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Notes: Abstract The increasing use of high-dose chemotherapy with autologous hematopoietic transplantation for the treatment of solid malignancies has raised concern about the role of tumor cells contaminating the grafts. Minimal residual disease (MRD) in autologous grafts has became a dynamic and intensively studied field in oncology. This review discusses the current status of MRD in breast cancer autografts and presents existing data on detection methodology, clinical relevance, biologic characteristics and purging techniques.

Notes: Abstract Purpose: To evaluate the activity and toxicity of the combination of paclitaxel given by three-hour infusion, and carboplatin as first-line chemotherapy in patients with advanced breast cancer (ABC). Background: Paclitaxel is an active agent in ABC. Furthermore, our group has shown that the combination of paclitaxel and carboplatin is effective in anthracycline-resistant ABC. Patients and methods: From January 1996 until March 1997, 66 women with ABC were treated with paclitaxel (175 mg/m2) by three-hour infusion followed by carboplatin at an AUC of 6 mg × min/ml every three weeks. The median age of the patients was 56 years (range 28–75). A total of 39 patients had received adjuvant chemotherapy and 22 of them were treated with an anthracycline or mitoxantrone-containing regimen. Results: A total of 324 cycles (median: six) were administered, 273 (85%) of them at full dose. The median number of delivered cycles was six. The median delivered dose intensity (DI) of paclitaxel was 55.1 mg/m2/week (range 30.5–69.3) and the relative DI was 0.95 (range 0.5–1.2). Eight patients (12%, 95% confidence interval (CI): 5%–22%) achieved complete and 28 (42%, 95% CI: 30%–55%) partial responses. Grade 3–4 toxicities included anemia (5%), granulocytopenia (24%), thrombocytopenia, nausea/vomiting and allergic reaction (3% each), myalgias/arthralgias and neurotoxicity (1,5% each). Febrile neutropenia occurred in eight (12%) patients. Alopecia was universal. After a median follow-up of 17.3 (range 0.07–24.5) months, 48 (72%) patients have demonstrated tumor progression and 24 (36%) have died. Median time to progression was 8.6 (range 0.07–23+) months and median survival 20.4 (range 0.07–24.5+) months. Conclusions: The combination of paclitaxel and carboplatin has moderate activity in ABC and can be easily delivered on an outpatient basis with manageable toxicity. This regimen may be useful especially in patients to whom anthracyclines or cisplatin administration is precluded because of other concomitant diseases.

Notes: Abstract Breast cancer is the second commonest primary tumour responsible for gastrointestinal metastases after malignant melanoma. The real incidence of gastrointestinal metastases in breast cancer patients is probably underestimated owing to the non-specific presenting symptoms and death of patients caused by other more obvious metastases. The predominant histological subtype of gastrointestinal metastases of breast cancer is invasive lobular carcinoma and the median interval from diagnosis of primary breast cancer to gastrointestinal metastases is five years. We report two cases of disseminated breast cancer with gastrointestinal involvement with a rather long survival.

Notes: Abstract Backgound: A prospective randomised trial was undertaken to evaluate the role of neoadjuvant chemoendocrine therapy prior to surgery in primary operable breast cancer. Patients and methods: Three hundred nine women (median age 56 years, range 27–70) with primary operable breast cancer confirmed on fine needle aspiration (FNA) cytology were recruited to this study. They were treated with a combination of mitozantrone and methotrexate (± mitomycin-C) combined with tamoxifen (2MT). Patients received eight cycles of 2MT (four prior to surgery in the neoadjuvant group) and tamoxifen for five years with appropriate surgery and radiotherapy. The two groups were comparable for age, menopausal status, stage and surgical requirements. Results: The clinical response rates to neoadjuvant therapy were as follows: 22% complete response (CR), 29% minimal residual disease (MRD), 33% partial response (PR), 15% no change (NC) and only two patients had clinical evidence of progressive disease. Surgical requirements were reduced from 31 patients (22%) of the adjuvant group having mastectomy to 14 (10%) in the neoadjuvant group (P 〈 0.003). At a median follow-up of 48 months (range 10–70 months) there is no statistically significant difference between the two groups in terms of local relapse, metastatic relapse or overall survival. Symptomatic and haematologic acute toxicity was low and similar for adjuvant and neoadjuvant therapy. Conclusion: This randomised trial has shown a significant reduction in the surgical requirements for mastectomy, after treatment with neoadjuvant chemoendocrine therapy, with no deterioration in local or distal relapse.

Notes: Abstract Background: A significant proportion of breast cancer patients receiving tamoxifen therapy relapse during treatment following acquisition of tamoxifen-resistant or oestrogen-independent phenotypes. The mechanism behind this rapid progression to oestrogen autonomy is at present unclear and further treatment modalities are limited. Suramin represents a novel potential second line therapy. The mechanism of the antineoplastic activity of suramin is not completely understood, although the drug binds to many growth factors including epidermal growth factor and insulin-like growth factors and can also dissociate growth factors from their receptors. In this study we have related suramin sensitivity to the expression of receptors for epidermal growth factor and insulin-like growth factor-I in a number of breast cancer cell lines including lines resistant to tamoxifen. Materials and methods: The anti-proliferative effects of suramin were investigated in two oestrogen dependent breast cancer lines (ZR-75-1 and MCF-7), oestrogen independent (ZR-PR-LT) and tamoxifen resistant (ZR-75-9a1) variants of ZR-75-1 and a tamoxifen resistant (LY2) variant of MCF-7. Full dose response curves were constructed and IC50values determined for each cell line. Sensitivity to suramin was correlated with the level of expressio n of receptors for epidermal growth factor (EGFR) and insulin-like growth factor-I (IGFR). On observing stimulation of cell proliferation by suramin in the tamoxifen resistant cell lines in the presence of tamoxifen we also investigated the possible role of suramin sequestration of transforming growth factor-β in mediating this effect. Results: All cell lines exhibited a dose- and time-dependent response to suramin treatment. Tamoxifen resistant ZR-75-9a1 cells (day 6 IC5085 µg ml−1) were more resistant to suramin than oestrogen independent ZR-PR-LT cells (day 6 IC5045 µg ml−1), and the parent ZR-75-1 line (day 6 IC5056 µg ml−1). Increased sensitivity to suramin was associated with increased expression of IGFR and decreased expression of EGFR. Tamoxifen resistant LY2 cells were significantly more sensitive to suramin (day 6 IC5070 µg ml−1) than MCF-7 cells (day 6 IC50350 µg ml−1). Both IGFR and EGFR expression by LY2 cells was lower than in the parent line. The antioestrogen-resistant ZR-75-9a1 and LY2 lines grown in the presence of 8 µM tamoxifen were growth stimulated by concentrations of the drug below 100 µg/ml. As growth stimulation observed in the presence of tamoxifen may have been due to suramin sequestration of tamoxifen induced TGF-β1 secretion we also investigated the response of the cells to this peptide in the presence and absence of suramin. All cell lines were growth inhibited by TGF-β1 except ZR-75-9a1 which was unresponsive. Responses to TGF-β1 were modified in the presence of 100 µg suramin ml−1 although TGF-β1 was unable to mimic the ability of tamoxifen to stimulate proliferation in the presence of suramin. Conclusions: These results suggest that for ZR-75-1 cells and variants, increased sensitivity to suramin is associated with an increase in expression of IGFR and a decrease in EGFR numbers. However, tamoxifen resistant LY2 cells, in which both IGFR and EGFR expression is reduced were considerably more sensitive than parental MCF-7 cells suggesting that there is no clear relationship between EGFR and IGFR expression and suramin sensitivity. The unexpected stimulation of cell proliferation of the tamoxifen resistant variants by suramin in the presence of tamoxifen could not be explained by suramin sequestration of transforming growth factor-β and the mechanism of this interaction remains unclear.

Notes: Abstract Purpose: To evaluate the clinical course of patients with a metastatic breast cancer (MBC) confined to the lungs and treated with doxorubicin/cyclophosphamide-containing chemotherapy (DC-CT). Patients and methods: Between 1973 and 1985, 1581 patients with MBC were treated with DC-CT at M.D. Anderson Cancer Center. Data for 88 patients (5.6%) with metastases confined to the lungs were reviewed to correlate various clinical characteristics with response to treatment and survival. Results: The overall response rate was 76% with 33% achieving complete response (CR). The median overall survival time was 22 months (range 1–210). The 10-year survival rate was 9%. The overall response and CR rates were higher for the patients with metastases confined to the lungs (76% and 33%, respectively) than for the remainder of MBC patients (64% and 14%; P 〈 0.01). The 10-year survival rate was also higher (9% versus 3%, P 〈 0.01), but there were no differences in median overall survival rate. Conclusions: This retrospective analysis demonstrated that patients with metastases confined to the lungs treated with DC-CT had a high objective response rate, especially high CR rates, and a median survival comparable to that of our entire population of MBC patients. A small but clinically significant percentage of patients had prolonged survival. Therefore, not all visceral sites are indicators of poor prognosis.

Notes: Abstract Background: Conventional dosages of cytostatics in mg/m2 will cause marked variations in systemic exposure, resulting in over- and under-treatment, at least with respect to side effects. Patients and methods: We are conducting a randomized adjuvant study for breast cancer patients younger than 60 years of age with ≥70% risk of recurrence within five years. The first 89 consecutive patients who have received nine courses q three weeks of individually dose-escalated and G-CSF (filgrastim)-supported FEC (5-fluorouracil (5-FU), epirubicin, and cyclophosphamide) therapy given with ciprofloxacin prophylaxis were included in this analysis. Six different FEC dose levels were used for treatment at equivalent haematological toxicity. Dose modifications were based on white blood cell and platelet counts on days 8, 11/12, 15, and 22. Results: Eighty-three of 89 patients completed all nine courses. The median epirubicin and cyclophosphamide doses were 782 mg/m2 (range 0–994 mg/m2) and 10.330 mg/m2 (range 0–14.460 mg/m2), respectively. Patients treated at the two highest dose levels experienced NCI grade 0 or 1 toxicities in 73% to 92% of the courses. Three patients have developed acute myeloid leukaemia, and two of them have demonstrated abnomalities compatible with topoisomerase II-poison-related karyotypic changes. Conclusions: Tailored adjuvant G-CSF-supported FEC polychemotherapy will make it possible for all patients to be treated at equivalent levels of haematological toxicity with significantly higher doses without a marked increase in other organ toxicities.

Notes: Abstract The first reported effective adjuvant combination regimen for patients withoperable breast cancer comprised oral cyclophosphamide (C) days 1–14with intravenous methotrexate (M) and fluorouracil (F) on days 1 and 8,repeated every 28 days (’classical‘ CMF). These drugs have since beenextensively used with or without endocrine therapies and/or other cytotoxics,as well as with radiation therapy to the chest wall yielding conflictingresults. Although doses and schedules have varied widely, the combination ofthese three drugs has been generically referred to as CMF. Evidence existsthat reducing the dose and/or altering the schedule of CMF (’modified‘ CMF)have compromised its efficacy in metastatic breast cancer. Reduction belowstandard dose of a similar regimen also gave inferior results in the adjuvantsetting. In fact, the recently reported improved outcome of adding radiationtherapy to CMF was only demonstrated in comparisons with a ’modified‘ CMF.Furthermore, trials in women with estrogen receptor-positive breast cancer,which did not demonstrate any significant benefit for the addition of adjuvantCMF to tamoxifen compared with tamoxifen alone, also used ’modified‘ CMF.Therefore, adherence to the ’classical‘ dose and schedule is recommended whenCMF is used in adjuvant therapy.

Notes: Abstract Background: We compared hematopoietic progenitor cell (HPC) collection and neoplastic cell contamination in breast cancer patients given cyclophosphamide (CTX) plus granulocyte-colony stimulating factor (G-CSF) or G-CSF alone for mobilization. Patients and methods: In 57 stage II–III breast cancer patients, CD34+ cells, colony-forming units-granulocyte macrophage (CFU-GM), early HPC and breast cancer cells were counted in HPC collections obtained after CTX plus G-CSF (n = 27) or G-CSF-alone mobilization (n = 30). Results: The CD34+ cell collection was about two-fold greater after CTX plus G-CSF mobilization (11.0 ± 7.9 vs. 5.8 ± 3.5 × 106/kg, P 〈 0.001). Similarly, the total number of CFU-GM, CD34+CD38− cells and of week-5 cobblestone area forming cells (CAFC) collected was significantly higher in patients mobilized with CTX plus G-CSF. Breast cancer cells were found in the apheresis products of 22% of patients mobilized with CTX plus G-CSF and in 10% of patients mobilized with G-CSF alone (P = 0.36). Of seven patients who failed G-CSF-alone mobilization and eventually underwent chemotherapy plus G-CSF mobilization, none had cytokeratin-positive cells after G-CSF mobilization, whereas four out of seven had cytokeratin-positive cells after chemotherapy plus G-CSF (P = 0.07 by χ2 test). Conclusion: The CTX plus G-CSF mobilization protocol was associated with a significantly higher HPC collection. However, this benefit was not accompanied by a reduction in the incidence of tumor-contaminated HPC graft.

Notes: Abstract Three areas of clinical research in breast cancer treatment led to news breaking presentations at the American Society of Clinical Oncology (ASCO) meeting, 1998, in Los Angeles. All three subjects represent important advances in cancer medicine. Prevention: Two related drugs, tamoxifen and raloxifene, were found in placebo controlled trials to significantly reduce the incidence of breast cancer for women at increased risk of developing the disease. Patterns of relapse showed that the reduced rate of breast cancer was exclusively observed for tumors expressing estrogen receptors, while the rate of tumors classified as estrogen-receptor negative was similar for the treatment and the control groups. This may indicate that the observed reduction in breast cancer incidence is due to a treatment effect on occult disease rather than its prevention. We certainly have no adequate information on mortality prevention. Adjuvant therapies: Taxol given every three weeks for four courses following an adjuvant treatment with four courses of doxorubicin and cyclophosphamide (AC) combination was found to be superior to not adding treatment after the four courses of AC in a trial involving 3170 patients. At 22 months of median follow-up, the quoted P-values were P = 0.0077 for disease-free survival and P = 0.039 for overall survival, but these did not cross the prospectively defined interim analysis boundaries for statistical significance at the 0.05 level. The difference was observed early during follow-up, and was exclusively seen in the 40% of patients who had ER-negative primaries and, therefore, did not receive tamoxifen following chemotherapy. One may thus argue that the early difference observed was primarily due to differences in the duration of the treatment regimens in the two groups and the early entry into the trial of patients with particularly aggressive neoplasia (e.g., ER-negative primaries) who would have benefited from a longer duration treatment. Treatment of advanced disease: The use of monoclonal antibodies to c-erb-B2 was found to induce responses in metastatic breast cancer. Patients with tumors expressing c-erb-B2 responded to weekly infusions of this biological agent. It was particularly impressive that the response rate for patients receiving infusion of the monoclonal antibodies together with the cytotoxics was superior to that with chemotherapy alone in a randomized trial. It is important to note that only patients with tumors overexpressing c-erbB-2 (the overall incidence is about 20%) were tested. It must still be demonstrated that the effect of these monoclonal antibodies is indeed confined to cells overexpressing c-erbB-2. Treatment related cardiac toxicity remains a problem, and the effects of treatment in various subsets of patients need to be defined before starting investigations in the adjuvant setting, which is a clear further objective of this specific research. The significant findings from clinical research opened several new questions, which must be answered before allowing them to be employed in routine patient care.

Notes: Abstract Background: Bone scan (BS), chest X-rays (CXR), liver ultrasonography (LUS) and laboratory parameters (LP) are frequently used as routine staging procedures for breast cancer patients. These procedures are not always appropriate in either clinical or research settings, regardless of the stage. The aim of this study was to identify groups of patients with differing risks for metastases in order to select more precise standard staging procedures. Patients and methods: The staging data relating to 406 breast cancer patients consecutively referred to our institution between November 1989 and October 1996 were analysed including pathological TNN grading and biological parameters. All of the cases with a positive or suspicious pre-operatory staging and who proved to have metastatic disease before surgery or during the first six months of follow-up were considered true- positive; all of the other cases with a positive or suspicious initial staging but with no evidence of distant metastasis before surgery and with a disease-free survival longer then six months were considered false-positive. In the same way all cases with negative initial staging who relapsed during the first six months of follow-up were considered false-negative and those with negative initial staging and with a disease-free survival longer then six months were considered true-negative. Statistical analysis was performed using Fisher's exact test. Results: BS, CXR and LUS, 388, 399 and 398 examinations respectively, were considered available, and 17 (4.38%), six (1.5%) and four (1%), respectively, proved to be true-positive. A statistically significant difference was observed when our cases were grouped according to T status (T4 vs. T1–T2–T3, P 〈 0.01) and nodal status (N0–N1 cases with less than three involved nodes and N1 with more than three positive lymph nodes N2 patients, P 〈 0.01). Conclusions: The present study suggests that breast cancer patients can be divided into three subgroups with different detection rates for distant metastases at staging (0.59%, 2.94% and 15.53%), and that the standard practice should be changed. In the first (T1N0 and T1N1 patients with ≤3 positive lymph nodes – 41.13% of the patients) and the second group (T2N0, T2N1 with ≤3 positive lymph nodes, T3N0 and T3N1 patients with ≤3 positive lymph nodes – 33.49% of the patients) there is no need for a complete set of staging procedures, whereas full procedural staging is needed in the third group of patients (T4, N1 with 〉3 lymph nodes and N2, 25.37% of the patients).

Notes: Abstract Clinical research for breast cancer is moving in three new directions following: 1) a critical analysis of three decades of randomized clinical trials for early disease; 2) increasing awareness of this lethal disease among women, generating women's associations which are pressing for improved breast cancer education, screening and treatment; 3) an exponential growth in our understanding of breast cancer molecular biology, leading to a number of innovative therapies with new targets in the cancer cell or its environment. It is the remarkable work of the Oxford Group which has finally vindicated the use of our three main weapons against breast cancer micro-metastases, namely tamoxifen, chemotherapy and ovarian ablation. There is now consensus that clinical research in the adjuvant setting may gain speed and efficiency through intergroup collaboration. Such an 'Intergroup' has been recently created in Europe and will collaborate with the American–Canadian Intergroup. Women's associations have only recently stepped forward to demand better care, and more effective therapies: they are becoming new partners in identifying critical issues in breast cancer research. Medical oncologists involved in breast cancer research are facing a new challenge: the optimal integration of traditional breast cancer therapies, namely endocrine treatments and chemotherapy, and entirely new strategies targeting signal transduction, apoptosis or angiogenesis. In view of the above, there is no doubt that we are entering a new and exciting era in breast cancer clinical research.

Notes: Abstract Background and purpose: Almost 10% of breast and ovarian cancers are familial, and the majority are linked to BRCA1 and BRCA2 germline mutations. Despite uncertainty about the management of female gene carriers, consensus guidelines have been established to assist practitioners and consultees in making health care decisions. Methodology: The Ad Hoc Committee was composed of 14 experts appointed by the French National Institute for Health and Medical Research, all of whom attended eleven workshops at which more than 3500 articles were systematically analyzed. Five additional experts critically analysed the first version of the report. Criteria and decision process: On a probability scale of the risk of developing breast or ovarian cancers, two thresholds were defined for use in determining whether an intervention would be worthwhile. The first is the threshold above which an intervention can be envisaged or recommended, and the second is the one below which an intervention can be ruled out; between the two, the decision has to be made on a case-by-case basis. Screening and preventive strategies analyzed: With respect to breast cancer: 1) hormonal interventions; 2) primary prevention (diet, family planning and chemoprevention); 3) screening (breast self-examination, clinician breast examination, tumor markers, imaging); 4) prophylactic mastectomy. With respect to ovarian cancer: 1) hormonal stimulation; 2) screening (clinical screening, ultrasound and tumor markers); 3) prophylactic oophorectomy. Main conclusions: For each strategy the following points were addressed: the information to be given to the consultee, the procedure and the indications. In addition, the committee's opinion about BRCA1 and BRCA2 mutation screening is that population-based, or even large-scale, implementation are not justified. Although no scientific evidence is available, the committee feels that specific management is indispensable and advocates the use of defined and evaluated procedures, and participation in clinical trials.

Notes: Abstract Background: The discovery of the multidrug resistance (MDR1) gene product P-glycoprotein (P-gp) has been widely seen as an important milestone in our understanding of the mechanisms underlying the clinical phenomenon of the emergence of resistant cells. MDR1 expression has been shown for numerous solid tumors and for virtually all hematologic malignancies. Nevertheless, results regarding MDR1/P-gp expression in human breast cancer have been controversial and the results of clinical trials on modulation of P-gp activity have not been encouraging. Patients and methods: MDR1/P-gp expression and the function of the P-gp pump were investigated in 61 tumor samples from patients with primary breast cancers by multiparameter analysis using MDR1-RT-PCR, immunohistochemistry with two MAbs (UIC2 and MRK 16) and the rhodamine 123 (Rh123) efflux assay. The cellular composition of the tumor cell suspension was analyzed by using specific MAbs against the P-gp expressing lymphocyte subsets CD4, CD8 and CD56, as well as against the HER-2/neu gene product, which was used to identify breast carcinoma cells. Results: UIC2 and MRK16 revealed a staining positivity in 72% and 75% of samples, respectively. A positive MDR1-RT-PCR signal was detected in 62% of the samples. Nevertheless, no correlation between immunohistochemistry and RT-PCR could be established. Furthermore, there was no correlation between HER-2/neu expression and MDR1-RT-PCR or P-gp immunohistochemical assays. A contamination by CD8+ and CD4+ lymphocytes was established in 100% and 84% of tumor cell suspensions, respectively. As assessed by the Rh123 efflux assay CD8+ and the CD4+ lymphocytes exhibited marked P-glycoprotein activity, whereas such activity was not detectable in a single instance for the breast carcinoma cells. In MDR1-RT-PCR positive samples, contamination by CD8 lymphocytes averaged 4.3%, while the contamination of CD8 cells in the MDR1 mRNA-negative samples was only 2.4% (P = 0.007). This signal vanished after elimination of the lymphocyte subpopulations by T-cell rosetting. Conclusions: In primary breast cancer detection of MDR1 gene expression by means of RT-PCR or immunohistochemical assays is not indicative for the MDR phenotype, since there is no evidence of significant activity of the P-gp pump.

Notes: Abstract The possible association between a high fat diet and increased breast cancer risk has remained controversial. This largely reflects the conflicting data obtained from migrant, case control and animal studies, which generally support this association, and cohort studies which often fail to show a link between fat and breast cancer. The mammary gland is particularly sensitive to estrogens during the fetal development, leading us to hypothesize that dietary fat levels during this period may significantly influence breast cancer risk. Using chemically-induced mammary tumors in rats as our experimental model, we have demonstrated the ability of a maternal diet, high in the polyunsaturated fatty acid (PUFA) linoleic acid, to alter mammary gland differentiation, accelerate the onset of sexual maturation, and increase breast cancer risk. The mammary glands of female rats exposed to a highfat diet in utero have more of the undifferentiated structures (terminal end buds) and fewer of the differentiated structures (alveolar buds) than the glands of rats exposed to a low-fat diet in utero. Furthermore, these mammary glands contain lower levels of total estrogen receptors and have reduced total protein kinase C activity. These effects appear to be mediated by an increase in tne serum estradiol levels of pregnancy, which are elevated at least 30% in pregnant dams fed a high fat diet. Furthermore, the administration of estradiol to pregnant dams produce effects on mammary gland development, onset of puberty and sensitivity to chemical carcinogenesis comparable to those seen in the offspring of rats fed a high fat diet during pregnancy. Our results, thus, support the hypothesis based on epidemiological data that high maternal estrogen levels increase daughters' breast cancer risk. The results also suggest that a high-fat diet may be an important factor in increasing pregnancy estrogenic activity.

Notes: Abstract We have investigated whether the raf-1 kinase, a downstream mediator of both receptor tyrosine kinase and protein kinase C signalling, is activated by estrogen (E2) in an estrogen receptor positive human breast cancer cell line. Autophosphorylation of raf-1 kinase was studied after treatment of MCF-7 cells with E2. E2-deprived cells contained low levels of raf-1 kinase activity. Treatment of cells for 1 min with E2 resulted in raf-1 autophosphorylation which was maximal within 5 min. Western blot analysis showed that raf-1 undergoes an electrophoretic mobility shift following E2 treatment. Egr-1 is a zinc finger-containing transcription factor which is expressed in association with raf-1 activation. Untreated MCF-7 cells expressed low levels of Egr-1 while E2 treatment resulted in an induction of egr-1 mRNA expression. These kinetics followed closely behind the E2 induction of c-myc mRNA. Egr-1 protein was similarly low in E2-deprived MCF-7 cells and was transiently increased following E2 treatment. Several studies have suggested that kinase activity may play a role in estrogen receptor (ER) activation. While activated v-raf failed to augment ER activation of transcription in transient transfection assays, a dominant negative mutant of raf-1 inhibited E2-induced transcription by 50% primarily as a result of increased baseline levels of E2 independent transcription. The results show that E2 can induce raf-1 kinase activity in MCF-7 breast cancer cells associated with the expression of an early growth response gene and modulation of ER signalling.

Notes: Abstract Objectives: A case-control-family study of breast cancer in women under the age of 40 was carried out in Melbourne and Sydney, Australia, from 1992 to 1995 to determine the risk factors for these women. Subjects included 467 incident cases identified by state cancer registries and 408 population-based controls. Methods: All participants completed a structured risk-factor questionnaire and family pedigree during an in-person interview. Where possible, cancers in first- and second-degree relatives were verified. Results: Multiple logistic regression analysis showed that the strongest risk factor for breast cancer was a family history of the disease - having at least one affected first-degree relative trebled the risk (relative risk [RR] = 3.3, 95 percent confidence interval [CI] = 1.9-5.8). Risk increased with height by three percent (standard error [SE] of one percent) per cm, and after adjusting for height, there was evidence for a decreased risk in women weighing 73 kg or more. There was an increased risk of breast cancer after the first full-term birth (RR = 1.8, CI = 1.0-3.5) but this risk fell by 30 percent (SE = 11 percent) with each subsequent livebirth. Conclusions: The effects of other reproductive factors and oral contraceptive use, although not nominally significant, were in accord with published findings from similar studies in young women. This study of Australian women has indicated that some risk factors for breast cancer in women under age 40 differ from those reported for older women either in direction (e.g., weight) or relative importance (e.g., family history).

Notes: Abstract Objectives: It is unclear whether physical activity is associated with a reduced risk of breast cancer. Some studies also suggest different effects between pre- and postmenopausal women, and lean and heavy women. Methods: We followed 1,566 University of Pennsylvania alumnae (mean age, 45.5 years), initially free of breast cancer, from 1962 until 1993. Physical activity at baseline was assessed by asking women about stairs climbed, blocks walked, and sports played. We estimated energy expenditure and categorized women into approximate thirds (〈 500, 500-999, 1,000+ kcal/wk). We identified 109 breast cancer cases during 35,365 person-years from follow-up questionnaires or from death certificates. Results: After adjustment for age and body mass index (BMI) (kg/m2), the relative risk (RR) of breast cancer was 0.92 (95 percent confidence interval [CI]=0.58-1.45) among women expending 500-999 kcal/wk and 0.73 (CI=0.46-1.14) for those expending 1,000+ kcal/wk, compared with women expending 〈 500 kcal/wk (P trend=0.17). This association was modified by menopausal status, but not BMI. For postmenopausal women, corresponding RRs were 0.95 (CI=0.58-1.57) and 0.49 (CI=0.28-0.86), respectively (P trend=0.015). Increased physical activity in premenopausal women was not significantly associated with decreased risk of breast cancer. Conclusions: These data support an inverse association between physical activity and breast cancer among postmenopausal women. Cancer Causes and Control 1998, 9, 433–439

Notes: Abstract Plasminogen activators are known to be involved in the metastatic spread of breast cancer. In the present study we examined the effects of antiestrogens [Analog II (1,1-dichloro-cis-2,3-diphenyl cyclopropane) (AII), ICI-182,780 (ICI) and tamoxifen (TAM)], on the in vitro release of uPA from estrogen receptor (ER)-positive MCF-7 (MCF) and ER-negative MDA-MB-231 (MDA) human breast cancer cell lines. Using a solid-phase radioassay, uPA activity was found to be higher in the culture medium from MDA cells compared to MCF cells. Aminocaproic acid, a specific plasmin inhibitor, produced a 50-60% reduction in the degradation of labeled substrate, in the solid phase assay, using culture medium from both cell lines, thus indicating that most of the proteolysis observed was due to uPA-mediated plasmin generation from plasminogen. In the absence of plasminogen, the enzyme activity was not detected in either the quantitative assay or by zymography. In MDA cells, uPA release was not altered by any of the antiestrogens used alone or in the presence of estradiol. In contrast, in MCF cells, ICI alone produced maximal inhibition (40%) of enzyme release, while estradiol alone produced a 120% increase in enzyme activity. When co-administered with estradiol, in MCF cultures, each antiestrogen reduced enzyme activity to control levels. Substrate gel zymography revealed that the urokinase-type PA is the predominant form of PA released by both cell lines. Comparison of the activity of all three antiestrogens used in this study indicates that ICI is the most potent inhibitor of enzyme activity in ER-positive MCF cells. © Rapid Science 1998

Notes: Abstract A case-cohort analysis of the association between diet and risk of benign proliferative epithelial disorders (BPED) of the breast was undertaken within a cohort of 56,537 women who were enrolled in the Canadian National Breast Screening Study (NBSS) and who completed a self-administered dietary questionnaire. (The NBSS is a randomized controlled trial of screening for breast cancer in women aged 40 to 59 years.) BPED are thought to have premalignant potential. Specific hypotheses were that risk of BPED would increase with increasing energy-adjusted fat intake and decrease with increasing energy-adjusted vitamin A and fiber intake. Additionally, we explored the association between calcium intake and risk of BPED. During the active follow-up phase of the NBSS, 657 women in the dietary cohort were diagnosed with biopsy-confirmed incident BPED. For comparative purposes, a subcohort consisting of a random sample of 5,581 women was selected from the full dietary cohort. After exclusions for various reasons, the analyses were based on 545 cases and 4,921 non-cases. Overall, the results were almost uniformly null, and provided little support for the study hypotheses. Rate ratios (95 percent confidence intervals [CI]) for the highest cf the lowest quintile levels for total fat, retinol, β-carotene, fiber, and calcium were 0.88 (CI = 0.65-1.20), 0.97 (CI = 0.71-1.31), 0.94 (CI = 0.70-1.27), 1.11 (CI = 0.82-1.50), and 0.81 (CI = 0.60-1.07), respectively. There were too few cases of atypical BPED for meaningful analysis, but results for those whose BPED showed no atypia were similar to the overall results. Further analyses conducted separately in the screened and control arms of the NBSS also failed to provide strong support for dietary associations, as did those conducted separately for screen-detected and interval-detected BPED.

Notes: Abstract We used Transwell chambers to study separately cellular motility and invasion. In order to assess the cellular motility, polycarbonate microporous filters were coated with extracellular matrix proteins which adsorbed on the filters without clogging the pores. To investigate the invasive behavior of tumor cells, filters were covered with a layer of Matrigel which clogged the pores. The motility and the invasion of breast cancer cell lines (MDA-MB-231, MCF-7/6 and MCF-7/AZ cells) were assessed quantitatively in different culture media: defined (serum-free), serum-containing and normal human fibroblast MRC-5 conditioned media. Inserum-containing medium, tumor cells migrated and invaded through the coated and covered filters. Their motility and invasion potentials were considerably lower in defined medium, whereas medium conditioned by MRC-5 fibroblasts stimulated both motility and invasion but not growth. The MRC-5 conditioned medium induced also the spreading of clusters of MCF-7 /6 cells grown on Matrigel-coated plates. © Rapid Science 1998

Notes: Abstract Hormone-independent growth and invasiveness represent phenotypic properties acquired during early progression of breast cancer. We compared human mammary adenocarcinoma cells, MCF-7, which are estrogen-dependent and poorly metastatic, with the estrogen-independent and highly metastatic subline, MCF7/LCC1, with regard to expression of tissue-degrading factors of the matrix metalloproteinase (MMP)-and urokinase (uPA)-dependent degradative pathways, as well as for their in vitro invasive properties. Both cell lines showed low constitutive mRNA expression of the MMP inhibitor TIMP-1. Baseline expression of TIMP-2 mRNA was also very low in MCF-7 cells, whereas the MCF7/LCC1 level was much higher (~10- fold). Furthermore, both cell lines revealed low constitutive capacity to migrate in an in vitro invasion assay. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA; 100 nM) induced the mRNAs for TIMP-1 as well as for MMP-1, MMP-9, the uPA receptor, and the uPA inhibitor PAI-1, am ongst which only the responses of MMP-9 and PAI-1 were cell-specific. The mRNA levels of MMP-9 and PAI-1 were ~10-fold and ~15-fold higher in MCF7/LCC1 cells compared to MCF-7 cells. The secretion of immuno-reactive PAI-1 was considerably elevated (. 20-fold) in TPA-treated MCF7/LCC1 cells, whereas the TPA-dependent level of 92-kDa MMP-9 was only ~2-fold higher in MCF7/LCC1 cells than in MCF-7 cells. In both cell lines treatment with TPA was associated with an increase (~10-fold) in in vitro migration, which in the MCF7/LCC1 cells was significantly attenuated by a reconstituted basement membrane extract (Matrigel). These data suggest that TPA-responsive in vitro invasive properties that are probably associ-ated with PAI-1 expression may co-vary with progression from hormone-dependent to -independent breast cancer. © Rapid Science 1998

Notes: Abstract Primary breast cancers were shown to overexpress CD44 v5 and v6 at the plasma membrane. However, the clinical significance of this overexpression remains unclear. Overexpression of CD44 v5 and v6 in primary breast cancers was found to correlate with metastasis and poor prognosis by some investigators, yet this correlation could not be confirmed by others using different antibodies. In this study the influence of metastatic disease, the site of metastasis, and the amount of CD44 v5 and v6 expression in the primary tumor on serum levels of the soluble forms of CD44 v5 and v6 (sCD44 v5 and v6) in breast cancer patients was investigated. Soluble CD44 v5 and v6 serum levels were measured by enzyme linked immuno sorbent assay in a group of breast cancer patients who developed metastases in various organs and in another group of patients with single organ metastasis. For control, sCD44 v5 and v6 levels were measured in breast cancer patients who remained free of metastasis and in healthy blood donors. Expression of plasma membrane bound CD44 v5 and v6 in the primary tumors of the patients with metastasis in various organs was correlated to sCD44 v5 and v6 levels in serum. Furthermore the size of sCD44 v6 was analyzed by immunoblot using a monoclonal antibody directed against CD44 v6. When metastases were detected, sCD44 v5 and v6 serum levels were increased as compared to levels measured one month after tumor surgery in patients free of metastases (p = 0.0025 and p = 0.0004). Six of 19 and 6 of 20 patients had sCD44 v5 and v6 serum levels above a cut-off level of 85 and 275 ng/mL, respectively. In these cases expression of CD44 v5 and v6 in the primary cancers was also elevated. Low sCD44 v5 and v6 serum levels were associated with weak expression of CD44 v5 and v6 in the respective primary cancers. As shown by statistical analysis of sCD44 v5 and v6 levels in 57 patients with single organ metastases, elevated sCD44 v6 levels but not sCD44 v5 levels were associated with metastases in liver or bone (p = 0.0025). Immunoblot analysis of soluble CD44 proteins in serum revealed two CD44 v6 specific signals of approximately 120 and 170 kDa. Increased sCD44 v5 and v6 serum levels in patients with breast cancer were influenced by the amount of CD44 v5 and v6 expression in the primary tumor by the site of metastasis. Elevated sCD44 v6 serum levels were preferentially found in patients with metastases in liver or bone.

Notes: Abstract In 1992, a special issue of Breast Cancer Research and Treatment was devoted to the insulin-like growth factors and breast cancer. In that issue, identification of the key components of the IGF system was reviewed and their potential role in breast cancer growth was described. In this issue, we revisit the IGF system with particular attention to data that further supports their role in the growth regulation of breast cancer. Several new facets of the IGF system are described, and several laboratories have more clearly defined how each individual component of the IGF system may influence breast cancer biology.

Notes: Abstract Purpose. In the past 15 years breast conserving therapy (BCT) has become an important treatment option for primary breast cancer. Thirty three angiosarcomas (AS) after BCT have been described in a total of 20 published reports. Limited follow-up data and the lack of information on incidence of AS prompted the authors to review the comprehensive experience in the Netherlands. Methods. Between 1987 and 1995 twenty-one patients with BCT-associated AS were diagnosed in the Netherlands. Follow-up after diagnosis of AS ranged from 6 to 82 months with a median of 24 months. Information on the total number of patients treated with BCT and on the numbers of angiosarcoma in the breast was obtained. Results. The median interval between BCT and AS was 74 months (range: 29–106) and appeared to decrease with higher age. Detection of skin changes followed by incisional biopsy provided the diagnosis. Two year overall (OS) and disease free survivals were 72% (s.e. 10.9) and 35% (s.e. 10.7), respectively. Two year OS after initial complete surgical resection was 86% (s.e. 9.3) compared to 0% after incomplete resection of the AS (P=0.04). The estimated incidence of AS after BCT is 0.16%. Conclusions. BCT-associated AS arises after a relatively short interval. Although the incidence of AS is low, the absolute number of patients at risk is increasing. This calls for vigilance concerning skin changes occurring after BCT. An incisional biopsy provides the only reliable diagnosis. The prognosis appears to be related to the completeness of surgical resection.

Notes: Abstract Monoclonal antibody (MAb) FC-2.15 recognizes Lewis × antigen (Le×-Ag) expressed on the cell surface of most human breast cancer cells. FC-2.15 displays important human complement (C′)-mediated cytotoxicity (CMC) against its target cells. In this study the reactivity of FC-2.15 against drug resistant-breast cancer cells was investigated, as well as the possibility to combine the antitumor activities of this MAb with adriamycin (Adr) or taxol. Since resistant clones with altered expression of tumor-associated antigens usually emerge after chemotherapy, the expression of Le×-Ag was analyzed in AdrR MCF-7 breast cancer cells (Adr resistant subline) and in tumor samples from nine patients with locally advanced breast carcinoma who were treated with FEC chemotherapy. A flow cytometry assay showed that most of AdrR MCF-7 cells, as well as wild type (WT) cells, expressed Le×-Ag; however, the Le× epitope is probably bound to different backbones in these cells. When the cytotoxic ability of FC-2.15 against WT and AdrR MCF-7 cells was compared, it was found that a 90 min treatment with FC-2.15 plus C′ induced similar CMC against both cell lines. An important cytolysis was obtained at 5 µg/ml FC-2.15, reaching a plateau at 25 µg/ml, at which cell population was diminished to 21.1% for WT and 27.9 for AdrR MCF-7 cells. Regarding human tumors, Le×-Ag expression was evaluated in samples obtained before and in most cases after chemotherapy, and it could be observed that: 1) before treatment, tumor samples from all patients analyzed (responders and non-responders to chemotherapy) were FC-2.15-positive; 2) the presence of Le×-Ag was not modified after treatment. The combined action of Adr or taxol with FC-2.15 was then evaluated. WT and AdrR MCF-7 cells were cultured with Adr or taxol followed by an incubation with different FC-2.15 concentrations plus C′. When the effect of Adr alone was determined, ID50 were 1 × 10-7 M for WT and 4.2 × 10-5 M for AdrR MCF-7 cells. The cytotoxic ability of taxol alone was also tested, and ID50 were 6.4 × 10-9 M for WT and 3.1 × 10-6 M for AdrR MCF-7 cells. When FC-2.15 was added to Adr or taxol, the cytotoxicity of the drug-FC-2.15 combined treatment was always higher than the isolated effects, showing additive cytotoxicity at the different concentrations tested and with both cell lines. Our results suggest that FC-2.15 may be a useful agent against breast tumor cells which survive chemotherapy with Adr or taxol.

Notes: Abstract Experimental laboratory data suggest that tumour growth is a balance between apoptosis and proliferation and that suppression of drug-induced apoptosis by oncogenes such as bcl-2 may be an important cause of intrinsic chemoresistance. The aims of this study were to assess the in vivo relationship of apoptosis to proliferation and Bcl-2 protein in human breast tumours both prior to chemotherapy and in the residual resistant cell population at the completion of treatment. We examined apoptotic index (AI), Ki67 and Bcl-2 protein expression in the tissue of 40 patients with operable breast cancer immediately before ECF preoperative chemotherapy, and in 20 of these patients with residual tumour, at the completion of treatment. There was a significant positive association between AI and Ki67 both before and after chemotherapy, and in their percentage change with treatment. In the residual specimens AI and Ki67 were significantly reduced compared with pre-treatment biopsies, while Bcl-2 expression showed a significant increase. No differences were seen in the pre-treatment levels of any of the variables measured between patients obtaining pathological complete response and those who did not, although numbers were small. These data suggest that apoptosis and proliferation are closely related in vivo. It is possible that the phenotype of reduced apoptosis and proliferation, and increased Bcl-2 may be associated with breast cancer cells resistant to cytotoxic chemotherapy, although this can only be proven by assessing larger numbers of patients in relation to pathological response.

Notes: Abstract Human mammary carcinoma xenografts (MCF-7) growing in nude mice were treated with natural interferon α (n-IFN-α) alone or conjugated to a humanized monoclonal antibody (MoAb) anti-breast mucin (HuBrE-3vl) or to irrelevant human IgG1κ. The IFN and the conjugates were administered as 20 intra-lesional (i.l.) injections to 1 of 2 xenografts in each mouse, or i.p. The growth inhibitory effects of HuBrE-3vl/nIFN-α were significantly greater than those of nIFN-α used as a single agent or conjugated to HuIgG1κ. These effects occurred locally in the tumors receiving i.l. injections and systemically, although to a slightly lesser extent, in the noninjected tumors of mice treated i.l. and in the xenografts of mice treated i.p. Biodistribution studies showed that the uptake of 125I-HuBrE-3vl/nIFN-α by the tumors 24 hours after i.l. or s.c. injection was greater than that of 125I-HuIgG1κ/nIFN-α,125 I-nIFN-α alone, or by normal tissues, documenting a tumor targeting effect and favorable tumor:normal tissues (T:NT) ratios. The targeting effects and the resulting tumor growth inhibition were favored by the IFN-mediated up-regulation of the HuBrE-3vl reactive antigen, which was more prominent after 3 weeks of treatment with HuBrE-3vl/nIFN-α. These results were superior to those we obtained previously with nIFN-α conjugated to another MoAb of the same group (Mc5). These studies point out the potential usefulness of HuBrE-3vl/nIFN-α for the local and systemic treatment of breast cancer lesions by providing a means of delivering high doses of IFN to the tumors while minimizing the amount of IFN binding to normal tissues.

Notes: Abstract The effect of EM-800, a new non-steroidal antiestrogen having pure antiestrogenic activity, was studied on chemical carcinogenesis induced by dimethylbenz(a)anthracene (DMBA) as well as on serum lipids and bone mass in the rat. Treatment with EM-800 orally, once daily, for 282 days (9 months), starting 3 days before DMBA administration, decreased the incidence of tumors from 95% in control animals to 60% (p 〈 0.01), 38% (p 〈 0.01), and 28% (p 〈 0.01) at the daily doses of 25 μg, 75 μg, and 250 μg, respectively. The average number of tumors per animal decreased from 4.5 ± 0.5 tumors in the control group to 0.9 ± 0.2 (p 〈 0.01), 0.5 ± 0.2 (p 〈 0.01), and 0.3 ± 0.1 (p 〈 0.01) tumors in the rats treated with the above-indicated doses of the antiestrogen. In addition, treatment with the increasing doses of EM-800 reduced serum cholesterol levels to 64%, 56%, and 48% of control, while serum triglycerides decreased to 31%, 28%, and 30% of control. Bone mineral content (BMC) and bone mineral density (BMD) of total skeleton, femur, and lumbar spine were not significantly affected following 282 days of treatment with EM-800. However, treatment with EM-800 inhibited the urinary ratio of hydroxyproline to creatinine (HP/Cr) from 14.0 ± 3.90 μmol/mmol in controls to 7.6 ± 0.8 (p 〈 0.05), 6.8 ± 0.8 (p 〈 0.01), and 6.8 ± 1.1 (p 〈 0.01) μmol/mmol, respectively, while the same treatment had no effect on serum total alkaline phosphatase (tALP) activity or urinary calcium and phosphorus excretion. The 25 μg, 75 μg, and 250 μg daily doses of EM-800 inhibited uterine weight by 35% (p 〈 0.01), 62% (p 〈 0.01), and 66% (p 〈 0.01), while vaginal weight was reduced by 8% (p 〈 0.05), 30% (p 〈 0.01), and 38% (p 〈 0.01), respectively. In agreement with the 27% increment (p 〈 0.05) in ovarian weight at the highest antiestrogen dose used, serum androstenedione (p 〈 0.05), androst-5-ene-3β,17β-diol (p 〈 0.01), testosterone (p 〈 0.05), and estradiol (p 〈 0.01) levels were increased. The present data show that EM-800 prevents the development of DMBA-induced mammary tumors while simultaneously inhibiting uterine and vaginal weight, reducing serum cholesterol and triglyceride levels, and having no adverse effect on bone mass following 9 months of treatment in the rat.

Notes: Abstract N,N-diethyl-2-[4-(phenylmethyl)phenoxy] ethanamine.HCl (DPPE) is a diphenylmethane analog of tamoxifen that antagonizes the intracellular binding of histamine to growth-regulatory sites, a proportion of which represents P450 enzymes, in microsomes and nuclei. We previously reported increased response rates and decreased myelotoxicity in patients with prostate and other cancers who received an intensive dose/schedule of DPPE plus single-agent chemotherapy. We now report the results of a study of DPPE combined with a standard dose/schedule of doxorubicin in twenty-three patients with metastatic breast cancer, sixteen of whom had received prior non-anthracycline chemotherapy. DPPE (6 mg/kg) was infused intravenously (IV) over 80 minutes. Doxorubicin (60 mg/m2) was administered IV over the last 20 minutes of the DPPE infusion. Treatment was repeated every 3 weeks (maximum, 7 cycles). Patients achieving complete response (CR) were followed off treatment until relapse. All patients were evaluable for toxicities and efficacy. Sixteen patients (69%; 95% C.I. = 47–87%) responded (7 CR and 9 PR). Eleven responders, including 6 with CR, had prior chemotherapy. Five responders (2CR, 3PR) had a poor (ECOG 3/4) performance status pre-treatment. Median CR duration was 11 (range 5–18) months. Hematological toxicity was low; GI toxicity (nausea/vomiting/dyspepsia) appeared somewhat higher than historical experience, but responded well to anti-emetics, ranitidine, and/or dexamethasone in most patients; a mean absolute drop in left ventricular ejection fraction of 8% occurred in 17 patients who received ⩾ 300 mg/m2 doxorubicin. The observed response rate in DPPE/doxorubicin-treated patients appeared to be higher than historically reported for doxorubicin alone in this setting, suggesting a chemopotentiating effect of DPPE. A multi-centre trial of this regimen in an additional 32 patients with early metastatic breast cancer has been conducted by the Clinical Trials Group, National Cancer Institute of Canada, and a phase 3 study is planned.

Notes: Abstract Introduction: The aims of the study were to assess the outcome among patients with early breast cancer operated on with wide local excision who developed a subsequent ipsilateral breast tumor recurrence, and to identify risk factors for uncontrolled local disease. Uncontrolled local disease (ULD) was defined as the appearance of clinically manifest invasive adenocarcinoma in the remaining breast or on the ipsilateral chest wall which could not be eradicated with salvage treatment during the period of follow-up (2–18 years). Patients and methods: Eighty-five patients in a cohort of 759 patients, treated for invasive Stage I–II breast cancer with breast-conserving surgery 1976–1985 in Stockholm, with a subsequent ipsilateral breast tumor recurrence (IBTR) were reviewed retrospectively. The majority of the patients were premenopausal (58%), node negative (72%), and had received postoperative radiotherapy (79%). Median follow-up time following breast-conserving surgery was 13 (9–19) years. Multivariate Cox's hazard regression was used in the statistical analysis to identify prognostic factors for ULD. Results: The majority (n = 61) of the IBTR's were located in the original tumor quadrant and showed the same histopathological features as the primary tumor. Salvage mastectomy (n = 65) or reexcision (n = 14) were performed in 79 (93%) of the patients. Twenty-one patients developed ULD. Five years following the diagnosis of IBTR the disease-free survival was 59%, the cumulative incidence for ULD was 24%, and for death in breast cancer 34%. In the cohort of 759 patients, patients who received radiotherapy following the primary breast-conserving surgery had 1% cumulative incidence of ULD following the diagnosis of IBTR compared to 4% among patients that received no postoperative radiotherapy. The cumulative incidence at 5 years of ULD following salvage mastectomy was 12% compared to 33% after salvage reexcision. Patients operated on with breast-conserving surgery with an original tumor size 〈 15 mm, who were treated with salvage mastectomy for IBTR, had in multivariate analysis the lowest relative risk for ULD. Adjuvant chemotherapy following IBTR treatment did not seem to improve local tumor control. Following the diagnosis of IBTR, 78% (n = 21) of the patients with ULD and/or regional recurrence (n = 27), died of a disseminated breast cancer in contrast to 10% (n = 6) among the remaining 58 patients. Conclusion: Uncontrolled local disease is an important outcome measure following breast-conserving surgery. In this cohort, salvage mastectomy provided a superior local control rate compared to salvage reexcision. A higher although not statistically significant rate of ULD was also seen in patients who had not received postoperative radiotherapy as part of their primary treatment.

Notes: Abstract A majority of studies have shown an increase in the risk of breast cancer among women previously diagnosed with fibroadenoma (FA). At present there is conflicting evidence whether some of the chromosome abnormalities frequently found in breast carcinoma, such as loss of heterozygosity (LOH), are already present in FAs and other types of benign breast disease and, if present, whether such abnormalities are associated with the observed increase in risk. Microsatellite instability (MSI) is also recognised as a marker of genetic damage and is thought to occur when there has been damage to the cell's mismatch repair (MMR) system. We have analysed 39 cases of FA obtained from paraffin-embedded tissue for the presence of MSI and LOH at 11 loci to determine if these types of genetic alterations occur in FA. The incidence of MSI and LOH found were 4 of 395 (1.0%) and 5 of 271 (1.8%) informative loci tested respectively. Approximately 8% of cases were positive for MSI and 10% were positive for LOH, with one specimen having multiple occurences of both MSI and LOH. We conclude that these forms of genetic alteration do occur in FAs but that the incidence is low.

Notes: Abstract Galectin-3 is a galactoside binding protein found at elevated levels in a wide variety of neoplastic cells and thought to be involved in cognitive cellular interactions during transformation and metastasis. Previously, we have shown that introduction of human galectin-3 (Mr 31,000) cDNA into the human breast cancer cells BT-549 which are galectin-3 null and non-tumorigenic in nude mice resulted in the establishment of four galectin-3 expressing clones. Three of them acquired tumorigenicity when inoculated in the mammary fat pad of nude mice. Here, we questioned what is the molecular difference between the nude mouse tumorigenic and non-tumorigenic galectin-3 expressing BT-549 cell clones. Differential display analysis and Northern blotting revealed that, unlike the tumorigenic clones, neither the parental cells nor the non-tumorigenic clone expressed a 6.5 Kb transcript. A 607 bp PCR (polymerase chain reaction) product from the differentially displayed mRNA revealed a 93% sequence homology with the human L1 retrotransposon previously suggested to play a role in the pathobiology of some breast cancers. In addition, we show that the two gene products, i.e., galectin-3 and L1, are co-expressed in breast carcinoma specimens and in other nude mouse tumorigenic cell lines.

Notes: Abstract Proliferation indices are used, along with other parameters, to estimate the risk of recurrence of breast cancer for individual patients. Because it is unlikely one index will be practical for all patients, it is important to understand the relationship between various indices of proliferation. For this reason, we compared a proliferation index based on in vivo labeling of S-phase tumor cells with the thymidine analog bromodeoxyuridine (BrdUrd), to a proliferation index based on an estimate of the growth fraction with the MIB-1 antibody to the Ki-67 antigen. With informed consent, we gave 145 patients 200 mg/m2 BrdUrd intravenously just prior to surgical removal of breast cancer. On histology sections, we visually counted S-phase cells which had incorporated BrdUrd using the Br-3 antibody which is specific to DNA-incorporated BrdUrd, and we counted cells in the growth fraction using the MIB-1 antibody to the Ki-67 antigen. We found that both indices were positively correlated with tumor size, number of positive nodes, and tumor grade, and both were negatively correlated with age and estrogen-progesterone receptor positivity. Using a linear functional relationship model, we found that the best (i.e. the maximal) fit between the two indices (correlation coefficient 0.79; p 〈 0.0001) occurred when each index was square root transformed, as is appropriate when counts follow a Poisson distribution. When we used the median as a cutpoint for each index, the classification of 19 percent of data pairs changed depending upon which index was used. We also estimated that the Ki-67 intercept (1.02 ± 0.25) was significantly greater than zero. We conclude that the BrdUrd index of DNA synthesis in S-phase correlates highly with the MIB-1 index of the growth fraction, and both indices correlate well with other parameters of tumor aggressiveness. Because this correlation is driven by concordance of the extremes of high and low counts, clinical comparison will be necessary to determine which is the better prognostic marker for human breast cancer.

Notes: Abstract Breast cancer cell lines display a wide variety of growth factor receptors, and considerable evidence implicates signaling from these receptors, especially ErbB2, in the important early stages of this disease, contributing to malignant progression. If this is true, then we would hypothesize that a useful prognostic indicator would be the level of activity of a second messenger protein used in common by these receptors. One such second messenger is the Shc adapter protein, which is activated when tyrosine phosphorylated by receptors. Therefore, one prediction from the hypothesis is that the level of tyrosine-phosphorylated Shc (PY-Shc) in breast cancer cell lines would correlate with total receptor tyrosine kinase activity. To begin to test this prediction, we examined Shc tyrosine phosphorylation in a diverse group of breast cancer cell lines that display varied levels of ErbB2. Using Shc immunoprecipitation and anti-phosphotyrosine immunoblotting analysis, we found a strong correlation between the level of ErbB2 overexpression (r=0.91, p 〈 0.0002) and PY-ErbB2 levels (r=0.89, p=0.0005) compared with the level of tyrosine phosphorylation of the p52 and p46 Shc isoforms. Consistent with Shc tyrosine phosphorylation being driven by ErbB2, an ErbB2-specific tyrosine kinase inhibitor markedly reduced Shc tyrosine phosphorylation. Unexpectedly, although all cell lines had comparable total amounts of p52 and p46 Shc, the amount of an inhibitory Shc isoform, p66, was inversely related to the level of ErbB2 expression (r=− 0.86, p=0.0013). This suggests that reduced p66 Shc expression may play a role in ErbB2-positive breast cancer. In summary, these data are consistent with our prediction that the cellular level of PY-Shc would correlate with the levels of activated ErbB2 displayed by cell lines derived from breast cancers.

Notes: Abstract Urokinase type plasminogen activator (uPA) and its inhibitors, plasminogen activator inhibitor type I (PAI-1) and type II (PAI-2), are supposed to be involved in the expression of the invasive and metastatic phenotype of cancer cells. However, clinical investigations on the prognostic significance of their levels in tumor tissue are difficult to realize because of the absence of a convenient method of measurement of these parameters. The aim of the present investigation was to set up a method allowing the measurement of these enzymes and of sex steroid receptor status in appropriate subcellular fraction(s) in conditions easily reproducible in routine. We found that a tissue homogenate prepared according to the method recommended [5] for current measurement of sex steroid receptors is appropriate for further distinct preparations. One aliquot is used for cytosol preparation; another can be treated by 2% Triton X-100 (vol/vol) and provide an extract containing the totality of uPA and PAI-1. The advantage of this procedure is that appropriate subcellular fractions can be derived from a unique homogenization step. Total uPA and PAI-1 are measured in a Triton extract with good performance as compared to previous investigations [4]. PAI-2 is measured in the same cytosol fraction used for sex steroid receptors and other parameters. Because of its simplicity and its high reliability, this method could be a useful tool in the investigation of uPA family proteases and analysis of their prognostic significance in early breast tumors.

Notes: Abstract The expression of estrogen (ER) and progesterone (PgR) receptors was analyzed in a retrospective series of 3000 patients who had operable primary breast cancer. Patients were stratified according to ER and PgR status and the study was focused on the two groups (ER−PgR+ and ER−PgR−) of patients whose tumors contained low levels of ER (〈 15 fmol/mg protein), regarding potential response to endocrine therapy. The comparison of clinical or histological characteristics between ER−PgR+ and ER−PgR− patients was analyzed as well as the disease-related death and survival. The mean follow-up was 86.3 months. Among the 529 ER−patients, 62 were PgR+ (12%), whereas 467 were PgR− (88%). The ER−PgR+ and ER−PgR− populations represented 2% and 15.6% of the overall population, respectively. In ER− tumors, the PgR status was significantly related to: age, menopausal status, tumor size, SBR grade, and histological type, but not to the type of surgical treatment or to lymph node involvement. ER−PgR+ tumors had smaller size (64% T1 vs 43%) (p=0.004) and were more frequently grade I (28% vs 12%) than ER−PgR− ones (p 〈 0.001). In addition, the patients with ER−PgR+ tumors were significantly younger (49.4 years vs 58.4 years; p 〈 0.0001), and were more frequently premenopausal (76% vs 36%; p 〈 0.001). The disease-free interval and the metastasis-free survival tended to be worse for ER−PgR− than for ER−PgR+ patients, but the difference was not statistically significant at 10 years. However, a small but significant difference in overall survival, in favor of the PgR+ group, was observed between the two groups during the first 5 years (p=0.03). We conclude that in combination with ER, PgR status defines a group of patients with clinical and biological specificity, which could be considered for specific endocrine therapy.

Notes: Abstract Steroidal aromatase inhibitors like formestane and exemestane are useful drugs for endocrine treatment of postmenopausal breast cancer. In addition, these drugs should be considered valuable probes to explore the biology of breast cancer with particular emphasis on possible relations between the degree of estrogen suppression and clinical efficacy and the possible role of intratumor estrogen synthesis. The fact that steroidal and non-steroidal aromatase inhibitors bind to different parts of the aromatase enzyme suggests these drugs may act in concert aggravating plasma estrogen suppression. Thus, use of a steroidal and a non-steroidal aromatase inhibitors in concert may be one way to improve breast cancer treatment and may also provide important information to a better understanding of the dose-response relationship between estrogen suppression and clinical effects. Further, the finding that patients progressing on non-steroidal aromatase inhibitors may respond to formestane as well as exemestane suggests these drugs may have differential effects, probably on the aromatization in the tumor tissue. Further studies are warranted to explore the influence of steroidal and non-steroidal aromatase inhibitors on intratumor aromatase activity and intratumor estrogen concentrations and to correlate these findings to intratumor drug concentrations. The findings that steroidal aromatase inhibitors may have clinical effects in patients progressing on treatment with the non-steroidal aromatase inhibitor aminoglutethimide is challenging, and suggest further studies to evaluate possible benefits of using different novel aromatase inhibitors in concert or sequence.

Notes: Abstract Vorozole (RivizorTM), is a triazole derivative and one of the new, third generation aromatase inhibitors. Vorozole causes reversible inhibition of cytochrome P450 aromatase with the majority of the aromatase inhibition activity attributable to the dextro-isomer. In vitro the IC50 against human placental aromatase and in cultured rat ovarian granulosa cells is 1.38 and 0.44 nM, respectively. Vorozole is selective and does not effect other cytochrome P450-dependent reactions at concentrations up to at least 500-fold the aromatase inhibiting concentration. In vitro vorozole, at concentrations of up to 10 μM, does not exhibit agonistic or antagonistic effects on steroid receptors including the estrogen, progestin, androgen and glucocorticoid receptors. In vivo vorozole produces dose-dependent inhibition of aromatase and reduces circulating estrogen levels. Vorozole has been shown to inhibit intratumoral aromatase activity in postmenopausal breast cancer patients pretreated for 7 days prior to undergoing mastectomy. Tissue estrone and estradiol levels were also shown to be decreased by 64% and 80%, respectively. In four phase II clinical trials, vorozole produced response rates of 18–33% corresponding to selective inhibition of estradiol. Vorozole has been examined in large, randomized multi-centre, controlled trials against both megestrol acetate (MA) and aminoglutethimide (AG) plus hydrocortisone. Against MA, response rates were comparable (10.5% vorozole; 7.6% MA) however, a trend towards improvement in median duration of response for vorozole (18.2 versus 12.5 months; p=0.07) was shown. No differences in time to progression or survival were noted. Significant and persistent weight gain associated with MA administration was the most notable difference in tolerability between the two agents. Against AG, vorozole showed a higher response rate (23% versus 18%) however this did not reach statistical significance (p=0.085). No differences in duration of response, time to progression and survival were noted. A significantly better Functional Living Index-Cancer (FLIC) quality of life score was associated with vorozole compared to AG. Vorozole is a specific, selective and potent aromatase inhibitor and useful for postmenopausal patients with advanced breast cancer.

Notes: Abstract The insulin-like growth factor (IGF) family of ligands, receptors, and binding proteins can regulate breast cancer cell proliferation in vitro, and interruption of these pathways inhibits IGF-mediated cell proliferation. If the IGF family members are key regulators of breast cancer growth and progression in vivo, we would expect their expression to be an indicator of the prognosis of the disease. Thus, measurement of IGF expression may provide an indicator of the growth effect within a tumor, and provide new targets for treatment of the disease. In this review we will summarize the data generated thus far indicating that IGF family members are indicators of prognosis of breast cancer, and that measurement of the whole IGF family in concert may provide useful information for treatment strategies of breast cancer.

Notes: Abstract Tamoxifen causes an objective response in about one-third of metastatic breast cancer and in only half of the breast cancer patients with estrogen receptor (ER) positive tumors. Steroid-receptor coactivator-1 (SRC-1) appears to be a general coactivator for steroid receptors and rate limiting factor necessary for efficient ER transactivation. We aimed to evaluate whether SRC-1 expression is an additional factor for prediction of response to first-line tamoxifen therapy in patients who developed recurrent disease. Here for the first time, we report on SRC-1 expression using a semi-quantitative RT-PCR in 21 primary breast tumors, seven mammary tumor cell-lines, 12 fibroblast cultures, and six normal breast tissues. The highest levels of SRC-1 were observed in normal tissues, intermediate levels in tumor tissues, and the lowest levels in breast tumor cell-lines. There was no relationship between the levels of SRC-1 in these primary tumors and the proportion of tumor cells within the surgical samples, nor with ER status. The median SRC-1 level was, however, lower in tumors from patients that did not respond to tamoxifen. Our findings suggest that high levels of SRC-1 indicate a favorable response to tamoxifen of patients with recurrent breast cancer. Abbreviations: PgR, progesterone receptor; ER, estrogen receptor; GR, glucocorticoid receptor; TR, thyroid hormone receptor; RXR, retinoid X receptor; SRC-1, steroid-receptor coactivator-1; EGF, epidermal growth factor; TGF, transforming growth factor; IGF, insulin like growth factor; UPA, urokinase-type plasminogen activator

Notes: Abstract Breast cancer is one of the most common cancers and is a leading cause of mortality in women. The TG.NK transgenic mouse line expresses the c-neu breast cancer oncogene under the control of an MMTV promoter and appears to be a useful animal model for evaluation of intervention strategies to delay/prevent breast cancer. Fiber-rich nonpurified diet (NTP-2000), as compared to a purified diet (AIN-76A), has previously been shown to significantly delay the development of mammary cancer in the TG.NK model. Four-week old hemizygous TG.NK female mice with MMTV/c-neu oncogene were fed NTP-2000 diet containing the retinoid analogue 4-hydroxyphenyl retinamide (4-HPR) at 5 mM/kg or an arotinoid Ro 40-8757 at 2 and 3 mmol/kg for 26 weeks. The 4-HPR at 5 mmol/kg diet delayed the development of palpable tumors up to 24 weeks, but by 26 weeks, the incidence was not significantly different from the NTP-2000 diet control group. However, the 4-HPR diet markedly decreased the average weight of the tumors at 26 weeks. The 4-HPR diet also caused a significant increase in body weight without an increase in food consumption. Arotinoid Ro-40-8757 at both doses inhibited the development of mammary tumors for the duration of the study. However, the Ro 40-8757 at 3 mmol/kg appeared to be toxic as indicated by a significant depression of the average body weight with alopecia and skin scaling in some mice. Our observations with TG.NK transgenic mouse and fiber-rich diet (NTP-2000) indicate that the arotinoid Ro 40-8757 has a markedly higher inhibitory effect on the development of mammary cancer than 4-HPR. Studies to evaluate genetic changes and expression of hormonal receptors and growth factors associated with the inhibition of mammary cancer development by the retinoid analogues are in progress.

Notes: Abstract Reconstitution of the p53-dependent apoptotic pathway by gene transfer of a recombinant wild-type p53 minigene leads to rapid apoptotic cell death in breast and other cancer cell types expressing null or mutant p53. Tumour cells expressing wild-type p53 have been reported to be more resistant to this treatment strategy, presumably as a result of mutations in downstream regulators of p53-dependent apoptotic signalling. The MCF-7 breast cancer cell line is representative of this class of tumour cell. Our recent observation of a p53-dependent apoptotic response following adenovirus-mediated HSV thymidine kinase gene transfer and gancyclovir treatment led us to reexamine recombinant p53 cytotoxicity in MCF-7 cells. Infection with a recombinant adenovirus expressing wild-type p53 resulted in a dramatic increase in p53 protein levels and was accompanied by an increase in p21WAF 1/CIP1 protein levels and G1 arrest within 24 hours post-infection. A significant decrease in MCF-7 cell viability was first observed at 5 days post-infection and coincided with the appearance of morphological and biochemical changes consistent with apoptotic cell death. By day 7 post-treatment, cell viability decreased to 45% and clonogenic survival was reduced to 12% of controls. The results demonstrate that persistent, high level expression of recombinant p53 can induce programmed cell death in MCF-7 cells. While the mechanism by which p53 overexpression overcomes the defect in downstream apoptotic signalling is not clear, our data suggests that this treatment strategy may be beneficial for the class of tumour cells represented by the MCF-7 cell line.

Notes: Abstract The plasma urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), and urokinase-type plasminogen activator receptor (uPAR) levels were measured in healthy volunteers and breast cancer patients. In pre-menopause healthy females, blood was sampled weekly during one menstruation cycle and menstruation phases (follicular, ovulatory, luteal) were determined by FSH/LH levels. uPA, PAI-1, and uPAR levels were at the nadir during ovulatory phase. uPA level was highest at follicular phase while PAI-1 level was highest at luteal phase. In comparison between pre- and post-menopause states, uPA and uPAR levels were higher in post-menopause state while PAI-1 level was higher in pre-menopause state. In breast cancer patients, uPA, PAI-1, and uPAR positive rates were low when we use the menopause-state-unmatched cut-off points. As we adjusted the cut-off points by menopause states, the PAI-1 positivity increased mainly in post-menopause cancer patients. These findings suggest that there is a minor but possible sequential change of these molecules during menstruation cycle which might blur the pathological positivity in pre-menopause cancer patients. The pathological elevation of PAI-1 was well detected in post-menopause cancer patients, but this elevation did not correlate with tumor burden such as number of metastatic sites or metastatic location. In conclusion, adjustment of physiological changes of uPA, PAI-1, and uPAR is required in determining pathological elevation of the plasma levels in cancer patients, especially in females.

Notes: Abstract Based on estrogen receptor (ER) and menopausal status, operable breast cancer (UICC stage I, II, III-a) patients were randomized for adjuvant endocrine therapy, chemotherapy, and chemoendocrine therapy, and the effects on the relapse-free survival (RFS) and overall survival (OS) were compared. Tamoxifen (TAM) 20 mg/day was administered orally for 2 years after mastectomy as an adjuvant endocrine therapy in postmenopausal patients. In premenopausal patients, oophorectomy (OVEX) was performed before TAM administration. In the chemotherapy arm (CHEM), patients were given 0.06 mg/kg of body weight of mitomycin C (MMC) intravenously, followed by an oral administration of cyclophosphamide (CPA) 100 mg/day in an administration of a 3-month period and a 3-month intermission. This 6-month schedule was repeated 4 times in 2 years. The chemoendocrine arm (CHEM + TAM) was composed of TAM with MMC + CPA chemotherapy. The patients were randomized according to ER and menopausal status. ER-positive patients were randomized to three arms: OVEX ± TAM, CHEM, and CHEM + TAM. For ER-negative patients there were two arms: CHEM and CHEM + TAM. 1579 patients entered the trial between September 1978 and December 1991, with median follow-up of 8.2 years. In ER-positive, premenopausal patients, there were no significant differences in RFS or OS among OVEX ± TAM, MMC + CPA, TAM + MMC + CPA arms. On the contrary, in ER-positive, postmenopausal patients, the chemoendocrine therapy showed a significantly higher RFS (p = 0.0400) and OS (p=0.0187) as compared with TAM to chemotherapy alone. There were no significant differences in RFS or OS by addition of TAM on the chemotherapy, in both pre- and post-menopausal ER-negative patients. It was concluded that in ER-positive premenopausal breast cancer, endocrine therapy alone may be equivalent in prolonging RFS and OS to chemotherapy or chemoendocrine therapy, and that ER-positive postmenopausal breast cancer may be better controlled with the combination of TAM and chemotherapy, as compared to TAM or chemotherapy alone. The importance of stratification of operable breast cancer by ER and menopausal status, as well as the direct comparisons of different treatments, were stressed.

Notes: Abstract Patients with an elevated level of cathepsin D in breast cancer tissue have an adverse prognosis. This study evaluated the prognostic relevance of cathepsin D detection in disseminated tumour cells in bone marrow. Bone marrow was sampled intraoperatively from both anterior iliac crests in 290 patients with primary breast cancer. Interphase cells were enhanced and stained immunocytologically with two antibodies: BM2, which detects tumour-associated glycoprotein TAG 12, which is typically expressed by almost all breast cancer cells, and the anti-cathepsin D antibody. 67 of 149 BM2-positive women (45%) developed metastatic disease (median follow-up time: 69 months). Of these, 15 were cathepsin D-positive (22%). Patients with cathepsin D-positive cells in bone marrow (n = 26; 9%) had a significantly shorter metastasis-free interval (38 months) compared with women who were cathepsin D-negative (64.5 months). The worst prognosis was seen in patients positive for both markers (30.5 months), followed by those who were cathepsin D-negative and BM2-positive (48 months). The detection of cathepsin D on disseminated tumour cells characterises a subgroup of patients with a poorer prognosis who should undergo more aggressive adjuvant systemic therapy.

Notes: Abstract This is the first report demonstrating an in vivo antitumor activity of antiprogestins (mifepristone, onapristone) alone and in combination with tamoxifen in the MCF-7 human breast cancer model. The MCF-7 cells produced progressive growing tumors in female nude mice supplemented with 17β-estradiol. Tumor regression was observed following either estrogen ablation alone or estrogen ablation in combination with tamoxifen. Monotherapy with tamoxifen or antiprogestins caused a retardation of estrogen-induced tumor progression. Complete inhibition or prevention of tumor growth occurred as a result of simultaneous administration of mifepristone and tamoxifen. The addition of mifepristone in this combination treatment was also effective in delaying or preventing tumor escape (relapse) from the antiestrogenic (antitumor) effect of tamoxifen. These results suggest a potential clinical benefit of adding an antiprogestin to antiestrogen therapy of breast cancer patients.

Notes: Abstract Neuroleptic drugs that bind sigma sites were tested for their ability to inhibit growth and radiosensitize MCF-7 human breast cancer cells. Inhibition of growth by ∼ 50% occurred in cells exposed to pimozide (0.6 μM), haloperidol (10 μM), and the sigma ligand DTG (1,3-di(2-tolyl)guanidine, 20 μM), but no growth inhibition occurred in cells exposed to clozapine, a neuroleptic drug lacking sigma binding activity, or dextromethorphan, a selective sigma 1 binding ligand. Pimozide (2.5 μM), but not haloperidol (3.6 μM), enhanced the sensitivity of MCF-7 cells to γ radiation in clonogenic survival assays. Pimozide significantly decreased MCF-7 clonogenic survival following a 5 or 8 Gy dose of γ radiation, and the dose of radiation required for 1% survival (survival enhancement ratio, SER) was decreased by a factor of 2. Exposure of normal WI-38 human embryonic lung cells to pimozide did not increase their sensitivity to γ radiation. Pimozide (2.5 μM) activated early apoptotic changes in MCF-7 cells that were detected by the uptake of Hoechst 33342 dye, and 10 μM pimozide activated a complete apoptotic pathway resulting in the death of 〉 90% of the cells within 24 hours. MCF-7 cells exposed to γ radiation alone (8 Gy) showed giant cell formation, mitotic arrest, and a limited degree of apoptosis and necrosis. Within 50 hours of treatment with a combination of radiation and pimozide, cell numbers were sharply reduced compared with cultures exposed to either radiation or pimozide alone. We conclude that pimozide augmented the sensitivity of MCF-7 cells to radiation-induced cell killing through a mechanism not shared by haloperidol, but suggest that concentration of pimozide in MCF-7 cells as a result of an enrichment of sigma 2 sites might target the radiosensitization.

Notes: Abstract The diagnostic value of a new tumor marker, c-erbB-2, was studied in the sera of 50 healthy subjects, 58 patients with benign breast diseases, and 413 patients with breast cancer (186 locoregional, 185 with advanced disease, and 42 with no evidence of disease). Using 15 U/ml as the cut-off, no healthy subjects or patients with benign diseases and only 2.4% of no evidence of disease patients had elevated serum levels. Abnormal c-erbB-2 levels were found in 29% (101/370) of the patients with breast carcinoma (locoregional 9%, metastases 45.4%). CEA (cut-off 5 U/ml) and CA 15.3 (cut-off 35 U/ml) sensitivity was 18% and 16% in patients with locoregional disease and 61% and 70% in those patients with advanced disease, respectively. A trend toward higher serum levels of all three tumor markers in patients with nodal involvement or greater tumor size was found, but was statistically significant only with CEA (p 〈 0.01). By contrast, c-erbB-2 was related to steroid receptors, in both locoregional and metastatic tumors. When the prognostic value of these markers was evaluated, patients with abnormally high presurgical CEA and c-erbB-2 had a worse prognosis than those patients with normal values, in both node-negative (p 〈 0.05 and p 〈 0.001, respectively) and node-positive patients (p 〈 0.556 and p 〈 0.001, respectively). By contrast, no relationship was found between CA 15.3 values and prognosis. Multivariate analysis showed that CEA and c-erbB-2 were also prognostic factors. The correlation between serum and tissue levels of c-erbB-2 was studied in the tumors of 161 patients. Significantly higher c-erbB-2 serum levels were found in patients with overexpression in tissue by immunohistochemistry, in both locoregional and advanced disease (p=0.0001). Serum concentrations in patients with advanced disease were related to the site of recurrence, with significantly higher values in patients with metastases (mainly in those with liver metastases) than in those with locoregional recurrence. In summary, c-erbB-2 serum levels seem to be a useful tumor marker in the prognosis of patients with breast cancer. Using all three tumor markers, sensitivity was 35% in patients with locoregional breast cancer and 88% in patients with recurrence.

Notes: Abstract Background: Kinetic resistance to cytotoxic drugs may account for the moderate responsiveness of breast cancer to chemotherapy. In the present study the in vitro effects of estradiol-mediated DNA stimulation on the cellular uptake of the DNA intercalating drug doxorubicin (DOX) were examined in MCF-7 human breast cancer cells. Methods: Using the fluorescent properties of the drug, the cellular uptake was investigated by high performance liquid chromatography (HPLC), and by flow cytometry. Results: The uptake of DOX (0.01–2 μg/ml) by MCF-7 cells in suspension, incubated for 1 and 6 h, showed a strong correlation between the incubation concentration of DOX and the cellular uptake of the drug as measured by HPLC and flow cytometry. Simultaneous exposure of MCF-7 cells, in monolayer culture, to DOX (0.04–0.2 μg/ml) and estradiol (1 nM) for 1–24 h showed no significant difference in uptake of the drug compared to control cultures exposed to DOX in the absence of estradiol. Neither was there a significant difference in uptake of DOX when MCF-7 cells were pretreated with estradiol (1 nM) for 16–24 h followed by a 0.5, 1, 6, and 21/23 h incubation with DOX (0.01–2 μg/ml). Pretreatment with estradiol did not affect the retention of DOX as measured 24 h after a 0.5 h incubation with DOX (2 μg/ml). Furthermore, fluorescence microscopy revealed no difference in the cellular DOX distribution pattern of estradiol-stimulated MCF-7 cultures compared to unstimulated cultures. Conclusion: From this study we can conclude that, for the human MCF-7 breast cancer cells in vitro, the uptake, retention, and cellular distribution of DOX are not influenced by estrogenic manipulation.

Notes: Abstract Because the proteolytic degradation of extracellular matrix is required for invasion and metastasis, it would appear that the important family of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) might be prognostic indicators of the invasive potential of a breast tumor. Nevertheless, there are few data demonstrating an independent prognostic value of any individual MMPs or TIMPs in primary breast cancer patients. It is possible, however, that the balance among levels of certain MMPs and their inhibitors will be more informative, since MMPs are clearly involved in paracrine tumor-stromal interactions and are associated with angiogenesis, which does appear to be prognostic.

Notes: Abstract IGF1 and IGF2 are circulating peptide hormones and locally-acting growth factors with both paracrine and autocrine functions. IGF1 and IGF2 signal through a common tyrosine kinase receptor, the insulin- like growth factor 1 receptor (IGF1R), and have mitogenic, cell survival, and insulin-like actions that are essential for embryogenesis, post-natal growth physiology, and breast development. The activities of IGF1 and 2 are tightly-regulated by a network of binding proteins and targeted degradation mechanisms. This complex regulatory system is disrupted in breast cancer, leading to excess IGF1R signaling. Evidence for this statement includes: a) breast cancers are infiltrated with IGF2 expressing stromal cells; b) mannose 6-phosphate/IGF2 receptor (M6P/IGF2R) is mutated in breast cancer, leading to loss of IGF2 degradation; c) IGF1R is overexpressed by malignant breast epithelial cells, and in some cases IGF1R is amplified; and d) complex changes in IGF binding protein expression occur during breast cancer progression which most likely also affect IGF1 and 2 signaling. The clinical importance of these epigenetic and genetic changes has recently been stressed by the finding that IGF1R signaling alters the apoptotic response of breast cancer cells to genotoxic stress and, in addition, IGF1R activation sensitizes cells to estrogen by inducing phosphorylation of the estrogen receptor. As a consequence of these findings, we propose that IGF analysis of breast cancer samples should shift from prognostic studies to an evaluation of IGF ligands, receptors, and binding proteins as resistance/sensitivity markers for radiation, chemotherapy, and endocrine therapy.

Notes: Abstract The present review focuses on the methodology and clinical significance of new diagnostic approaches to identify micrometastatic breast cancer cells present in bone marrow (BM), as a frequent site of overt metastases. Using monoclonal antibodies (mAbs) to epithelial cytokeratins (CK) or tumor-associated cell membrane glycoproteins, individual carcinoma cells can be detected on cytologic BM preparations at frequencies of 10-5 to 10-6. Prospective clinical studies have shown that the presence of these immunostained cells is prognostically relevant with regard to relapse-free and overall survival. The current interest in autologous bone marrow transplantation in patients with solid tumors further underlines the need for screening methods that allow the detection of minute numbers of residual tumor cells in the transplant. Although the development of new molecular detection methods based on the amplification of a marker mRNA species by the polymerase chain reaction technique is a very exciting area of research, the clinical significance of this approach needs to be demonstrated in prospective studies. The immunocytochemical assays may be, therefore, used to improve tumor staging with potential consequences for adjuvant therapy. Another promising clinical application is monitoring the response of micrometastatic cells to adjuvant therapies, which, at present, can only be assessed retrospectively after an extended period of clinical follow-up. The extremely low frequency of BM tumor cells greatly hampers approaches to obtain more specific information on their biological properties. The available data indicate that these cells represent a selected population of cancer cells which, however, still express a considerable degree of heterogeneity with regard to the expression of MHC class I antigens, adhesion molecules (EpCAM), growth factor receptors (EGF receptor, erb-B2, transferrin receptor), or proliferation-associated markers (Ki-67, p120). Regardless of the detection technique applied, there is an urgent demand for large multicentre trials, in which standardized methods are related to specified clinical outcomes.

Notes: Abstract Very few tumor markers have been recommended for routine clinical care of patients with breast cancer [1]. A framework to determine the clinical utility of tumor markers is required. In a previous publication, a "Tumor Marker Utility Grading System" (TMUGS) was proposed [2]. TMUGS included a semi-quantitative grading scale (0-3+) which can be used to assign a score to a given tumor marker for a given outcome. Only those markers that are felt to be sufficiently strong to influence a therapeutic decision that results in improved clinical outcome for the patient are recommended. The studies from which data are used to assign a TMUGS grade can be placed into one of five Levels of Evidence (LOE). An extension of TMUGS ("TMUGS-Plus") is now proposed in which the relative strength of a prognostic or predictive factor can be estimated and expressed in terms of a risk ratio (RR) for prognostic factors or benefit ratio (BR) for predictive factors. Three categories of prognostic factors and three categories of predictive factors are proposed (strong, moderate, and weak). It is recommended that only LOE type I studies (prospective, highly powered studies of the tumor marker, or meta-analysis of LOE II or III datasets), be used to estimate the RR or BR of a given factor. Finally, a matrix, based on assumptions of acceptable absolute benefits relative to risks, is proposed in which any given tumor marker can be assessed for its clinical utility. TMUGS-Plus should aid in the assessment of published data regarding clinical utility of tumor markers. Perhaps more important, clinical investigators can use TMUGS-Plus to design tumor marker studies that will fulfill criteria for clinical utility, resulting in more rapid acceptance of tumor markers for routine clinical use.

Notes: Abstract A quantitative nucleic acid hybridization assay for determination of estrogen receptor (ER) mRNA in breast carcinoma is described. The assay, which is based on the branched DNA (bDNA) technology, requires 20 mg of tissue, is simple, highly specific, and reproducible, and correlates reasonably well with an established methodology (r=0.87). The assay has a dynamic range of 3 × 103 – 6 × 107 copies of ER mRNA per well. ER message as high as 2.5 × 106 copies per well could be detected in normal breast tissues. Thus a sensitivity of 3 × 103 ER copies per well was sufficient to analyze clinical specimens. In the present studies, accurate measurement of tissue weight enabled direct reporting of the ER mRNA values as the end point results. The bDNA assay provides a useful tool for the detection and quantitation of ER mRNA in research and routine clinical laboratories.

Notes: Abstract The incidence of breast cancer in the U.S. is lower among Hispanic women than non-Hispanic white women. However, population-based studies show that Hispanic women are more likely to be diagnosed at a later stage than non-Hispanic whites. We aimed to determine whether: 1) a lower proportion of breast cancer was diagnosed at early vs. late stages in Hispanic compared to non-Hispanic white women from 1988–93 in San Diego County, and 2) lower income is related to later stage at diagnosis for both groups. All incident cases of breast cancer in San Diego County from the California Cancer Registry (10, 161 cases) were stratified by ‘early’ (in situ or localized) or ‘late’ (regional or distant) stage, and by race/ethnicity. Annual average age-adjusted incidence rates/100,000 (AAIR) were calculated. Incidence rate ratios (IRR) (AAIR for early stages divided by AAIR for late stages) were used as a surrogate of early detection. AAIRs for early and late stage disease were significantly higher for non-Hispanic whites (89.3, 42.3) than Hispanic women (46.7, 27.2). The IRR was significantly higher for non-Hispanic whites than Hispanics, (2.11 vs 1.72, p=0.01). This difference was greatest among women under 50 years old (IRR difference 0.63), and not apparent for women 65 or older (IRR difference 0.06). There was also an association between increasing census tract per capita income and higher rates of early stage disease among non-Hispanic whites but not Hispanics. Results suggest that Hispanic women and lower income women should be targeted for early detection.

Notes: Abstract Estrogen and progesterone receptors (ER and PR) have now been studied in clinical breast cancer for more than 20 years. Positive receptor status correlates with favorable prognostic features including a lower rate of cell proliferation and histologic evidence of tumor differentiation. During the first several years after diagnosis, patients with ER-positive tumors tend to have a lower recurrence rate; however, this is balanced by a higher recurrence rate in subsequent years so that the overall prognostic significance of receptor status is modest. ER and PR have their greatest utility in predicting response to hormonal therapy, both in the adjuvant setting and for advanced disease. When the assay is done properly and cut-offs for ER-negativity and positivity are defined by clinical studies of patients treated with endocrine therapy, receptor status is very helpful in identifying groups of patients who are very unlikely to benefit from hormonal therapy. Tumors that express both ER and PR have the greatest benefit from hormonal therapy, but those containing only ER or only PR still have significant responses. Two types of estrogen receptors, ERα and ERβ, have now been identified. Although there is considerable homology between these receptor forms, they appear to have important structural and functional differences that may be important for tissue and promoter- specific regulation of gene expression. These receptor forms, as well as ER variants and mutants, may also contribute to hormonal sensitivity and resistance. PR also exists in two forms, PRA and PRB. PRA appears to have repressor functions on both PRB and ERα, and the ratio of PRA to PRB in clinical breast tumors needs to be studied for its possible clinical relevance. Expression of receptor- interacting proteins can also modulate ER transcriptional activity, and these too need additional study to determine if they are markers of hormonal sensitivity or resistance. In summary, ER and PR status are important biomarkers that help physicians individualize therapy.

Notes: Abstract In the last few decades, much effort has been directed towards identifying the phenotypic or functional aspect of tumor cells which can contribute to a biofunctional staging for improving the accuracy of pathologic staging used for identifying patients at different risk. Among known biologic factors, the proliferative capacity of the tumor cell population, a feature common to all tumors, has been widely investigated. Several approaches have been used to measure different aspects of the cell cycle. Among these, the thymidine labeling index (TLI) represents the fraction of cells in S-phase cell fraction and is based on the active incorporation of labelled thymidine into DNA. From basic studies conducted on several thousands of patients, the TLI of primary breast cancers appears closely related to steroid receptor status and generally unrelated to pathologic stage. Retrospective analyses performed on large series of patients treated with local regional therapy alone have consistently shown the relevance of TLI value to clinical aggressiveness in terms of relapse-free survival and overall survival. Moreover, TLI is a prognostic indicator which is independent of tumor size, steroid receptors, and p53 and bc12 protein expression, and which, together with patient age and tumor size, is able to identify patients at different risk of loco-regional or distant metastases. Recently, a direct relationship between TLI and response to polychemotherapy has been shown in patients with operable and advanced breast cancers. This finding, derived from retrospective and recently confirmed in prospective clinical studies, has led to the activation of cell kinetics based therapeutic protocols for patients with node-negative and one to three node-positive operable breast cancers.

Notes: Abstract The prognostic and predictive value of p53 has been extensively studied in breast cancer. p53 serves a multifunctional role as a transcriptional regulator, genomic stabilizer, inhibitor of cell cycle progression, facilitator of apoptosis, and also perhaps an inhibitor of angiogenesis. Abrogation of its function should therefore lead to a more aggressive breast cancer phenotype and a worse clinical outcome, and indeed the preponderance of studies confirm this, with the risk of recurrence and death increasing by 50% or more if p53 is abnormal. Lack of unanimity of results may be due to differences in technique, study design, or population, as well as the subjectivity inherent in some approaches; however, the complexity and random nature of genomic change present in cancer cells may well also contribute to the lack of unanimity. Because many anticancer agents may exert a therapeutic effect through genomic damage and subsequent triggering of apoptosis, and because p53 can respond to genomic damage and facilitate apoptosis, it can be hypothesized that an intact p53 would predict sensitivity to therapy. Present data in breast cancer, however, does not clearly indicate that this is the case. There are several potential explanations. Study designs to accurately test the predictive value of a molecular marker are more exacting and difficult to achieve than prognostic studies. There may also be multiple alternative pathways, not involving p53, that play a part in determining the therapeutic effect of a treatment. The prognostic value of a downstream effector of p53 has also been assessed, though less extensively. p21 is transcriptionally upregulated by p53 and is an inhibitor of cyclin-dependent kinases and thus of cell cycle progression. Higher levels of p21 might indicate a more indolent type of breast cancer. However, data from a number of clinical studies is very conflicting, and at present p21 is not a promising prognostic factor in breast cancer.

Notes: Abstract Growth, progression, and metastasis of breast cancer, as well as of most of the other tumors, are angiogenesis-dependent processes. Several pro-angiogenic growth factors and endogenous inhibitors of angiogenesis have been identified and sequenced, and experimental studies suggest that angiogenic activity of a tumor may result from down-regulation of inhibitors of angiogenesis or up-regulation of endothelial growth factors. The mechanisms leading to the alteration of the balance between positive and negative modulators of angiogenesis are only partially known. We are at the beginning of research to identify the more active angiogenic factors in human breast cancer, and little information is presently available on their clinical significance. Preliminary results suggest that among the known angiogenic peptides, both vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor / thymidine phosphorylase (PD-ECGF/TP) have promising prognostic and, perhaps, predictive value. No data are available on the clinical value of co-determination of positive and negative regulators of angiogenesis to look at the angiogenic balance of each single tumor. Only a few studies have assessed the role of endogenous inhibitors of angiogenesis in human breast cancer, with results available only on thrombospondin-1 and -2 (TSP-1, -2). Finally, the determination of some integrins such as α6 and αvβ3 and of some other endothelial-adhesion molecules seems to be of potential prognostic value. Recognizing which are the more biologically active positive and negative angiogenic factors is the key for the identification not only of new prognostic markers but also of targets for antiangiogenic therapy in human breast cancer.

Notes: Abstract Some prognostic factors, such as steroid receptors, appear strongly related to outcome in early studies with short follow-up, but as follow-up matures the relationships appear to weaken. We investigated this phenomenon for several factors (tumor size, axillary lymph nodes, S-phase fraction, estrogen receptor (ER) status, and adjuvant therapy) in a large sample of breast cancer cases (N=2,873) with up to 17 years of follow-up for disease-free survival (DFS). Subjects in the study were identified from patients who had hormone receptor assays performed in our laboratory. Analysis of DFS included fitting a multivariate Cox proportional hazards model, testing for nonproportionality, and examining diagnostic plots. The assumption of proportional hazards was violated for several factors including ER, tumor size, and S-phase fraction. For ER, the hazard ratio was initially less than 1.0, indicating a good effect on prognosis, but increased at later times to values greater than 1.0, indicating a bad effect on prognosis. In contrast, the hazard ratios for tumor size and S-phase were initially high and decreased asymptotically toward 1.0 over time. Analysis of p53 expression in a subset of cases yielded qualitatively similar results. We conclude that several standard prognostic factors (ER, tumor size, S-phase fraction) and possibly other investigational factors have important but nonproportional effects on hazard. It is likely that violation of proportional hazards is common and not limited to breast cancer. Failure to recognize violations of proportional hazards can lead to both over- and under-estimation of the effects of important prognostic factors.

Notes: Abstract Background: Insulin and insulin-like growth factor I (IGF-I) are important mitogens in vitro and in vivo. It has been hypothesized that these factors may play an important role in the development of breast cancer. Methods: A case-control study comparing plasma insulin levels in 99 premenopausal women with newly diagnosed node-negative invasive carcinoma of the breast and 99 age-matched controls with incident biopsied non-proliferative breast disease (NP) was conducted. Women with known diabetes were excluded. Results: For the entire study group, mean age was 42.6 ± 5.1 years and mean weight was 62.9 ± 10.3 kg. After adjustment for age and weight, elevated insulin levels were significantly associated with breast cancer, Odds Ratio (OR) for women in the highest insulin quintile versus the lowest quintile=2.83 (95% Confidence Interval [CI] 1.22–6.58). There were no statistically significant differences between cases and controls for IGF-I and IGFBP-1 levels. However, after adjustment for age, the association between plasma levels of insulin-like growth factor binding protein 3 (IGFBP-3) and breast cancer approached statistical significance; OR for highest quintile versus lowest quintile of IGFBP-3 being 2.05 (95% CI, 0.93–4.53). All results were independent of diet and other known risk factors for breast cancer. Conclusion: Circulating insulin levels and possibly IGFBP-3 levels are elevated in women with premenopausal breast cancer. This association may reflect an underlying syndrome of insulin resistance that is independent of obesity.

Notes: Abstract Aim: To determine the effects of tamoxifen on the levels of hormone receptors and proliferation markers in the early phase of treatment and the relationship of the changes with tumor response in patients with primary breast cancer. Methods: Twenty-one women with primary, operable breast carcinomas were treated with tamoxifen 20 mg daily. Fine needle aspiration (FNA) was used to obtain samples prior to the start and at 14 days and 8-weeks post-treatment. From these samples estrogen receptor (ER), progesterone receptor (PgR), and Ki67 levels were determined using immunocytochemistry and ploidy and S-phase fraction (SPF) using flow cytometry. Tumor response was measured clinically according to UICC criteria. Results: There were 12 responders (2 CR, 10 PR) and 9 non-responders (2 NC, 7 PD). Responders were more likely to be ER + (p=0.002), PgR + (p=0.006), and low SPF (p=0.06). At 14 days post-tamoxifen, the median decrease in Ki67 (% cells staining) for responders was − 4.8 and for non-responders − 0.15 (p=0.005). This decrease was seen predominantly in ER + tumours. The difference in SPF was not significant. A decrease in ER was seen in 3/15 patients all of whom were responders. A rise in PgR was seen in 7/17 patients and all but one were responders. Similar changes for ER and PgR were seen at 8-weeks post-tamoxifen, although the reductions in Ki67 and SPF at that time point were not related to response. Conclusion: We have observed a decrease in Ki67 and ER and a rise in PgR after 14 days of treatment with tamoxifen that was related to subsequent response. This is the first study in which an early decrease in a proliferation marker has been shown to relate to subsequent clinical response.

Notes: Abstract Previous studies revealed that prostate-specific antigen (PSA) is present in 〉 30% of human breast tumor cytosols. Survival analysis showed that patients with PSA-producing tumors have a reduced risk for relapse, suggesting PSA to be an independent favorable prognostic marker for a large subset of breast cancer patients. The present investigation established an in vivo model for the induction of PSA in human breast cancer tumors growing as xenografts in severe combined immunodeficient (SCID) mice. The human mammary cancer cell-line T47D was grown i.m. in female mice. When the tumor and leg diameter reached 10 mm, the mice were stimulated daily with norgestrel for either 5 or 7 days to produce PSA, and sacrificed on day 8. The prostate cancer cell-line LNCaP was grown in male mice and functioned as a positive control for PSA production. After T47D and LNCaP mice were sacrificed, a highly sensitive immunofluorometric assay was used to analyze the PSA concentration in the tumor, muscle, liver, and kidney cytosols. Norgestrel-stimulated T47D mice showed significantly more PSA in the tumors compared to tumors of the control mice. However, PSA levels in tumors of the stimulated mice were significantly lower than those in the LNCaP xenografts. No PSA levels above background were present in the blood and normal tissue of the norgestrel-stimulated or control T47D xenografts. This mouse model will be a valuable tool for investigating and screening new therapies for a subgroup of breast cancer patients who have significant PSA concentrations in their tumors.

Notes: Abstract Hypoxic tumor cells may represent a fraction of cells that are not susceptible to radiation or chemotherapy. Intratumoral oxygen partial pressure (pO2) is the result of oxygen delivery and consumption. Cell proliferation is one factor to effect oxygen consumption and we therefore studied the correlation between tumor pO2 and histological parameters. Patients and methods: In 36 women and one man (age range 29–80 years) with suspected breast cancer. Before tumor resection, intralesional pO2 was determined with a polarographic needle electrode. Under ultrasound control, 200 tumor measurements were obtained; Hb levels, Hk, arterial blood gas parameters, and tissue temperature were determined. The median of pO2 values and the percentage of hypoxic areas (pO2 〈 10 mmHg) were calculated and correlated with the histological type, grading, ER, PR, and the expression of Ki-67, p53, EGFR, pS2, and c-erb-B2. Results: The overall median pO2 was 44 mmHg, and 1024 measurements (13.8%) represented hypoxic areas. Ductal and lobular invasive cancers showed median pO2 of 41 mmHg. The mean pO2 of G1 tumors was 59 mmHg and the hypoxic fraction 8%, in contrast to G2 tumors with 43 mmHg and 17%, and G3 tumors with 36 mmHg and 20.4% (p 〈 0.01). We observed a correlation with tumor size and an increased rate of hypoxic areas in T3–4 lesions (p 〈 0.02). Also tumors with negative nodes or positive ER had significantly higher pO2 values, as did tumors with an overexpression of c-erb-B2, p53, and cathepsin D. Conclusion: Oxygenation of human breast cancers can safely be measured in patients prior to surgical therapy. pO2 values correlate both with prognostic markers examined histologically and with molecular growth factors. As the efficacy of preoperative or adjuvant treatment in individuals may depend on oxygen partial pressure, efforts to manipulate tumor pO2 for therapeutic purpose could be promising.

Notes: Abstract Previous work from our laboratory demonstrated aneuploidy for several chromosomes by interphase fluorescence in situ hybridization (FISH) in a high proportion of breast cancer specimens. In the literature, only limited data are available concerning chromosome 8 anomalies in breast cancer. To determine chromosome 8 ploidy status in primary and metastatic specimens from 81 breast cancer patients, FISH analysis with a DNA probe recognizing chromosome 8 centromeres was performed. In all primary tumor specimens (n=30), significant proportions of cells were aneuploid exhibiting gain of chromosome 8 copy numbers; in 75% of effusion specimens previously classified as malignant by cytology and/or FISH for various chromosomes (n=40), cell populations aneuploid for chromosome 8 were detected; effusions previously classified non-malignant (n=11) were diploid in 10 cases, whereas one specimen contained rare hyperdiploid cells. Among these cells complex chromosomal aneuploidy could be demonstrated by two-color FISH, suggesting malignancy. Trisomic and tetrasomic clones were predominant in the majority of samples, but a marked intratumor cytogenetic heterogeneity was observed in most cases. Primary tumors and corresponding positive axillary lymph nodes revealed similar distributions of chromosome 8 copy numbers, analogous to previous findings with other chromosomes. This implies that, by using suitable FISH probes after examination of the respective primary tumor, an efficient search for (micro)metastasis might be feasible.

Notes: Abstract Many genetic abnormalities disclosed even in somatic cells like peripheral blood lymphocytes may mark footprint(s) of malignancy(ies). The present cytogenetic study on peripheral blood lymphocytes of sporadic breast cancer patients (n = 20) and their first degree relatives (n = 39) reports abnormalities of chromosomes 16, 5, 12, and 17 respectively in 17.59%, 8.33%, 6.48%, and 5.57% cells of patients and 15.83%, 8.33%, 7.5%, and 5% cells of their first degree relatives. These common chromosomal abnormalities pave the way to assume why first degree relatives of sporadic breast cancer patients are at increased risk of developing the same or other malignancies.

Notes: Abstract The phenomenon of tumor angiogenesis is an important aspect of understanding tumor biology. Studies in breast carcinoma have shown microvessel density (MVD) assessed by immunohistochemistry to be of prognostic importance in primary breast cancer. On the other hand, recently developed highly sensitive color-coded Doppler techniques offer a noninvasive method to examine neovascularisation in breast tumors. The purpose of this study was to determine the relationship between Doppler flow parameters and microvessel count assessed by immunohistochemistry. Fifty-three patients with primary breast cancer were examined preoperatively with color-coded Doppler ultrasound. The obtained Doppler frequency spectra were analyzed for peak systolic flow velocity (Vmax). Following surgery, paraffin-embedded microsections were immunohistochemically stained for factor VIII-related antigen. Tumor angiogenesis was assessed by microvessel count under light microscopy. Undifferentiated tumors correlated with high MVD (p=0.009) whereas other clinicopathological parameters were not associated with MVD. Color Doppler signals were detected in 50 out of 53 breast tumors. Evaluation of tumor flow velocity with various clinicopathological parameters showed a significant correlation with tumor size (p=0.0001) and lymph node metastasis (p=0.02). However, there was no significant correlation between MVD and intratumoral blood flow velocity assessed by color-coded Doppler. Our findings showed that Doppler flow measurement did not correlate with the extent of tumor angiogenesis of breast cancer. The present data give circumstantial evidence that microvessel count assessed by immunohistochemistry reflects the microvascular network, whereas tumor vasculature documented by Doppler ultrasound supplies information on the macrovasculature.

Notes: Abstract One hundred and six patients with operable breast cancer were studied at intervals one day before surgery, and at six weeks and one year post-operatively by means of taped clinical interviews and self-report questionnaires (Impact of Event Scale (IES) and General Health Questionnaire (GHQ-28)). A year after surgery, 9%reported a high level of intrusive symptoms and 10% of avoidance symptoms compared to 18% and 14% after six weeks, respectively. Based on questionnaire data, a year after surgery, thirteen patients (12%) were estimated to have a posttraumatic stress disorder (PTSD) compared to fifteen patients (14%) after six weeks. Severity of posttraumatic stress symptoms (PTSS) after one year was significantly associated with impaired psychosocial functioning over the last year previous to surgery (p 〈 0.05), negative life events during the year before surgery (p 〈 0.05), health problems during the previous ten years (p 〈 0.01), and a personality trait characterized by high emotional reactivity (p 〈 0.001). Crisis support in the acute situation, type of surgery, axillary-node metastases, and postoperative adjuvant chemotherapy did not predict subsequent PTSS. Premorbid health variables, personality, and level of distress six weeks after surgery were most important risk factors for persistent PTSS in our patients with operable breast cancer stage I and II.

Notes: Abstract Local environmental signals regulate the growth and development of both normal and malignant breast epithelium. Members of the insulin-like growth factor (IGF) family likely influence both of these processes. The localization of IGF2 to stroma specifically surrounding malignant breast epithelium indicates that this growth factor may play a critical role in the genesis or maintenance of this transformed phenotype. Recent studies have sought to understand the mechanism by which IGF2 expressing fibroblasts are localized to the periphery of malignant breast cancer cells. In addition, the consequences of the expression of IGF-signaling components likely expand beyond their direct effects on mitogenesis. Indirect effects predominantly associated with the IGF2 receptor could also influence the invasive potential of breast tumor cells.

Notes: Abstract The effects of antiestrogens, tamoxifen and ICI 182,780, and aromatase inhibitors, arimidex (anastrozole ZD1033) and letrozole (CGS 20,267), on the growth of tumors were studied in nude mice. In this model, estrogen dependent MCF-7 human breast cancer cells stably transfected with the aromatase gene were inoculated in four sites per mouse. Sufficient estrogen is produced from aromatization of androstenedione supplement (0.1 mg/mouse/day) by the cells to stimulate their proliferation, tumor formation, and maintain the uterus similar to that of the intact mouse. Once the tumors reached a measurable size, the mice were injected with antiestrogen or inhibitor for 35–56 days. Tumor volumes were measured weekly. At autopsy, the tumors were removed, cleaned, and weighed. Statistical data was determined from tumor weights. Both antiestrogens were effective in reducing tumor growth in these mice. Tamoxifen appears to be more effective than ICI 182,780, although the former stimulated the uterine weight whereas the pure antiestrogen did not. However, both aromatase inhibitors were more effective than the antiestrogens. Tumor regression was observed with letrozole. Thus, after-treatment tumor weights were less than those of a group of mice at the start of treatment. The aromatase inhibitors also reduced the weight of the uterus, suggesting that these compounds, as well as the pure antiestrogen, may not cause endometrial proliferation, unlike tamoxifen. These aromatase inhibitors may not only benefit patients who have relapsed from tamoxifen, but may be more effective in patients as first line agents for suppressing the effects of estrogen.

Notes: Abstract To determine whether canine malignancies share common genetic lesions with their human counterparts, and are thus potentially interesting model systems in which to pose questions regarding tumor etiology and progression, we have elucidated the entire exon/intron structure of the canine p53 gene. A search for p53 gene abnormalities in mammary tumor tissue was undertaken utilizing single strand conformation polymorphism analysis. Mutations were detected in exons 4, 5, 6, and 7 of the p53 gene and consisted of nonsense, splicing, and frameshift mutations. None of 11 benign tumors and 6 of 40 primary carcinomas (15%) were found to harbor subtle p53 mutations. In 14 carcinomas examined the results in primary tumors and metastases were the same. These findings implicate involvement of this gene in the genesis of some malignant canine tumors, in a fashion similar to their human counterparts.

Notes: Abstract The typical high fat, low fibre diet of the industrialised West, particularly when associated with inadequate exercise, is likely to advance the onset of puberty. This will manifest in girls as an earlier menarche, earlier onset of breast development, and an earlier growth spurt. Both earlier menarche and adult tallness are markers of increased risk to breast cancer. Earlier menarche in the West is usually associated with earlier onset of hyperinsulinaemia, and multiple case-control studies report that hyperinsulinaemia too is a marker of increased breast cancer risk. Although the Western diet is linked both to earlier menarche and also to earlier hyperinsulinaemia, the mechanism involved is not necessarily the same. While menarche is likely to be triggered by a threshold level of fatness, manifestation of insulin resistance is genetically-determined and strongly influenced by the fatty acid profile of the diet. The putative mechanisms by which they influence mammary carcinogenesis also differ. Early menarche is reported to be associated with a raised oestradiol level persisting into early adult life. On the other hand, hyperinsulinaemia is commonly associated with abnormal aromatase activity in the ovaries. In addition, the concomitant increase in bioactive levels of insulin-like growth factor-I may synergise with oestrogen in stimulating proliferative activity in mammary epithelium. Dietary modification and exercise regimens are proposed in families at high risk to breast cancer. The measures have been shown to reduce insulin levels in both children and adults, and serial monitoring of insulin and sex steroid levels could be used to detect a metabolic-endocrine effect.

Notes: Abstract High-dose therapy followed by peripheral blood stem cell (PBSC) support was performed in 29 patients with primary high-risk (Group I) or chemoresponsive metastatic (Group II) breast cancer patients. Group I patients had received PBSC mobilization within 4 weeks of modified radical mastectomy. Group II patients had to achieve minimal residual disease (MRD) by induction chemotherapy before being considered eligible for PBSC mobilization and high-dose therapy. An innovative FE120C regimen (5-FU 600 mg/m2, i.v., day 1; epirubicin 120 mg/m2, i.v., day 1; cyclophosphamide 600 mg/m2, i.v., day 1) plus G-CSF (300 μg/day, subcutaneous injection for 9 days, from day 4 post-FE120 C) was used to mobilize PBSCs. After high-dose CTCb (cyclophosphamide 6,000 mg/m2, thiothepa 500 mg/m2, carboplatin 800 mg/m2, in 4 days), patients received PBSC infusion and daily C-CSF 300 μg sub cutaneous injection. There were 19 and 16 patients enrolled into Group I and Group II, respectively. Ten of the Group II patients had achieved minimal residual disease (MRD) after induction chemotherapy. The median numbers of mobilized total CD34 + cells for Group I and Group II patients were 27.3 (9.2 to 114.1) × 106/kg and 17.1 (5.9 to 69.1) × 106/kg respectively. The median time to neutrophil recovery (ANC ≥ 500/μL) was 8 and 9 days in Group I and II, respectively. The median time to platelet recovery (≥ 50,000/μL) was 10 and 15 days in Group I and II, respectively. No major treatment-related toxicities were noted. In Group I, 13 out of 19 patients (68.4%; 43–87%, 95% C.I.) remained recurrence-free with a median follow-up of 31 months (6 + to 55 + months). In Group II, 3 out of 10 patients (30%; 7–65%, 95% C.I.) remained progression-free at 33 +, 35 +, 39 + months from induction therapy. We suggest that the FE120C plus G-CSF is an effective and innovative regimen for PBSC mobilization in breast cancer patients, and high-dose CTCb therapy with PBSC support is a safe and well-tolerated treatment modality.

Notes: Abstract Aromatase activity is increased in breast tumors and it is possible that this results from stimulation by cytokines and/or prostaglandin E2 (PGE2) which regulate the activity of this enzyme. As different promoters can be used to control aromatase gene expression in normal and malignant breast tissues, which are regulated by different factors, we are currently investigating the relative roles of cytokines and PGE2 in controlling breast tumor aromatase activity. No significant effect of PGE2 on aromatase activity in MCF-7 breast cancer cells has so far been detected. However, preliminary evidence was obtained, from co-cultures of MCF-7 cells and breast tumor-derived fibroblasts, that MCF-7 cells secrete a factor, possibly a cytokine, which can act in a paracrine manner to enhance aromatase activity in stromal cells. Understanding the complex regulation of aromatase activity in breast tumors could lead to novel therapies for specifically inhibiting tumor estrogen synthesis.

Notes: Abstract Evidence to support the contention that estrogen biosynthesis in breast cancers is of clinical significance has been sought by relating activity to (i) clinical response to aromatase inhibitors and (ii) tumor concentrations of estrogens. Significant correlations have been reported between the presence/high levels of tumor aromatase in vitro and likelihood of response to aminoglutethimide in patients with advanced breast cancer, but the association is not absolute and it has been more difficult to demonstrate similar relationships in patients with earlier stages of cancers treated with other more potent inhibitors. There are however data to suggest that in vitro measurements of aromatase may not reflect in situ estrogen synthesis. For example mammary adipose tissue fibroblasts preincubated with reversible aromatase inhibitors may paradoxically display elevated in vitro aromatase activity. Similar enhanced in vitro activity may be observed in breast material taken from patients treated neo-adjuvantly with aromatase inhibitors such as aminoglutethamide and letrozole. That this is an artifact of in vitro systems can be demonstrated by performing in situ assessments of aromatase activity in patients before and after treatment with aromatase inhibitors. Thus it can be shown that letrozole markedly inhibits in situ aromatase and reduces tumor levels of estrogens.

Notes: Abstract The plasma levels of free estradiol are very low in postmenopausal women. However, concentrations of estrogens within breast tissue have been reported to be higher than in plasma and similar to plasma concentrations in premenopausal women. One mechanism by which this may occur is for breast cells to synthesize estrogens themselves and produce high concentrations locally. Thus, tumor aromatase may be a significant source of estrogen which stimulates tumor growth. To address the question of the importance of this pathway, we have investigated the expression of aromatase within the normal breast and breast cancers. Because conventional biochemical assays for measuring aromatase activity require relatively large amounts of tissue, we developed an immunocytochemical method using a monoclonal antibody to determine the expression of aromatase. The method can be applied to sections of tumors embedded in paraffin blocks as routinely prepared for pathology. Since we have previously shown that mRNA for aromatase (P450 arom) and the protein are expressed in the same cells of the human placenta, we used in situ hybridization of sequence specific probes to P450 arom mRNA in breast tissue as one method to verify the specificity of the immunocytochemical detection of the enzyme. Both immunocytochemistry and in situ hybridization identified aromatase enzyme and mRNA expression in the epithelial cells of the terminal ductal lobula units (TDLU) and surrounding stromal cells of the normal human breast, and in the tumor epithelial cells and stromal cells of breast cancers. In addition, evidence for the functional significance of tumor aromatase was indicated by a correlation between aromatase activity and expression of proliferating cell nuclear antigen (PCNA) in the tumor, and by increased thymidine incorporation into DNA in response to testosterone in tumors in histoculture which had high aromatase activity but not in those with low activity. The findings suggests that estrogen produced locally is important in enhancing proliferation of the tumor.

Notes: Abstract Lovastatin, a fungal antibiotic used in the treatment of hypercholesterolemia, is an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the key regulatory enzyme in the mevalonate pathway of cholesterol synthesis. We examined the antitumor properties of lovastatin on the F3II sarcomatoid mammary carcinoma, a highly invasive and metastatic murine tumor model. Female BALB/c inbred mice were inoculated subcutaneously with F3II tumor cells and injected i.p. daily with 10 mg/kg body weight of lovastatin or administered p.o. at a level corresponding to the human dosage of 1–2 mg/kg/day. Treatment significantly prolonged tumor latency and reduced tumor formation and metastatic dissemination to the lungs from established mammary tumors. In vitro, antitumor properties of lovastatin were strongly associated with inhibition of tumor cell attachment and migration. These actions were prevented by addition of mevalonate but not by equivalent concentrations of farnesyl pyrophosphate. In accordance, Western blot assays showed that lovastatin effects did not appear to be related to modifications in Ras oncoproteins in our model. The present data indicate that lovastatin could be an antitumor agent with potentially useful clinical applications in breast cancer.

Notes: Abstract This review summarizes the cytogenetic information on benign breast lesions of various histologies, i.e., fibrocystic lesions from women with and without a known hereditary predisposition to breast cancer, fibroadenomas, phyllodes tumors, and papillomas, and relate the chromosomal features with those in breast carcinoma. In general, the frequency of chromosome abnormalities is lower in benign lesions than in breast cancer, and seems to correlate with the histologic features of the tissue, and the corresponding risk of developing invasive mammary carcinoma; aberrations are more common in proliferative than in nonproliferative lesions. The karyotypes are generally less complex than those detected in invasive carcinoma, and more often involve balanced rearrangements. No lesion-specific aberration has so far been detected; on the contrary, changes repeatedly encountered in breast cancer samples can be found in benign lesions as well, e.g., gain of 1q, interstitial deletion of 3p, and trisomies 7, 18, and 20. Especially intriguing is the prevalence of rearrangements of the short arm of chromosome 3, with the minimally deleted bands 3p13–14, in proliferative lesions from prophylactic mastectomies in breast cancer families. The potential tumor suppressor gene(s) in this region remains, however, to be identified.

Notes: Abstract A clearer picture of the role of adrenal androgens in the etiology of breast cancer is beginning to emerge. Women who develop breast cancer in premenopausal years tend to have subnormal serum levels of adrenal androgens, while subjects who develop the disease in postmenopausal years have supranormal levels of these hormones. Androgens, by acting via the androgen receptor, oppose estrogen-stimulated cell growth in premenopausal years. In postmenopausal women, elevated adrenal androgen levels stimulate cell growth by the action of the unique adrenal androgen 5-androstene-3β,17β-diol, also termed hermaphrodiol, via its combination with the estrogen receptor in a hormone milieu lacking, or having low concentrations of, the classical estrogen 17β-estradiol.

Notes: Abstract We first recall the observational and theoretical facts that constitute the so-called 3He problem. We then review the chemical anomalies that could be related to the destruction of 3He in red giants stars. We show how a simple consistent mechanism can lead to the destruction of 3He in low mass stars and simultaneously account for the low 12C/13C ratios and low lithium abundances observed in giant stars of different populations. This process should both naturally account for the recent measurements of 3He/H in galactic HII regions and allow for high values of 3He observed in some planetary nebulae. We propose a simple statistical estimation of the fraction of stars that may be affected by this process.

Notes: Abstract Tumour markers correlate strongly with prognosis based on tumour burden and surgical resectability. If chemotherapy is extremely effective in certain stage of the disease, the sensitive marker may be of great use in monitoring disease response and drug treatment. Hence, this study was launched to evaluate the changes in tumour marker enzymes like lactate dehydrogenase (LDH), glumate oxaloacetate transaminase (SGOT), glutamate pyruvate transaminase (SGPT), alkaline phosphatase, and acid phosphatase in before and after 3 and 6 months tamoxifen treated breast cancer patients. In addition, the changes in serum glycoproteins viz., hexose, hexosamine, and sialic acid and lysosomal enzymes such as N-acetyl-beta-D-glucosaminidase, beta-D-galactosidase, and beta-D-glucuronidase were analysed in these patients. These values were compared with their age matched healthy control subjects. At 6 months evaluation, the tamoxifen treated postmenopausal breast cancer women showed a statistically significant decreased (p 〈 0.001, 0.05 respectively) levels of LDH, SGOT, SGPT, alkaline and acid phosphatases than their baseline values. Similarly, the levels of hexose, hexosamine, and sialic acid and N-acetyl-beta-D-glucosaminidase, beta-D-galactosidase, and beta-D-glucuronidase were decreased significantly (p 〈 0.001 ) in tamoxifen received postmenopausal women. The result of this study suggested that tamoxifen potentially retard the metastasis of breast cancer as well as the bone demineralisation in postmenopausal breast cancer women. Thus, tamoxifen may also have its antitumour activity through its beneficial effects on tumour marker enzymes and serum proteins in breast cancer women.

Notes: Abstract This study examined the helping process that occurred when 26 breast cancer patients (the disclosers) talked about their illness-related concerns with their partner and, in a separate conversation, with a fellow patient (the volunteer helpers). The conversations were rated by trained observers and by the disclosers in terms of several process and outcome variables. From the observers' perspective, the volunteer helpers were more helpful, empathic, and supportive, less critical, and used more self-disclosure than the partners. Disclosers did not differentiate between the two types of helper, and gave generally high ratings to both conversations. Strengths and weaknesses of each type of helper were identified. Findings are discussed in relation to the literature on formal and informal helping, and implications for training nonprofessional helpers are suggested.

Notes: Abstract A Phase II study of paclitaxel in patients with primary advanced or metastatic breast cancer was conducted by a cooperative study group consisting of 16 institutions in Japan. Paclitaxel at a dose of 210 mg/m2 was intravenously infused over 3 hours, along with premedication to prevent hypersensitivity reactions. The course was repeated at 21-day intervals. Of 62 eligible patients, 60 were evaluable for toxicity and 59 were evaluable for efficacy. Forty-five patients were previously treated with anthracyclines. Twenty-one of 59 patients (35.6%) had a major objective response including 2 CRs and 19 PRs (95% confidence interval, 23.6–49.1%). A response rate of 35.5% (CR1, PR10) was observed in 31 patients refractory to the anthracyclines containing prior metastatic chemotherapy. Median (range) time was 41 (6–100) days to onset of and median duration of response was 125 (36–305) days. Toxicities included leukopenia (grade 3, 4: 67%), anemia (grade 1–3: 80%), thrombocytopenia (grade 1: 8%), alopecia (grade 3: 43%), peripheral neuropathy (grade 1–3: 93%), arthralgia (59%), myalgia (46%), nausea and vomiting (40%), fever (33%), allergic reaction (grade 3: 2%) and hypotension (grade 3: 5%). All toxicities were tolerable and manageable. Paclitaxel intravenously infused over 3 hours demonstrated a significant antitumor activity for metastatic breast cancer. Furthermore, paclitaxel exhibited non-cross resistance to anthracycline. Paclitaxel administered as a convenient 3-hour infusion is effective for patients with metastatic breast cancer and has an acceptable toxicity profile.

Notes: Abstract New human breast cell lines were developed from metastatic breast cancer tissues and normal breast tissues. Primary cultures were initiated from cellular outgrowths of explanted tissues or from mechanically isolated cells in two serum-free media. Cell cultures derived from both cancer and normal tissues were immortalized with pRSV-T plasmid to generate permanent breast cell lines that exhibited an epithelial morphology. Cell lines generated in this study were characterized with respect to morphology, growth rate, karyotype, presence of specific genes, and the expression of epithelial and breast markers. The cell lines expressed the epithelial cell markers, cytokeratins 8 and 18, and retained the capacity to produce human milk fat globulin. They also express the BRCA-1, erbB2, and EGF receptor genes and possess the H-ras, K-ras, and p53 genes. Preliminary data showed that one of the new cancer cell lines was highly sensitive to the cytotoxic action of taxol. It is envisioned that the new breast cell lines will be useful as targets for identification of therapeutic agents against breast cancer and as models for carcinogenesis studies.

Notes: Abstract As a model system for the identification of genes involved in the progression of human breast cancer, differential gene expression in cell lines MCF-7 and MCF-7ADR was investigated. The latter cell line is derived from the former. Cell line MCF-7 is estrogen receptor-positive, vimentin-negative and uninvasive in the Matrigel outgrowth assay and in the nude mouse, while MCF-7ADR is estrogen receptor-negative, hormone-resistant, vimentin-positive, invasive in the Matrigel outgrowth assay and in the nude mouse and resistant to adriamycin due to overexpression of glycoprotein gp170. We have shown that tumor progression in this model system is mediated by transcriptional regulation of mitochondria-related genes, proteases, transmembrane receptors and cell cycle-related gene proteins. Among the genes differentially regulated at the transcriptional level in the cell lines MCF-7 and MCP-7ADR are a new mitochondrial transcript, mitochondrial creatine kinase, matrix metalloproteinase-1, stromelysin-3, urokinase and its receptor, tissue factor, E-cadherin, epidermal growth factor receptor, transmembrane proteins Mat-8 and progression associated protein (PAP), cyclin E, cyclin-dependent kinase-2 and cell cycle inhibitory proteins p16, p21 and p27.

Notes: Abstract A new murine cell line, named MG1361, was established from mammary adenocarcinomas arising in a MMTV-neu transgenic mouse lineage where breast tumors develop in 100% of females, due to the over-expression of the activated rat neu oncogene in the mammary gland. The MG1361 cell line shows an epithelial-like morphology, has a poor plating efficiency, low clonogenic capacity, and a doubling time of 23.8 hours. Karyotype and flow cytometry analysis revealed a hypotetraploid number of chromosomes, whereas cell cycle analysis showed 31.2% of cells to be in the G1 phase, 21.4% in S and 47.4% in G2 + M. This cell line maintains a high level of neu expression in vitro. The MG1361 cell line was tumorigenic when inoculated in immunodeficient (nude) mice and the derived tumors showed the same histological features as the primary tumors from which they were isolated. MG1361 cells were positive for specific ER and PgR binding which was competed by tamoxifen, making this cell line useful for the evaluation of endocrine therapy. Moreover, they were sensitive to etoposide treatment, suggesting that they could be a model for the study of chemotherapy-induced apoptosis. As the tumors arising in MMTV-neu transgenic mice have many features in common with human mammary adenocarcinomas (Sacco et al., Gene Therapy 1995; 2: 493–497), this cell line can be utilized to perform basic studies on the role of the neu oncogene in the maintenance of the transformed phenotype, and to test novel protocols of therapeutic strategies.

Notes: Abstract Experimental evidence suggests an important role of the type I IGF receptor (IGF-IR) in breast cancer development. Breast tumors and breast cancer cell lines express the IGF-IR. IGF-IR levels are higher in cancer cells than in normal breast tissue or in benign mammary tumors. The ligands of the IGF-IR are potent mitogens promoting monolayer and anchorage-independent growth of breast cancer cells. Interference with IGF-IR activation, expression, or signaling inhibits growth and induces apoptosis in breast cancer cells. In addition, recent studies established the involvement of the IGF-IR in the regulation of breast cancer cell motility and adhesion. We have demonstrated that in MCF-7 cells, overexpression of the IGF-IR promotes E-cadherin-dependent cell aggregation, which is associated with enhanced cell proliferation and prolonged survival in three-dimensional culture. The expression or function of the IGF-IR in breast cancer cells is modulated by different humoral factors, such as estrogen, progesterone, IGF-II, and interleukin-1. The IGF-IR and the estrogen receptor (ER) are usually co-expressed and the two signaling systems are engaged in a complex functional cross-talk controlling cell proliferation. Despite the convincing experimental evidence, the role of the IGF-IR in breast cancer etiology, especially in metastatic progression, is still not clear. The view emerging from cellular and animal studies is that abnormally high levels of IGF-IRs may contribute to the increase of tumor mass and/or aid tumor recurrence, by promoting proliferation, cell survival, and cell-cell interactions. However, in breast cancer, except for the well established correlation with ER status, the associations of the IGF-IR with other prognostic parameters are still insufficiently documented.

Notes: Abstract The aim of this study was to test the prognostic contribution of estrogen (ER) and progesterone (PgR) receptor status to an index consisting of the number of positive lymph nodes, the mean nuclear area of the breast cancer cells (MNA), and tumour diameter. This index is compared with a Danish index, which includes the same factors but uses histological grade instead of MNA. The Danish index has been developed from the Nottingham Prognostic Index (NPI). In the present study of 1629 breast cancer patients the Cox proportional hazard method is used to examine the time-dependency of the index, and to test for interaction between the index and the hormone receptors. The index sorts the patients into groups with low, intermediate, and high risk of dying. Logistic regression analysis is used to report the sensitivity and specificity of the index with and without ER and PgR. Our index gave information comparable to that of the Danish group. However, the information given by our index is time-dependent, its strength being weaker after 5-year of follow-up. PgR and ER add information to high risk patients, but only in the first 5-year period. High risk patients with positive hormone receptors have a prognosis similar to intermediate risk ones. PgR increases the ability of the index to predict breast cancer deaths correctly by 5 percent in high risk patients. In conclusion, PgR and ER act differently in groups of patients with different risk levels when time-dependency is considered. This indicates biological differences in subgroups as defined by the index.

Notes: Abstract Local recurrence rates play an important role in the evaluation and judgement of local treatment modalities in breast cancer. In the medical literature different methods for the calculation of these rates have been used. In this note we discuss the methods, the observed differences, and the interpretation of the resulting estimates.

Notes: Abstract From May 1975 until May 1980, 128 operable breast cancer patients, clinical stage I–II, had a core bone marrow biopsy (BMB) from the posterior iliac crest as a part of the routine diagnostic work-up at the time of initial diagnosis. The mean age of the patients was 56 years, range 26–93. In a previous study on this material, 10 patients (7.8 per cent) were positive for tumor cells and 118 negative by conventional histopathology of BMB [1]. In 1996 we reexamined all BMB separately at two laboratories, using monoclonal antibodies against cytokeratins AE1—AE3, KL1, CAM 5–2 (DOP), and DC10, BA17 (MCI). The number of extrinsic cells in the bone marrow was graded positive for micrometastases when ≥ 5 cells or suspicious when 1–4 cells per ∼2 × 106 bone marrow cells were found, using high power field magnification. Micrometastases were detected in 17 patients (13.3 per cent) and another 8 patients were classified as suspicious. The presence of micrometastases was correlated to the axillary lymph node stage and primary tumor location. Median follow-up was 20 years. All 17 micrometastatic patients relapsed and died within 6 years of disease progression with evident osseous metastases. There was one disease-free survivor of the 8 patients with suspicious BMB after 17 years of follow-up. The median overall survival was significantly shorter in tumor-cell positive patients, being 1.9 years compared to 11.7 years in the BMB negative and BMB suspicious groups (p 〈 0.0001). Immunohistochemical analysis of core BMB taken postoperatively may be useful in predicting the prognosis in patients with breast cancer clinical stage I–II.

Notes: Abstract Background: It is important to reduce local residual cancer to avoid local recurrence after breast conserving treatment. We therefore tried to detect the Backgroundintraductal components and small invasive foci of breast cancers by contrast-enhanced helical computed tomography (CE-CT). Methods: In 122 women whose breasts were examined by CE-CT preoperatively, intraductal spread detected on ultrasound (US), mammography (MMG), and CE-CT, and extensive intraductal components (EICs) detected by histological examination were analyzed for correlations among the extent and subtypes of intraductal components, and deviations in tumor size. Results: EICs were present in 44 patients. The sensitivities of EIC detection by US, MMG, and CE-CT were 35%, 61%, and 88%, respectively, and the corresponding specificities were 83%, 86%, and 79%, respectively. The sensitivities of detecting EIC and small invasive foci were 34%, 57%, and 91%, respectively. In 5 patients, EIC could only be visualized by CE-CT. The median deviation of the size of intraductal spread revealed by CE-CT from pathological EIC was 0.0 cm (range + 3.0 to − 1.7 cm. Conclusions: CE-CT is useful for visualizing intraductal spread and small invasive foci of breast cancer.

Notes: Abstract Mammography has become the mainstay of breast cancer screening. However, widespread mammography has led to an increase of the number of breast biopsies done for benign disease. Therefore, a method to better discriminate benign from malignant lesions is needed. Fractal analysis is a mathematical method which can quantify complex shapes. It has been previously shown retrospectively that the composite fractal dimensions, D, of malignant mammographic masses is higher than for benign lesions. A prospective study of 75 patients who were recommended to undergo needle localized breast biopsy by independent radiologists had the composite D calculated. Fractal analysis was done without knowledge of the biopsy results. The mean composite D of malignant lesions was higher than benign lesions, 2.545 ± 0.067 vs. 1.936 ± 0.144 (p=0.00004). Calculation of a receiver-operating characteristic curve showed that a cutoff value of 2.067 had a 100% sensitivity and 63% specificity (i.e., false positive rate of 37%). Mean D for fibroadenomas was 2.087 ± 0.054, fibrocystic disease was 1.877 ± 0.167, DCIS was 2.261 ± 0.069, and invasive cancer was 2.634 ± 0.039 (1-way ANOVA, p=0.00007). These data imply that fractal analysis may be beneficial in discriminating between benign and malignant lesions. However, further study in a larger number of patients with a variety of lesions is needed.

Notes: Abstract The presence of fibratic fows (FF) in infiltrating ductal carcinoma (IDC) has been shown to be an important histological factor associated with high tumor aggressiveness, or early tumor recurrence or death. However, the clinicopathological significance of FF for predicting the long-term survival of the patients with IDC has not been fully investigated. In order to elucidate this aspect, we divided 140 IDCs with at least 10 years of follow up into tumors with FF and those without. IDC with FF showed significantly higher histologic grade (P=0.02), higher frequency of tumor necrosis (P=0.02), higher frequency of cases with more than three positive lymph node metastases (P=0.04), higher T classification (P=0.009), and higher pathological stage (P=0.0002) than those without FF. Relative risk (RR) of tumor recurrence and death was significantly higher in tumors with FF than in those without (RR=4.5, P 〈 0.00001 and RR=5.6, P 〈 0.00001, respectively). In cases of early stage cancer (stages I, IIA, and IIB), or in those with less than four lymph node metastases, IDCs with FF demonstrated a significantly higher risk than those without. Multivariate adjustments for other pathological factors did not change the RRs significantly. These results indicate that in long-term follow up the presence of FF is a significant prognostic parameter for IDC, and therefore strongly suggest that IDCs must be divided into those with and without FF.

Notes: Abstract Estradiol stimulates the growth of breast tumor cells in both pre- and post menopausal women. Following the menopause, the levels of estradiol in breast tumor tissues are similar to those from tumors obtained prior to cessation of ovarian function, even though plasma estrogen levels are 10–50 fold lower in post- than in premenopausal women. These observations suggested the possibility of enhanced estradiol uptake from plasma or in situ synthesis in post-menopausal women. We systematically studied these possibilities in a series of model systems. Initially we demonstrated a very high affinity estradiol binding site in tissues from castrated rats. Enhanced uptake occurred under conditions of low plasma estrogen levels when compared to animals with higher estradiol levels. In situ synthesis also occurred both through the sulfatase and aromatase pathways. In further studies, we compared uptake from plasma with in situ synthesis via aromatase in a nude mouse model. Under the conditions utilized, in situ synthesis resulted in much higher tissue estradiol levels and tumor growth rates than did uptake from plasma. During these studies we demonstrated that tumors deprived of estradiol developed mechanisms rendering them more sensitive to estrogen. This involved the ability of cells to adapt to estradiol deprivation to allow them to be responsive to four log lower amounts of estrogen than when studied under wild type conditions. In addition, cells adapted by increasing their level of aromatase and thus developing the capability to become more sensitive to estrogen precursors. Taken together, these studies demonstrate that breast cancer tissue is highly plastic and can adapt to conditions of estrogen deprivation via a variety of mechanisms.

Notes: Abstract A definition of the theoretical components of an ideal aromatase inhibitor is developed, one aspect of which is completeness of enzyme inhibition. The three triazole inhibitors, letrozole, vorozole and anastrozole all inhibit whole body aromatisation by 〉 96% at their clinically used doses and vorozole and letrozole were more effective in Phase III clinical trials than aminoglutethimide (250 mg bd) which achieves 〈 90% inhibition. The possibility is considered that the apparent small differences between the triazoles may be associated with differences in clinical effectiveness. These new compounds merit consideration for studies of breast cancer prevention.