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Browse Health

Credentials

Education ?

Medical School Score Rankings
Yale University (1968)
  •  
Top 25%

Awards & Distinctions ?

Awards  
Castle Connolly America's Top Doctors® (2002, 2004 - 2008, 2010 - 2015)
Castle Connolly America's Top Doctors® for Cancer (2005 - 2007, 2009 - 2012, 2014 - 2015)
Patients' Choice Award (2015)
Compassionate Doctor Recognition (2014)
On-Time Doctor Award (2015)
Appointments
Georgetown University School Of Medicine
PROF OF ONCOLOGY
University Of Miami School Of Medicine
PROFESSOR AND CHAIR
University Of Michigan Medical School
PROFESSOR AND CHAIR
Associations
American Board of Internal Medicine

Affiliations ?

Dr. Lippman is affiliated with 8 hospitals.

Hospital Affiliations

Score

Rankings

  • Jackson Memorial Hospital
    1611 NW 12th Ave, Miami, FL 33136
    •  
    Top 50%
  • University of Miami Hospital & Clinics
    1475 NW 12th Ave, Miami, FL 33136
    •  
  • Cedars Medical Center
    Medical Oncology
    1400 NW 12th Ave, Miami, FL 33136
    •  
  • University of Miami Hosp & ClinicsSylvester Comprehensive Cancer Cntr
  • University of Miami Miller School of Medicine Faculty
  • U. Of M. Hospitals & Clinics - Ncch
  • University of Miami Hospital Medical Staff
  • UMHC/Sylvester Comprehensive Cancer Center
  • Publications & Research

    Dr. Lippman has contributed to 178 publications.
    Title Anti-vascular Therapy: a New Approach to Cancer Treatment.
    Date June 2010
    Journal The Western Journal of Medicine
    Title An Expression Signature of Estrogen-regulated Genes Predicts Disease-free Survival in Tamoxifen-treated Patients Better Than Progesterone Receptor Status.
    Date November 2008
    Journal Transactions of the American Clinical and Climatological Association
    Excerpt

    We have used data derived from hormonal responses in human breast cancer cell lines to develop a panel of 36 genes which can robustly identify patients who will or will not benefit from tamoxifen treatment. These genes had the following characteristics: 1) induction by 17beta-estradiol (E2) in estrogen receptor (ER)-positive breast cancer cell lines in vitro, 2) under-expression in ER-negative breast tumors, and 3) correlation with PR mRNA expression in breast tumors. The average expression of these 36 genes was used as a "risk index" for assessing disease-specific survival in two independent tumor profile datasets of 60 and 67 patients treated with tamoxifen (these data not having been used to initially select the 36 genes), with a high risk group in each dataset defined as those with the bottom 25% of risk index values. This combined biological and informatic analysis is potentially applicable to many other cancer therapeutics.

    Title A Transcriptional Fingerprint of Estrogen in Human Breast Cancer Predicts Patient Survival.
    Date February 2008
    Journal Neoplasia (new York, N.y.)
    Excerpt

    Estrogen signaling plays an essential role in breast cancer progression, and estrogen receptor (ER) status has long been a marker of hormone responsiveness. However, ER status alone has been an incomplete predictor of endocrine therapy, as some ER+ tumors, nevertheless, have poor prognosis. Here we sought to use expression profiling of ER+ breast cancer cells to screen for a robust estrogen-regulated gene signature that may serve as a better indicator of cancer outcome. We identified 532 estrogen-induced genes and further developed a 73-gene signature that best separated a training set of 286 primary breast carcinomas into prognostic subtypes by stepwise cross-validation. Notably, this signature predicts clinical outcome in over 10 patient cohorts as well as their respective ER+ subcohorts. Further, this signature separates patients who have received endocrine therapy into two prognostic subgroups, suggesting its specificity as a measure of estrogen signaling, and thus hormone sensitivity. The 73-gene signature also provides additional predictive value for patient survival, independent of other clinical parameters, and outperforms other previously reported molecular outcome signatures. Taken together, these data demonstrate the power of using cell culture systems to screen for robust gene signatures of clinical relevance.

    Title Effect of Raloxifene on the Incidence of Invasive Breast Cancer in Postmenopausal Women with Osteoporosis Categorized by Breast Cancer Risk.
    Date November 2007
    Journal Clinical Cancer Research : an Official Journal of the American Association for Cancer Research
    Excerpt

    PURPOSE: To assess the effect of raloxifene, indicated for osteoporosis treatment and prevention, on invasive breast cancer in subgroups of postmenopausal women defined by risk factors for breast cancer. EXPERIMENTAL DESIGN: Data from the 4-year Multiple Outcomes of Raloxifene Evaluation (MORE) trial (N=7,705) and a follow-up study, the 4-year Continuing Outcomes Relevant to Evista (CORE) trial (N=4,011), were analyzed. Prespecified subgroups were defined by age (>or=65 versus<65 years), age at menopause (>or=49 versus<49 years), body mass index (>or=25 versus<25 kg/m2), family history of breast cancer (yes/no), serum estradiol level (5-10 versus<5, >10 versus<5 pmol/L), prior estrogen therapy (yes/no), and bone mass at MORE baseline, and 5-year predicted risk, assessed using the modified Gail model (>or=1.67 versus<1.67%), at CORE baseline. Time-to-first invasive breast cancer was analyzed using Cox proportional hazards models. RESULTS: In the placebo group, older age, higher estradiol level, and a family history of breast cancer were associated with an increased breast cancer risk (P<0.05). Raloxifene therapy was associated with a reduced breast cancer risk in both women at lower and those at higher breast cancer risk. Hazard ratio point estimates were 0.11 to 0.67, corresponding to a 33% to 89% reduction in breast cancer risk with raloxifene versus placebo. The therapy by family history interaction was significant (P=0.04). CONCLUSIONS: Raloxifene therapy was associated with a reduced risk of invasive breast cancer in postmenopausal women irrespective of the presence/absence of risk factors; its effect was greater in women with a family history of breast cancer.

    Title A Prospective Study of Adjuvant Cmf in Males with Node Positive Breast Cancer: 20-year Follow-up.
    Date August 2007
    Journal Breast Cancer Research and Treatment
    Excerpt

    PURPOSE: To determine the long-term overall survival of male patients with stage II node positive breast cancer treated with adjuvant chemotherapy. PATIENTS AND METHODS: Between 1974 and 1988, 31 male breast cancer patients were prospectively enrolled on study MB-82 in the National Cancer Institute. Following mastectomy, patients were treated with 12 cycles of cyclophosphamide, methotrexate, and fluorouracil (CMF) chemotherapy. RESULTS: Median patient age was 61 years (38-74 years). Twenty-one patients (68%) had 1-3 positive axillary lymph nodes while ten patients (32%) had four or more positive nodes. Estrogen receptor status was positive in 22 (71%), negative in 1 (3%), and unknown in 8 (26%) tumors. Progesterone receptor status was positive in 18 (58%), negative in 3 (10%), and unknown in 10 (32%) tumors. Median potential follow-up for all patients is 22.5 years with a median survival of 16.3 years. Twenty-one of 31 patients have died; one from a treatment-related complication, nine patients from recurrent breast cancer, five from other cancers, one from non-cancer related causes, and five from unknown causes. Ten patients remain alive at a median of 19.2 years. The overall survival probability at 10 years is 64.5% (95% CI: 46.9-78.9%), at 15 years is 51.6% (95% CI: 34.8-68%), and at 20 years is 42.4% (95% CI: 25.8-60.8%). CONCLUSION: To our knowledge, 20-year prospective data with adjuvant chemotherapy in male breast cancer has never been reported. Adjuvant chemotherapy may benefit male breast cancer patients with positive nodes.

    Title New Guidelines for Reporting of Tumor Marker Studies in Breast Cancer Research and Treatment: Remark.
    Date July 2007
    Journal Breast Cancer Research and Treatment
    Title Genes Regulated by Estrogen in Breast Tumor Cells in Vitro Are Similarly Regulated in Vivo in Tumor Xenografts and Human Breast Tumors.
    Date September 2006
    Journal Genome Biology
    Excerpt

    BACKGROUND: Estrogen plays a central role in breast cancer pathogenesis. Although many studies have characterized the estrogen regulation of genes using in vitro cell culture models by global mRNA expression profiling, it is not clear whether these genes are similarly regulated in vivo or how they might be coordinately expressed in primary human tumors. RESULTS: We generated DNA microarray-based gene expression profiles from three estrogen receptor alpha (ERalpha)-positive breast cancer cell lines stimulated by 17beta-estradiol (E2) in vitro over a time course, as well as from MCF-7 cells grown as xenografts in ovariectomized athymic nude mice with E2 supplementation and after its withdrawal. When the patterns of genes regulated by E2 in vitro were compared to those obtained from xenografts, we found a remarkable overlap (over 40%) of genes regulated by E2 in both contexts. These patterns were compared to those obtained from published clinical data sets. We show that, as a group, E2-regulated genes from our preclinical models were co-expressed with ERalpha in a panel of ERalpha+ breast tumor mRNA profiles, when corrections were made for patient age, as well as with progesterone receptor. Furthermore, the E2-regulated genes were significantly enriched for transcriptional targets of the myc oncogene and were found to be coordinately expressed with Myc in human tumors. CONCLUSION: Our results provide significant validation of a widely used in vitro model of estrogen signaling as being pathologically relevant to breast cancers in vivo.

    Title Greb1 is a Novel Androgen-regulated Gene Required for Prostate Cancer Growth.
    Date June 2006
    Journal The Prostate
    Excerpt

    BACKGROUND: Gene regulated in breast cancer 1 (GREB1) is a novel estrogen-regulated gene shown to play a pivotal role in hormone-stimulated breast cancer growth. GREB1 is expressed in the prostate and its putative promoter contains potential androgen receptor (AR) response elements. METHODS: We investigated the effects of androgens on GREB1 expression and its role in androgen-dependent prostate cancer growth. RESULTS: Real-time PCR demonstrated high level GREB1 expression in benign prostatic hypertrophy (BPH), localized prostate cancer (L-PCa), and hormone refractory prostate cancer (HR-PCa). Androgen treatment of AR-positive prostate cancer cells induced dose-dependent GREB1 expression, which was blocked by anti-androgens. AR binding to the GREB1 promoter was confirmed by chromatin immunoprecipitation (ChIP) assays. Suppression of GREB1 by RNA interference blocked androgen-stimulated LNCaP cell proliferation. CONCLUSIONS: GREB1 is expressed in proliferating prostatic tissue and prostate cancer, is regulated by androgens, and suppression of GREB1 blocks androgen-induced growth suggesting GREB1 may be critically involved in prostate cancer proliferation.

    Title Greb 1 is a Critical Regulator of Hormone Dependent Breast Cancer Growth.
    Date October 2005
    Journal Breast Cancer Research and Treatment
    Excerpt

    BACKGROUND: Estrogen plays a central role in breast cancer pathogenesis and many potent risk factors for the development of the disease can be explained in terms of increased lifetime exposure to estrogen. Although estrogen regulated genes have been identified, those critically involved in growth regulation remain elusive.METHODS. To identify candidate genes involved in estrogen stimulated breast cancer growth, DNA microarray based gene expression profiles were generated from three estrogen receptor alpha (ER alpha) positive breast cancer cell lines grown under multiple stimulatory and inhibitory conditions.RESULTS: Only three genes were significantly induced by 17beta-estradiol (E2) relative to control in all three cell lines: GREB 1, stromal cell-derived factor 1 (SDF-1) and trefoil factor 1 (pS2). Quantitative real-time PCR assays confirmed that in all three cell lines, GREB 1 was induced by E2, but not by the antiestrogens tamoxifen (TAM) or ICI 182,780. GREB 1 expression level was strongly correlated with ER alpha positivity in 39 breast cancer cell lines of known ER alpha expression status. GREB 1 induction by E2 was rapid (7.3 fold by 2 h for MCF-7) and mirrored the fraction of cells entering S-phase when released from an estrogen deprivation induced cell arrest. Suppression of GREB 1 using siRNA blocked estrogen induced growth in MCF-7 cells and caused a paradoxical E2 induced growth inhibition.CONCLUSION: These data suggest that GREB 1 is critically involved in the estrogen induced growth of breast cancer cells and has the potential of being a clinical marker for response to endocrine therapy as well as a potential therapeutic target.

    Title Consensus Statement: Expedition Inspiration 2004 Breast Cancer Symposium 'breast Cancer--the Development and Validation of New Therapeutics'.
    Date May 2005
    Journal Breast Cancer Research and Treatment
    Title Long-term Follow-up for Locally Advanced and Inflammatory Breast Cancer Patients Treated with Multimodality Therapy.
    Date November 2004
    Journal Journal of Clinical Oncology : Official Journal of the American Society of Clinical Oncology
    Excerpt

    PURPOSE: To determine long-term event-free (EFS) and overall survival (OS) for patients with stage III breast cancer treated with combined-modality therapy. PATIENTS AND METHODS: Between 1980 and 1988, 107 patients with stage III breast cancer were prospectively enrolled for study at the National Cancer Institute and stratified by whether or not they had features of inflammatory breast cancer (IBC). Patients were treated to best response with cyclophosphamide, doxorubicin, methotrexate, fluorouracil, leucovorin, and hormonal synchronization with conjugated estrogens and tamoxifen. Patients with pathologic complete response received definitive radiotherapy to the breast and axilla, whereas patients with residual disease underwent mastectomy, lymph node dissection, and radiotherapy. All patients underwent six additional cycles of adjuvant chemotherapy. RESULTS: OS and EFS were obtained with a median live patient follow-up time of 16.8 years. The 46 IBC patients had a median OS of 3.8 years and EFS of 2.3 years, compared with 12.2 and 9.0 years, respectively, in stage IIIA breast cancer patients. Fifteen-year OS survival was 20% for IBC versus 50% for stage IIIA patients and 23% for stage IIIB non-IBC. Pathologic response was not associated with improved survival for stage IIIA or IBC patients. Presence of dermal lymphatic invasion did not change the probability of survival in clinical IBC patients. CONCLUSION: Fifteen-year follow-up of stage IIIA and inflammatory breast cancer is rarely reported; IBC patients have a poor long-term outlook.

    Title Genotyping for Polymorphic Drug Metabolizing Enzymes from Paraffin-embedded and Immunohistochemically Stained Tumor Samples.
    Date April 2004
    Journal Pharmacogenetics
    Excerpt

    OBJECTIVES: Paraffin-embedded tumor samples are valuable in the study of cancer for routine staging, tumor marker analysis, and in retrospective studies to test new prognostic and predictive biomarkers. Their utility in retrospective pharmacogenetic analysis of clinical trials has yet to be evaluated. We set out to establish genotyping methods for relevant genes from archival tumor samples and determine if fixation, processing or somatic changes in the tumor might affect our ability to identify germ-line polymorphisms. METHODS AND RESULTS: To establish the assays, paraffin blocks were made using pellets prepared from eight tumor cell lines. DNA was isolated from viable cells and from sections from these blocks, and genotyped for polymorphisms in CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A5 and MDR1 using conventional PCR-RFLP assays. This demonstrated that fixation and processing did not alter the genotypes obtained (100% concordance). Next, sections were obtained from paraffin-embedded archival breast samples from 10 patients for whom gDNA isolated from peripheral blood was available for comparison. Concordance was complete with the same genotype being obtained for 100% of the samples tested. Attempts to extend these methods for the study of hematoxylin/eosin or immunohistochemically stained sections were not successful since the staining inhibited the PCR reactions. Only 25 of 50 samples were successfully amplified and of those only 14 produced accurate genotypes. CONCLUSIONS: Accurate genetic testing for polymorphisms in several genes of pharmacogenetic importance can be obtained from archival paraffin-embedded tumor samples. Thus, pharmacogenetic analysis can be applied to existing cancer therapy trials to test associations between these polymorphisms and treatment response.

    Title Acceptability of Diagnostic Tests for Breast Cancer.
    Date November 2003
    Journal Breast Cancer Research and Treatment
    Excerpt

    PURPOSE: To assess the acceptability of new non-invasive breast cancer diagnostic tests intended to triage women in need of biopsy. METHODS: Women who had abnormal screening tests and had been recommended to have a biopsy were invited to receive digital mammography, magnetic resonance imaging (MRI), and nuclear medicine evaluation (Tc-99m-sestamibi scanning) before biopsy. Participants completed a questionnaire about satisfaction and acceptability of the procedures. Satisfaction measured women's overall and test-specific satisfaction. Acceptability was measured by self-reported discomfort, embarrassment and women's preference in terms of willingness to pay to avoid a biopsy. RESULTS: Women were satisfied with all of the potential diagnostic triage procedures. Most found the tests more comfortable than a routine mammogram (47, 50, and 66% undergoing MRI, digital mammography, and sestamibi scanning, respectively). Women who provided a response to willingness to pay questions (N = 43) were willing to pay an average of 611 dollars to have a test instead of a biopsy, if the test was as accurate as biopsy. The willingness to pay significantly decreased to 308 dollars if the test only had 95% accuracy. Those who had prior benign breast disease were less willing to pay for a test with 95% accuracy than those without this history. CONCLUSION: Instead of immediate biopsy after an abnormal screening, these results suggest that women would find non-invasive triage tests acceptable, or preferable to biopsy if they were equally accurate or nearly equally accurate as a biopsy. New technologies to diagnose breast cancer should focus on decreasing discomfort as well as increasing test accuracy.

    Title Eighteen-year Results in the Treatment of Early Breast Carcinoma with Mastectomy Versus Breast Conservation Therapy: the National Cancer Institute Randomized Trial.
    Date August 2003
    Journal Cancer
    Excerpt

    BACKGROUND: Between 1979-1987, the National Cancer Institute conducted a randomized, prospective study of mastectomy (MT) versus breast conservation therapy (BCT) in the treatment of patients with early-stage breast carcinoma. After a median potential follow-up of 18.4 years, the authors present the updated results. METHODS: After informed consent was obtained from each patient, 237 evaluable women with clinical AJCC Stage I and Stage II breast carcinoma were enrolled on an institutionally reviewed protocol and randomly assigned to undergo modified radical MT (116 patients) or BCT (121 patients), which was comprised of lumpectomy, axillary lymph node dissection, and radiation therapy. Negative surgical margins in the lumpectomy arm were not required. The 237 randomized patients were followed for a median potential follow-up of 18.4 years. The primary endpoints were overall survival and disease-free survival. RESULTS: At a median follow-up of 18.4 years, there was no detectable difference with regard to overall survival between patients treated with MT and those treated with BCT (58% vs. 54%; P = 0.67 overall). Twenty-seven women in the BCT arm (22%) experienced an in-breast event. After censoring in-breast events in the BCT arm that were salvaged successfully by MT, disease-free survival also was found to be statistically similar (67% in the MT arm vs. 63% in the BCT arm; P = 0.64 overall). There was no statistically significant difference with regard to contralateral breast carcinoma between the two treatment arms (P = 0.70). CONCLUSIONS: After nearly 20 years of follow-up, there was no detectable difference in overall survival or disease-free survival in patients with early-stage breast carcinoma who were treated with MT compared with those treated with BCT. For BCT patients, long-term in-breast failures continued to occur throughout the duration of follow-up. There was no statistically significant difference in the incidence of contralateral breast carcinoma between the two treatment groups.

    Title Mobilization of Peripheral Blood Stem Cells with Paclitaxel and Rhg-csf in High-risk Breast Cancer Patients.
    Date July 2003
    Journal Journal of Hematotherapy & Stem Cell Research
    Excerpt

    Preclinical studies have demonstrated the rapid and efficient mobilization of hematopoietic peripheral blood stem cells (PBSC) in a mouse model using the combination of paclitaxel with recombinant human granulocyte colony-stimulating factor (rhG-CSF). On the basis of these results, a clinical trial was initiated using rhG-CSF with paclitaxel for PBSC mobilization in high-risk breast cancer patients. The mobilized PBSC were evaluated for CD34(+) cell number, mononuclear cell content, and clonogenic potential. One-hundred and seventeen breast cancer patients received paclitaxel (300 mg/m(2)) administered as a 24-h continuous intravenous infusion. Forty-eight hours after completing paclitaxel, rhG-CSF (5 microg/kg) was initiated and continued until completion of PBSC collection. Leukapheresis was initiated once the white blood cell count reached 1.0 x 10(9)/L. Each collection was evaluated for the numbers of mononuclear cells (MNC) and CD34(+) cells. Clonogenic potential was enumerated using colony-forming units-granulocyte-macrophage (CFU-GM) and burst-forming units-erythroid (BFU-E). Patients receiving paclitaxel with rhG-CSF mobilized a large number of mononuclear cells/apheresis (mean, 3.7 x 10(8); range, 3.3-4.1) and CD34(+) cells/apheresis (mean, 7.2 x 10(6); range, 6.1-8.4). The average number of leukophereses needed was 1.8 (mean, range 1.6-2.0). Colony growth was normal with 178.9 x 10(5) and 214.8 x 10(5) colonies counted in CFU-GM and BFU-E assays, respectively. Patients engrafted platelets and neutrophils on day 10 following transplantation. In conclusion, PBSC mobilization with paclitaxel and rhG-CSF results in a large number of mononuclear cells and CD34(+) cells with normal clonogenic potential. The cells engraft normally following high-dose chemotherapy and autologous stem cell transplantation in high-risk breast cancer patients. These results demonstrate that paclitaxel with rhG-CSF is an efficient mobilizing agent in high-risk breast cancer patients.

    Title Dietary Modulation of Pregnancy Estrogen Levels and Breast Cancer Risk Among Female Rat Offspring.
    Date June 2003
    Journal Clinical Cancer Research : an Official Journal of the American Association for Cancer Research
    Excerpt

    Against the hypothesis that high estrogen levels in utero increase the risk of developing breast cancer in later life are data showing that pregnancy estrogen levels are significantly higher in Asian women who have low breast cancer risk than in Caucasian women. We investigated whether maternal dietary intake of genistein or n-3 polyunsaturated fatty acids (PUFAs), which are typical to Asian but not Caucasian diet, affect pregnancy estrogen levels and susceptibility to mammary tumorigenesis among offspring.

    Title Transcriptome Analysis of Her2 Reveals a Molecular Connection to Fatty Acid Synthesis.
    Date March 2003
    Journal Cancer Research
    Excerpt

    HER2 (erbB2/neu) is a member of the erbB family of receptor tyrosine kinases and is involved in regulating the growth of several types of human carcinomas. HER2 represents a successful therapeutic target of the biotechnology era as exemplified by the drug Herceptin (trastuzumab), which has clinical activity in a subset of breast cancer patients. Using DNA microarrays, we identified a cohort of genes that are differentially regulated by HER2 in breast epithelial cells. One of the HER2-regulated genes discovered was fatty acid synthase (FAS), which has been shown to be overexpressed in breast cancer as well as other cancers. FAS is implicated in tumorigenesis through its role in cell proliferation and membrane lipid incorporation of neoplastic cells. Here, we demonstrate that HER2-mediated induction of FAS is inhibitable by Herceptin and tyrosine kinase inhibitors of HER2. Through a phosphatidylinositol 3'-kinase-dependent pathway, HER2 stimulates the FAS promoter and ultimately mediates increased fatty acid synthesis. Interestingly, pharmacological inhibition of FAS preferentially induced apoptosis of HER2-overexpressing breast epithelial cells relative to matched vector control cells. These studies characterize a molecular connection between two genes individually implicated in tumorigenesis but never linked together.

    Title Development and Validation of a Method for Using Breast Core Needle Biopsies for Gene Expression Microarray Analyses.
    Date October 2002
    Journal Clinical Cancer Research : an Official Journal of the American Association for Cancer Research
    Excerpt

    PURPOSE: Gene expression microarray technologies have the potential to define molecular profiles that may identify specific phenotypes(diagnosis), establish a patient's expected clinical outcome (prognosis), and indicate the likelihood of a beneficial effect of a specific therapy (prediction). We wished to develop optimal tissue acquisition, processing, and analysis procedures for exploring the gene expression profiles of breast core needle biopsies representing cancer and noncancer tissues. EXPERIMENTAL DESIGN: Human breast cancer xenografts were used to evaluate several processing methods for prospectively collecting adequate amounts of high-quality RNA for gene expression microarray studies. Samples were assessed for the preservation of tissue architecture and the quality and quantity of RNA recovered. An optimized protocol was applied to a small study of core needle breast biopsies from patients, in which we compared the molecular profiles from cancer with those from noncancer biopsies. Gene expression data were obtained using Research Genetics, Inc. Named Genes cDNA microarrays. Data were visualized using simple hierarchical clustering and a novel principal component analysis-based multidimensional scaling. Data dimensionality was reduced by simple statistical approaches. Predictive neural networks were built using a multilayer perceptron and evaluated in an independent data set from snap-frozen mastectomy specimens. RESULTS: Processing tissue through RNALater preserves tissue architecture when biopsies are washed for 5 min on ice with ice-cold PBS before histopathological analysis. Cell margins are clear, tissue folding and fragmentation are not observed, and integrity of the cores is maintained, allowing optimal pathological interpretation and preservation of important diagnostic information. Adequate concentrations of high-quality RNA are recovered; 51 of 55 biopsies produced a median of 1.34 microg of total RNA (range, 100 ng to 12.60 microg). Snap-freezing or the use of RNALater does not affect RNA recovery or the molecular profiles obtained from biopsies. The neural network predictors accurately discriminate between predominantly cancer and noncancer breast biopsies. CONCLUSIONS: The approaches generated in these studies provide a simple, safe, and effective method for prospectively acquiring and processing breast core needle biopsies for gene expression studies. Gene expression data from these studies can be used to build accurate predictive models that separate different molecular profiles. The data establish the use and effectiveness of these approaches for future prospective studies.

    Title Rifampin is a Selective, Pleiotropic Inducer of Drug Metabolism Genes in Human Hepatocytes: Studies with Cdna and Oligonucleotide Expression Arrays.
    Date December 2001
    Journal The Journal of Pharmacology and Experimental Therapeutics
    Excerpt

    We used expression microarrays to test the effects of rifampin on the overall pattern of mRNA expression of multiple metabolic enzymes in primary human hepatocytes. Two microarrays were utilized, a cDNA-based array and one that is oligonucleotide-based. The cDNA-based expression arrays showed that rifampin caused a 7.7 +/- 6.6-fold induction in CYP2A6 and a 4.0 +/- 2.0-fold increase in the CYP2C family of enzymes while having little effect on CYP2E1 or CYP2D6. Many non-P450 enzymes were also induced including FMO-4 and -5, UGT-1A, MAO-B, and GST-P1. The oligonucleotide-based array made it possible to detect different levels of induction within the CYP2C family, with rifampin causing a 6.5-fold increase in expression of CYP2C8 and a 3.7-fold increase in CYP2C9 while having no effect on the level of CYP2C18 mRNA. Rifampin also induced other CYP enzymes including CYP2B6 and all three members of the CYP3A family, with CYP3A4 showing the highest level of induction at 55.1-fold. RNase protection assays were used to validate results from the arrays and a comparison of all three methods of mRNA detection showed qualitatively similar results. These data make it clear that rifampin treatment brings about broad changes in the pattern of gene expression, rather than increased expression of a small number of metabolic enzymes. Clinicians and researchers who use and study rifampin and other drugs that induce drug metabolism should be alert to the possibility of multiple effects.

    Title Indicators of Lifetime Estrogen Exposure: Effect on Breast Cancer Incidence and Interaction with Raloxifene Therapy in the Multiple Outcomes of Raloxifene Evaluation Study Participants.
    Date July 2001
    Journal Journal of Clinical Oncology : Official Journal of the American Society of Clinical Oncology
    Excerpt

    To test the hypothesis that risk factors related to lifetime estrogen exposure predict breast cancer incidence and to test if any subgroups experience enhanced benefit from raloxifene.

    Title Continued Breast Cancer Risk Reduction in Postmenopausal Women Treated with Raloxifene: 4-year Results from the More Trial. Multiple Outcomes of Raloxifene Evaluation.
    Date June 2001
    Journal Breast Cancer Research and Treatment
    Excerpt

    Raloxifene, a selective estrogen receptor modulator approved for the prevention and treatment of postmenopausal osteoporosis, has shown a significant reduction in breast cancer incidence after 3 years in this placebo-controlled, randomized clinical trial in postmenopausal women with osteoporosis. This article includes results from an additional annual mammogram at 4 years and represents 3,004 additional patient-years of follow-up in this trial. Breast cancers were ascertained through annual screening mammograms and adjudicated by an independent oncology review board. A total of 7,705 women were enrolled in the 4-year trial; 2,576 received placebo, 2,557 raloxifene 60 mg/day, and 2,572 raloxifene 120 mg/day. Women were a mean of 66.5-years old at trial entry, 19 years postmenopause, and osteoporotic (low bone mineral density and/or prevalent vertebral fractures). As of 1 November 1999, 61 invasive breast cancers had been reported and were confirmed by the adjudication board, resulting in a 72% risk reduction with raloxifene (relative risk (RR) 0.28, 95% confidence interval (CI) 0.17, 0.46). These data indicate that 93 osteoporotic women would need to be treated with raloxifene for 4 years to prevent one case of invasive breast cancer. Raloxifene reduced the risk of estrogen receptor-positive invasive breast cancer by 84% (RR 0.16, 95% CI 0.09, 0.30). Raloxifene was generally safe and well-tolerated, however, thromboembolic disease occurred more frequently with raloxifene compared with placebo (p=0.003). We conclude that raloxifene continues to reduce the risk of breast cancer in women with osteoporosis after 4 years of treatment, through prevention of new cancers or suppression of subclinical tumors, or both. Additional randomized clinical trials continue to evaluate this effect in postmenopausal women with osteoporosis, at risk for cardiovascular disease, and at high risk for breast cancer.

    Title Adjuvant Therapy for All Patients with Breast Cancer?
    Date March 2001
    Journal Journal of the National Cancer Institute
    Title Immunoconjugates of Geldanamycin and Anti-her2 Monoclonal Antibodies: Antiproliferative Activity on Human Breast Carcinoma Cell Lines.
    Date December 2000
    Journal Journal of the National Cancer Institute
    Excerpt

    BACKGROUND: HER2 is a membrane receptor whose overexpression is strongly associated with poor prognosis in breast carcinomas. Inhibition of HER2 activity can reduce tumor growth, which led to the development of Herceptin, an anti-HER2 monoclonal antibody (MAb) that is already in clinical use. However, the objective response rate to Herceptin monotherapy is quite low. HER2 activity can also be inhibited by the highly cytotoxic antibiotic geldanamycin (GA). However, GA is not used clinically because of its adverse toxicity. Our purpose was to enhance the inhibitory activity of anti-HER2 MAb by coupling it to GA. METHODS: We synthesized 17-(3-aminopropylamino)GA (17-APA-GA) and conjugated it to the anti-HER2 MAb e21, to form e21 : GA. The noninternalizing anti-HER2 MAb AE1 was used as a control. Internalization assays and western blot analyses were used to determine whether the anti-HER2 MAbs and their immunoconjugates were internalized into HER2-expressing cells and reduced HER2 levels. All statistical tests were two-sided. RESULTS: The immunoconjugate e21 : GA inhibited the proliferation of HER2-overexpressing cell lines better than unconjugated e21 (concentration required for 50% inhibition = 40 versus 1650 microg/mL, respectively). At 15 microg/mL, e21 : GA reduced HER2 levels by 86% within 16 hours, whereas unconjugated e21, 17-APA-GA, or AE1 : GA reduced HER2 levels by only 20%. These effects were not caused by release of 17-APA-GA from the immunoconjugate because immunoconjugates containing [(3)H]GA were stable in serum at 37 degrees C. Furthermore, e21 : GA did not significantly inhibit proliferation of the adult T-cell leukemia cell line HuT102, which is HER2 negative yet highly sensitive to GA. CONCLUSIONS: Our findings suggest that conjugating GA to internalizing MAbs enhances the inhibitory effect of the MAbs. This approach might also be applied in cellular targeting via growth factors and may be of clinical interest.

    Title Expression and Function of Angiopoietin-1 in Breast Cancer.
    Date December 2000
    Journal British Journal of Cancer
    Excerpt

    Angiopoietin-1 (Ang1) has been shown to act as an angiogenic promoter in embryonic angiogenesis by promoting vascular branching, pericyte recruitment and endothelial survival. We have investigated the role of Ang1 in tumour neovascularization under clinical conditions and in animal models. The expression of Ang1 in clinical breast cancer specimens was analysed by using laser-capture microdissection and reverse transcriptase-linked polymerase chain reaction (RT-PCR) on RNA isolated from the samples. Despite the expression of Ang1 in many human breast cancer cell lines, the gene was expressed in only three of 21 breast cancer clinical specimens, even though its receptor, Tie2, is abundant in the vasculature of all of these tumours. Ang1 was then overexpressed in a human breast cancer cell line (MCF-7) on its own and in conjunction with FGF1, an angiogenic factor shown to be able to increase the tumorigenicity of MCF-7 cells. High concentrations of Ang1 were produced in the conditioned media of the transfected cells (range 156-820 ng ml(-1)). However, in contrast to its physiological role as promoter of angiogenesis, overexpression of Ang1 did not enhance tumour growth, but instead caused up to a 3-fold retardation of tumour growth (P = 0.003).

    Title Endoscopic Mapping and Surrogate Markers for Better Surveillance in Barrett Esophagus. A Study of 700 Biopsy Specimens.
    Date November 2000
    Journal American Journal of Clinical Pathology
    Excerpt

    Surveillance methods in Barrett esophagus (BE) using light microscopic examination of random biopsy specimens may miss focal dysplasia. In addition, dysplastic foci identified initially may not be relocated subsequently, making chemoprevention studies difficult. By using a special gastroscope, systematic mapping (4-quadrant biopsy specimens at 1-cm intervals) was performed in 22 patients (33 total mappings yielding 700 biopsy specimens). H&E, immunohistochemistry, and DNA ploidy analysis were performed. c-erbB-2 and positive Ki-67 were detected only in dysplastic sites; thus, their detection did not precede morphologically identifiable dysplasia. On the other hand, aneuploidy and p53 were detected in dysplastic and nondysplastic areas. p53 was correlated with dysplasia, and S-phase narrowly missed correlation, while aneuploidy was not correlated. PCNA and bcl-2 were ubiquitous, limiting their usefulness. On second maps, epithelial type was reidentified with 81% accuracy. A significant correlation was found between p53 and dysplasia. Sites of dysplasia and abnormal biomarkers could be relocated accurately by using endoscopic mapping. Therefore, mapping combined with biomarker studies may provide better surveillance and serve as a useful technique in chemoprevention studies.

    Title Phase Ii Evaluation of Thalidomide in Patients with Metastatic Breast Cancer.
    Date July 2000
    Journal Journal of Clinical Oncology : Official Journal of the American Society of Clinical Oncology
    Excerpt

    PURPOSE: To determine the efficacy, safety, pharmacokinetics, and effect on serum angiogenic growth factors of two dose levels of thalidomide in patients with metastatic breast cancer. PATIENTS AND METHODS: Twenty-eight patients with progressive metastatic breast cancer were randomized to receive either daily 200 mg of thalidomide or 800 mg to be escalated to 1,200 mg. Fourteen heavily pretreated patients were assigned to each dose level. Each cycle consisted of 8 weeks of treatment. Pharmacokinetics and growth factor serum levels were evaluated. RESULTS: No patient had a true partial or complete response. On the 800-mg arm, 13 patients had progressive disease at or before 8 weeks of treatment and one refused to continue treatment. The dose was reduced because of somnolence to 600 mg for five patients and to 400 mg for two and was increased for one to 1,000 mg and for four to 1,200 mg. On the 200-mg arm, 12 patients had progressive disease at or before 8 weeks and two had stable disease at 8 weeks, of whom one was removed from study at week 11 because of grade 3 neuropathy and the other had progressive disease at week 16. Dose-limiting toxicities included somnolence and neuropathy. Adverse events that did not require dose or schedule modifications included constipation, fatigue, dry mouth, dizziness, nausea, anorexia, arrhythmia, headaches, skin rash, hypotension, and neutropenia. Evaluation of circulating angiogenic factors and pharmacokinetic studies failed to provide insight into the reason for the lack of efficacy. CONCLUSION: Single-agent thalidomide has little or no activity in patients with heavily pretreated breast cancer. Further studies that include different patient populations and/or combinations with other agents might be performed at the lower dose levels.

    Title Epidermal Growth Factor Receptor Viii Enhances Tumorigenicity in Human Breast Cancer.
    Date June 2000
    Journal Cancer Research
    Excerpt

    Epidermal growth factor receptor vIII (EGFRvIII) is a tumor-specific, ligand-independent, constitutively active variant of the EGFR. Its expression has been detected in gliomas and various other human malignancies. To more fully characterize the function and potential biological role of EGFRvIII in regulating cell proliferation and in tumorigenesis, we transfected EGFRvIII cDNA into a nontumorigenic, interleukin 3 (IL-3)-dependent murine hematopoietic cell line (32D cells). We observed 32D cells expressing high levels of EGFRvIII (32D/EGFRvIII P5) to be capable of abrogating the IL-3-dependent pathway in the absence of ligands. In contrast, the parental cells, 32D/EGFR, 32D/ErbB-4, and 32D/ErbB-2+ErbB-3 cells, all depended on IL-3 or EGF-like ligands for growth. 32D/EGFRvIII P5 cells subjected to long-term culture conditions in the absence of IL-3 revealed further elevation of EGFRvIII expression levels. These results suggested that the IL-3-independent phenotype is mediated by EGFRvIII. The level of expression is a critical driving force for the IL-3-independent phenotype. Dose-response analysis revealed 32D/EGFRvIII cells to require 500-fold higher concentrations (50 ng/ml) of EGF to further stimulate the EGF-mediated proliferation than in the 32D/EGFR cells (100 pg/ml). Similar effects were also observed in beta-cellulin-mediated proliferation. Moreover, 32D cells expressing high levels of EGFRvIII formed large tumors in nude mice, even when no exogenous EGF ligand was administered. In contrast, no tumors grew in mice injected with 32D/EGFR, 32D/ErbB-4, and 32D/ErbB-2+ErbB-3 cells or low-expressing clone 32D/EGFRvIII C2 cells or the parental 32D cells. The changes of the ligand specificity support the notion for an altered conformation of EGFRvIII to reveal an activated ligand-independent oncoprotein with tumorigenic activity analogous to v-erbB. These studies clearly demonstrate that EGFRvIII is capable of transforming a nontumorigenic, IL-3-dependent murine hematopoietic cell line (32D cells) into an IL-3-independent and ligand-independent malignant phenotype in vitro and in vivo. To delineate the biological significance of EGFRvIII in human breast cancer, we expressed EGFRvIII in the MCF-7 human breast cancer cell line. Expression of EGFRvIII in MCF-7 cells produced a constitutively activated EGFRvIII receptor. Expression of EGFRvIII in MCF-7 cells also elevated ErbB-2 phosphorylation, presumably through heterodimerization and cross-talk. These MCF-7/EGFRvIII transfectants exhibited an approximately 3-fold increase in colony formation in 1% serum with no significant effect observed at higher percentages of serum. A similar result was also seen in anchorage-dependent assays. Furthermore, EGFRvIII expression significantly enhanced tumorigenicity of MCF-7 cells in athymic nude mice with P < 0.001. Collectively, these results provide the first evidence that EGFRvIII could play a pivotal role in human breast cancer progression.

    Title Angiopoietin-1 and Its Receptor Tie-2 Participate in the Regulation of Capillary-like Tubule Formation and Survival of Endothelial Cells.
    Date December 1999
    Journal Microvascular Research
    Excerpt

    Angiopoietin-1 (Ang-1) and its receptor Tie-2, a trans-membrane tyrosine kinase uniquely expressed by endothelial cells, are shown by null mutation studies to be essential to developmental angiogenesis. The phenotypic abnormalities in these knockout animals suggest that Tie-2 signaling is necessary for the maintenance and expansion of the primitive capillary network. We present in vitro evidence indicating that the Ang-1/Tie-2 system participates in the regulation of capillary tubule formation and is necessary for the survival of confluent endothelial cells. Although recombinant Ang-1, which induces Tie-2 phosphorylation, has no effect on the proliferation of endothelial cells, treatment of confluent adult bovine aortic endothelial cells (ABAE) cells grown on collagen gels with Ang-1 (100 ng/ml) causes the cells to migrate into the collagen gel and form capillary-like tubules. The tubule-forming effect of Ang-1 is similar to the effect caused by FGF-2. A soluble form of the Tie-2 extracellular domain, in fivefold molar excess, blocks Ang-1-induced tubule formation. Specific elimination of Tie-2 protein expression in cultured ABAE cells as a result of transfection with an antisense oligonucleotide causes cell death in a dose-dependent manner (IC(50) = 50 nM). The antisense treatment has no effect on cells that do not express Tie-2. Cells treated with antisense oligonucleotide show a sixfold increase in the rate of apoptosis as assessed by in situ end labeling of fragmented DNA. These findings are consistent with the view that Ang-1/Tie-2 signaling is essential for both angiogenesis and endothelial cell survival.

    Title The Effect of Raloxifene on Risk of Breast Cancer in Postmenopausal Women: Results from the More Randomized Trial. Multiple Outcomes of Raloxifene Evaluation.
    Date June 1999
    Journal Jama : the Journal of the American Medical Association
    Excerpt

    CONTEXT: Raloxifene hydrochloride is a selective estrogen receptor modulator that has antiestrogenic effects on breast and endometrial tissue and estrogenic effects on bone, lipid metabolism, and blood clotting. OBJECTIVE: To determine whether women taking raloxifene have a lower risk of invasive breast cancer. DESIGN AND SETTING: The Multiple Outcomes of Raloxifene Evaluation (MORE), a multicenter, randomized, double-blind trial, in which women taking raloxifene or placebo were followed up for a median of 40 months (SD, 3 years), from 1994 through 1998, at 180 clinical centers composed of community settings and medical practices in 25 countries, mainly in the United States and Europe. PARTICIPANTS: A total of 7705 postmenopausal women, younger than 81 (mean age, 66.5) years, with osteoporosis, defined by the presence of vertebral fractures or a femoral neck or spine T-score of at least 2.5 SDs below the mean for young healthy women. Almost all participants (96%) were white. Women who had a history of breast cancer or who were taking estrogen were excluded. INTERVENTION: Raloxifene, 60 mg, 2 tablets daily; or raloxifene, 60 mg, 1 tablet daily and 1 placebo tablet; or 2 placebo tablets. MAIN OUTCOME MEASURES: New cases of breast cancer, confirmed by histopathology. Transvaginal ultrasonography was used to assess the endometrial effects of raloxifene in 1781 women. Deep vein thrombosis or pulmonary embolism were determined by chart review. RESULTS: Thirteen cases of breast cancer were confirmed among the 5129 women assigned to raloxifene vs 27 among the 2576 women assigned to placebo (relative risk [RR], 0.24; 95% confidence interval [CI], 0.13-0.44; P<.001). To prevent 1 case of breast cancer, 126 women would need to be treated. Raloxifene decreased the risk of estrogen receptor-positive breast cancer by 90% (RR, 0.10; 95% CI, 0.04-0.24), but not estrogen receptor-negative invasive breast cancer (RR, 0.88; 95% CI, 0.26-3.0). Raloxifene increased the risk of venous thromboembolic disease (RR, 3.1; 95% CI, 1.5-6.2), but did not increase the risk of endometrial cancer (RR, 0.8; 95% CI, 0.2-2.7). CONCLUSION: Among postmenopausal women with osteoporosis, the risk of invasive breast cancer was decreased by 76% during 3 years of treatment with raloxifene.

    Title Inhibition of Angiogenesis and Breast Cancer Xenograft Tumor Growth by Vegi, a Novel Cytokine of the Tnf Superfamily.
    Date June 1999
    Journal International Journal of Cancer. Journal International Du Cancer
    Excerpt

    Recently, we reported a novel protein of the tumor necrosis factor (TNF) superfamily, named vascular endothelial cell growth inhibitor (VEGI), which is expressed predominantly in endothelial cells. When a secreted form of this new protein was overexpressed in mouse colon cancer cells, the growth of tumors formed by these cells in black mice was inhibited. We now report that recombinant VEGI inhibits the proliferation of endothelial cells but not that of other types of cells examined. The protein also inhibits formation of capillary-like structures by endothelial cells in collagen gels, and the growth of capillaries into collagen gels placed on the chick chorioallantoic membrane. The anticancer potential of VEGI was examined in a breast cancer xenograft tumor model in which the cancer cells were co-injected with Chinese hamster ovary cells overexpressing a secreted form of the protein. The co-injection resulted in potent inhibition of xenograft tumor growth. Our findings are consistent with the view that VEGI is an endothelial cell-specific negative regulator of angiogenesis.

    Title Immunotherapy with Interleukin-2 and Alpha-interferon After Il-2-activated Hematopoietic Stem Cell Transplantation for Breast Cancer.
    Date May 1999
    Journal Bone Marrow Transplantation
    Excerpt

    We previously demonstrated findings suggestive of autologous GVHD in patients receiving IL-2-activated peripheral blood stem cells (PBSC) with IL-2 after transplantation. A pilot study was designed to test tolerability, feasibility and frequency of autologous GVHD and engraftment using IL-2 and alpha-IFN post-transplantation. After cyclophosphamide (6 g/m2) and carboplatin (1800 mg/m2), patients with high-risk stage II or III breast cancer received chemotherapy and rhG-CSF mobilized autologous PBSC that had been cultured in IL-2 for 24 h. Subcutaneous administration of IL-2 began on day 0 at 6 x 10(5) IU/m2/day for 5 of 7 days each week and continued for 4 weeks. Once engraftment occurred, alpha-IFN was initiated at a dose of 1 x 10(6)/m2/day subcutaneously for 30 days. Thirty-four consecutive patients with stage II (n=20), IIIA (n=6) and IIIB (n=8) disease were treated. All patients were without evidence of disease at the time of transplantation. The average time required for the ANC to reach 500/mm3 was 10 days (range: 8-11 days) and for platelets to reach 20000/mm3 was 10.7 days (range: 6-21 days). Forty-seven percent of patients (n=16) completed the full course of immunotherapy; the remaining patients received attenuated doses due to patient's request (n=6), development of temperature >38 degrees C (n=3), development of neutropenia (n=3), serious infection (n=1) and miscellaneous reasons (n=5). Four patients experienced transient moderate toxicities (level 3) including elevated liver function tests, nausea, rash and capillary leak syndrome. Pathological findings suggestive of skin GVHD developed in 43% of patients (12/28 patients) when skin biopsies were evaluated in a blinded fashion. At 13 months post-transplant (median; range: 5-24 months), 28 patients (82%) remain disease-free. These results demonstrate the feasibility and toxicity of this regimen along with pathological findings compatible with autologous GVHD of the skin.

    Title Cripto-1 Indirectly Stimulates the Tyrosine Phosphorylation of Erb B-4 Through a Novel Receptor.
    Date April 1999
    Journal The Journal of Biological Chemistry
    Excerpt

    Cripto-1 (CR-1) is a recently discovered protein of the epidermal growth factor family that fails to directly bind to any of the four known erb B type 1 receptor tyrosine kinases. The present study demonstrates that CR-1 indirectly induces tyrosine phosphorylation of erb B-4 but not of the epidermal growth factor-related receptors erb B-2 and erb B-3 in different mouse and human mammary epithelial cell lines. In addition, down-regulation of erb B-4 in NMuMG mouse mammary epithelial cells and in T47D human breast cancer cells, using an anti-erb B-4 blocking antibody or a hammerhead ribozyme vector targeted to erb B-4 mRNA, impairs the ability of CR-1 to fully activate mitogen-activated protein kinase. Finally, chemical cross-linking of 125I-CR-1 to mouse and human mammary epithelial cell membranes results in the labeling of two specific bands with a molecular weight of 130 and 60 kDa, suggesting that the CR-1 receptor represents a novel receptor structurally unrelated to any of the known type I receptor tyrosine kinases. In conclusion, these data demonstrate that CR-1, upon binding to an unknown receptor, can enhance the tyrosine kinase activity of erb B-4 and that a functional erb B-4 receptor is required for CR-1-induced MAPK activation.

    Title Science, Medicine, and the Future. Antivascular Therapy: a New Approach to Cancer Treatment.
    Date April 1999
    Journal Bmj (clinical Research Ed.)
    Title Erbb-2 and Response to Doxorubicin in Patients with Axillary Lymph Node-positive, Hormone Receptor-negative Breast Cancer.
    Date September 1998
    Journal Journal of the National Cancer Institute
    Excerpt

    BACKGROUND: Overexpression of the erbB-2 protein by breast cancer cells has been suggested to be a predictor of response to doxorubicin. A retrospective study was designed to test this hypothesis. METHODS: In National Surgical Adjuvant Breast and Bowel Project protocol B-11, patients with axillary lymph node-positive, hormone receptor-negative breast cancer were randomly assigned to receive either L-phenylalanine mustard plus 5-fluorouracil (PF) or a combination of L-phenylalanine mustard, 5-fluorouracil, and doxorubicin (PAF). Tumor cell expression of erbB-2 was determined by immunohistochemistry for 638 of 682 eligible patients. Statistical analyses were performed to test for interaction between treatment and erbB-2 status (positive versus negative) with respect to disease-free survival (DFS), survival, recurrence-free survival (RFS), and distant disease-free survival (DDFS). Reported P values are two-sided. RESULTS: Overexpression of erbB-2 (i.e., positive immunohistochemical staining) was observed in 239 (37.5%) of the 638 tumors studied. Overexpression was associated with tumor size (P=.02), lack of estrogen receptors (P=.008), and the number of positive lymph nodes (P=.0001). After a mean time on study of 13.5 years, the clinical benefit from doxorubicin (PAF versus PF) was statistically significant for patients with erbB-2-positive tumors--DFS: relative risk of failure (RR)=0.60 (95% confidence interval [CI]=0.44-0.83), P=.001; survival: RR=0.66 (95% CI=0.47-0.92), P =.01; RFS: RR=0.58 (95% CI=0.42-0.82), P=.002; DDFS: RR=0.61 (95% CI=0.44-0.85), P=.003. However, it was not significant for patients with erbB-2-negative tumors-DFS: RR=0.96 (95% CI=0.75-1.23), P=.74; survival: RR =0.90 (95% CI=0.69-1.19), P=.47; RFS: RR=0.88 (95% CI=0.67-1.16), P=.37; DDFS: RR=1.03 (95% CI=0.79-1.35), P=.84. Interaction between doxorubicin treatment and erbB-2 overexpression was statistically significant for DFS (P=.02) and DDFS (P=.02) but not for survival (P= .15) or RFS (P=.06). CONCLUSIONS: These data support the hypothesis of a preferential benefit from doxorubicin in patients with erbB-2-positive breast cancer.

    Title Erbb-4 Ribozymes Abolish Neuregulin-induced Mitogenesis.
    Date August 1998
    Journal Cancer Research
    Excerpt

    The epidermal growth factor-like receptor tyrosine kinase (ErbB) family is frequently overexpressed in a variety of human carcinomas, including breast cancer. To assist in characterizing the role of ErbB-4 in breast cancer, we generated three specific hammerhead ribozymes targeted to the ErbB-4 mRNA. These ribozymes, Rz6, Rz21, and Rz29, efficiently catalyzed the specific cleavage of ErbB-4 message in a cell-free system. We demonstrated that the neuregulin-induced mitogenic effect was abolished in ribozyme Rz29- and Rz6-transfected 32D/ErbB-4 cells. Inhibition of mitogenesis was characterized by ribozyme-mediated down-regulation of ErbB-4 expression. In addition, we provide the first evidence that different threshold levels of ErbB-4 expression and activation correlate with different responses to neuregulin stimulation. High levels of ErbB-4 expression, phosphorylation, and homodimerization are necessary for neuregulin-stimulated, interleukin 3-independent cell proliferation in the 32D/E4 cells. In the case of Rz29-transfected 32D/E4 cells, low levels of ErbB-4 expression allowed neuregulin-induced phosphorylation but were insufficient to couple the activated receptor to cellular signaling. Furthermore, expression of the functional ErbB-4 ribozyme in T47D human breast carcinoma cells led to a down-regulation of endogenous ErbB-4 expression and a reduction of anchorage-independent colony formation. These studies support the use of ErbB-4 ribozymes to define the role of ErbB-4 receptors in human cancers.

    Title Stromal Igf-ii Messenger Rna in Breast Cancer: Relationship with Progesterone Receptor Expressed by Malignant Epithelial Cells.
    Date July 1998
    Journal Journal of Endocrinological Investigation
    Excerpt

    In breast cancer, insulin-like growth factor II (IGF-II) is stromal in origin and is considered an important regulator of tumour epithelium growth. The presence of progesterone receptor (PR) is expression of an intact oestrogen regulatory pathway of breast malignant epithelial cells and represents a parameter of cell differentiation in breast cancer. In this study we have examined the relationship between IGF-II mRNA expression and ER, PR content in 75 breast cancer. Formalin-fixed paraffin-embedded tissue sections were used to preserve histological details. IGF-II mRNA was evaluated by in situ hybridisation method and ER, PR by immunohistochemistry. IGF-II mRNA was scored semi-quantitatively: 2.6% breast tumour specimen expressed no IGF-II mRNA, 46.7% had low levels of expression (IGF-II-) and 50.7% had moderate or high IGF-II mRNA content (IGF-II+). IGF-II mRNA was found in the stroma fibroblasts surrounding malignant lesions and no signal was detected in malignant epithelial cells. In contrast, ER and PR were expressed only by neoplastic epithelial cells and no immunoreactivity was found in the stroma: 50/75 (66.6%) breast cancer specimens were positive for ER (ER+) and 35 (46.6%) for PR (PR+). Both, IGF-II mRNA and PR were directly correlated with the stromal proliferation (p < 0.05 and p < 0.001, respectively). No relationship was found between IGF-II RNA and ER. In contrast 24/35 (73.5%) PR breast cancer tissues were IGF-II+ (p < 0.01) and a strong correlation was found between epithelial PR immunostaining and stromal IGF-II mRNA content (p < 0.003). Our data indicate that in breast cancer IGF-II mRNA is generally expressed by stromal cells and ER and PR by epithelial cancer cells, and that IGF-II mRNA expression is strongly related with both percentage and staining intensity of PR+ epithelial cancer cells. These data support the hypothesis that IGF-II produced by the fibroblasts may exert a paracrin effect on malignant epithelium regulating its differentiation.

    Title Recombinant Heregulin-pseudomonas Exotoxin Fusion Proteins: Interactions with the Heregulin Receptors and Antitumor Activity in Vivo.
    Date May 1998
    Journal Clinical Cancer Research : an Official Journal of the American Association for Cancer Research
    Excerpt

    Growth factor receptors provide unique opportunities for development of targeted anticancer therapy. Members of the type I receptor tyrosine kinase family, including epidermal growth factor (EGF) receptor (EGFR) and ErbB-2/neu, are often overexpressed in various human cancer cells, including breast. Recently, it has been shown that both ErbB-3 and ErbB-4 are receptors for heregulin (HRG)/Neu differentiation factor. Eight chimeric toxins composed of the extracellular and EGF-like domains of four different HRG isoforms and truncated Pseudomonas exotoxin (PE38KDEL) were constructed. The fusion proteins exhibited activity similar to the native HRG in inducing ErbB receptors phosphorylation. The EGF-like domain of HRG13 and HRGbeta2 fused to PE38KDEL showed the highest cytotoxic activity, with a IC50 of < or = 0.001 ng/ml. The alpha isoforms that were fused to PE38KDEL were 100-fold less active than the beta isoforms. The HRG-Pseudomonas exotoxin (PE) toxins show extremely high activity against cells expressing ErbB-4 receptor, alone or together with other members of the ErbB receptor family. Cells that do not express ErbB-4 but express ErbB-3 receptor, together with the ErbB-2 or EGFR, exhibited moderate sensitivity to HRG-PE toxins. HRG-PE toxins have little or no activity against cells expressing EGFR, ErbB-2, or ErbB-3 alone. More than an 80% tumor regression was achieved by intratumor injection of 1 microg of fusion proteins per day for 5 days. Continuous i.p. administration of EGF-like domain of HRGbeta1-PE38KDEL for 7 days via a miniosmotic pump at a dose of 40 microg/kg/day inhibited the growth of ErbB-4 receptor positive but not ErbB-4 receptor negative cell lines in athymic nude mice. We conclude that there is therapeutic potential of HRG-PE toxins in the therapy of cancers overexpressing the ErbB-4 or ErbB-2 plus ErbB-3 receptors.

    Title Interleukin-2-activated Hematopoietic Stem Cell Transplantation for Breast Cancer: Investigation of Dose Level with Clinical Correlates.
    Date January 1998
    Journal Bone Marrow Transplantation
    Excerpt

    Incubating hematopoietic stem cells with IL-2 in vitro for 24 h generates cytotoxic T cells. When infused into patients, these cells may stimulate a graft-versus-tumor (GVT) effect. This clinical trial was designed to assess the ability of IL-2 activated peripheral blood stem cells (PBSC) to reconstitute hematopoiesis, to investigate dose levels and dose-limiting toxicities of IL-2, and to evaluate clinical results and preliminary laboratory effects using a combination of IL-2-activated autologous PBSC followed by IL-2 after transplantation. Sixty-one women with stage II-IV breast cancer were treated. After the administration of carboplatin (200 mg/m2/day for 3 days) and cyclophosphamide (2 g/m2/day for 3 days), patients received autologous PBSC that were cultured in IL-2 for 24 h followed by parenteral administration of IL-2 beginning the day of transplantation. Three escalating doses of IL-2 were evaluated with increasing duration up to 4 weeks. Of the 57 patients receiving IL-2 after tranplantation, 19 patients (33.3%) were unable to complete the planned course of IL-2 therapy due to persistent fevers (n = 9), diarrhea (n = 2), pulmonary capillary leak syndrome (n = 3), development of a rash (n = 1), atrial fibrillation (n = 1), or patient's request (n = 3). One death occurred during hospitalization. Engraftment of neutrophils occurred on day 11.5 (mean; range 8-21 days) and platelets on day 11.7 (mean; range 7-33 days). The maximal tolerated dose of IL-2 was 6 x 10(5) IU/m2/day for 4 weeks. Disease-free survival rates for all stages were comparable to current reports in the literature. Preliminary laboratory evaluations include FACScan analysis of the IL-2 activated PBSC demonstrating an increased percentage of CD3+, CD25+, HLA-DR+ T cells. Phenotypically similar cells were present in peripheral blood samples of patients when tested 15 days after transplantation. This study demonstrates successful engraftment with IL-2-activated PBSC after high-dose chemotherapy for women with stage II-IV breast cancer. The regimen is feasible and, although toxicities are common, they are manageable and correlate with increasing dose and duration of IL-2.

    Title Coexpression of Stromelysin-3 and Insulin-like Growth Factor Ii in Tumors of Ectodermal, Mesodermal, and Endodermal Origin: Indicator of a Fetal Cell Phenotype.
    Date July 1997
    Journal The Journal of Clinical Endocrinology and Metabolism
    Excerpt

    Stromelysin-3 (ST-3) is thought to play an important role in invasion and tumor progression. We have analyzed ST-3 expression in fibroblasts with defined topographical relations to breast cancers. We demonstrate that these fibroblasts exhibit the same distinctive pattern of messenger ribonucleic acid (mRNA) expression that we have previously shown for insulin-like growth factor II (IGF-II). Tumor-derived fibroblasts and skin fibroblasts produce abundant ST-3 mRNA. Fibroblasts from normal breast stroma distant from the malignant tumor in the same patient express considerably less ST-3 mRNA. When we analyzed ST-3 and IGF-II gene expression in sarcomas, we found a similar pattern of coexpression. Immunohistochemical analysis of IGF-II and ST-3 protein expression in sarcomas and breast tumors confirmed the mRNA data. ST-3 mRNA expression was also seen in most colon cancer cell lines, again matching reports of IGF-II gene expression. As the two proteins are known to play an important role during fetal growth and development, their coexpression in fibroblasts from malignant tumors of ectodermal (breast cancer) and mesodermal (sarcoma) origin and in epithelial cells of endodermal origin (colon cancer) implies a more primitive cellular phenotype. The regained ability to express such developmentally regulated proteins might, therefore, be a more general marker indicating a fetal-type phenotype of cells in a malignant tumor.

    Title Affinity for the Insulin-like Growth Factor-ii (igf-ii) Receptor Inhibits Autocrine Igf-ii Activity in Mcf-7 Breast Cancer Cells.
    Date April 1997
    Journal Molecular Endocrinology (baltimore, Md.)
    Excerpt

    We have investigated the autocrine regulation of insulin-like growth factor-II (IGF-II) signaling by the insulin-like growth factor-I receptor (IGF-IR) and the insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-IIR) in MCF-7 breast cancer cells, employing retroviruses encoding both IGF-I, IGF-II, and IGF-I and II mutants with reductions in affinity for either the IGF-IR or the IGF-IIR. These studies revealed reciprocal roles for IGF-IR and IGF-IIR affinity in the regulation of autocrine IGF-II activity. IGF-IR affinity was required for serum-free proliferation but also for efficient IGF-II secretion. In contrast, cellular proliferation, receptor tyrosine kinase-dependent signaling, and extracellular IGF-II protein accumulation were all reduced in the presence of IGF-IIR affinity. Inhibition of IGF-II signaling appeared to be the sole consequence of IGF-IIR affinity, as no cellular responses attributable to selective IGF-IIR binding by a reduced IGF-IR affinity IGF-II mutant could be detected. By operating as an IGF-II antagonist, the IGF-IIR has tumor suppressor-like properties, a suggestion consistent with reports of loss of heterozygosity at the IGF-IIR locus in a variety of human malignancies.

    Title Breast Cancer Risk in Rats Fed a Diet High in N-6 Polyunsaturated Fatty Acids During Pregnancy.
    Date January 1997
    Journal Journal of the National Cancer Institute
    Excerpt

    Women who took the synthetic estrogen diethylstilbestrol during pregnancy exhibit an elevated risk of breast cancer, whereas those who suffered from preeclampsia, which is associated with low circulating pregnancy estrogens, exhibit a reduced risk. Since a high-fat diet may increase circulating estrogen levels and possibly breast cancer risk, dietary factors during pregnancy could influence the risk of developing this disease.

    Title Regulation of Estrogen Receptor Concentration and Activity by an Erbb/her Ligand in Breast Carcinoma Cell Lines.
    Date December 1996
    Journal Endocrinology
    Excerpt

    Expression of the erbB-2 oncogene in breast cancer patients correlates with poor prognosis and failure of hormonal therapy. In this study, the effects of a putative erbB/HER ligand, gp30, on estrogen receptor (ER) concentration and activity was investigated in the estrogen receptor positive human breast cancer cells, BT474 and MCF-7, which express either high or low levels of erbB-2 and erbB-4, respectively. Treatment of cells with gp30 resulted in a decrease in the steady-state level of estrogen receptor protein by approximately 70-80%. The effect of gp30 on the concentration of ER was independent of serum in the media and was not inhibited by an epidermal growth factor receptor blocking antibody. In addition to the effect on ER protein, gp30 decreased the steady-state level of ER messenger RNA. Transcription run on experiments demonstrated that the decrease in ER expression was mediated by a decrease in ER gene transcription. The effect of gp30 on estrogen receptor activity was also investigated in this study. Treatment of cells with gp30 blocked estradiol induction of progesterone receptor. Inhibition was observed at the level of progesterone receptor protein, messenger RNA, and gene transcription. gp30 also blocked estradiol induction of pS2 gene transcription. In addition to its effects on progesterone receptor and pS2, gp30 blocked activation of an estrogen response element in a transient transfection assay and inhibited ER binding to its response element in a DNA mobility shift assay, suggesting a direct effect on the estrogen receptor. The effects of gp30 on estrogen receptor concentration and activity were independent of the level of erbB-2 and erbB-4 in the cell. These data show that gp30 regulates the concentration of ER and modulates ER activity.

    Title Intensive Outpatient Adjuvant Therapy for Breast Cancer: Results of Dose Escalation and Quality of Life.
    Date June 1996
    Journal Journal of Clinical Oncology : Official Journal of the American Society of Clinical Oncology
    Excerpt

    PURPOSE: A dose-escalation study was conducted to determine the maximum-tolerated dose (MTD) and dose-limiting toxicities (DLTs) of cyclophosphamide (CY) in combination with granulocyte colony-stimulating factor (G-CSF0 and doxorubicin (DOX) given every 2 weeks for eight cycles as outpatient adjuvant therapy for node-positive breast cancer. A pilot study to assess quality of life (QOL) was performed. PATIENTS AND METHODS: From March 1991 to April 1993, 19 patients were entered. Patients received escalating doses of CY intravenously (i.v.) (1,000 mg/m2, 1,500 mg/m2, 2,000 mg/m2, or 2,500 mg/m2) with DOX 40 mg/m2, G-CSF 10 micrograms/kg/d on days 2 to 12, and mesna, every 2 weeks for eight cycles. QOL was measured by the Profile of Mood States (POMS), the Psychosocial Adjustment to Illness Scale-Self Report (PAIS-SR), and a 27-item QOL scale. RESULTS: The CY dose of 2,500 mg/m2 every 2 weeks elicited toxicities that required dose reductions secondary to a combination of thrombocytopenia, hematuria, and anemia that required transfusion. The dose of 2,000 mg/m2 resulted in an acceptable toxicity profile. Ninety-two percent of cycles at the 2,000-mg/m2 dose were delivered on schedule and 77% without hospitalization. QOL assessments indicated high levels of distress measured by POMS in 47%, poor overall quality of life in 40%, and significant problems with physical symptoms in less than 27% of all patients for any given cycle. CONCLUSION: A dose of CY at 2,000 mg/m2 can be administered every 2 weeks with DOX and G-CSF for eight cycles in the outpatient setting with manageable toxicity. The majority of women described levels of physical symptoms and emotional distress as tolerable during treatment.

    Title Early Life Affects the Risk of Developing Breast Cancer.
    Date January 1996
    Journal Annals of the New York Academy of Sciences
    Title Phase I Trial of Pentosan Polysulfate.
    Date January 1996
    Journal Investigational New Drugs
    Excerpt

    Pentosan polysulfate is a semisynthetic pentasaccharide heparinoid derived from beechwood shavings. A total of nineteen patients with various adult solid tumors were treated with three dose levels (15, 22.5, and 30 mg/m2/dose) of subcutaneous pentosan polysulfate every 6 hours. The dose limiting toxicities were thrombocytopenia and elevated transaminases at the dose of 30 mg/m2 every 6 hours. The recommended starting dose for phase II trials is 22.5 mg/m2 given every 6 hours. There was an increase in anticoagulant activity as measured by activated partial thromboplastin time (aPTT) at the dose of 22.5 mg/m2 every 6 hours in most patients. There were no objective responses and three patients had stable disease lasting 16, 19 and 76 weeks.

    Title Regulation of Retinoblastoma Gene Expression in Hormone-dependent Breast Cancer.
    Date December 1995
    Journal Endocrinology
    Excerpt

    Studies have shown an increased risk for breast cancer in the mothers of children suffering from retinoblastoma and osteosarcoma, suggesting a role for the retinoblastoma susceptibility (Rb) gene product in breast cancer. We now show that estradiol decreases the expression of Rb at the level of protein and messenger RNA (mRNA) in estrogen-dependent breast cancer cell lines. Treatment of MCF-7 cells with 10(-9) M estradiol for 48 h resulted in a 70% decrease in the level of Rb protein. Ribonuclease protection assays showed a 50% decrease in the steady state levels of Rb mRNA by 12 h and a 70% decrease in Rb mRNA by 24 h. Treatment with estradiol had no effect on the rate of Rb gene transcription or on Rb mRNA stability, but resulted in an increase in the steady state level of Rb mRNA in the nucleus. The effect of estradiol was inhibited by 10(-7) M 4-hydroxytamoxifen. In the absence of estradiol, the antiestrogens 4-hydroxytamoxifen and ICI 164,384 increased Rb mRNA by 50% over that in estrogen-depleted conditions. Estradiol regulation of Rb mRNA also occurred in other estrogen-dependent breast cancer cell lines. Insulin-like growth factor I, insulin, progestins, and epidermal growth factor had no effect on Rb expression. In summary, these results show that estradiol specifically regulates the expression of the Rb susceptibility gene product in hormone-dependent breast cancer by a posttranscriptional mechanism that occurs in the nucleus. The results from this study suggest that the negative regulation of Rb expression by estradiol, rather than Rb loss or mutation, may play an important role in breast carcinogenesis.

    Title Malignant Breast Epithelium Selects for Insulin-like Growth Factor Ii Expression in Breast Stroma: Evidence for Paracrine Function.
    Date June 1995
    Journal Cancer Research
    Excerpt

    Paracrine interactions between stromal and epithelial cells are important influences on the growth and malignant behavior of breast cancers. Insulin-like growth factors I and II (IGF-I and II) are expressed by fibroblasts in benign and malignant breast lesions, and both are strong mitogens for a number of breast cancer epithelial cell lines in vitro. We have analyzed the stromal mRNA expression of IGF-I and IGF-II in matched sets of fibroblast cell lines derived from three locations in the affected breast of eight patients with breast cancer: (a) the breast tumor itself; (b) surrounding normal breast tissue; and (c) overlying breast skin. IGF-I expression was easily detected in all fibroblasts derived from normal breast tissue. In general, lesser amounts of IGF-I mRNA were detected in fibroblasts derived from breast tumors or skin. In contrast, IGF-II expression was detected at very low levels in only 3 of 8 normal breast fibroblasts, but was present in 6 of 8 tumor fibroblasts. IGF-II mRNA was expressed in all skin fibroblasts tested. IGF-II-negative stromal fibroblasts from normal breast, which were plated at low density and allowed to grow to confluence in the presence of MCF-7 breast tumor epithelial cells, demonstrated a marked increase in IGF-II mRNA expression. IGF-II in situ hybridization studies confirmed that IGF-II expression is seen at high levels in stroma of many invasive breast cancers but not normal breast. We conclude that paracrine influences, mediated by soluble factors released by breast tumor epithelium, are able to specifically increase expression of IGF-II in breast stroma, most likely by a process of clonal selection.

    Title Hormone Dependence of Breast Cancer Cells and the Effects of Tamoxifen and Estrogen: 31p Nmr Studies.
    Date June 1995
    Journal Breast Cancer Research and Treatment
    Excerpt

    Many breast tumors appear to progress from estrogen-dependent growth to a more malignant phenotype characterized by estrogen-independent growth, antiestrogen resistance, and a high metastatic potential. Utilizing 31P NMR spectroscopy on human breast cancer cells growing in vitro, we have investigated the effects of 17 beta-estradiol and tamoxifen on the metabolic/bioenergetic spectra of a series of human breast cancer cells that vary in their estrogen and antiestrogen responsiveness. A comparison of baseline spectra associates higher levels of phosphodiesters and UDP-glucosides (e.g. UDP-glucose, UDP-N-acetylglucosamine), and lower phosphocholine/glycerylphosphocholine and phosphocholine/phosphoethanolamine ratios, with the acquisition of estrogen-independent growth in estrogen receptor expressing cells. No metabolic changes are clearly associated with the metastatic phenotype. Whilst estrogen treatment produces no consistently significant spectral changes in any of the cell lines, the estrogen-independent and estrogen-responsive MCF7/MIII cell line responds to tamoxifen treatment by significantly increasing all spectral resonances 30%-40% above baseline values. This may reflect a tamoxifen-induced change to a more differentiated or apoptotic phenotype, or an attempt by the cells to reverse the inhibitory effects of the drug. The ability to detect metabolic changes in response to tamoxifen by NMR spectroscopy may provide a novel means to identify those tumors that are responsive to antiestrogen therapy.

    Title Antiestrogen Resistance in Er Positive Breast Cancer Cells.
    Date April 1995
    Journal Breast Cancer Research and Treatment
    Excerpt

    Acquisition of the antiestrogen resistance by breast cancer cells in vivo may result from a variety of mechanisms. The main pathway appears to involve loss of estrogen receptor (ER) expression or selection for ER negative cells among heterogenous population of tumor cells. However, clinical data suggest that, in about 30% of the cases, antiestrogen resistance arises even in the presence of estrogen receptors. Postulated mechanisms leading to the latter phenotype include selection for variant receptor forms during treatment, development of novel metabolic pathways for the drug, loss of nuclear co-factors, or activation of signal transduction pathway that cross activate ER signals. We have used an in vitro experimental system utilizing LY-2 cell line, an ER positive and antiestrogen resistant MCF-7 cell variant, to study the mechanism of antiestrogen resistance in the presence of functional ER. Result from a complementation experiment suggests that LY-2 phenotype is a recessive trait. Cloning of the genetic defect in the LY-2 cells would provide further insight for the mechanism of antiestrogen resistance in ER positive breast cancer cells.

    Title Ten-year Results of a Comparison of Conservation with Mastectomy in the Treatment of Stage I and Ii Breast Cancer.
    Date April 1995
    Journal The New England Journal of Medicine
    Excerpt

    BACKGROUND. Breast-conservation therapy for early-stage breast cancer is now an accepted treatment, but there is still controversy about its comparability with mastectomy. Between 1979 and 1987, the National Cancer Institute conducted a randomized, single-institution trial comparing lumpectomy, axillary dissection, and radiation with mastectomy and axillary dissection for stage I and II breast cancer. We update the results of that trial after a median potential follow-up of 10.1 years. METHODS. Two hundred forty-seven patients with clinical stage I and II breast cancer were randomly assigned to undergo either modified radical mastectomy or lumpectomy, axillary dissection, and radiation therapy. The 237 patients who actually underwent randomization have been followed for a median of 10.1 years. The primary end points were overall survival and disease-free survival. RESULTS. At 10 years overall survival was 75 percent for the patients assigned to mastectomy and 77 percent for those assigned to lumpectomy plus radiation (P = 0.89). Disease-free survival at 10 years was 69 percent for the patients assigned to mastectomy and 72 percent for those assigned to lumpectomy plus radiation (P = 0.93). The rate of local regional recurrence at 10 years was 10 percent after mastectomy and 5 percent after lumpectomy plus radiation (P = 0.17) after recurrences successfully treated by mastectomy were censored from the analysis. CONCLUSIONS. In the management of stage I and II breast cancer, breast conservation with lumpectomy and radiation offers results at 10 years that are equivalent to those with mastectomy.

    Title Detectable Inhibition of Heparin-binding Growth Factor Activity in Sera from Patients Treated with Pentosan Polysulfate.
    Date July 1993
    Journal Journal of the National Cancer Institute
    Excerpt

    BACKGROUND: Previous studies indicate that the heparinoid pentosan polysulfate (PPS) can inhibit heparin-binding growth factors (HBGFs) released from tumor cells and thus block tumor growth in animal models. However, because of its heparin-like activity, the major toxic effect expected for PPS is its inhibition of coagulation. PURPOSE: Our purpose was to determine if anti-HBGF activity could be achieved in patients without causing complications from anticoagulation. METHODS: We initiated a phase I trial in cancer patients and developed a cell proliferation assay to detect PPS in human serum based on its antigrowth factor activity. Blood samples from six healthy volunteers were collected in tubes containing different concentrations of PPS (FIBREZYM; concentration range, 0-10 micrograms/mL). Additional samples were obtained from four patients in the phase I trial before and after subcutaneous treatment with 15 mg/m2 of PPS. The activated partial thromboplastin time (aPTT), which is associated with coagulation, was measured in all blood samples. Serum prepared from the blood samples was heat inactivated and then incubated for 4-5 days with proliferating SW-13 cells, allowing determination of antigrowth factor activity. RESULTS: PPS added to blood samples increased aPTT only at concentrations above 1 microgram/mL, whereas HBGF-dependent proliferation was inhibited at less than 0.1 microgram/mL. Sera obtained from patients up to 4 hours after PPS treatment specifically inhibited HBGF-dependent cell proliferation by more than 65% even at a 1:10 dilution. At the same time, the aPTT was not altered in these patients, indicating no significant effect on coagulation by this dose of the heparinoid. CONCLUSIONS: HBGF-inhibitory concentrations of PPS can be achieved in patients' sera without significant effects on coagulation. IMPLICATION: The assay presented here could be useful to determine doses and scheduling of treatment in studies evaluating PPS as an antitumor agent.

    Title Effect of Endocrine Therapy on Growth of T61 Human Breast Cancer Xenografts is Directly Correlated to a Specific Down-regulation of Insulin-like Growth Factor Ii (igf-ii).
    Date March 1993
    Journal European Journal of Cancer (oxford, England : 1990)
    Excerpt

    Insulin-like growth factors I and II (IGF-I and IGF-II) are potent mitogens for some human breast cancer cell lines, and expression of IGF-II mRNA in the oestrogen receptor-positive (ER+) and oestradiol (E2) stimulated human breast cancer cell line T47D is increased by E2, suggesting a role for IGF-II in the mitogenic response to E2. Very little information is available from the literature on the relation between growth inhibition by endocrine therapy and cellular production of IGF-II. Here we report on the effect of E2 and tamoxifen (TAM) on IGF-II mRNA and protein expression in the ER+T61 human breast cancer xenograft. Growth of the T61 tumour is inhibited by treatment with E2 and TAM. Ribonuclease (RNAse) protection assays with human- and mouse-specific IGF-II antisense probes were used to study the regulation of IGF-II mRNA by E2 and TAM in the tumour. IGF-II protein expression was studied by radioimmunoassay. Untreated T61 tumours have a high baseline expression of IGF-II mRNA. TAM treatment of T61 tumours, which results in inhibition of tumour growth without tumour regression, reduced IGF-II mRNA expression approximately 10-fold after 48 h of treatment. E2 treatment of T61 tumours, which results in tumour regression, was accompanied by a more pronounced decrease in IGF-II mRNA expression in the tumour cells; 96 h after initiation of E2 treatment, there was almost no detectable IGF-II mRNA. Analyses of IGF-II protein showed that both treatments significantly reduced the concentration of IGF-II protein in the tumours. This down-regulation was found to be specific for IGF-II, since analyses of the effect of E2 on the expression of IGF-I mRNA, 36B4 mRNA, transforming growth factor alpha(TGF-alpha) mRNA, and epidermal growth factor (EGF) receptor mRNA in T61 tumours did not reveal any down-regulation. To further study the relation between inhibition of tumour growth and down-regulation of IGF-II, we exposed T61 tumours to a monoclonal antibody, alpha-IR3, which abolishes the physiological effect of IGF-I and IGF-II by blocking the binding of both growth factors to the type I IGF receptor. Treatment with alpha-IR3 resulted in inhibition of tumour growth during treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

    Title Clinical Significance of Erbb2 Protein Overexpression.
    Date January 1993
    Journal Cancer Treatment and Research
    Title Tyrosine Kinase Receptor--nuclear Protooncogene Interactions in Breast Cancer.
    Date January 1993
    Journal Cancer Treatment and Research
    Excerpt

    In summary, evidence is beginning to accumulate in support of a major role for tyrosine kinase receptors (and their activating growth factors) and steroid hormones and their receptors in normal development and differentiation of the mammary gland. A point of intersection of their mechanisms of action in growth control appears to be the induction of nuclear protooncogenes such as c-myc. When c-myc is amplified, as it is in many breast cancers, EGF and FGF receptor tyrosine kinase action becomes transforming, not simply mitogenic. A source of the transforming factors could be either stromal or epithelial. This mechanism could function early in the progression of breast cancer. c-erbB-2 and EGF receptor overexpression and amplification, when they occur, appear to render tumors even more malignant and of especially poor prognosis. These mechanisms could function late in the progression of breast cancer. Transgenic mouse studies have begun to echo these themes. They have established that a growth factor (TGF-alpha) and its receptor (EGF receptor), which appear to be important in normal mouse and human proliferation and gland development, and a protooncogene (c-myc), commonly amplified and overexpressed in human and mouse breast cancer, can each contribute to mammary carcinogenesis. The mechanisms of the two are likely to be distinct. myc is likely to be acting as a tumor initiator in combination with normal proliferative factors, whereas TGF-alpha is likely to be acting as a hyperproliferative (promotional) factor in combination with a normal background of mutational events. The role of unmutated but amplified erbB-2 in the transgenic mouse is not yet known.

    Title Stromal-epithelial Interactions in Breast Cancer.
    Date January 1993
    Journal Cancer Treatment and Research
    Title Purification and Characterization of a Novel Growth Factor from Human Breast Cancer Cells.
    Date September 1992
    Journal Biochemistry
    Excerpt

    We have purified and characterized a novel 30-kDa glycoprotein (gp30) with TGF alpha-like properties secreted from the estrogen receptor negative breast cancer cell line MDA-MB-231. This factor was immunoprecipitated by an anti-TGF alpha polyclonal antibody and also had TGF alpha-like biological activity, as assayed by EGF radioreceptor assay and anchorage-independent assays. In addition, the novel growth factor stimulated phosphorylation of the EGF receptor and erbB-2 receptor. However, the novel growth factor, unlike EGF and TGF alpha, bound to heparin-Sepharose. Purification of gp30 was obtained to apparent homogeneity by heparin affinity chromatography and subsequent reversed-phase chromatography. Tunicamycin treatment in vivo or N-glycanase deglycosylation in vitro revealed a putative precursor of approximately 22 kDa molecular mass in contrast to the "normal" 16-kDa precursor species for TGF alpha. In vitro translation of total mRNA from MDA-MB-231 cells confirmed the size of the putative precursor. Biochemical characterization of gp30 was begun by V8 protease digestion of the deglycosylated polypeptide and the translated products. Peptide mapping of V8-digested, immunoprecipitated material suggests that the amino acid sequence of this unique protein is distinct from mature TGF alpha and not the result of a posttranslational modification of the precursor. We conclude that this TGF alpha-like (gp30) polypeptide is a novel growth factor with agonistic activity for both EGF and erbB-2 receptors.

    Title Mastectomy Versus Breast-conserving Therapy in the Treatment of Stage I and Ii Carcinoma of the Breast: a Randomized Trial at the National Cancer Institute.
    Date June 1992
    Journal Journal of Clinical Oncology : Official Journal of the American Society of Clinical Oncology
    Excerpt

    PURPOSE: Mastectomy versus excisional biopsy (lumpectomy) plus radiation for the treatment of stage I and II breast cancer was compared in a prospective randomized study. PATIENTS AND METHODS: From 1979 to 1987, 247 women were randomized and 237 were treated on this study. All patients received a full axillary dissection and all node-positive patients received adjuvant chemotherapy with cyclophosphamide and doxorubicin. Radiation consisted of external-beam therapy to the whole breast with or without supraclavicular nodal irradiation followed by a boost to the tumor bed. RESULTS: The minimum time on the study was 18 months and the median time on the study was 68 months. No differences in overall survival or disease-free survival were observed. Actuarial estimates at 5 years showed that 85% of mastectomy-treated patients were alive compared with 89% of the lumpectomy/radiation patients (P2 = .49; 95% two-sided confidence interval [CI] about this difference, 0% to 9% favoring lumpectomy plus radiation). The probability of failure in the irradiated breast was 12% by 5 years and 20% by 8 years according to actuarial estimates. Of 15 local breast failures, 14 were treated with and 12 were controlled by mastectomy; the ultimate local-regional control was similar in both arms of the trial. CONCLUSION: These data add further weight to the conclusion that breast conservation using lumpectomy and breast irradiation is equivalent to mastectomy in terms of survival and ultimate local control for stage I and II breast cancer patients.

    Title Comparison of Urinary Transforming Growth Factor-alpha in Women with Disseminated Breast Cancer and Healthy Control Women.
    Date April 1992
    Journal Cancer Detection and Prevention
    Excerpt

    In an effort to explore the use of polypeptide growth factors as potential markers for cancer detection, we have identified the presence of transforming growth factor-alpha (TGF-alpha) in pooled urine of patients with metastatic breast cancer by a commercial radioimmunoassay (RIA) based on a rabbit antiserum raised to the C-terminal 17aa synthetic fragment of rat TGF-alpha. This TGF-alpha RIA detected both high molecular weight (HMW) and low molecular weight (LMW) forms of TGF-alpha in the conditioned media of a breast cancer cell line (MDA-MB-231) and in the urine of healthy women and those with breast cancer. The ratio of HMW to LMW species of TGF-alpha by RIA after Bio-Gel P-100 chromatography was approximately equal in pooled urine samples from both healthy women and those with breast cancer, and in the conditioned media from the cell line MDA-MB-231. Using established procedures for concentrating urinary proteins from 24-h urine samples by adsorption onto methyl-bonded microparticulate silica and selective elution by acetonitrile, TGF-alpha RIA results from women with disseminated breast carcinoma were compared with those of healthy pre- and post-menopausal control women. Analysis indicated a median TGF-alpha value of 981 ng/g urinary creatinine for urine samples from cancer patients (range 608 to 1737) and 642 ng/g creatinine (range 417 to 941) for control urine samples. Although the difference was statistically significant (p less than 0.05), urinary TGF-alpha detection with this assay method appears to have limited usefulness as a diagnostic marker for metastatic human adenocarcinoma of the breast.(ABSTRACT TRUNCATED AT 250 WORDS)

    Title Association of Increased Basement Membrane Invasiveness with Absence of Estrogen Receptor and Expression of Vimentin in Human Breast Cancer Cell Lines.
    Date March 1992
    Journal Journal of Cellular Physiology
    Excerpt

    Lack of estrogen receptor (ER) and presence of vimentin (VIM) associate with poor prognosis in human breast cancer. We have explored the relationships between ER, VIM, and invasiveness in human breast cancer cell lines. In the matrigel outgrowth assay, ER+/VIM- (MCF-7, T47D, ZR-75-1), and ER-/VIM- (MDA-MB-468, SK-Br-3) cell lines were uninvasive, while ER-/VIM+ (BT549, MDA-MB-231, MDA-MB-435, MDA-MB-436, Hs578T) lines formed invasive, penetrating colonies. Similarly, ER-/VIM+ cell lines were significantly more invasive than either the ER+/VIM- or ER-/VIM- cell lines in the Boyden chamber chemoinvasion assay. Invasive activity in nude mice was only seen with ER-/VIM+ cell lines MDA-MB-231, MDA-MB-435 and MDA-MB-436. Hs578T cells (ER-/VIM+) showed hematogenous dissemination to the lungs in one of five mice, but lacked local invasion. The ER-/VIM+ MCF-7ADR subline was significantly more active than the MCF-7 cells in vitro, but resembled the wild-type MCF-7 parent in in vivo activity. Data from these cell lines suggest that human breast cancer progression results first in the loss of ER, and subsequently in VIM acquisition, the latter being associated with increased metastatic potential through enhanced invasiveness. The MCF-7ADR data provide evidence that this transition can occur in human breast cancer cells. Vimentin expression may provide useful insights into mechanisms of invasion and/or breast cancer cell progression.

    Title Insulin-like Growth Factor-ii Overexpression in Mcf-7 Cells Induces Phenotypic Changes Associated with Malignant Progression.
    Date March 1992
    Journal Molecular Endocrinology (baltimore, Md.)
    Excerpt

    It has been proposed that the insulin-like growth factors (IGFs) can act as autocrine and/or paracrine growth promoters in breast cancer. To investigate this hypothesis, we infected early passage MCF-7 cells with a retroviral vector containing the coding sequence for the IGF-II preprohormone along with a constitutive cytomegalovirus promoter sequence. These cells do not normally express IGF-I or IGF-II. After infection with the retroviral vector, several single cell clones were analyzed. Seven of nine isolated clones expressed very high levels of IGF-II mRNA. Biologically active IGF-II protein was easily detectable in the medium conditioned by the IGF-II-expressing clones, and IGF receptors were down-regulated in these. All IGF-II-expressing clones showed marked morphological changes in anchorage-dependent culture, growing in large clumps and as free-floating colonies. The cells also cloned in soft agar in the absence of estrogen, while the wild-type MCF-7 cells and control cells infected with an irrelevant DNA sequence showed none of these properties. alpha IR-3, an antibody that blocks the type I IGF receptor, inhibited the growth of IGF-II-expressing clones in serum-free medium. This model demonstrates that IGF-II can serve as an autocrine growth stimulant in breast cancer epithelial cells and that IGF-II overexpression may be capable of mediating malignant progression in human breast cancer.

    Title Post-transcriptional Destabilization of Estrogen Receptor Mrna in Mcf-7 Cells by 12-o-tetradecanoylphorbol-13-acetate.
    Date October 1991
    Journal The Journal of Biological Chemistry
    Excerpt

    The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the regulation of the estrogen receptor (ER) was investigated in this study. After treatment with 100 nM TPA the concentration of receptor protein was measured using an enzyme immunoassay. By 24 h the receptor protein declined by about 80% from a level of approximately 236 fmol of ER/mg of protein in control cells to 50 fmol of ER/mg of protein in cells treated with TPA. Similar results were obtained with an estrogen receptor ligand binding assay. After removal of TPA, the level of ER returned to control values. 4-alpha-Phorbol, a compound related to TPA, had no effect on ER. The effects of TPA on ER expression appear to be mediated by activation of protein kinase C as H-7, an inhibitor of protein kinase C, blocks these effects. In addition to the effect on ER protein, TPA treatment also resulted in a decrease in the steady-state level of ER mRNA as determined by a RNase protection assay. The metabolic inhibitor cycloheximide was unable to prevent the TPA-induced decrease in ER mRNA. Transcription run-off experiments demonstrated that TPA had no effect on ER gene transcription. A half-life study demonstrated that TPA decreased ER mRNA half-life by a factor of 6 from approximately 4 h in control cells to 40 min in TPA-treated cells. These data suggest that the decline in ER expression is mediated by post-transcriptional destabilization of ER mRNA.

    Title Growth Factor Messenger Rna Expression by Human Breast Fibroblasts from Benign and Malignant Lesions.
    Date October 1991
    Journal Cancer Research
    Excerpt

    Breast tumors are a complex mix of epithelial, stromal, and vascular elements. We examined primary cultures of breast fibroblasts derived from benign and malignant lesions for expression of various growth factors. All fibroblast cultures, regardless of whether they were derived from benign or malignant lesions, expressed platelet-derived growth factor A chain, basic fibroblast growth factor, fibroblast growth factor 5, and transforming growth factor beta 1 mRNA. None expressed platelet-derived growth factor B chain or transforming growth factor alpha mRNA. However, examination of mRNA expression for the insulin-like growth factors revealed that 7 of 8 fibroblasts derived from benign lesions expressed insulin-like growth factor I (IGF-I) mRNA, while only 1 of 9 fibroblasts derived from malignancies expressed IGF-I mRNA. The opposite picture was seen for insulin-like growth factor II (IGF-II) mRNA expression, in which 1 of 9 benign-derived fibroblasts expressed IGF-II mRNA, while 5 of 9 malignant-derived fibroblasts expressed IGF-II. This correlated with previous in situ hybridization data, which showed IGF-I mRNA expression confined to the stroma of benign breast tissue. PDGF treatment of tumor fibroblasts resulted in a 3-fold increase in IGF-II mRNA. Thus there was an apparent dichotomy between IGF-I mRNA expression in the majority of fibroblasts derived from benign lesions and IGF-II mRNA expression in the majority of tumor-derived fibroblasts. Since the insulin-like growth factors are potent mitogens for breast tumor epithelial cells, this further supports the notion of a paracrine growth-promoting role for the insulin-like growth factors in breast lesions and suggests that IGF-II may be the more important growth promoter in malignant lesions.

    Title Insulin-like Growth Factors in Human Breast Cancer.
    Date September 1991
    Journal Breast Cancer Research and Treatment
    Excerpt

    Several protooncogenes and suppressor genes and a variety of growth factors and their receptors have been shown to be mutated, deleted, or activated in human breast cancer. These changes may account for the unregulated growth of breast carcinoma cells. Insulin-like growth factors I and II (IGF-I, IGF-II) belong to a family of polypeptides with growth promoting properties and structural homology to insulin. They exert their mitogenic effects by binding to the IGF-I receptor and activating its tyrosine protein kinase. Other proteins that specifically bind the IGFs include the plasma membrane IGF-II receptor, which also binds lysosomal hydrolases, and several IGF-binding proteins which may serve to modulate IGF interactions with receptors. Breast cancer cell lines express IGF-I and IGF-II receptors and different patterns of binding proteins. IGF-I and IGF-II are each mitogenic for subsets of breast cancer cell lines. This effect is inhibited by antibodies directed against the IGF-I receptor. In breast tumors, IGF-I is expressed by stromal cells, but not carcinoma cells; it is not expressed by breast cancer cell lines. IGF-I is therefore a potential paracrine regulator of breast cancer cell growth. Similarly, IGF-II is expressed in breast tumors, predominantly in stromal cells, but sometimes also in carcinoma cells and in a subset of cell lines. Thus, IGF-II is also a potential paracrine regulator of breast cancer cell growth; in addition, it can be an autocrine regulator in some breast cancer cells.

    Title Identification of Insulin-like Growth Factor Binding Proteins in Breast Cancer Cells.
    Date August 1991
    Journal Breast Cancer Research and Treatment
    Excerpt

    The insulin-like growth factors (IGFs) are potent mitogens for some breast cancer cell lines. Recent evidence suggests that IGF-induced mitogenesis may be influenced by specific IGF binding proteins (IGFBPs). In this study, breast cancer cell lines were examined for IGFBP protein and mRNA expression. Western ligand blot examination of conditioned media from breast cancer cell lines suggested that the IGFBP protein expression was heterogeneous. Although all breast cancer cell lines expressed a 24 kDa binding protein, MCF-7, an estrogen receptor positive (ER+) cell line, expressed a IGFBP compatible with reported sizes for IGFBP-2. Estrogen receptor negative (ER-) cells (MDA-MD-231, Hs578T) secreted IGFBPs consistent with sizes reported for IGFBP-1 and -3. Examination of mRNA expression supported these findings; IGFBP-2 was seen in all (4/4) ER+ cell lines while high levels of IGFBP-3 were found in ER- cell lines (3/5), although lower levels of IGFBP-3 mRNA could be found in some ER+ cell lines. In MCF-7 cells, steady state levels of IGFBP-3 mRNA were decreased by estradiol, while IGFBP-2 mRNA levels were slightly increased. These data suggest that IGFBP expression by breast cancer cells is heterogeneous, that the pattern of IGFBP expression is different between ER+ and ER- cell lines, and that in ER+ cells IGFBP mRNA may be regulated by estrogens. Thus, the IGFBPs may play an important role in mediating the mitogenic response of breast cancer cells to the IGFs.

    Title Quantification of Erbb-2/neu Levels in Tissue.
    Date August 1991
    Journal Methods in Enzymology
    Title Tumor Growth Dependent on Kaposi's Sarcoma-derived Fibroblast Growth Factor Inhibited by Pentosan Polysulfate.
    Date June 1991
    Journal Journal of the National Cancer Institute
    Excerpt

    A neoangiogenic response is critical for the unrestricted growth of solid tumors beyond a few millimeters in diameter. Release of adequate growth-stimulating activity from tumor cells is obviously required for the stimulation of blood vessel growth, and blockade of such stimulatory activity should repress tumor growth at the microscopic level. To test this hypothesis and to study appropriate inhibitors, we used a human adrenal cancer cell line (SW-13/K-fgf) engineered to secrete Kaposi's sarcoma-derived fibroblast growth factor (K-FGF), which we previously showed to induce growth of highly vascularized subcutaneous tumors in animals by autocrine and paracrine stimuli. In the present study, we tested different polysulfates for their selective inhibition of proliferation induced by K-FGF versus proliferation independent of K-FGF. Suramin and dextran sulfate showed slight selective inhibition of K-FGF-induced proliferation, ie, inhibition three- and five-fold greater, respectively, than the inhibition of proliferation independent of K-FGF. In contrast, heparin was inactive. The heparin analogue pentosan polysulfate (PPS), however, showed selective inhibition that was more than 2000-fold greater. The inhibitory effects of PPS on growth of SW-13/K-fgf cells, as well as endothelial cells, were fully reversible by an excess of added FGF. Daily intraperitoneal injections of PPS were tolerated well by athymic nude mice and prevented growth of subcutaneous SW-13/K-fgf tumor xenografts. PPS will be a useful tool to elucidate the effects of FGFs in vitro and in vivo and appears to be a prototype for the development of tumoricidal therapy based on targeting of growth factors.

    Title Autocrine Growth Stimulation by Secreted Kaposi Fibroblast Growth Factor but Not by Endogenous Basic Fibroblast Growth Factor.
    Date May 1991
    Journal Cell Growth & Differentiation : the Molecular Biology Journal of the American Association for Cancer Research
    Excerpt

    We studied the different potentials of a secreted and a nonsecreted member of the fibroblast growth factor (FGF) family to induce autocrine growth stimulation in human adrenal cortex carcinoma cells (SW-13). These epithelial cells express basic FGF (bFGF) cell surface receptors, and picomolar concentrations of bFGF suffice to induce anchorage-independent growth. The requirement for exogenously added bFGF contrasts with the intracellular storage of biologically active bFGF in SW-13 cells greater than 10,000-fold in excess of the concentration needed to stimulate anchorage independent growth. To study whether the expression of a secreted FGF would alter the growth phenotype of these cells, we transfected them with an expression vector coding for the Kaposi-fgf (K-fgf) oncogene. In contrast to controls, K-fgf-transfected cells secrete significant amounts of biologically active K-fgf protein into the growth media, show up to 50-fold increased colony formation in soft agar, and grow into rapidly progressing, highly vascularized tumors in athymic nude mice. A reversible inhibition of the autocrine growth stimulation in vitro is brought about by the polyanionic compound suramin. We conclude that FGF has to be released from SW-13 cells to function fully as a growth stimulator in vitro and in vivo.

    Title Reimbursement for Solid Tumor Autologous Bone Marrow Transplantation Trials: a Strategy for Ensuring Continuation of a Promising Therapy.
    Date May 1991
    Journal Cancer Investigation
    Title The Role of Estrogens in Growth Regulation of Breast Cancer.
    Date April 1991
    Journal Critical Reviews in Oncogenesis
    Title Transforming Growth Factor-beta Induces Platelet-derived Growth Factor (pdgf) Messenger Rna and Pdgf Secretion While Inhibiting Growth in Normal Human Mammary Epithelial Cells.
    Date March 1991
    Journal Molecular Endocrinology (baltimore, Md.)
    Excerpt

    Platelet-derived growth factor (PDGF) is a potent mitogen in human serum which specifically stimulates the proliferation of mesenchymal cells. We have now examined normal human mammary epithelial cells (HMEC) derived from reduction mammaplasties and grown in a serum-free defined medium. Medium conditioned by HMEC contained a PDGF-like activity that competed with [125I]PDGF for binding to PDGF receptors in normal human fibroblasts. When conditioned media were incubated with antiserum specific for either PDGF-A or PDGF-B, only PDGF-A antiserum was capable of inhibiting binding of conditioned media to PDGF receptors. Using an RNase protection assay, mRNA from normal HMEC was probed for both the PDGF-A and PDGF-B chains. Little or no PDGF-B was found in HMEC strains, while a strong signal was seen with the PDGF-A probe. When HMEC were grown in the presence of transforming growth factor-beta (TGF beta) for 48 h, inhibition of growth was observed in association with a 20- to 40-fold stimulation of PDGF-B mRNA and a 2-fold stimulation of PDGF-A mRNA. This mRNA induction was extremely rapid (within 1 h), and secreted PDGF activity was induced 2- to 3-fold. Two other HMEC growth inhibitors and differentiating agents, sodium butyrate and phorbol ester 12-O-tetradecanoylphorbol-13-acetate, had no effect on PDGF mRNA regulation. The current study suggests that PDGF gene induction is an extremely rapid and specific indicator of TGF beta function regardless of whether TGF beta is acting in a growth stimulatory or inhibitory manner. Any role of PDGF-B in TGF beta modulation of differentiation of normal or malignant mammary gland remains to be determined.

    Title Stromal Influences on Transformation of Human Mammary Epithelial Cells Overexpressing C-myc and Sv40t.
    Date January 1991
    Journal Journal of Cellular Physiology
    Excerpt

    The proto-oncogene c-myc and the oncogene SV40T, both of which have been implicated in the process of cellular immortalization in vitro, have been introduced via amphotropic retroviral expression vectors into the human mammary epithelial cell (HMEC) line 184A1N4 (A1N4). Two stable cell lines were established by growth in selective medium and were found to overexpress either c-myc (A1N4-myc) or SV40T antigen (A1N4-T). Neither the A1N4, A1N4-myc, or A1N4-T cells will grow in soft agar or form tumors in nude mice. However, A1N4-T or A1N4-myc cells, but not the parental A1N4 cells, form colonies in soft agar in response to either epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), or basic fibroblast growth factor (bFGF). Like EGF and TGF alpha, bFGF is moderately mitogenic for the anchorage-dependent growth (ADG) of all three cell lines. Further, co-cultivation of A1N4-T or A1N4-myc cells with primary diploid mammary fibroblasts can also induce the anchorage-independent growth (AIG) and stimulate the ADG of A1N4-T or A1N4-myc. In addition, conditioned medium obtained from these mammary fibroblasts also stimulated the AIG of the A1N4-T and A1N4-myc cells and was found to contain immunoreactive TGF alpha and bioactive FGF. The mammary fibroblasts express specific mRNA transcripts for bFGF and acidic FGF (aFGF). These results suggest that growth factors such as TFG alpha or FGF, which may be derived from the adjacent mammary stroma, might influence in a paracrine manner the phenotypic characteristics of a population of human mammary epithelial cells toward transformation.

    Title Endocrine Therapy of Human Breast Cancer Cells: the Role of Secreted Polypeptide Growth Factors.
    Date October 1990
    Journal Cancer Cells (cold Spring Harbor, N.y. : 1989)
    Excerpt

    Endocrine therapy is an important modality in the treatment of breast cancer. However, the precise molecular mechanisms underlying the growth inhibitory effect of endocrine therapy are unknown. Recently, it has been shown that breast cancer cells express and secrete polypeptide growth factors that can regulate the growth of the cells through autocrine and/or paracrine pathways. These growth factors are thought to be involved in the response to endocrine therapy. Three different mechanisms have been suggested: (1) stimulation of growth inhibitory peptides; (2) repression of mitogenic peptides; and (3) stimulation of mitogenic growth factors in cells overexpressing the corresponding receptor. This article reviews the scientific evidence on which these hypotheses are based.

    Title Systemic Therapy of Locally Advanced Breast Cancer: Review and Guidelines.
    Date September 1990
    Journal Oncology (williston Park, N.y.)
    Excerpt

    For patients with locally advanced breast cancer, mastectomy and/or radiation therapy achieve poor survival rates. However, the addition of cytotoxic chemotherapy optimizes outcome by treating micrometastases that are present at diagnosis. Current recommendations specify maximum doses of systemic therapy, preferably a doxorubicin-containing regimen, prior to local therapy with radiation and/or surgery. The authors review randomized studies of chemotherapy, radiation, and various combinations given pre- or postoperatively, as well as nonrandomized studies using combination chemotherapy regimens. They also discuss the possible future use of high-dose chemotherapy with autologous bone marrow rescue and the potential of manipulating growth factors which regulate breast cancer.

    Title The Effects of Hormonal and Chemotherapy on Tumoral and Nonneoplastic Breast Tissue.
    Date April 1990
    Journal Human Pathology
    Excerpt

    One hundred four sequential pretherapy and posttherapy breast tissue specimens from 57 patients with locally advanced and metastatic breast cancer were evaluated in an attempt to define the effects of systemic chemotherapeutic agents on the tumors and residual nonneoplastic breast tissue. The patients were treated uniformly at the National Cancer Institute on an experimental protocol combining systemic chemotherapy with attempted hormonal synchronization. Tumors were sampled prior to and following several cycles of chemotherapy to a maximum objective clinical response (average number of cycles, 7). In 38 cases, the posttreatment biopsy was positive for tumor. The most striking histologic change was extreme vacuolization of tumor cells that often resembled histiocytes. Atrophy of the terminal duct lobular unit (TDLU) and atypia of epithelial cells in TDLU and large ducts were also seen. Severe degrees of epithelial atypia occasionally proved to be difficult to distinguish from residual intraductal carcinoma. Breast biopsies were stained with antibodies to cytokeratin, epithelial membrane antigen (EMA), B72.3, lactalbumin, and SP1 using immunoperoxidase techniques. The number of cases showing immunoreactivity with antibodies to cytokeratin, EMA, and B72.3 remained approximately the same before and after therapy, while SP1 expression decreased and lactalbumin expression increased after therapy. Recognition of chemotherapeutic changes in breast tissue is important since systemic chemotherapy plays an important role in the management of breast cancer.

    Title Regulation of Breast Cancer Cells by Hormones and Growth Factors: Effects on Proliferation and Basement Membrane Invasiveness.
    Date March 1990
    Journal Hormone Research
    Excerpt

    The current understanding of the regulation of breast cancer cell proliferation and invasiveness by hormones and growth factors is reviewed. It has been shown that polypeptide growth factors are involved in hormone-independent breast cancer, and are sometimes oestrogen-regulated in hormone-responsive models. Basement-membrane invasiveness, relating to the metastatic potential of these cells, is also stimulated by oestrogen in hormone-dependent models, elevated in hormone-independent models, and is growth factor sensitive. Further understanding of the differential effects of growth factors on breast cancer cell proliferation and invasiveness should facilitate better therapeutic exploitation of regulation at this level.

    Title Pathologic Findings from the National Surgical Adjuvant Breast and Bowel Project: Prognostic Significance of Erbb-2 Protein Overexpression in Primary Breast Cancer.
    Date February 1990
    Journal Journal of Clinical Oncology : Official Journal of the American Society of Clinical Oncology
    Excerpt

    In order to investigate the prognostic significance of erbB-2 overexpression, immunohistochemical staining for the erbB-2 protein was performed on sections from paraffin blocks of 292 primary invasive breast cancers obtained from women enrolled in the National Surgical Adjuvant Breast and Bowel Project (NSABP) protocol B-06. Positive reaction indicative of erbB-2 overexpression was observed on tumor cells in 62 (21%) samples. Women whose cancers were judged to have erbB-2 overexpression had a significantly worse overall survival (P = .0012) with twice the mortality rate of women without detectable erbB-2 expression. No statistically significant effect was evident for disease-free survival (P = .22). In multivariate analysis, detection of erbB-2 overexpression was the second most predictive independent variable for survival after nodal status. Overexpression of erbB-2 was more common among tumors of poor nuclear grade (29%) than those of good nuclear grade (12%). The association of erbB-2 overexpression with decreased survival was evident only among women with tumors of good nuclear grade. In this subgroup, erbB-2 overexpression was associated with an approximately fivefold increase in mortality rate (P = .00001). The combined predictive value of erbB-2 overexpression and nuclear grade was evident regardless of their lymph node status. These results provide evidence that detection of erbB-2 overexpression may be an independent prognostic variable for patient survival. Moreover, when combined with evaluation of nuclear grade, it may be possible to use immunostaining for erbB-2 protein to identify patients at increased risk from within a relatively low-risk group.

    Title Role of an Estrogen Receptor-dependent Mechanism in the Regulation of Estrogen Receptor Mrna in Mcf-7 Cells.
    Date February 1990
    Journal Molecular Endocrinology (baltimore, Md.)
    Excerpt

    We have previously demonstrated that regulation of estrogen receptor (ER) expression in MCF-7 breast cancer cells is a complex process involving transcriptional and posttranscriptional regulation by estradiol. Treatment of MCF-7 cells with estradiol results in the down-regulation of receptor expression; posttranscriptional suppression of receptor mRNA appears to be the predominant mechanism. To determine whether posttranscriptional regulation of ER gene expression is mediated by an ER-dependent mechanism independent of protein synthesis, we have used the competitive estrogen antagonist, 4-hydroxytamoxifen, and the inhibitor of protein synthesis, cycloheximide, to study regulation of ER mRNA by estradiol. 4-Hydroxytamoxifen had no effect on the steady-state level of receptor mRNA and effectively blocked the suppression of ER mRNA by estradiol. The metabolic inhibitor, cycloheximide, was unable to prevent the estrogen induced decrease in ER mRNA. These data provide evidence that the posttranscriptional suppression of ER expression through estradiol is mediated through the ER independent of protein synthesis. A study of the effects of estradiol on the steady-state levels of nuclear and cytoplasmic receptor mRNA suggest that posttranscriptional suppression is a nuclear event.

    Title Anti-epidermal Growth Factor Receptor Antibodies Inhibit the Autocrine-stimulated Growth of Mda-468 Human Breast Cancer Cells.
    Date February 1990
    Journal Molecular Endocrinology (baltimore, Md.)
    Excerpt

    The response of malignant and nonmalignant human breast cell lines to the growth inhibitory effects of monoclonal antibodies against the epidermal growth factor (EGF) receptor was studied. A series of human breast cell lines, which express EGF receptor, were used: MDA-468, MDA-231, and Hs578T human breast cancer cells and the transformed human mammary epithelial cell lines 184A1N4 and 184A1N4-T that have been benzo[a]pyrene immortalized and further transformed with SV40T, respectively. Four antibodies of two different classes were tested: 225 immunoglobulin G (IgG), 108.4 IgG, 96 immunoglobulin M (IgM), and 42 IgM. All four antibodies inhibited the anchorage-dependent and -independent, EGF-stimulated growth of 184A1N4 and 184A1N4-T cells, respectively, and this growth inhibition could be reversed by the addition of increasing concentrations of EGF. In contrast, the antibodies inhibited the anchorage-dependent and -independent growth of MDA-468 cells in the absence of exogenous EGF suggesting that the antibodies were acting to block access of an endogenously produced ligand to the EGF receptor. In the presence of antibody and increasing concentrations of EGF, MDA-468 cell growth was first stimulated then inhibited as the EGF concentration increased, thus, uncovering the growth stimulatory potential of low concentrations of EGF in these cells. Data is presented that indicates MDA-468 cells secrete a transforming growth factor with autocrine growth stimulatory capabilities. The growth of MDA-231 and Hs578T cells, which contain activated ras oncogenes, was not inhibited by the antibodies and the growth of these cell lines was not stimulated by EGF. Of the cell lines studied only MDA-468 cells appear to possess an autocrine growth stimulatory capacity.

    Title Expression of the Transforming Growth Factor-alpha/epidermal Growth Factor Receptor Pathway in Normal Human Breast Epithelial Cells.
    Date February 1990
    Journal Endocrinology
    Excerpt

    To better understand the possible roles and interactions of transforming growth factor-alpha (TGF alpha) and its receptor, the epidermal growth factor (EGF) receptor in human breast epithelium, we have studied the expression of TGF alpha and the EGF receptor in a series of normal human mammary epithelial cells derived from reduction mammoplasty before in vitro propagation, during short term proliferation in vitro, and after immortalization. Increased TGF alpha mRNA expression coincided with conversion of the cells to a proliferative state in vitro. After establishment, propagation, and proliferation in vitro, the cells expressed high levels of both TGF alpha and EGF receptor mRNAs. Addition of diverse growth inhibitory agents, including 12-O-tetradecanoylphorbol-13-acetate (TPA), TGF beta, and sodium butyrate, to one of these rapidly proliferating cell populations (no. 184) failed to reduce the expression of either TGF alpha or the EGF receptor. Likewise, cessation of growth associated with both senescence and confluence of the 184 cells did not result in reduced expression. However, regulation of TGF alpha mRNA could be demonstrated by withdrawal of EGF from the medium or by antibody-mediated blockade of the EGF receptor in 184 cells. Antibody-mediated EGF receptor blockade also results in inhibition of growth and [3H]thymidine labeling. An autoregulatory autocrine loop appears operant in proliferating breast epithelial cells. Both growth and levels of TGF alpha mRNA expression are controlled by binding of ligand to the EGF receptor. These studies suggest a role for the TGF alpha/EGF receptor pathway in normal breast cell physiology.

    Title Effect of Preoperative Chemotherapy on Mastectomy for Locally Advanced Breast Cancer.
    Date February 1990
    Journal The American Surgeon
    Excerpt

    Mastectomy is frequently performed after intensive chemotherapy for locally advanced breast cancer. The effects of preoperative chemotherapy on the postoperative course and the timing of subsequent adjuvant therapy, however, have not been defined. We therefore reviewed the perioperative course of 54 patients undergoing mastectomy after combination (CAMFPT) chemotherapy for stage IIIA,B (IIIA - 25 pts; IIIB noninflammatory - 5 pts; IIIB inflammatory-24 patients) breast cancer. A median of 7 cycles (6 months) of chemotherapy was administered preoperatively. Mastectomy was performed a median of 20 days after last chemotherapy; white blood cell count (WBC) and platelet counts returned to normal limits preoperatively. Total mastectomy with or without axillary node dissection was performed in 53 patients, and a Halsted radical mastectomy in 1 patient. Negative margins on breast and/or axillary tissue were achieved in 47 patients (87.0%). Postoperative complications included skin flap necrosis in 8 patients (14.8%), seroma formation in 5 patients (9.3%), and wound infection in 1 patient (1.9%). Median operative blood loss (550 cc), hospital stay (8 days), and duration of wound catheter drainage (6 days) were comparable to published reports for modified radical mastectomy without preoperative chemotherapy. Systemic chemotherapy was resumed a median of 16 days after mastectomy, and radiotherapy started a median of 33 days after mastectomy. These findings indicate that intensive preoperative chemotherapy does not increase the hospital course or the postoperative complications of mastectomy for locally advanced breast cancer. In view of the current interest in treatment of stage I and II breast cancer with preoperative chemotherapy, this information may be useful in their management as well.

    Title Insulin-like Growth Factor Receptor Expression and Function in Human Breast Cancer.
    Date January 1990
    Journal Cancer Research
    Excerpt

    The insulin-like growth factors IGF-I and IGF-II are potent mitogens for several breast tumor cell lines in culture. Additionally, both IGF-I and IGF-II mRNAs are easily detected in the majority of breast tumor specimens examined, while no breast cancer epithelial cell lines we have studied express authentic IGF-I mRNA, and few lines express IGF-II mRNA. Although receptors for insulin, IGF-I, and IGF-II have been described, there is significant cross-reactivity between the various receptors and ligands in the insulin/insulin-like growth factor family, and it is not clear which receptor or receptors are responsible for the biological effects of these growth factors in this system. Using an RNase protection assay, we examined breast tumor specimens and breast cancer epithelial cell lines for expression of mRNA encoding the type I and type II IGF receptors as well as the insulin receptor. Virtually all of the specimens examined expressed mRNA for all three receptors. We then examined estrogen-dependent MCF-7 cells for the mitogenic effects of IGF-I and II in the presence of antibodies to both the type I and type II receptors. alpha IR-3, a monoclonal antibody which blocks the type I receptor, abolished the mitogenic effects of both IGF-I and IGF-II. It did not, however, block the mitogenic effects of insulin. We conclude that type I and type II IGF receptors are ubiquitously expressed in breast cancer, and our experiments with MCF-7 cells suggest the mitogenic effects of both IGF-I and IGF-II are mediated via the type I IGF receptor.

    Title Estrogen Regulation of Protein Synthesis and Cell Growth in Human Breast Cancer.
    Date January 1990
    Journal Vitamins and Hormones
    Title Expression of Insulin-like Growth Factor-ii Mrna in Fetal Kidney and Wilms' Tumor. An in Situ Hybridization Study.
    Date December 1989
    Journal Laboratory Investigation; a Journal of Technical Methods and Pathology
    Excerpt

    The pattern of insulin-like growth factor II (IGF-II) mRNA expression in developing kidney and Wilms' tumor was examined with in situ hybridization. In developing kidney, IGF-II was primarily expressed in blastemal cells and lost with their differentiation. In triphasic Wilms' tumor, a similar relationship was found. But in a monomorphous Wilms', tumor cells with epithelial differentiation expressed IGF-II mRNA. These data suggest that IGF-II may be involved in fetal nephrogenesis, that its expression is inversely coupled to normal epithelial differentiation, and that this differentiation may be aberrantly regulated in Wilms' tumor.

    Title Production of and Responsiveness to Transforming Growth Factor-beta in Normal and Oncogene-transformed Human Mammary Epithelial Cells.
    Date December 1989
    Journal Cancer Research
    Excerpt

    Transforming growth factors-beta (TGF beta) are a family of closely related, ubiquitously expressed growth factors with the common properties of induction of growth inhibition and expression of differentiation-related markers in epithelial cells. We investigated the role of TGF beta 1 in growth regulation of normal human mammary epithelial cells and in benzo(a)pyrene immortalized sublines further transformed by oncogenes in retroviral vectors. The normal cells were markedly growth inhibited by TGF beta 1, produced TGF beta in a latent form, and expressed TGF beta receptors. In the immortalized cells, both TGF beta-induced growth inhibition and TGF beta receptor binding were reduced. With the single oncogenes v-Ha-ras, v-mos, and SV40 T, growth sensitivity to TGF beta 1 increased, but TGF beta production or TGF beta receptor expression was not altered. Transformation to full malignancy by both SV40 T and v-Ha-ras led to escape from growth inhibition by TGF beta under anchorage-independent, but not anchorage-dependent, conditions without affecting TGF beta production or receptor characteristics. Thus, modulation of TGF beta growth responsiveness in these normal and oncogene transformed human mammary epithelial cells apparently occurs at a level distal to TGF beta receptor binding and is not solely correlated to expression of transforming oncogenes. Further, modulation of TGF beta production is not an indicator of malignant transformation in this system.

    Title Transforming Growth Factors Type Beta 1 and Beta 2 Are Equipotent Growth Inhibitors of Human Breast Cancer Cell Lines.
    Date November 1989
    Journal Journal of Cellular Physiology
    Excerpt

    At least one member of the TGF-beta family, TGF-beta 1, has been previously shown to inhibit the anchorage-independent growth of some human breast cancer cell lines (Knabbe et al., 1987; Arteaga et al., 1988). Members of the TGF-beta family might, therefore, provide new strategies for breast cancer therapy. We have studied the inhibitory effects of TGF-beta 1 and TGF-beta 2 on the anchorage-independent growth of the oestrogen receptor-negative cell lines MDA-MB-231, SK-BR-3, Hs578T, MDA-MB-468, and MDA-MB-468-S4 (an MDA-MB-468 clone not growth inhibited by EGF) and the estrogen receptor-positive cell lines MCF7, ZR-75-1, T-47D. TGF-beta 1 and TGF-beta 2 caused a 75-90% growth inhibition of MDA-MB-231, SK-BR-3, Hs578T, and MDA-MB-468 cells and a 50% growth inhibition of ZR-75-1 and early passage (less than 100) MCF7 cells. T-47D cells responded to TGF-beta only in serum-free conditions in the presence of IGF-1 or EGF. The growth of MDA-MB-468-S4 cells and late passage (greater than 500) MCF7 cells was not inhibited by TGF-beta 1 or TGF-beta 2. TGF-beta-sensitive MCF7 and MDA-MB-231 cells did not respond to Muellerian inhibiting substance (MIS), a TGF-beta-related polypeptide. TGF-beta 1 or TGF-beta 2 were mutually competitive for receptor binding with a similar affinity (Kd 25-130 pM, 1,000-13,000 sites per cell). To determine the time course of the TGF-beta effect, an anchorage-dependent growth assay was carried out using MDA-MB-231 cells. Growth inhibition occurred at 6 days, and cell-cycle changes were seen 12 hr after the addition of TGF-beta. Cells accumulated in the G1 phase and were thus inhibited from entering the S-phase. These data indicate that TGF-beta is a potent growth inhibitor in most breast cancer cell lines and provide a basis for studying TGF-beta effects in vivo.

    Title Transformation of Mouse Mammary Epithelial Cells with the Ha-ras but Not with the Neu Oncogene Results in a Gene Dosage-dependent Increase in Transforming Growth Factor-alpha Production.
    Date September 1989
    Journal Febs Letters
    Excerpt

    An enhanced expression of transforming growth factor-alpha (TGF alpha) was demonstrated in two clones of NOG-8 mouse mammary epithelial cells, NOG-8 SR1 and NOG-8 SR2, that have been transformed by a v-Ha-ras oncogene. The amount of TGF alpha production in NOG-8 SR1 and NOG-8 SR2 cells was dependent on the level of p21ras expression in these clones, which directly correlated with their cloning efficiency in soft agar. There was also a decrease in the number of epidermal growth factor (EGF) receptors on the NOG-8 SR1 and NOG-8 SR2 cells that is proportional to the amount of TGF alpha secreted. These effects were specific for ras because neu-transformed NOG-8 cells grew in soft agar at a comparable level to NOG-8 SR2 cells yet did not show any increase in TGF alpha production or change in EGF receptor expression.

    Title Modulation by Estrogen and Growth Factors of Transforming Growth Factor-alpha and Epidermal Growth Factor Receptor Expression in Normal and Malignant Human Mammary Epithelial Cells.
    Date September 1989
    Journal Recent Results in Cancer Research. Fortschritte Der Krebsforschung. Progrès Dans Les Recherches Sur Le Cancer
    Title Heterogeneous Expression of Erbb-2 Messenger Rna in Human Breast Cancer.
    Date August 1989
    Journal Cancer Research
    Excerpt

    Amplification and mRNA expression of the erbB-2 gene was analyzed in 61 samples of primary human breast carcinoma. In the 57 samples where RNA could be isolated four different expression level groups were identified. Comparison of hybridization signal with that for beta-actin revealed that erbB-2 mRNA could not be detected in 6 of 57 samples (11%), was detected at normal levels in 32 of 57 samples (56%), showed 4- to 8-fold overexpression in 8 of 57 samples (14%), and showed 16- to 128-fold overexpression in 11 of 57 samples (19%). Examination of the DNA of the same set of samples revealed 6 of 61 samples (10%) with distinct gene amplification and 6 of 61 samples (10%) with possible gene amplification. The highest levels of erbB-2 overexpression were associated with gene amplification. Samples with 4- to 16-fold overexpression of the erbB-2 mRNA occurred without evident gene abnormalities. There was no association of erbB-2 expression or gene amplification with clinical stage of breast carcinoma or axillary lymph node involvement. The clear amplification of the erbB-2 gene may be associated with a significantly shorter time to treatment failure.

    Title Analysis of Insulin-like Growth Factor I Gene Expression in Malignancy: Evidence for a Paracrine Role in Human Breast Cancer.
    Date August 1989
    Journal Molecular Endocrinology (baltimore, Md.)
    Excerpt

    Insulin-like growth factor I (IGF-I) activity has been reported to be produced by several human cancers. Identification of RNAs transcribed from the IGF-I gene has been complicated by the detection of multiple hybridizing bands on Northern analysis. To determine if any of these RNAs are transcribed from the IGF-I gene, we have used a sensitive and specific ribonuclease (RNAse) protection assay for IGF-I. We have also studied the breast cancer tissue expression of IGF-I using in situ hybridization histochemistry. We have found no IGF-I mRNA in breast (zero of 11) or colon cancer (zero of 9) cell lines; both of these tumors have been previously reported to express IGF-I mRNA. However, three of three neuroepithelioma and one of two Ewing's sarcoma cell lines express IGF-I mRNA; therefore, in these tumors IGF-I may be an autocrine growth factor. In contrast to breast cancer cell lines, RNA extracted from breast tissues has easily detectable IGF-I mRNA. In situ hybridizations show that IGF-I mRNA is expressed in the stromal cells, and not by normal or malignant epithelial cells. These findings suggest that although IGF-I is not produced by breast epithelial cells it may function as either a paracrine stimulator of epithelial cells or an autocrine stimulator of stromal cells.

    Title Fluorouracil and High-dose Leucovorin in Previously Treated Patients with Metastatic Breast Cancer.
    Date July 1989
    Journal Journal of Clinical Oncology : Official Journal of the American Society of Clinical Oncology
    Excerpt

    The efficacy and toxicity of leucovorin 500 mg/m2 administered intravenously (IV) over 30 minutes daily for five days followed in one hour by fluorouracil (5-FU) 375 mg/m2 administered IV daily for five days, each given every 3 weeks, was assessed in 54 previously treated patients with metastatic breast cancer. An overall objective response rate of 24% was achieved (95% confidence interval, 13% to 38%), with an additional 56% of patients maintaining stable disease. Eleven of 12 patients who responded had received previous 5-FU therapy. Toxicity of this regimen included grade 3 diarrhea in 13%, grade 3 or 4 mucositis in 33%, grade 3 or 4 granulocytopenia in 65%, and grade 3 or 4 thrombocytopenia in 19%. Delay of treatment was required for hematologic toxicity in 44 patients. Thirty-eight patients required dose reductions due to toxicity. Biochemical evaluation of tumor biopsy specimens obtained from 17 patients used as their own controls with and without leucovorin was performed. These studies reveal an increased stabilization of the 5-fluorodeoxyuridylate (FdUMP)-thymidylate synthase (TS) folate ternary complex with the addition of leucovorin. There was a 71% +/- 14% occupancy or inhibition of the enzyme with the use of both 5-FU and leucovorin, v 30% +/- 13% for 5-FU alone (P2 less than .037). The percent TS bound in responding patients was substantially higher than in those patients with progressive disease. Finally, the mean total tumor TS pre-therapy in seven patients was 31 fmol/mg compared with a mean of 81 fmol/mg in these same seven patients 24 hours after therapy. This 2.6-fold increase suggests that there is an induction of the enzyme, TS, with 5-FU treatment.

    Title Progression of Human Breast Cancer Cells from Hormone-dependent to Hormone-independent Growth Both in Vitro and in Vivo.
    Date June 1989
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    We have isolated a series of sublines of the hormone-dependent MCF-7 human breast cancer cell line after selection both in vivo and in vitro for growth in the presence of subphysiological concentrations of estrogens. These sublines represent a model system for study of the processes leading to hormonal autonomy. The cells form growing tumors in ovariectomized athymic nude mice in the absence of estrogen supplementation but retain some responsivity to estrogen as determined by stimulation of the rate of tumor growth in vivo and by induction of progesterone receptor. An ovarian-independent but hormone-responsive phenotype may occur early in the natural progression to hormone-independent and unresponsive growth in breast cancer. We observed no change in the affinity or decrease in the level of expression of estrogen receptors and progesterone receptors among the sublines and the parental cells. Epidermal growth factor receptors are not overexpressed in ovarian-independent cells. Thus, altered hormone receptor expression may be a late event in the acquisition of a hormone-independent and unresponsive phenotype. Sublines isolated by in vivo but not in vitro selection are more invasive than the parental cells both in vivo and across an artificial basement membrane in vitro. Thus, as yet unknown tumor-host interactions may be important in the development of an invasive phenotype. Furthermore, acquisition of the ovarian-independent and invasive phenotypes can occur independently.

    Title Treatment of Patients with Inflammatory Breast Cancer.
    Date June 1989
    Journal Important Advances in Oncology
    Title Breast Conservation Versus Mastectomy: Distress Sequelae As a Function of Choice.
    Date April 1989
    Journal Journal of Clinical Oncology : Official Journal of the American Society of Clinical Oncology
    Excerpt

    Between 1981 and 1984, 93 stage I and II breast cancer patients were entered onto a trial at the National Cancer Institute (NCI) randomizing patients to excisional biopsy plus radiation v mastectomy. Between 1984 and 1987, 98 stage I and II breast cancer patients were entered onto a behavioral study in Pittsburgh, approximately 70% of whom elected to have breast conservation surgery. Patients at both sites were assessed three to five days postsurgery, and again at 3-month's follow-up, using a well-validated mood measure, the Profile of Mood States (POMS). There were no demographic or disease differences between the two samples. In the Pittsburgh sample, using a repeated measures multivariate analysis of covariance (MANCOVA) analysis, after adjusting for menopausal status and radiotherapy and chemotherapy toxicity, the conservation group was psychologically worse off (F = 2.7, P less than .03). For example, they were significantly more distressed over time (F = 5.5, P less than .02), and more depressed in general (F = 9.2, P less than .005). Using Karnofsky ratings, the two groups were identical in terms of disability at 3-month's follow-up. In contrast, for the NCI patients participating in the randomized trial, after adjusting for chemotherapy and radiotherapy treatments, reported overall distress decreased over time (F = 17.4, P less than .0001) for all patients, irrespective of treatment group, and the between-groups MANCOVA was not significant. Thus, when comparing the two samples, when "choice" played a major role, the conservation patients were psychologically worse off--at least at 3-month's follow-up.(ABSTRACT TRUNCATED AT 250 WORDS)

    Title Transforming Growth Factor Alpha Production and Epidermal Growth Factor Receptor Expression in Normal and Oncogene Transformed Human Mammary Epithelial Cells.
    Date March 1989
    Journal Molecular Endocrinology (baltimore, Md.)
    Excerpt

    We have characterized the expression of transforming growth factor alpha (TGF alpha) and its receptor, the epidermal growth factor receptor (EGF-R), in normal and malignantly transformed human mammary epithelial cells. Human mammary epithelial cells were derived from a reduction mammoplasty (184), immortalized by benzo-a-pyrene (184A 1N4), and further transformed by the oncogenes simian virus 40 T (SV40 T), v-Ha-ras, and v-mos alone or in combination using retroviral vectors. 184 and 184A 1N4 cells require EGF for anchorage-dependent clonal growth. In mass culture, they secrete TGF alpha at high concentrations and exhibit an attenuated requirement for exogenous EGF/TGF alpha. SV40 T transformed cells have 4-fold increased EGF-R, have acquired the ability to clone in soft agar with EGF/TGF alpha supplementation, but are not tumorigenic. Cells transformed by v-mos or v-Ha-ras are weakly tumorigenic and capable of both anchorage dependent and independent growth in the absence of EGF/TGF alpha. Cells transformed by both SV40 T and v-Ha-ras are highly tumorigenic, are refractory to EGF/TGF alpha, and clone with high efficiency in soft agar. The expression of v-Ha-ras is associated with a loss of the high (but not low) affinity binding component of the EGF-R. Malignant transformation and loss of TGF alpha/EGF responsiveness did not correlate with an increase in TGF alpha production. Thus, TGF alpha production does not appear to be a tumor specific marker for human mammary epithelial cells. Differential growth responses to EGF/TGF alpha, rather than enhanced production of TGF alpha, may determine the transition from normal to malignant human breast epithelium.

    Title Regulation of the Estrogen Receptor in Mcf-7 Cells by Estradiol.
    Date March 1989
    Journal Molecular Endocrinology (baltimore, Md.)
    Excerpt

    The role of estradiol in the regulation of its cognate receptor in MCF-7 cells was investigated in this study. After treatment with 10(-9) M estradiol, the level of receptor protein was measured using an enzymeimmunoassay. By 6 h, the receptor protein declined by about 60% from a level of approximately 3.6 to 1.2 fmol/micrograms DNA. The level of receptor remained suppressed for 24-48 h. Similar results were obtained with an estrogen receptor (ER) binding assay. The steady state level of ER mRNA was determined by an RNase protection assay. Estrogen treatment resulted in a maximum suppression of mRNA by 6 h. Receptor mRNA remained depressed for 48 h. Transcription run on experiments demonstrated a transient decrease of about 90% in ER transcription after 1 h. By 3-6 h transcription increased approximately 2-fold and remained elevated for at least 48 h. These data suggest that estrogen down-regulates ER mRNA by inhibition of ER gene transcription at early times and by a posttranscriptional effect on receptor mRNA at later times.

    Title Tamoxifen and Fluoxymesterone Versus Tamoxifen and Danazol in Metastatic Breast Cancer--a Randomized Study.
    Date January 1989
    Journal Breast Cancer Research and Treatment
    Excerpt

    A prospective randomized trial of tamoxifen and fluoxymesterone versus tamoxifen and danazol in metastatic breast cancer was conducted from December 1980 to September 1985. Patients were eligible regardless of site of disease, estrogen receptor status, or age. Sixty-two of sixty-three randomized patients were evaluable for response. Overall response for tamoxifen and fluoxymesterone was 11% with 61% stabilization of disease, versus 12% response rate for tamoxifen and danazol with 59% stabilization. Toxicities with tamoxifen and fluoxymesterone were greater with an increase in masculinization. We conclude that the response rates to the combinations of tamoxifen and fluoxymesterone or tamoxifen and danazol reported are equivalent in this study but that the increased toxicity with tamoxifen and fluoxymesterone would make tamoxifen and danazol the treatment of choice if a combination were to be used.

    Title Insulin-like Growth Factor Ii Mrna Expression in Human Breast Cancer.
    Date December 1988
    Journal Cancer Research
    Excerpt

    Insulin-like growth factor II is a growth factor important in fetal development. Several cancer tissues and cell lines have been reported to express IGF-II and rat IGF-II is mitogenic for breast cancer cell lines. Using Northern analysis and ribonuclease protection assays, IGF-II mRNA was detected in normal fibroblasts and in the established breast cancer cell line, T47D. In this cell line, steady state levels of IGF-II message were increased by treatment with estradiol. 10 nM IGF-II, purified from human serum, was mitogenic for breast cancer cell lines. In vitro, IGF-II may act as an autocrine growth factor for some cell lines. RNA derived from breast cancer, pathologically normal breast tissue, and benign breast disease also contained IGF-II mRNA. When paired samples of normal and cancer tissue were obtained from the breast of the same patient, the level of IGF-II mRNA expression in the normal tissue was at least that found in the cancer. This is consistent with previous observations that show IGF-II is expressed in mesenchyme. These findings suggest that in breast cancer IGF-II is produced by stromal tissue elements and potentially by the malignant epithelial cells. Therefore, IGF-II may function as an autocrine or a paracrine growth factor in different breast tumors.

    Title Expression of Transforming Growth Factor Alpha and Its Messenger Ribonucleic Acid in Human Breast Cancer: Its Regulation by Estrogen and Its Possible Functional Significance.
    Date October 1988
    Journal Molecular Endocrinology (baltimore, Md.)
    Excerpt

    We have studied the estrogenic regulation and the potential autocrine role of transforming growth factor alpha (TGF alpha) in the human breast cancer cell line MCF-7. A biologically active apparent mol wt 30 k TGF alpha was identified by gel filtration chromatography in medium conditioned by MCF-7 breast cancer cells. We previously reported induction of TGF alpha levels in medium by 17 beta-estradiol. We now report correlated increases in TGF alpha mRNA, by Northern and slot blot analysis, after estrogen treatment of MCF-7 cells in vitro. In vivo experiments confirmed these data: estrogen withdrawal from MCF-7 tumor-bearing nude mice resulted in a decline in tumor size and TGF alpha mRNA levels. To explore the functional significance of TGF alpha in MCF-7 cells, anti-TGF alpha antibody was added to MCF-7 soft agar cloning assays. Inhibition of MCF-7 growth resulted, supporting an autocrine role for TGF alpha. Further experiments using an anti-EGF receptor antibody expanded this data, demonstrating inhibition of estrogen-stimulated monolayer MCF-7 cell growth. Examining the generality of TGF alpha expression, 4.8 kilobase TGF alpha mRNAs were seen in three other human breast cancer cell lines, MDA-MB-231, ZR 75B, and T47D. Expression of TGF alpha mRNA was detected in 70% of estrogen receptor positive and negative primary human breast tumors from 40 patients when examined by slot blot and Northern analysis. Thus, we have demonstrated broad expression of TGF alpha in human breast cancer, its hormonal regulation in an estrogen-responsive cell line, and its possible functional significance in MCF-7 cell growth.

    Title Epidermal Growth Factor Receptor Gene Expression in Estrogen Receptor-positive and Negative Human Breast Cancer Cell Lines.
    Date August 1988
    Journal Molecular Endocrinology (baltimore, Md.)
    Excerpt

    Expression of epidermal growth factor (EGF) receptor by human breast cancer tissues has an inverse relationship with expression of the estrogen receptor and may be associated with a poor clinical response. We have studied the regulation of EGF receptor expression in a series of human breast cancer cell lines with varying degrees of estrogen responsiveness. Three estrogen receptor-positive lines, MCF-7, ZR-75-1, and T47D, were found to have less than 70,000 EGF binding sites per cell by radioreceptor assay and were growth stimulated in vitro by EGF. Four estrogen receptor-negative lines, MDA-MB-231, Hs578T, EVSA-T, and BT-20, contained greater than 70,000 EGF binding sites per cell and showed no in vitro growth stimulation by EGF. In all cell lines EGF receptor number was correlated with the amount of EGF receptor protein and RNA. Differences in EGF receptor expression between the cell types was not due to amplification of the EGF receptor gene. Rather, variations in EGF receptor expression between lines were due, at least in part, to differences in the rate of EGF gene transcription as determined by nuclear run-off studies. Our data confirm the previously described inverse relationship between expression of EGF and estrogen receptors. We show here that the absence of estrogen receptor expression in human breast cancer cell lines is associated with higher levels of functional EGF receptor protein and mRNA.

    Title Alterations in Phosphoinositide Metabolism Associated with 17 Beta-estradiol and Growth Factor Treatment of Mcf-7 Breast Cancer Cells.
    Date August 1988
    Journal Molecular Endocrinology (baltimore, Md.)
    Excerpt

    Steady-state levels of phosphatidyl inositol (PtdIns) turnover are examined in MCF-7 human breast cancer cells in response to estradiol treatment. Elevated levels of PtdIns are observed 12-24 h after estradiol treatment, occur at estradiol concentrations as low as 10(-12) M, and are competitively blocked by the antiestrogen LY117018. MCF-7 cells secrete a transforming growth factor (TGF) alpha-like material which can partly replace estradiol in conferring tumorgenicity in nude mice. We show that acute or chronic treatment of MCF-7 cells with TGF alpha results in elevated PtdIns turnover and that chronic treatment increases growth rate. In contrast TGF beta is growth inhibitory and blocks estradiol-induced increases in PtdIns turnover. A phosphatidyl inositol 4,5-bisphosphate specific phospholipase-C activity has been identified and is elevated in association with estradiol treatment. These data are consistent with estradiol-induced autocrine growth factors, including TGF alpha, acting through the PtdIns turnover pathway as part of their mechanism of action.

    Title Multihormonal Regulation of Insulin-like Growth Factor-i-related Protein in Mcf-7 Human Breast Cancer Cells.
    Date August 1988
    Journal Molecular Endocrinology (baltimore, Md.)
    Excerpt

    MCF-7 human breast cancer cells have been studied for hormonal regulation of secretion of an insulin growth factor-I (IGF-I)-related growth factor. 17 beta-Estradiol, which is required for tumorigenesis of the cell line in the nude mouse and which stimulates proliferation in vitro, was able to significantly induce IGF-I secretion at 10(-13) M, with maximal induction at 10(-11) M. Under optimal conditions IGF-I could be induced 4-fold after 4 days. Demonstration of estrogenic stimulations required removal of phenol red, a weak estrogen, from the cell culture medium. In addition to estrogen, insulin, epidermal growth factor, and transforming growth factor alpha induce both cellular proliferation and IGF-I secretion, while growth inhibitory antiestrogens, transforming growth factor beta, and glucocorticoids have the opposite effect. In each case, modulations in IGF-I secretion preceeded effects on cellular proliferation. IGF-I was not regulated by human GH, basic fibroblast growth factor, platelet-derived growth factor, or PRL, none of which affected proliferation rate. Thus, regulation of IGF-I secretion in human breast cancer is controlled by different hormones from those previously reported in human fibroblasts. Regulation of IGF-I by neither estrogen nor antiestrogen was associated with changes in steady-state mRNA levels; thus regulation may occur at a step beyond mRNA. We conclude that IGF-I production is tightly coupled to growth regulation by estrogens, antiestrogens, and other hormones and may contribute to autocrine and/or paracrine growth regulation by these agents in breast cancer.

    Title Growth Regulatory Peptide Production by Human Breast Carcinoma Cells.
    Date August 1988
    Journal Journal of Steroid Biochemistry
    Excerpt

    The mechanisms by which human breast cancers regulate their own growth have been studied by us in an in vitro model system. We showed that specific growth factors (IGF-I, TGF alpha, PDGF) are secreted by human breast cancer cells. A variety of experiments suggest that they are involved in tumor growth and progression. These activities are induced by estradiol in hormone-dependent breast cancer cells and secreted constitutively by estrogen-independent cells. Concentrates of conditioned medium derived from breast cancer cells can induce the growth of hormone-dependent cells in vivo in athymic nude mice. Hormone-dependent breast cancer cells also secrete TGF beta. TGF beta is growth inhibitory. Growth inhibitors such as antiestrogens or glucocorticoids increase TGF beta secretion. An antiestrogen-resistant mutant of MCF-7 cells does not secrete TGF beta when treated with antiestrogen, but is growth inhibited when treated with exogenous TGF beta. Thus, TGF beta functions as a negative autocrine growth regulator and is probably responsible for some of the growth inhibitory effects of antiestrogens.

    Title The Cellular Response of Human Breast Cancer to Estrogen.
    Date June 1988
    Journal Progress in Clinical and Biological Research
    Title Effects of Steroid Hormones and Peptide Growth Factors on Protooncogene C-fos Expression in Human Breast Cancer Cells.
    Date March 1988
    Journal Cancer Research
    Excerpt

    To investigate if the estrogen control of the tumorigenic phenotype of breast cancer cells was mediated through activation of the c-fos protooncogene, we examined the expression of this oncogene in MCF-7 cells. In cells synchronized by double thymidine blockade, the peptide growth factors transforming growth factor alpha and epidermal growth factor increased c-fos mRNA levels 6-fold above controls after 30 min of treatment. The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, increased c-fos mRNA levels 4- to 5-fold above control. 17 beta-Estradiol, a growth stimulator, increased c-fos mRNA levels less than 2-fold above control levels, while progesterone, vitamin D3, dihydrotestosterone, and dexamethasone had little effect on c-fos mRNA levels. In contrast, 17 beta-estradiol treatment initially diminished the c-myc RNA level after 30 min of treatment and resulted in an elevation of c-myc by 2.5 h after initiation of treatment. We conclude that c-fos induction in these cells is growth related and accompanies stimulation by transforming growth factor alpha and epidermal growth factor. 17 beta-Estradiol, on the other hand, induced much smaller increases in c-fos mRNA levels, suggesting an alternative or more complex mechanism of cellular stimulation.

    Title Preliminary Results of a Phase Ii Trial for the Treatment of Metastatic Breast Cancer with 5-fluorouracil and Leucovorin.
    Date March 1988
    Journal Nci Monographs : a Publication of the National Cancer Institute
    Excerpt

    The active metabolite of FUra, 5-fluorodeoxyuridine monophosphate (5-FdUMP), requires the presence of reduced folates to form a covalent ternary complex with the target enzyme thymidylate synthase (TS). In vitro and in vivo studies have demonstrated a potentiation of the cytotoxic effects of FUra when combined with the reduced folate, leucovorin. We have applied this concept to the treatment of metastatic breast cancer in a phase II trial, as recent clinical studies on patients with colorectal carcinoma have suggested an enhanced efficacy for the combination of FUra plus leucovorin. Patients entered on the present study are undergoing treatment with a 5-day daily regimen of leucovorin (500 mg/m2, iv) followed by FUra (375 mg/m2, iv). Toxicity and response data are currently being collected on patients who have failed "standard" combination regimens that included FUra. In patients with accessible tumor, serial biopsies are being obtained during treatment with the combination of FUra and leucovorin and during therapy with FUra alone to assess the degree of 5-FdUMP binding to the target enzyme, TS, in the presence and absence of exogenously administered leucovorin. Preliminary results from the biochemical studies suggest an enhanced saturation of TS by the fluorinated pyrimidine when administered with leucovorin.

    Title Stimulation of Breast Cancer with Estrogens: How Much Clinical Value?
    Date December 1987
    Journal European Journal of Cancer & Clinical Oncology
    Title Growth Regulation of Human Breast Carcinoma Occurs Through Regulated Growth Factor Secretion.
    Date November 1987
    Journal Journal of Cellular Biochemistry
    Excerpt

    We describe studies on human breast cancer in which it is shown that specific growth factors (IGF-I, TGF alpha, PDGF) are secreted by human breast cancer cells and likely to be involved in tumor growth and progression. These activities are regulated by estradiol in hormone-dependent breast cancer and secreted constitutively by hormone-independent cells. These growth factor activities can induce the growth of hormone-dependent cells in vivo in athymic nude mice. Hormone-dependent breast cancer cells also secrete TGF beta, a growth-inhibitory substance, when treated with antiestrogens. TGF beta functions as a negative autocrine growth regulator and is responsible for some of the growth-inhibitory effects of antiestrogens.

    Title Neoadjuvant Chemotherapy in the Combined Modality Approach of Locally Advanced Nonmetastatic Breast Cancer.
    Date August 1987
    Journal Cancer Research
    Excerpt

    We have treated 76 patients with locally advanced breast cancer, 31 with stage IIIA, 41 with stage IIIB, and 4 with stage IV disease, with primary induction chemotherapy including an attempted hormonal synchronization in 70 patients. All were treated to maximum objective clinical response before proceeding to any local therapy. Patients achieving a complete response with a negative repeat biopsy generally received radiation therapy while patients with residual disease, partial response (PR) or no change (NC) status received debulking surgery prior to radiation therapy. Regardless of response to induction chemotherapy, patients received at least 6 additional months of chemotherapy following local therapy. Initial doses of combination chemotherapy were escalated to targeted myelosuppression. The objective response rate to induction chemotherapy was 93% with 49% complete response (CR), 44% PR, and 7% NC. The median numbers of cycles of chemotherapy to achieve a CR, PR, or NC were 5, 3, and 5, respectively. Three patients who currently have PRs are still on chemotherapy with continued tumor regression. Of 37 patients achieving a CR to chemotherapy, 35 were assessed by biopsies to determine pathological evidence of response. Twenty-three of the 37 patients (62%) were proven to be complete responders with negative biopsies. Twenty-four patients have relapsed, 6 with stage IIIA, 16 with stage IIIB, and 2 with stage IV. Five patients have had locoregional relapses alone, 4 locoregional and distant, and 15 distant alone. Median time to progression is 35.9 months for stage IIIA and 34.2 months for stage IIIB. Median survival is 35.3 months for stage IIIB and is indeterminate for stage IIIA. This aggressive primary chemotherapy regimen with hormonal synchronization followed by local therapy appears to provide excellent local control and encouraging early results on systemic disease control.

    Title [125i]17-alpha-iodovinyl 11-beta-methoxyestradiol Interaction in Vivo with Estrogen Receptors in Hormone-independent Mcf-7 Human Breast Cancer Transfected with the V-rash Oncogene.
    Date June 1987
    Journal Cancer Research
    Excerpt

    [125I]17-alpha-Iodovinyl 11-beta-methoxyestradiol [( 125I]MIVE2) has been evaluated as a potential radiotracer for the diagnostic imaging of estrogen receptor (ER)-positive human breast cancer. In vivo distribution experiments with athymic ovariectomized nude mice bearing human breast tumors revealed an apparent correlation between uptake of 125I-labeled compound and estrogen receptor concentration in the tumors. At 4 h after i.v. injection of [125I]MIVE2, HS578T (ER negative), ZR75-B (intermediate ER), and MCF-7ras (high ER) tumors accumulated 0.320 +/- 0.186, 0.679 +/- 0.467, and 2.6163 +/- 1.0121% injected dose/g, respectively. With coinjection of unlabeled 17-beta-estradiol, levels of radioactivity in MCF-7ras tumors were decreased to 0.4859 +/- 0.1424% injected dose/g, indicating a receptor-mediated process. Peak activity of radioligand in MCF-7ras tumors and uteri was observed at 2 h and was retained for the 8-h time course. Blood and nontarget tissue, such as muscle, revealed a rapid clearance of 125I-labeled compound by 8 h. Eight hours after injection, uterus and tumor-to-blood ratios were calculated to be 225 and 21, respectively. Also, MCF-7ras tumors were shown to accumulate 6.5-fold more radioactivity than muscle. These data suggest that [125I]MIVE2 has the capability of interacting specifically and with high affinity with estrogen receptors in human breast tumors in nude mice and may possibly be used for imaging receptor-positive tumors in breast cancer patients with very low serum estrogen levels. Selective uptake of compound in MCF-7ras tumors emphasizes the usefulness of an estrogen receptor-positive tumor model which has a unique ability to grow in a host system without circulating estrogens.

    Title Controversies in the Treatment of Cancer of the Breast.
    Date June 1987
    Journal Annual Review of Medicine
    Excerpt

    There are many important controversial areas in the management of breast cancer. This chapter focuses on adjuvant hormonal and chemotherapy for early-stage breast cancer. Standard care for node-positive premenopausal women is chemotherapy. However, there are still unanswered questions revolving around the administration of the treatment such as optimal timing, treatment duration, specific drugs, and dose intensity. Also, the questions of adjuvant chemotherapy for postmenopausal women, adjuvant endocrine therapy, and therapy for node-negative women are reviewed.

    Title Activation of Growth Factor Secretion in Tumorigenic States of Breast Cancer Induced by 17 Beta-estradiol or V-ha-ras Oncogene.
    Date March 1987
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    The MCF-7 human breast cancer cell line responds to estrogen stimulation in vitro by increased secretion of growth factors and proliferation and in vivo by tumor formation in the nude mouse. To test a possible role of growth factor secretion in expression of the tumorigenic phenotype, we stably transfected MCF-7 cells with the v-Ha-ras oncogene to produce the MCF-7ras cell line. The MCF-7ras cell line was tumorigenic in the absence of estrogens and secreted 3- to 5-fold elevated levels of a high molecular weight form of a type alpha transforming growth factor-like growth factor, type beta transforming growth factor, and insulin-like growth factor I. MCF-7ras cells, in contrast to MCF-7, were less sensitive to further growth stimulation by estrogen, type alpha transforming growth factor, and insulin-like growth factor I and showed little change in receptor levels for these hormones. Conditioned medium from MCF-7ras cells as well as two of its component growth factors (insulin-like growth factor I and type alpha transforming growth factor) replaced estrogen in stimulating MCF-7 colony formation in vitro. A coordinate increase in growth factor secretion by human breast cancer may contribute to its escape from estrogen dependence.

    Title National Institutes of Health Consensus Development Conference on Adjuvant Chemotherapy and Endocrine Therapy for Breast Cancer. Editorial Overview.
    Date December 1986
    Journal Nci Monographs : a Publication of the National Cancer Institute
    Title Secretion of an Insulin-like Growth Factor-i-related Protein by Human Breast Cancer Cells.
    Date September 1986
    Journal Cancer Research
    Excerpt

    Somatomedin activity is required for proliferation of normal cells; recently somatomedin activity in the cellular environment was shown to be necessary for expression of the transformed phenotype. We demonstrate here that authentic serum-derived insulin-like growth factor-I (IGF-I) stimulates the proliferation of four human breast cancer cell lines, MCF-7, MDA-MB-231, ZR-75-1, and Hs578T in serum-free monolayer culture and that each of these lines produces and secretes an IGF-I-related growth factor. The two highly tumorigenic estrogen-independent cell lines, MDA-MB-231 and Hs578T, produced 2- to 10-fold more IGF-I than did two estrogen responsive cell lines, MCF-7 and ZR-75-1, which are not tumorigenic in the absence of estrogen. These breast cancer cells also secrete a Mr 50,000 binding activity which partially obscured detection of IGF-I by radioimmunoassay. Acid-ethanol extraction allowed dissociation of the high molecular weight complex; whereupon, fully immunoreactive IGF-I comigrated on acid gel exclusion chromatography with authentic human serum-derived IGF-I. Radioimmunoassay displacement curves for breast cancer cell line-derived IGF-I were parallel to those for authentic IGF-I. Northern blot analysis of mRNA from four breast cancer cell lines demonstrated specific hybridization with a human IGF-I probe corresponding to one of the two major transcripts seen in human liver mRNA. These data suggest that breast cancer cell line-derived IGF-I is similar to liver-synthesized, serum-derived IGF-I.

    Title 8th San Antonio Breast Cancer Symposium--plenary Lecture. Autocrine and Paracrine Growth Regulation of Human Breast Cancer.
    Date August 1986
    Journal Breast Cancer Research and Treatment
    Excerpt

    We consider the hypothesis that estrogen control of hormone dependent breast cancer is mediated by autocrine and paracrine growth factors secreted by the breast cancer cells themselves. Though we show direct, unmediated effects of estrogen on specific cell functions, we also provide evidence that human breast cancer cells secrete a collection of growth factors (IGF-I, TGF alpha, TGF beta, a PDGF-like competency factor, and at least one new epithelial colony stimulating factor). Some of these are estrogen-regulated in hormone dependent cells, and are constitutively increased in cells which acquire independence either spontaneously or by ras transfection. Collectively, the secreted growth factors are capable of promoting tumor formation by MCF-7 cells in nude mice, though not to the same extent as estrogens. There would seem to be potential for clinical intervention in the autocrine and paracrine control of breast cancer cells, including some cells which are no longer dependent on estrogens.

    Title Complete Axillary Lymph Node Dissection for Stage I-ii Carcinoma of the Breast.
    Date June 1986
    Journal Journal of Clinical Oncology : Official Journal of the American Society of Clinical Oncology
    Excerpt

    We reviewed the complete axillary dissection specimens of 136 patients with stage I-II breast cancer to clarify the distribution of axillary lymph node metastases in this disease. Our series included 71 patients undergoing axillary dissection as part of a modified radical mastectomy (MRM) and 65 patients undergoing axillary dissection in conjunction with conservative surgery of the breast and definitive postoperative breast radiotherapy (CAD). These two groups of patients were comparable according to age, menopausal status, tumor size, and clinical stage. In all patients the pectoralis minor muscle was excised and all axillary tissue removed. Each specimen contained a median of 23 lymph nodes. The axillary levels (I, II, III) were determined according to the relationship of axillary tissue to the pectoralis minor muscle (lateral, inferior, medial). Thirty-nine percent of the lymph nodes were contained in level I, 41% in level II, and 20% in level III. There were no significant differences noted in the number of lymph nodes or in the distribution of lymph nodes according to axillary level between dissections performed as part of the MRM or those done as a single procedure (CAD). Sixty-five patients (47.8%) had one or more positive lymph nodes in their axillary specimen. The clinical and pathologic stage was determined and compared for all patients. Among patients judged to have a clinically negative axilla, 37.6% had histologically positive lymph nodes (clinical false-negative rate). For patients with a clinically positive axilla, 11.1% had, histologically, no evidence of metastatic disease (clinical false-positive rate). When the distribution of lymph node metastases according to axillary level was studied, it was found that 29.2% of lymph node-positive patients (or 14.0% of all patients) had metastases only to level II and/or III of the axilla, with level I being negative (skip metastases). This incidence of skip metastases was greater among clinically node-negative than among clinically node-positive patients, but was not related to the size or location of the primary tumor in the breast. In addition, it was found that 20.0% of lymph node-positive patients (or 9.6% of all patients) were converted from three or fewer to four or more positive nodes by analysis of lymph nodes contained in levels II and III. This conversion from three or fewer to four or more positive nodes was due primarily to information contained in level II, with level III contributing to a smaller degree.(ABSTRACT TRUNCATED AT 400 WORDS)

    Title Binding Characteristics and Biological Activity of 17 Alpha-[125i]iodovinyl-11 Beta-methoxyestradiol, an Estrogen Receptor-binding Radiopharmaceutical, in Human Breast Cancer Cells (mcf-7).
    Date May 1986
    Journal Cancer Research
    Excerpt

    17 alpha-[125I]Iodovinyl-11 beta-methoxyestradiol ([125I]MIVE2), a gamma-emitting analogue of estradiol, previously shown to bind to rat uterine estradiol receptor, was studied to determine the binding characteristics and biological activity in human breast cancer cells. In vitro determination of receptor binding by dextran-coated charcoal assays indicates that [125I]-MIVE2 binds specifically and with a high affinity to cytosolic estrogen receptors in the human breast cancer cell line, MCF-7. [3H]Estradiol binds to the receptor with approximately four times the affinity of [125I]-MIVE2 (Kd = 2.55 X 10(-9) M for [125I]MIVE2; Kd = 6.4 X 10(-10) M for [3H]estradiol). Unlabeled MIVE2 produces estrogenic effects similar to those of estradiol such as progesterone receptor induction and increases in thymidine incorporation in MCF-7 cells in culture. Cytosolic progesterone receptor levels were elevated 2.8-fold over control levels by 6 X 10(-9) M MIVE2. Stimulation of thymidine incorporation (approximately 300% above control levels) was observed after exposure to 1 X 10(-9) M MIVE2. Preliminary data show receptor-mediated uptake by the uterus in biodistribution studies in athymic nude mice given injections of [125I] MIVE2 (32-34 microCi). At 4 h, uterus:blood ratios are 20.5 and target tissue:nontarget tissue ratios are 12.9. In light of the fact that this compound can be prepared with a high specific activity, [125I]MIVE2 may have potential as a radiotracer for imaging estrogen receptor-positive breast tumors or metastatic lesions in human breast cancer patients.

    Title Transcriptional Control of Thymidine Kinase Gene Expression by Estrogen and Antiestrogens in Mcf-7 Human Breast Cancer Cells.
    Date May 1986
    Journal The Journal of Biological Chemistry
    Excerpt

    The mechanism by which estrogen and antiestrogens modulate cytoplasmic thymidine kinase (TK) activity has been studied in MCF-7 cells. Using a cloned cDNA probe for human TK, we have identified a single 1500-nucleotide transcript as the cytoplasmic TK-mRNA in MCF-7 cells. In normally cycling or synchronously growing cells, the level of this mRNA maximally increased 2-3-fold after 24 h of estradiol-17 beta (E2) stimulation and decreased below control level in the presence of antiestrogens. Neither E2 nor antiestrogens altered the size of TK-mRNA. Hormonal regulation of TK-mRNA paralleled changes in TK enzyme activity and [3H]thymidine incorporation. Using endogenous nuclear run-off transcription to investigate the expression of the human TK gene, we demonstrate that TK-mRNA levels were regulated by transcriptional control. Modulation of TK gene activity during the process of estrogen stimulation or antiestrogen inhibition was not accompanied by changes in the methylation pattern at internal sites of the TK gene. These results suggest a transcriptional control of human TK gene by E2 and antiestrogens in MCF-7 cells.

    Title Characterization of Estrogen Responsive Transforming Activity in Human Breast Cancer Cell Lines.
    Date April 1986
    Journal Cancer Research
    Excerpt

    We have characterized the production of transforming growth factor (TGF) activities by five human breast cancer cell lines in culture. The presence of TGF-alpha and -beta activity in medium conditioned by these cells was detected by induction of anchorage-independent colony formation of normal rat kidney cells in soft agar and by epidermal growth factor and TGF-beta receptor competition studies. In MCF-7, an estrogen-receptor positive cell line which requires estrogen for tumorigenesis in vivo, 17 beta-estradiol induced a 2-5-fold increase in a TGF-alpha-like activity (apparent molecular weight, 68,000 and 30,000 by column chromatography). An estrogen induction of lower magnitude (1.5-2.5-fold) was also seen in two other estrogen responsive cell lines, ZR-75-B and T47D. TGF-beta was not induced by 17 beta-estradiol in any of the three cell lines and was decreased by up to 50% of control in the MCF-7 and T47D cell lines. TGF-beta was not detectable by radioreceptor assay in the ZR-75-B cell line. Two hormone-independent and highly tumorigenic cell lines were studied. The MDA-MB-231 cell line produced large amounts of both TGF-alpha-like (Mr 30,000) and TGF-beta activities. In the HS578T cell line, little TGF-alpha was detectable, while large amounts of TGF-beta were produced. No simple correlations between the tumorigenicity of the cell lines in nude mice and production of either TGF activity could be demonstrated.

    Title Use of Two Mcf-7 Cell Variants to Evaluate the Growth Regulatory Potential of Estrogen-induced Products.
    Date April 1986
    Journal Cancer Research
    Excerpt

    Two variants of the human estrogen-responsive breast cancer cell line MCF-7, were utilized to study the expression of an estrogen-induced gene, pS2, and an estrogen-induced Mr 52,000 protein. One variant cell line, I13, is growth inhibited after chronic exposure to estrogen. Both the pS2 gene product and the Mr 52,000 protein were produced at maximal levels at a time when I13 growth was inhibited by estrogen. The variant cell line, LY2, selected for its resistance to the growth-inhibitory effects of the antiestrogen, LY117018, grew normally in the presence of this drug, although both pS2 expression and Mr 52,000 protein production were inhibited. These results confirm that the pS2 gene and Mr 52,000 protein are estrogen-regulated elements, but the lack of correlation between their activities and variant cell growth suggests that they are not major autocrine growth-stimulatory agents.

    Title The Management of Nonmetastatic Locally Advanced Breast Cancer Using Primary Induction Chemotherapy with Hormonal Synchronization Followed by Radiation Therapy with or Without Debulking Surgery.
    Date December 1985
    Journal World Journal of Surgery
    Title Mastectomy Versus Radiotherapy As Treatment for Stage I-ii Breast Cancer: a Prospective Randomized Trial at the National Cancer Institute.
    Date December 1985
    Journal World Journal of Surgery
    Title Clonogenic and Nonclonogenic in Vitro Chemosensitivity Assays.
    Date September 1985
    Journal Cancer Treatment Reports
    Excerpt

    We need practical laboratory methods for predicting the chemosensitivity of human neoplasms. Over the past 20 years, numerous investigators have implied or stated with increasing certitude that clonogenic assays are the most valid (or only valid) approach to predictive chemosensitivity testing. We feel that this point of view may have insufficient scientific validity and may be harmful to progress in this area. In addition to well-known technical problems, there are serious theoretical problems with clonogenic assays. These include the disruption of normal cell-to-cell interactions, the possibility that true tumor stem cells may be largely nondividing (G0) cells, while cells forming colonies are exclusively dividing cells, the possibility that clonogenic cells may largely represent cells which are not true stem cells, and the fact that clonogenic assays have the ability to measure cell kill over a narrow log range, while meaningful clinical responses require a multiple-log cell kill. This latter fact mandates the use of unrealistically low drug concentrations to avoid excessive false-positive results. Neither theoretical concepts, direct experimental data, nor clinical correlations support the alleged superiority of clonogenic assays. Clonogenic assays may not be advantageous compared to other more practical methods of estimating the general chemosensitivity of proliferating cells. In contrast, there is a growing body of literature which indicates that early evidence of cell damage in the entire tumor cell population may accurately predict for a multiple-log stem cell kill and meaningful clinical response. Future studies should continue to develop and test assays based on alternative methods for detecting cell kill in the proliferating and total tumor cell populations.

    Title Plasma Melatonin and the Hormone-dependency of Human Breast Cancer.
    Date August 1985
    Journal Journal of Clinical Oncology : Official Journal of the American Society of Clinical Oncology
    Excerpt

    We studied the 24-hour plasma melatonin profile in three groups of women: normal individuals, women with breast cancer, and women at high risk for breast cancer, to determine the relationship of plasma melatonin to this malignancy. The mean daytime (nadir) and mean nighttime (peak) plasma levels for the normal subjects were 9.1 pg/mL and 70.9 pg/mL, respectively. The mean daytime and nighttime plasma levels, and the range of melatonin day to night differences for women with breast cancer and women at high risk for breast cancer were comparable to each other and to the normal subjects, with no statistically significant differences noted. The patients with breast cancer demonstrated a striking correlation between the melatonin diurnal rhythm and the steroid receptor content of the primary tumor. Women with estrogen (ER) or progesterone (PR) receptor-positive tumors had a significantly lower mean plasma melatonin day to night difference than did patients with ER- or PR-negative tumors. Further, a strong inverse correlation was observed between the plasma melatonin concentration and the quantities of ER and PR in the primary tumor: the lower the plasma melatonin concentration the greater the amount of either receptor in the primary tumor. Plasma melatonin did not correlate with tumor glucocorticoid receptor content or stage of breast cancer among these patients, or with menopausal status, age, parity, or the plasma levels of estrone, estradiol, progesterone, follicle-stimulating hormone (FSH), or luteinizing hormone (LH) among all individuals studied. Plasma melatonin was also independent of the degree of risk for breast cancer among the high-risk patients. These findings suggest an important relationship between the plasma melatonin diurnal rhythm and the hormone dependency of human breast cancer, and may have implications for both the prognosis and treatment of this malignancy.

    Title Transfection of V-rash Dna into Mcf-7 Human Breast Cancer Cells Bypasses Dependence on Estrogen for Tumorigenicity.
    Date May 1985
    Journal Science (new York, N.y.)
    Excerpt

    The natural history of estrogen-responsive breast cancers often involves a phenotypic change to an estrogen-unresponsive, more aggressive tumor. The human breast cancer cell line, MCF-7, which requires estradiol for tumor formation in vivo and shows growth stimulation in response to estradiol in vitro, is a model for hormone-responsive tumors. The v-rasH onc gene was transfected into MCF-7 cells. The cloned MCF-7ras transfectants, which expressed the v-rasH messenger RNA and v-rasH p21 protein (21,000 daltons), were characterized. In contrast to the parental cell line, MCF-7ras cells no longer responded to exogenous estrogen in culture and their growth was minimally inhibited by exogenous antiestrogens. When tested in the nude mouse, the MCF-7ras cells were fully tumorigenic in the absence of estrogen supplementation. Thus, cells acquiring an activated onc gene can bypass the hormonal regulatory signals that trigger the neoplastic growth of a human breast cancer cell line.

    Title Effects of Estrogen and Tamoxifen on the Regulation of Dihydrofolate Reductase Gene Expression in a Human Breast Cancer Cell Line.
    Date April 1985
    Journal Cancer Research
    Excerpt

    We have studied the effects of estrogen and the antiestrogen tamoxifen on the regulation of dihydrofolate reductase (DHFR) gene expression in a methotrexate-resistant (MTXR) human breast cancer cell line MCF-7, which contains a 50-fold increase in the level of DHFR enzyme and amplified DHFR gene sequences. Despite their selection for methotrexate resistance, the MTXR cells have retained many characteristics of the parental MCF-7 cell line. Concentrations of estrogen receptors as well as their binding affinity to estradiol are identical in both cell lines. MTXR MCF-7 cells remain sensitive to estrogen and respond to estradiol with an induction of progesterone receptors, as well as increases in the rate of DNA synthesis and cell growth. Incubation of MTXR MCF-7 cells with estradiol results in an additional 1.5- to 3.0-fold increase in their already elevated level of DHFR. The hormone-induced increases in DNA synthesis and DHFR levels are similar both with respect to the time course of inductions, as well as their dose response to estradiol. However, these two estrogen-induced effects are not coupled, since the induction of DHFR occurs even in the absence of concomitant DNA synthesis. Estradiol has no effect on DHFR enzyme stability; thus, the entire effect of estrogen on DHFR levels results from the increased synthesis of this housekeeping enzyme. In contrast, treatment of MTXR MCF-7 cells with the antiestrogen tamoxifen reduces the rate of DHFR enzyme synthesis, resulting in lower cellular levels of DHFR. These MTXR MCF-7 cells represent a useful model in which to study the mechanisms involved in the modulation of DHFR gene expression by estrogen and tamoxifen. Since the level of DHFR is a critical determinant of methotrexate cytotoxicity understanding, the regulation of DHFR gene expression may have clinical implications for the use of hormonal therapy in combination with chemotherapy for the treatment of breast cancer.

    Title Biologic Implications of Steroid Hormone Receptors in Cancers of the Colon.
    Date April 1985
    Journal Southern Medical Journal
    Excerpt

    In follow-up investigations on 41 patients with primary cancer of the colon that had been assayed for the presence of steroid-binding activity, six of eight patients whose tumors showed steroid-binding activity for at least one steroid were free of disease one to three years postoperatively. In contrast, only two of 12 patients who had negative binding assay (16%) were free of disease in the same interval. The finding of steroid receptors in human colon cancers does not immediately imply therapeutic advantage. These binding activities may not be receptors, and even if they are, cytoplasmic receptors are a necessary, but not sufficient condition for hormone response. There are, however, sufficient epidemiologic, etiologic, and nutritional observations that seem to link breast and colon cancers to indicate further investigation into the biologic significance of the association of steroid-binding protein in human colon cancer.

    Title Response to Doxorubicin of Cultured Normal and Cancerous Human Mammary Epithelial Cells.
    Date April 1985
    Journal Journal of the National Cancer Institute
    Excerpt

    Epithelial cells were isolated and cultured from a number of human mammary specimens of both cancerous and noncancerous origin. Doxorubicin (Dx) sensitivity was measured at second passage with the use of a highly efficient clonogenic assay. For 23 different tumor specimens derived from patients without previous chemotherapy, the drug concentrations required to kill 50% of the cells varied approximately 35-fold. In contrast, for 11 tumor specimens from patients who relapsed after regimens containing Dx, the drug concentration for 50% survival varied only fivefold and the dose-response curves for these specimens clustered at the more resistant end of the spectrum. A wide range of sensitivities was also observed among 13 noncancerous mammary specimens; however, tumor tissue and noncancerous tissue from the same donor were similar. When cultures were subjected to drug incubation periods of 1 and 4 hours, dose-response curves were superimposable when plotted as a function of drug concentration multiplied by time.

    Title Tamoxifen Flare in Advanced Endometrial Carcinoma.
    Date March 1985
    Journal Journal of Clinical Oncology : Official Journal of the American Society of Clinical Oncology
    Excerpt

    Tamoxifen citrate has recently been shown to be effective palliative therapy in advanced endometrial carcinoma. Flare reactions from tamoxifen are well known in treatment of metastatic breast carcinoma and are not an indication for stopping therapy. We report a patient being treated with tamoxifen for advanced endometrial cancer who developed acute abdominal pain as a manifestation of a flare reaction and the significance of this observation.

    Title Isolation and Characterization of an Estrogen-inhibited Variant Derived from the Mcf-7 Breast Cancer Cell Line.
    Date September 1984
    Journal Cancer Research
    Excerpt

    The mechanism by which pharmacological concentrations of estrogen can paradoxically inhibit the growth of human breast cancer is unknown. We have selected for a variant line of MCF-7 which may help to understand this process. The variant was selected by exposing MCF-7 cells to high-specific-activity 16 alpha-[125I]iodoestradiol. These cells were viably frozen for two isotopic half-lives, defrosted once, then reexposed to 16 alpha-[125I]iodoestradiol to allow maximal radiation damage mediated by isotope associated with binding sites. This cell line (113) is one of 55 lines cloned from MCF-7 cells that survived this treatment. The growth response to estradiol of the 113 breast cancer cells grown in monolayer is normal for 4 to 6 days, and then the cell number plateaus as the cells appear to round up and detach. Concomitantly, a decrease in [3H]thymidine incorporation occurs. The cells cannot be rescued by removing estradiol from the medium. The inhibition is dose dependent and can be seen in concentrations of estradiol as low as 10(-10) M. The 113 cells are also inhibited by antiestrogens. They have normal levels of estrogen receptors which bind to DNA cellulose with activation. Progesterone receptors are estrogen inducible, although the levels are one-third that of wild-type MCF-7 cells. The morphological changes determined by electron microscopy of estrogen-treated cells are typical of degenerative cells. To investigate the possibility that inhibitory factors are secreted into the medium by 113 cells, conditioned medium from estrogen-exposed 113 cells is added to normal MCF-7 and 113 cells. No decrease in [3H]thymidine incorporation compared to controls is observed. When the secreted proteins are labeled with [35S]methionine and analyzed by sodium dodecyl sulfate-acrylamide gels, no major differences are apparent in the 113 and MCF-7 cells. Thus, the source of the defect is still unknown. It remains to be seen if the growth-inhibitory effects of 17 beta-estradiol on this cell line are receptor mediated or related to specific gene products which can be identified.

    Title Relationship Between Estrogen-receptor Proteins and Response to Chemotherapy in Breast Cancer.
    Date May 1984
    Journal Cancer Treatment Reports
    Title Breast Cancer Management: Recent Advances and Recommendations.
    Date May 1984
    Journal Advances in Internal Medicine
    Title Characterization of Insulin Regulation of Lipid Synthesis in Mcf-7 Human Breast Cancer Cells.
    Date May 1984
    Journal Breast Cancer Research and Treatment
    Excerpt

    Stimulation of lipid synthesis by insulin in MCF-7 human breast cancer cells is characterized by an increase in acetate incorporation into long-chain fatty acids. The effects occurs in the absence of an increase in glucose uptake by the cells, and cannot be explained by a decrease in turnover of cellular fatty acids. Differential substrate experiments as well as direct measurement of enzyme activities indicate that insulin stimulates increases in activity of the first enzyme of the de novo pathway, acetyl CoA carboxylase. [32Pi] incorporation into phospholipids is also stimulated by insulin. Thin layer chromatography reveals five peaks of [32Pi]-labeled phospholipids corresponding in mobility to the following standards: lysophosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. [32Pi] incorporation into each of these peaks is stimulated, although the degree of stimulation varies.

    Title The Impact of Primary Irradiation Treatment of Localized Breast Cancer on the Ability to Administer Systemic Adjuvant Chemotherapy.
    Date April 1984
    Journal Journal of Clinical Oncology : Official Journal of the American Society of Clinical Oncology
    Excerpt

    The impact of primary irradiation of localized breast cancer on the ability to administer Adriamycin-cytoxan adjuvant chemotherapy to patients with stage II breast cancer was examined. Patients were prospectively randomized to receive either irradiation or mastectomy as local therapy and did not differ with respect to other prognostic variables that might influence tolerance to chemotherapy. All of the patients received chemotherapy dose escalations (or reductions) until maximal tolerated drug doses were established. Patients receiving irradiation had minimally greater myelosuppression which was nearly totally explainable by lymphopenia. Irradiated patients required dose reduction nearly twice as often as mastectomy patients although commonly their dose could be reescalated. Patients managed with radiotherapy received slightly less drug than patients treated with mastectomy when treated to an identical degree of bone marrow suppression. The primary management of breast cancer by irradiation does not induce substantial changes in the ability of patients to tolerate adjuvant chemotherapy.

    Title Psychosocial and Physical Outcomes of Primary Breast Cancer Therapy: Mastectomy Vs Excisional Biopsy and Irradiation.
    Date March 1984
    Journal Breast Cancer Research and Treatment
    Excerpt

    Thirty-eight patients treated for primary breast cancer as part of a prospective randomized clinical trial were questioned retrospectively as to their psychosocial adaptation to treatment. Twenty patients had received mastectomy and eighteen had received excisional biopsy plus radiation of the intact breast. Aside from body image concerns, there were no marked psychosocial differences detected between these groups. Previous studies emphasizing serious psychological problems in mastectomy patients and fewer such problems in nonmastectomy patients may be influenced by biases that are not present in a randomized study design.

    Title Protein-associated Intercalator-induced Dna Scission is Enhanced by Estrogen Stimulation in Human Breast Cancer Cells.
    Date November 1983
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    Estrogen-responsive human breast cancer cells (MCF-7) displayed a higher frequency of intercalator-induced protein-associated DNA scission after treatment with 17 beta-estradiol (E2) than did cells that had not received estrogen treatment. This effect was dependent on estrogen concentration (maximum enhancement at approximately equal to 1 nM E2) and time (maximum effect seen approximately equal to 24 hr after E2 addition). Human breast cancer cells lacking estrogen receptors did not display the enhanced response. Antiestrogens produced a slight decrease in intercalator-induced DNA scission, whereas insulin produced an enhanced effect. The DNA breaks produced by the intercalators 5-iminodaunorubicin and 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) in these cells were undetectable without enzymatic deproteinization of cell lysates prior to quantification by alkaline elution. Intercalator-induced DNA-protein crosslinking also was enhanced in E2-treated MCF-7 cells. Studies with m-[14C]AMSA revealed no estrogen-associated increases in drug uptake. The data suggest that E2 treatment, either by specifically and directly increasing active transcription in chromatin or through secondary effects on DNA that accompany alterations in cell growth or cell cycle distribution, alters the susceptibility of DNA to intercalator-induced protein-associated DNA scission. If this enhanced protein-associated scission is selectively localized to transcriptionally active chromatin, the adsorption of the DNA-bound proteins to membrane filters (DNA-protein crosslinking) may allow identification and isolation of estrogen-regulated gene sequences.

    Title Melatonin Increases Oestrogen Receptor Binding Activity of Human Breast Cancer Cells.
    Date November 1983
    Journal Nature
    Excerpt

    Breast cancer is frequently a hormone-dependent tumour, and several studies have suggested that the pineal gland hormone melatonin may influence the growth and development of this malignancy. Subcutaneous injections of melatonin have been shown to inhibit, and pinealectomy to enhance, the development of dimethyl benz(a)anthracene (DMBA)-induced mammary tumours in rats. Use of the psychotropic drug thorazine, which increases plasma melatonin levels, has been associated with a decreased incidence of breast cancer in psychiatric patients. Calcification of the pineal gland has been correlated with an increased incidence of breast cancer in women. While the mechanism by which melatonin influences these tumours is unknown, both human breast cancer and DMBA-induced tumours contain oestrogen receptors (ER) and respond to changes in the oestrogen milieu. We therefore wondered whether melatonin might be altering ER binding activity of these tumours. We report here that in vitro incubation of MCF-7 human breast cancer cells with melatonin in physiological conditions increased the cytoplasmic and nuclear ER activity of these cells within 40 min, giving no change in the equilibrium dissociation constant (Kd) of the receptor. This induction was blocked by cycloheximide, and thus requires continuous protein synthesis. The modulation of ER binding activity of breast cancer by another endogenous hormone may be important for understanding the behaviour and treatment of this disease, and may provide insight into the factors regulating the synthesis and metabolism of steroid hormone receptors.

    Title Melatonin-induced Increase in Cytoplasmic Estrogen Receptor Activity in Hamster Uteri.
    Date August 1983
    Journal Endocrinology
    Excerpt

    The pineal gland hormone, melatonin, has been shown to influence the growth and metabolism of uterine tissue in the Syrian hamster. Because the uterus is an estrogen-responsive target tissue, we studied the effect of melatonin on the unoccupied estrogen receptor (ER) activity of immature hamster uteri in vivo and in vitro. A single sc injection of 2.5 micrograms melatonin increased the ER activity of uterine cytosol (Rc) 30% within 60 min [from 147.4 +/- 14.1 (SE), to 191.8 +/- 18.5 fmol/mg protein]. Scatchard analysis showed the induced population of receptor to be homogeneous and of high affinity, with an equilibrium dissociation constant (Kd = 0.08 nM) similar to that for uteri for vehicle-treated animals. The induction of Rc by melatonin persisted for 90 min, but Rc returned to control levels by 2 h after injection. Scatchard analysis of unoccupied nuclear receptor showed no significant differences either in the quantity of receptor (vehicle = 379.7 +/- 46.7, melatonin = 396.9 +/- 76.7 fmol/mg protein) or in the Kd (vehicle = 0.75 nM, melatonin = 0.60 nM) between these two groups. In vitro incubation of fresh whole uteri for 60 min in media containing 10(-5) M melatonin increased Rc by 83% (from 41.3 +/- 10.7 to 75.4 +/- 11.0 fmol/mg protein), supporting a direct effect of melatonin on uterine tissue. The Rc induced by melatonin both in vivo and in vitro was sensitive to the degree of homogenization used in preparation of the cytosol. Whereas conservative homogenization (two 10-sec bursts with an intermittent 40-sec cooling period) of uteri from melatonin-treated animals was associated with a significant increase in Rc, homogenization for 60 sec without intermittent cooling showed a significant decrease (from 251.1 +/- 17.4 to 182.7 +/- 5.8 fmol/mg protein) as compared with uteri from vehicle-treated animals. Whole organ analysis of uteri from animals injected with melatonin followed by [3H]estradiol, in which no homogenization was used, demonstrated a 26% increase in Rc specific binding, confirming induction as the primary response to melatonin. These findings demonstrate the ability of melatonin to modulate ER activity in hamster uteri, and may help explain the effects of melatonin on uterine growth and metabolism.

    Title Clonal Variation of Mcf-7 Breast Cancer Cells in Vitro and in Athymic Nude Mice.
    Date May 1983
    Journal Cancer Research
    Title Evaluation of Tamoxifen Doses with and Without Fluoxymesterone in Advanced Breast Cancer.
    Date March 1983
    Journal Annals of Internal Medicine
    Excerpt

    From a group of 108 female patients with measurable and/or evaluable metastatic breast carcinoma, 52 were randomized to receive tamoxifen and 56 to receive tamoxifen and fluoxymesterone. The fluoxymesterone dose, given orally twice a day, was 7 mg/m2 body surface area. The tamoxifen dose per patient, also given orally twice a day, ranged from 2 to 100 mg/m2 body surface area. Eighty-five percent of the patients had received previous chemotherapy and 60% previous hormone therapy. The complete and partial remission rate was better with the tamoxifen and fluoxymesterone regimen (p = 0.016), with remission rates of 15% for tamoxifen alone and 38% for the combination. The tamoxifen and fluoxymesterone regimen appeared to have higher remission rates in all subsets of pretreatment variables. Duration of remission with each regimen was similar, but the overall time to treatment failure for tamoxifen and fluoxymesterone was longer than for tamoxifen alone (180 versus 64 days, p = 0.01). Median survival with the combination was 380 days compared to 330 days for tamoxifen. No significant dose-response relationships emerged. Side effects were not different between dose levels or regimens except for the androgen effects in the tamoxifen and fluoxymesterone combination. These results suggest that there is no major dose-response effect for doses ranging from 2 to 100 mg/m2 body surface area given twice daily in this largely (94%) postmenopausal pretreated patient group, and that the tamoxifen and fluoxymesterone regimen is superior to tamoxifen alone.

    Title Dihydrofolate Reductase Gene Amplification and Possible Rearrangement in Estrogen-responsive Methotrexate-resistant Human Breast Cancer Cells.
    Date February 1983
    Journal The Journal of Biological Chemistry
    Excerpt

    Methotrexate-resistant (MTXR) human breast cancer cells have been obtained which are 1000-fold less sensitive to this drug than the wild type MCF-7 cells from which they were derived. The resistant cells contain approximately a 25-fold increase in the activity of the target enzyme dihydrofolate (DHF) reductase. Enzyme inhibition studies and methotrexate affinity studies fail to reveal any difference in the DHF reductase present in the MTXR cells compared to wild type MCF-7 cells. Cytogenetic analysis demonstrates the presence of elongated marker chromosomes in the resistant cells which are not found in the parental cell line. Analysis of the DNA from MTXR and wild type MCF-7 cells using Southern blot hybridization indicates that the MTXR MCF-7 cells contain more copies of DHF reductase gene sequences than do wild type MCF-7 cells. These experiments also suggest that the amplified DHF reductase gene sequences in MTXR cells may have undergone a uniform structural rearrangement involving the 5' flanking sequences during the process of amplification. MTXR MCF-7 cells respond to estradiol by increasing cell growth, and the level of DHF reductase in the MTXR cells is further induced following administration of estradiol. Radiolabeling studies demonstrate that estrogen stimulates the actual synthesis of DHF reductase in human breast cancer cells.

    Title Comparison of Dye Exclusion Assays with a Clonogenic Assay in the Determination of Drug-induced Cytotoxicity.
    Date January 1983
    Journal Cancer Research
    Excerpt

    The following factors must be considered when dye exclusion assays are interpreted. (a) It may require several days for lethally damaged cells to lose their membrane integrity following a cytotoxic insult. (b) During this time, the "surviving" cells may continue to proliferate. (c) Also during this time, some lethally damaged cells may undergo an early disintegration, so that they are not present to be stained with dye at the end of the culture period. Factors b and c may cause an underestimate of cell kill when the results of the assay are based upon the traditional "percent viability" expression. In order to overcome these problems, an internal standard was developed and tested. This was based upon the addition of a constant number of permanently fixed duck erythrocytes to the cultures of cells from two different established tumor cell lines. Results were based upon comparisons of the ratios of "viable" tumor cells to duck erythrocytes on permanent cytocentrifuge slides prepared from the cultures. This novel "ratio" method was found to be a more sensitive index of drug-induced cell kill than the traditional percent viability method. A standard agar cloning assay gave somewhat higher estimates of cell kill than the ratio method, although both assays were in qualitative agreement for the drugs tested. All three assays demonstrated a clear dose-effect relationship for most of the drugs tested. Dye exclusion assays may have a useful role in chemosensitivity testing in vitro.

    Title A New Model System for Studying the Phosphatidylinositol Cycle.
    Date October 1982
    Journal Journal of Cellular Physiology
    Excerpt

    An early manifestation of the response of WRK-1 rat mammary tumor cells to vasopressin is an increase in incorporation of (32P)Pi into phospholipids. Incorporation into all classes of phospholipids is stimulated; however, incorporation into phosphatidylinositol (PI) is increased to the greatest degree (3- to 10-fold as compared with 1.3- to 2-fold for the other phospholipids). Furthermore, increased incorporation into PI is accompanied by an increased rate of PI turnover; turnover rates of the other phospholipids are unaffected by vasopressin.

    Title Phase Ii Trial of Chlorozotocin in Malignant Melanoma, Breast Cancer, and Other Solid Tumors.
    Date August 1982
    Journal Cancer Treatment Reports
    Excerpt

    We have conducted a broad phase II clinical trial of chlorozotocin in 74 patients including 28 with malignant melanoma, 18 with breast cancer, nine with non-Hodgkin's lymphoma, six with nonseminomatous testicular cancer, five with ovarian cancer, four with sarcoma, three with non-beta islet cell carcinoma of the pancreas, and one with anaplastic carcinoma of the thyroid. Objective responses were noted only in 15% of the patients with melanoma and in 11% of the patients with non-Hodgkin's lymphoma. Significant leukopenia and thrombocytopenia were observed only in previously treated patients. Chlorozotocin does not appear to offer clinically significant advantages over other currently available nitrosoureas.

    Title Vasopressin: Action on Wrk-1 Rat Mammary Tumor Cells.
    Date May 1982
    Journal Journal of the National Cancer Institute
    Excerpt

    WRK-1, a cell line in long-term culture derived from a 7, 12-dimethylbenz[a]anthracene-induced rat mammary tumor, responds to physiologic concentrations of vasopressin with increased precursor incorporation into phospholipids and with increased protein accumulation. Because vasopressin has been reported to be a potent mitogen for Hela cells and 3T3 cells, a study was conducted to determine whether it could act as a mitogen for WRK-1 cells. Under no conditions was a clear-cut mitogen response to vasopressin demonstrated.

    Title Specific Estrogen Receptor Binding and Biological Effects of 16 Alpha-iodoestradiol on Human Breast Cancer Cells.
    Date September 1981
    Journal Cancer Research
    Excerpt

    16 alpha-Iodoestradiol was evaluated as an estrogen in MCF-7 and ZR-75-1 human breast cancer cells. 16 alpha-[125I]Iodoestradiol binds to equivalent numbers of specific estrogen receptors with a comparable binding affinity and similar sucrose density gradient behavior. 16 alpha-Iodoestradiol translocates receptor sites to the nucleus and induces a full range of estrogenic effects including thymidine incorporation, cell growth, and progesterone receptor. Because of its high specific activity, this compound may have unique applications on imaging of estrogen receptor-containing cells and specific receptor-mediated cytotoxicity.

    Title Purification and Properties of Estrogen-responsive Cytoplasmic Thymidine Kinase from Human Breast Cancer.
    Date March 1981
    Journal Cancer Research
    Excerpt

    The effect of 17 beta-estradiol on cytoplasmic thymidine kinase activity was studied in MCF-7, a human breast cancer cell line in culture which responds to estrogens with an increase in the rate of growth. Levels of 17 beta-estradiol which maximally stimulate [3H]thymidine incorporation into DNA also maximally stimulate thymidine kinase activity. The Vmax for thymidine increased while the Km was not affected by estrogen stimulation when performed on nonpurified enzyme. Tamoxifen, an antiestrogen, decreased the specific activity of the enzyme. To further study its hormonal regulation, cytoplasmic thymidine kinase was purified greater than 2000-fold by affinity column chromatography. The purified preparation migrated in one band to a pI of 8.5 on an isoelectric focusing gel. The purified thymidine kinase was further characterized by examining its molecular weight, pH optimum, heat stability, utilization of phosphate donors, inhibition by nucleotides, and the effect of pyrimidine nucleoside analogs.

    Title The Use of Immunocytochemical Techniques for the Detection of Steroid Hormones in Breast Cancer Cells.
    Date March 1981
    Journal Cancer
    Excerpt

    Indirect immunofluorescence and immunoperoxidase assays were developed to detect estradiol and progesterone in breast cancer cells. Appropriate controls were used to confirm immunologic specificity. Studies of estradiol binding by human breast cancer cells identified three groups: no detectable binding (25%); all tumor cells exhibiting binding although to different degrees (4%); and tumors with varying numbers of positive and negative cells (71%). Similar observations were made with respect to progesterone binding. The percentage of cells with estradiol binding was correlated with the amount of estrogen receptors (ER) present in the tumor specimens. Post-hormone binding events e.g., nuclear binding of estradiol, were also evaluated. Some tumor cells showing cytoplasmic binding of estradiol did not show nuclear binding of estradiol; such tumors lacked detectable diethylstilbestrol under routine assay conditions, and relatively high concentrations of estradiol were needed to observe estradiol-specific staining. The results suggest that the immunocytochemical assays detect hormone-specific binding, but that the binding is probably due to multiple classes of steroid-binding sites.

    Title Neurohypophysial-hormone-responsive Cell Line Derived from a Dimethylbenzanthracene-induced Rat Mammary Tumour.
    Date September 1980
    Journal The Biochemical Journal
    Excerpt

    WRK-1, a cloned cell line derived from a rat mammary tumour, responds to physiological concentrations of vasopressin and pharmacological concentrations of oxytocin with increased incorporation of [14C]acetate into lipids and increased protein accumulation. The presence of pharmacological concentrations of insulin, which itself is active on the WRK-1 cells, further enhances the effects of the neurohypophysial hormones. Unlike the action of vasopressin on other responsive tissues, the stimulation of acetate incorporation by WRK-1 cells is not observed until 24 h after the addition of the hormone. The lipids synthesized in response to the hormones are predominantly polar lipids, rather than the triclyclycerold characteristic of the differentiated mammary gland. [1-Deaminocysteine, 8-D-arginine] vasopressin, a vasopressin analogue that lacks pressor activity, has no effect on WRK-1 cells.

    Title The Role of Intracellular Equilibria and the Effect of Antiestrogens on Estrogen-receptor Dissociation Kinetics from Perfused Cultures of Human Breast Cancer Cells.
    Date September 1980
    Journal Endocrinology
    Title Combined Effects of Methotrexate and 5-fluoropyrimidine on Human Breast Cancer Cells in Serum-free Culture.
    Date September 1980
    Journal European Journal of Cancer
    Title Steroid Hormone Receptors and Melanoma.
    Date August 1980
    Journal The Journal of Investigative Dermatology
    Title Estrogen Receptor Status: an Important Variable in Predicting Response to Endocrine Therapy in Metastatic Breast Cancer.
    Date July 1980
    Journal European Journal of Cancer
    Title Clinical Correlations of Steroid Receptors and Male Breast Cancer.
    Date May 1980
    Journal Cancer Research
    Excerpt

    Estrogen receptors (ER) were present in tumor specimens from 29 of 34 cases of male breast cancer. There was a significant negative correlation of ER concentration with age. The quantity of ER tended to correlate directly with progesterone receptor levels, disease-free interval, and response duration among responders, but not to a statistically significant extent. In 13 patients for whom response data were available, no significant correlation was observed between ER levels and either frequency or duration of orchiectomy response. Among the six patients with tumor ER levels of less than 30 fmol per mg of protein, however, only two brief responses to orchiectomy occurred that were of little clinical benefit, while three of seven patients with higher ER responded more favorably. Thus, although this suggests that a relationship between low ER and unfavorable orchiectomy response may emerge as more patients are studied, currently available data do not justify basing therapeutic intervention on ER status of a biopsy in a manner analogous to that used for female breast cancer. Nine of 14 male breast cancer patients had positive progesterone receptor assays and several had androgen or glucocorticoid receptors. Tissue from only three of ten men with gynecomastia had measurable ER, and these were limited to the 4S component on sucrose gradients.

    Title Association Between Steroid Hormone Receptor Status and Disease-free Interval in Breast Cancer.
    Date November 1979
    Journal Cancer Treatment Reports
    Excerpt

    The possibility of an association between steroid hormone receptor status and disease-free interval was examined in 292 patients with breast cancer. Estrogen receptor positivity was associated with a prolonged disease-free interval. This association was independent of age, menopausal status, tumor size, or nodal status. There was no association between the presence or absence of progesterone, androgen, or glucocorticoid receptor and disease-free interval.

    Title Direct Inhibition of Growth and Antagonism of Insulin Action by Glucocorticoids in Human Breast Cancer Cells in Culture.
    Date August 1979
    Journal Cancer Research
    Excerpt

    We have examined the interaction of dexamethasone with the ZR75-1 human breast cancer cell line to determine if glucocorticoids might directly inhibit growth of breast cancer cells. Growth of these cells in serum-free medium was stimulated significantly by physiological concentrations of insulin (0.1 to 1.0 nM). Pharmacological concentrations of dexamethasone (10 nM) reduced cell number below that found in controls and nearly abolished the effect of insulin after several days in culture. Thymidine and uridine, but not leucine, incorporation into macromolecules or acetate incorporation into fatty acids were similarly inhibited by dexamethasone in the presence of absence of insulin. Dexamethasone did not inhibit insulin effects by altering insulin receptor affinity or concentration, as determined by Scatchard analyses of insulin binding. Net thymidine uptake into the trichloroacetic acid-soluble fraction of the cell was stimulated by insulin and inhibited by dexamethasone also inhibited thymidine kinase activity multiple potential sites of glucocorticoid action that directly oppose the effects of insulin. They also suggest that glucocorticoids have a direct inhibitory effect on proliferation of human breast cancer cells, which may help explain breast tumor regression following pharmacological glucocorticoid therapy.

    Title Relationship Between the Progesterone, Androgen, and Glucocorticoid Receptor and Response Rate to Endocrine Therapy in Metastatic Breast Cancer.
    Date August 1979
    Journal Cancer Research
    Title Hepatic Estrogen and Progesterone Receptors in an Estrogen-associated Hepatic Neoplasm.
    Date July 1979
    Journal Cancer Chemotherapy and Pharmacology
    Excerpt

    A patient with a benign hepatic neoplasm developing after treatment with estrogenic hormones is described. After excision, the neoplastic tissue was analyzed for the presence of cytosol estrogen and progesterone binding proteins. The neoplasm was classified as focal nodular hyperplasia of the liver and was demonstrated to contain high-affinity cytosol estrogen and progesterone hormone receptors. The estrogen-binding affinity of the neoplasm was three times greater than that of normal liver. Further investigation of cytosol hormone receptors in estrogen associated hepatic neoplasms will be required to define the role of these binding proteins in the possible etiology of certain liver tumors.

    Title Steroid Hormone Receptors in Human Colon Cancers.
    Date June 1979
    Journal Cancer
    Excerpt

    Tumors from patients with primary colon cancer were studied for the presence of steroid hormone receptors for estrogen (E2), progesterone (Prog), dihydrotestosterone (DHT) and glucocorticoid. Ten of 33 (30%) tumors contained high affinity E2 receptors. Four were males and six females with positive assays predominantly from the left colon. Twenty-three of these tumors were also assayed for DHT and Prog and six (26%) contained all three receptors. An additional twelve tumors had at least one receptor, so that 70% of the tumors studied contained one or more receptors. Five of 22 (23%) samples were positive for glucocorticoid receptors. Common etiological factors associated with colon and breast cancer were briefly discussed. These factors, along with the presence of hormone receptors in primary colon malignancies suggest that some large bowel cancers may be endocrine-dependent.

    Title Distribution, Frequency, and Quantitative Analysis of Estrogen, Progesterone, Androgen, and Glucocorticoid Receptors in Human Breast Cancer.
    Date June 1979
    Journal Cancer Research
    Excerpt

    The distribution and frequency of steroid hormone receptors are described 329 patients with breast cancer. The distribution of each of the steroid hormone receptors is unimodal with a progressive increase in the proportion of patients positive at lower receptor values. Receptor values expressed as fmol/mg cytoplasmic protein are well correlated with values expressed as fmol/mg breast tumor. Estrogen receptor was positive in 53% of the patients; progesterone receptor was positive in 38% of the patients; glucocorticoid receptor was positive in 52% of the patients; and androgen receptor was positive in 31% of the patients. The type of tissue assayed did not affect steroid hormone receptor positivity. For primary tumors, there was no correlation between steroid hormone receptor positivity and location of the tumor in the breast, size of the tumor, or extent of the disease. Each of the steroid hormone receptors was positively associated with each of the other steroid hormone receptors. Estrogen receptor was correlated with menopausal status and axillary nodal status, but these correlations did not exist for the other steroid hormone receptors. Estrogen receptor was not correlated with age after adjustment for menopausal status. The other steroid hormone receptors were not correlated with age.

    Title Association Between Steroid Hormone Receptors and Response Rate to Cytotoxic Chemotherapy in Metastatic Breast Cancer.
    Date December 1978
    Journal Cancer Treatment Reports
    Excerpt

    The influence of steroid hormone receptors on response rate to cytotoxic chemotherapy in 70 patients with metastatic breast cancer was determined in a retrospective study. We have previously reported that 34 of 45 patients with tumors containing low or absent estrogen-receptor values had objective responses to chemotherapy while three of 25 patients with positive estrogen-receptor tumors responded. In the present study, 22 of 34 patients with low or absent progesterone-receptor tumors had an objective response to cytotoxic chemotherapy, while none of eight patients with a positive progesterone-receptor tumor responded (P less than 0.05). Patients having tumors with a negative estrogen receptor and a negative progesterone receptor had a response rate of 88% (21 of 24 patients). There were three patients whose tumors were estrogen-receptor negative but progesterone-receptor positive; none had a response to chemotherapy. Chemotherapy response was not associated with the presence or absence of either androgen or glucocorticoid receptor. We conclude that progesterone-receptor values in addition to estrogen-receptor status may prove to be important correlates of response to cytotoxic chemotherapy in metastatic breast cancer. Androgen- and glucocorticoid-receptor analyses are not helpful in predicting response to chemotherapy.

    Title Establishment and Characterization of Three New Continuous Cell Lines Derived from Human Breast Carcinomas.
    Date November 1978
    Journal Cancer Research
    Excerpt

    Three continuous lines of mammary tumor cells (ZR-75-1, ZR-75-27, and ZR-75-30) have been established from malignant effusions of two women with breast cancer. Differentiated properties expressed by each cell line include: (a) epithelial morphology (by light and electron microscopy) resembling that of the parental tumors; (b) presence of receptors for estrogen and other steroid hormones; and (c) growth responsiveness to estrogen and/or progesterone. All three cell lines possess human karyotypes that differ from one another in modal chromosome number as well as in characteristic marker chromosomes. Two of the cultures (ZR-75-27 and ZR-75-30), although derived from the same patient, have stable differences in their karyotypes.

    Title Failure of Breast Cancer Cells in Long-term Culture to Aromatize Androstenedione.
    Date August 1978
    Journal Steroids
    Excerpt

    The ability of breast cancer cells in long-term tissue culture to aromatize androstenedione to estrone and estradiol was examined. (3H) androstenedione (1.1 x 10(-7)M) was incubated with 9 cell lines of human breast cancer and one line derived from a dimethylbenz(a)anthracene induced rat mammary tumor. No conversion to estrone or estradiol was detected. The findings are discussed in light of previous studies showing aromatization of androgens in breast cancer tissues.

    Title The Relation Between Estrogen Receptors and Response Rate to Cytotoxic Chemotherapy in Metastatic Breast Cancer.
    Date July 1978
    Journal The New England Journal of Medicine
    Excerpt

    In a retrospective study we determined the relation between estrogen receptors and the response rate to cytotoxic chemotherapy in 70 patients with metastatic breast cancer. Thirty-four of 45 patients with low or absent estrogen-receptor values (less than 10 fmol per milligram of cytoplasmic protein) had objective responses to chemotherapy, whereas only three of 25 patients with higher values (greater than 10 fmol per milligram of cytoplasmic protein) responded (P less than 0.0001). There were no statistically significant differences between the two groups in age, menopausal status, disease-free interval, Karnofsky index or prior therapy. Differences in sites of involvement or type of chemotherapy did not account for the increased response rate in receptor-negative patients. We conclude that estrogen-receptor values are an important predictor of response to cytotoxic chemotherapy in metastatic breast cancer.

    Title Correlation Among Insulin Binding, Degradation, and Biological Activity in Human Breast Cancer Cells in Long-term Tissue Culture.
    Date February 1978
    Journal Cancer Research
    Title Insulin Stimulation of Fatty Acid Synthesis in Human Breast Cancer in Long Term Tissue Culture.
    Date November 1977
    Journal Endocrinology
    Excerpt

    The effect of various steroid and peptide hormones on the rate of incorporation of (14C)acetate into fatty acids was studied in MCF-7 cells, a human breast cancer cell line in continuous tissue culture. Physiologic concentrations of insulin elicited an increase in the rate of precursor incorporation. Addition of human placental lactogen and dexamethasone with insulin caused no further increment. Other hormones, such as 17beta-estradiol and 15a-dihydrotestosterone, had no effect on the rate of net labeled-acetate incorporation, although they stimulated the rate of general macromolecular synthesis. The stimulation by insulin appeared to be independent of glucose concentration, since the effect was seen in the absence of glucose. The insulin-induced increase was observed as early as 1 h following hormone addition and appeared to be maximal by 4 h. Inhibitors of RNA and protein synthesis had no effect on the insulin-mediated stimulation of precursor incorporation into fatty acids. Thus, these human cell lines in long term tissue culture may be of value as a model system in studying the action of physiologic concentrations of insulin.

    Title Glucocorticoid Receptors in Subpopulations of Childhood Acute Lymphocytic Leukemia.
    Date September 1977
    Journal Cancer Research
    Title Insulin Stimulation of Fatty Acid Synthesis in Human Breast Cancer Cells.
    Date July 1977
    Journal Journal of the National Cancer Institute
    Excerpt

    Four human breast cancer cell lines were tested for their response to hormones with respect to the lactational function, fatty acid synthesis. Physiologic concentrations of insulin enhanced the incorporation of [14C]acetate into fatty acids in two of four cell lines tested. All cell lines had specific, high-affinity insulin receptors; therefore, the failure of two lines to respond could not be attributed to the absence of receptor. The effect of insulin involved an increase in the maximun velocity of incorporation rather than a decrease in the Michaelis constant.

    Title Steroid Hormone Receptors in Normal Human Lymphocytes. Induction of Glucocorticoid Receptor Activity by Phytohemagglutinin Stimulation.
    Date June 1977
    Journal The Journal of Biological Chemistry
    Excerpt

    The presence of specific steroid hormone receptors in human lymphocytes was investigated in unstimulated and phytohemagglutinin-stimulated glass wool column-purified peripheral blood lymphocytes. Specific steroid binding in intact cells was determined by a whole cell competitive binding assay. Non-phytohemagglutinin-stimulated lymphocytes had about 2700 specific glucocorticoid binding sites per cell; phytohemagglutinin stimulation induced a 2 to 3-fold increase in glucocorticoid receptor activity within 16 h of culture. No estrogen, androgen, or progestin binding sites were detected in either unstimulated or phytohemagglutinin-stimulated peripheral blood lymphocytes. Scatchard analysis of glucocorticoid binding was consistent with a single class of receptor sites with a dissociation constant (Kd) of about 5.5 x 10(-9) M (correlation coefficient r = -0.96). Significant competition for radiolabeled dexamethasone binding was not observed with steroids lacking glucocorticoid activity. There was good agreement between relative binding affinities of various steroids to glucocorticoid receptor in lymphocytes and ability of these steroids to inhibit phytohemagglutinin-stimulated thymidine incorporation.

    Title Evaluation of Tamoxifen Dose in Advanced Breast Cancer: a Progress Report.
    Date May 1977
    Journal Cancer Treatment Reports
    Excerpt

    The results of an ongoing trial randomizing patients with progressive, metastatic breast carcinoma between tamoxifen (Tam, NSC-180973) and Tam plus fluoxymesterone (Flu) (7 mg/m2 bid) are reported. Each patient received a single dose level of Tam in the range of 2-100 mg/m2 bid. The combination had a higher response rate overall (45% vs 28%) and when only the patients' soft tissue sites were analyzed (54% vs 9%, P=0.04). The time to treatment failure was longer for the combination among those patients with a response or disease stabilization (P=0.08). Response rates with Tam doses less than 12 mg/m2 bid were also higher than with doses greater than or equal to 12 mg/m2 for all patients in the study (62% vs 30%, P=0.025) and for those where only soft tissue sites were evaluable (43% vs 29%, P=0.07). Side effects were mild and consisted primarily of transient hematologic suppression, nausea, masculinization, hepatic enzyme elevations, and edema. The latter three were observed only with the Flu regimen. Leukopenia and thrombocytopenia were more frequent at Tam doses less than 12 mg/m2 bid whereas nausea was more common at higher doses. Tam doses as high as 100 mg/m2 bid were well tolerated. Tam amy be more effective at low doses, has only mild side effects, and is well tolerated at doses up to 100 mg/m2 bid. Combining Tam with Flu appears to enhance the therapeutic effectiveness.

    Title Evaluation of an Intermittent Schedule of Dibromodulcitol in Breast Cancer.
    Date May 1977
    Journal Cancer Treatment Reports
    Excerpt

    Dibromodulcitol was administered orally on Days 1-10 every 3-4 weeks to 29 patients with metastatic breast carcinoma refractory to previous combination chemotherapy. Initial doses between 70 and 280 mg/m2/day were utilized. The dose was escalated as tolerated in subsequent cycles in individual patients. Hematosuppression was dose-limiting. At doses of greater than 200 mg/m2/day leukopenia (greater than 1000 cells/mm3) and thrombocytopenia (greater than 25,000 platelets/mm3) occurred in one of 28 cycles and two of 27 cycles respectively. In contrast, at doses of less than or equal to 200 mg/m2/day leukopenia and thrombocytopenia occurred in four of 18 cycles and five of 18 cycles respectively. Recovery of leukocytes (less than 4000 cells/mm3) and platelets (less than 100,000 platelets/mm3) by Day 29 after the start of therapy was also delayed at higher doses. Responses were observed in three of 29 evaluable patients and subjective improvement of osseous disease in one additional patient. A dose of 180 mg/m2/day X 10 Every 28 days is recommended in previously treated patients to avoid severe hematologic side effects.

    Title Receptors for Steroid Hormones in Human Melanoma.
    Date April 1977
    Journal Surgical Forum
    Title Casein Production by Human Breast Cancer.
    Date April 1977
    Journal Cancer Research
    Excerpt

    Casein was measured in the sera of breast cancer patients, in breast cancer tumors, and in breast cancer cells in long-term tissue culture using a sensitive and specific radioimmunoassay. Levels present in breast cancer sera were not elevated above control values. Eight of forty-seven (17%) of the tumor samples tested were positive for casein, the highest level representing 0.003% of the soluble protein. When seven human breast cancer cell lines were assayed for casein, the results were uniformly negative even under conditions of stimulation by lactogenic hormones. In addition, direct immunoprecipitation of labeled cellular protein supported the negative result of the radioimmunoassay. Thus it appears that casein production is not a common characteristic of most human breast cancers.

    Title Hormone Responsive Human Breast Cancer in Long-term Tissue Culture: Effect of Insulin.
    Date February 1977
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    The mechanisms of steroid and peptide hormone action in human breast cancer are poorly understood. We have previously characterized a cell line of human breast cancer in long-term tissue culture that possesses various steroid hormone receptors and responses, providing a model for the study of steroid hormone action. The present studies describe a human breast cancer in vitro that responds to physiologie concentrations of insulin with an increased rate of macromolecular synthesis and growth. Thymidine and uridine incorporation in cells in serum-free medium are stimulated by 10(-11) M insulin and are maximal with 10(-8) M. Leucine incorporation is stimulated by 5 X 10(-11) M insulin and is maximal with 10(-9) M. Significant stimulation of uridine and leucine incorporation is evident by 3 hr and maximal by 10 hr. A 10-hr lag period exists for insulin stimulation of thymidine incorporation, which is maximal form 14 to 24 hr. The effect of 10(-8) M insulin on macromolecular synthesis is accompanied by a 69% increase above controls in the number of cells after 24 hr. The effect on macromolecular synthesis is observed in glucose-free medium. Insulin's effect on protein synthesis is not blocked by inhibition of RNA synthesis with actinomycin D. Glucocorticoids partially inhibit the action of insulin in these cells. This system provides a model for studying insulin action, and suggests that some human breast cancer may show growth regulation by insulin.

    Title Oestrogen Receptors in Human Malignant Melanoma.
    Date October 1976
    Journal Lancet
    Excerpt

    16 of 35 patients (46%) with malignant melanoma were found to have cytoplasmic oestrogen-receptor activity in biopsy specimens. OEstrogen-receptor activity was detected in primary lesions, lymph-node metastases, skin metastases, and visceral metastases. Equal percentages of males and females had positive assays. Scatchard analysis of binding in one case was consistent with a single class of high affinity receptor sites.

    Title Mediastinoscopy: a Diagnostic Aid in Metastatic Carcinoma of the Breast.
    Date July 1976
    Journal Cancer
    Excerpt

    Six patients with metastatic carcinoma of the breast underwent mediastinoscopy to obtain tissue for estrogen receptor analysis and pathologic confirmation of metastatic tumor. The indication for mediastioxcopy was an abnormal mediastinal accumulation of gallium in five patients, only two of whom had an abnormality noted on tomography. All six patients had tumor recovered by mediastinoscopy as demonstrated by pathologic examination and/or estrogen receptor assay, Therefore, the diagnosis of mediastinal metastases in breast cancer may be suggested by either chest roentgenograms, mediastinal tomography, or gallium scintigraphy. Mediastinoscopy is a safe, effective procedure capable of establishing the diagnosis of metastatic carcinoma of the breast and of procuring sufficient tissue for estrogen receptor analysis in patients without more readily accessible sites of metastases.

    Title A Quantitative Study of Muramidase Distribution in Normal and Nitrogen Mustard-treated Rats.
    Date January 1973
    Journal The Yale Journal of Biology and Medicine
    Title Bone Marrow Transplantation from Hl-a Matched Donors to Patients with Acute Leukemia. Toxicity and Antileukemic Effect.
    Date August 1972
    Journal Transplantation
    Title The Androgen Metabolite 5alpha-androstane-3beta,17beta-diol (3betaadiol) Induces Breast Cancer Growth Via Estrogen Receptor: Implications for Aromatase Inhibitor Resistance.
    Date
    Journal Breast Cancer Research and Treatment
    Excerpt

    The aromatase inhibitors (AIs) are used to treat estrogen receptor-positive (ER+) breast tumors in post-menopausal women, and function by blocking the conversion of adrenal androgens to estrogens by the enzyme CYP19 aromatase. Breast cancer patients receiving AI therapy have circulating estrogen levels below the level of detection; however, androgen concentrations remain unchanged. We were interested in studying the effects of androgens on breast cancer cell proliferation under profound estrogen-deprived conditions. Using in vitro models of estrogen-dependent breast cancer cell growth we show that the androgens testosterone and 5alpha-dihydrotestosterone induce the growth of MCF-7, T47D and BT-474 cells in the absence of estrogen. Furthermore, we demonstrate that under profound estrogen-deprived conditions these breast cancer cells up-regulate steroidogenic enzymes that can metabolize androgens to estrogens. Lastly, we found that the downstream metabolite of 5alpha-dihydrotestosterone, 5alpha-androstane-3beta,17beta-diol (3betaAdiol), is estrogenic in breast cancer cells, and induces growth and ER-signaling via activation of ERalpha. In conclusion, our results show that breast cancer cells deprived of estrogen up-regulate steroidogenic enzymes and metabolize androgens to estrogen-like steroids. The generation of estrogen-like steroids represents a potential mechanism of resistance to aromatase inhibitors.

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