Jeffrey R. Marks - US grants

Dramatic differences have been identified in the ability of breast cancer cells to respond to retinoic acid (RA) both in their ability to continue proliferation and to specifically induce transcription mediated by a retinoic acid response element (RARE). Given the multitude of RA receptors (RAR), isoforms, and other interacting proteins in the RA pathway, this proposal seeks to identify the mechanism of aberrant response using a functional rather than purely genetic or DNA sequence based approach. Initially, efforts will concentrate on four cell lines, two RA high responders and two low responders. 1) Stable transfection of an RARE-chloramphenicol acetyl transferase (CAT) gene into each of these cell lines will provide targets for RA induction assays without the variability associated with transient transfections. A functional RAR will also be stably introduced into these cell lines to explore whether non-responders can be converted to responding cells. 2.) In responsive and nonresponsive cells, RAR transcripts will be examined by hybridization. Expression of receptor proteins will be assesed by immunoblots using recently developed antisera. RA binding to cytosolic and nuclear extracts will be measured by radiolabeled ligand binding studies. 3.) The binding of human RARalpha to specific DNA elements will be measured by gel-shift assays and the effect of adding nuclear and cytosolic extracts from responsive and nonresponsive cells will be investigated. 3) In order to identify genes that are regulated by RA in breast cancer and in normal mammary epithelial cells, differential display polymerase chain reactions will be performed comparing RA responsive, non-responsive, and normal cells. These messages will be cloned and sequenced to determine their identity. Expression of these transcripts will be investigated in primary tissues before and after all-trans RA treatment in a Phase I-II study underway in the Duke Breast Oncology Clinic. In this way, we hope to identify surrogate markers of RA responsiveness which may guide therapy. These studies may identify alterations in gene regulation which are involved in tumor initiation, progression, and which can by used in breast cancer therapy.

Identification of the hereditary breast and ovarian cancer susceptibility gene, BRCA1, has led to increased awareness regarding the disease, increased surveillance in women with a family history, and the advent of genetic testing for mutations. However, our knowledge regarding the role of this gene in sporadic cancers and its function in normal and neoplastic growth and development is sparse. This proposal is a continuation of studies performed over the past two years designed to investigate these issues. The following experiments are outlined in this proposal: 1) Since BRCA1 is rarely a target for somatic mutation breast cancer, is it inactivated by loss of expression? Using microdissected normal epithelium, carcinoma in situ, and invasive cancers, both expression and promoter methylation will be evaluated employing PCR based methodology. 2) BRCA1 mRNA expression is tightly regulated through the cell cycle. Alterations in expression, either the levels of temporal nature, may find its function. Therefore, the basal and cell cycle regulated promoter elements will be identified. Using the minimal promoter fragment, the identity of trans-acting factors that complex with specific promoter sequences will be established. These experiments will complement and extend those proposed in the first aim. 3) It has been difficult to derive cell lines that over- express BRCA1 in order to study phenotypic and molecular events associated with the protein. In order to overcome this problem, tetracycline inducible cell lines have been established. BRCA1 under the test-inducible promoter will be introduced into these cells to study phenotypic effects of BRCA1 over-expression including cell cycle parameters, apoptosis, response to DNA damage, and response to differentiating agents. 4) Using these same cell lines, genes that are induced by BRCA1 expression will be identified using representational difference analysis (RDA). From the promoter elements of these genes, common BRCA1 response elements will be distinguished.

The SPORE in Breast Cancer at Duke University is entering its third and final year. We are answering an RFA to renew the Duke SPORE for two additional years, bringing our Program into cycle with other NCI SPORE sites. Our renewal includes six major Projects, four Core Resources, a Pilot Project and a Career Development Award. The emphasis of our SPORE is in two major areas of research: the genetics of breast cancer susceptibility and the development of new therapeutics. Three Projects related to susceptibility genetics. Project 2 investigate the function the function of BRCA1 and its potential role in sporadic breast cancer. Expression of BRCA1 will be assessed in clinical cancer, modulated in experimental cell lines and examined during progression of the cell cycle. Project 3 will conclude a randomized trial of counseling which compares standard informed consent materials to education information tailored and the individual patient. Baseline descriptive data and the results of the randomized trial will be analyzed in years four and five. Project 4 competes for inclusion in the SPORE; this project was disallowed after the first year. The Project developed a statistical algorithm to calculate the carrier probability for breast and ovarian susceptibility based upon characteristics of the family history. This algorithm will be improve, validated and translated into clinical utility during our last two years. Core 1 has developed a family ascertainment procedure, including an epidemiological questionnaire and data management system. A genetic testing laboratory can determine sequence alterations in BRCA1/2 rapidly and at low cost, providing free testing to Project 3 subprojects. Three Projects are concerned with new therapies for breast cancer. Project 1 has discovered a new estrogen antagonist with improve therapeutic characteristics compared to tamoxifen. A clinical trial is planned. Laboratory work concerns the pre-clinical efficacy and mechanism of action of this new drug. Project 5 continues to characterize anti-tumor cellular reactivities in breast cancer patients and proposes a vaccination trial in cancer patients. Project 6 has chosen to focus on inhibitors of endothelial growth factor receptors, working toward small molecule inhibitors that have clinical utility. Biomarkers of angiogenesis will be developed and clinically tested. Core 2 for Information Management has developed the Family Cancer Database, the Tissue Resources Database and provides statistical support for major Projects, in particular the large amount of data generated in Project 3. Core 4 provides frozen and paraffin embedded material from more than 100 patients and operates a modern preclinical animal testing facility used by Projects 1 and 6 for large scale therapeutic trials. A Pilot Project Program and Cancer Development Award are described. Core 3 administers the SPORE and includes a strong emphasis on education, continuous improve on research and involvement of Patient Advocates.

This proposal will attempt to identify expression-based markers for breast cancer and develop assays using these markers to detect circulating breast cancer cells. Our application has been developed as a collaborative effort between the Duke Breast Cancer Program and Abbott Diagnostic Breast Cancer Venture Group and will utilize the considerable strengths of each institution. The basic approach for marker identification takes advantage of ongoing (and separately funded) efforts using powerful new genomics tools. At Duke, SAGE (serial analysis of gene expression) libraries of breast cancer and normal breast epithelium are being constructed as part of CGAP. Also at Duke, a series of 20 primary breast cancers will be analyzed using high-density Affymetrix expression chips (30,000 genes/ESTs will be sampled). Finally, at Abbott Diagnostics, markers have already been identified by using the Incyte Corporation expression libraries. These three sources of expression data will be compared electronically and the most promising markers that are expressed at high levels in a tissue or cancer specific manner will be identified. After the in silico analysis, the lead markers will be put through a validation algorithm that will include northern blotting, multiple tissue RNA dot blots, RT-PCR, and in situ cytohybridization. A panel of breast cell lines and a series of 50 primary breast cancers will be used to determine how commonly expressed these markers are in the target tissue. After these initial validation steps, a series of reconstitution experiments will be performed by spiking normal whole blood with known numbers of breast cancer cells, derived from cell lines and malignant effusions. Epithelial cells will be purified using magnetic bead/antibody technology and the resulting purified samples will be analyzed by flow cytometry, histology, and quantitative RT-PCR. Towards increasing sensitivity, a separate and parallel screen will focus on markers that are highly overexpressed in a subset of breast cancers. PCR multiplexing approaches will be explored in this context and will also include using telomerase gene (TERT) expression as a cancer specific marker in conjunction with other lead markers. Antibodies to the most promising candidates will be developed, particularly for gene products that may be secreted or presented on the cell surface. Finally, a collection of whole blood and serum samples will be obtained from women newly diagnosed with breast cancer at Duke. This bank will constitute a primary validation set for markers and approaches that have proven to be the most robust.

DESCRIPTION (provided by applicant): We propose to create a Clinical Validation Center within the Early Detection Research Network that is focused on breast and gynecologic cancers. Our group brings a combination of depth of experience and clinical resources that would be difficult to match. This Center will be comprised of two primary sites: Duke University Medical Center (DUMC) and Johns Hopkins Medical Institute (JHMI). Each of these sites will contribute clinical resources including specimens and data and will collaborate and coordinate on biomarker validation trials. The primary mission of our group will be to compare promising biomarkers for their utility in specific clinical applications for breast, ovarian, and cervical cancer. We propose that this will include studies of overall risk, early detection, likelihood of disease progression, and disease monitoring. We not expect that all such studies will be performed for all diseases; however we are prepared to perform several such studies at the onset of the program. These studies include markers of high risk lesions in cervical dysplasia, markers of progression in pre-invasive breast cancer, and predictors of outcome in serous ovarian cancer. The resources that we will use to accomplish these goals include: 1) High and consistent patient volume and subject recruitment, 2) Large and mature collections of clinical specimens including serum, plasma, DNA, and tissue all linked and updated regularly, 3) Large subsets of specimens are already annotated with molecular data including gene expression, CGH, SNP, methylation, and proteomic analyses, 4) A large population based case control study in ovarian cancer, 5) Access to other large data and clinical resources including the Ovarian Cancer Association Consortium and first contributor information from The Cancer Genome Atlas (TCGA) for both breast and ovarian cancer. Our expertise comes from biomarker discovery and development in these diseases and is augmented by the various rigors of population science, statistics, and clinical research. Our Center will also participate fully in all activities of the Network and seek additional collaborations within and outside the Network as needed to advance the work.

Abstract Mammography is an early detection modality for breast cancer that is implemented widely in the United States, has established benchmarks of performance, and in most studies throughout the world has been demonstrated to reduce mortality due to the disease. This relatively inexpensive x-ray imaging of the breast also provides a location that can be directly sampled through needle biopsy which leads generally to an unambiguous pathologic diagnosis of invasive cancer, carcinoma in situ, or benign findings. No system is perfect and mammographic screening, particularly in the US, prompts over 1.6 million biopsies per year detecting approximately 230,000 invasive and 60,000 non-invasive cancers for a positive predictive value of less than 20%. There may be substantial room to improve on this and reduce the number of biopsies but this improvement must not sacrifice detection rates so the negative predictive value (NPV, identification of true negatives) must remain very high. In this Biomarker Development Laboratory application, we propose to test whether a combination of mammographic feature analysis and candidate biomarkers that we have identified can achieve an NPV that would be acceptable to patients and providers to prevent unnecessary breast biopsies. One of the biomarkers is a type of circulating giant cell termed ?Cancer Associated Macrophage Like? (CAML) that can only be detected using freshly drawn whole blood, we propose to conduct a prospective trial at Duke University in women undergoing breast cancer diagnosis. Our realistic goal is to accrue ~1000 women over the course of 4 years for which full field digital mammography has been performed. The images will undergo feature extraction for decision modeling. Blood will be analyzed for the presence and type of CAML cells, immunosignaturing using the high density peptide arrays developed by Stephen Johnston at Arizona State, and measurements of two specific analytes that have the highest sensitivity and specificity for basal type cancers, CA125 and TP53 autoantibodies. As feature analysis from imaging alone can achieve, at least for masses on mammography, an AUC of ~0.9, the study is designed to determine whether the biomarkers have sufficient complementary information to the imaging and each other to increase the AUC to 0.95 allowing us to identify a threshold where there is a 98% NPV. We will make use of the most careful and consistent standard operating procedures, the best candidate biomarkers, and the most well developed imaging algorithms to make this a definitive study.