Roland Sedivy

The EndoPredict score provides prognostic information on late distant metastases in ER+/HER2- breast cancer patients.

Background:ER+/HER2− breast cancers have a proclivity for late recurrence. A personalised estimate of relapse risk after 5 years of endocrine treatment can improve patient selection for extended hormonal therapy.Methods:A total of 1702 postmenopausal ER+/HER2− breast cancer patients from two adjuvant phase III trials (ABCSG6, ABCSG8) treated with 5 years of endocrine therapy participated in this study. The multigene test EndoPredict (EP) and the EPclin score (which combines EP with tumour size and nodal status) were predefined in independent training cohorts. All patients were retrospectively assigned to risk categories based on gene expression and on clinical parameters. The primary end point was distant metastasis (DM). Kaplan–Meier method and Cox regression analysis were used in an early (0–5 years) and late time interval (>5 years post diagnosis).Results:EP is a significant, independent, prognostic parameter in the early and late time interval. The expression levels of proliferative and ER signalling genes contribute differentially to the underlying biology of early and late DM. The EPclin stratified 64% of patients at risk after 5 years into a low-risk subgroup with an absolute 1.8% of late DM at 10 years of follow-up.Conclusion:The EP test provides additional prognostic information for the identification of early and late DM beyond what can be achieved by combining the commonly used clinical parameters. The EPclin reliably identified a subgroup of patients who have an excellent long-term prognosis after 5 years of endocrine therapy. The side effects of extended therapy should be weighed against this projected outcome.

EndoPredict improves the prognostic classification derived from common clinical guidelines in ER-positive, HER2-negative early breast cancer

Background In early estrogen receptor (ER)-positive/HER2-negative breast cancer, the decision to administer chemotherapy is largely based on prognostic criteria. The combined molecular/clinical EndoPredict test (EPclin) has been validated to accurately assess prognosis in this population. In this study, the clinical relevance of EPclin in relation to well-established clinical guidelines is assessed. Patients and methods We assigned risk groups to 1702 ER-positive/HER2-negative postmenopausal women from two large phase III trials treated only with endocrine therapy. Prognosis was assigned according to National Comprehensive Cancer Center Network-, German S3-, St Gallen guidelines and the EPclin. Prognostic groups were compared using the Kaplan–Meier survival analysis. Results After 10 years, absolute risk reductions (ARR) between the high- and low-risk groups ranged from 6.9% to 11.2% if assigned according to guidelines. It was at 18.7% for EPclin. EPclin reassigned 58%–61% of women classified as high-/intermediate-risk (according to clinical guidelines) to low risk. Women reclassified to low risk showed a 5% rate of distant metastasis at 10 years. Conclusion The EPclin score is able to predict favorable prognosis in a majority of patients that clinical guidelines would assign to intermediate or high risk. EPclin may reduce the indications for chemotherapy in ER-positive postmenopausal women with a limited number of clinical risk factors.

Inhibition of tumor cell growth by antibodies induced after vaccination with peptides derived from the extracellular domain of Her‐2/neu

The anti Her‐2/neu monoclonal antibody Trastuzumab has strong inhibiting effects on tumor growth in vitro and in vivo and is therefore used for immunotherapy in breast cancer patients. Due to necessity of frequent applications, however, cost intensiveness of Trastuzumab treatment and its limited duration of affectivity, an active immunization inducing a perhaps preventive and long‐term immunity to Her‐2/neu remains a desirable goal. We attempted to induce anti Her‐2/neu antibodies by peptide vaccination and to test their efficacy in inhibiting tumor cell growth in vitro. By computer aided analyses, 7 putative B cell epitopes of Her‐2/neu were defined and synthesized. These peptide epitopes were coupled to tetanus toxoid and used for immunization in BALB/c mice. Among these peptides, immunizations with 2 single peptides or a combination of 2 peptides induced anti‐peptide antibody levels, primarily of the IgG1 isotype. These antibodies were also directed against the native Her‐2/neu antigen, as shown in precipitation assays and ELISA with cell lysates of the Her‐2/neu overexpressing breast cancer cell line SK‐BR‐3. Isolated IgG fractions from immune sera incubated with SK‐BR‐3 cells led to a moderate inhibition of the tumor cell growth in vitro, as well as to complement dependent cell lyses comparable to that achieved by incubation with Trastuzumab. Moreover, peptide immunization in rabbits generated anti‐Her 2‐neu IgG that, in contrast to mouse sera, were able to mediate a 31–46% lysis of SK‐BR‐3 cells in ADCC experiments. We conclude from our data that immunization with Her‐2/neu peptides successfully induced humoral immune response with anti‐tumor activity in an animal model.

Dissecting progressive stages of 5-fluorouracil resistance in vitro using RNA expression profiling

Resistance to anticancer drugs such as the widely used antimetabolite 5‐fluorouracil (FU) is one of the most important obstacles to cancer chemotherapy. Using GeneChip arrays, we compared the expression profile of different stages of FU resistance in colon cancer cells after in vitro selection of low‐, intermediate‐ and high‐resistance phenotypes. Drug resistance was associated with significant changes in expression of 330 genes, mainly during early or intermediate stage. Functional annotation revealed a majority of genes involved in signal transduction, cell adhesion and cytoskeleton with subsequent alterations in apoptotic response, cell cycle control, drug transport, fluoropyrimidine metabolism and DNA repair. A set of 33 genes distinguished all resistant subclones from sensitive progenitor cells. In the early stage, downregulation of collagens and keratins, together with upregulation of profilin 2 and ICAM‐2, suggested cytoskeletal changes and cell adhesion remodeling. Interestingly, 6 members of the S100 calcium‐binding protein family were suppressed. Acquisition of the intermediate‐resistance phenotype included upregulation of the well‐known drug resistance gene ABCC6 (ATP‐binding cassette subfamily C member 6). The very small number of genes affected during transition to high resistance included the primary FU target thymidylate synthase. Although limited to an in vitro model, our data suggest that resistance to FU cannot be explained by known mechanisms alone and substantially involves a wide molecular repertoire. This study emphasizes the understanding of resistance as a time‐depending process: the cell is particularly challenged at the beginning of this process, while acquisition of the high‐resistance phenotype seems to be less demanding.

Apoptosis in human colorectal tumours: ultrastructure and quantitative studies on tissue localization and association with bak expression

Abstract Apoptotic cell death in human tumours has been demonstrated by electron and light microscopy. In adenomas, fragmented and apoptotic nuclei and signs of phagocytosis have been observed close to the basement membrane. In carcinomas the characteristic structures were apoptotic bodies with small fragments of chromatin. DNA fragmentation was shown by in situ end-labelling. Quantitative assessment of apoptosis and proliferation revealed a high apoptotic index (AI) in all types of adenoma (tubular: 1.77±0.35%, tubulovillous: 2.38± 0.41%; villous: 3.3±0.39%) as well as loss of compartmentalization of proliferating and dying cells. In carcinomas a shift towards proliferation was evident, as shown by lower AIs than in adenomas (0.9±0.68% and 1.1±0.12% for moderately and poorly differentiated tumours), higher Ki67 indices (38.32±2.23% and 57± 3.89%, respectively) and higher mitosis (0.9±0.56% and 1.21±0.17%, respectively). However, apoptosis was observed in all tumours and is available as a target for therapeutic intervention. Expression of the apoptosis related proteins bcl-2 and bak also reflected loss of compartmentalization. While bcl-2 did not show a consistent relationship to AI in tumour specimens, bak was positively correlated with apoptosis in 4 of 8 adenomas and 4 of 7 carcinomas, suggesting a role for this protein in the induction of apoptosis in a subset of tumours.

Short‐term rhythmic proliferation of human breast cancer cell lines: surface effects and fractal growth patterns

Kinetic studies of cell proliferation rates shed light on the growth dynamics of cancer. Most such studies are based on measurements of cell numbers that were evaluated in time intervals of about 12 h. Studies of the initial tumour growth with short measuring intervals are rare. This study was therefore designed with 1 h measuring intervals over a 24 h period. Human breast cancer cell lines (ZR‐75‐1, SK‐BR‐3, MCF‐7) and a benign cell line (HBL‐100) were used to study the hourly thymidine uptake as a measure of cells in synthesis. In parallel experiments, the same cell lines were also exposed to tumour necrosis factor alpha (TNF‐α) to explore the effect of an apoptosis‐inducing substance on initial tumour growth kinetics. In time‐evolution plots, there was an oscillation of the labelling index of thymidine uptake for all investigated cell lines, with and without TNF‐α. Based on the results obtained, a mathematical model was developed mimicking the real experiment. To describe the system dynamically a cellular automaton model was studied. The growth kinetics revealed by the simulation were in accordance with our experimental data. Two‐ and three‐dimensional growth simulations of this computer model yielded objects morphologically similar to real images of human breast cancer. Almost identical fractal dimensions of the virtual and real tumours further supported this visual similarity. The cellular automata models could, therefore, be seen as a bridge towards realistic in vivo scenarios. From a clinical point of view, the results obtained may be applicable not only to primary tumours, but even to tumour cell microfoci and small metastases, which are a major concern in early metastasizing tumours such as breast cancer. Copyright

Anomalous diffusion on dynamical networks: a model for interacting epithelial cell migration

We propose a model for cell migration where epithelial cells are able to detect trajectories of other cells and try to follow them. As cells move along in 2D cell culture, they mark their paths by loosing tiny parts of cytoplasm. Any cell moving on a surface where other cells have moved before faces a network of cell trajectories, which it tries to restrict its motion onto. With the Tsallis modification of classical thermodynamics one can solve the relevant Fokker–Planck like equation and obtain experimentally testable distribution functions. We compare the model to experimental data of normal mammary epithelial cells and cells which have been genetically manipulated to change their cell–cell interaction.

Histopathology of columnar-lined esophagus in patients with gastroesophageal reflux disease

ZusammenfassungHINTERGRUND: Es herrscht Uneinigkeit darüber, ob ein endoskopisch normal erscheinender gastroösophagealer Übergang bei Patienten mit gastroösophagealer Refluxkrankheit biopsiert werden soll. Wir beschäftigten uns mit dieser Frage und setzten Videoendoskopie und Histopatholgie ein. METHODEN: Bei 114 aufeinander folgenden Patienten (58 männliche) mit Symptomen der gastroösophagealen Refluxkrankheit wurde die Endoskopie inklusive Biopsien aus dem gastroösophagealen Übergang prospektiv dokumentiert. Das Vorliegen einer magenartigen Schleimhaut proximal des Anstiegs der Magenfalten wurde als endoskopisch sichtbares Zylinderepithelsegment definiert. Die Histopathologie wurde entsprechend der Paull-Chandrasoma Klassifikation durchgeführt. ERGEBNISSE: 85 Patienten (74,6%) hatten ein endoskopisch sichtbares Zylinderepithelsegment mit Längen von ≤0,5 cm (n = 82), 1 cm (n = 2) und 7 cm (n = 1). 29 Patienten (25,4%) hatten eine endoskopisch normal erscheinende Schleimhautgrenze. Histopathologisch hatten alle Patienten einen Zylinderepithel-Ösophagus. Eine intestinale Metaplasie und eine niedrig-gradige Dysplasie wurden bei 26 (22,8%) bzw. bei 5 (4,4%) identifiziert. Die Häufigkeit der intestinalen Metaplasie und der Dysplasie war zwischen endoskopisch sichtbarem Zylinderepithel-Ösophagus und normalem Übergang statistisch nicht signifikant unterschiedlich (p = 0,408 bzw. p = 0,775). Das Auftreten der intestinalen Metaplasie war unabhängig von einer Ösophagitis und einer Hiatushernie (p = 0,398 bzw. p = 0,405). ZUSAMMENFASSUNG: Ein Zylinderepithel-Ösophagus kann durch die Endoskopie nicht ausgeschlossen werden. Bei Patienten mit gastrosösophagealer Refluxkrankheit werden zum histopathologischen Ausschluss einer intestinalen Metaplasie und einer niedriggradigen Dysplasie Biopsien aus einem normal erscheinenden Schleimhautübergang empfohlen.SummaryBACKGROUND AND AIMS: The question of whether an endoscopically normal-appearing esophagogastric junction should be biopsied in patients with gastroesophageal reflux disease is controversial. We have addressed this issue using endoscopy and histopathology. METHODS: A total of 114 consecutive patients (58 males) with symptoms of gastroesophageal reflux disease prospectively underwent endoscopy, including biopsy sampling from the esophagogastric junction. Endoscopically visible columnar-lined esophagus was defined by the presence of gastric-type mucosa above the level of the rise of the gastric folds. Histopathology was conducted using the Paull-Chandrasoma classification. RESULTS: Of the 114 patients, 85 (74.6%) had endoscopically visible columnar-lined esophagus of length ≤0.5 cm (n = 82), 1 cm (n = 2) and 7 cm (n = 1); 29 patients (25.4%) had a normal endoscopic junction. All patients had histopathologic columnar-lined esophagus. Intestinal metaplasia and low-grade dysplasia was identified in 26 (22.8%) and 5 (4.4%) individuals, respectively, and was not statistically different in endoscopically normal vs. abnormal junction (P = 0.408 for intestinal metaplasia, P = 0.775 for low grade dysplasia). Intestinal metaplasia was independent from endoscopic esophagitis (P = 0.398) and hiatal hernia (P = 0.405). CONCLUSIONS: Columnar-lined esophagus cannot be excluded by endoscopy. In patients with gastroesophageal reflux disease, biopsy sampling of normal-appearing junction is recommended for histopathologic exclusion of intestinal metaplasia and low-grade dysplasia.

Quantitative measurement of cell migration using time-lapse videomicroscopy and non-linear system analysis

Epithelial cells of the mammary gland possess the inherent capacity to form epithelial monolayers in vitro. This requires coordination of cell migration, cell–cell contact formation, and cell proliferation. Using time-lapse phase contrast videomicroscopy we have observed mammary gland epithelial cells over different time scales. We show the generation of a complete polarized epithelial monolayer in real-time, starting from a few cells. We subsequently concentrated on the early stages of this process by tracking epithelial cells during phases of polarized migration. We performed migration analysis using fractal measures. With this technology the structure of seemingly random processes not accessible to the usual methods of linear analysis can be measured. As a control and proof of principle approach we applied infection of cells with an adenoviral vector, which is used as a gene targeting vector for many applications. Infection markedly influenced the patterns of migratory behavior. We, therefore, believe that time-lapse videomicroscopy in combination with fractal analysis can contribute to differential characterization of distinct cellular migration patterns. This will be useful in situations of long-term alterations in cell culture systems.

Atherosclerosis as a paradigmatic disease of the elderly: role of the immune system.

When a new hypothesis about the etiology and pathogenesis of a disease is developed, there is always the danger that it will be presented as the only acceptable explanation for the occurrence of a given pathologic condition. In view of the well-proven multifactoral pathogenesis of atherosclerosis, we would like to emphasize that we are not postulating that immunity to HSP60 is the only cause of atherogenesis, especially in the later stages where there are clinically-apparent sequelae, such as myocardial infarction, stroke, and other atherosclerosis-dependent symptoms. In this article, we summarized some of the experimental and clinical data that we and others have collected in support of the concept that atherosclerosis is a good example of pleotropic antagonism, and postulated that age-dependent diseases are the price we pay for genetic traits established by natural selection to assure maximum survival until the age of reproduction, the effects of which may, however, become deleterious later in life. In the present case, the cost we pay for protective immunity to microbial and altered autologous HSP60 is the risk of cross-reactivity with HSP60 expressed by arterial endothelial cells that are subjected to stress factors already known as classical atherosclerosis risk factors. We showed that the first inflammatory stage of atherosclerosis starts early in life, long before it becomes clinically apparent. More severe lesions that lead to atherosclerosis-dependent organ-specific or systemic symptoms will only occur if classical atherosclerosis risk factors, especially those involving the cholesterol metabolism, remain present.