Torsten Kroll

Monitoring the Response of Circulating Epithelial Tumor Cells to Adjuvant Chemotherapy in Breast Cancer Allows Detection of Patients at Risk of Early Relapse

PURPOSE To demonstrate that it is possible to monitor the response to adjuvant therapy by repeated analysis of circulating epithelial tumor cells (CETCs) and to detect patients early who are at risk of relapse. PATIENTS AND METHODS In 91 nonmetastatic primary breast cancer patients, CETCs were quantified using laser scanning cytometry of anti-epithelial cell adhesion molecule-stained epithelial cells from whole unseparated blood before and during adjuvant chemotherapy. RESULTS Numbers of CETCs were analyzed before therapy, before each new cycle, and at the end of chemotherapy. The following three typical patterns of response were observed: (1) decrease in cell numbers (> 10-fold); (2) marginal changes in cell numbers (< 10-fold); and (3) an (sometimes saw-toothed) increase or an initial decrease with subsequent reincrease (> 10-fold) in numbers of CETCs. Twenty relapses (22%) were observed within the accrual time of 40 months, including one of 28 patients from response group 1, five of 30 patients from response group 2, and 14 of 33 patients from response group 3. The difference in relapse-free survival was highly significant for CETC (hazard ratio = 4.407; 95% CI, 1.739 to 9.418; P < .001) between patients with decreasing cell numbers and those with marginal changes and between patients with marginal changes and those with an increase of more than 10-fold (linear Cox regression model). CONCLUSION These results show that peripherally circulating tumor cells are influenced by systemic chemotherapy and that an increase (even after initial response to therapy) of 10-fold or more at the end of therapy is a strong predictor of relapse and a surrogate marker for the aggressiveness of the tumor cells.

Gene expression profiling reveals effects of Cimicifuga racemosa (L.) NUTT. (black cohosh) on the estrogen receptor positive human breast cancer cell line MCF-7

BackgroundExtracts from the rhizome of Cimicifuga racemosa (black cohosh) are increasingly popular as herbal alternative to hormone replacement therapy (HRT) for the alleviation of postmenopausal disorders. However, the molecular mode of action and the active principles are presently not clear. Previously published data have been largely contradictory. We, therefore, investigated the effects of a lipophilic black cohosh rhizome extract and cycloartane-type triterpenoids on the estrogen receptor positive human breast cancer cell line MCF-7.ResultsBoth extract and purified compounds clearly inhibited cellular proliferation. Gene expression profiling with the extract allowed us to identify 431 regulated genes with high significance. The extract induced expression pattern differed from those of 17β-estradiol or the estrogen receptor antagonist tamoxifen. We observed a significant enrichment of genes in an anti-proliferative and apoptosis-sensitizing manner, as well as an increase of mRNAs coding for gene products involved in several stress response pathways. These functional groups were highly overrepresented among all regulated genes. Also several transcripts coding for oxidoreductases were induced, as for example the cytochrome P450 family members 1A1 and 1B1. In addition, some transcripts associated with antitumor but also tumor-promoting activity were regulated. Real-Time RT-PCR analysis of 13 selected genes was conducted after treatment with purified compounds – the cycloartane-type triterpene glycoside actein and triterpene aglycons – showing similar expression levels compared to the extract.ConclusionNo estrogenic but antiproliferative and proapoptotic gene expression was shown for black cohosh in MCF-7 cells at the transcriptional level. The effects may be results of the activation of different pathways. The cycloartane glycosides and – for the first time – their aglycons could be identified as an active principle in black cohosh.

Sugar-Selective Enrichment of a D-Glucose-Substituted Ruthenium Bipyridyl Complex Inside HepG2 Cancer Cells

Ruthenium bipyridyl (bpy) complexes display luminescent properties and a rich photochemistry associated with the visible-light-induced formation of a long-lived MLCT state (t up to 1 ms), which can be efficiently quenched by oxygen under the formation of singlet oxygen (F=0.86). These properties render the complexes interesting candidates for fluorescent sensors and photodynamic-therapy applications. However, the in vivo potential of these complexes depends heavily upon both the molecular (sensor applications) and the cellular (photodynamic therapy applications) recognition of the luminescent probes. In addition, Ru(bpy)3 -type complexes are positively charged ions and therefore possess only limited cellmembrane permeability for intact cells ; this significantly limits their potential applications. It has been shown recently that the latter limitation can be overcome by using more lipophilic derivatives such as Ru(DIP)2dppz 2+ (DIP=4,7-diphenyl-1,10phenanthroline, dppz=dipyridophenazine). Cellular uptake into HeLa cells by nonspecific passive diffusion could be observed by using confocal laser scanning microscopy (CLSM) and flow cytometry. 6] A dinuclear Ru complex was observed to bind to DNA in cellulo. Modification of bipyridyl ruthenium complexes in the 4,4’-position can influence their allocation in cellular matrices as shown for a recently reported 4,4’-di(N,Ndiethyl-amino)-substituted homoleptic compound that selectively binds to the cell membrane. One further example towards the design of biosensors is the modification of bipyridyl ligands with biotin, which acts as a binding site for avidin. Carbohydrate-modified complexes present a highly interesting alternative, as it has been shown that their presence enhances the specificity of the interaction with biological probes. These luminescent sensors for the recognition of lectins were tested in vitro as probes for the influenza virus and show different lectin affinities and amplified luminescence depending on the spacer used. Other advantages of the introduction of these groups include increased biocompatibility, a reduction of toxicity and utilization of the targeting properties of sugar-specific receptors or metabolic pathways. Sugar-containing metal complexes are therefore increasingly examined. Recently, mannosylated bipyridyl based ligands with branched spacers possessing a dendrimeric structure were used to obtain Ru complexes. Under irradiation these complexes were able to produce singlet oxygen and to act as lectin biosensors, in particular if an appropriately attached quencher was displaced by the binding lectin. However, while molecular selectivity can be introduced, cellular selectively has so far not been demonstrated. In this contribution we present the synthesis of new ruthenium polypyridyl complexes with peripherally attached sugar substituents. These complexes show, for the first time, a distinct uptake into cancer cells but still retain their luminescent properties. In all known examples the linkages between saccharides and metal-chelating units was realized by using a biodegradable amide or O-glycosidic bonds with flexible spacers; these compounds focused on lectin binding and did not address cellular uptake. To avoid lectin binding in serum or on cell surfaces, the carbohydrates in the compounds reported in this work were bound close to the bipyridine. For biodistribution studies of the intact glycoconjugates we synthesized S-glycosylated disubstituted bipyridines (L1–L3) because it is known that Sglycosidic bonds resist endogenous hydrolysis catalysed by ubiquitous glycosidases. The symmetric ligands were complexed to Ru by using Ru(DMSO)2Cl2 under reflux in water. This results in the formation of compact “ball-shaped” ruthenium complexes of the general formula Ru(L)3Cl2 (Scheme 1). After purification utilizing a Sephadex LH-20 column, the complexes were obtained as red powders. For comparison, the complex Ru(dmbpy)3Cl2 from 4,4’-dimethyl-2,2’-bipyridine (dmbpy) was synthesized by using the same reaction conditions. [a] Dr. M. Gottschaldt, Prof. U. S. Schubert Laboratory for Organic and Macromolecular Chemistry, Friedrich-Schiller-University Jena Humboldtstrasse 10, 07743 Jena (Germany) Fax: (+49)3641-948202 E-mail : michael.gottschaldt@uni-jena.de [b] Prof. U. S. Schubert Laboratory of Macromolecular Chemistry and Nanoscience, Eindhoven University of Technology Den Dolech 2, 5600MB Eindhoven (The Netherlands) [c] Prof. S. Rau Friedrich Alexander Universit t Erlangen–N rnberg Department for Chemistry and Pharmacy Egerlandstrasse 1, 91058 Erlangen (Germany) [d] Prof. S. Yano Innovative Collaboration Center, Kyoto University Nishikyo-ku, Kyoto-daigaku Katsura, Kyoto 615-8520 (Japan) [e] Prof. J. G. Vos Solar Energy Conversion SRC, School of Chemical Sciences Dublin City University Dublin 9 (Ireland) [f] Dr. T. Kroll, Dr. J. Clement Clinics of Internal Medicine, University Hospital Jena Erlanger Allee 101, 07747 Jena (Germany) [g] Prof. I. Hilger Institute of Diagnostic and Interventional Radiology University Hospital Jena Erlanger Allee 101, 07747 Jena (Germany) E-mail : ingrid.hilger@med.uni-jena.de Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.200900769.

Effects of Leuzea carthamoides on human breast adenocarcinoma MCF-7 cells determined by gene expression profiling and functional assays.

Products derived from roots of Leuzea carthamoides (Maral root) are being promoted as dietary supplements with anti-aging, adaptogenic and anabolic activity, without much scientific evidence. We investigated the effects of a lipophilic Leuzea root extract and the major phytoecdysteroid, 20-hydroxyecdysone, in human breast adenocarcinoma MCF-7 cells. Cell proliferation was inhibited by the extract (IC50 = 30 microg/mL) but not by 20-hydroxyecdysone. Genome-wide expression profiling using Affymetrix HG U133 Plus 2.0 microarrays was carried out to analyse effects at the transcriptional level. 241 genes appeared to be differentially expressed after Leuzea treatment, more than after treatment with either 17beta-estradiol or tamoxifen. Transcripts linked to cell cycle regulation and DNA replication were highly over-represented and regulated in an anti-proliferative manner. Genes involved in apoptosis were regulated in a pro-apoptotic manner. Expression levels of several oxidoreductase transcripts were strongly induced, most prominent CYP1A1, known to be regulated via the aryl hydrocarbon receptor pathway. An XRE-dependent reporter gene assay confirmed the AhR-agonistic activity of the Leuzea root extract, whereas 20-hydroxyecdysone was not active. Leuzea extract also inhibited 5alpha-reductase, type II. While the extract significantly modulates cellular activities, the phytoecdysteroids, are most likely not the active principles of L. carthamoides.

Monitoring circulating epithelial tumor cells (CETCs) during primary systemic chemotherapy including trastuzumab for early prediction of outcome in patients with Her2/neu-positive tumors

In Her2/neu growth factor overexpressing poor prognosis breast cancers trastuzumab has shown to improve disease-free survival as well as overall survival in metastatic and primary diseases. On the basis of these positive results, trastuzumab has also been included into studies of neo-adjuvant therapy of Her2/neu-positive T2 tumors [1].

Monitoring Therapy with Gene Expression Profiling Reveals Physiological Differences in Drug Action

Gene expression profiling has become a versatile tool for biomedical research, which allows the assessment of a wide variety of basic questions in cellular regulation, in particular when a large number of molecular parameters have changed. There are various applications in drug research for which gene expression profiling is a very suitable approach. This includes: target identification, target validation, validation of drug specificity and monitoring of drug action during therapy. The focus of this article is the therapy monitoring and the interpretation of the gene expression profiles in respect to physiological differences of drug action. As an example, we will discuss changes in gene expression in blood samples from CML patients treated with the tyrosine kinase inhibitor (imatinib mesylate) and compare the observed effects on gene expression with the effects of IFNalpha treatment. In comparison with other examples of therapy monitoring the potential of this application of gene expression profiling for optimizing individual therapy will be discussed.

Abstract P1-07-16: Liver derived epithelial cells as source of false positive circulating tumor cells in early breast cancer

The MAINTRAC technique as introduced by our coworkers from Jena (RBC lysis, fluorometric detection and analysis on Olympius ScanR) detects more circulating epithelial cells than techniques using enrichment. Also cells with a low EPCAM expression are detected and not only the typical cells with bright expression found after immunomagnetic enrichment. The relative cheapness and reproducibility allows frequent monitoring during and after therapy Using 3 colour detection (EPCAMfitc, DAPI, Vimentin PE) living and dead circulating epithelial cells in EMT, or cells in EMT with stemcell markers (EPCAMfitc, Vimentin-PE, CD44PacBlue) can be detected. In early breast cancer (n = 135) cells can be found in 60% of patients and in 40% higher cell counts (>100 ml are detectable. A control population(n = 100) showed low numbers in 98% (e.g ( Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-07-16.

Abstract A2: Circulating epithelial tumor cells (CETC) for molecular analysis, chemo sensitivity testing in vitro and therapy monitoring in vivo

Most breast cancer patients do not die of their primary tumor but from metastases developing sometimes years after the primary tumor has been removed. The first spread of tumor cells detectable with conventional methods is in the lymph nodes and patients with lymph node positive disease have a poorer disease free survival than patients without affected lymph nodes. Cells from the tumor seem also to be disseminated continuously into blood during tumor growth and an increase in circulating epithelial tumor cell (CETC) numbers during adjuvant therapy is correlated with an 11- 16 fold increase in hazard of relapse. A reincrease in numbers indicates increasing resistance to the applied drugs. Therefore, it would be desirable to better characterize these cells, test them for alternative drugs and monitor the actual response to the therapy decided on. For this purpose we have developed a method to isolate individual CETC from blood from breast cancer patients and to successfully perform molecular characterization. Cells from the same sample were subject to chemo sensitivity testing and the drugs with the highest effectivity defined. Patients were subsequently treated according to guide lines. The molecular characteristics, sensitivity to the applied drugs and response of the CETC to these drugs were subsequently compared to clinical outcome. The response of CETC to therapy correlated highly with the previously tested chemo sensitivity and with the reduction of an index metastasis in metastatic breast cancer patients and progression free survival. The molecular characteristics of these cells are under investigation. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A2

Abstract P3-10-37: Molecular Analysis of Single Circulating Tumor Cells for Characterization of the Targets of Systemic Breast Cancer Therapy as Chance to Individualize Therapy

Background: In breast cancer therapy predictive and prognostic markers are derived from the primary tumor but systemic therapy aims at eliminating the remnant tumor cells left in the body after surgery which the cells circulating in peripheral blood are part of. For individualized therapy, therefore, it would be advantageous to better characterize this remnant disease for more targeted therapeutic approaches. Materials and Methods: 10 to 20 single live epithelial antigen positive cells were isolated from peripheral blood of 50 newly diagnosed breast cancer patients using the automated capillary cell isolation system of the MMI CellEctor and individually deposited under visual control onto Ampli Grid slides (Advalytics). The mRNA was the reverse transcribed and amplified using primers for 6 different tumor associated targets among them EpCAM and Her2/neu. Results: Expression profiling could be successfully performed from more than 80% of all individually deposited isolated cells demonstrating the epithelial nature of these cells but also heterogeneity among the cells of in individual patients with respect to other genes. Thus we show that circulating epithelial cells from breast cancer patients can be individually deposited and these single celsl can subsequently be subject to expression profiling. Discussion: Further analysis by high through-put profiling of the remnant tumor burden in breast cancer patients after surgery comprising the cells circulating in peripheral blood may contribute to better characterize the targets of systemic therapy in order to individualize cancer therapy. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-37.

Circulating Epithelial Cells as a Tool for Monitoring Treatment Success of Primary Chemotherapy with Simultaneous or Sequential and without Trastuzumab.

In spite of ample prognostic markers available in breast cancer, still a considerable proportion of patients with good prognostic markers suffers relapse whereas patients with poor prognostic markers may remain disease free. It would, therefore, be desirable to control, at the individual patient level, whether the applied therapy is effective. Our previous work indicates, that in cancer patients most of the epithelial cells circulating in peripheral blood (CETC) are part of the tumor and that the response of these cells reflects the response of the tumor to the applied therapies.Therefore monitoring the decrease or increase in numbers of these cells providing a unique tool for therapy surveillance was used to monitor neoadjuvant chemotherapy in 26 her2/neu positive breast cancer patients with either IHC3+ or FISH confirmed Her2/neu positive breast cancer. Patients were prospectively analysed for the number of CETC before therapy, before each new cycle of chemotherapy and during maintenance therapy at each visit initially every three months and subsequently at more extended intervals. 1ml of blood was drawn into EDTA vials, red blood cells lysed and the white blood cell pellet stained with FITC-labelled anti-Epcam. Green fluorescent cells were detected by image analysis and dead cells excluded due to red PI fluorescence.After an initial variable reduction of CETC during neoadjuvant chemotherapy, tightly connected to tumor reduction we regularly observed a massive release of cells from the shrinking tumor. Although these cells may not be able to settle in tumors with low metastatic potential, in 4/4 patients with Her2/neu positive tumors who did not receive trastuzumab the number of CETCs further increased after termination of therapy and surgery followed by rapid distant relapse indicating that the cells released in these patients may be highly aggressive with a high potential to settle and grow into metastases.It is, however not clear, whether sequential or simultaneous addition of trastuzumab to chemotherapy is preferential. In our hands 65% of the patients receiving simultaneous trastuzumab showed increasing CETCs and all have suffered relapse whereas all patients with decreasing CETCs are still in complete remission indicating that in the neoadjuvant situation during maintenance therapy an increase in CETC is the earliest indicator of imminent relapse.6/6 patients who received trastuzumab sequentially to the neoadjuvant treatment all showed decreasing numbers with all of them still without signs of disease after 4 years of follow up. Thus, trastuzumab was highly effective in this treatment and even if the tumor cells were not eliminated immediately, trastuzumab contributed to prevent them from settling and growing into metastases and CETC monitoring favours sequential trastuzumab. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3016.