Andrea Sassen

Cytogenetic analysis of HER1/EGFR, HER2, HER3 and HER4 in 278 breast cancer patients

IntroductionThe HER (human EGFR related) family of receptor tyrosine kinases (HER1/EGFR (epidermal growth factor receptor)/c-erbB1, HER2/c-erbB2, HER3/c-erbB3 and HER4/c-erbB4) shares a high degree of structural and functional homology. It constitutes a complex network, coupling various extracellular ligands to intracellular signal transduction pathways resulting in receptor interaction and cross-activation. The most famous family member is HER2, which is a target in Herceptin™ therapy in metastatic status and also in adjuvant therapy of breast cancer in the event of dysregulation as a result of gene amplification and resulting protein overexpression. The HER2-related HER receptors have been shown to interact directly with HER2 receptors and thereby mutually affect their activity and subsequent malignant growth potential. However, the clinical outcome with regard to total HER receptor state remains largely unknown.MethodsWe investigated HER1–HER4, at both the DNA and the protein level, using fluorescence in situ hybridisation (FISH) probes targeted to all four receptor loci and also immunohistochemistry in tissue microarrays derived from 278 breast cancer patients.ResultsWe retrospectively found HER3 gene amplification with a univariate negative impact on disease-free survival (hazard ratio 2.35, 95% confidence interval 1.08 to 5.11, p = 0.031), whereas HER4 amplification showed a positive trend in overall and disease-free survival. Protein expression revealed no additional information.ConclusionOverall, the simultaneous quantification of HER3 and HER4 receptor genes by means of FISH might enable the rendering of a more precise stratification of breast cancer patients by providing additional prognostic information. The continuation of explorative and prospective studies on all HER receptors will be required for an evaluation of their potential use for specific therapeutic targeting with respect to individualised therapy.

Presence of HER4 associates with increased sensitivity to Herceptin™ in patients with metastatic breast cancer

IntroductionHER2 overexpression, or rather HER2 gene amplification, is indicative for Herceptin therapy in both metastatic and pre-metastatic breast cancer patients. Patients individual sensitivity to Herceptin treatment, however, varies enormously and spans from effectual responsiveness over acquired insensitivity to complete resistance from the outset. Thus no predictive information can be deduced from HER2 determination so that molecular biomarkers indicative for Herceptin sensitivity or resistance need to be identified. Both ErbB receptor-dependent signalling molecules as well as HER2-related ErbB receptor tyrosine kinases, known to mutually interact and to cross-regulate each other are prime candidates to be involved in cellular susceptibility to Herceptin.MethodsUsing immunohistochemistry and fluorescence in situ hybridisation, we retrospectively investigated primary breast cancer tissues from 48 patients who were under Herceptin treatment. We quantified the gene copy numbers of all HER receptors and evaluated their coexpression profile. Moreover the HER2 phosphorylation state, the ratio of native to truncated HER2, p27(kip1) and PTEN expression were objects of this study.ResultsAbove all markers investigated in this study Kaplan-Meier and Cox regression analysis revealed a significant positive impact of HER4 (co-)expression on overall survival from beginning of antibody therapy. Both HER4 expression and HER4 gene amplification emerged as independent prognostic markers in Herceptin-treated breast cancer patients and responsiveness to Herceptin turned out to be more efficient if tumour cells show HER4 expression.ConclusionsAlthough HER4 is known to potentially exert a tumour cell killing activity and in turn to have a favourable impact in breast cancer patients we demonstrate here the first time that HER4 expression prolongs overall survival in Herceptin-treated patients. Elucidating HER4 receptor function in the context of Herceptin treatment will advance the design of highly efficient receptor targeting. By then we need to extend the analysis of breast cancer by allowing for HER2/HER4 coexpression by which valuable additional prognostic and predictive information might possibly be revealed.

Trägt Nikotin zur Krebsentstehung im oberen Aerodigestivtrakt bei

BACKGROUND It is accepted that nicotine in tobacco smoke causes addiction via nicotinic acetylcholine receptors in the central nervous system. For a long time, the tumorigenic potential of smoking was attributed to compounds other than nicotine. However, more recently data have accumulated which suggest that nicotine may add to the cancer risk by stimulating cellular growth via non-neuronal acetylcholine receptors, by suppressing apoptosis, and by inducing angiogenesis not only in atheromatous plaques but also in tumors. In the present study the possible direct genotoxic effects of nicotine on DNA were investigated in human target cells of carcinogenesis in the upper aerodigestive tract. PATIENTS AND METHODS Human nasal mucosa, lymphatic tissue of the palatine tonsils, supraglottic epithelium of the larynx, and peripheral lymphocytes were exposed to rising concentrations of nicotine. DNA damage was investigated by alkaline single-cell microgel electrophoresis (Comet) assay. Cytotoxicity was assessed by trypan blue exclusion. RESULTS Nicotine induced dose-dependent DNA damage in all cell types at low cytotoxic concentrations that allowed viabilities well above 80%. The lowest nicotine concentrations eliciting a significant increase in DNA migration were 1 mM for tonsillar cells and 0.25 mM for all other cell types. CONCLUSION Nicotine induces genotoxic effects in human target cells of carcinogenesis in the upper aerodigestive tract at relevant concentrations. Thus, nicotine may contribute directly to tumor initiation resulting from smoking.

[Mini-organ cultures of human nasal mucosa. A model for eco-genotoxicological investigations].

ZusammenfassungHintergrundFür inhalative und ingestive Umwelt- und Arbeitsstoffe ist das Epithel des oberen Aerodigestivtrakts das primäre Kontaktorgan. In diesem Zielgewebe entstehen durch Xenobiotika induzierte Karzinome. Über ein Modell epithelialer Miniorgankulturen zur Untersuchung genotoxischer Effekte wird hier berichtet.MethodeHumane Mukosafragmente der unteren Nasenmuschel wurden als Miniorgane kultiviert und ein-, zwei- und dreifach gegenüber den potenziell mutagenen Substanzen N-Nitrosodiethylamin (NDEA), Benzo[a]pyren-7,8-dihydrodiol-9,10-epoxid (BPDE) und Natriumdichromat (Na2Cr2O7) mit anschließenden Reparaturphasen exponiert. Untersucht wurde die strukturelle Integrität der Miniorgankulturen (inverse Mikroskopie, Histologie), das Auftreten von DNA-Strangbrüchen und Reparaturaktivitäten mittels Einzelzellmikrogelelektrophorese (Comet Assay), die Apoptoseinduktion über Phosphatidylserinverlagerung (Annexin V Assay) sowie die Bildung der Entzündungsparameter Interleukin 8 und Granulozyten-Makrophagen-Kolonie-stimulierender-Faktor (ELISA).ErgebnisseWährend einer zweiwöchigen Kultivierung blieben die Miniorgane strukturell intakt. Mehrfache Expositionen gegenüber NDEA und BPDE waren mit gesteigerter DNA-Migration verbunden. Na2Cr2O7 verursachte mit jeder weiteren Exposition höhere DNA-Fragmentierungen. Eine Reduzierung der DNA-Schäden im Sinne einer Reparatur war lediglich nach Exposition mit Na2Cr2O7 zu beobachten. Nach NDEA-Exposition zeigte sich keine, nach dreifacher BPDE- und Na2Cr2O7-Belastung jedoch eine signifikante Apoptoseinduktion. Entzündungsreaktionen über Veränderungen der Zytokinfreisetzung von Miniorgankulturen wurden nach NDEA-Belastung nicht nachgewiesen. Mehrfache BPDE- und Na2Cr2O7-Expositionen verursachten aber einen Rückgang der GM-CSF-Produktion, Na2Cr2O7 zusätzlich eine Reduzierung der IL-8-Freisetzung von Miniorganen.SchlussfolgerungDas dreidimensionale Miniorgankulturmodell humaner nasaler Mukosazellen konnte potenzielle Gefahrenstoffe in einer in-vivo-nahen Situation charakterisieren. Es erwies sich nicht nur für genotoxikologische, sondern auch für zytologische und immunologische Untersuchungen als geeignet und erlaubte mehrfache Expositionen mit anschließenden Reparaturphasen. Die metabolische Kompetenz dieser Organkulturen konnte durch die Produktion von Entzündungsparametern gezeigt werden.AbstractBackgroundVolatile and ingestive xenobiotics may induce cancer in the mucosa of the upper aerodigestive tract. A new model is presented combining mini-organ cultures of human mucosa and the Comet assay that allows investigation of tumor initiation steps in vitro.MethodSpecimens of human mucosa of the inferior nasal turbinates were cultured as mini-organs and exposed to xenobiotics once, twice or three times with consecutive repair intervals. The cultures were monitored for structural integrity (inverse microscopy, histology), DNA fragmentation and repair activity (Comet assay), induction of apoptosis (annexin V assay), and production of IL-8 and GM-CSF (ELISA).ResultsMini-organ cultures showed a good structural integrity during the whole culture period. Exposure to N-nitrosodiethylamine (NDEA) and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) induced significant DNA fragmentation. Sodium dichromate (Na2Cr2O7) had an additive DNA fragmentation effect with repetitive exposure. Significant DNA repair was seen after strand break induction by Na2Cr2O7, only. Apoptosis was seen after three exposures to BPDE und Na2Cr2O7,but not NDEA. Inflammatory cytokine release was unaltered by NDEA. However, BPDE and Na2Cr2O7 reduced GM-CSF and Na2Cr2O7 reduced IL-8 excretion.ConclusionThis three dimensional mini-organ culture system proved to be very helpful in characterizing volatile and ingestive xenobiotics potentially hazardous to humans. Beside the information concerning genotoxicity, it allows cytological and immunological studies. In contrast to investigations with fresh specimens, repetitive or chronic exposure to xenobiotics is possible in mucosal cells with their epithelial structural integrity. Therefore, mini-organ cultures of human upper aerodigestive tract epithelia represent a model closely resembling the in vivo situation.

Does nicotine add to the carcinogenic strain of tobacco smoke

BACKGROUND It is accepted that nicotine in tobacco smoke causes addiction via nicotinic acetylcholine receptors in the central nervous system. For a long time, the tumorigenic potential of smoking was attributed to compounds other than nicotine. However, more recently data have accumulated which suggest that nicotine may add to the cancer risk by stimulating cellular growth via non-neuronal acetylcholine receptors, by suppressing apoptosis, and by inducing angiogenesis not only in atheromatous plaques but also in tumors. In the present study the possible direct genotoxic effects of nicotine on DNA were investigated in human target cells of carcinogenesis in the upper aerodigestive tract. PATIENTS AND METHODS Human nasal mucosa, lymphatic tissue of the palatine tonsils, supraglottic epithelium of the larynx, and peripheral lymphocytes were exposed to rising concentrations of nicotine. DNA damage was investigated by alkaline single-cell microgel electrophoresis (Comet) assay. Cytotoxicity was assessed by trypan blue exclusion. RESULTS Nicotine induced dose-dependent DNA damage in all cell types at low cytotoxic concentrations that allowed viabilities well above 80%. The lowest nicotine concentrations eliciting a significant increase in DNA migration were 1 mM for tonsillar cells and 0.25 mM for all other cell types. CONCLUSION Nicotine induces genotoxic effects in human target cells of carcinogenesis in the upper aerodigestive tract at relevant concentrations. Thus, nicotine may contribute directly to tumor initiation resulting from smoking.

Miniorgankulturen humaner nasaler Mukosa

ZusammenfassungHintergrundFür inhalative und ingestive Umwelt- und Arbeitsstoffe ist das Epithel des oberen Aerodigestivtrakts das primäre Kontaktorgan. In diesem Zielgewebe entstehen durch Xenobiotika induzierte Karzinome. Über ein Modell epithelialer Miniorgankulturen zur Untersuchung genotoxischer Effekte wird hier berichtet.MethodeHumane Mukosafragmente der unteren Nasenmuschel wurden als Miniorgane kultiviert und ein-, zwei- und dreifach gegenüber den potenziell mutagenen Substanzen N-Nitrosodiethylamin (NDEA), Benzo[a]pyren-7,8-dihydrodiol-9,10-epoxid (BPDE) und Natriumdichromat (Na2Cr2O7) mit anschließenden Reparaturphasen exponiert. Untersucht wurde die strukturelle Integrität der Miniorgankulturen (inverse Mikroskopie, Histologie), das Auftreten von DNA-Strangbrüchen und Reparaturaktivitäten mittels Einzelzellmikrogelelektrophorese (Comet Assay), die Apoptoseinduktion über Phosphatidylserinverlagerung (Annexin V Assay) sowie die Bildung der Entzündungsparameter Interleukin 8 und Granulozyten-Makrophagen-Kolonie-stimulierender-Faktor (ELISA).ErgebnisseWährend einer zweiwöchigen Kultivierung blieben die Miniorgane strukturell intakt. Mehrfache Expositionen gegenüber NDEA und BPDE waren mit gesteigerter DNA-Migration verbunden. Na2Cr2O7 verursachte mit jeder weiteren Exposition höhere DNA-Fragmentierungen. Eine Reduzierung der DNA-Schäden im Sinne einer Reparatur war lediglich nach Exposition mit Na2Cr2O7 zu beobachten. Nach NDEA-Exposition zeigte sich keine, nach dreifacher BPDE- und Na2Cr2O7-Belastung jedoch eine signifikante Apoptoseinduktion. Entzündungsreaktionen über Veränderungen der Zytokinfreisetzung von Miniorgankulturen wurden nach NDEA-Belastung nicht nachgewiesen. Mehrfache BPDE- und Na2Cr2O7-Expositionen verursachten aber einen Rückgang der GM-CSF-Produktion, Na2Cr2O7 zusätzlich eine Reduzierung der IL-8-Freisetzung von Miniorganen.SchlussfolgerungDas dreidimensionale Miniorgankulturmodell humaner nasaler Mukosazellen konnte potenzielle Gefahrenstoffe in einer in-vivo-nahen Situation charakterisieren. Es erwies sich nicht nur für genotoxikologische, sondern auch für zytologische und immunologische Untersuchungen als geeignet und erlaubte mehrfache Expositionen mit anschließenden Reparaturphasen. Die metabolische Kompetenz dieser Organkulturen konnte durch die Produktion von Entzündungsparametern gezeigt werden.AbstractBackgroundVolatile and ingestive xenobiotics may induce cancer in the mucosa of the upper aerodigestive tract. A new model is presented combining mini-organ cultures of human mucosa and the Comet assay that allows investigation of tumor initiation steps in vitro.MethodSpecimens of human mucosa of the inferior nasal turbinates were cultured as mini-organs and exposed to xenobiotics once, twice or three times with consecutive repair intervals. The cultures were monitored for structural integrity (inverse microscopy, histology), DNA fragmentation and repair activity (Comet assay), induction of apoptosis (annexin V assay), and production of IL-8 and GM-CSF (ELISA).ResultsMini-organ cultures showed a good structural integrity during the whole culture period. Exposure to N-nitrosodiethylamine (NDEA) and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) induced significant DNA fragmentation. Sodium dichromate (Na2Cr2O7) had an additive DNA fragmentation effect with repetitive exposure. Significant DNA repair was seen after strand break induction by Na2Cr2O7, only. Apoptosis was seen after three exposures to BPDE und Na2Cr2O7,but not NDEA. Inflammatory cytokine release was unaltered by NDEA. However, BPDE and Na2Cr2O7 reduced GM-CSF and Na2Cr2O7 reduced IL-8 excretion.ConclusionThis three dimensional mini-organ culture system proved to be very helpful in characterizing volatile and ingestive xenobiotics potentially hazardous to humans. Beside the information concerning genotoxicity, it allows cytological and immunological studies. In contrast to investigations with fresh specimens, repetitive or chronic exposure to xenobiotics is possible in mucosal cells with their epithelial structural integrity. Therefore, mini-organ cultures of human upper aerodigestive tract epithelia represent a model closely resembling the in vivo situation.

Multichromatic phenotyping of HER receptor coexpression in breast tumor tissue samples using flow cytometry--possibilities and limitations.

The prognostic significance of HER2 expression in human breast carcinomas is beyond dispute nowadays. The HER family of receptor tyrosine kinases comprises four members (HER1/ErbB1/EGFR, HER2/ErbB2, HER3/ErbB3, and HER4/ErbB4) that act in concert via transactivation and consequently compose a functional signaling unit. Besides HER2 overexpression, coexpression of other HER receptors has substantial impact on course of disease and potential therapeutic benefit. This observation is substantiated by numerous preclinical studies and retrospective studies done on patients with breast cancer. Against this background, the quantification of all HER receptor expressions at the same time would significantly extend the information content revealed by routine diagnosis of breast cancer tissues. Moreover, the knowledge of HER receptor coexpression profiles in primary tumor samples could provide the basis to design and develop highly specific antireceptor treatment strategies. Here, we report on a simultaneous flow cytometric detection of all four HER receptors on carcinoma cells isolated from primary breast cancer tissues and separated from nonepithelial cells by cytokeratin staining. Combined with DNA, i.e. ploidy quantification, the approach resulted in a six‐parameter assay that could complement the diagnosis of a variety of diseases in which HER receptor expression has a pivotal impact on the degree of malignancy.

Trägt Nikotin zur Krebsentstehung im oberen Aerodigestivtrakt bei?@@@Does nicotine add to the carcinogenic strain of tobacco smoke?

BACKGROUND It is accepted that nicotine in tobacco smoke causes addiction via nicotinic acetylcholine receptors in the central nervous system. For a long time, the tumorigenic potential of smoking was attributed to compounds other than nicotine. However, more recently data have accumulated which suggest that nicotine may add to the cancer risk by stimulating cellular growth via non-neuronal acetylcholine receptors, by suppressing apoptosis, and by inducing angiogenesis not only in atheromatous plaques but also in tumors. In the present study the possible direct genotoxic effects of nicotine on DNA were investigated in human target cells of carcinogenesis in the upper aerodigestive tract. PATIENTS AND METHODS Human nasal mucosa, lymphatic tissue of the palatine tonsils, supraglottic epithelium of the larynx, and peripheral lymphocytes were exposed to rising concentrations of nicotine. DNA damage was investigated by alkaline single-cell microgel electrophoresis (Comet) assay. Cytotoxicity was assessed by trypan blue exclusion. RESULTS Nicotine induced dose-dependent DNA damage in all cell types at low cytotoxic concentrations that allowed viabilities well above 80%. The lowest nicotine concentrations eliciting a significant increase in DNA migration were 1 mM for tonsillar cells and 0.25 mM for all other cell types. CONCLUSION Nicotine induces genotoxic effects in human target cells of carcinogenesis in the upper aerodigestive tract at relevant concentrations. Thus, nicotine may contribute directly to tumor initiation resulting from smoking.