Timothy P. Fleming

Circulating Tumor Cells in Patients with Breast Cancer Dormancy

Purpose: The purpose of this study was to test the hypothesis that circulating tumor cells (CTCs) are present in patients many years after mastectomy without evidence of disease and that these CTCs are shed from persisting tumor in patients with breast cancer dormancy. Experimental Design: We searched for CTCs in 36 dormancy candidate patients and 26 age-matched controls using stringent criteria for cytomorphology, immunophenotype, and aneusomy. Results: Thirteen of 36 dormancy candidates, 7 to 22 years after mastectomy and without evidence of clinical disease, had CTCs, usually on more than one occasion. Only 1 of 26 controls had a possible CTC (no aneusomy). The statistical difference of these two distributions was significant (exact P = 0.0043). The CTCs in patients whose primary breast cancer was just removed had a half-life measured in 1 to 2.4 hours. Conclusions: The CTCs that are dying must be replenished every few hours by replicating tumor cells somewhere in the tissues. Hence, there appears to be a balance between tumor replication and cell death for as long as 22 years in dormancy candidates. We conclude that this is one mechanism underlying tumor dormancy.

Differential Protective Activity of αA- and αB-crystallin in Lens Epithelial Cells

αA- and αB-crystallins are molecular chaperones expressed at low levels in lens epithelial cells, and their expression increases dramatically during differentiation to lens fibers. However, the functions of αA- and αB-crystallins in lens epithelial cells have not been studied in detail. In this study, the relative ability of αA- and αB-crystallin, in protecting lens epithelial cells from apoptotic cell death was determined. The introduction of αA-crystallin in the transformed human lens epithelial (HLE) B-3 lens epithelial cell line (which expresses low endogenous levels of αB-crystallin) led to a nearly complete protection of cell death induced by staurosporine, Fas monoclonal antibody, or the cytokine tumor necrosis factor α. To further study the relative protective activities of αA- and αB-crystallins, we created a cell line derived from αA−/−αB−/− double knockout mouse lens epithelia by infecting primary cells with Ad12-SV40 hybrid virus. The transformed cell line αAαBKO1 derived from αA/αB double knockout cells was transfected with αA- or αB-crystallin cDNA contained in pCIneo mammalian expression vector. Cells expressing different amounts of either αA-crystallin or αB-crystallin were isolated. The ability of αA- or αB-crystallin to confer protection from apoptotic cell death was determined by annexin labeling and flow cytometry of staurosporine- or UVA- treated cells. The results indicate that the anti-apoptotic activity of αA-crystallin was two to three-fold higher than that of αB-crystallin. Our work suggests that comparing the in vitro annexin labeling of lens epithelial cells is an effective way to measure the protective activity of αA- and αB-crystallin. Since the expression of αA-crystallin is largely restricted to the lens, its greater protective effect against apoptosis suggests that it may play a significant role in protecting lens epithelial cells from stress.

Uteroglobin/Clara cell 10-kDa family of proteins: Nomenclature committee report

COMMITTEE CHAIR: J. KLUGa,b COMMITTEE MEMBERS: H. M. BEIER,c A. BERNARD,d B. S. CHILTON,e T. P. FLEMING, f R. I. LEHRER,g L. MIELE,h N. PATTABIRAMAN,i AND G. SINGHj bInstitut für Molekularbiologie und Tumorforschung (IMT), Philipps-Universität Marburg, D-35033 Marburg, Germany cDepartment of Anatomy and Reproductive Biology, RWTH University of Aachen, D-52057 Aachen, Germany dUnit of Industrial Toxicology and Occupational Medicine, Faculty of Medicine, Catholic University of Louvain, B-1200 Bruxelles, Belgium eDepartment of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, USA fDepartment of Surgery, Washington University School of Medicine, St. Louis, Missouri 63110, USA gDepartment of Medicine and Molecular Biology Institute, University of California (Los Angeles) School of Medicine, Los Angeles, California 90095-1690, USA hCardinal Bernardin Cancer Center and Department of Pathology, Loyola University Medical Center, Maywood, Illinois 60153, USA iAdvanced Biomedical Computing Center, SAIC-NCI/FCRDC, Frederick, Maryland 21702-1201, USA jDepartment of Pathology, University of Pittsburgh School of Medicine and VA Medical Center, Pittsburgh, Pennsylvania 15240, USA

Mammaglobin, a breast-specific gene, and its utility as a marker for breast cancer.

Abstract: The mammaglobin gene encodes a 10‐kDa glycoprotein that is distantly related to a family of proteins that includes rat estramustine binding protein (EMBP)/prostatein and human Clara cell 10‐kDa protein (CC10)/uteroglobin. Among normal adult tissues, mammaglobin mRNA expression has been detected only in the mammary gland. As an initial step to determine mammaglobins clinical utility as a breast tumor marker, we evaluated the frequency and specificity with which mammaglobin expression could be detected in primary breast tumors, metastatic breast tumors, and breast tumor cells present in the peripheral circulation. Approximately 80% of all primary and metastatic breast tumors examined were strongly immunopositive for mammaglobin protein, and staining was independent of tumor grade. Among peripheral stem cell collections from breast cancer patients, mammaglobin mRNA could be detected in 60% of cases. Recent work has identified the secreted mammaglobin protein in the sera of some breast cancer patients using both Western blot and ELISA. This study demonstrates that the detection of mammaglobin protein and mRNA in clinical samples may be a useful marker for primary, metastatic, and occult breast cancer.

Isolation and molecular profiling of bone marrow micrometastases identifies TWIST1 as a marker of early tumor relapse in breast cancer patients.

Purpose: Micrometastatic cells detected in the bone marrow have prognostic significance in breast cancer. These cells are heterogeneous and likely do not exhibit uniform biological behavior. To understand the molecular diversity of disseminated cancer cells that reside in bone marrow, we enriched this cell population and did global gene expression profiling in the context of a prospective clinical trial involving women with clinical stage II/III breast cancer undergoing neoadjuvant chemotherapy. Experimental Design: Enrichment of TACSTD1 (EpCAM)–expressing cells from bone marrow of breast cancer patients was achieved using immunomagnetic beads. Gene expression profiles were compared between enriched cell populations and whole bone marrow from 5 normal volunteers and 23 breast cancer patients after neoadjuvant chemotherapy treatment. Enriched cells from bone marrow samples of breast cancer patients before treatment or at 1 year follow-up were also analyzed (total of 87 data sets). The expression of transcripts specifically detected in enriched cell populations from breast cancer patients was correlated with 1-year clinical outcome using quantitative reverse transcription-PCR in an independent cohort of bone marrow samples. Results: Analysis of EpCAM-enriched bone marrow cells revealed specific expression of a subgroup of transcripts, including the metastasis regulator, TWIST1. Most transcripts identified, including TWIST1, were not expressed in enriched populations of bone marrow from normal volunteers, suggesting that this expression profile reflects a signature of breast cancer bone marrow micrometastases that persist after chemotherapy. In an independent set of bone marrow samples obtained before any treatment, TWIST1 expression correlated with early disease relapse. Conclusions: Disseminated breast cancer cells present in bone marrow after chemotherapy possess unique transcriptional signatures. Genes whose expression is overrepresented in these cell populations, such as TWIST1, may prove to be excellent markers of early distant relapse in breast cancer patients.

Transcriptional repression of epithelial cell adhesion molecule contributes to p53 control of breast cancer invasion.

p53 is a tumor suppressor gene with well-characterized roles in cell cycle regulation, apoptosis, and maintenance of genome stability. Recent evidence suggests that p53 may also contribute to the regulation of migration and invasion. Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that is overexpressed in the majority of human epithelial carcinomas, including breast and colorectal carcinomas. We show by chromatin immunoprecipitation assays that p53 interacts with a candidate p53 binding site within the EpCAM gene. p53-mediated transcriptional repression of EpCAM was confirmed in gain-of-function and loss-of-function experimental systems. Induction of wild-type p53 was associated with a significant dose-dependent decrease in EpCAM expression; conversely, specific ablation of p53 was associated with a significant increase in EpCAM expression. At the functional level, specific ablation of p53 expression is associated with increased breast cancer invasion, and this effect is abrogated by concomitant specific ablation of EpCAM expression. Taken together, these biochemical and functional data are the first demonstration that (a) wild-type p53 protein binds to a response element within the EpCAM gene and negatively regulates EpCAM expression, and (b) transcriptional repression of EpCAM contributes to p53 control of breast cancer invasion.

Lens epithelial cells derived from αB-crystallin knockout mice demonstrate hyperproliferation and genomic instability

B‐crystallin is a member of the small heat shock protein family and can act as a molecular chaperone preventing the in vitro aggregation of other proteins denatured by heat or other stress conditions. Expression of αB‐crystallin increases in cells exposed to stress and enhanced in tumors of neuroectodermal origin and in many neurodegenerative diseases. In the present study, we examined the properties of lens epithelial cells derived from mice in which the αB‐crystallin gene had been knocked out. Primary rodent cells immortalize spontaneously in tissue culture with a frequency of 10 –5 to 10 –6. Primary lens epithelial cells derived from aB‐crystaUin–/– mice produced hyperproliferative clones at a frequency of 7.6 × 10–2, four orders of magnitude greater than predicted by spontaneous immortalization (1). Hyperproliferative αB‐crys‐tallin–/– cells were shown to be truly immortal since they have been passaged for more than 100 population doublings without any diminution in growth potential. In striking contrast to the wild‐type cells, which were diploid, the αB‐crystallin–/– cultures had a high proportion of tetraploid and higher ploidy cells, indicating that the loss of αB‐crystallin is associated with an increase in genomic instability. Further evidence of genomic instability of αB‐crystallin–/– cells was observed when primary cultures were infected with Ad12– SV40 hybrid virus. In striking contrast to wild‐type cells, αB‐crystallin–/– cells expressing SV40 T antigen exhibited a widespread cytocidal response 2 to 3 days after attaining confluence, indicating that SV40 T antigen enhanced the intrinsic genomic instability of αB‐crystallin–/– lens epithelial cells. These observations suggest that the widely distributed molecular chaperone αB‐crystallin may play an important nuclear role in maintaining genomic integrity.—Andley, U. P., Song, Z., Wawrousek, E. F., Brady, J. P., Bassnett, S., Fleming, T. P. Lens epithelial cells derived from αB‐crystallin knockout mice demonstrate hyperproliferation and genomic instability. FASEB J. 15, 221–229 (2001)

Identification of HLA-A3-restricted CD8+ T cell epitopes derived from mammaglobin-A, a tumor-associated antigen of human breast cancer.

Mammaglobin‐A is highly overexpressed in breast cancer cell lines and primary breast tumors. This pattern of expression is restricted to mammary epithelium and metastatic breast tumors. Thus, mammaglobin‐A‐specific T cell immune responses may provide an important approach for the design of breast cancer‐specific immunotherapy. The purpose of our study was to define the T cell‐mediated immune response to mammaglobin‐A. We determined that the frequency of mammaglobin‐A‐reactive CD8+ and CD4+ T cells in breast cancer patients is significantly higher than that observed in healthy female controls using limiting dilution analyses (p = 0.026 and p = 0.02, respectively). We identified 8 mammaglobin‐A‐derived 9‐mer peptides with the highest binding affinity for the HLA‐A3 molecule (Mam‐A3.1–8) using a computer‐assisted analysis of the mammaglobin‐A protein sequence. Subsequently, we determined that CD8+ T cells from breast cancer patients reacted to peptides Mam‐A3.1 (23–31, PLLENVISK), Mam‐A3.3 (2–10, KLLMVLMLA), Mam‐A3.4 (55–63, TTNAIDELK) and Mam‐A3.8 (58–66, AIDELKECF) using an IFN‐γ enzyme‐linked immunospot assay. A CD8+ T cell line generated in vitro against HLA‐A*0301‐transfected TAP‐deficient T2 cells loaded with these peptides showed significant cytotoxic activity against the Mam‐A3.1 peptide. This CD8+ T cell line showed a significant HLA‐A3‐restricted cytotoxic activity against mammaglobin‐A‐positive but not mammaglobin‐A‐negative breast cancer cells. In summary, our study identified four HLA‐A3‐restricted mammaglobin‐A‐derived epitopes naturally expressed by breast cancer cells, indicating the immunotherapeutic potential of this novel antigen for the treatment and prevention of breast cancer.

Photoacoustic microscopy of tyrosinase reporter gene in vivo

Photoacoustic tomography is a hybrid modality based on optical absorption excitation and ultrasonic detection. It is sensitive to melanin, one of the primary absorbers in skin. For cells that do not naturally contain melanin, melanin production can be induced by introducing the gene for tyrosinase, the primary enzyme responsible for expression of melanin in melanogenic cells. Optical resolution photoacoustic microscopy was used in the ex vivo study reported here, where the signal from transfected cells increased by more than 10 times over wild-type cells. A subsequent in vivo experiment was conducted to demonstrate the capability of photoacoustic microscopy to spectrally differentiate between tyrosinase-catalyzed melanin and various other absorbers in tissue.

Recognition of HLA-A2-restricted mammaglobin-A-derived epitopes by CD8+ cytotoxic T lymphocytes from breast cancer patients.

A breast cancer-associated antigen, mammaglobin-A, is specifically expressed in 80% of primary breast tumors. The definition of immune responses against this highly expressed breast cancer-specific antigen should be of great value in the development of new therapeutic strategies for breast cancer. Thus, the purpose of this study was to identify HLA-A2-restricted mammaglobin-A-derived epitopes recognized by CD8+ cytotoxic T lymphocytes (CTL). We identified seven mammaglobin-A-derived candidate epitopes that bind the HLA-A2 molecule (Mam-A2.1-7) by means of a HLA class I-peptide binding computer algorithm from the Bioinformatics & Molecular Analysis Section of the National Institutes of Health. Subsequently, we determined that CD8+ CTLs from breast cancer patients reacted to the Mam-A2.1 (83–92, LIYDSSLCDL), Mam-A2.2 (2–10, KLLMVLMLA), Mam-A2.3 (4–12, LMVLMLAAL), Mam-A2.4 (66–74, FLNQTDETL), and Mam-A2.7 (32–40, TINPQVSKT) epitopes using an IFN-c ELISPOT assay. Interestingly, healthy individuals also showed high reactivity to the Mam-A2.2 epitope. Two CD8+ CTL lines generated in vitro against TAP-deficient T2 cells loaded with the candidate epitopes showed significant cytotoxic activity against the Mam-A2.1-4 epitopes. These CD8+CTL lines recognized a HLA-A2+breast cancer cell line expressing the Mam-A2.1 epitope. In addition, DNA vaccination of HLA-A2+/human CD8+ double-transgenic mice with a DNA construct encoding the Mam-A2.1 epitope and the HLA-A2 molecule induced a significant expansion of epitope-specific CD8+ CTLs that recognize the same HLA- A2+/Mam-A2.1+ breast cancer cell line. In conclusion, these results demonstrate the immunotherapeutic potential of mammaglobin-A for the treatment and prevention of breast cancer.