Search

1) Background: It is well known that solid tumors have the ability to form metastases. The development of distant metastases is due to the primary tumor shedding cells that travel to distant sites via the blood vessels. Platelets specifically promote tumor cells survival in the bloodstream. To investigate the interplay between platelets and circulating tumor cells, we implemented our approach to label simultaneously circulating epithelial tumor cells and platelets. 2) Methods: 40 breast cancer patients were enrolled into the study. Blood samples were collected in EDTA tubes without fixatives and processed at 3 timepoints (0h, 24h and 48h) using the maintrac® approach. In order to visualize platelets we used anti-CD36 antibody staining. 3) Results: We observed at 0h post-blood draw platelet aggregates strictly attached to single cells that did not stain with anti-EpCAM antibody. After keeping blood samples at room temperature for 24h platelet aggregates could still be detected, but the anti-EpCAM antibody became accessible to the underlying cells. CTCs were detected in 94% of patients on day 1 post-blood draw. At 48h following initial blood drawing, platelets had almost completely disappeared from the cell surface and the number of detected CTCs remains stable between 24h and 48h. Furthermore, the number of CTCs correlates with clinical stage of disease. Patients with stage I/II have significantly less CTCs as compared to patients with stage III/IV (median 5 vs. 19 CTCs/100µl cell suspension, p< 0.05). 4) Conclusion: Our results suggest that platelets play a key role in masking circulating tumor cells. Masking may explain the difficulties in detection of these cells and prevention of their elimination by the immune system. Citation Format: Dorothea Schott, Monika PIZON, Katharina Pachmann. Interaction between CTCs and platelets complicate their detection by masking surface epitopes [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-26-12.

Background: Circulating cancer stem cells (cCSCs) are a small aggressive subset of circulating tumor cells with cancer stem cells features: resistance to diverse cancer treatments and the capacity for generating new metastases. Patient-derived xenografts (PDX) are an increasingly accepted 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 xenotransplant assessment of potential new therapeutic targets in cancers. However, animal models do not necessarily represent the real world scenario and are costly and time consuming. An attractive alternative to such animal experiments is the chicken chorioallantoic membrane (CAM) assay. CAM assay is increasingly used as a rapid cost-effective in vivo drug-testing platform that recapitulates many aspects of human cancers. Methods: In this study, primary cultures from circulating cancer stem cells were established using sphere-forming assays. Subsequently, tumorspheres were transplanted onto the CAM membrane of fertilized chicken eggs to form secondary microtumors. Histopathological analyses were performed to confirm that the CAM tumor had the original morphological profile of the patient tumor. Results: We have developed an innovative reliable in vitro platform for cultivation of CSCs from peripheral blood of breast cancer patients. The number of tumorspheres increased significantly with tumor progression. Patients with metastatic disease had statistically more tumorspheres as compared to patients without metastasis (30 vs 10/100µl blood, p< 0.05). Patients with multiple metastases had more tumorspheres compared to patients with single metastases (60 vs 30/100µl blood, p< 0.05). The number of tumorspheres was positively correlated with Ki-67, Her2 status and grade score in primary breast tumors. Tumorspheres showed self-renewal, growth potential, invasion and differentiation in vivo. Tumorspheres could be successfully grafted onto the CAM and grafting positively correlated with aggressiveness and proliferation capacity of the primary tumor. These tumors growing on the CAM pathologically closely resembled the primary tumor. Conclusion: The number of tumorspheres cultured from peripheral blood of cancer patients and the success rate of establishing PDX directly reflect the aggressiveness and proliferation capacity of the primary tumor. Our results support the CAM model using cCSC as a valuable alternative for xenotransplant models. It allows the establishment of new PDXs and provides a fast, cost-effective, and easy to use preclinical platform for cancer biology research, new drug development, treatment individualization, and biomarker discovery. Citation Format: Monika PIZON, Dorothea Schott, Ulrich Pachmann, Katharina Pachmann. Establishing Breast Cancer Patient-derived Xenografts from circulating cancer stem cells in the chorioallantoic membrane (CAM) Model [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-08-11.

Intrauterine development is a key period in human life. The foetal progress largely depends on the function of the placenta, whose responsibility is transportation and biosynthesis of fatty acids. Desaturation enzymes play a key role in placental fatty acid metabolism. Expression of genes coding for desaturases may be associated with pregnancy abnormalities. The objective of this study was to determine the transcriptional activity of the placental genes Fatty Acid Desaturases 1, 2 and 3 (FADS 1, 2 and 3) in women who gave birth to the infants appropriate for gestational age, large for gestational age, small for gestational age, with intrauterine growth restriction and born preterm. 34 pregnant women aged 21–37 years old participated in the study. The placental samples were taken from a site located 2–3 cm away from the umbilical cord attachment. The collected tissue sections were stored in RNAlater according to the manufacturer’s protocol, until required for molecular analysis. The expression profiles of FADS1, FADS2 and FADS3 were determined with RT-qPCR. There was no difference in FADS1 and FADS2 expression between the groups. However, the differences in the expression of the FADS3 were found. Analysis of the FADS1, FADS2 and FADS3 transcription showed significant differences between most of the examined groups. Our findings suggest that the transcriptional activity of FADS genes changes with the severity of intrauterine disorders and is associated with foetal lipid disorders linked to a greater accumulation of fat in the foetal tissues.

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.

Solid tumors are notorious for their ability to form lethal metastases, sometimes several decades following initial cancer diagnosis. Development of distant metastases is a result of the primary tumor shedding cells that travel via lymphatics and the blood to distant sites where they can form metastases. Platelets are known to specifically enhance tumor cells’ survival in the bloodstream by as yet poorly understood mechanisms. To study the interplay of platelets with circulating tumor cells, we implemented our published approach to label both circulating epithelial tumor cells and platelets. Blood samples were collected avoiding fixation from patients with non-metastatic cancer diagnoses and processed at 4 time points following blood collection. Circulating epithelial tumor cells were undetectable directly after blood collection but became visible after overnight storage at room temperature presumably due to release of platelets from the tumor cells. Our results suggest that platelets play a key role in masking circulating tumor cells. Masking may explain the difficulties in detection of these cells and prevention of their elimination by the immune system. Our unmasking approach detects sufficient numbers of circulating tumor cells to monitor the effect on blood tumor cells of different therapeutic measures, thus contributing to improved systemic therapies for cancer.

Background: Circulating cancer stem cells (cCSCs) are a small aggressive subset of circulating tumor cells with cancer stem cells features: resistance to diverse cancer treatments and the capacity for generating new metastases. Patient-derived xenografts (PDX) are an increasingly accepted 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 xenotransplant assessment of potential new therapeutic targets in cancers. However, animal models do not necessarily represent the real world scenario and are costly and time consuming. An attractive alternative to such animal experiments is the chicken chorioallantoic membrane (CAM) assay. Methods: In this study, primary cultures from circulating cancer stem cells were established using sphere-forming assays. Subsequently, tumorspheres were transplanted onto the CAM membrane of fertilized chicken eggs to form secondary microtumors. Results: We have developed an innovative reliable in vitro platform for cultivation of CSCs from peripheral blood of cancer patients. The number of tumorspheres increased significantly with tumor progression. Patients with metastatic disease had statistically more tumorspheres as compared to patients without metastasis (30 vs 10/100µl blood, p Conclusion: The number of tumorspheres cultured from peripheral blood of cancer patients and the success rate of establishing PDX directly reflect the aggressiveness and proliferation capacity of the primary tumor. Our results support the CAM model using cCSC as a valuable alternative for xenotransplant models. It allows establishing new PDXs and providing a fast, low-cost, easy to use preclinical platform for cancer biology research, new drug development, treatment individualization, and biomarker discovery. Citation Format: Monika Pizon, Dorothea Schott, Ulrich Pachmann, Rainer Schobert, Katharina Pachmann. Circulating cancer stem cells as a tool for generating patient-derived xenografts on chick chorioallantoic membrane (CAM) in cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3119.

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

Highlights • CETCs have the same molecular properties as the cells in the primary tumor. • Circulating cancer stem cells can be successfully identified in the blood of breast cancer patients and monitored during RT. • Patients after neoadjuvant chemotherapy had the highest number of CETCs prior to RT but the CETC counts decreased significantly during RT indicating that this group of patients could most likely benefit from adjuvant RT. • Increase in cCSC numbers during RT could be a predictor for early recurrence of cancer disease., 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.

Background Cancer stem cells (CSCs) are the critical subpopulation of cancer cells involved in tumor formation, progression, metastasis and drug resistance. CSCs also have been considered as the main cause of cancer recurrence. The exact role of CSCs in escape of cancer from immunosurveillance is still unknown, but recent studies revealed that enhanced PDL-1 expression in cancer stem cells is linked to immune evasion and could crucially contribute to the maintenance of CSC self-renewal. Understanding the mechanisms behind this PDL-1 overexpression in cancer stem cells is critical for developing more effective anti-PD-1/PD-L1 therapy. Therefore the aim of the study was to determine the number of tumorspheres and expression of PD-L1 on tumorspheres cultured from the peripheral blood in patients with breast cancer.Methods:110 patients with breast cancer in different stages of disease were included in this study. The determination of circulating tumor cells and circulating cancer stem cells (cCSC) was performed using maintrac and tumorsphere forming assay, respectively. Additionally anti-PDL-1 antibody staining was applied to examine PDL-1 expression on breast tumorspheres.Results:We developed an innovative in vitro platform for cultivation of CSCs from peripheral blood of solid cancer patients and confirmed for the first time that tumorspheres are positive for PDL-1 in breast cancer patients. Our data also show a considerable heterogeneity in the PDL-1 expression of tumorspheres. We confirmed once again that the number of tumorspheres increased significantly with tumor progression, especially with presence of metastasis. Patients with metastatic disease had statistically more tumorspheres as compared to patients without metastasis (30 vs 10/100µl blood, p Citation Format: Monika Pizon, Dorothea Schott, Ulrich Pachmann, Katharina Pachmann. Determination of PD-L1 expression on tumorspheres cultured from the peripheral blood in patients with breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-04-08.

The present state of cancer chemotherapy is unsatisfactory. New anticancer drugs that marginally improve the survival of patients continue to be developed at an unsustainably high cost. The study aimed to elucidate the effects of insulin (INS), an inexpensive drug with a convincing safety profile, on the susceptibility of colon cancer to chemotherapeutic agents: 5-fluorouracil (FU), oxaliplatin (OXA), irinotecan (IRI), cyclophosphamide (CPA) and docetaxel (DOC). To examine the effects of insulin on cell viability and apoptosis, we performed an in vitro analysis on colon cancer cell lines Caco-2 and SW480. To verify the results, we performed in vivo analysis on mice bearing MC38 colon tumors. To assess the underlying mechanism of the therapy, we examined the mRNA expression of pathways related to the signaling downstream of insulin receptors (INSR). Moreover, we performed Western blotting to confirm expression patterns derived from the genetic analysis. For the quantification of circulating tumor cells in the peripheral blood, we used the maintrac method. The results of our study show that insulin-pretreated colon cancer cells are significantly more susceptible to commonly used chemotherapeutics. The apoptosis ratio was also enhanced when INS was administered complementary to the examined drugs. The in vivo study showed that the combination of INS and FU resulted in significant inhibition of tumor growth and reduction of the number of circulating tumor cells. This combination caused a significant downregulation of the key signaling substrates downstream of INSR. The results indicate that the downregulation of PIK3CA (phosphatidylinositol 3-kinase catalytic subunit alpha), which plays a critical role in cell signaling and GRB2 (growth factor receptor-bound protein 2), a regulator of cell proliferation and differentiation may be responsible for the sensitizing effect of INS. These findings were confirmed at protein levels by Western blotting. In conclusion, these results suggest that INS might be potentially applied to clinical use to enhance the therapeutic effectiveness of chemotherapeutic drugs. The findings may become a platform for the future development of new and inexpensive strategies for the clinical chemotherapy of tumors.

// Monika Pizon 1 , Dorothea Schott 1 , Ulrich Pachmann 1 , Katharina Pachmann 1 1 Transfusion Center Bayreuth, 95448, Bayreuth, Germany Correspondence to: Monika Pizon, email: mpizon@simfo.de Keywords: breast cancer, circulating epithelial tumor cells, circulating cancer stem cells, predictor for presence of metastasis Received: January 27, 2016 Accepted: June 02, 2016 Published: June 20, 2016 ABSTRACT 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.

Background: Breast malignancies continuously shed tumor cells which may enter the circulation, spread to other tissue and initiate metastases, but only a small subpopulation among CETCs is capable of metastasis formation. The subpopulation of CETC capable of forming tumorspheres in vitro and carrying stem cells properties is assumed to have metastatic potential. The aim of this study was to characterize the pattern of expression of the tumorspheres as compared to CETCs. Methods: Blood samples were obtained from patients diagnosed with breast tumors. CETCs were enumerated with maintrac method and subsequently cultivated in stem cell-selective medium. The expression of stem cell marker genes EpCAM, Nanog, Okt4 Sox2, ALDH and CD133 were detected by RT-PCR in single CETCs and tumorspheres. Results: 0.1- 10% of CETCs could form non-adherent tumorspheres under stem cell- selective conditions after a period of 14 days. Array qRT-PCR analysis revealed that putative stem cell markers, such as Oct4, Sox2, Nanog, EpCAM, ALDH1 and CD133 are overexpressed in relation to house-keeping genes RPL13a and GAPDH in tumor spheres in contrast to the significantly lower expression level of these stem cell markers in individually isolated CETCs. High expression level of pluripotency genes in tumorspheres was associated with aggressive tumor behaviour in terms of tumor progression and type of cancer. Conclusion: Thus, differential expression of stem cell markers in CETCs and tumorspheres substantiates the hypothesis that only a small population of circulating tumor cells possess the stem cell properties and are responsible for metastasis formation. Citation Format: Zimon D, Pizon M, Mayer V, Pachmann K. Stem cell markers are overexpressed in tumorspheres cultured from circulating epithelial tumor cells (CETCs) in patients with breast cancer. [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 P3-07-59.

Background: Cancer stem cells (CSCs) are the critical subpopulation of cancer cells involved in tumor formation, progression, metastasis, and drug resistance. CSCs also have been considered the main cause of cancer recurrence, and the identification of targets and biomarkers is still under development. In this study, primary cultures from peripheral blood were established, using sphere-forming assays for CSC enrichment, and correlated with clinicopathologic features. Methods: Circulating cancer stem cells were cultured under conditions favoring growth of tumorspheres from 72 patients with breast cancer, including a subpopulation of 23 patients with metastatic disease. Cell viability, stem cell marker expression, and ALDH 1 activity were evaluated by fluorescence scanning microscope (Olympus Scan®R). Results: Sphere formation was observed in 79% of patients with breast cancer. In the current study we found that the number of tumorspheres depended on stage of disease. Patients with metastatic disease had statistically significantly more tumorspheres compared to patients without metastasis (median 30 vs. 10; p=0.002). Furthermore, patients with multiple metastases had more tumorspheres as compared to patients with single metastasis (median 30 vs. 60; p Conclusion: Identifying the circulating cancer stem cell may provide more clinically useful prognostic information and can be the basis for developing novel therapies. Citation Format: Monika Pizon, Dorothea Schott, Ulrich Pachmann, Katharina Pachmann. The number of tumorspheres cultured from peripheral blood is directly related to presence of metastasis in breast cancer patients [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B34.

Background: Preclinical studies have suggested that radiotherapy (RT) followed by immunotherapy could be a promising strategy for synergistic enhancement of treatment efficacy. RT induces immunogenic changes in cancer cells that can lead to adaptive upregulation of PD-L1 expression. Moreover, RT not only reduces local recurrence but also improves overall survival by preventing distant metastases, which indicates its influence on the remaining occult tumor, such as circulating tumor cells. For this reason, the aim of this study was to determine the PD-L1 status on CETCs before and during RT. Methods: CETCs were determined in 54 primary breast cancer patients in early and locally advanced stages. The number of CETCs and their expression of PDL-L1 were investigated using the maintrac method. The fraction of PD-L1 positive CETCs was assessed prior to (baseline), 3 and 6 weeks after the start of RT. Results: We found a correlation between the PD-L1 status of CETCs and aggressiveness of cancer disease prior to radiotherapy. Patients with positive lymph node status presented more PD-L1 positive CETCs compared to patients with negative lymph nodes (mean 73% vs. 50%, p Conclusion: Although the number of CETCs and the fraction of PD-L1 positive CETCs were higher in patients who had received neoadjuvant chemotherapy, radiotherapy reduced both measured biomarkers. In the future PD-1/PD-L1 checkpoint inhibitors after radiotherapy might have additional benefit in breast cancer patients. Citation Format: Dorothea Schott, Monika Pizon, Katharina Pachmann, Ulrich Pachmann, Matthias Maeurer. Changes in the number of residual circulating epithelial tumor cells (CETCs) and their programmed cell death ligand 1 (PD-L1) status during radiotherapy in breast cancer patients [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B39.

Background: Preclinical studies have suggested that radiotherapy followed by immunotherapy could be a promising strategy for synergistic enhancement of treatment efficacy. Radiation induces immunogenic changes in cancer cells which can lead to adaptive up-regulation of PD-L1 expression. Moreover, radiotherapy reduces not only local recurrence but even more distant relapse indicating its influence also on the residual occult tumor such as circulating tumor cells. For this reason the aim of this study was to determine the PD-L1 status on CETCs before and during radiotherapy. Methods: CETCs were determined in 54 primary breast cancer patients in early and locally advanced stages. The number of CETCs and their expression of PDL-L1 were investigated using the maintrac method. The fraction of PD-L1 positive CETCs was assessed prior to (baseline), 3 and 6 weeks after the start of radiotherapy. Results: We found an association between the PD-L1 status on CETCs and aggressiveness of cancer disease before radiotherapy. Patients with positive lymph node status had more PD-L1 positive CETCs as compared to patients with negative lymph nodes (mean 73 vs. 50, p Conclusion: The number of CETCs and the fraction of PD-L1 positive CETCs were higher in patients who had received neoadjuvant chemotherapy and radiotherapy reduced both measured biomarkers. In the future PD-1/PD-L1 checkpoint inhibitors after radiotherapy might have additional benefit in breast cancer patients. Citation Format: Dorothea Schott, Monika Pizon, Ulrich Pachmann, Katharina Pachmann, Matthias Mäurer. Influence of radiotherapy on residual circulating epithelial tumor cells (CETCs) and their programmed cell death ligand 1 (PD-L1) status in breast cancer patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-01-14.

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-67‑positive CETCs was significantly higher in patients who had received radiation therapy compared to patients who had not received irradiation. B7-H3‑positive CETCs seemed to be more aggressive as the percentage of B7-H3‑positive CETCs correlated with the percentage of cells positive for the proliferation marker, Ki-67 (r=0.72 P

Background: A major obstacle in the successful treatment of cancer is the occurrence of metastasis. The presence of CETCs is closely related to tumor recurrence, but the mechanisms through which CETCs promote metastasis are still unclear. The aim of this study was to determine the proliferative capacity of CETCs by analyzing the frequency of mammosphere formation with subsequent phenotypic characterization of the spheres arising in breast cancer patients. Methods: CETCs were cultured under condition favoring growth of mammospheres from 38 patients with breast cancer, including a subpopulation of 13 patients with metastatic disease. Cell viability, stem cell marker expression and ALDH 1 activity was evaluated by fluorescence scanning microscope (Olympus Scan®R). Results: Sphere formation was observed in 74 % of patients with breast cancer. Patients with distant metastasis had higher numbers of mammospheres (median 13,0 vs 1,0; p≤0,001) compared to patients without metastasis. In multivariate analysis, a high number of mammospheres was associated with the presence of metastasis. The mammospheres area under the ROC curve was 0,99. Six or more spheres classified metastatic disease with a sensitivity and specificity of 96 % and 100 %, respectively. These results suggest that above cut-off six in the number of mammospheres is statistically highly indicator for disease progression. Analysis of surface marker expression profile of mammospheres showed that spheres cultured from CETCs had typical phenotype of cancer stem cells with a high enzymatic activity for ALDH 1 with the ALDEFLUOR assay. There was no sphere formation in a control group with 50 healthy donors. Conclusion This study demonstrates that a small fraction of CETCs has proliferative activity. Identifying the CETC subset with cancer stem cells properties may provide more clinically useful prognostic information and may be a new indicator for the presence of metastases. Citation Format: Monika Pizon, Dorothea Zimon, Ulrich Pachmann, Katharina Pachmann. Mammospheres derived from circulating epithelial tumor cells are an indicator for presence of metastasis in patient with breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-06-06.

// Dorothea Sonja Schott 1 , Monika Pizon 1 , Ulrich Pachmann 1 and Katharina Pachmann 1 1 Transfusion Center Bayreuth, Bayreuth, Germany Correspondence to: Dorothea Sonja Schott, email: dschott@simfo.de Keywords: circulating epithelial tumor cells, programmed cell death ligand 1, programmed cell death ligand 2, checkpoint inhibitors Received: November 24, 2016 Accepted: July 11, 2017 Published: August 18, 2017 ABSTRACT 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

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. Material and 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 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 responsible to resistance to endocrine therapy this may provide a new biomarker to select patients who might benefit from combination treatment of ER and AR inhibitors. Note: This abstract was not presented at the meeting. Citation Format: Monika Pizon, Dorothea Schott, Daniel Lux, Ulrich Pachmann, Katharina Pachmann. Influence of endocrine therapy on the ratio of androgen receptor (AR) to estrogen receptor (ER) positive circulating epithelial tumor cells (CETCs) in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2295.

Background: Breast cancer is one of the most common types of cancer in women worldwide. It has been demonstrated that even localized tumors without clinically apparent metastases give rise to circulating epithelial tumor cells (CETCs). Recent studies have provided strong support for the circulating cancer stem cell (cCSC) hypothesis, which suggests that a more aggressive subpopulation of circulating tumor cells are the source of metastatic spread from the primary tumor. Measuring CETCs in blood of patients has emerged as a non-invasive diagnostic procedure for screening patients who may be at high risk for developing metastatic cancers. However, accurate detection of CETCs may provide erroneous results, since CETCs undergoing EMT during metastasis are down-regulated for the expression of epithelial cell markers. Methods: 26 breast cancer patients were included into the study. The determination of CETCs and cCSC was performed using maintrac® method and tumorsphere forming assay, respectively. Cell viability, surface marker expression and ALDH 1 activity of the cells in the spheres were evaluated by fluorescence scanning microscope. Results: Sphere formation was observed in 80 % of patients. We found that the number of tumorspheres depended on stage of disease. The number of tumorspheres increased significantly with tumor progression, especially with the presence of metastases. Tumorsphere formation was observed in all metastatic patients (median of 30 tumorspheres/100μl of blood), although only 27% of them had detectable CETCs. Analysis of surface marker expression profile showed that the cells in the spheres had typical phenotype of cancer stem cells. Sphere formation was not observed in healthy subjects (n=20). Conclusion: There is a high correlation between the numbers of tumorspheres cultured from peripheral blood with clinical stage of disease. This may be an indicator of tumor aggressiveness, especially presence of metastases and may provide clinically useful prognostic information. Citation Format: Pizon M, Schott D, Pachmann U, Pachmann K. Detection of circulating cancer stem cells: A novel approach as a predictive marker for breast cancer metastases. This may be especially useful in patients with undetectable circulating epithelial tumor cells [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-01-12.

Background: Breast cancer is one of the leading causes of cancer death for women worldwide. Major hurdles for a successful treatment are cancer metastases, resistance to therapy and disease recurrence. The presence of circulating epithelial tumor cells (CETCs) is closely related to metastasis formation, but the mechanisms through which CETCs promote recurrence of disease are still unclear. CD36 is found in the cell membrane of tumor cells and responsible for taking up fatty acid. It is known that CD36 is a general marker of metastatic cells and its amplification specifically correlated with metastasis in a large number of human tumors. Therefore, the aim of this study was to determine the metastatic marker CD36 on CETCs and to correlate it's frequency with clinicopathological features in breast cancer patients. Methods: CETCs were determined from blood of 42 patients suffering from breast cancer. The number of vital CETCs and their expression of CD 36 was investigated using the maintrac® method. Results: CD36 expressing CETCs were detected in 95 % of breast cancer patients. Breast cancer patients with metastatic disease had significantly more CD36 positive CETCs as compared to patients without metastases (median 72% vs. 48%; p Conclusion: We showed for the first time that breast cancer patients have detectable CETCs with a high frequency of CD36 which highly correlated with progression of cancer disease. Our findings suggest that using a CD36 antibody treatment may be an effective strategy to fight cancer. Citation Format: Monika Pizon, Dorothea Schott, Ulrich Pachmann, Katharina Pachmann. A subpopulation of circulating epithelial tumor cells positive for fatty acid receptor CD36 is linked to aggressiveness of breast cancer disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5193.

Background: Recently the hypothesis has been proposed that a more aggressive subpopulation of circulating tumor cells, circulating cancer stem cells, are the source of metastatic spread from the primary tumor. These cells are considered to be responsible for treatment relapse and have therefore become a major target in cancer research. The antibiotic salinomycin isolated from Streptomyces albus is the best established cancer stem cell inhibitor and has been shown to reduce the expression of markers for stemness and spheroid-forming ability. The mechanisms of the anticancer effects remain unclear. In this study, we evaluated the cytotoxic effect of salinomycin on CETCs and tumorspheres cultured from cCSC. Methods: 20 patients with breast cancer in different stages of disease were included. The determination of CETCs and cCSC was performed using Maintrac® method and tumorsphere forming assay, respectively. To evaluate the cytotoxic effect of salinomycin on CETCs and spheroids, they were exposed to different concentrations of salinomycin in short time culture for different periods of time. Results: 79% of patients had detectable CETCs and sphere formation was observed in 74% of breast cancer patients. Interestingly, growth of spheroids was detected also in patients without CETCs. The number of tumorspheres increased significantly with tumor progression, especially with presence of metastasis. Treatment with salinomycin showed an 80% reduction in spheroid formation compared with control. Salinomycin reduced the viability of spheroids in a dose-dependent manner. Salinomycin had a stronger cytotoxic effect on CETCs in nonmetastatic as compared to metastatic patients (median of dead CETCs 85% vs. 34%, p Citation Format: Monika Pizon, Dorothea Schott, Ulrich Pachmann, Katharina Pachmann, Norbert Szalus. Salinomycin kills not only circulating epithelial tumor cells (CETCs) but also circulating cancer stem cells (cCSC) and may potentially prevent progression in patients with breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A067.

Background Epicardial ablation concomitant to cardiac surgery is an easy and safe approach to treat atrial fibrillation (AF), but its efficacy in longstanding persistent (LsPe) AF remains intermediate. Although larger left atrial size has been associated with worse outcome after ablation, biochemical predictors of success are not well established. The aim of this study was to evaluate relationship between biochemical marker, echo-characteristic and cardiac rhythm in 6 months follow-up after epicardial ultrasound (HIFU) ablation. Methods We included 78 consecutive patients, who underwent elective cardiac surgery. 42 patients with AF (11.9% paroxysmal, 23.8% persistent, 64.3% LsPeAF) underwent concomitant HIFU ablation (AF ablation group), 16 with AF underwent cardiac surgery without ablation (AF control) and 20 had preoperatively normal sinus rhythm (SR control). We measured plasma ANP secretion before, on postoperative day (POD) 1, POD 7 as well as 3 and 6 months after surgery. Moreover, we estimated cardiac rhythm and atrial mechanical function by Atrial Filling Fraction (AFF) and A-wave velocity in follow-up. Results Baseline ANP levels were higher in patients with LsPeAF, as compared to the paroxysmal and permanent AF and to the SR control group. Patients with LsPeAF (n = 27) who converted to SR had preoperatively smaller left atrial diameter (LAD) and LA area (p < 0.05) and higher ANP level (p = 0.009) than those who remained in AF at 6 months after ablation. Multivariate regression analysis revealed that only preoperative ANP level was an independent predictor of cardiac rhythm after ablation. Patients with LsPeAF and preoperative ANP >7.5 nmol/l presented with SR in 80%, in contrast to those with ANP

BackgroundCirculating epithelial tumor cell (CETC) analysis is a promising diagnostic field for estimating the risk for metastatic relapse and progression in patients with malignant disease. CETCs characterization can be used as a liquid biopsy for prognostic and predictive purposes in breast and other cancers. IGF-IR and VEGFR-2 play an important role in tumor growth and the progression of cancer disease. The purpose of the current study was therefore to investigate their expression on CETCs.MethodsCETCs were determined from the blood of 50 patients suffering from breast cancer. The number of vital CETCs and the expression of IGF-IR and VEGFR-2 were investigated using the maintrac® method.ResultsIGF-IR and VEGFR-2 expression on the surface of CETCs were detected in 84% of patients. A statistically high correlation was found between IGF-IR and VEGFR-2 (r = 0.745 and pSummaryIn this study, a parallel and co-expression of IGF-IR and VEGFR-2 was examined on the surface of CETCs in breast cancer patients for the first time. Characterization of CETCs may be a promising approach for the rational design of targeted anticancer therapies.

Background: Solid malignancies continuously shed tumor cells which may enter the circulation, spread to other tissues and initiate metastases, but it is assumed that only a small subpopulation among CETCs is capable of metastasis formation, the subpopulation of CETCs capable of forming tumorspheres in vitro and carrying stem cells properties. The aim of this study was to characterize the pattern of expression of the tumorspheres as compared to CETCs. Methods: Blood samples were obtained from patients diagnosed with solid tumors. CETCs were enumerated with the maintrac method and subsequently cultivated in epithelial stem cell-selective medium. Immunofluorescence and qRT-PCR were performed to examine the metastatic ability of tumorspheres in vitro. Results: Analysis of surface markers in non-adherent tumorspheres forming from CETCs under stem cell- selective conditions after a period of 14 days showed typical phenotype for cancer stem cells depending on type of cancer. Furthermore, spheres had high enzymatic activity for ALDH 1. Array qRT-PCR analysis revealed that putative stem cell markers, such as Oct4, Sox2, Nanog, EpCAM, ALDH1 and CD133 are overexpressed in relation to house-keeping genes RPL13a and GAPDH in tumor spheres in contrast to the significantly lower expression level of these stem cell markers in individually isolated CETCs. High expression level of pluripotency genes in tumorspheres was associated with aggressive tumor behaviour in terms of tumor progression and type of cancer. Conclusion: Here we show that stem cell markers are overexpressed in tumorspheres as compared to CETCs. Obviously only a small population of CETCs can be grown into spheres which possess stem cell properties and most probably are responsible for recurrence and treatment failure. Precise characterization of CETCs and tumorspheres on the single cell and sphere level will contribute to a better understanding of metastasis formation and lead to development of cancer stem cell based cancer therapy. Citation Format: Katharina Pachmann, Monika Pizon, Dorothea Schott, Ulrich Pachmann. Tumorspheres cultured from circulating epithelial tumor cells (CETCs) overexpress stem cell markers in patients with solid cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2498.

Background: Analysis of CETCs is a promising diagnostic field for estimating the risk for metastatic relapse and progression. The phenotypic characterization of CETCs may provide real time information and can be of great value in therapy monitoring. Programmed cell death ligand 1 (PDL-1) is an important protein frequently upregulated in a number of different cancers. Cancer cells expressing PDL-1 inhibit immune-modulatory T-cell activation allowing disease progression. Therefore this immune checkpoint has emerged as an important target for immune therapy. The purpose of the current study was to identify potential patients who may benefit from PDL-1 targeted immunotherapies. Methods: CETCs were determined from blood of 22 (69%) non-metastatic and 10 (31%) metastatic breast cancer patients. The number of vital CETCs and the expression of PDL-1 were investigated using the maintrac® method. Results: PDL-1 expressing CETCs were detected in 94% of breast cancer patients; however the fraction varied between 30 to 100% in individual patients. There was no association between the frequency of PDL-1 positive CETCs and chemotherapy. Interestingly, we found a relationship between the frequencies of PDL-1 positive CETCs and progression of disease. Patients with metastatic disease had more PDL-1 positive CETCs as compared to patients without metastasis. Conclusion: Metastatic breast cancer patients have an elevated fraction of PDL-1 positive CETCs as compared to non-metastatic patients. These cells have the capacity to block the immune system and therefore may be a promising target for anticancer therapies. Monitoring the frequency of PDL-1 positive CETCs could contribute to individual patient's response for to anti-PDL-1 therapy. Citation Format: Monika Pizon, Dorothea Zimon, Ulrich Pachmann, Katharina Pachmann. PDL-1 is more frequently expressed on circulating epithelial tumor cells (CETCs) in metastatic than in non-metastatic breast cancer patients and could be a crucial factor in the inhibition of immune response to CETCs during metastasis formation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B95.

Background: Among the cells that are disseminated from a malignant tumor only very few are capable to resettle in distant organs and grow into life-threatening metastases. Therefore, the question arises how and whether such cells which have the potential to grow into metastases can be detected. It has been shown that a subpopulation of cells from breast cancer tissue can form so-called mammospheres with stem cell features. Here we show that such tumor spheres can also be grown from peripherally circulating tumor cells from breast cancer patients in different stages of disease Materials and Methods: Using a nondissipative approach with only one enrichment step of red blood cell lysis, the cells from the pellet, containing the white blood cells together with the putative tumor cells were cultured under conditions favoring the growth of epithelial cells. At 7, 14 and 21days the cell cultures were inspected for the appearance of spheroids staining with anti-EpCAM, anti-CD24 and anti-CD44 antibody and.expressing ALDH1. Results: Peripherally circulating cells from patients with malignant tumors in different stages of disease were analyzed for the presence of circulating epithelial tumor suspect cells and the frequencies of tumorspheres. Tumorspheres could so far be grown from 79% of 36 patients in whom more than 1700/ml epithelial tumor suspect cells were detected. Numbers of tumorspheres varied from 1 to 29 /ml and correlated with the aggressiveness of the tumor. Surprsingly the numbers were highest in patients after surgery who had not yet received any systemic therapy. The size of the spheres increased from day 7 to day 21. The spheres were negative for CD24 and positive for CD44. They highly express ALDH1 and thus exhibite typical features of stem cells. Conclusion: Here, we demonstrate that the circulating tumor cells, detected in our approach contain a subpopulation with stem cell-like properties capable of growing into tumorspheres. The frequency and growth potential of cells capable of forming spheres seems to be dependent from the properties of the primary tumor. The possibility to grow tumorspheres from peripherally circulating tumor cells may open up a new field, where the relevant cells with stem cell properties from individual patients can now be specifically analysed further for genetic endowment, transcriptional activity, heterogeneity and stem cell markers. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD6-1.

Background: The detection of tumor cells circulating in the peripheral blood of patients with breast cancer is a sign that cells have been able to leave the primary tumor and survive in the circulation. However, in order to form metastases they require additional properties such as the ability to adhere, self renew and grow. Here we present data that a variable fraction among the circulating tumor cells detected by the maintrac® approach express mRNA of the stem cell gene nanog and of the adhesion molecule vimentin and are capable of forming tumor spheres a property ascribed to TIC. Patients and methods: Between 10 to 50 circulating epithelial antigen positive cells detected by the maintrac® approach were selected randomly from each of 20 patients with breast cancer before and after surgery and isolated using automated capillary aspiration and deposited individually onto slides for expression profiling. In addition, the circulating tumor cells were cultured without isolation among the white blood cells from 5 patients with breast cancer in different stages of disease using culture methods favoring growth of epithelial cells. Results: Although no EpCAM mRNA positive cells expressing stem cell genes or the adhesion molecule vimentin were detected before surgery, 10 to 20% of the cells were found to be positive for mRNA of these genes after surgery. Most surprisingly, it was possible to grow tumor spheres (Fig 1) from these circulating cells without previous isolation in all patients in a fraction comprising between 1% of the epithelial antigen positive cells in patients with newly diagnosed tumor up to 10% in a patient with advanced breast cancer. Conclusion: We here show, that among the peripherally circulating tumor cells a variable fraction is able to express stem cell and adhesion properties and can be grown into tumor spheres, a property ascribed to cells capable of initiating tumors and metastases. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-03-13.