Influence of endocrine therapy on the ratio of androgen receptor (AR) to estrogen receptor (ER) positive circulating epithelial tumor cells (CETCs) in breast cancer.

BACKGROUND: The androgen receptor (AR) is expressed in the majority of breast cancers and across the main breast cancer subtypes. Despite the high frequency of AR expression in breast cancer its appraisal remains controversial because its role is complex, dependent on the hormonal milieu. The aim of the current study was to investigate the frequency of AR and ER positive CETCs in breast cancer patients. METHODS: The number of vital CETCs was determined from blood of 66 patients suffering from breast cancer and the expression of AR and ER on these cells was investigated using the maintrac method. RESULTS: Numbers of CETCs/mL blood were significantly higher in patients with advanced disease as compared to patients with early stage disease. The fraction of AR positive CETCs was significantly higher than the fraction of ER positive CETCs (90% vs. 50%; P < 0.001). Patients with positive lymph nodes had less AR positive CETCs as compared to patients with negative lymph node status. The AR:ER ratio was higher in patients receiving tamoxifen therapy as compared to patients without tamoxifen therapy whereas treatment with aromatase inhibitor had no influence on AR:ER ratio. CONCLUSIONS: The ratio of AR to ER positive CETCs, obviously, is influenced by endocrine therapy, more specifically therapy with tamoxifen. Since AR expression seems to be one of the possible mechanism of resistance to endocrine therapy this may provide a new biomarker to select patients who might benefit from combination treatment of ER and AR inhibitors.

NSAID analgesic ketorolac used perioperatively may suppress early breast cancer relapse: particular relevance to triple negative subgroup.

To explain a bimodal relapse hazard among early stage breast cancer patients treated by mastectomy we postulated that relapses within 4 years of surgery resulted from something that happened at about the time of surgery to provoke sudden exits from dormant phases to active growth. Relapses at 10 months appeared to be surgery-induced angiogenesis of dormant avascular micrometastases. Another relapse mode with peak about 30 months corresponded to sudden growth from a single cell. Late relapses were not synchronized to surgery. This hypothesis could explain a wide variety of breast cancer observations. We have been looking for new data that might provide more insight concerning the various relapse modes. Retrospective data reported in June 2010 study of 327 consecutive patients compared various perioperative analgesics and anesthetics in one Belgian hospital and one surgeon. Patients were treated with mastectomy and conventional adjuvant therapy. Follow-up was average 27.3 months with range 13-44 months. Updated hazard as of September 2011 for this series is now presented. NSAID ketorolac, a common analgesic used in surgery, is associated with far superior disease-free survival in the first few years after surgery. The expected prominent early relapse events are all but absent. In the 9-18 month period, there is fivefold reduction in relapses. If this observation holds up to further scrutiny, it could mean that the simple use of this safe and effective anti-inflammatory agent at surgery might eliminate most early relapses. Transient systemic inflammation accompanying surgery could be part of the metastatic tumor seeding process and could have been effectively blocked by perioperative anti-inflammatory agents. In addition, antiangiogenic properties of NSAIDs could also play a role. Triple negative breast cancer may be the ideal group with which to test perioperative ketorolac to prevent early relapses.

Demasking of epithelial cell adhesion molecule (EpCAM) on circulating epithelial tumor cells by Tween®20 treatment in breast cancer patients.

BACKGROUND: The epithelial cell adhesion molecule (EpCAM) embedded in the plasma membrane of circulating epithelial tumor cells (CETC) is used for detection and enrichment of circulating tumor cells in peripheral blood and as a target for anti-epithelial antibodies elicited during immune response in anti-tumor immunization. Although an efficient immune response against EpCAM can be generated, the clinical application of such approaches has not been successful so far and the detection of circulating epithelial cells is highly variable. One reason for these discrepancies may be that not all circulating tumor cells are equally accessible for the specific antibody. A possible reason might be masking of EpCAM by glycoproteins or membrane lipoproteins preventing antibody binding. METHODS: We have tested the application of detergents as demasking agents known to be successful in demasking red blood cell epitopes and determined how and in which way they affect integral membrane proteins and membrane lipids. RESULTS: The results showed that the polysorbate Tween®20, a non-ionic detergent like organic solvent is able to demask EpCAM on CETC and makes it better accessible to its specific antibody retaining at the same time full cell viability. CONCLUSIONS: The data presented in this study suggest that EpCAM is present on part of circulating tumor cells in a masked form and that it is possible to demask EpCAM on CETC of breast cancer patients using Tween®20 treatment. But further studies are needed to elucidate the mechanism of demasking.

A study of Docetaxel-induced effects in MCF-7 cells by means of Raman microspectroscopy.

Chemotherapies feature a low success rate of about 25%, and therefore, the choice of the most effective cytostatic drug for the individual patient and monitoring the efficiency of an ongoing chemotherapy are important steps towards personalized therapy. Thereby, an objective method able to differentiate between treated and untreated cancer cells would be essential. In this study, we provide molecular insights into Docetaxel-induced effects in MCF-7 cells, as a model system for adenocarcinoma, by means of Raman microspectroscopy combined with powerful chemometric methods. The analysis of the Raman data is divided into two steps. In the first part, the morphology of cell organelles, e.g. the cell nucleus has been visualized by analysing the Raman spectra with k-means cluster analysis and artificial neural networks and compared to the histopathologic gold standard method hematoxylin and eosin staining. This comparison showed that Raman microscopy is capable of displaying the cell morphology; however, this is in contrast to hematoxylin and eosin staining label free and can therefore be applied potentially in vivo. Because Docetaxel is a drug acting within the cell nucleus, Raman spectra originating from the cell nucleus region were further investigated in a next step. Thereby we were able to differentiate treated from untreated MCF-7 cells and to quantify the cell-drug response by utilizing linear discriminant analysis models.

Chick Chorioallantoic Membrane (CAM) Assays as a Model of Patient-Derived Xenografts from Circulating Cancer Stem Cells (cCSCs) in Breast Cancer Patients.

BACKGROUND: cCSCs are a small subset of circulating tumor cells with cancer stem cell features: resistance to cancer treatments and the capacity for generating metastases. PDX are an appreciated tool in oncology, providing biologically meaningful models of many cancer types, and potential platforms for the development of precision oncology approaches. Commonly, mouse models are used for the in vivo assessment of potential new therapeutic targets in cancers. However, animal models are costly and time consuming. An attractive alternative to such animal experiments is the chicken chorioallantoic membrane assay. METHODS: In this study, primary cultures from cCSCs were established using the sphere-forming assay. Subsequently, tumorspheres were transplanted onto the CAM membrane of fertilized chicken eggs to form secondary microtumors. RESULTS: We have developed an innovative in vitro platform for cultivation of cCSCs from peripheral blood of cancer patients. The number of tumorspheres increased significantly with tumor progression and aggressiveness of primary tumor. The number of tumorspheres was positively correlated with Ki-67, Her2 status, and grade score in primary breast tumors. The grafting of tumorspheres onto the CAM was successful and positively correlated with aggressiveness and proliferation capacity of the primary tumor. These tumors pathologically closely resembled the primary tumor. CONCLUSIONS: The number of tumorspheres cultured from peripheral blood and the success rate of establishing PDX directly reflect the aggressiveness and proliferation capacity of the primary tumor. A CAM-based PDX model using cCSC provides a fast, low-cost, easy to handle, and powerful preclinical platform for drug screening, therapy optimization, and biomarker discovery.

Increased Circulating Epithelial Tumor Cells (CETC/CTC) over the Course of Adjuvant Radiotherapy Is a Predictor of Less Favorable Outcome in Patients with Early-Stage Breast Cancer.

BACKGROUND: Adjuvant radiotherapy (RT) is an integral component of a multidisciplinary treatment strategy for early-stage breast cancer. It significantly reduces the incidence of loco-regional recurrence but also of distant events. Distant events are due to tumor cells disseminated from the primary tumor into lymphatic fluid or blood, circulating epithelial tumor cells (CETC/CTC), which can reach distant tissues and regrow into metastases. The purpose of this study is to determine changes in the number of CETC/CTC in the course of adjuvant RT, and to evaluate whether they are correlated to local recurrence and distant metastases in breast cancer patients. METHODS: Blood from 165 patients irradiated between 2002 and 2012 was analyzed 0-6 weeks prior to and 0-6 weeks after RT using the maintrac® method, and patients were followed over a median period of 8.97 (1.16-19.09) years. RESULTS: Patients with an increase in CETC/CTC numbers over the course of adjuvant RT had a significantly worse disease-free survival (p = 0.004) than patients with stable or decreasing CETC/CTC numbers. CETC/CTC behavior was the most important factor in predicting subsequent relapse-free survival. In particular, patients who had received neoadjuvant chemotherapy were disproportionately more likely to develop metastases when cell counts increased over the course of RT (p = 0.003; hazard ratio 4.886). CONCLUSIONS: Using the maintrac® method, CETC/CTC were detected in almost all breast cancer patients after surgery. The increase in CETC/CTC numbers over the course of RT represents a potential predictive biomarker to judge relative risk/benefit in patients with early breast cancer. The results of this study highlight the need for prospective clinical trials on CETC/CTC status as a predictive criterion and for individualization of treatment. CLINICAL TRIAL REGISTRATION: The trial is registered (2 May 2019) at trials.gov under NCT03935802.

Monitoring of circulating epithelial tumor cells using the Maintrac® method and its potential benefit for the treatment of patients with colorectal cancer.

Circulating tumor cells are an important link between primary tumors and metastases. A longitudinal monitoring of their numbers and properties can provide valuable information on therapy response and disease progression for patients with colorectal cancer. As several techniques for the detection of circulating tumor cells are notorious for yielding low detection rates in patients with non-metastatic colorectal cancer, the present study aimed to perform a proof-of-principle study using the Maintrac® approach for an assessment of circulating epithelial tumor cells (CETCs) in patients with colorectal cancer receiving neoadjuvant and/or adjuvant radio/chemotherapy (R/CT). CETCs in the peripheral blood of 22 patients with colorectal cancer were quantified by fluorescence image analysis (Maintrac®) before and after the first cycle of a neoadjuvant and/or adjuvant R/CT, as well as before and after surgical resection of the primary tumor. To determine that blood-borne CETCs originate from tumor tissues, spheres were cultured from CETCs as well as from primary tumor tissue and compared with the expression of tumor-specific antigens. Within the scope of this study, it was demonstrated that the Maintrac® method allows for the precise detection and characterization of CETCs in the blood of patients with colorectal cancer independent of tumor stage. Furthermore, correlations between CETC parameters and patients' response to neoadjuvant and/or adjuvant R/CT that have been described in previous literature could be reproduced. Whether the observed trends are of a general nature and suitable as an auxiliary criterion for prognosis and treatment decisions remains to be shown. Patients with rectal cancer may benefit from CETC monitoring as a method to select suitable patients for adjuvant therapy.

Prospective Monitoring of Circulating Epithelial Tumor Cells (CETC) Reveals Changes in Gene Expression during Adjuvant Radiotherapy of Breast Cancer Patients.

Circulating epithelial tumor cells (CETC) are considered to be responsible for the formation of metastases. Therefore, their importance as prognostic and/or predictive markers in breast cancer is being intensively investigated. Here, the reliability of single cell expression analyses in isolated and collected CETC from whole blood samples of patients with early-stage breast cancer before and after radiotherapy (RT) using the maintrac® method was investigated. Single-cell expression analyses were performed with qRT-PCR on a panel of selected genes: GAPDH, EpCAM, NANOG, Bcl-2, TLR 4, COX-2, PIK3CA, Her-2/neu, Vimentin, c-Met, Ki-67. In all patients, viable CETC were detected prior to and at the end of radiotherapy. In 7 of the 9 (77.8%) subjects examined, the CETC number at the end of the radiotherapy series was higher than before. The majority of genes analyzed showed increased expression after completion of radiotherapy compared to baseline. Procedures and methods used in this pilot study proved to be feasible. The method is suitable for further investigation of the underlying molecular biological mechanisms occurring in cells surviving radiotherapy and possibly the development of radiation resistance.

Influence of adjuvant radiotherapy on circulating epithelial tumor cells and circulating cancer stem cells in primary non-metastatic breast cancer.

BACKGROUND: There is an unmet need to identify biomarkers that directly reflect response to adjuvant radiotherapy (RT). Circulating epithelial tumor cells (CETCs) represent the liquid component of solid tumors and are responsible for metastatic relapse. CETC subsets with cancer stem cell characteristics, circulating cancer stem cells (cCSCs), play a pivotal role in the metastatic cascade. Monitoring the most aggressive subpopulation of CETCs could reflect the aggressiveness of the remaining tumor burden. There is limited data on the detection and monitoring changes in CETC and cCSC numbers during RT in early breast cancer. METHODS: CETC numbers were analyzed prior to, at midterm and at the end of RT in 52 primary non-metastatic breast cancer patients. Hormone receptor status was determined in CETCs prior to and at the end of RT. For the identification of cCSCs cell suspensions from the peripheral blood of patients were cultured in vitro under conditions favoring growth of tumorspheres. RESULTS: Hormone receptor status in CETCs before RT was comparable to that in primary tumor tissue. Prior to RT numbers of CETCs correlated with aggressiveness of primary tumors. cCSCs could be successfully identified and monitored during RT. Prior to RT patients treated with neoadjuvant chemotherapy had significantly higher numbers of CETCs and tumorspheres compared to patients after adjuvant chemotherapy. During RT, the number of CETCs decreased continuously in patients after neoadjuvant chemotherapy but not after adjuvant chemotherapy. CONCLUSION: Monitoring the number of CETCs and the CETC subset with cancer stem cell properties during RT may provide additional clinically useful prognostic information.

High depletion of breast cancer cells from the peripheral blood with the method of non-specific separation.

Circulating epithelial tumour cells (CETCs) play an important role in the formation of metastases in breast cancer patients. The depletion of such CETCs from peripheral blood of breast cancer patients using non-specific separation (without antibodies) of tumour cells from normal blood leucocytes might contribute to reduce the load of the patient's blood with tumour cells and subsequently reduce the probability of metastasis formation. This method is based on cell type-specific interaction of living cells with Carboxymethyl Dextrane (CMD) coated magnetic nanoparticles. We have developed a mild flow separation method using CMD-coated magnetic nanoparticles (core size ca. 25 nm) along with a low-field gradient magnetic separator and an external separation column (blood bag). The ability of tumour cells to preferentially bind such particles and to separate tumour cells from the white blood cells from blood samples of 25 breast cancer patients (fresh and 24-hour stored blood samples) were tested. The circulating tumour cells were quantified before and after separation by maintrac analysis. We achieved a very high depletion rate of tumour cells to < 3% remaining in the investigated 24 hours stored blood samples and ≤14% in all fresh blood samples concurrent with maintaining 56% ± 4% of vital leukocytes in all fresh blood samples.

[Unmittelbare Remission eines mit großzelligem B-Non-Hodgkin-Lymphom befallenen inguinalen Lymphknotens unter alleiniger homöopathischer Behandlung mit Conium: Wann ist eine alleinige adjuvant-homöopathische Tumortherapie zulässig und sinnvoll?] / Immediate Remission of an Inguinal Lymph Node Afflicted with Large-Cell B-Non-Hodgkin's Lymphoma Under Sole Homeopathic Treatment with Conium: When Is a Sole Adjuvant-Homeopathic Tumor Therapy Permissible and Useful?

Bei einer 63-jährigen Patientin wird mittels Biopsie eines linksinguinalen Lymphknotens ein großzelliges B-Non-Hodgkin-Lymphom diagnostiziert. Unmittelbar nach Beginn einer homöopathischen Therapie mit Conium C 30 beginnt sich der Lymphknoten in der linken Leiste zurückzubilden. Bei Exzision des Lymphknotens vierzehn Tage nach Therapiebeginn können histologisch keine Residuen des Tumors mehr nachgewiesen werden und es darf von einer vollständigen Remission ausgegangen werden. Die Patientin bleibt in der Folge rezidivfrei. Das homöopathische Mittel Conium (Schierling) kommt in der adjuvanten homöopathischen Tumortherapie und bei vergrößerten Lymphknoten als häufig indiziertes Mittel zur Anwendung.A large-cell B-cell non-Hodgkin Lymphoma (LCBCL) was diagnosed bioptically in a female patient (age 63 years) in one left inguinal lymph node. Immediately after beginning homeopathic treatment with Conium C 30, the lymph node started to show a reduction in size. Two weeks after starting homeopathic therapy, histological examination of the excised lymph node showed no evidence of a residual tumor ­ suggestive of a complete remission. The patient remains disease free until now. The homeopathic remedy Conium (hemlock) is frequently applied for adjuvant homeopathic tumor therapy as well as for the treatment of enlarged lymph nodes.

The Value of Monitoring the Behavior of Circulating Tumor Cells at the End of Endocrine Therapy in Breast Cancer Patients.

After five years of endocrine therapy, patients with ER+ (estrogen receptor positive) breast cancer face the question of the benefit of further treatment. Ten years of endocrine therapy has been demonstrated to improve survival compared to five years. However, the individual benefit of continuation remains unclear. Therefore, markers for predicting benefit from endocrine treatment and extended endocrine treatment are desperately needed. In this study the dynamics over time of the tumor cells circulating in peripheral blood of patients, circulating tumor cells/ circulating epithelial tumor cells (CTC/CETC), as the systemic part of the tumor were investigated in 36 patients with ER+ primary breast cancer. CTC/CETCs were monitored serially during and after endocrine therapy. After termination of endocrine therapy 12 patients showed an increase in CTC/CETCs, with 8 of 12 suffering relapse. No change or a reduction was observed in 24 patients, with 2 of 24 suffering relapse. Initial tumor size was marginally prognostic (p = 0.053) but not nodal status nor the mere number of CTC/CETCs. Only the trajectory of CTC/CETCs was a statistically significant predictor of relapse free survival (increasing cell numbers: mean = 940 days vs. stable/decreasing cell numbers mean not reached). Individual cases demonstrated that an increase of CTC/CETCs after discontinuation of tamoxifen therapy could be stopped by resuming the endocrine therapy.

B7-H3 on circulating epithelial tumor cells correlates with the proliferation marker, Ki-67, and may be associated with the aggressiveness of tumors in breast cancer patients.

Circulating epithelial tumor cells (CETCs) in peripheral blood are a prerequisite for the development of metastases. B7-H3 is an important immune checkpoint member of the B7 family and inhibits T-cell mediated antitumor immunity. Its expression is associated with a negative prognosis and a poor clinical outcome. Based on the clinical success of inhibitory immune checkpoint blockade, monoclonal antibodies (mAbs) against B7-H3 appear to be a promising therapeutic strategy. The proliferation biomarker, Ki-67, is used as a prognostic factor for breast cancer and reflects the proliferative potential of the tumor. In order to better understand the role of B7-H3 and Ki-67 in cancer development, in this study, we used a real-time biopsy for determining both biomarkers on CETCs in breast cancer patients. Blood from 50 patients suffering from breast cancer was analyzed for CETCs and the expression of B7-H3 and Ki-67 using the maintrac® method. B7-H3 expression on CETCs was found in 82% of the patients. The frequency of B7-H3- and Ki-67­positive CETCs was significantly higher in patients who had received radiation therapy compared to patients who had not received irradiation. B7-H3­positive CETCs seemed to be more aggressive as the percentage of B7-H3­positive CETCs correlated with the percentage of cells positive for the proliferation marker, Ki-67 (r=0.72 P<0.001). A significant association between the Ki-67 and B7-H3 expression level on the CETCs and nodal status was observed. On the whole, the findings of this study indicate that breast cancer patients have detectable CETCs with a high frequency of B7-H3 expression regardless of the stage of the disease. B7-H3 seems to be an important factor in immune evasion and may thus be a promising target for anticancer therapies. Radiation may lead to an upregulation of B7-H3 expression on CETCs, which could be a possible mechanism of acquired radio-resistance.

Circulating epithelial tumor cells as a prognostic tool for malignant melanoma.

Although immune therapies with checkpoint inhibitors have gained increasing attention in advanced and metastatic melanoma, interferon-α remains a standard therapy for nonmetastatic malignant melanoma with risk factors. Interferons can successfully prevent relapse; however, the response rate is still not as high as would be desired. Prognostic tools to predict the response are required, which could lead to more individualized treatment regimens. In numerous studies over the past decade, circulating epithelial tumor cells (CETCs) have been shown to be a promising biomarker for estimating the risk of metastatic relapse, and we sought to determine whether they can also be used for this purpose in malignant melanoma. To establish a prognostic tool for patients with melanoma, we quantified CETCs over the course of interferon treatment in 49 patients. Patients were categorized into two groups according to the behavior of their circulating tumor cells during the interferon treatment: those with increasing and those with decreasing numbers of circulating tumor cells. Patients with increasing numbers of circulating tumor cells had a significantly higher risk of relapse. Kaplan-Meier survival analysis showed a significant difference between patients with increasing CETC numbers (mean survival time: 2.6 years) and patients with decreasing or stable CETC numbers (mean survival time: 12.6 years) (P=0.001). Quantification of CETCs could prove to be a prognostic marker for patients with melanoma receiving interferon immunotherapy. Further studies should determine whether these results are applicable to other immunotherapies, for example, immune checkpoint inhibition.

Quantification of circulating tumour cells for the monitoring of adjuvant therapy in breast cancer: an increase in cell number at completion of therapy is a predictor of early relapse.

Treatment efficiency of adjuvant therapy in breast cancer is revealed after several years by statistical evaluation, but this gives no answer for the individual patient. Having shown that circulating epithelial tumour cells (CETC) respond to neoadjuvant therapy in exactly the same way as the tumour, we monitored adjuvant therapy in 25 non-metastatic breast cancer patients. Nineteen patients with a decline or no change in number of CETC showed no relapse whereas six patients with a more than ten-fold increase had five distant and one local relapse, indicating that the dynamic of CETC in the individual patient is predictive of outcome.

Sensitive detection of PD-L1 expression on circulating epithelial tumor cells (CETCs) could be a potential biomarker to select patients for treatment with PD-1/PD-L1 inhibitors in early and metastatic solid tumors.

BACKGROUND: The current cancer research strongly focuses on immune therapies, where the PD-1, with its ligands plays an important role. It is known that PD-L1 is frequently up-regulated in a number of different cancers and the relevance of this pathway has been extensively studied and therapeutic approaches targeting PD-1 and PD-L1 have been developed. We used a non-invasive, real-time biopsy for determining PD-L1 and PD-L2 expression in CETCs of solid cancer patients. METHODS: CETCs were determined from blood of 128 patients suffering from breast (72), prostate (27), colorectal (18) and lung (11) cancer. The number of vital CETCs and the expression of PD-L1 and PD-L2 were investigated using the maintrac® method. RESULTS: PD-L1 expressing CETCs were detected in 94.5% of breast, 100% of prostate, 95.4% of colorectal and 82% of lung cancer patients whereas only 75% of breast cancer patients had PD-L2 positive CETCs. In the PD-L1 and PD-L2 expressing patients the cell fraction of PD-L1 positive CETCs is significantly higher than the fraction of PD-L2 positive CETCs (54.6% vs. 28.7%; p<0.001). Breast cancer patients with metastatic disease had significantly more PD-L1 positive CETCs as compared to patients without metastasis (median 75% vs. 61.1%; p<0.05). CONCLUSION: PD-L1 seems to be a major factor in immune evasion and is highly expressed on CETCs regardless of the type of cancer. Monitoring the frequency of PD-L1 positive CETCs could reflect individual patient's response for an anti-PD-1/PD-L1 therapy and may be a promising target of anticancer treatment.

Seeding of epithelial cells into circulation during surgery for breast cancer: the fate of malignant and benign mobilized cells.

BACKGROUND: Surgery of malignant tumors has long been suspected to be the reason for enhancement of growth of metastases with fatal outcome. This often prevented surgeons from touching the tumor if not absolutely necessary. We have shown in lung cancer patients that surgery, itself, leads to mobilization of tumor cells into peripheral blood. Some of the mobilized cells finding an appropriate niche might grow to form early metastases. Monitoring of tumor cell release during and the fate of such cells after surgery for breast cancer may help to reveal how metastases develop after surgery. METHOD: We used the MAINTRAC analysis, a new tool for online observation of circulating epithelial cells, to monitor the number of epithelial cells before, 30 min, 60 min, three and seven days after surgery and during subsequent variable follow up in breast cancer patients. RESULTS: Circulating epithelial cells were already present before surgery in all patients. During the first 30-60 min after surgery values did not change immediately. They started increasing during the following 3 to 4 days up to thousand fold in 85% of treated patients in spite of complete resection of the tumor with tumor free margins in all patients. There was a subsequent re-decrease, with cell numbers remaining above pre-surgery values in 58% of cases until onset of chemotherapy. In a few cases, where no further therapy or only hormone treatment was given due to low risk stage, cell numbers were monitored for up to three years. They remained elevated with no or a slow decrease over time. This was in contrast to the observation in a patient where surgery was performed for benign condition. She was monitored before surgery with no cells detectable. Epithelial cells increased up to more than 50,000 after surgery but followed by a complete reduction to below the threshold of detection. CONCLUSION: Frequently before but regularly during surgery of breast cancer, epithelial cells are mobilized into circulation. Part of these cells, most probably normal or apoptotic cells, are cleared from the circulation as also shown to occur in benign conditions. After resection even if complete and of small tumors, cells can remain in the circulation over long times. Such cells may remain "dormant" but might settle and grow into metastases, if they find appropriate conditions, even after years.

The number of tumorspheres cultured from peripheral blood is a predictor for presence of metastasis in patients with breast cancer.

BACKGROUND: Tumor metastases are the major cause of cancer morbidity and mortality. A subpopulation of tumor cells with stem-like properties is assumed to be responsible for tumor invasion, metastasis, heterogeneity and therapeutic resistance. This population is termed cancer stem cells (CSCs). We have developed a simple method for identification and characterization of circulating cancer stem cells among circulating epithelial tumor cells (CETCs). METHODS: CETCs were cultured under conditions favoring growth of tumorspheres from 72 patients with breast cancer, including a subpopulation of 23 patients with metastatic disease. CETCs were determined using the maintrac® method. Gene expression profiles of single CETCs and tumorspheres of the same patients were analyzed using qRT-PCR. RESULTS: Sphere formation was observed in 79 % of patients. We found that the number of tumorspheres depended on stage of disease. Furthermore, the most important factor for growing of tumorspheres is obtaining chemotherapy. Patients with chemotherapy treatment had lower numbers of tumorspheres compared to patients without chemotherapy. Patients with HER2 positive primary tumor had higher number of tumorspheres. Analysis of surface marker expression profile of tumorspheres showed that cells in the spheres had typical phenotype of cancer stem cells. There was no sphere formation in a control group with 50 healthy donors. CONCLUSIONS: This study demonstrates that a small fraction of CETCs has proliferative activity. Identifying the CETC subset with cancer stem cell properties may provide more clinically useful prognostic information. Chemotherapy is the most important component in cancer therapy because it frequently reduces the number of tumorspheres.

Treatment of advanced solid tumours with NSAIDs: Correlation of quantitative monitoring of circulating tumour cells and positron emission tomography-computed tomography imaging.

The detection and characterisation of tumour-derived circulating epithelial tumor cells (CETCs) or circulating tumor cells (CTCs) have been a main focus of basic oncological research over previous years. Numerous studies in the past decade have shown that CTCs are a promising tool for the estimation of the risk for metastatic relapse. The present observational study describes treatment results using tumour imaging and the quantification of CTCs. A group of 14 patients with advanced carcinomas was followed during their anticancer treatments. CTC numbers were serially detected and treatment success was estimated by positron emission tomography-computed tomography. A connection was found between tumour remission and a decreasing CTC count in 83%, a connection between stable disease and stable CTC numbers in 78% and a connection between progressive disease (PD) and an increase in CTC count in 50% of cases. In the patients with PD, an incomplete response was observed affecting the CTCs, but not the solid region of the tumour. As a result of this study, it may be concluded that patients with solid tumours benefit from serial quantification of CTCs in addition to imaging, as this combination of techniques provides a more sensitive result than imaging alone.

Quantification of the response of circulating epithelial cells to neodadjuvant treatment for breast cancer: a new tool for therapy monitoring.

INTRODUCTION: In adjuvant treatment for breast cancer there is no tool available with which to measure the efficacy of the therapy. In contrast, in neoadjuvant therapy reduction in tumour size is used as an indicator of the sensitivity of tumour cells to the agents applied. If circulating epithelial (tumour) cells can be shown to react to therapy in the same way as the primary tumour, then this response may be exploited to monitor the effect of therapy in the adjuvant setting. METHOD: We used MAINTRAC analysis to monitor the reduction in circulating epithelial cells during the first three to four cycles of neoadjuvant therapy in 30 breast cancer patients. RESULTS: MAINTRAC analysis revealed a patient-specific response. Comparison of this response with the decline in size of the primary tumour showed that the reduction in number of circulating epithelial cells accurately predicted final tumour reduction at surgery if the entire neoadjuvant regimen consisted of chemotherapy. However, the response of the circulating tumour cells was unable to predict the response to additional antibody therapy. CONCLUSION: The response of circulating epithelial cells faithfully reflects the response of the whole tumour to adjuvant therapy, indicating that these cells may be considered part of the tumour and can be used for therapy monitoring.