely functional relevance.
Project 4 Pleiotrophin (PTN) is a secreted growth factor that is mitogenic for a number of cell types (endothelial, epithelial, fibroblast) but can also induce neurite outgrowth. It functions as a growth factor during neonatal brain development, for primate trophoblast formation [1] and during tumor invasion, angiogenesis and metastasis [1,2,7]. In studies of the regulation of the gene we detected a retroviral insertion into the human PTN gene that generates a tissue-specific promoter not present in the murine genome [1,5]. The same retrovirus is as also inserted into human BRCA-1 [5]. Over the past few years we showed in functional studies with ribozyme-targeting that PTN can play a rate-limiting role for tumor angiogenesis, invasion and metastasis [1-3,7,8]. We originally proposed to test the hypothesis that the secreted, heparin-binding growth factor pleiotrophin (PTN) plays a major role in mammary carcinogenesis. We have recently indentified a receptor for PTN and have focussed our research on the understanding of PTN signal transduction and biologic effects in breast cancer via this receptor. In work over the past three years we found that PTN stimulates tyrosine phosphorylation of two proteins in the 190 to 215 kDa range and induces phosphorylation of the MAP kinase and PI3kinase pathways [4]. We now cloned a candidate receptor for PTN (PTN-R) using phage-display of human cDNA libraries. This is a transmembrane orphan tyrosine kinase receptor of a predicted MM of 170 kDa and runs at 210 kDa when transfected into COS-7 cells due to glycosylation. Signal transduction upon stimulation with PTN of transiently transfected cells induces phosphorylation of several proteins similar to the ones identified with the endogenous receptor [4]. Overexpression of the receptor enhances and reduction of the endogenous receptor with ribozymes reduces the stimulation by PTN. Expression studies via immunohistochemistry and RT-PCR show expression in a number of breast cancer cell lines as well as a one-half of primary tumors. Further characterization of the receptor signal transduction, expression and generation of blocking agents is planned under this proposal over the next two years.
Project 5 Recent studies by many investigators have identified representatives of all four major classes of proteases in breast cancer. However, the exact roles of these proteases in tumor angiogenesis growth, and metastasis are not yet clarified. We initially hypothesized in this project that the matrix metalloproteases (including MMP-1, MMP-2, and MMP-9) play a pivotal role in tumor invasion and metastasis of breast cancer. We also proposed that the serine/threonine protein kinase family termed PKC is also important in this process due, at least in part, to its regulation of MMP synthesis and secretion. To test these ideas we designed preclinical laboratory studies and clinical trials of several inhibitors of MMPs and PKC. Subsequently, we discovered a new serine protease (termed matriptase) that we believe also interacts with the MMP system and critically modulates breast tumor invasion and metastasis. Over the course of this project, 9 clinical trials have been completed or are nearing completion of accrual. These have included 7 Phase I and 3 Phase II trials. Studies with the MMP inhibitor Batimastat (BB-94) were completed and published in prior years of the grant. Studies with the improved, orally available MMP inhibitor Marimastat (BB-2516) were also completed and published earlier, but the serum samples from these trials were banked and undergoing current studies in comparison to serum samples from our most recent anti-MMP trial, using the new inhibitor BMS-275-291. BMS-275-291 is thought to be further improved over Marimastat relative to its inflammatory toxicity profile; our initial results were presented at the most recent EORTC meeting. We are now completing a full comparison of sera from BMS-275-271 - and Marimastat - treated patients in order to test the hypothesis that the former is less toxic in association with less modulation of levels of serum TNF, TNFRII, and Her-2 extracellular domain (all thought to be targets of ADAM family metalloproteases, undesirable targets of Marimastat). In terms of laboratory studies, we previously published results of studies demonstrating growth inhibitory effects of Batimastat on the MDA-435/LCC6 tumor xenograft model; we did not observe anti-invasion effects of the drug. We have now completed and submitted for publication a follow-up study demonstrating the induction of urokinase at the stromal-tumor interface of Batimastat-treated MPA-435/LCC6 tumors in nude mice. As noted above, a second area of study of this grant has been to evaluate the role of PKC in regulation of metalloproteases. Two Phase I clinical trials with the PKC-directed drug Bryostatin were carried out; a presentation was made at the most recent ASCO meeting on the combination of Bryostatin with cisplatin, and a manuscript has just been submitted on the same topic. From our basic science studies, we have now published our characterization of the effects of anti PKC agents on in vivo and in vitro models of breast cancer invasion/metastasis; inhibition of PKC blocks MCF-7 substrate adhesion through modulation of a2b1 integrin . In addition, we published a paper demonstrating that inhibition of PKC blocks MCF-7 motility, invasion, and production of multiple classes of proteases. Local invasion and metastasis of breast cancer have been proposed to require imbalanced or unregulated expression of proteases at invading edges of carcinoma cells. However, to date, most of the proteases found associated with the disease (for example, most metalloproteases and urokinase) have been observed to be synthesized in the tumor stroma, as a host reaction to the disease. In addition, an indirect mechanism has been proposed for certain of these proteases whereby the protease is recruited to the surfaces of cancer cells and activated. In contrast, in our most recent studies, we have characterized in breast cancer a novel, integral membrane serine protease, matriptase, and its cognate Kunitz-type inhibitor, hepatocyte growth factor activator inhibitor-1 (HAI-1) system. While matriptase was isolated based on its ability to degrade gelatin, we have observed that it is capable of activation of urokinase (but not plasminogen) and of hepatocyte growth factor (HGF); MMP studies are underway. Future studies are focussed on establishing the molecular basis and prognostic significance of disregulated activation and expression of matriptase in breast cancer. Molecular modeling and biochemical studies also are currently addressing potential small molecule and natural product inhibitors of matriptase for therapeutic application to breast cancer.
Project 6 Preliminary laboratory and clinical data indicate that ex vivo IL-2 activation of hematopoietic stem cells from patients with breast cancer will induce a syndrome compatible with autologous graft vs. host disease when infused following high dose chemotherapy and parenteral IL-2. Correlative pre-clinical studies suggest an associated graft vs. tumor effect. A Phase III Clinical Trial evaluating immune therapy post-transplant was initiated with the primary clinical endpoints of determining disease-free and overall survival. To date, sixty-one patients have been registered from 3 centers. All patients received cyclophosphamide (6 gms/m2), carboplatin (800 mg/m2) and Thiotepa (500 mg/m2) for 4 days. Two days later, autologous stem cells (SC) were infused. Patients randomized to the immune therapy arm received autologous SC activated in vitro with IL-2 (1000 I.U./ml) for 24 hrs. and parenteral IL-2 (6 x 105 I.U./m2 SQ qd) beginning on the day of transplantation and continuing for 30d. To date, 59 patients with a mean age of 49.2 yrs. (range 34-68 yrs.) have received transplantation (ST II/IIIA n = 36; Stage IIIB n = 14; Stage IV n = 11). Engraftment of neutrophils occurred on day 10 (mean; range 8-12d) and platelets on day 9.6 (mean; range 6-15d). There have been no treatment-related deaths. Six of 29 patients were removed from parenteral IL-2 due to infection (n = 5) or patient's desire (n = 1). No difference in toxicities was observed in the two treatment arms. Level 3 (moderate) toxicities have been transient and include infection (n = 3), diarrhea (n = 1), mucositis (n=3), metabolic abnormalities (n = 5), nausea (n=1) or cardiac arrhythmia (n = 1). One patient not receiving IL-2 developed Level 4 (severe) esophagitis and rash. Laboratory correlates demonstrated an increase in the percentage of CD3+ T-cells (> 3 x control) and CD56+ NK cells (>2 x control) at 3-4 weeks post-transplant in the peripheral blood of patients receiving IL-2 (controls = patients not receiving IL-2 therapy). When patients' peripheral blood lymphocytes were evaluated in a standard 51Cr-release assay (E: T 100:1), there was increased cytotoxicity against human breast cancer cell lines in those patients who received IL-2 compared to controls. This effect persisted for 4 weeks after transplantation. ELISA demonstrated a 2-fold increase in serum IL-2 receptor levels in patients receiving IL-2 that persisted for 8 weeks following transplant. The trial continues to accrue patients and clinical outcomes are being monitored. Additional evaluations are examining the presence of autologous graft versus host disease and a possible graft versus tumor effect.
Project 7 Vascular endothelial growth inhibitor (VEGI) is a new member of the TNF family (Zhai et al., IJC 82:131, 1999). The VEGI gene is expressed predominantly in endothelial cells. The gene product exhibits potent antiangiogenic and anticancer activities. Recombinant VEGI induces growth arrest and apoptotic death of endothelial cells. We have now identified a number of new isoforms of VEGI that apparently are differential splicing products of a common gene transcript. The tissue distribution profiles of these isoforms are distinctly different. The C-terminal segment of 152 residues of each of the isoforms is identical to that of the originally identified VEGI, which is 174-residue long and is now renamed VEGI174. A recombinant protein consisting of this C-terminal segment fully retains the antiangiogenic activity. The N-terminal segments of the isoforms share no sequence homology. One of the isoforms, VEGI251, consists of 251 amino acid residues and contains a secretion signal-like hydrophobic domain. Transduction of human umbilical cord vein endothelial cells (HUVEC) with a lentiviral vector carrying the VEGI251 cDNA resulted in cell death. Similar results were obtained if the virus was carrying a cDNA encoding a secreted form of VEGI in which the activity domain was linked to a secretion signal peptide derived from interleukin-6. No cell death was induced, however, by the virus carrying VEGI174. Microscopic analysis of HUVEC cells transiently transfected with VEGI251 revealed an intracellular trafficking pattern of the protein consistent with that of a secreted protein. When VEGI251 was overexpressed in human breast cancer MDA-MB-231 cells, the protein was located in the conditioned media of the cell cultures. The VEGI251-overexpressing cancer cells exhibited markedly decreased tumor growth rates when implanted into the mammary fat pads of female athymic nude mice, as compared with parental cells and vector-transfected cells. Overexpression of VEGI174 in the cancer cells, in contrast, had no effect on their tumorigenicity. These findings are consistent with the view that VEGI251 is secreted by endothelial cells and negatively regulates the growth of the same cells through an autocrine mechanism.
Project 8 We have taken three approaches to block the HER-2/neu signaling pathway based on a site of action. First is binding site inhibitors which compete directly with ligand at that site; second is kinase inhibitors which compete with ATP binding site or interact with the regulatory or substrate binding and, third, inhibitors that interfere with other downstream signal transduction elements. We have identified small molecules that inhibit these actions at these three sites in the transmission of abnormal growth signals through the HER-2/neu signaling pathway. The first approach aims at preventing activation, whereas the latter two approaches aim at preventing signal transduction. Antagonists of Growth Factor Heregulin - We have used a structure-based strategy towards the discovery of small molecules as potential specific heregulin (HRG) antagonists. Three classes of lead compounds were discovered and we have demonstrated previously proof of the principle that small molecules discovered through structure and computer pharmacophore search can block HRG binding and inhibit the biological activity of HRG receptors. The lead compound SMA1 (RA7) is orally active in the in vivo anti-tumor studies and could enhance the sensitivity of Cisplatin both in vitro and in vivo in human breast cancer cell line MDA-MB-231. Moreover, inhibition of HRG-induced phosphorylation of HRG receptors can lead to inhibition of MAP kinase activation and protein kinase B/Akt phosphorylation both in vitro and in vivo. HER-2/neu selective kinase inhibitor - Using the 3D structure of HER-2 kinase domain, we have performed a structure-based database search of 500,000 small molecules using the MCDOCK program. Compounds that are able to fit into the binding pocket and have favorable interaction energy with HER-2 are considered as potential lead compounds. To date, six selective HER-2 kinase inhibitors have been discovered and one such inhibitor has a sub-micromolar potency (IC50) in MDA-453 cells over-expressing HER-2 and over 100-fold selectivity for HER-2 over EGFR. When tested in pilot animal studies, the lead inhibitor B17 demonstrated in vivo activity in blocking the HER-2 kinase when administered in tumor-bearing mice. More recently, in animal experiments, the lead inhibitor B17 significantly blocked the tumor cell proliferation of the established human breast carcinoma. Inhibitor of Adapter Protein Grb2 - An early intermediate in the signaling transduction in the HER-2 pathway is the adapter protein Grb2. Blocking interactions between activated HER-2 tyrosine kinase and Grb2 SH2 domains has the potential to shut down signaling through this mitogenically important pathway. For this reason, Grb2 SH2 domain inhibitors could potentially constitute attractive agents for the treatment of breast cancer where HER-2 receptors have been shown to play important roles. Using a beta-bend mimicking tripeptide motif previously described and in collaboration with Dr. Burke at NCI, we recently found non-phosphate-containing analogues that are able to inhibit Grb2 SH2 domain binding with IC50 values down to the low nanomolar range. When administered to cells in culture, effective inhibition of intracellular Grb2 binding to cognate HER-2 protein and oblation of MAP kinase signaling is also observed. Adding to the therapeutic value of these agents is their cytostatic effects, which may help to delay the growth of cancer cells, and their lack of cytotoxicity when administered to HER-2 overexpressing cancer cells. In studies in which inhibitors were used in combination with the treatments taxotere, 5-FU, or doxorubicin, greater inhibition of growth was observed than with any agent alone. Because of the high relevance of HER-2 in breast cancer, the discovery of potent and selective HER-2 signaling inhibitors will represent the first but very important step to develop HER-2 targeted drug candidates as effective therapies for the treatment of breast cancer, either used alone or in combination with other therapies. We believe that the combined efforts of this multi?disciplinary team will bring significantly improved small, non?peptidal molecule drugs into a phase I clinical trial in the near future.
Project 9 The tumor suppressor gene p53 has been shown to be involved in the control of DNA damage-induced apoptosis. Loss or malfunction of this p53-mediated apoptotic pathway has been proposed as one mechanism by which tumors become resistant to chemotherapy or radiation.
Core A The Breast Cancer Cell Line Resource exists to provide support to SPORE investigators in all aspects of their tissue culture needs. This it does in several ways: 1) Breast Cancer Cell Collection - The Resource manages the Lombardi Cancer Center Cancer Cell Line Repository - an extensive collection of normal and tumor cell lines stored in a series of liquid nitrogen freezers. These are made available to investigators either as frozen vials of cells or as growing cultures. Liquid nitrogen storage space is also available for investigator's cells. Breast cancer cell lines are made available to other SPOREs' investigators for the cost of shipping. 2) Shared Culture Space- Three separate shared tissue culture rooms are equipped and maintained by the Resource. These rooms provide culture facilities to investigators do not have such facilities in their own lab. 3) Quarantine Lab and Mycoplasma Testing - A 200 square foot quarantine laboratory is provided for investigators to culture cells prior to mycoplasma testing, and the Resource provides a mycoplasma testing service. 4) Helper Virus Assay - A helper virus assay service is offered for investigators working with retroviruses. 5) EBV Transformation -To support those investigators engaged in studies of the genetics of breast cancer, the Resource provides an EBV immortalization service. Blood from patients is used to generate lymphoblastoid cell lines to provide an unlimited supply DNA for genetic testing. 6) Culture Reagents - Batches of various tissue culture reagents such as fetal calf serum are tested by the Resource, which then uses its considerable purchasing power to obtain large quantities these reagents at highly competitive prices. These reagents are then provided to investigators at cost. 7) Primary Cultures - The preparation of primary cultures of both normal and tumor cells and the generation of cell lines from these cultures is another service that the facility provides. 8) General Services - A number of other services are offered. These include the preparation of charcoal stripped serum, the culture of large numbers of cells for animal experiments, verification of the identity of the cells by DNA finger printing, freezing large numbers of cells for distribution to other investigators, and the preparation of cell pellets and lysates for various purposes.
Core B The Breast Cancer Tumor Bank Core is part of the Histopathology and Tissue Shared Resource which is IRB approved. Dr. Baljit Singh is the director of the resource. Ms. Susan Constable is the manager and supervises three research assistants. The primary objectives of this shared resource are threefold: (1) To provide comprehensive tissue procurement, histologic, and tissue processing services, (2) To make available pertinent frozen and fixed tumor and normal tissues from patients with follow-up information for use by Lombardi Cancer Center investigators and (3) To provide expert assistance in conducting experiments using specimens from human and animal subjects, including assistance in interpreting immunohistochemical and in-situ hybridization studies.. A dedicated research assistant collects breast tumor and corresponding normal specimens from the surgical pathology suite. The bank contains more than 150 frozen breast cancer specimens. The pathology archives contain paraffin blocks from all the patients operated at Georgetown University Medical Center and these are available for investigative purposes. The pathology index was computerized in 1991 and natural work searches can be done to locate cases from that period onward. Demographic information is never revealed to the investigators and all specimens released for research have fictitious identifiers, which can not be linked to the patient. Approximately forty-five investigative laboratories utilized the shared resource services annually. All protocols requiring human material must have IRB approval or qualify for exemption. The Scientific Research Committee reviews large requests for tissue for scientific approval and statistical validity. In 1999, The Core acquired Laser Capture Microdissection capability by installing the Arcturus pixcellTM. A histopathology slide (without cover slip) is placed on the translation stage of a microscope, and the user selects an area of interest in the center of the field of view. Next, a transport arm allows the user to place a transparent thermoplastic transfer film, which is bonded to the bottom of a cap, in direct apposition to the selected section. Upon activation of an infrared laser, which is integrated with the microscope, mild thermal transients allow the thermoplastic film to rapidly melt and form a bond with the tissue that is stronger than the bond between tissue and the slide. The cells of interest are now transferred to the thermoplastic film. The Core has also acquired a "Tissue Arrayer" (Beecher Instruments). This technology has the capability of creating a paraffin block with up to 1,200 cores of tissue embedded in it. This allows for the creation of "Tissue microarrays", which can be used for comprehensive molecular profiling of cancer. An array of "normal structures" could be used to study expression of novel proteins in various organs. This is a powerful research tool that significantly enhances our capability to ask "tissue" related questions simply and at a substantially reduced cost. A tissue microarray of 54 breast carcinoma has been prepared with five-to-ten cores of tissue from each cancer. An analysis of representing heterogeneity of tissues in a microarray was presented at the US and Canadian Academy of Pathology meeting at New Orleans, March 25-31, 2000 and the SPORE workshop in June 2000 at Chantilly, VA.
Core C The overall purpose of the Clinical Research Core is to provide a bidirectional source of clinical material for performance of translational research studies in breast cancer. The Core Director, Dr. Claudine Isaacs has considerable experience in design and conduct of clinical trials, and she is the Director of the Cancer Assessment and Risk Evaluation (CARE) program. The Clinical Breast Cancer Core contains eight medical oncologists, two surgical oncologists, a radiation oncologist, and a breast cancer pathologist who devote part or all of their clinical effort to care of breast cancer patients. The Clinical Breast Cancer Program is a fully coordinated and comprehensive unit designed to place patients on clinical trials that will address issues of risk and genetics, chemoprevention, primary evaluation and therapy, adjuvant therapy, prognosis, and treatment of metastatic disease. The clinical trial portfolio consists of "in-house" studies, cooperative group (CALGB, NSABP) trials, and pharmaceutical investigations. Meetings are held twice a week with all clinicians involved in LCC breast cancer trials. One of these is designed to discuss individual new patients and changes in clinical course of current patients to assess trial eligibility, and to serve as a forum for review of ongoing protocols (accrual, toxicities, etc) and discussion of new protocol concepts. The second meeting serves partly as a "tumor board," but perhaps more importantly as a forum for didactic interactions regarding important clinical issues surrounding evaluation and treatment of breast cancer. Once a month, laboratory-based scientists are asked to present their data at this meeting, increasing the potential collaborative interaction between clinical and basic scientists in the SPORE. Dr. Isaacs meets on a rotating fashion with individual SPORE investigators to determine how to further facilitate collaborations between the clinical and laboratory programs. In regards to clinical trials, the Georgetown SPORE investigators are conducting a prospective randomized trial, of ex vivo treatment of PBSC's with IL2, followed by parenteral IL2, for patients undergoing high dose chemotherapy (see Dr. Meehan's abstract for details). Dr. Isaacs and her predecessor, Dr. Daniel Hayes, have collaborated closely with Dr. Meehan in the design of the completed pilot, in which the optimal dose of IL2 was determined. Accrual to the randomized trial began in late 1997 and is proceeding at the expected rate. The clinical core has also played a major role in the revision of Dr. Clarke's Project in which pre-treatment breast cancer tissue will be obtained for gene expression analysis prior to preoperative "neo-adjuvant" hormone and chemotherapy. Core biopsy samples will be provided to Dr. Clarke's laboratory for expression array studies, and the results will be correlated with clinical and pathological response (see Dr. Clarke's abstract for full details). Dr. Isaacs has been involved with Dr. Shields and Dr. Singh in further development of LCC specimen repositories linked to clinical information. They have planned a system, designated "Clinical Outcomes Research Database (CORD), which is linked to the Breast Cancer Biomarker Resource (Core E). Breast Cancer patients seen in the Lombardi Cancer Center clinic are now asked to participate in the CORD/Biomarker resource project, in order to develop a bank of specimens for future translational research. This system will dramatically enhance the ability of SPORE investigators to identify important diagnostic and prognostic factors generated in their laboratories. Under Director for Lombardi's Cancer Prevention, Control and Outcomes Program Dr. Jeanne Mandelblatt's direction, these investigators have also incorporated a quality of life questionnaire into the CORD database. This strategy will permit correlation between patient's disease status, outcomes, biomarkers, and quality of life. In summary, the clinical core is enthusiastically participating in several SPORE projects. It is anticipated that during the upcoming year several collaborations between Dr. Isaacs and her clinical colleagues and the laboratory scientists will continue to generate important data.
Core D The Transgenic Shared Resource exists to assist SPORE investigators in the use of transgenic technology in their research. To this end the Resource provides a number of services related to the use of existing transgenic mouse models and to the development of new models. These include providing advice and assistance with the genotyping of transgenic animals, the design and construction of DNA constructs for making new transgenic models, and the production of new transgenic mice by pro-nuclear injection of DNA constructs. An embryo freezing service allows investigators to cryo-preserve transgenic models they are not currently working with and services under development include an embryonic stem-cell culture service that will facilitate the development of targeted mutant mouse models. The Resource has a collection of targeting vectors commonly used in the production of transgenic models, particularly relating to mammary cancer and can assist investigators in the identification and procurement of a suitable vector for their work. Core E The Breast Cancer Serum Biomarker Resource (BCBR) core is responsible for maintaining and expanding a bank of sequential serum, plasma, and lymphocyte samples linked to a computer based with associated clinical and follow-up data. The core facility provides SPORE investigators with access to patient samples and consultation for design of molecular clinical correlation studies. The BCBR includes samples from breast cancer patients, and healthy women with a normal or high risk of breast cancer. The core facility allows molecules under investigation by SPORE researchers to be evaluated clinically as markers of early breast cancer, tumor burden, and for predicting response to specific therapies. Measuring such markers in serum or plasma as opposed to tumor tissue allows serial measurements to be made during the course of treatment and follow-up. Serial changes in markers values from baseline can be correlated with disease progression and treatment failure. Once tests become available for determining carrier status for breast cancer predisposing mutations (e.g. BRCA1), the lymphocyte samples will be extremely valuable for studying the pathogenesis of heritable breast cancer. The BCBR was initially funded as a component of the Clinical Breast Cancer Research Core in the LCC Breast Cancer SPORE in 1992. At that time it was a newly developed resource, with samples to be obtained from new and continuing LCC Patients. Subsequent to the funding of the LCC SPORE, an extensive serum/plasma bank and clinical database with more than 18,000 serial samples and long-term follow-up on more that 1500 patients was acquired from the University of Wisconsin Cancer Center (UWCC). Breast cancer patients treated and response data, and follow-up data were maintained on a computerized database. Because of the large size and comprehensive nature of the UWCC serum bank, the scope of operations of the BCBR increased considerably, necessitating the establishment of the BCBR as a separate SPORE core facility. It is a primary goal of the BCBR to make these samples and the associated database accessible and available to other breast cancer SPORE centers. |
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