T. Fehm

Progestogens and membrane-initiated effects on the proliferation of human breast cancer cells

ABSTRACT Objectives Evidence is accumulating that progestogens may play a crucial role in the development of breast cancer under contraception and hormone therapy in reproductive and menopausal women. Progesterone receptor membrane component 1 (PGRMC1) expressed in breast cancer may be important in tumorigenesis and thus may increase breast cancer risk. The aim of this project was to investigate the influence of progesterone and nine synthetic progestins on MCF-7 breast cancer cells overexpressing PGRMC1. Methods MCF-7 cells were stably transfected with PGRMC1 expression plasmid (WT-12). To test the effects of progestogerone (P) and the synthetic progestins chlormadinone acetate (CMA), desogestrel (DSG), drospirenone (DRSP), dydrogesterone (DYD), levonorgestrel (LNG), medroxyprogesterone acetate (MPA), nomegestrol (NOM) and norethisterone (NET) on cell proliferation, MCF-7 and WT-12 cells were stimulated with different concentrations (0.01–1 µmol/l). Results In MCF-7 cells, DRSP, DSG, DYD, LNG and NET increased the proliferation at 1 µmol/l, the effect being highest for NET with about 20%. In WT-12 cells, the same progestins, but additionally MPA, showed a significant increase, which was much higher (30–245%) than in MCF-7 cells. Here again, NET showed the highest proliferative effect. No effect was found for CMA, NOM and P. Conclusion Some synthetic progestins trigger a proliferative response of PGRMC1-overexpressed MCF-7 cancer cells. The effect of progestogens on breast cancer tumorigenesis may clearly depend on the specific pharmacology of the various synthetic progestins.

Possible role of PGRMC1 in breast cancer development

Abstract Hormone therapy may increase the risk of breast cancer. Thus, especially the addition of synthetic progestins may play a decisive role according to the results of clinical studies. Overexpression of a special receptor, i.e. the progesterone receptor membrane component-1 (PGRMC1), may offer a potential new pathway to explain the observed increase in breast cancer risk in the combined arm of the Womens Health Initiative. PGRMC1 is expressed in breast cancer tissue and may be important in tumorigenesis. The expression of PGRMC1 in breast cancer tissue is significantly different from that in normal mammary glands. Certain synthetic progestins can increase the proliferation of PGRMC1-overexpressing breast cancer cells and may thus be involved in tumorigenesis, while progesterone and certain synthetic progestins such as nomegestrol or chlormadinone acetate react neutrally. Our investigations point towards an important role of estrogen receptor-α in the signaling cascade, resulting in the proliferative effect induced by progestins. Thus, activation of PGRMC1 may explain the increased breast cancer risk observed during treatment with certain progestins. Very recently, PGRMC1 was investigated in serum samples of lung cancer patients and matched healthy patients; significantly higher concentrations were shown in the cancer patients. Therefore, PGRMC1 might be a predictor for other cancers as well but, according to clinical trials, its importance for a possible screening tool, particularly for breast cancer risk during hormone therapy, seems of interest.

Dormancy in breast cancer

Tumor dormancy describes a prolonged quiescent state in which tumor cells are present, but disease progression is not yet clinically apparent. Breast cancer is especially known for long asymptomatic periods, up to 25 years, with no evidence of the disease, followed by a relapse. Factors that determine the cells decision to enter a dormant state and that control its duration remain unclear. In recent years, considerable progress has been made in understanding how tumor cells circulating in the blood interact and extravasate into secondary sites and which factors might determine whether these cells survive, remain dormant, or become macrometastases. The mechanisms of tumor cell dormancy are still not clear. Two different hypotheses are currently discussed: tumor cells persist either by completely withdrawing from the cell cycle or by continuing to proliferate at a slow rate that is counterbalanced by cell death. Because dormant disseminated tumor cells may be the founders of metastasis, one hypothesis is that dormant tumor cells, or at least a fraction of them, share stem cell-like characteristics that may be responsible for their long half-lives and their suggested resistance to standard chemotherapy. Therefore, knowledge of the biology of tumor cell dormancy may be the basis from which to develop innovative targeted therapies to control or eliminate this tumor cell fraction. In this review, we discuss biological mechanisms and clinical implications of tumor dormancy in breast cancer patients.

Comparison between circulating and disseminated tumor cells in breast cancer.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 AbstractsnnAbstract #301 nnBackground: The presence of disseminated tumor cells (DTC) in the bone marrow (BM) of breast cancer patients is an independent prognostic factor. The role of circulating tumor cells (CTC) in blood is not yet defined. Since BM aspiration is less accepted by patients compared to blood drawing, it would be highly desirable to replace BM aspiration by blood analysis. Therefore, the purpose of the present study was: (1) to examine the presence of CTC in peripheral blood (2) to assess the correlation between CTC in blood and DTC in BM and (3) since adjuvant therapy targets minimal residual disease (MRD), to compare the expression profile of therapeutic relevant markers between CTC (a potential surrogate marker for MRD) and the primary tumor. Materials and Methods: 282 blood samples from primary breast cancer patients were collected. All samples underwent immunomagnetic enrichment using the AdnaTest BreastCancerSelect (AdnaGen AG, Germany) within 4 hours after blood withdrawal followed by RNA isolation and subsequent gene expression analysis by reverse transcription and Multiplex-PCR in separated tumor cells using the AdnaTest BreastCancerDetect. CTC were analyzed for the three breast cancer associated markers: GA733-2, Muc-1, Her-2 and β–actin as an internal PCR control. Expression of the estrogen (ER) and progesterone (PR) receptor was assessed in an additional RT-PCR. The analysis of PCR products was performed by capillary electrophoresis on the Agilent Bioanalyzer 2100. Furthermore, two BM aspirates from each of these patients were analyzed by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3. Evaluation of DTC was performed with the ACIS system (Chromavision) and the ARIOL system (Applied Imaging) according to the ISHAGE evaluation criteria. Results: 282 patients could be included into this study. 44/282 (16%) of these patients had detectable CTC in the bloodstream. BM samples could be analyzed in 267/282 patients. DTC were found in 64/267 patients, resulting in a positivity rate for DTC in the BM of 23%. BM status did not correlate with the presence of CTC (p=0.112) supporting the theory of different clinical relevance. MUC-1 and GA 73.3 were expressed by 57% and 61% of the CTC, respectively. CTC were HER2 positive in 39% of patients whereas only 11% of the corresponding primary tumor overexpressed HER2. Most of the patients had ER und PR positive tumors (84% and 73%, respectively). However, the majority of the CTC were ER and PR negative (75% and 98%, respectively). Conclusions: (1) These data indicate that due to the discordance between CTC and DTC the clinical relevance may be different. (2) The expression profile between CTC (a possible surrogate marker for MRD) and the primary tumor (the current gold standard for selecting targeted therapy against MRD) differs. Thus, the consequence for selecting HER2 targeted or anti - endocrine therapy has to be evaluated.nnCitation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 301.

New Tumor Biomarkers in Ovarian Cancer and Its Prognostic and Clinical Relevance

In Europe and the United States, ovarian cancer is currently the major cause of death from gynecological malignancy. Up to 60% ovarian cancer patients die from locally advanced disease. Nonetheless, even patients treated with optimal cytoreduction may subsequently suffer from metastatic disease. Since treatment strategies are developed to control locoregional cancer growth, it may be anticipated that more patients will die of distant metastases. The lack of early disease signals further contributes to the fact that only onefourth of ovarian cancers are identified at stage I. Ovarian cancer remains thus undetected due to late symptoms and the lack of reliable and clinically applicable screening tools. Further, the identification of new biomarkers could optimize prediction and monitoring of anticancer therapies and provide insights into ovarian cancer progression. Presence of disseminated tumor cells (DTC) in bone marrow is a common phenomenon observed in solid epithelial tumors. As shown by a multi-center analysis of bone marrow (BM) specimens from more than 4,700 patients, DTC detection at the time of breast cancer diagnosis is strongly correlated with poor clinical outcome (level-I-evidence) [1]. There is growing evidence that hematogenous tumor cell dissemination may occur in other tumors, such as prostate, colon and gynecologic malignancies. DTC, as surrogate parameter for occult hematogenous spread, are routinely detected in 22-51% ovarian cancer patients. Interestingly, ovarian metastases to the bone are only rarely observed [2], [3], [4]. Whether bone marrow serves in these patients as a temporary compartment from where persistent DTC are able to migrate, remains unclear. It has been demonstrated that dissemination of isolated tumor cells to secondary sites occurs as early as in FIGO stage I disease [4]. Single tumor cells acquire thus the potential to disseminate to extraperitoneal compartments early in the process of the disease. Of all prognostic factors, monitoring of minimal residual disease is the only one available after the tumor has been removed. Beside monitoring of tumor markers, there is currently a major effort to identify other biological markers which can be assessed with minimally invasive methods and persist beyond surgery. We previously reported on a significant correlation of positive bone marrow status with shortened relapse-free survival in ovarian cancer patients [5]. DTC persistence after completion of platinum-based chemotherapy was also found to be prognostically relevant [6]. Further, the identification of molecular biomarkers may represent excellent targets for new treatment strategies for chemoresistant

Differenziell aktivierte Signaltransduktionswege im platinresistenten Ovarialkarzinom

Zielsetzung: 75% der Patientinnen mit Ovarialkarzinom zeigen einen rezidivierenden Krankheitsverlauf aufgrund einer Resistenz gegenuber einer Platin-basierten Chemotherapie. Die molekularen Ursachen der Resistenzentwicklung sind bisher unbekannt. Ziel der Studie war, Unterschiede in der Proteinexpression in zugrunde liegenden Signaltransduktionswegen zwischen Platin-sensiblen und Platin-resistenten Ovarialkarzinomen zu identifizieren. Methoden: 24 kryokonservierte Tumorresektate wurden histologisch auf ihren Tumorgehalt uberpruft, wobei 12 anhand der klinischen Daten als Platin-sensibel, 12 als Platin-resistent klassifiziert wurden. Die lysierten Gewebe wurden via DIGI-West analysiert. Beim DIGI-West handelt es sich um eine neue, Antikorper-basierte Detektionsmethode, die eine Quantifizierung von 500 Proteinen und somit die Erstellung von Proteinprofilen ermoglicht. Ergebnisse: Differentiell exprimierte Proteine bzw. Phosphorylierungsmuster wurden in drei bzw. 10 Fallen detektiert. Die Ergebnisse wurden in einem in-vitro Modell, basierend auf der Zelllinie A2780 und dem Platin-resistenten Aquivalent A2780cis, verifiziert. Die Carboplatin- sowie Paclitaxelsensitivitat wurde im ATP-Zellproliferationsassay uberpruft. Anschliesend wurden zwei auffallige Proteinen, die mit Zellproliferation und Zellzyklus assoziiert sind, mittels Immunblot analysiert. Die Ergebnisse stutzen die Hypothese, dass deutliche Veranderungen in den Proteinmodifikationen dieser Proteine nach unterschiedlicher Behandlungsdauer eine Rolle in der Resistenzentwicklung spielen konnten. Zusammenfassung: Platinbasierte Chemotherapie benotigt proliferative Zellen um wirksam zu sein. Grundlegende, regulatorische Signalwege der Proliferation und Apoptose mussen funktional sein, um die Wirksamkeit der Therapeutika auf Tumorzellen zu ermoglichen. Die Hypothese, dass nicht nur Proteinexpressionen, sondern auch Proteinmodifikationen bei der Entwicklung der Platinresistenz im Ovarialkarzinom von Bedeutung sind, bedarf weitergehender Untersuchungen.

Nachweis disseminierter Tumorzellen im Knochenmark von Männern mit Mammakarzinom im Frühstadium

Hintergrund: Brustkrebs ist bei Mannern eine seltene Erkrankung. Therapie und Diagnostik orientieren sich daher an der Behandlung von Frauen mit Mammakarzinom. Hier ist der Nachweis disseminierter Tumorzellen (DTZ) im Knochenmark mit einer schlechteren Prognose vergesellschaftet. Ziel dieser Studie war daher die Analyse der Pravalenz und prognostischen Bedeutung von DTZs bei Mannern, die an einem Mammakarzinom im Fruhstadium erkrankt sind. Methodik: Bei mannlichen Patienten, die zwischen Januar 2001 und April 2015 an Brustkrebs im Fruhstadium (T1 – 4, N0 – 2, M0) erkrankt sind wurde eine Knochenmarks-Biopsie im Rahmen der Primaroperation durchgefuhrt. DTZs wurden mittels Immunzytochemie (Panzytokeratin Antikorper A45/B-B3) und anhand zytomorphologischer Kriterien identifiziert. Ergebnisse: 24 Patientin konnten in die retrospektive Analyse eingeschlossen werden. DTZs wurden in vier Fallen identifiziert (17%). Es gab keinen signifkanten Zusammenhang zwischen dem DTZ-Status und anderen klinisch-pathologischen Faktoren. Ein signifikanter Einfluss der DTZs auf das Uberleben wurde ebensowenig festgestellt. Zusammenfassung: DTZs lassen sind auch bei Mannern, die an einem Mammakarzinom erkrankt nachweisen. Die Detektionsrate ist ahnlich hoch wie bei Frauen. Es wurde keine Korrelation zwischen dem Vorhandensein von DTZs und anderen Tumoreigenschaften oder der Prognose festgestellt, am ehesten ist dies auf die geringe Fallzahl der vorliegenden Untersuchung zuruckzufuhren. Kunftige klinische Anwendungen von DTZs bei Frauen konnten allerdings auch bei Mannern von Bedeutung sein.

Abstract P2-02-13: EpCAM-independent enrichment approach for isolation of circulating tumor cells (CTCs) in breast cancer - What can be found in the EpCAM-depleted fraction?

Circulating tumor cells (CTCs) are the potential precursors of metastatic disease. Many assays have been established for the enumeration of CTCs. However, major limitations include the reliance on the expression of the cell surface marker epithelial cell adhesion molecule (EpCAM). These approaches may not detect CTCs that either express no/low levels of EpCAM or undergo epithelial-to-mesenchymal transition (EMT). We present an enrichment strategy combining different antibodies specific for surface proteins and extracellular matrix (ECM) components to capture EpCAMneg cell lines and EpCAMneg CTCs from EpCAM-depleted breast cancer blood samples. Expression of proteins (Trop2, CD49f, cMet, CK8, CD44, ADAM8, CD146, TEM8, CD47) was verified by immunofluorescence on EpCAM-positive (e.g. MCF7, SKBR3) and -negative (MDA-MB-231) breast cancer cell lines; antibodies and ECM proteins (e.g. hyaluronic acid (HA), collagen I, laminin) were further spotted in a single- and multi-arrayed format onto glass slides (Schott, NEXTERION® AL) and coupled to immunomagnetic beads (Dynabeads/Adembeads). Tumor cell adhesion of EpCAMpos/neg cell lines was visualized by Coomassie/MitoTracker; EpCAMneg CTCs enriched via functionalized Adem-/Dynabeads were identified by immunofluorescence staining for anti-pan-Cytokeratin(CK)-FITC/anti-CD45 AF647/DAPI and quantified manually by microscopy. Regarding cell lines, marginal binding of EpCAMneg MDA-MB-231 cells to EpCAM-antibodies could be observed. Efficient adhesion/capturing of EpCAMneg cells could be achieved via HA and immobilized antibodies against CD49f and Trop2. By analyzing 29 EpCAM-depleted fractions from 25 metastatic breast cancer patients, we were able to identify EpCAMneg CTCs in 69% of the samples [range 1-24] applying Trop2, CD49f, cMet, CK8 and/or HA magnetic enrichment. Accessorily, EpCAMneg dual-positive (CKpos/CD45pos) cells could be traced in 28 out of 29 samples [range 1-480]. Herein, we demonstrate an enhanced enrichment strategy to optimize capturing of EpCAMneg CTCs by targeting various cell surface antigens with antibody mixtures and ECM components. Thereby, potential relevant CTCs can be gathered and subjected to subsequent molecular analysis. Citation Format: Fehm T, Schneck H, Gierke B, Pawlak M, Templin M, Niederacher D, Neubauer H. EpCAM-independent enrichment approach for isolation of circulating tumor cells (CTCs) in breast cancer - What can be found in the EpCAM-depleted fraction?. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-13.

Abstract P4-09-09: Verification of the breast cancer progression-associated miRNA hsa-miR-199a-5p using NanoString® platform

A fundamental and clinically important step during breast tumourigenesis is the transition from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC). Improved knowledge of this transition from pre-invasive to invasive breast cancer will pave the way for novel preventative and therapeutic strategies. We have previously reported on differential expression of the miRNA hsa-miR-199a-5p in 15 matched pairs of DCIS and IDC areas isolated by laser capture microdissection (LCM) from formalin fixed and paraffin embedded (FFPE) breast cancer tissues using Illumina miRNA BeadChip microarray platform. Differential expression of hsa-miR-199a-5p was validated by quantitative RT-PCR in additional independent DCIS/IDC sample pairs from 25 breast cancer patients. Knock down of hsa-miR-199a-5p in invasive MDA-MB-231 and TMX2-28 breast cancer cells using a specific inhibitor significantly reduced invasiveness by approx. 73% and 71%, respectively (P Now we report on experiments to validate differential expression of hsa-miR-199a-5p using a new platform – NanoString® (nCounter® miRNA Expression Assay, NanoString Technologies®). The NanoString® System is an automated, digital detection and counting system which uses a novel barcoding technology to directly profile up to 800 miRNAs simultaneously from a single sample. Total RNA from 6 DCIS/IDC FFPE tumours was used for miRNA expression analysis. This analysis resulted in 10 differentially expressed miRNAs including hsa-miR-199a-5p which is upregulated in IDC (P In this project we identified candidate progression-associated miRNAs which are differentially expressed between DCIS and IDC. Hsa-miR-199a-5p was validated in an independent sample cohort and its expression was further verified using the new miRNA expression analysis platform NanoString®. Hsa-miR-199a5-p is influencing in vitro cell invasiveness and may therefore be a potential drugable regulator of tumour progression and invasion in breast cancer. Citation Format: Fehm T, Schultz S, Bartsch H, Petat-Dutter K, Kahlert S, Sotlar K, Niederacher D, Neubauer H. Verification of the breast cancer progression-associated miRNA hsa-miR-199a-5p using NanoString® platform. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-09-09.

Various Factors Contributing to Tumor Dormancy: Therapeutic Implications in Breast Cancer

Cancer dormancy describes a stage in tumor progression where tumor cells survive in a quiescent state. Breast cancer is especially known for prolonged asymptomatic periods (up to 15–20 years) followed by a recurrence. Two main mechanisms of tumor cell dormancy are under discussion: tumor cells cease dividing completely or persist proliferating at a slow rate counterbalanced by apoptosis. In the last decades, major efforts have been made to understand the process of interaction between circulating tumor cells (CTCs) in the bloodstream and their extravasation into distant sites, where CTCs may survive in a dormant state or acquire the ability to build metastases. Despite remarkable progress in this field, factors that determinate the fate of a single tumor cell remain to be clarified.