BREAST CANCER RESEARCH

Breast cancer remains one of the most common malignancies among women, second only in incidence to lung cancer. For the first time since 1950, breast cancer death rates for American women declined nearly 5% between 1989 and 1992. Two plausible explanations for the decline are an increase in breast cancer awareness leading to early detection, and adjuvant systemic therapy for both node positive and node negative breast cancer. In an effort to contribute to the reduction of both the incidences of breast cancer and the morbidity and mortality due to this disease, we have developed a multidisciplinary program encompassing basic, clinical, and prevention and control research.

The focus of the research by this group is the identification and characterization of populations that are at an increased risk of developing breast cancer using epidemiologic, genetic, and molecular studies. Under the direction of Dr. Daly, the Fox Chase Network Breast Cancer Risk Registry has been developed to allow for the transfer of cancer control expertise and technology to the community. The registry will increase the scientific database for future genetic and epidemiologic research. Dr. Godwin made substantial progress in his pilot study on the role of the ovarian cancer locus 1 (OVCA1) gene in the development of human breast cancer. This project is now competitively funded. Dr. Sauter has established a program to study nipple aspirate cytologies in women at high risk for breast cancer. These efforts focus on potential intermediate biomarkers for future breast cancer prevention studies.

This research program examines four main behavioral aspects of breast cancer risk and disease. Firstly, we are evaluating a population of women whose family histories indicate that they are at heritable risk for breast/ovarian cancer. After receipt of this feedback, these women can show signs of chronic stress similar to that seen following traumatic medical events, and often demonstrate the dis-organizing effects of loss of control over one's life in a predictable manner. Our group is developing a program for an enhanced genetic counseling protocol that tests the efficacy of different types of appeals tailored to affected and unaffected women. The goal is to enhance adjustment and coping, as well as decision-making about preventive options (e.g., prophylactic surgery, chemoprevention). We are also extending this work to develop protocols that can be delivered on an outreach basis, through the Cancer Information Service.

In a second context, we are exploring the efficacy of a brief counseling intervention designed to enhance decision making and adjustment in early stage breast cancer. Thirdly, we are evaluating a population of breast cancer survivors who need to initiate and maintain cancer-screening regimens, including routine screening and follow-up diagnostic testing. These health-promoting regimens can, however, reactivate deep anxieties about the cancer recurring or progressing, as well as other associated fears. To address these needs, we are developing a project based on the cognitive-social model to identify and modify maladaptive thoughts and behaviors that interfere with consistent long-term health-protective behaviors. In these studies, we are focusing particularly on the role of information seeking, since we have identified distinctive differences among women in coping with breast cancer and related risks. In our prior work, we have shown that patients benefit from different types of psychosocial interventions, tailored to their own psychological profile.

The fourth population we study is women who do not adhere to screening, prevention, and treatment regimens. These women become more vulnerable to cancer because of this lack of compliance. The two types of barriers that have the largest effect in this population are demographic and psychological barriers. In one study, we are focusing on women with limited education and low socio-economic levels--conditions that are common among inner-city, minority populations. The intervention is targeted to psychological and practical barriers to promote access to educational interventions and health-care community resources for coping with cancer risk. In another study, we are exploring the efficacy of time-and-cost-effective outreach strategies for nonadherence, in a corporate context.

In addition, Dr. Manne is studying the impact of BRCA1 and BRCA2 genetic testing affects on marriage. She is also investigating whether the husbands of patients with breast cancer could benefit from psychosocial intervention.

Overall, the breast cancer research group has been involved in overcoming obstacles to effective therapy in breast cancer. The areas of investigation include drug resistance, dose intensity, novel therapeutic agents, and targeted immunotherapy.

Drug Resistance. Ongoing clinical laboratory studies in the area of drug resistance include a phase II study using Taxol modulated by PSC 833 to overcome drug resistance mediated by the multi-drug resistance (MDR) gene. This study is being conducted in col-laboration with Stanford University and the Eastern Cooperative Oncology Group. Another novel MDR inhibitor will soon be in trial that, unlike PSC 833, does not alter the pharmacokinetics of cytotoxic agents.

Dose Intensity. We are participating in two Philadelphia Bone Marrow Consortium studies. We have completed a phase III study comparing conventional chemotherapy to high dose chemotherapy and autologous bone marrow transplant after responsiveness to standard induction chemotherapy in patients with metastatic breast cancer. This data will be critical in defining the role of high dose chemotherapy in breast cancer.

A phase II study of induction doxorubicin followed by high dose chemotherapy with peripheral blood stem cell support for patients with locally advanced and inflammatory breast cancer (PBT-3) is also in progress. This study examines dose intensity and will allow for sequential tumor biopsies to evaluate expression of MDR1 and other drug resistance genes.

Novel Therapeutic Agents. We have continued clinical investigation of paclitaxel dosing, scheduling, and use in combination with other chemotherapeutic agents. We have completed an adjuvant study for node positive breast cancer patients. This study used paclitaxel after adjuvant adriamycin and cyclophosphamide (AC). The study is also designed to investigate the role of dose intensity of adriamycin as an adjuvant treatment. Preliminary results indicate that the sequential addition of paclitaxel to AC as post-operative adjuvant therapy significantly improves disease free, as well as overall, survival. However, there is no effect of dose intensity of adriamycin. Dr. Goldstein is the principal investigator of the current Intergroup adjuvant trial investigating adriamycin and cyclophosphamide versus adriamycin and docetaxel.

In collaboration with Drs. I. and J. Russo, we will be studying the role of human chorionic gonadotropin (hCG) in the treatment and prevention of breast cancer. The rationale for these studies is the Russos' finding that hCG can inhibit mouse mammary carcinogenesis.

Targeted Immunotherapy. In collaboration with Drs. Weiner and Bookman, we have continued phase I studies of the bi-specific antibody 2B1, which has specificity for the HER2/neu (c-erb B2) gene product and of the recombinant SGN-10 immunotoxin, which targets the BR96 (Le^x) tumor antigen, respectively. We have also activated a phase I study for patients with advanced breast cancer using a fusion antibody targeted toward breast cancer cells and cytotoxic T cells. Furthermore, we are investigating the activity of herceptin, a monoclonal antibody that targets HER2/neu, in combination with docetaxel.

MDR1 expression confers pleiotropic drug resistance in tumor cells. Recently, the multidrug resistance-associated protein gene, MRP, which encodes another ATP-binding cassette (ABC) protein, has been associated with non-P-glycoprotein (PGP) drug resistance in tumor cells. The clinical significance of MRP expression is not clear; furthermore, its relationship to MDR1 expression is not known. Initial results from a retrospective study suggest that MRP expression may be significant in breast cancer. We are currently involved in a prospective study of MDR1 and MRP expression in breast cancer. We are using several assays to assess MDR1 expression levels, including quantitative-cDNA reverse transcription-polymerase chain reaction (QRT-PCR).We have also developed a QRT-PCR assay to measure MRP mRNA levels.Our data show that MRP mRNA levels can be quantitatively assessed and directly compared to MDR1 expression levels.

MDR1 and MRP expression have been shown in vitro to be important mediators of multiple drug resistance. In vivo analyses have also indicated that these two genes may play critical roles in tumor responsiveness in some cancers. To evaluate the clinical significance of drug resistance mechanisms in breast cancer, we examined the expression of MDR1 and MRP in primary breast carcinoma and normal adjacent tissue (NAT) (1). In addition, we are examining the concordance among different detection methods. To this end, we have developed a highly reproducible QRT-PCR assay to measure the expression of MDR1 and MRP in breast biopsies. In the current study, we examined the expression of both genes in primary breast carcinoma and NAT by QRTPCR (n=74) and immunohistochemistry (n=32). Expression of both genes was observed in all specimens examined, both tumor and NAT. The expression of MDR1 in these samples was, however, 25 times less than the drug-resistant control cell line, KB 85. Using monoclonal anti-PGP antibodies, JSB1 and UIC2, immuno-histochemical analysis of PGP corroborated the PCR results with only 6% (2/31) being positive for JSB1 staining and 0/32 for UIC2. MRP expression in the specimens did not exceed control cell line levels and MRP immunohistochemical detection using monoclonal anti-MRP antibodies, MRPm6 and QCRL-1, detected moderate levels of expression in all cases. MDR1 expression was independent of grade, stage, tumor size, nodal status, metastasis, and estrogen and progesterone receptor status. There was, however, a significant correlation of MDR expression with age and histology. Approximately twice the expression of MDR1 was observed in the <50 age group compared to the >50 age group and lobular carcinoma had 4 times the expression of MDR1 as other histological types; these data indicate that MDR1 may contribute to poor prognosis associated with young age and lobular histology. MRP expression was independent of all clinical parameters. Thus, these results show that while MDR1 expression is detectable in primary breast carcinoma by PCR, this expression when measured quantitatively is extremely low and may not be significant. We are currently using these quantitative assays to assess the significance of expression of MDR1 and MRP in the context of clinical trials where sequential samples, pre- and post-treatment, are being evaluated.

We are analyzing tissue samples from advanced breast cancer patients enrolled in a phase II study of patients with locally advanced and inflammatory breast cancer (PBT-3). MDR1 and MRP expression in the samples were determined by QRT-PCR. Immunohistochemistry using a panel of monoclonal antibodies specific for MDR, as well as MRP, is in progress. Results from these analyses will be correlated with clinical response and resistance to adriamycin and Taxol.

We have previously shown that low-level multidrug resistance in a mitoxantrone-selected subline of MCF7 cells was associated with an accumulation defect not mediated by MDR1 or MRP. In addition to mitoxantrone, this subline exhibited cross-resistance to doxorubicin and several camptothecin analogues, namely topotecan, but not camptothecin or cisplatin. Furthermore, confocal analysis of drug uptake indicated that these cells also had altered subcellular drug distribution with enhanced vesicular accumulation of mitoxantrone and topotecan, but not doxorubicin. We have investigated the nature of this vesicular compartment and now report that this resistant subline has an altered golgi network compared to the sensitive parental line. Furthermore, we have shown that decreased expression of the tuberous sclerosis 2 (TSC2) gene, whose function appears to be integral to proper endocytosis, is down-regulated in these resistance lines. We have also examined the expression pattern of other known ABC transporters, including ABC-C, canalicular multispecific organic anion-transporter (cMOAT[MRP2]), and MRP3-5 and have found only cMOAT (MRP2) and MRP3 to be overexpressed in the resistant subline. This resistance and subcellular drug distribution, however, cannot be reversed by buthionine sulphoximine. We are currently exploring the activity of other potential transport inhibitors, as well as assessing the role of cMOAT and MRP3 in this resistance. We are further exploring other genetic alterations using RNA fingerprinting techniques.

1.   DEXTER, D.W., REDDY, R.K., GELES, K.G., BANSAL, S., MYINT, M.A., ROGATKO, A., LEIGHTON, J.L., GOLDSTEIN, L.J. Quantitative reverse transcriptase-polymerase chain reaction measured expression of MDR1 and MRP in primary breast carcinoma. Clin. Cancer Res. 4:1533-1592, 1998.

FREEDMAN, G.M., FOWBLE, B.L., HANLON, A.L., MYINT, M.A., HOFFMAN, J.P., SIGURDSON, E.R., EISENBERG, B.L., GOLDSTEIN, L.J., FEIN, D.A. A close or positive margin after mastectomy is not an indication for chest wall irradiation except in women aged fifty or younger. Int. J. Radiat. Oncol. Biol. Phys. 41(3):599-605, 1998.

FOWBLE, B., HANLON, A., FREEDMAN, G., PATCHEFSKY, A., KESSLER, H., NICOLAOU, N., HOFFMAN, J., SIGURDSON, E., BORAAS, M., GOLDSTEIN, L. Postmenopausal hormone replacement therapy (HRT): Effect on diagnosis and outcome in early stage invasive breast cancer treated with conservative surgery and radiation. J. Clin. Oncol. (in press).

FOWBLE, B., HANLON, A., FREEDMAN, G., PATCHEFSKY, A., KESSLER, H., NICOLAOU, N., HOFFMAN, J., SIGURDSON, E., BORAAS, M., GOLDSTEIN, L. Postmenopausal hormone replacement therapy (HRT): Effect on diagnosis and outcome in early stage invasive breast cancer treated with conservative surgery and radiation. J. Clin. Oncol. (in press).

FOWBLE, B., HANLON, A.L., PATCHEFSKY, A., FREEDMAN, G., HOFFMAN, J.P., SIGURDSON, E.R., GOLDSTEIN, L.J. The presence of proliferative breast disease with atypia does not significantly influence outcome in early stage invasive breast cancer treated with conservative surgery and radiation. Int. J. Radiat. Oncol. Biol. Phys. 42:105-115, 1998.

^a   D.W. Dexter: Present address--Pennsylvania State University College of Medicine, Hershey, PA 17033

^b   C.Kim: Temple University Medical School, Philadelphia, PA 19140

Illustrations or unpublished data in these reports should not be used without permission of the author.

Fox Chase Cancer Center

Scientific Report 1998