Prognostic impact of breast cancer subtypes in elderly patients.

We aimed to analyse the impact of breast cancer (BC) subtypes on the clinical course of disease with special emphasis on the occurrence of brain metastases (BM) and outcome in an elderly BC population. A total number of 706 patients ≥65 years receiving treatment for BC from 2007 to 2011 were identified from a BC database. 62 patients diagnosed with DCIS and 73 patients with incomplete datasets were excluded, leaving 571 patients for this analysis. Patient characteristics, biological tumour subtypes, and clinical outcome including overall survival (OS) were obtained by retrospective chart review. 380/571 (66, 5 %) patients aged 65-74 years were grouped among the young-old, 182/571 (31.9 %) patients aged 75-84 years among the old-old, and 29/571 (5.1 %) patients aged ≥85 years among the oldest-old. 392/571 (68.8 %) patients presented with luminal BC, 119/571 (20.8 %) with HER2-positive, and 59/571 (10.3 %) with triple-negative BC (TNBC). At 38 months median follow-up, 115/571 (20.1 %) patients presented with distant recurrence. A higher recurrence rate was observed in the HER2-positive subtype (43/119 (36.1 %)), as compared to TNBC (15/59 (25.4 %)) and luminal BC (57/392 (14.5 %); p < 0.001). BM were detected at a significantly higher rate in HER2-positive BC patients (9/119 (7.6 %)), as compared to TNBC (2/59 (3.4 %)) and luminal BC patients (6/392 (1.5 %); p = 0.003). Diagnosis of metastatic disease (HR 7.7; 95 % CI 5.2-11.4; p < 0.001) as well as development of BM (HR 3.5; 95 % CI 1.9-6.4; p < 0.001) had a significantly negative impact on OS in a time-dependent covariate cox regression model. In contrast to younger BC patients, outcome in this large cohort of elderly patients suggests that HER2-positive disease-not TNBC-featured the most aggressive clinical course with the highest rates of metastatic spread and BM. In-depth analysis regarding a potentially distinct biology of TNBC in elderly is therefore warranted.

Port-a-Cath extravasation of vesicant cytotoxics: surgical options for a rare complication of cancer chemotherapy.

BACKGROUND: Although implantation of a central venous device such as a Port-a-Cath was initially considered safe, extravasation rates up to 4.7% have been reported. Therefore, the objective of this study was to propose a structured procedure for the management of extravasation of a cytotoxic treatment. METHODS: A total of eight patients were evaluated after port extravasation of epirubicin (n = 3), platinum compounds (n = 3), paclitaxel (n = 1), or trabectedin (n = 1) into the subcutaneous space. Immediate explantation of the port was performed in combination with a "Subcutaneous Wash-Out Procedure" (SWOP). When removal of the port was delayed, débridement and flap coverage were performed as necessary. Epirubicin concentrations present in the samples obtained during surgical intervention were subsequently analysed using high-performance liquid chromatography (HPLC). Patients were followed for at least six months and were examined for sequelae such as pain, induration, redness, and limited movement. RESULTS: All three patients whose extravasation event was detected during chemotherapy administration benefited from SWOP with acceptable side effects (e.g., erythema). The analysis of epirubicin concentrations demonstrated the active removal of relevant amounts of the compound by wound rinsing. In contrast, late detection of extravasation led to major débridement and flap coverage in four out of five patients. A high body mass index (BMI) value was associated with all of the patients that experienced port extravasation. CONCLUSION: Depending on when Port-a-Cath extravasations into subcutaneous tissue are detected, different treatments are appropriate. When extravasation is detected early, the SWOP was found to be beneficial.

Fulvestrant induces resistance by modulating GPER and CDK6 expression: implication of methyltransferases, deacetylases and the hSWI/SNF chromatin remodelling complex.

BACKGROUND: Breast cancer is the leading cause of cancer death in women living in the western hemisphere. Despite major advances in first-line endocrine therapy of advanced oestrogen receptor (ER)-positive breast cancer, the frequent recurrence of resistant cancer cells represents a serious obstacle to successful treatment. Understanding the mechanisms leading to acquired resistance, therefore, could pave the way to the development of second-line therapeutics. To this end, we generated an ER-positive breast cancer cell line (MCF-7) with resistance to the therapeutic anti-oestrogen fulvestrant (FUL) and studied the molecular changes involved in resistance. METHODS: Naive MCF-7 cells were treated with increasing FUL concentrations and the gene expression profile of the resulting FUL-resistant strain (FR.MCF-7) was compared with that of naive cells using GeneChip arrays. After validation by real-time PCR and/or western blotting, selected resistance-associated genes were functionally studied by siRNA-mediated silencing or pharmacological inhibition. Furthermore, general mechanisms causing aberrant gene expression were investigated. RESULTS: Fulvestrant resistance was associated with repression of GPER and the overexpression of CDK6, whereas ERBB2, ABCG2, ER and ER-related genes (GREB1, RERG) or genes expressed in resistant breast cancer (BCAR1, BCAR3) did not contribute to resistance. Aberrant GPER and CDK6 expression was most likely caused by modification of DNA methylation and histone acetylation, respectively. Therefore, part of the resistance mechanism was loss of RB1 control. The hSWI/SNF (human SWItch/Sucrose NonFermentable) chromatin remodelling complex, which is tightly linked to nucleosome acetylation and repositioning, was also affected, because as a stress response to FUL treatment-naive cells altered the expression of five subunits within a few hours (BRG1, BAF250A, BAF170, BAF155, BAF47). The aberrant constitutive expression of BAF250A, BAF170 and BAF155 and a deviant stress response of BRG1, BAF170 and BAF47 in FR.MCF-7 cells to FUL treatment accompanied acquired FUL resistance. The regular and aberrant expression profiles of BAF155 correlated directly with that of CDK6 in naive and in FR.MCF-7 cells corroborating the finding that CDK6 overexpression was due to nucleosome alterations. CONCLUSION: The study revealed that FUL resistance is associated with the dysregulation of GPER and CDK6. A mechanism leading to aberrant gene expression was most likely unscheduled chromatin remodelling by hSWI/SNF. Hence, three targets should be conceptually addressed in a second-line adjuvant therapy: the catalytic centre of SWI/SNF (BRG1) to delay the development of FUL resistance, GPER to increase sensitivity to FUL and the reconstitution of the RB1 pathway to overcome resistance.

Brain metastases free survival differs between breast cancer subtypes.

BACKGROUND: Brain metastases (BM) are frequently diagnosed in patients with HER-2-positive metastatic breast cancer; in addition, an increasing incidence was reported for triple-negative tumours. We aimed to compare brain metastases free survival (BMFS) of breast cancer subtypes in patients treated between 1996 until 2010. METHODS: Brain metastases free survival was measured as the interval from diagnosis of extracranial breast cancer metastases until diagnosis of BM. HER-2 status was analysed by immunohistochemistry and reanalysed by fluorescent in situ hybridisation if a score of 2+ was gained. Oestrogen-receptor (ER) and progesterone-receptor (PgR) status was analysed by immunohistochemistry. Brain metastases free survival curves were estimated with the Kaplan-Meier method and compared with the log-rank test. RESULTS: Data of 213 patients (46 luminal/124 HER-2/43 triple-negative subtype) with BM from breast cancer were available for the analysis. Brain metastases free survival differed significantly between breast cancer subtypes. Median BMFS in triple-negative tumours was 14 months (95% CI: 11.34-16.66) compared with 18 months (95% CI: 14.46-21.54) in HER-2-positive tumours (P=0.001) and 34 months (95% CI: 23.71-44.29) in luminal tumours (P=0.001), respectively. In HER-2-positive patients, co-positivity for ER and HER-2 prolonged BMFS (26 vs 15 m; P=0.033); in luminal tumours, co-expression of ER and PgR was not significantly associated with BMFS. Brain metastases free survival in patients with lung metastases was significantly shorter (17 vs 21 months; P=0.014). CONCLUSION: Brain metastases free survival in triple-negative breast cancer, as well as in HER-2-positive/ER-negative, is significantly shorter compared with HER-2/ER co-positive or luminal tumours, mirroring the aggressiveness of these breast cancer subtypes.

Impact of anti-HER2 therapy on overall survival in HER2-overexpressing breast cancer patients with brain metastases.

BACKGROUND: Trastuzumab-based therapy after diagnosis of brain metastases (BM) may improve survival due to prolonged systemic disease control. We investigated whether lapatinib may yield additional survival benefit. METHODS: Eighty patients with BM from HER2-positive breast cancer were identified. Karnofsky Performance Score (KPS) of at least 70 was required. We included a control group of 37 patients treated before 2003, when continuation of trastuzumab after diagnosis of BM was not yet recommended. Remainders received either trastuzumab or lapatinib and trastuzumab (either concomitantly or sequentially) with or without chemotherapy. RESULTS: Median overall survival (OS) in patients receiving trastuzumab after diagnosis of BM was 13 months; corresponding numbers were 9 months in patients treated with chemotherapy, and 3 months with radiotherapy alone. Median OS was not reached in the lapatinib group. Addition of lapatinib prolonged OS over trastuzumab alone (P=0.002). After correction for potential confounders, lapatinib therapy remained an independent positive predictor for survival (HR 0.279; P=0.012). INTERPRETATION: This retrospective single-centre study suggests that the introduction of lapatinib improved survival in patients with BM from HER2-positive breast cancer. Patients with KPS ≥70 may benefit when treated with lapatinib in addition to trastuzumab after completion of local therapy.

Long-term persistence of acquired resistance to 5-fluorouracil in the colon cancer cell line SW620.

Treatment resistance to antineoplastic drugs represents a major clinical problem. Here, we investigated the long-term stability of acquired resistance to 5-fluorouracil (FU) in an in vitro colon cancer model, using four sub-clones characterised by increasing FU-resistance derived from the cell line SW620. The resistance phenotype was preserved after FU withdrawal for 15weeks (~100 cell divisions) independent of the established level of drug resistance and of epigenetic silencing. Remarkably, resistant clones tolerated serum deprivation, adopted a CD133(+) CD44(-) phenotype, and further exhibited loss of membrane-bound E-cadherin together with predominant nuclear ß-catenin localisation. Thus, we provide evidence for a long-term memory of acquired drug resistance, driven by multiple cellular strategies (epithelial-mesenchymal transition and selective propagation of CD133(+) cells). These resistance phenomena, in turn, accentuate the malignant phenotype.

Serum levels of tumour necrosis factor-alpha and interleukin-6 and their correlation with body mass index, weight loss, appetite and survival rate--preliminary data of Viennese outpatients with metastatic cancer during palliative chemotherapy.

The serum cytokine levels (in particular interleukine-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha)) of 61 advanced stage cancer patients receiving palliative chemotherapy as outpatients were determined with quantikine immunoassays. The values were correlated with body mass index (BMI), weight loss and appetite. Furthermore cytokine levels of patients who have died within one year were compared with those of patients who have survived more than a year. Serum levels of IL-6 (median: 1.93 pg/ml, range: 0.32-42.87) and of TNF-alpha (median: 2.55 pg/ml, range: 1.03-34.06) did not correlate with BMI, weight loss and appetite. Serum IL-6 levels of patients with survival time less than one year were significantly higher than the levels of patients who survived more than one year, no significant differences in TNF-alpha serum levels were evident. The data of this observation are consistent with current literature. Due to changes in serum levels of proinflammatory cytokines in response to chemotherapy and additional therapy, it is unlikely that IL-6 and TNF-alpha can be used as independent indicators for weight loss and appetite. Nevertheless, high serum levels of IL-6 correlate with short-time mortality.

Monitoring, removal and risk assessment of cytostatic drugs in hospital wastewater.

Cytostatic agents are applied in cancer therapy and subsequently excreted into hospital wastewater. As these substances are known to be carcinogenic, mutagenic and toxic for reproduction, they should be removed from wastewater at their source of origin. In this study the fate and effects of the cancerostatic platinum compounds (CPC) cisplatin, carboplatin, oxaliplatin, 5-fluorouracil (5-FU) and the anthracyclines doxorubicin, daunorubicin and epirubicin were investigated in hospital wastewater. Wastewater from the in-patient treatment ward of a hospital in Vienna was collected and monitored for the occurrence of the selected drugs. A calculation model was established to spot the correlation between administered dosage and measured concentrations. To investigate the fate of the selected substances during wastewater treatment, the oncologic wastewater was treated in a pilot membrane bioreactor system (MBR) and in downstream advanced wastewater treatment processes (adsorption to activated carbon and UV-treatment). Genotoxic effects of the oncologic wastewater were assessed before and after wastewater treatment followed by a risk assessment. Monitoring concentrations of the selected cytostatics in the oncologic wastewater were in line with calculated concentrations. Due to different mechanisms (adsorption, biodegradation) in the MBR-system 5 - FU and the anthracyclines were removed < LOD, whereas CPC were removed by 60%. In parallel, genotoxic effects could be reduced significantly by the MBR-system. The risk for humans, the aquatic and terrestrial environment by hospital wastewater containing cytostatic drugs was classified as small in a preliminary risk assessment.

Fate of 5-fluorouracil, doxorubicin, epirubicin, and daunorubicin in hospital wastewater and their elimination by activated sludge and treatment in a membrane-bio-reactor system.

Antineoplastic agents are applied in cancer therapy and end up in hospital wastewater by human excretions. In this study, the raw wastewater of the sewer of the oncologic in-patient treatment ward of the Vienna University Hospital was monitored for 98 d over 2 years for the cytostatics 5-fluorouracil (5-FU), doxorubicin (DOX), epirubicin, and daunorubicin. In a next step, the elimination of the drugs by a membrane-bio-reactor system was investigated. In addition, their fate in wastewater and elimination by activated sludge was investigated with radio-labelled substances. During the monitoring periods, concentration levels ranging from <8.6 to 124 microgl(-1) for 5-FU and from <0.26 to 1.35 microgl(-1) for DOX were determined. The concentrations analysed fitted the lower ranges calculated by an input-output model. Treatment of oncologic wastewater in the membrane bio-reactor as well as the analysis of the effluents of the Vienna University Hospital resulted in concentrations below the limit of detection. Investigations with radio-labelled compounds showed that 5-FU is eliminated from the liquid phase below the limit of detection. But, up to 25% of radio-labelled equivalents of the drug's amount were found in the gaseous phase and only a marginal part in the solid phase, this indicates that at least one part of the drug is biodegraded. For the anthracyclines more than 90% was eliminated from the liquid phase. In this case, adsorption to suspended solids seems to be the major elimination pathway, as up to 30% of the radio-labelled equivalents of the drug's amount was detected in the solid phase. Our results indicate that the investigated anticancer drugs are eliminated by sewage treatment plants, either by biodegradation or adsorption.

Analysis of mechanisms contributing to AraC-mediated chemoresistance and re-establishment of drug sensitivity by the novel heterodinucleoside phosphate 5-FdUrd-araC.

Chemoresistance is a biological response of cells to survive toxic stress. During cancer treatment the development of chemoresistance is a major problem. The mechanisms how cells become insensitive, and which downstream pathways are affected are not completely understood. Since it has not been well analysed which and how many regulative disorders are subsummised under the term "chemoresistance", we examined and measured arabinosylcytosine (AraC)-mediated desensitation of two mechanisms relevant for tissue homeostasis, cell cycle inhibition and apoptosis induction. MCF-7 cells harbouring ectopic mutated p53 were suitable for this investigation because they activated these mechanisms subsequently and became insensitive to AraC with regard to cell cycle inhibition and apoptosis induction. The major causal mechanism of acquired resistance against AraC was most likely through the inhibition of the first step of AraC phosphorylation within the cell, which is rate limiting for its activation. With regard to cell cycle inhibition AraC-resistant cells were also resistant against 5-fluorodeoxyuridine (5-FdUrd), but fully responsive to 5-FdUrd-induced apoptosis, evidencing that cell cycle and apoptosis are independent of each other. Apoptosis correlated with AIF-activation and was independent of Caspase 7, whereas cell cycle inhibition correlated with cyclinD1 expression but not with induction of p21 or p27. The phosphate conjugated 5-FdUrd-araC heterodimer (5-Fluoro-2'-desoxyuridylyl-(3'-->5')-Arabinocytidine), which is a prodrug of AraC-monophosphate, reactivated AIF and down-regulated cyclin D1 in AraC-resistant cells and circumvented resistance to apoptosis and to cell cycle inhibition. Also, cells which were resistant to 5-FdUrd or doxorubicin were sensitive to 5-FdUrd-araC. This investigation demonstrates that chemoresistance affects apoptosis induction and cell cycle inhibition independently and that detailed knowledge about the affected downstream pathways would enable the design of targeted intervention with small molecules to restore chemosensitivity.

Cytotoxic and apoptotic effects of novel heterodinucleoside phosphates consisting of 5-fluorodeoxyuridine and Ara-C in human cancer cell lines.

In search for possible alternatives in the treatment of human malignancies we investigated several new heterodinucleoside phosphates consisting of 5-Fluorodeoxyuridine (5-FdUrd) and Arabinofuranosylcytosine (Ara-C). We show that all dimers tested inhibited the number of colonies of CCL228, CCL227, 5-FU resistant CCL227 and HT-29 human colon tumor cells with IC50 values ranging from 0.65 to 1 nM. Dimer # 2 inhibited the number of sensitive and Ara-C resistant H9 human lymphoma cells with IC50 values ranging from 200 to 230 nM. Since no significant difference in the cytotoxicity of the dimers could be observed between sensitive and resistant cells, these compounds might be used in the treatment of 5-FU and Ara-C resistant tumors.

Penetration of capecitabine and its metabolites into malignant and healthy tissues of patients with advanced breast cancer.

Capecitabine is an oral prodrug of 5-fluorouracil (FU). Since FU concentrations achieved in malignant lesions are an important determinant of efficacy, we investigated the intratumoral transcapillary transfer of capecitabine and its metabolites in vivo. A total of 10 patients with skin metastases from breast cancer received a daily dose of 2500 mg m(-2) capecitabine administered orally in two divided doses for 2 weeks. Microdialysis probes were inserted into a cutaneous metastasis and subcutaneous connective tissue to evaluate the interstitial tissue pharmacokinetics of capecitabine and its metabolites 5'-deoxy-5-fluorocytidine (DFCR), 5'-deoxy-5-fluorouridine (DFUR), and FU by capillary electrophoresis. As intended with the prodrug design of capecitabine, FU was present in low concentrations in tumour interstitium (median c(max): 0.26 microg ml(-1)) when compared with capecitabine, DFCR, and DFUR (median c(max): 2.66, 4.22, and 2.13 microg ml(-1), respectively). Capecitabine and its metabolites easily penetrated malignant and healthy tissue and equilibrated within 45 min between plasma and tissue interstitium. Considering tissue exposure at the extracellular level, no significant differences between healthy and malignant tissues were observed. Our data show that absorption and metabolism determined the tissue pharmacokinetics of capecitabine. There was no evidence of drug tolerance, which may be attributed to impaired transcapillary transfer into tissue, even after repeated administration as shown for three patients.

On-line solid-phase extraction and determination of paclitaxel in human plasma.

The application of coupled-column liquid chromatographic analysis to pharmacokinetic studies eliminates the need for sample clean-up from plasma. Considering lipophilic antineoplastic agents, we tested this approach to analyze paclitaxel under unfavourable circumstances (i.e., weekly low-dose regimen, plasma protein binding >90%, UV detection at 229 nm). The excellent quality control data (recovery: 95.6-100.7%, inter-assay relative standard deviation on 5 days: 1.3-3.2%, accuracy: 0.9-2.7%) and the detection limit of 19 nM indicates the usefulness of this method for the analysis of paclitaxel in plasma using on-line solid-phase extraction.