Vaginal estriol to overcome side-effects of aromatase inhibitors in breast cancer patients.

OBJECTIVE: Aromatase inhibitors are essential as endocrine treatment for hormone receptor-positive postmenopausal breast cancer patients. Menopausal symptoms are often aggravated during endocrine treatment. We investigated whether vaginal estriol is a safe therapeutic option to overcome the urogenital side-effects of aromatase inhibitors. Serum hormone levels were used as the surrogate parameter for safety. METHODS: Fasting serum hormone levels of ten postmenopausal breast cancer patients receiving aromatase inhibitors were prospectively measured by electro-chemiluminescence immunoassays and gas chromatography/mass spectrometry before and 2 weeks after daily application of 0.5 mg vaginal estriol (Ovestin® ovula), respectively. RESULTS: Two weeks of daily vaginal estriol treatment did not change serum estradiol or estriol levels. However, significant decreases in levels of serum follicle stimulating hormone (p = 0.01) and luteinizing hormone (p = 0.02) were observed. Five out of six breast cancer patients noticed an improvement in vaginal dryness and/or dyspareunia. CONCLUSIONS: The significant decline in gonadotropin levels, indicating systemic effects, has to be kept in mind when offering vaginal estriol to breast cancer patients receiving an aromatase inhibitor.

Factors influencing the identification rate of the sentinel node in breast cancer.

Sentinel node biopsy is a widely accepted alternative to primary axillary lymph node dissection for ipsilateral nodal assessment in breast cancer. We have performed a retrospective chart review in 713 consecutive patients with primary, operable breast cancer who underwent sentinel node biopsy in order to identify factors that determine the sentinel node identification rate. Chi-squared test, univariate and multivariate analyses were used to evaluate the influence of different factors on the sentinel identification rate. Among the factors investigated, tumour size was correlated with sentinel lymph nodes detection rates (multiple logistic regression, P= 0.002). In addition, the patient's age showed to be a significant influencing factor (multiple logistic regression, P= 0.006). Body mass index and grade only exhibited a significant correlation with the identification rate in the univariate (P= 0.041, P= 0.025), but not in the multivariate analysis (P= not significant). All associations were found to be independent of the site of injection. Interestingly, surgeons with intermediate expertise (11-20 prior dissections) had the highest detection rates (P= 0.004). We conclude that sentinel identification rates are higher in larger tumours and in younger patients, independent of the injection site. Surgical experience in sentinel node dissection is not linearly correlated with higher identification rates.

Proteomics in mammary cancer research.

During the past few years, the intensified detection of small (mammary) carcinomas causes an increase in the number of mammary cancers. Cancer of the mammary tissues has an almost individually unpredictable behavior and aggressiveness. Therefore, a better insight in the molecular biological defects, which are responsible for initiation and progressive aggressiveness of mammary cancer, is necessary. Proteomics are an alternative to identify proteins which initiate carcinogenesis and can be useful to predict cancer prognosis. Today, the most commonly used technique for large-scale protein identification in clinical samples is two-dimensional electrophoresis (2-DE) in combination with image analysis and MS. Using these techniques, qualitative and quantitative information can be achieved regarding protein forms and post-translational modifications. In the following article we review proteomic techniques that are now commonly used in order to elucidate the role of proteins in breast cancer.

Prevalence of pre-malignant and malignant lesions in prophylactic mastectomy specimens of BRCA1 mutation carriers: comparison with a control group.

PURPOSE: BRCA1 mutation carriers are at high risk for breast cancer (BC). The risk management strategy may include radiological investigations for early detection or prophylactic mastectomy (PM). For a mutation carrier, PM may be more significant than surveillance alone when pre-malignant and malignant changes occur increasingly in mastectomy specimens, given normal findings on radiological investigations. In the present study we retrospectively investigated the differences between histological findings in PM specimens of BRCA1 carriers and those of a control group. METHODS: Twenty-four healthy and 28 affected carriers in the presence of normal preoperative radiological findings were included in the study. To compare the frequency of pre-malignant and malignant lesions in PM specimens, a control group matched for age and disease status was included. T-tests for independent samples and Wilcoxon's signed-rank test were used for comparison of groups. RESULTS: The entire study group differed significantly from the control group (42.3 vs. 5.8%; P < 0.001) in terms of the occurrence of pre-malignant and malignant lesions. Both, the sub-group comparison of healthy mutation carriers as well as diseased carriers with their controls, showed a significant difference in terms of the occurrence of pre-malignant and malignant changes (45.8 vs. 0%; P = 0.002; 39.3 vs. 10.7%; P = 0.03). In PM specimens of mutation carriers, carcinomas were identified in 5.8% (3/52) and pre-malignant changes in 36.5% (19/52). CONCLUSIONS: BRCA1 mutation carriers should be informed of the fact that pre-malignant and even malignant changes are frequently found in PM specimens despite normal radiological findings.

Estetrol, a pregnancy-specific human steroid, prevents and suppresses mammary tumor growth in a rat model.

Estetrol (E4) is a pregnancy-specific D-ring metabolite of estradiol (E2) and estriol (E3) produced by the human fetal liver and present in both male and female fetuses. In adults, female exposure is restricted to the gestational period. We report that E4, dose-dependently, prevents the growth of chemically induced (7,12-dimethylbenz(a)anthracene, DMBA) mammary tumors in female Sprague-Dawley rats and that E4 has the potential to reduce the number and size of pre-existing mammary tumors. We performed two prevention studies and one intervention study. In the prevention studies, we investigated the effect of oral doses of E4 over a dose range of 0.5-3.0 mg/kg. The intervention study used oral dose levels of 1, 3 and 10 mg/kg E4. The antiestrogen tamoxifen was used as reference compound in all three studies and ovariectomy and ethinylestradiol, at estrogenic doses pharmacologically equipotent to E4, acted as control treatments in the second prevention study and in the intervention study. Rats treated with DMBA develop estrogen-responsive breast tumors. This model has become the standard pharmacological model to investigate the effect of new compounds on breast tumors. When DMBA-induced rats were co-treated with E4 for 8 weeks, this resulted in a dose-dependent reduction in the number and size of tumors, an effect that appeared equally effective as tamoxifen treatment or ovariectomy and was not seen with ethinylestradiol. When E4 was administered to rats in which tumors had already developed, a significant decrease in the number and size of tumors was observed after 4 weeks. This decrease was dose-dependent, comparable to tamoxifen-treated animals, and at high dose levels E4 was as effective as ovariectomy.

Intratumoral IGF-I protein expression is selectively upregulated in breast cancer patients with BRCA1/2 mutations.

BRCA1/2 mutations predispose to early onset breast and ovarian cancers. The phenotypic expression of mutant alleles, however, is thought to be modified by factors that are also involved in the pathogenesis of sporadic breast cancer. One such protein is IGF-I, one of the strongest mitogens to breast cancer cells in vitro. We have utilized immunohistochemistry to compare the intratumoral IGF-I and IGF-I receptor (IGF-IR) protein expression in 57 BRCA1/2 mutation carriers and 102 matched breast cancer patients without a family history in a nested case-control study. BRCA1 silencing by siRNA was used to investigate the effect of BRCA mutations on IGF-I protein expression. IGF-I protein expression was detected in tumoral epithelium and surrounding stroma, and was significantly upregulated in tumors of BRCA mutation carriers when compared with matched sporadic tumors (epithelial: 87.7% vs 61.8%, P=0.001; stromal: 73.7% vs 34.3%, P<0.001). By contrast, IGF-IR protein expression was confined to malignant epithelium and was unchanged in mutation carriers (52.6% vs 39.2%, P=0.310). While in mutation carriers IGF-IR protein expression was significantly correlated with both epithelial (P=0.003) and stromal IGF-I (P=0.02), this association was less pronounced in sporadic breast cancer (P=0.02 respectively). siRNA-mediated downregulation of BRCA1 in primary human mammary gland cells triggered upregulation of endogenous intracellular IGF-I in vitro. The increased intratumoral IGF-I protein expression in BRCA mutation carriers suggests an involvement of the IGF-I/IGF-IR axis in the biological behavior of breast cancers in this population and could define a potential therapeutic target.

Safety of tibolone in the treatment of vasomotor symptoms in breast cancer patients--design and baseline data 'LIBERATE' trial.

Many patients with a history of breast cancer (BC) will suffer from vasomotor symptoms, which can be induced or exacerbated by treatment with tamoxifen or aromatase inhibitors. The LIBERATE trial was designed as a randomized, double-blind, multicenter trial to demonstrate that tibolone 2.5mg/day (Livial) is non-inferior to placebo regarding BC recurrence in women with vasomotor symptoms surgically treated for primary BC within the last 5 years. Secondary objectives are effects on vasomotor symptoms as well as overall survival, bone mineral density and health-related quality of life. Mean age at randomization was 52.6 years, and the mean time since surgery was 2.1 years. The mean daily number of hot flushes and sweating episodes was 7.3 and 6.1, respectively. For the primary tumor, Stage IIA or higher was reported for >70% of the patients. In subjects whose receptor status was known, 78.2% of the tumors were estrogen receptors positive. At randomization, tamoxifen was given to 66.2% of all patients and aromatase inhibitors to 7%. Chemotherapy was reported by 5% at randomization. The adjuvant tamoxifen use in LIBERATE allows a comparison with the Stockholm trial (showing no risk of BC recurrence associated with hormone therapy), which was stopped prematurely subsequent to HABITS. The LIBERATE trial is the largest, ongoing, well-controlled study for treatment of vasomotor symptoms in BC patients.

Efficacy of primary systemic chemotherapy regimen containing anthracyclines with/without taxanes in comparison to CMF in women with early breast cancer.

Neoadjuvant chemotherapy is the treatment of choice for locally-advanced breast cancer and leads to down staging and improved breast-conserving therapy (BCT) rates. While its efficacy is well established, considerably less is known about the most effective regimen. We have performed a retrospective analysis of 132 breast cancer patients who had undergone neoadjuvant chemotherapy at our institution. Patients had either received a) anthracyclines ("A", n=35), b) anthracyclines and taxanes ("AT", n=55), or c) neither of the two compounds ("NoA/T", n=42). Clinical response, pathological response and survival were evaluated in each arm. While all three regimens resulted in significant tumor regression, AT was most effective with a mean tumor shrinkage of 39% (ultrasound) and 41% (mammography) (Kruskal-Wallis, p=0.004, and p=0.027). Breast conservation was achieved in 75% by AT, in 49% by A, and in 19% by NoA/T (Kruskal-Wallis, p<0.001). The treatment groups did not differ in respect to pathological complete response (pCR) (chi2-test, p=0.068), although higher cumulative anthracycline doses were predictive of pCR in multivariate analyses (p=0.022). While the mammographic but not the ultrasound-determined tumor diameter determined whether a woman underwent BCT, only an ultrasound-determined size reduction was predictive for disease-free survival (DFS) and overall survival (OS) (log rank, p=0.0093, and p=0.044, respectively). Other parameters that affected BCT rates were age (p= 0.003), year of diagnosis (p=<0.001), presence of multifocal disease (p= 0.032) and the cumulative anthracycline dose (p= <0.001). While the combination of anthracyclines and taxanes is most effective in achieving clinical remission and BCT, the cumulative anthracycline dose appears most important for achieving pCR.

Jun and Fos family protein expression in human breast cancer: correlation of protein expression and clinicopathological parameters.

OBJECTIVES: The activator protein-1 (AP-1) is a dimeric transcription factor formed by members of the Jun and Fos protein family. AP-1 plays a role in a variety of physiological functions including cell proliferation and differentiation, although both c-Jun and c-Fos have also been implicated in oncogenic transformation and tumor progression. To further elucidate the role of AP-1 in breast cancer, we have investigated the expression of the AP-1 proteins c-Jun, JunB, JunD, phosphorylated c-Jun, c-Fos, Fral, Fra2 and the tumor supressor protein p53. METHODS: Protein expression was evaluated on a breast cancer tissue microarray with 58 lymph node positive or negative breast cancer specimens, 29 corresponding lymph node metastases, and 11 tissue samples from surrounding tumor-free tissue, each cored as triplicate. Jun and Fos protein family expression was evaluated by immunohistochemistry and was correlated with clinicopathological parameters. RESULTS: High expression levels were observed for c-Jun, JunD, c-Fos and Fra2, whereas JunB and Fral exhibited lower staining. c-Jun protein expression was correlated to Fral staining (p = 0.007, Kendall's Tau) and Fral was further associated with c-Fos (p < 0.001), JunD (p = 0.001) and Fra2 (p = 0.011) expression. JunD expression correlated with c-Fos (p < 0.001), JunB (p = 0.035) and c-Jun (p = 0.05). Activated c-Jun correlated with c-Fos expression (p = 0.041). JunB was negatively correlated to tumor stage, (p = 0.093, corr coeff. = -0.293, Spearman's correlation) but was significantly increased in nodal negative tumors (p = 0.004, Mann Whitney test). In addition, increased Fral expression showed a trend towards an increased overall survival (p = 0.077, RR = 0.534, Cox regression). CONCLUSION: Our results suggest an important role for JunB and Fral in the biological behavior of malignant breast tumors.

Enzyme immunoassay and Scatchard plot estimation of estrogen receptor in gynecological tumors.

Tissue samples of three endometrial carcinomas, seven ovarian carcinomas, and 24 mammary carcinomas were analyzed for estrogen receptor (ER) by enzyme immunoassay (EIA) and a conventional dextran-coated charcoal (DCC) method. In addition, ER and progesterone receptor were assayed by DCC only in 68 ovarian carcinoma specimens. All three endometrial cancer specimens showed elevated ER values by both assays. As with mammary cancers the ER-EIA values tend to be higher than DCC values. It was intriguing to note that negative Scatchard plot data resulted in residual ER levels in the EIA system. Also four ovarian cancer specimens with negative ER values by the DCC assay had detectable levels by ER-EIA, and three of these four had ER-EIA values less than or equal to 10 fmol/mg of protein. Of the ten breast cancers with negative DCC values, seven were less than or equal to 10 fmol/mg of protein by the ER-EIA. Good correlation (r = 0.88) between EIA and Scatchard plot data was calculated from ER data of 24 mammary carcinoma tissue samples. Receptor assays in 68 ovarian cancer patients indicate that ER determinations should become a useful tool in the management of patients bearing this carcinoma. In addition, receptor determinations may improve the possibility of predicting which well differentiated Stage I ovarian carcinomas are likely to recur. Present data combine to suggest that ER-EIA may become a useful diagnostic laboratory tool.

HER-2 amplification, steroid receptors and epidermal growth factor receptor in primary breast cancer.

Amplification of HER-2 oncogene was analysed in DNAs obtained from 291 primary human mammary carcinomas. 52/291 (18%) were found to contain amplified HER-2 oncogene. Moderate amplification (2- to 5-fold) was noted in 36/291 (12%). Thirteen tumors (4.5%) had a copy number of 5 to 10. A 10- to 20-fold and greater than 20-fold amplification was observed in 2 and 1 patient, respectively. Sample sizes allowed the determination of estrogen receptor (ER) and progesterone receptor (PgR) levels in 253/291 primary breast cancers. HER-2 gene amplification was noted in 14% of ER+ patients and in 28% of ER- patients, respectively (P = 0.02). Similarly a significantly greater number of PgR- primary mammary carcinoma exhibited an amplification of the HER-2 gene compared to PgR+ cases (22% vs. 16%, P = 0.01). Although statistically not significant, tumors with HER-2 gene amplification were found to have lower levels of ER and PgR. No association of HER-2 amplification with the androgen receptor and EGF receptor was observed. Present data combine to suggest that tumor progression is more stringently controlled by the oncogene upon loss of hormone dependency. Differences found in HER-2 amplification between steroid receptor positive and negative tumors could be helpful to define a specific subset of women to whom adjuvant therapy should be directed.

Determination of HER-2/neu amplification and expression in tumor tissue and cultured cells using a simple, phenol free method for nucleic acid isolation.

A rapid, simple and non-toxic procedure for the simultaneous isolation of DNA and RNA from tumor tissue and cells grown in vitro is described. Guanidinium isothiocyanate was used for homogenization of tumor tissue and for cell lysis. Separation of proteins, DNA and RNA was carried out by isopycnic centrifugation in cesium trifluoroacetate. DNA was further purified by salting out residual protein. Nucleic acids prepared by this method from 47 primary human carcinomas and 17 human cell lines were analysed for amplification and expression of the HER-2/neu proto-oncogene. 2- to 10-fold amplification of HER-2/neu was noted in 7/22 mammary carcinomas (32%) and in 4/14 ovarian carcinomas (28%). No amplification of the proto-oncogene was found in 4 laryngeal carcinomas, 1 pharyngeal carcinoma, 2 retrolingual carcinomas, 3 gastric carcinomas and 1 kidney carcinoma. HER-2/neu overexpression was observed in 6/22 of mammary carcinomas (27%) and 7/14 of ovarian carcinomas (50%). No overexpression was found in all other carcinomas studied. Concordance between amplification and overexpression was noted in 3 mammary and 4 ovarian carcinomas, respectively. 3 mammary and 3 ovarian carcinomas showed overexpression without amplification. 5 human mammary carcinoma cell lines showed both amplification and overexpression of HER-2/neu. In two mammary carcinoma cell lines (MDA MB-453 and ZR 75-1) overexpression was noted without amplification of the proto-oncogene. These data combine to suggest that mechanisms other than gene amplification may also lead to overexpression of the HER-2/neu protooncogene in cancer cells.

Randomized trial of low-dose chemotherapy added to tamoxifen in patients with receptor-positive and lymph node-positive breast cancer.

PURPOSE: To evaluate the outcome in patients with stage II hormone receptor-positive breast cancer treated or not treated with low-dose, short-term chemotherapy in addition to tamoxifen in terms of disease-free and overall survival. PATIENTS AND METHODS: A total of 613 patients were randomized to receive either low-dose chemotherapy (doxorubicin 20 mg/m(2) and vincristine 1 mg/m(2) on day 1; cyclophosphamide 300 mg/m(2); methotrexate 25 mg/m(2); and fluorouracil 600 mg/m(2) on days 29 and 36 intravenously) or no chemotherapy in addition to 20 mg of tamoxifen orally for 2 years. A third group without any treatment (postmenopausal patients only) was terminated after the accrual of 79 patients due to ethical reasons. RESULTS: After a median follow-up period of 7.5 years, the addition of chemotherapy did not improve the outcome in patients as compared with those treated with tamoxifen alone, neither with respect to disease-free nor overall survival. Multivariate analysis of prognostic factors for disease-free survival revealed menopausal status, in addition to nodal status, progesterone receptor, and histologic grade as significant. Both untreated postmenopausal and tamoxifen-treated premenopausal patients showed identical prognoses significantly inferior to the tamoxifen-treated postmenopausal cohort. Prognostic factors for overall survival in the multivariate analysis showed nodal and tumor stage, tumor grade, and hormone receptor level as significant. CONCLUSION: Low-dose chemotherapy in addition to tamoxifen does not improve the prognosis of stage II breast cancer patients with hormone-responsive tumors. Tamoxifen-treated postmenopausal patients show a significantly better prognosis than premenopausal patients, favoring the hypothesis of a more pronounced effect of tamoxifen in the older age groups.

Messenger RNA determination of estrogen receptor, progesterone receptor, pS2, and plasminogen activator inhibitor-1 by competitive reverse transcription-polymerase chain reaction in human breast cancer.

Estrogen receptor (ER), progesterone receptor (PR), the estrogen-inducible protein pS2, and plasminogen activator inhibitor-1 (PAI-1) are important prognostic factors in primary breast cancer. The protein concentrations of these factors in breast tumors have been well documented. However, few data about the mRNA expression of ER, PR, pS2, and PAI-1 in breast cancer are available, which is mostly due to the limitations of conventional techniques for mRNA analysis. We have described a competitive reverse transcription-PCR system for the simultaneous quantification of ER, PR, pS2, and PAI-1 mRNA in tumor samples. Here, we evaluated 100 tumor biopsies from breast cancer patients for the mRNA expression of ER, PR, pS2, and PAI-1. The results were analyzed for correlations with protein status and with clinical data. Significant correlations between mRNA expression levels and protein concentrations of all tested markers were found. In only a few cases was there an obvious discordance between the measurable amounts of mRNA and protein, especially for ER and PR. In addition, ER, PR, and pS2 mRNA levels correlated significantly with each other. No correlation between PAI-1 mRNA amount and the expression of the other markers was found. With respect to clinical data, ER and PR mRNA levels were found to be inversely correlated to tumor size and histological grade but not to the lymph node status. pS2 and PAI-1 mRNA expression were not correlated with tumor size, grade, or lymph node involvement. In conclusion, competitive reverse transcription-PCR may be used as an alternative for the study of prognostic factors in human breast cancer and other malignancies. However, before mRNA expression is measured for diagnostics, a presumed concordance of mRNA and protein expression must be evaluated very carefully for every gene.

Evaluation of the United States Food and Drug Administration-approved scoring and test system of HER-2 protein expression in breast cancer.

PURPOSE: The purpose of this study was to investigate the prognostic significance of assessment of human epidermal growth factor receptor (HER)-2 oncogene protein overexpression of breast cancer tissue by the United States Food and Drug Administration (FDA)-approved HercepTest and grading system (negative, 0 or 1+; weakly positive, 2+; strongly positive, 3+). Furthermore, results of the HercepTest were correlated with immunohistochemical results obtained using different antibodies and protocols and with HER-2 oncogene gene amplification assessed by fluorescence in situ hybridization (FISH). EXPERIMENTAL DESIGN: HER-2 status in 303 patients with lymph node-positive breast cancer was investigated by using a rabbit polyclonal antibody (DAKO) by conventional immunohistochemistry and by applying the HercepTest. Furthermore, the monoclonal antibody CB-11 was used in conventional immunohistochemistry and with the NexES automatic stainer, which is also under consideration for FDA approval for determination of eligibility for Herceptin therapy. Results were compared with FISH analysis performed in all 2+ and 3+ specimens (103 cases) and 104 HER-2-negative specimens. RESULTS: 3+ positive carcinomas were found in 8.9-15.7% of specimens. FISH revealed that almost exclusively 3+ positive cases were amplified, with the HercepTest and the NexES automatic stainer giving the best results. In univariate analysis, staining with the HercepTest revealed a significantly worse prognosis in 3+ cases. Also, 3+ cases were significantly associated with lower estrogen receptor levels and histological grade III tumors. CONCLUSIONS: This study shows that the results of the FDA-approved HER-2 grading and test system correlated strongly with findings in FISH. Furthermore, HercepTest proved to be of prognostic relevance. Strict adherence to the given protocols is critical.

Expression of cathepsin D in head and neck cancer.

To determine overexpression of cathepsin D in head and neck tumours we examined cytosols from 53 primary tumours, nine cytosols of lymph node metastases and 12 cytosols from adjacent normal tissue. We found a significantly lower concentration in normal tissue compared with tumour cytosol as well as with metastases, even when we compared tumours and corresponding metastases pairwise. In addition, we found a significantly higher concentration of cathepsin D in five lymph node metastases than in the corresponding tumours. We conclude that the reported role of cathepsin D is not restricted to breast cancer but could also be important in head and neck cancer.

Very low-dose adjuvant chemotherapy in steroid receptor negative stage I breast cancer patients. Austrian Breast Cancer Study Group.

A randomised clinical trial was performed to test whether or not low-dose chemotherapy lasting only 35 days improves the outcome of breast cancer patients with stage I disease and negative oestrogen and progesterone receptors (ER-, PgR-). Between 1984 and 1990, 277 stage I breast cancer patients with tumours negative for both oestrogen and progesterone receptors were randomised to receive either low-dose short-term chemotherapy or no chemotherapy. Chemotherapy consisted of one cycle of doxorubicin, vincristin (AV) and one cycle of cyclophosphamide, methotrexate, fluorouracil (CMF). Patients were stratified for tumour stage, type of surgery, menopausal status and participating centre. Results were analysed both by univariate and multivariate statistical. After a median length of follow-up of 84 months, disease-free (DFS) and overall survival (OS) did not differ significantly between patients having received adjuvant chemotherapy and the control group. Uni- and multivariate analysis did not show any significant prognostic or therapy related factor. A low-dose short-term adjuvant chemotherapy is insufficient to improve the prognosis of patients with breast cancer stage I with ER-, PgR-tumours.

Prognostic significance of mutations in the p53 gene, particularly in the zinc-binding domains, in lymph node- and steroid receptor positive breast cancer patients. Austrian Breast Cancer Study Group.

The aim of our study was to evaluate if p53 mutations, especially those in the L2/L3 domains of the p53 gene, add prognostic information for node-positive and steroid receptor positive breast cancer patients. Two hundred and five tumour samples from a randomised clinical trial of 596 lymph node- and steroid receptor positive breast cancer patients were included. All patients had been randomly allocated to receive 20 mg of adjuvant tamoxifen (TAM) daily for 2 years or TAM plus one cycle of low-dose, short-term chemotherapy. For detection of p53 mutations we used in vitro amplification by polymerase chain reaction and consecutively performed temperature gradient gel electrophoresis (PCR-TGGE) and direct sequencing. We found p53 mutations in 42/205 (20%) cases: 16/42 (38%) p53 mutations occurred within the L2/L3 domains of the p53 gene, and 26/42 (62%) outside the L2/L3 domains. p53 mutation served as a statistically significant parameter in predicting disease-free survival in univariate (P = 0.02) and multivariate (P = 0.009) analysis. For overall survival, no significant differences were observed. Patients with tumours that had p53 mutations within the L2/L3 domains of the gene showed no significant difference to those with mutations outside the L2/L3 domains for disease-free survival. For overall survival, mutations in the L2/L3 domains showed a marginally significant difference (P = 0.05) in multivariate analysis, but not in univariate analysis (P = 0.13). We conclude that mutation in the L2/L3 domains of the p53 gene is not an independent prognostic indicator of disease outcome for patients suffering from breast cancer with lymph node metastases and positive steroid receptors.

Use of 99mTc-furifosmin scintigraphy--planar and SPECT--to evaluate suggestive palpable and nonpalpable breast lesions.

UNLABELLED: Our objective was to evaluate the role of 99mTc-furifosmin scintigraphy--planar and SPECT--in discriminating benign from malignant breast disease. METHODS: The trial was prospective, open, and diagnostic. We recruited 30 consecutive patients with 14 palpable and 16 nonpalpable breast lesions. After receiving informed consent, we injected 555-640 MBq 99mTc-furifosmin intravenously in the arm contralateral to the breast lesion. Planar imaging and SPECT were performed. All patients underwent excision of the tumor within 2 wk. Using histology as the gold standard, we calculated sensitivity, specificity, and positive and negative predictive values for 99mTc-furifosmin in planar and SPECT technique. RESULTS: For 18 malignant and 12 benign breast lesions, a sensitivity of 50% for planar imaging and 72% for SPECT was seen. Specificity and positive and negative predictive values were 83%, 82%, and 53%, respectively, for planar imaging and 50%, 68%, and 55%, respectively, for SPECT. For the 14 palpable tumors (10 malignant, 4 benign), which averaged 17+/-10 mm in size (size range, 4-45 mm), a sensitivity of 60% for planar imaging and 80% for SPECT was achieved. Sixteen lesions were not palpable (median size, 9+/-3 mm [size range, 4-13 mm]). In this subgroup, 99mTc-furifosmin scintigraphy yielded a sensitivity of 37% for planar and 62% for SPECT technique (P>0.05). CONCLUSION: 99mTc-furifosmin scintigraphy is not a potent competitor to established scintigraphic procedures. In comparing this tracer with 99mTc-sestamibi and 99mTc-tetrofosmin, we cannot recommend 99mTc-furifosmin for the diagnosis of breast cancer.

Growth inhibition by dehydrothyrsiferol - a non-Pgp modulator, derived from a marine red alga - in human breast cancer cell lines.

The novel marine terpenoid dehydrothyrsiferol (DHT) has been isolated from a Canarian red alga Laurencia viridis sp. nov (Ceramiales, Rhodomelaceae) (1). Its cytotoxicity against three human breast cancer cell lines, namely T47D, ZR-75-1, and Hs578T was examined and compared with the chemotherapeutic compound doxorubicin and the mitosis-inhibitor colchicine. Primary breast carcinomas exhibit MDR1 gene expression (3). As the investigated mammary cell lines did not exhibit rhodamine 123 efflux we proved in a P-glycoprotein (Pgp) overexpressing human epidermoid cancer cell line that the marine metabolite does not modulate Pgp mediated drug transport. Therefore, it could be used in Pgp expressing cancer cells without interference.