Efficacy of Lapatinib in Patients with HER2-Negative Metastatic Breast Cancer and HER2-Positive Circulating Tumor Cells-The DETECT III Clinical Trial.

BACKGROUND: The phenotypes of tumor cells change during disease progression, but invasive rebiopsies of metastatic lesions are not always feasible. Here we aimed to determine whether initially HER2-negative metastatic breast cancer (MBC) patients with HER2-positive circulating tumor cells (CTCs) benefit from a HER2-targeted therapy. METHODS: The open-label, interventional randomized phase III clinical trial (EudraCT Number 2010-024238-46, CliniclTrials.gov Identifier: NCT01619111) recruited from March 2012 until September 2019 with a follow-up duration of 19.5 months. It was a multicenter clinical trial with 94 participating German study centers. A total of 2137 patients with HER2-negative MBC were screened for HER2-positive CTCs with a final modified intention-to-treat population of 101 patients. Eligible patients were randomized to standard therapy with or without lapatinib. Primary study endpoints included CTC clearance (no CTCs at the end of treatment) and secondary endpoints were progression-free survival, overall survival (OS), and safety. RESULTS: In both treatment arms CTC clearance at first follow-up visit-although not being significantly different for both arms at any time point-was significantly associated with improved OS (42.4 vs 14.1 months; P = 0.002). Patients treated additionally with lapatinib had a significantly improved OS over patients receiving standard treatment (20.5 vs 9.1 months, P = 0.009). CONCLUSIONS: DETECT III is the first clinical study indicating that phenotyping of CTCs might have clinical utility for stratification of MBC cancer patients to HER2-targeting therapies. The OS benefit could be related to lapatinib, but further studies are required to prove this clinical observation. ClinicalTrials.gov Registration Number: NCT01619111.

A workflow for the enrichment, the identification, and the isolation of non-apoptotic single circulating tumor cells for RNA sequencing analysis.

Circulating tumor cells (CTCs) are constantly shed by tumor tissue and can serve as a valuable analyte for a gene expression analysis from a liquid biopsy. However, a high proportion of CTCs can be apoptotic leading to rapid mRNA decay and challenging the analysis of their transcriptome. We established a workflow to enrich, to identify, and to isolate single CTCs including the discrimination of apoptotic and non-apoptotic CTCs for further single CTC transcriptome analysis. Viable tumor cells-we first used cells from breast cancer cell lines followed by CTCs from metastatic breast cancer patients-were enriched with the CellSearch system from diagnostic leukapheresis products, identified by immunofluorescence analysis for neoplastic markers, and isolated by micromanipulation. Then, their cDNA was generated, amplified, and sequenced. In order to exclude early apoptotic tumor cells, staining with Annexin V coupled to a fluorescent dye was used. Annexin V staining intensity was associated with decreased RNA integrity as well as lower numbers of total reads, exon reads, and detected genes in cell line cells and CTCs. A comparative RNA analysis of single cells from MDA-MB-231 and MCF7 cell lines revealed the expected differential transcriptome profiles. Enrichment and staining procedures of cell line cells that were spiked into blood had only little effect on the obtained RNA sequencing data compared to processing of naïve cells. Further, the detection of transcripts of housekeeping genes such as GAPDH was associated with a significantly higher quality of expression data from CTCs. This workflow enables the enrichment, detection, and isolation of single CTCs for individual transcriptome analyses. The discrimination of apoptotic and non-apoptotic cells allows to focus on CTCs with a high RNA integrity to ensure a successful transcriptome analysis.

Progesterone-induced progesterone receptor membrane component 1 rise-to-decline changes are essential for decidualization.

BACKGROUND: Decidualization of endometrial cells is the prerequisite for embryo implantation and subsequent placenta formation and is induced by rising progesterone levels following ovulation. One of the hormone receptors contributing to endometrial homeostasis is Progesterone Receptor Membrane Component 1 (PGRMC1), a non-classical membrane-bound progesterone receptor with yet unclear function. In this study, we aimed to investigate how PGRMC1 contributes to human decidualization. METHODS: We first analyzed PGRMC1 expression profile during a regular menstrual cycle in RNA-sequencing datasets. To further explore the function of PGRMC1 in human decidualization, we implemented an inducible decidualization system, which is achieved by culturing two human endometrial stromal cell lines in decidualization-inducing medium containing medroxyprogesterone acetate and 8-Br-cAMP. In our system, we measured PGRMC1 expression during hormone induction as well as decidualization status upon PGRMC1 knockdown at different time points. We further conferred proximity ligation assay to identify PGRMC1 interaction partners. RESULTS: In a regular menstrual cycle, PGRMC1 mRNA expression is gradually decreased from the proliferative phase to the secretory phase. In in vitro experiments, we observed that PGRMC1 expression follows a rise-to-decline pattern, in which its expression level initially increased during the first 6 days after induction (PGRMC1 increasing phase) and decreased in the following days (PGRMC1 decreasing phase). Knockdown of PGRMC1 expression before the induction led to a failed decidualization, while its knockdown after induction did not inhibit decidualization, suggesting that the progestin-induced 'PGRMC1 increasing phase' is essential for normal decidualization. Furthermore, we found that the interactions of prohibitin 1 and prohibitin 2 with PGRMC1 were induced upon progestin treatment. Knocking down each of the prohibitins slowed down the decidualization process compared to the control, suggesting that PGRMC1 cooperates with prohibitins to regulate decidualization. CONCLUSIONS: According to our findings, PGRMC1 expression followed a progestin-induced rise-to-decline expression pattern during human endometrial decidualization process; and the correct execution of this expression program was crucial for successful decidualization. Thereby, the results of our in vitro model explained how PGRMC1 dysregulation during decidualization may present a new perspective on infertility-related diseases.

Ultra-sensitive CTC-based liquid biopsy for pancreatic cancer enabled by large blood volume analysis.

The limited sensitivity of circulating tumor cell (CTC) detection in pancreatic adenocarcinoma (PDAC) stems from their extremely low concentration in the whole circulating blood, necessitating enhanced detection methodologies. This study sought to amplify assay-sensitivity by employing diagnostic leukapheresis (DLA) to screen large blood volumes. Sixty patients were subjected to DLA, with a median processed blood volume of ~ 2.8 L and approximately 5% of the resulting DLA-product analyzed using CellSearch (CS). Notably, DLA significantly increased CS-CTC detection to 44% in M0-patients and 74% in M1-patients, yielding a 60-fold increase in CS-CTC enumeration. DLA also provided sufficient CS-CTCs for genomic profiling, thereby delivering additional genomic information compared to tissue biopsy samples. DLA CS-CTCs exhibited a pronounced negative prognostic impact on overall survival (OS), evidenced by a reduction in OS from 28.6 to 8.5 months (univariate: p = 0.002; multivariable: p = 0.043). Additionally, a marked enhancement in sensitivity was achieved (by around 3-4-times) compared to peripheral blood (PB) samples, with positive predictive values for OS being preserved at around 90%. Prognostic relevance of CS-CTCs in PDAC was further validated in PB-samples from 228 PDAC patients, consolidating the established association between CTC-presence and reduced OS (8.5 vs. 19.0 months, p < 0.001). In conclusion, DLA-derived CS-CTCs may serve as a viable tool for identifying high-risk PDAC-patients and aiding the optimization of multimodal treatment strategies. Moreover, DLA enables comprehensive diagnostic profiling by providing ample CTC material, reinforcing its utility as a reliable liquid-biopsy approach. This high-volume liquid-biopsy strategy presents a potential pathway for enhancing clinical management in this malignancy.

Dynamics of CXCR4 positive circulating tumor cells in prostate cancer patients during radiotherapy.

Ablative radiotherapy is a highly efficient treatment modality for patients with metastatic prostate cancer (PCa). However, a subset of patients does not respond. Currently, this subgroup with bad prognosis cannot be identified before disease progression. We hypothesize that markers indicative of radioresistance, stemness and/or bone tropism may have a prognostic potential to identify patients profiting from metastases-directed radiotherapy. Therefore, circulating tumor cells (CTCs) were analyzed in patients with metastatic PCa (n = 24) during radiotherapy with CellSearch, multicolor flow cytometry and imaging cytometry. Analysis of copy-number alteration indicates a polyclonal CTC population that changes after radiotherapy. CTCs were found in 8 out of 24 patients (33.3%) and were associated with a shorter time to biochemical progression after radiotherapy. Whereas the total CTC count dropped after radiotherapy, a chemokine receptor CXCR4-expressing subpopulation representing 28.6% of the total CTC population remained stable up to 3 months. At once, we observed higher chemokine CCL2 plasma concentrations and proinflammatory monocytes. Additional functional analyses demonstrated key roles of CXCR4 and CCL2 for cellular radiosensitivity, tumorigenicity and stem-like potential in vitro and in vivo. Moreover, a high CXCR4 and CCL2 expression was found in bone metastasis biopsies of PCa patients. In summary, panCK+ CXCR4+ CTCs may have a prognostic potential in patients with metastatic PCa treated with metastasis-directed radiotherapy.

Comparative analysis of EpCAM high-expressing and low-expressing circulating tumour cells with regard to their clonal relationship and clinical value.

BACKGROUND: Circulating tumour cells (CTCs) are mainly enriched based on the epithelial cell adhesion molecule (EpCAM). Although it was shown that an EpCAM low-expressing CTC fraction is not captured by such approaches, knowledge about its prognostic and predictive relevance and its relation to EpCAM-positive CTCs is lacking. METHODS: We developed an immunomagnetic assay to enrich CTCs from metastatic breast cancer patients EpCAM independently using antibodies against Trop-2 and CD-49f and characterised their EpCAM expression. DNA of single EpCAM high expressing and low expressing CTCs was analyzed regarding chromosomal aberrations and predictive mutations. Additionally, we compared CTC-enrichment on the CellSearch system using this antibody mix and the EpCAM based enrichment. RESULTS: Both antibodies acted synergistically in capturing CTCs. Patients with EpCAM high-expressing CTCs had a worse overall and progression-free survival. EpCAM high- and low-expressing CTCs presented similar chromosomal aberrations and mutations indicating a close evolutionary relationship. A sequential enrichment of CTCs from the EpCAM-depleted fraction yielded a population of CTCs not captured EpCAM dependently but harbouring predictive information. CONCLUSIONS: Our data indicate that EpCAM low-expressing CTCs could be used as a valuable tumour surrogate material-although they may be prognostically less relevant than EpCAM high-expressing CTCs-and have particular benefit if no CTCs are detected using EpCAM-dependent technologies.

Implementing microwell slides for detection and isolation of single circulating tumor cells from complex cell suspensions.

Cell loss during detection and isolation of circulating tumor cells (CTCs) is a challenge especially when label-free pre-enrichment technologies are used without the aid of magnetic particles. Although microfluidic systems can remove the majority of "contaminating" white blood cells (WBCs), their remaining numbers are still impeding single CTC isolation, thus making additional separation steps needed. This study aimed to develop a workflow from blood-to-single CTC for complex cell suspensions by testing two microwell formats. In the first step, different cell lines were used to compare the performances of Sievewell™ 370 K (TOK, Japan) and CellCelector™ Nanowell U25 (ALS Automated Lab Solutions, Germany) slides for cell labelling and single-cell micromanipulation. Confounding levels of auto-fluorescence inherent to different plastic materials used to cast the microwells, staining recovery rates, and cell isolation rates were determined. In the second step, three different blood preservation tubes were tested for RNA analysis. Lastly, the established workflow was applied to isolate CTCs from peripheral blood samples obtained from metastasized breast cancer (mBC) patients for single-cell DNA and RNA analysis. The detection of CTCs in Sievewell slides profit from better signal-to-noise ratios in the fluorescence channels mainly used for CTC detection. In addition, due to its design, Sievewell supports direct in situ CTC labelling, which minimizes cell loss and leads to single-cell recovery rates after staining of approx. 94%. Detection of PIK3CA mutations in single CTCs verified the applicability of the workflow for the analysis of genomic DNA of CTCs. Furthermore, combined with blood preservation up to 48 h at room temperature in LBguard tubes, panel RT-PCR transcript analysis was successful for single cell line cells and CTCs, respectively. The combined use of Sievewell microwell slides and CellCelector™ automated micromanipulation system improves single CTC detection, labelling and isolation from complex cell suspensions. This approach is especially valuable when samples of high cellular content are processed.

Progesterone receptor membrane component 1 regulates lipid homeostasis and drives oncogenic signaling resulting in breast cancer progression.

BACKGROUND: PGRMC1 (progesterone receptor membrane component 1) is a highly conserved heme binding protein, which is overexpressed especially in hormone receptor-positive breast cancer and plays an important role in breast carcinogenesis. Nevertheless, little is known about the mechanisms by which PGRMC1 drives tumor progression. The aim of our study was to investigate the involvement of PGRMC1 in cholesterol metabolism to detect new mechanisms by which PGRMC1 can increase lipid metabolism and alter cancer-related signaling pathways leading to breast cancer progression. METHODS: The effect of PGRMC1 overexpression and silencing on cellular proliferation was examined in vitro and in a xenograft mouse model. Next, we investigated the interaction of PGRMC1 with enzymes involved in the cholesterol synthesis pathway such as CYP51, FDFT1, and SCD1. Further, the impact of PGRMC1 expression on lipid levels and expression of enzymes involved in lipid homeostasis was examined. Additionally, we assessed the role of PGRMC1 in key cancer-related signaling pathways including EGFR/HER2 and ERα signaling. RESULTS: Overexpression of PGRMC1 resulted in significantly enhanced proliferation. PGRMC1 interacted with key enzymes of the cholesterol synthesis pathway, alters the expression of proteins, and results in increased lipid levels. PGRMC1 also influenced lipid raft formation leading to altered expression of growth receptors in membranes of breast cancer cells. Analysis of activation of proteins revealed facilitated ERα and EGFR activation and downstream signaling dependent on PGRMC1 overexpression in hormone receptor-positive breast cancer cells. Depletion of cholesterol and fatty acids induced by statins reversed this growth benefit. CONCLUSION: PGRMC1 may mediate proliferation and progression of breast cancer cells potentially by altering lipid metabolism and by activating key oncogenic signaling pathways, such as ERα expression and activation, as well as EGFR signaling. Our present study underlines the potential of PGRMC1 as a target for anti-cancer therapy.

Protein complexes including PGRMC1 and actin-associated proteins are disrupted by AG-205.

PGRMC1 is a protein from the MAPR family with a range of cellular functions. PGRMC1 has been described to play a role in fertility, neuroprotection, steroidogenesis, membrane trafficking and in cancer cell biology. PGRMC1 represents a likely key regulator of cell metabolism and proliferation, as well as a potential target for anti-cancer therapies. To further understand the functions of PGRMC1 and the mechanism of the small molecule inhibitor of PGRMC1, AG-205, proteins differentially bound to PGRMC1 were identified following AG-205 treatment of MIA PaCa-2 cells. Our results suggest that AG-205 influences PGRMC1 interactions with the actin cytoskeleton. The binding of two PGRMC1-associated proteins that support this, RACK1 and alpha-Actinin-1, was reduced following AG-205 treatment. The biology associated with PGRMC1 binding partners identified here merits further investigation.

Clinical relevance of circulating tumor cells in ovarian, fallopian tube and peritoneal cancer.

PURPOSE: Presence of circulating tumor cells (CTCs) is associated with impaired clinical outcome in several solid cancers. Limited data are available on the significance of CTCs in gynaecological malignancies. The aims of the present study were to evaluate the dynamics of CTCs in patients with ovarian, fallopian tube and peritoneal cancer during chemotherapy and to assess their clinical relevance. METHODS: 43 patients with ovarian, fallopian tube and peritoneal cancer were included into this prospective study. Patients received chemotherapy according to national guidelines. CTC analysis was performed using the CellSearch system prior to chemotherapy, after three and six cycles. RESULTS: In 26% of the patients, ≥ 1CTC per 7.5 ml of blood was detected at baseline (17% of patients with de novo disease, compared to 35% in recurrent patients). Presence of CTCs did not correlate with other factors. After three cycles of therapy, CTC positivity rate declined to 4.8%. After six cycles, no patient showed persistent CTCs. Patients with ≥ 1 CTC at baseline had significantly shorter overall survival and progression-free survival compared to CTC-negative patients (OS: median 3.1 months vs. not reached, p = 0.006, PFS: median 3.1 vs. 23.1 months, p = 0.005). When only the subgroup with newly diagnosed cancer was considered, the association between CTC status and survival was not significant (OS: mean 17.4 vs. 29.0 months, p = 0.192, PFS: 14.3 vs. 26.9 months, p = 0.085). Presence of ≥ 1 CTC after three cycles predicted shorter OS in the entire patient cohort (p < 0.001). CONCLUSIONS: Hematogenous tumor cell dissemination is a common phenomenon in ovarian, fallopian tube and peritoneal cancer. CTC status before start of systemic therapy correlates with clinical outcome. Chemotherapy leads to a rapid decline in CTC counts; further research is needed to evaluate the clinical value of CTC monitoring after therapy.

Label-Free Enrichment and Molecular Characterization of Viable Circulating Tumor Cells from Diagnostic Leukapheresis Products.

INTRODUCTION: Circulating tumor cells (CTCs) may be used to improve cancer diagnosis, prognosis, and treatment. However, because knowledge regarding CTC biology is limited and the numbers of CTCs and CTC-positive cancer patients are low, progress in this field is slow. We addressed this limitation by combining diagnostic leukapheresis (DLA) and microfluidic enrichment to obtain large numbers of viable CTCs from metastasized breast cancer patients. METHODS: DLA was applied to 9 patients, and 7.5 mL of peripheral blood was drawn. CTCs were enriched with the Parsortix™ system. The quality of CTCs from fresh and cryopreserved DLA products was tested, and CTCs were cultured in vitro. Single uncultured and cultured CTCs were isolated by micromanipulation to determine different parameters, such as genomic aberrations and mutation profiles of selected tumor-associated genes. Expression levels of estrogen receptor and HER2/neu were monitored during in vitro culture. RESULTS: Viable CTCs from peripheral blood and fresh or frozen DLA products could be enriched. DLA increased the likelihood of successful CTC culture. Cryopreserved DLA products could be stored with minimal CTC loss and no overt reduction in the tumor cell quality and viability during an observation period of up to 3 years. The analyzed parameters did not change during in vitro culture. DLA samples with high CTC numbers and lower ratios of apoptotic CTCs were more likely to grow in culture. CONCLUSIONS: The increased CTC numbers from fresh or cryopreserved DLA products facilitate multiple functional and molecular analyses and, thus, could improve our knowledge of their biology.

Determination of the androgen receptor status of circulating tumour cells in metastatic breast cancer patients.

BACKGROUND: The prognostic relevance of circulating tumour cells (CTCs) in metastatic breast cancer (MBC) patients has been confirmed by several clinical trials. However, predictive blood-based biomarkers for stratification of patients for targeted therapy are still lacking. The DETECT studies explore the utility of CTC phenotype for treatment decisions in patients with HER2 negative MBC. Associated with this concept is a plethora of translational projects aiming to identify potential predictive biomarkers. The androgen receptor (AR) is expressed in over 70% of hormone receptor-positive and up-to 45% of triple-negative tumours. Studies has indicated the promising nature of AR as a new therapy target with a clinical benefit rate for anti-AR treatment in MBC patients up to 25% The aim of this analysis was the characterization of CTCs regarding the expression of the AR using immunofluorescence. METHODS: MBC patients were screened for the HER2-status of CTCs in the DETECT studies. In a subset of CTC-positive patients (n = 67) an additional blood sample was used for immunomagnetic enrichment of CTCs using the CellSearch® Profile Kit prior to transfer of the cells onto cytospin slides. Establishment of immunofluorescence staining for the AR was performed using prostate cancer cell lines LNCaP and DU145 as positive and negative control, respectively. Staining of DAPI, pan-cytokeratin (CK) and CD45 was applied to identify nucleated epithelial cells as CTCs and to exclude leucocytes. RESULTS: Co-staining of the AR, CK and CD45 according to the above mentioned workflow has been successfully established using cell lines with known AR expression spiked into the blood samples from healthy donors. For this translational project, samples were analysed from 67 patients participating in the DETECT studies. At least one CTC was detected in 37 out of 67 patients (56%). In 16 of these 37 patients (43%) AR-positive CTCs were detected. In eight out of 25 patients (32%) with more than one CTC, AR-positive and AR-negative CTCs were observed. CONCLUSION: In 43% of the analysed CTC samples from patients with MBC the AR expression has been detected. The predictive value of AR expression in CTCs remains to be evaluated in further trials.

From in vitro to ex vivo: subcellular localization and uptake of graphene quantum dots into solid tumors.

Among various nanoparticles tested for pharmacological applications over the recent years, graphene quantum dots (GQDs) seem to be promising candidates for the construction of drug delivery systems due to their superior biophysical and biochemical properties. The subcellular fate of incorporated nanomaterial is decisive for transporting pharmaceuticals into target cells. Therefore a detailed characterization of the uptake of GQDs into different breast cancer models was performed. The demonstrated accumulation inside the endolysosomal system might be the reason for the particles' low toxicity, but has to be overcome for cytosolic or nuclear drug delivery. Furthermore, the penetration of GQDs into precision-cut mammary tumor slices was studied. These constitute a far closer to reality model system than monoclonal cell lines. The constant uptake into the depth of the tissue slices underlines the systems' potential for drug delivery into solid tumors.

Toward a real liquid biopsy in metastatic breast and prostate cancer: Diagnostic LeukApheresis increases CTC yields in a European prospective multicenter study (CTCTrap).

Frequently, the number of circulating tumor cells (CTC) isolated in 7.5 mL of blood is too small to reliably determine tumor heterogeneity and to be representative as a "liquid biopsy". In the EU FP7 program CTCTrap, we aimed to validate and optimize the recently introduced Diagnostic LeukApheresis (DLA) to screen liters of blood. Here we present the results obtained from 34 metastatic cancer patients subjected to DLA in the participating institutions. About 7.5 mL blood processed with CellSearch® was used as "gold standard" reference. DLAs were obtained from 22 metastatic prostate and 12 metastatic breast cancer patients at four different institutions without any noticeable side effects. DLA samples were prepared and processed with different analysis techniques. Processing DLA using CellSearch resulted in a 0-32 fold increase in CTC yield compared to processing 7.5 mL blood. Filtration of DLA through 5 µm pores microsieves was accompanied by large CTC losses. Leukocyte depletion of 18 mL followed by CellSearch yielded an increase of the number of CTC but a relative decrease in yield (37%) versus CellSearch DLA. In four out of seven patients with 0 CTC detected in 7.5 mL of blood, CTC were detected in DLA (range 1-4 CTC). The CTC obtained through DLA enables molecular characterization of the tumor. CTC enrichment technologies however still need to be improved to isolate all the CTC present in the DLA.

Diagnostic leukapheresis for CTC analysis in breast cancer patients: CTC frequency, clinical experiences and recommendations for standardized reporting.

Diagnostic leukapheresis (DLA) is based on continuous centrifugation that collects mononuclear cells from peripheral blood with a density of 1.055-1.08 g/ml. As epithelial cells have a similar density, DLA cocollects circulating tumor cell (CTCs) along with the targeted mononuclear cells. Here, we report on our single center experience applying DLA in 40 nonmetastatic and metastatic breast cancer patients and its impact on CTC detection. We found that the use of just 5% of the DLA product (corresponding to a median peripheral blood volume of around 60 ml) in the CellSearch® assay already leads to a significant increase in CTC detection frequency and yield. The implementation of the method was unproblematic, and we did not observe any adverse events in our patient cohort. Extrapolating the CTC counts in the DLA samples to the whole DLA product indicated that enormous CTC numbers could be harvested by this approach (around 205x more CTCs than in the 7.5 ml blood sample in M1 patients). In conclusion, DLA is a clinically safe method to collect CTCs from liters of blood enabling a real liquid biopsy. Yet, further technical developments are required to process whole DLA products and exploit the full potential of this approach. As it is foreseeable that DLA will be used by several groups, and hopefully ultimately brought to the patients in a routine setting, we discuss recommendations on the minimum of required information for reporting on DLAs to allow comparison across different approaches. © 2018 International Society for Advancement of Cytometry.

Filtration based assessment of CTCs and CellSearch® based assessment are both powerful predictors of prognosis for metastatic breast cancer patients.

BACKGROUND: The assessment of circulating tumor cells (CTCs) has been shown to enable monitoring of treatment response and early detection of metastatic breast cancer (MBC) recurrence. The aim of this study was to compare a well-established CTC detection method based on immunomagnetic isolation with a new, filtration-based platform. METHODS: In this prospective study, two 7.5 ml blood draws were obtained from 60 MBC patients and CTC enumeration was assessed using both the CellSearch® and the newly developed filtration-based platform. We analyzed the correlation of CTC-positivity between both methods and their ability to predict prognosis. Overall survival (OS) was calculated and Kaplan-Meier curves were estimated with thresholds of ≥1 and ≥5 detected CTCs. RESULTS: The CTC positivity rate of the CellSearch® system was 56.7% and of the filtration-based platform 66.7%. There was a high correlation of CTC enumeration obtained with both methods. The OS for patients without detected CTCs, regardless of the method used, was significantly higher compared to patients with one or more CTCs (p < 0.001). The median OS of patients with no CTCs vs. ≥ 1 CTC assessed by CellSearch® was 1.83 years (95% CI: 1.63-2.02) vs. 0.74 years (95% CI: 0.51-1.52). If CTCs were detected by the filtration-based method the median OS times were 1.88 years (95% CI: 1.74-2.03) vs. 0.59 years (95% CI: 0.38-0.80). CONCLUSIONS: The newly established EpCAM independently filtration-based system is a suitable method to determine CTC counts for MBC patients. Our study confirms CTCs as being strong predictors of prognosis in our population of MBC patients.

Membrane-initiated effects of Serelys® on proliferation and apoptosis of human breast cancer cells.

Herbal extracts used for the alleviation of postmenopausal symptoms might have a lower risk of breast cancer development than hormone therapy. Serelys® is a product composed of purified pollen cytoplasm extracts. Recent experimental data revealed that estrogens might trigger a further proliferative effect on breast cancer cells via the progesterone receptor membrane component-1 (PGRMC1) in addition to the proliferative effect via intracellularly located receptors. MCF-7 and T47D cells were stably transfected with PGRMC1. Different concentrations of the extract alone and in combination with fixed concentrations of estradiol or a growth factor mixture were tested. Proliferation of treated cells was determined by the 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl-tetrazolium bromide (MTT)-test and apoptosis was determined using a Cell Death Detection ELISA kit (CDD). Serelys® was neutral in the cell lines transfected or not transfected with PGRMC1. It was also neutral in combination with estradiol or growth factors in terms of cell proliferation and cell apoptosis. Thus in contrast to hormone therapy Serelys® appears to trigger no further breast cancer risk when applied in the post menopause to women, who do or do not overexpress PGRMC1. Overall Serelys® may be an effective alternative for alleviating postmenopausal symptoms without increasing breast cancer risk.

Navigation through inter- and intratumoral heterogeneity of endocrine resistance mechanisms in breast cancer: A potential role for Liquid Biopsies?

The majority of breast cancers are hormone receptor positive due to the expression of the estrogen and/or progesterone receptors. Endocrine therapy is a major treatment option for all disease stages of hormone receptor-positive breast cancer and improves overall survival. However, endocrine therapy is limited by de novo and acquired resistance. Several factors have been proposed for endocrine therapy failures, which include molecular alterations in the estrogen receptor pathway, altered expression of cell-cycle regulators, autophagy, and epithelial-to-mesenchymal transition as a consequence of tumor progression and selection pressure. It is essential to reveal and monitor intra- and intertumoral alterations in breast cancer to allow optimal therapy outcome. Endocrine therapy navigation by molecular profiling of tissue biopsies is the current gold standard but limited in many reasons. "Liquid biopsies" such as circulating-tumor cells and circulating-tumor DNA offer hope to fill that gap in allowing non-invasive serial assessment of biomarkers predicting success of endocrine therapy regimen. In this context, this review will provide an overview on inter- and intratumoral heterogeneity of endocrine resistance mechanisms and discuss the potential role of "liquid biopsies" as navigators to personalize treatment methods and prevent endocrine treatment resistance in breast cancer.

High Level of Progesteron Receptor Membrane Component 1 (PGRMC 1) in Tissue of Breast Cancer Patients is Associated with Worse Response to Anthracycline-Based Neoadjuvant Therapy.

PGRMC1 is known to be highly expressed in breast cancer tissue and is associated with chemoresistance in breast cancer cells. However, its role in breast cancer signaling is not fully understood yet. In the present study, the expression status of PGRMC1 and its phosphorylated version (pPGRMC1) in breast cancer tissue and surrounding stroma before and after neoadjuvant therapy was examined to find a possible association to therapy response. Tissue biopsies of 69 breast cancer patients were analyzed by immunohistochemistry for expression levels of PGRMC1 and pPGRMC1. Expression status of PGRMC1 and pPGRMC1 in tumor tissue was compared with expression status of progesterone receptor (PR), estrogen receptor α (ERα), total estrogen receptor ß (ERß), ERß1, ERß2, the proliferation marker Ki-67, and human epidermal growth factor receptor 2 (HER2/neu). Correlations were calculated for expression of PGRMC1 and pPGRMC1 before and after neoadjuvant-therapy. PGRMC1 and pPGRMC1 were highly abundant in every breast cancer tissue sample. Considerably lower signals were detected in surrounding tissue. Further, PGRMC1 and pPGRMC1 abundance was found to correlate with ERß expression. A lower level of pPGRMC1 could be found in post-therapy surgical specimens compared to specimens before treatment. Interestingly, patients with high PGRMC1 tumor levels showed worse response to anthracycline-based therapy as patients with lower PGRMC1 levels. These new findings demonstrate that PGRMC1 might play an important role in progression and therapy resistance of human breast tumors and could offer an interesting target for anticancer therapy.

Enrichment, Isolation and Molecular Characterization of EpCAM-Negative Circulating Tumor Cells.

The presence of EpCAM-positive circulating tumor cells (CTCs) in the peripheral blood is associated with poor clinical outcomes in breast, colorectal and prostate cancer, as well as the prognosis of other tumor types. In addition, recent studies have suggested that the presence of CTCs undergoing epithelial-to-mesenchymal transition and, as such, may exhibit reduced or no expression of epithelial proteins e.g. EpCAM, might be related to disease progression in metastatic breast cancer (MBC) patients. Analyzing the neoplastic nature of this EpCAM-low/negative (EpCAM-neg) subpopulation remains an open issue as the current standard detection methods for CTCs are not efficient at identifying this subpopulation of cells. The possible association of EpCAM-neg CTCs with EpCAM-positive (EpCAM-pos) CTCs and role in the clinicopathological features and prognosis of MBC patients has still to be demonstrated. Several technologies have been developed and are currently being tested for the identification and the downstream analyses of EpCAM-pos CTCs. These technologies can be adapted and implemented into workflows to isolate and investigate EpCAM-neg cells to understand their biology and clinical relevance. This chapter will endeavour to explain the rationale behind the identification and analyses of all CTC subgroups, as well as to review the current strategies employed to enrich, isolate and characterize EpCAM-negative CTCs. Finally, the latest findings in the field will briefly be discussed with regard to their clinical relevance.