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Length-weight relationships (LWRs) are pivotal for comparative life-history studies, conservation strategies and ecosystem modelling among regions. They provide essential information on the growth, fitness and wellbeing of a population in an ecosystem. Length and weight relationships and descriptive statistics for eight Chondrichthyes, caught off the Madeira archipelago between 2004 and 2019 from depths ranging from 350 to 2500 m, are herein reported. A total of 767 specimens was studied and it was observed that the parameter b(relative growth rate) oscillated between 2.558 for males of Centrophorus squamosusand 3.494 for females of Etmopterus princeps. This study is the first to include the LWRs for these 8 Chondrichthyes species in Madeira waters.

A list of phytoplankton taxa off Madeira Island (Northeast Atlantic) is provided, including historical records dating back to 1845. A total of 473 taxa was reported, with diatoms representing the main component of the phytoplankton community around Madeira Island with 55%, followed by the dinoflagellates with 33%, the haptophytes with 11%, cyanophytes with 1% and one planktonic chlorophyte. Compared to species numbers of the World Ocean, where diatoms equals more or less dinoflagellates, a discrepancy is observed. This could be due to the still scarce number of taxonomic studies dealing with phytoplankton. However, it provides a baseline for further studies of the regional marine phytoplankton community composition. It also constitutes valuable basic information for future assessments within management instruments like the European Water Framework Directive, the Marine Strategy Framework Directive and national regulations concerning marine food safety in light of first reports of Harmful Algae Blooms and related intoxications.

The ability to predict tumor sensitivity toward radiotherapy may significantly impact the selection of patients for preoperative combined-modality therapy. The aim of the present study was to test the predictive value of Polo-like kinase 1 (PLK1) in rectal cancer patients and to investigate whether PLK1 plays a direct role in mediating radiation sensitivity. PLK1 expression was evaluated by immunohistochemistry (n= 76) or Affymetrix HG133 microarray (n= 20) on pretreatment biopsies of patients with advanced rectal cancer. Expression was correlated with both tumor regression in the resected specimen and long-term clinical outcome. Furthermore, we used small interfering RNAs (siRNAs) to down-regulate PLK1 expression in colorectal cancer cells and analyzed the effects of PLK1-specific siRNAs by Western blot and quantitative real-time PCR analysis, FACScan analysis, caspase 3/7 assays, and colony-forming assays. We observed that increased PLK1 protein expression was significantly related to a poorer tumor regression and a higher risk of local recurrence in uni- and multivariate analysis. A significant decrease of PLK1 expression by siRNAs in combination with ionizing radiation induced an increased percentage of apoptotic cells and increased caspase 3/7 activity. Furthermore, enhanced G2-M levels, decreased cellular viability, and reduced clonogenic survival were demonstrated, indicating a radiosensitizing effect of PLK1 depletion. Therefore, PLK1 may be a novel predictive marker for radiation response as well as a promising therapeutic target in rectal cancer patients.

Soil compaction affects physical soil condition, in particular aeration, soil strength, and water availability and has adverse effects on plant growth. Bulk density is the most frequently used indicator to describe the state of compaction of a soil. However, this parameter lacks a direct functional relationship with plant growth. Various indicators have been proposed to simultaneously characterize the state of compaction of agricultural soil and its suitability for plant growth. This paper examines and compares the critical limits for crop plant growth based on three of these indicators: packing density, least limiting water range, and S parameter the latter is the slope of the soil waterretention curve in the inflexion point. In a first step, we reviewed the literature for published optimum and limiting values of bulk density and found that these values were highly dependent on clay and silt content. Converting them into corresponding values of packing density composite index of bulk density and clay content, a value of 1.70 was found to effectively distinguish between optimum and limiting soil conditions for plant growth. In a second step, the packing density of 59 soil horizons sampled in N Switzerland was compared with the least limiting water range and the S parameter of these soil horizons both determined by means of pedotransfer functions taken from the literature. A linear relationship between the three parameters was found, which allowed for a comparison of the published critical limits for plant growth based on these parameters. The critical limits of the three indicators, which had been postulated independently of each other in the literature, were found to agree well with each other. This means that all of them could equally be used to describe the compaction state of a soil and its physical suitability for plant growth. However, the proposed critical limits of packing density, least limiting water range, and S parameter still need further validation by field studies relating plant growth to soil compaction.

Human polo-like kinase 1 (Plk1), a key regulator of mitosis, is over-expressed in various human tumors. It is a negative prognostic factor for cancer patients and a measure for the aggressiveness of a tumor. Thus, targeting Plk1 might be a promising approach for cancer therapy. Kinase inhibitors are divided in type I inhibitors, targeting the highly conserved active conformation, and the more selective type II inhibitors, targeting the inactive conformation of kinases. We analyzed our previously identified type II Plk1 inhibitor SBE13 which is able to inhibit Plk1 activity. To determine its ability to induce cell death in cancer cells, we applied kinase assays, Western blot analyses, FACScan analyses, Caspase assays and immunofluorescence studies. We detected decreased cell proliferation, delayed progression through the cell cycle in lower SBE13 concentrations, a G2/M arrest using higher SBE13 concentrations followed by apoptosis, and abnormal mitotic figures. Notably, SBE13 did not influence activity of other kinases (Plk2, Plk3, Aurora A), indicating the selectivity of this type II Plk1 inhibitor. This study suggests that Plk1 kinase inhibitors targeting the inactive conformation of Plk1 may be considered for the development of cancer therapeutics.

Reliable predictive and prognostic markers for routine diagnostic purposes are needed for breast cancer patients treated with neoadjuvant chemotherapy. We evaluated protein biomarkers in a cohort of 116 participants of the GeparDuo study on anthracycline/taxane-based neoadjuvant chemotherapy for operable breast cancer to test for associations with pathological complete response (pCR) and disease-free survival (DFS). Particularly, we evaluated if interactions between hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) expression might lead to a different clinical behavior of HR+/HER2+ co-expressing and HR+/HER2- tumors and whether subgroups of triple negative tumors might be identified by the help of Ki67 labeling index, cytokeratin 5/6 (CK5/6), as well as cyclooxygenase-2 (COX-2), and Y-box binding protein 1 (YB-1) expression. Expression analysis was performed using immunohistochemistry and silver-enhanced in situ hybridization on tissue microarrays (TMAs) of pretherapeutic core biopsies. pCR rates were significantly different between the biology-based tumor types (P= 0.044) with HR+/HER2+ and HR-/HER2- tumors having higher pCR rates than HR+/HER2- tumors. Ki67 labeling index, confirmed as significant predictor of pCR in the whole cohort (P= 0.001), identified HR-/HER- (triple negative) carcinomas with a higher chance for a pCR (P= 0.006). Biology-based tumor type (P= 0.046 for HR+/HER2+ vs. HR+/HER2-), Ki67 labeling index (P= 0.028), and treatment arm (P= 0.036) were independent predictors of pCR in a multivariate model. DFS was different in the biology-based tumor types (P< 0.0001) with HR+/HER2- and HR+/HER2+ tumors having the best prognosis and HR-/HER2+ tumors showing the worst outcome. Biology-based tumor type was an independent prognostic factor for DFS in multivariate analysis (P< 0.001). Our data demonstrate that a biology-based breast cancer classification using estrogen receptor (ER), progesterone receptor (PgR), and HER2 bears independent predictive and prognostic potential. The HR+/HER2+ co-expressing carcinomas emerged as a group of tumors with a good response rate to neoadjuvant chemotherapy and a favorable prognosis. HR+/HER2- tumors had a good prognosis irrespective of a pCR, whereas patients with HR-/HER- and HR-/HER+ tumors, especially if they had not achieved a pCR, had an unfavorable prognosis and are in need of additional treatment options. ClinicalTrials.gov identifier: NCT00793377

Lymphocyte infiltration (LI) is often seen in breast cancer but its importance remains controversial. A positive correlation of human epidermal growth factor receptor 2 (HER2) amplification and LI has been described, which was associated with a more favorable outcome. However, specific lymphocytes might also promote tumor progression by shifting the cytokine milieu in the tumor. Affymetrix HG-U133A microarray data of 1,781 primary breast cancer samples from 12 datasets were included. The correlation of immune system-related metagenes with different immune cells, clinical parameters, and survival was analyzed. A large cluster of nearly 600 genes with functions in immune cells was consistently obtained in all datasets. Seven robust metagenes from this cluster can act as surrogate markers for the amount of different immune cell types in the breast cancer sample. An IgG metagene as a marker for B cells had no significant prognostic value. In contrast, a strong positive prognostic value for the T-cell surrogate marker (lymphocyte-specific kinase (LCK) metagene) was observed among all estrogen receptor (ER)-negative tumors and those ER-positive tumors with a HER2 overexpression. Moreover ER-negative tumors with high expression of both IgG and LCK metagenes seem to respond better to neoadjuvant chemotherapy. Precise definitions of the specific subtypes of immune cells in the tumor can be accomplished from microarray data. These surrogate markers define subgroups of tumors with different prognosis. Importantly, all known prognostic gene signatures uniformly assign poor prognosis to all ER-negative tumors. In contrast, the LCK metagene actually separates the ER-negative group into better or worse prognosis.

Polo-like kinase 1 (Plk1) is overexpressed in tumor tissues and its expression level is tightly associated with the malignancy of tumors and prognosis of tumor patients. Thus, Plk1 is considered as one of the most attractive molecular targets for anticancer therapy. Recently, several small molecule inhibitors of Plk1 have been identified and characterized, and the first generation of Plk1 inhibitors has been investigated in clinical trials. However, the long-term effect of the downregulation of Plk1 on tumor cells has not yet been studied. In this work we have investigated the phenotype of HeLa cells, in which Plk1 is continuously downregulated by constitutive expression of shRNA. The data demonstrate that the long-term suppression of Plk1 increases the levels of cyclin-dependent kinase inhibitor p21WAF1/CIP, which is partially induced by the elevated tumor suppressor p73 in p53-inactivated HeLa cells. The increased kinase inhibitor p21WAF1/CIP1localizes in both cyctoplasm as well as in nucleus and interacts directly with Cdk1/cyclin B1. Moreover, the knockdown of Plk1 leads to a decreased oncoprotein MDM2 and an elevated pro-apoptotic protein Bax in HeLa cells. Importantly, HeLa cells with reduced level of Plk1, which induces an increase of p21, p73 and Bax, are more sensitive to some chemotherapeutic agents, such as cisplatin.

Despite the fact that people older than 65 years of age have the highest incidence of developing breast cancer, these patients are excluded from clinical trials in most cases. Furthermore, most physicians tend towards therapy regimens without the use of dose-dense, highly active taxane-based treatments because of a lack of data regarding toxicities of these compounds in older patients. Pooled side-effect data were analyzed from four prospective, randomized clinical trials in which patients of different age groups (< 60 years, between 60 and 64 years, and > 64 years) with primary breast cancer received taxane-based chemotherapy. Dose delays, dose reductions, hospitalization, and therapy discontinuation increased with age. Hematologic toxicities and some nonhematologic toxicities were generally more common in older patients. Leucopenia increased from 55.3% in patients aged < 60 years to 65.5% in patients aged > 64 years (P< 0.001), and neutropenia increased from 46.9% to 57.4% (P< 0.001). There was no difference, however, in clinically more relevant febrile neutropenia between the different age groups. Thrombopenia shows a similar age-dependent increase, whereas there is no difference between the age groups concerning anemia. Hot flushes and elevated liver enzymes decreased with increasing age. The present pooled analysis of a substantial cohort of older primary breast cancer patients demonstrates that taxane-containing (neo)adjuvant chemotherapy is feasible in older patients and that toxicity can be reduced by sequential therapy regimens.

To investigate the predictive value of clinical and biological markers for a pathological complete remission after a preoperative dose-dense regimen of doxorubicin and docetaxel, with or without tamoxifen, in primary operable breast cancer. Patients with a histologically confirmed diagnosis of previously untreated, operable, and measurable primary breast cancer (tumour (T), nodes (N) and metastases (M) score: T2-3(≥ 3 cm) N0-2 M0) were treated in a prospectively randomised trial with four cycles of dose-dense (bi-weekly) doxorubicin and docetaxel (ddAT) chemotherapy, with or without tamoxifen, prior to surgery. Clinical and pathological parameters (menopausal status, clinical tumour size and nodal status, grade, and clinical response after two cycles) and a panel of biomarkers (oestrogen and progesterone receptors, Ki-67, human epidermal growth factor receptor 2 (HER2), p53, bcl-2, all detected by immunohistochemistry) were correlated with the detection of a pathological complete response (pCR). A pCR was observed in 9.7% in 248 patients randomised in the study and in 8.6% in the subset of 196 patients with available tumour tissue. Clinically negative axillary lymph nodes, poor tumour differentiation, negative oestrogen receptor status, negative progesterone receptor status, and loss of bcl-2 were significantly predictive for a pCR in a univariate logistic regression model, whereas in a multivariate analysis only the clinical nodal status and hormonal receptor status provided significantly independent information. Backward stepwise logistic regression revealed a response after two cycles, with hormone receptor status and lymph-node status as significant predictors. Patients with a low percentage of cells stained positive for Ki-67 showed a better response when treated with tamoxifen, whereas patients with a high percentage of Ki-67 positive cells did not have an additional benefit when treated with tamoxifen. Tumours overexpressing HER2 showed a similar response to that in HER2-negative patients when treated without tamoxifen, but when HER2-positive tumours were treated with tamoxifen, no pCR was observed. Reliable prediction of a pathological complete response after preoperative chemotherapy is not possible with clinical and biological factors routinely determined before start of treatment. The response after two cycles of chemotherapy is a strong but dependent predictor. The only independent factor in this subset of patients was bcl-2. NCT00543829

Conditional gene silencing in mammalian cells, via the controlled expression of short hairpin RNAs (shRNAs), is an effective method for studying gene function, particularly if the gene is essential for cell survival or development. Here we describe a simple and rapid protocol for the generation of tetracycline (Tet)-inducible vectors that express shRNAs in a time- and dosage-dependent manner. Tet-operator (TetO) sequences responsive to occupation by the Tet-repressor (TetR) were inserted at alternative positions within the wild-type H1 promoter and cloned into a eukaryotic expression vector. Additional cloning sites downstream of the promoter enable the insertion of shRNA sequences. This Tet-inducible shRNA expression system can be used for both transient and stable RNA interference (RNAi) approaches to control gene function in a spatiotemporal fashion. The entire protocol (preparation of constructs, generation of stable cell lines and functional analysis) can be completed in 3 months.

The transcriptional cofactor FHL2 interacts with a broad variety of transcription factors and its expression is often deregulated in various types of cancer. Here we analyzed for the first time the molecular function of FHL2 in breast cancer. FHL2 is overexpressed in almost all human mammary carcinoma samples tested but not in normal breast tissues and only low levels of FHL2 expression were present in four premalignant ductal carcinoma in situ (DCIS). Cell cycle analysis revealed an upregulation of endogenous FHL2 towards G2/M in MDA-MB 231 cells and an accelerated G2/M transition when FHL2 expression was suppressed in these cells. In search for G2/M specific target genes regulated by FHL2, we found that expression of the cell cycle inhibitor p21Cip1/Waf1 (hereafter p21) is dependent on FHL2 in MDA-MB 231 breast cancer cells. Downregulation of FHL2 by shRNA abrogated the cell cycle dependent upregulation of p21 as well as the induction of p21 in response to treatment with the DNA damaging agent doxorubicin. FHL2-dependent p21 expression occurs in a p53-independent manner and p21 expression can be downregulated by specific inhibition of mitogen-activated protein kinases (MAPKs), implicating an involvement of MAPK signaling in this regulation. Analysis of FHL2 contribution to the MAPK signaling identified FHL2 as an important downstream effector of MAPKs in breast cancer cells, capable of transactivating endogenous AP1 target genes as well as AP1 dependent reporter genes. Finally, downregulation of FHL2 reduces the ability of MDA-MB 231 cells to form colonies in soft agar, while FHL2 overexpression enhances colony formation of breast cancer cells. Thus, our findings indicate that overexpression of the transcriptional cofactor FHL2 contributes to breast cancer development by mediating transcriptional activation of MAPK target genes known to be involved in cancer progression, such as p21.

For almost three decades, tamoxifen has been the mainstay of hormonal therapy in breast cancer patients, but now the third-generation aromatase inhibitors (AIs), anastrozole, letrozole and exemestane, are emerging as potential alternatives, associating greater clinical efficacy with a more favourable overall safety profile than tamoxifen. AIs are associated with a lower incidence of thromboembolic events and vaginal bleeding compared with tamoxifen, although they are known to affect bone turnover and possibly lipid metabolism. As the available AIs have similar efficacy, it is likely that safety and tolerability profiles will have an impact on agent selection in clinical practice. Therefore, it is important that differences in the safety profiles of the third-generation AIs are understood.

Trastuzumab—a humanised monoclonal antibody against HER2—has been shown to improve disease-free survival after chemotherapy in women with HER2-positive early breast cancer. We investigated the drug's effect on overall survival after a median follow-up of 2 years in the Herceptin Adjuvant (HERA) study.

Breast carcinoma during pregnancy (BCP) is a difficult clinical situation, as it appears to put the health of the mother in conflict with that of the fetus.An international expert meeting was conducted to form guidelines on how to diagnose and treat women with BCP.The goal for treatment of the pregnant woman with breast carcinoma is the same as that of the nonpregnant breast carcinoma patient: local control of disease and prevention of systemic metastases. However, certain treatment modalities need to be modified because of the potential for adverse effects on the fetus. There is evidence to support the safety of anthracycline‐based chemotherapy during the second and third trimesters of pregnancy (Oxford Level of Evidence [LOE] 2b). Because of the lack of evidence, the expert opinion was not to recommend the routine use of newer cytotoxic drugs like the taxanes during pregnancy (LOE 5).The recommendations provided should help to reach informed decision making by the patient. The ongoing prospective collection of data on BCP, such as that at the University of Texas M.D. Anderson Cancer Center (UTMDACC) and that of the German Breast Group/Breast International Group (GBG/BIG), is necessary to further our knowledge regarding the treatment of this unique group of breast carcinoma patients. Cancer 2006. © 2005 American Cancer Society.

Introduction ScFv(FRP5)-ETA is a recombinant antibody toxin with binding specificity for ErbB2 (HER2). It consists of an N-terminal single-chain antibody fragment (scFv), genetically linked to truncated Pseudomonas exotoxin A (ETA). Potent antitumoral activity of scFv(FRP5)-ETA against ErbB2-overexpressing tumor cells was previously demonstrated in vitro and in animal models. Here we report the first systemic application of scFv(FRP5)-ETA in human cancer patients.Methods We have performed a phase I dose-finding study, with the objective to assess the maximum tolerated dose and the dose-limiting toxicity of intravenously injected scFv(FRP5)-ETA. Eighteen patients suffering from ErbB2-expressing metastatic breast cancers, prostate cancers, head and neck cancer, non small cell lung cancer, or transitional cell carcinoma were treated. Dose levels of 2, 4, 10, 12.5, and 20 μg/kg scFv(FRP5)-ETA were administered as five daily infusions each for two consecutive weeks.Results No hematologic, renal, and/or cardiovascular toxicities were noted in any of the patients treated. However, transient elevation of liver enzymes was observed, and considered dose limiting, in one of six patients at the maximum tolerated dose of 12.5 μg/kg, and in two of three patients at 20 μg/kg. Fifteen minutes after injection, peak concentrations of more than 100 ng/ml scFv(FRP5)-ETA were obtained at a dose of 10 μg/kg, indicating that predicted therapeutic levels of the recombinant protein can be applied without inducing toxic side effects. Induction of antibodies against scFv(FRP5)-ETA was observed 8 days after initiation of therapy in 13 patients investigated, but only in five of these patients could neutralizing activity be detected. Two patients showed stable disease and in three patients clinical signs of activity in terms of signs and symptoms were observed (all treated at doses ≥ 10 μg/kg). Disease progression occurred in 11 of the patients.Conclusion Our results demonstrate that systemic therapy with scFv(FRP5)-ETA can be safely administered up to a maximum tolerated dose of 12.5 μg/kg in patients with ErbB2-expressing tumors, justifying further clinical development.

ScFv(FRP5)-ETA is a recombinant antibody toxin with binding specificity for ErbB2 (HER2). It consists of an N-terminal single-chain antibody fragment (scFv), genetically linked to truncated Pseudomonasexotoxin A (ETA). Potent antitumoral activity of scFv(FRP5)-ETA against ErbB2-overexpressing tumor cells was previously demonstrated in vitroand in animal models. Here we report the first systemic application of scFv(FRP5)-ETA in human cancer patients. We have performed a phase I dose-finding study, with the objective to assess the maximum tolerated dose and the dose-limiting toxicity of intravenously injected scFv(FRP5)-ETA. Eighteen patients suffering from ErbB2-expressing metastatic breast cancers, prostate cancers, head and neck cancer, non small cell lung cancer, or transitional cell carcinoma were treated. Dose levels of 2, 4, 10, 12.5, and 20 μg/kg scFv(FRP5)-ETA were administered as five daily infusions each for two consecutive weeks. No hematologic, renal, and/or cardiovascular toxicities were noted in any of the patients treated. However, transient elevation of liver enzymes was observed, and considered dose limiting, in one of six patients at the maximum tolerated dose of 12.5 μg/kg, and in two of three patients at 20 μg/kg. Fifteen minutes after injection, peak concentrations of more than 100 ng/ml scFv(FRP5)-ETA were obtained at a dose of 10 μg/kg, indicating that predicted therapeutic levels of the recombinant protein can be applied without inducing toxic side effects. Induction of antibodies against scFv(FRP5)-ETA was observed 8 days after initiation of therapy in 13 patients investigated, but only in five of these patients could neutralizing activity be detected. Two patients showed stable disease and in three patients clinical signs of activity in terms of signs and symptoms were observed (all treated at doses ≥ 10 μg/kg). Disease progression occurred in 11 of the patients. Our results demonstrate that systemic therapy with scFv(FRP5)-ETA can be safely administered up to a maximum tolerated dose of 12.5 μg/kg in patients with ErbB2-expressing tumors, justifying further clinical development.

Gonadotropin-releasing hormone analogs are, alongside tamoxifen, one of the most commonly used drugs in the treatment of pre-/perimenopausal endocrine-responsive breast cancer. Goserelin, as a principal agent of this class of drugs, is mainly investigated in clinical trials. The indirect comparison of goserelin with tamoxifen as a single drug in the adjuvant setting showed similar efficacy. Furthermore, goserelin is as effective as cyclophosphamide, methotrexate and 5-fluorouracil chemotherapy, and total endocrine blockade as a combination of gonadotropin-releasing hormone analog and tamoxifen showed a comparable benefit with anthracycline-containing adjuvant chemotherapy. Goserelin administered after cessation of chemotherapy leads to a further improvement and may be equieffective as tamoxifen or a combination of both. Data concerning taxane-based and dose-dense chemotherapy as well as combination of gonadotropin-releasing hormone analogs with third-generation aromatase inhibitors are still lacking (ongoing suppression of ovarian function, tamoxifen and exemestane, and premenopausal endocrine-responsive chemotherapy trials). Moreover, duration of therapy with gonadotropin-releasing hormone analogs (2–3 years or longer) is still a matter of debate. Palliative endocrine treatment is standard in the first-line therapy of patients without life-threatening disease and endocrine-responsive breast cancer. Treatment decisions depend upon adjuvant endocrine pretreatment. Clinical data regarding ovarian protection by synchronous use of gonadotropin-releasing hormone in young breast cancer patients receiving chemotherapy are incoherent.

The purpose of this study was to evaluate the potential of the new 8G stereotactic vacuum-assisted breast biopsy (ST-driver, Mammotome; Ethicon Endosurgery) in the histologic evaluation of BI-RADS IV microcalcifications.

Two hundred and fifty-six (256) patients (72% preoperative, 28% pre-Mammotome) were prospectively examined with EIS using the TS 2000 (TransScan Research and Development Center, Israel; temporarily distributed by Siemens, Erlangen) with the “LOS”-software (level of suspicion). All exams were performed with the targeted scan probe, the observer knowing all clinical and imaging facts. The area of the lesions was examined with EIS at least with 5 single scans. The evaluation included a scaling of lesions from 1 (surely benign) up to 5 (highly suggestive for malignancy) as well as the additional notification of spots. Results of EIS were based upon the automatic scaling which is provided by the software and were compared with mammography and histology. Furthermore the influence of the histology, size of lesions, and presence/absence of spots on the EIS results were analyzed.Histology revealed benign results in 138 lesions and malignant results in 118 lesions (DCIS=61, ID-Ca=51, IL-Ca=5, mucinous Ca=1). Mammography as expected yielded high values with 91% sensitivity and 62% specificity. Overall sensitivity of EIS was 75.4%, specificity 42.03%, negative predictive value 66.7% and positive predictive value 52.7% (89 TP, 58 TN, 80 FP, 29 FN). EIS was false negative in 20 ID-Ca, 3 IL-Ca, 1 IDL-Ca, 4 DCIS, and 1 mucinous carcinoma. Sensitivity and specificity of EIS did not differ for the different histological differentiations neither for the degree of invasion. Also the additional notification of “spots” didn't show a correlation to malignancy. There were significant differences of the sensitivity of EIS regarding the tumor size. While EIS correctly diagnosed 85% of lesions <10 mm in size, only 64% of lesions >10 mm were detected. Most frequent lesion types for false positives were mastopathy (55/80 FP) and fibroadenoma (21/80 FP). Patient acceptance of EIS was perfect and there were no drop outs because of movement artifacts.In conclusion the “LOS”-software clearly improved the clinical performance of the TS 2000 as compared to the initial software. The high sensitivity of EIS in small cancers which was found in our study may indicate an advantage of this method. However, the overall sensitivity and specificity with this setup of EIS is still far too low. Further improvements especially including the measurement of higher frequencies should be realized.

Timing of systemic treatment in primary operable breast cancer is subject to extensive investigation, suggesting that pathologic complete remission (pCR) might improve survival in this setting. The German Adjuvant Breast Cancer Group previously demonstrated the feasibility of a dose-dense biweekly schedule of 4 cycles doxorubicin 50 mg/m2and docetaxel 75 mg/m2(ddAT) ± tamoxifen in the neoadjuvant setting to yield a pCR of 9.7% (Gepardo trial). Patients assigned to ddAT received prophylactic granulocyte colony-stimulating factor support (5 νg/kg days 5–10). The current study (GeparDUO) was designed to assess whether the pCR rate, including no viable invasive and preinvasive tumor cells, achieved with ddAT was equivalent to sequential administration of doxorubicin/ cyclophosphamide followed by docetaxel (AC-DOC) over 24 weeks in primary operable breast cancer. From June 1999 to September 2001, 913 patients were enrolled in this trial. In total, 395 patients randomized before August 1, 2000, were included in the second interim analysis. Safety data were available from 369 patients (ddAT, n = 191; AC-DOC, n = 178) demonstrating that toxicity of both regimens was tolerable. Grade 3/4 neutropenia occurred in 39.8% of patients receiving ddAT and in 69.3% of patients treated with AC-DOC. Efficacy data were available in 378 patients. A pCR occurred in 14.8% of the primary breast tumors. According to the recommendations of the data monitoring committee, recruitment to the study was halted as of September 2001 (n = 913/1000) due to the significant difference in pCR rates observed between the treatment arms. Surgery was documented in 380 patients. Breast conservation was possible in 288 cases (75.8%). The application of both schedules is safe and feasible in an outpatient setting. Although, results obtained from this interim analysis are encouraging, caution is recommended until the results obtained show statistical difference in pCR.

Recent studies demonstrated that tumors overexpressing the epidermal growth factor receptor (EGF-R, erbB-1) are associated with poor clinical outcome. This led to the development of a variety of monoclonal antibodies targeting the extracellular domain of this receptor tyrosine kinase. The aim of our study was the evaluation of anti-EGF-R antibody EMD 55900 therapy for treatment of breast cancer. On the basis of 299 tumor specimens derived from breast cancer patients, we investigated EGF-R expression and generated a collective of primary xenotransplants in athymic nude mice. The animals received therapy in 2 treatment schedules to investigate the therapeutic response in early stages of tumor formation as well as on well established tumors. Using 6 different tumors with EGF-R expression levels between 10–300 fmol/mg total protein, we found a therapeutic effect when the EGF-R expression of the tumors was at least 40 fMol/mg. On the basis of these experimental data and our EGF-R expression analysis of breast cancer specimens, we conclude that up to 15% of breast cancer patients could benefit from this monotherapy with EMD 55900. © 2002 Wiley-Liss, Inc.

Nitric oxide (NO) is present in various human solid tumors and tumor cell lines, and it is believed that NO plays an important role in tumor growth. An increased NO concentration catalyzed by NO synthase (NOS) is cytotoxic and can promote apoptosis. The expression of endothelial NOS (e-NOS) and induced NOS (i-NOS) was examined in various breast tumors. Immunohistochemical staining with a monoclonal antibody (Ab) against e-NOS and a polyclonal Ab against i-NOS was performed on paraffin embedded tissues from 41 benign breast lesions, 9 in situ breast lesions, and 54 invasive breast lesions. Functionality was confirmed by detection of NO using spin-trapping electron paramagnetic resonance (EPR) spectroscopy. e-NOS expression was found in 2 benign lesions (5%; 1 fibroadenoma and 1 proliferative mastopathy), in 5 in situ lesions (56%), and in 33 invasive lesions (61%). None of the benign lesions was positive for i-NOS, but 6 in situ lesions (67%) and 33 invasive lesions (61%) showed tumor cell staining. In particular, capillaries that were embedded in lymphocytic stroma showed a positive reaction for e-NOS. The functionality of NOS was demonstrated by direct NO formation using the EPR spin-trapping method. Tumors that were positive for e-NOS were observed more often in younger patients (P = 0.05). These tumors more frequently were highly differentiated or moderately differentiated and more often showed invasive ductal subtypes and a lower proliferation rate. Tumors that were positive for both e-NOS and i-NOS were more likely to be lymph node negative tumors. Both i-NOS-expressing lesions and e-NOS-expressing lesions showed strong coexpression (P = 0.00001). NOS is detected predominantly in in situ lesions and invasive breast lesions but rarely in benign lesions. NOS is found more frequently in invasive carcinomas with low malignancy. Using the spin-trapping EPR method, this study demonstrates direct NO formation in human breast tumors for the first time. Cancer 2002;95:1191–8. © 2002 American Cancer Society. DOI 10.1002/cncr.10817

For many tumuors, pathological subclasses exist which have to be further defined by genetic markers to improve therapy and follow‐up strategies. In this study, cDNA array analyses of breast cancers have been performed to classify tumuors into categories based on expression patterns. Comparing purified normal ductal epithelial cells and corresponding tumour tissues, the expression of only a small fraction of genes was found to be significantly changed. A subset of genes repeatedly found to be differentially expressed in breast cancers was subsequently employed to perform a classification of 82 normal and malignant breast specimens by cluster analysis. This analysis identifies a subgroup of transcriptionally related tumours, designated class A, which can be further subdivided into A1 and A2. Correlation with classical clinicopathological parameters revealed that subgroup A1 was characterized by a high number of node‐positive tumours (14 of 16). In this subgroup there was a disproportionate number of patients who had already developed distant metastases at the time of diagnosis (25% in this subgroup, compared with 5% among the rest of the samples). Taken together, the use of these differentially expressed marker genes in conjunction with sample clustering algorithms provides a novel molecular classification of breast cancer specimens, which facilitates the identification of patients with a higher risk of recurrence. Copyright © 2001 John Wiley & Sons, Ltd.

Fibroblast growth factor receptors (FGFRs) play crucial roles in signal transduction of adult tissues and during embryonic development. To study the transcriptional control, we isolated and characterized the promoter of human FGFR4. Two transcription initiation sites were identified. The deletion analysis in different cell types defined a core promoter reaching from −9 to −198, lacking TATA and CCAAT boxes but displaying high GC content (77%) in a stretch of 300 bp upstream of the major mRNA start. This region harbors multiple binding motifs for transcription factors. Moreover, the region between −1085 and −1140 contains a potential repressor element, which downregulates transcriptional activity. To identify conserved regulatory elements, we isolated and analyzed also the murine FGFR4 promoter. Only one transcription start was identified using RNase protection assays. Sequence alignment of human and mouse shows a striking similarity in the core promoter region of both genes, encompassing conserved transcription factor binding sites and a splice acceptor site. Furthermore, the region containing the putative repressor element is also conserved suggesting a functional role for gene expression.

We compared the efficacy and safety of the oral aromatase inactivator exemestane (EXE) with megestrol acetate (MA) in women with metastatic breast cancer. This phase III randomized, double-blind, multicenter study was conducted in 769 postmenopausal women who had experienced tamoxifen failure. Treatment arms consisted of EXE 25 mg once daily (n = 366) or MA 40 mg four times daily (160 mg daily; n = 403). Peerreviewed, intent-to-treat analyses demonstrated that EXE induced a trend toward higher rates of complete response (CR) + partial response (PR) (15.0% vs. 12.4%) and of CR + PR + stable disease (SD) ≥ 24 weeks (37.4% vs. 34.6%), but differences were not statistically significant. Statistically significant differences favoring EXE were seen in median duration of CR + PR + SD ≥ 24 weeks (60.1 vs. 49.1 weeks; P= 0.025), time to tumor progression (20.3 vs. 16.6 weeks; P= 0.037), time to treatment failure (16.3 vs. 15.7 weeks; P= 0.042), and overall survival (not reached vs. 123.4 weeks; P= 0.039). Both treatments were well tolerated, but MA was associated with more grade 3 or 4 weight gain (8% vs. 17%, P= 0.001); the pain score was similar in both groups. There was a trend toward superiority in treatment-related signs and symptoms (TRSS) with EXE. There was greater improvement in the pain score and TRSS in patients achieving an objective response with EXE vs. MA. Quality of life improved or was similar for EXE in most domains. Exemestane offers an important new treatment option for postmenopausal women with hormone-responsive breast cancer.

Objective: To compare the diagnostic value of intercellular adhesion molecule (ICAM)-1 with that of C-reactive protein (CRP) and white blood cell (WBC) count for detecting histologic chorioamnionitis in serum of women with preterm labor.

Previous data have shown that the mRNA-expression of the serinlthreonine-kinase polo-like kinase (PLK) is closely correlated with the survival of patients suffering from a subset of malignant tumors. PLK-mRNA and protein-expression are restricted to cells in the cell cycle. PLK-mRNA-transcripts are highly abundant in proliferating cells; no gene expression is found in G0-phase cells. Here we investigated the mRNA- and protein-expression of PLK- and estrogen-receptor (ER) in human breast-carcinoma by northern-blotting, RT PCR and immunohistochemistry. The expression of MIB-I was determined on serial sections. Analysis of the immunohistochemical data revealed a close correlation between the ER and PLK-expression (r = 0.677; p = 0.001, n = 30). No relationship between the mRNA-expression of ER and PLK was found. Furthermore, no correlation for the protein expression of PLK and MIB-I exists. The influence of estrogen (ES) is known to have proliferative potential. The expression of ER correlates with the ES-plasma-level. In addition, the hormone cycle of premenopausal women undergoes rapid vacillations with varying effects on the proliferating tumor cells, e.g., growth induction. Our results therefore show that ERexpression is not only of therapeutic value for the clinician, but it may also be a tool for determining the tumor proliferation index more precisely by integrating the hormone-mediated proliferation stimulus.

The monoclonal antibody (MAb) 2E11 is commonly used for detection of microdisseminated epithelial cells in bone marrow of cancer patients. Surprisingly, in an earlier report 2E11 was shown to bind to mononuclear cells in bone marrow in 61% of healthy donors. In the present study we tested whether this cross-reaction with non-epithelial bone-marrow cells can be characterized further. In addition, we analyzed the influence of 2E11 concentration on the staining of mononuclear cells. We performed immunocytochemical double stainings of bone-marrow aspirations from breast-cancer patients using 2E11/A45-B/B3 (MAb against cytokeratin 8, 18, 19) and 2E11/CD45 (MAb against CD45-leukocyte common antigen), while tumor cell lines MCF-7 and K526 as well as bone marrow from breast-cancer patients were treated with different concentrations of 2E11. A portion of 2E11-positive cells was characterized as hematopoietic cells by CD-45-binding, while others were identified as epithelial cells by A45-B/B3-binding. We defined a concentration of 2E11 to immunolabel epithelial cells and distinguish hematopoietic cells. Higher concentrations of 2E11 enhance staining of hematopoietic cells, to match that of epithelial cells. We conclude that 2E11 shows cross-reactivity to epitopes displayed by hematopoietic cells. However, specific staining of epithelial cells can be achieved. As long as there is no antibody available which is highly specific for epithelial cells, detection of microdisseminated tumor cells in bone marrow by antigen-antibody reaction should be verified morphological criteria. Int. J. Cancer (Pred. Oncol.) 84:502–505, 1999. © 1999 Wiley-Liss, Inc.

Objective:Ovarian cancer antigen CA 125 is used widely for diagnostic and therapeutic evaluation of ovarian cancer. Although the initial description of CA 125 was in 1981, its function is still unknown. In collagen gel system the invasiveness of the endometriotic cell line EEC 145 is enhanced by a proteinaceous factor in peritoneal fluid mimicking molecular features of CA 125. Therefore we hypothesize that this factor is CA 125.Methods:The influence of heat-treated peritoneal fluid on the invasiveness of the endometriotic cell line EEC 145 and several human carcinoma cell lines (EJ28, RT112, AN3 CA, RL95-2, HEC-1-A, HeLa, MCF7, T-47D, and SK-OV-3) was investigated in a collagen gel invasion assay by addition of (1); 10% heat-treated peritoneal fluid, (2); peritoneal fluid after immunoprecipitation with antibodies directed to CA 125, and (3); purified CA 125.Results:The invasion index of the endometriotic cell line EEC 145 significantly increased from 4.78 ± 0.98 to 6.57 ± 1.58 (P = .0001) by addition of 10% heat-treated peritoneal fluid to the culture medium. The invasion-promoting effect of peritoneal fluid was abolished by reductions of the CA 125 concentration through immunoprecipitation and was mimicked in a dose-dependent manner by addition of CA 125 to the culture medium. The invasiveness of the investigated human carcinoma cell lines was not affected by heat-treated peritoneal fluid or CA 125.Conclusion:The results are consistent with the hypothesis that CA 125 can influence invasiveness in a benign endometriotic cell line.

Objective: Ovarian cancer antigen CA 125 is used widely for diagnostic and therapeutic evaluation of ovarian cancer. Although the initial description of CA 125 was in 1981, its function is still unknown. In collagen gel system the invasiveness of the endometriotic cell line EEC 145 is enhanced by a proteinaceous factor in peritoneal fluid mimicking molecular features of CA 125. Therefore we hypothesize that this factor is CA 125. Methods: The influence of heat-treated peritoneal fluid on the invasiveness of the endometriotic cell line EEC 145 and several human carcinoma cell lines (EJ28, RT112, AN3 CA, RL95-2, HEC-1-A, HeLa, MCF7, T-47D, and SK-OV-3) was investigated in a collagen gel invasion assay by addition of (1); 10% heat-treated peritoneal fluid, (2); peritoneal fluid after immunoprecipitation with antibodies directed to CA 125, and (3); purified CA 125. Results: The invasion index of the endometriotic cell line EEC 145 significantly increased from 4.78 ± 0.98 to 6.57 ± 1.58 (P= .0001) by addition of 10% heat-treated peritoneal fluid to the culture medium. The invasion-promoting effect of peritoneal fluid was abolished by reductions of the CA 125 concentration through immunoprecipitation and was mimicked in a dose-dependent manner by addition of CA 125 to the culture medium. The invasiveness of the investigated human carcinoma cell lines was not affected by heat-treated peritoneal fluid or CA 125. Conclusion: The results are consistent with the hypothesis that CA 125 can influence invasiveness in a benign endometriotic cell line.

Evidence to date indicates that structurally abnormal estrogen receptor (variant ER) can be detected in some human breast tumors. Based onin vitro ability to bind DNA sequences containing the cognate estrogen response element (ERE), these variant receptors may be categorized into DNA-binding ER (Type-1 variants) and non-DNA-binding ER (Type-2 variants). To look for Type-2 variants of normal size (67 kDa ER) that lack the ability to form immunoreactive ER-ERE complexes, a panel of 40 cryopreserved primary breast tumors were extracted and analyzed by enzyme immunoassay (ER-EIA), gel-shift, and Western blot techniques. For the 33 tumor extracts containing =10 fmol/mg ER (by ER-EIA), the amount of 67 kDa ER detectable by D75 anti-ER monoclonal antibody under fully denatured and reduced assay conditions (Western blotting) did not correlate well with the presence or intensity of D75 immunoreactive ER-ERE bands seen under native conditions by gel-shift assay. Overall, 30% (10 of 33) of these extracts containing 67 kDa ER failed to produce immunoreactive ER-ERE complexes, with this frequency varying from over 40% in tumor samples with lower ER content (10-49 fmol/mg) to 11% in tumor samples with the highest ER content (>100 fmol/mg). These results indicate that Type-2 variant receptors characterized as non-DNA-binding 67 kDa ER may be present in a significant fraction of ER-positive primary breast tumors; preliminary evidence suggests that further study of abnormalities in ER tertiary or quaternary structure, such as those produced by intracellular oxidation of ER thiol groups, is warranted.

Splice variants of CD44 expressed in a metastasizing cell line derived from a rat pancreatic adenocarcinoma have been shown recently to confer metastatic potential onto non-metastasizing rat pancreatic carcinoma and sarcoma cell lines. Homologues of these variants have also been detected in a variety of human malignancies. Using antibodies raised against a bacterially expressed fusion protein containing variant CD44 sequences, we have explored the expression of variant CD44 glycoproteins on tumors of the female breast. The material examined included normal tissue, hyperplastic lesions, 103 primary invasive mammary carcinomas, 10in situ carcinomas, 12 local recurrences and 18 lymph node metastases. Using a polyclonal serum directed against several variant CD44 epitopes, normal mammary epithelia as well as ductal hyperplasias were negative for these splice variants, while the variant CD44 epitopes were detectable in all but six of the primary invasive carcinomas. From the reaction with various monoclonal antibodies and polyclonal sera specific for individual epitopes it is obvious that the tumors predominantly express CD44 variants encoded by exons v5 to v7. Interestingly, all investigated lymph node metastases reacted positively with the variant-specific antibodies, in contrast to primary tumors which reacted in 54% to 86% of the cases, depending on the antibody used. Statistical analysis revealed a significant correlation between expression of variant exons v3/v4 and v6 and increased tumor grade (p = 0.001 and p < 0.05, respectively; Fisher's exact test). Exon v6 is carried by the variants which confer metastatic capability in the rat. These results indicate that the expression of the CD44 variants is upregulated in mammary carcinomas and is closely linked to tumor anaplasia.

Gonadotropin-releasing hormone analogs (GnRH-A) have been added to the armentarium in the therapy of hormone-dependent breast cancer in premenopausal women. The effect of chronic GnRH-A-treatment in premenopausal women is based on the suppression of the hypothalamus-pituitary-ovarian axis and the reduction of sex-steroid serum levels. In addition, a number of experimental and clinical data have been accumulated indicating a direct action of GnRH-A on breast cancer cells and tissue. In this study we analyzed 235 human breast cancer biopsies for specific GnRH-A-binding. We demonstrate high affinity GnRH-A binding sites in human breast cancer tissues. The evaluation of clinical data showed no correlation of the level of GnRH-A-binding with classical tumor parameters.

Tissue samples from 49 women with seven benign and 42 malignant ovarian tumors were examined by means of DNA flow cytometry (FCM). The FCM data (DNA-ploidy and the number of S-phase fractions) were compared with the International Federation of Gynecology and Obstetrics (FIGO) stage, the histologic grade of differentiation and in some cases with the clinical outcome. The benign ovarian tumors were all diploid with a mean of 2.1 (± 1.66) S-phase fractions. Adenocarcinomas with the histologic grade 1 were diploid and had a mean of 2.1 (± 1.17) S-phase fractions. Grade 2 adenocarcinomas were aneuploid in eight of nine cases and revealed an increased proliferative activity (7.7 ± 2.67 S-phase fractions). A high number of aneuploid cases (nine of 13) and an increased DNA synthesis were found in grade 3 adenocarcinomas (12.0 ± 5.72 S-phase fractions). Four of six malignant non-epithelial tumors also had high numbers of S-phase fractions (9.7–14.5). A significant correlation between the histologic grade and the DNA synthesis, FIGO stage, and DNA-ploidy was found. DNA-FCM may be used as an additional diagnostic tool supplementing routine histopathologic examination of ovarian tumors for better biological characterization, especially for those with uncertain grading, for grade 2 neoplasms, and for malignant nonepithelial tumors.

Summary The object of the study was to evaluate the effectiveness of ifosfamide/etoposide and mesna therapy in advanced breast cancer. A total of 44 patients with breast cancer were included in the trial. Eligibility criteria included measurable, refractory disease; prior anthracycline therapy (or its contraindication); a life expectancy of at least 3 months; and adequate hepatic, renal, CNS and bone marrow function. All patients were=70 years of age and had a Karnofsky performance status of=50%. There were 36 evaluable cases. Sites of metastatic disease included bone (19), skin (18), liver (9), lung (14), lymph node (19), and miscellaneous (7). Treatment consisted of 1,500 mg/m2 ifosfamide given i.v. on days 1–5, 120 mg/m2 etoposide given i.v. on days 1–3, and 400 mg i.v. mesna given with and at 4 and 8 h after ifosfamide. Cycles were repeated every 28 days. Initial doses were reduced by 25% or 50% in patients who had previously undergone both chemotherapy and radiotherapy. A median of 4 cycles (range, 2–8) were given. The myelotoxicity was marked: WHO grades 3/4 leukopenia (n=37), grades 3/4 thrombocytopenia (n=12), and grades 2/3 anemia (n=13). Due to myelotoxicity, dose reduction or prolongation of treatment-free intervals was necessary in 28 cases. Alopecia was seen in 35 patients and CNS toxicity, in 8. Partial remission (PR) was obtained in five cases and complete remission (CR), in three. Sites of response included the lung (5), skin (4), lymph node (5), and peritoneum (1). The duration of response was 4 (n=2) and 8 (n=1) months for CR and 2 (n=2), 6 (n=2), and 10 (n=1) months for PR. We conclude that the ifosfamide/etoposide and mesna regimen is effective, but its myelotoxicity is treatment-limiting.

Summary There may exist an association between the genetically determined oxidation status of the antihypertensive agent debrisoquine (DEB) and the propensity to develop tumours. The metabolism of DEB is extensive in 90% of healthy subjects (metabolic ratio=MR=0–12.6; MR=% DEB excreted divided by % 4-hydroxy-DEB excreted) and poor in 10% (MR >12.6). In patients with cancer of the lung, urinary bladder, and gastrointestinum, the percentage of high metabolizers is increased to >98%. The poor metabolizer mode is almost devoid of cancer patients. It was investigated whether breast cancer patients show a similar association with respect to the oxidative status of DEB. 108 breast cancer patients and 123 women with benign gynecologic disorders received 1 tablet of 10 mg DEB orally in the evening. Urine was collected for the subsequent 8 hrs and analysed for its content of DEB and its main urinary metabolite 4-OH-DEB by means of HPLC. No decreased amount of poor metabolizers was seen in the cancer group.

This study compares the two oxazaphosphorine compounds ASTA Z 7557 (AZ) and cyclophosphamide (CP) in their therapeutic activity as well as in their myelotoxicity and DNA damage being induced after a single intraperitoneal injection. Therapeutic activity was determined towards methylnitrosourea-induced rat mammary carcinomas in vivo and in vitro, resulting in comparable efficacy of both compounds at their optimal doses, respectively, with the sensitivity of individual tumors being reflected by the degree of inhibition of 3H-thymidine uptake of these cells in vitro.