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.

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.

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.

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.

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.

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.

Insulin-like growth factor receptor I (IGF-IR) and vascular endothelial growth factor receptor 2 (VEGFR-2) are expressed on the circulating epithelial tumor cells of breast cancer patients.

BACKGROUND: Circulating 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. METHODS: CETCs 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. RESULTS: IGF-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 p<0.001) on the CETCs. The co-expression of both receptors was confirmed in some experiments and ranged between 70% and 100%. Statistically significant correlations were observed between the number of CETCs and IGF-IR (r = 0.315 and p<0.05) and VEGFR-2 (r = 0.310 and p<0.05) expression. The presence of CETCs and the level of IGF-IR and VEGFR-2 expression were not associated with tumor stage, hormone receptor status or nodal/distant metastasis. SUMMARY: In 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.

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.

Monitoring the response of circulating epithelial tumor cells to adjuvant chemotherapy in breast cancer allows detection of patients at risk of early relapse.

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

An increase in cell number at completion of therapy may develop as an indicator of early relapse: quantification of circulating epithelial tumor cells (CETC) for monitoring of adjuvant therapy in breast cancer.

PURPOSE: Treatment efficiency of adjuvant therapy in breast cancer is only revealed after several years by statistical evaluation and gives no answer for the individual patient. We here present a method to analyze the response to adjuvant chemotherapy online in individual patients. METHODS/RESULTS: In 25 consecutive non-metastatic primary breast cancer patients adjuvant fluorouracil/epirubicin/cyclophosphamid (FEC) or EC followed by taxane (EC-T) or cyclophosphamid/methotrexate/fluorouracil (CMF) therapy were given. Circulating epithelial tumor cells (CETC) were quantified before and after each second cycle of the therapy regimen, between the anthracycline and the taxane block of the regimen and in some cases repeatedly during CMF treatment. Independent of the initial cell number CETC numbers showed a decline, no change or a minor increase in 15 patients of which 14 remained in complete remission and 1 suffered local relapse. Ten patients showed an increase at the end of therapy of which 4 have relapsed during the observation time of between 2 months and up to 54 months. This patient group was compared to a previously published group of 25 patients who have all reached a follow-up of 4.5 years or until relapse. CONCLUSION: As in the previous report, Kaplan-Meier analysis revealed a high correlation between the response of CETC to therapy and relapse (p < 0.0001) and curves of both patient groups were super imposable. Multivariate analysis revealed the response of CETC to therapy to be an independent predictive marker for relapse.

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.

Influence of isotypes of disease-associated autoantibodies on the expression of natural autoantibody repertoires in humans.

Current understanding of immune network interactions mediated by immunoglobulins focuses on the role of idiotypes expressed on antibody variable regions. Idiotype interactions account significantly for the functional integrity of natural self-reactive antibody repertoires, whereas immunoglobulin isotypes are not considered to direct natural autoimmunity. Autoimmune thrombocytopenic purpura (AITP), a bleeding disorder caused by clonally restricted platelet-specific autoantibodies of the IgM or IgG isotype, is an excellent model to investigate the impact of isotype differences of immunoglobulins on the selection of natural self-reactive antibody repertoires in humans. Using specific analytical techniques to characterize the natural self-reactive antibody repertoire (i.e. quantitative immunoblotting, affinity biosensor technology), we here demonstrate that isotype differences of disease-associated autoantibodies are associated with altered natural self-reactive antibody repertoires in humans. Our data suggest that regulation of natural autoreactivity by antibody isotype might occur under certain conditions. The control of natural self-reactive antibody repertoires by immunoglobulin isotypes at a supraclonal level may provide a structural basis for non-organ-specific broad alterations of natural self-reactive antibody repertoires in organ-specific autoimmune diseases.

Evidence that human autoimmune thrombocytopenia mediated by both immunoglobulin isotypes IgM and IgG is an independent disease entity.

Autoimmune thrombocytopenic purpura (AITP) is a bleeding disorder caused by clonally restricted self-reactive antibodies with specificity for platelet glycoproteins. Anti-platelet autoantibodies in AITP mainly belong to the IgG class. The occurrence of anti-platelet autoantibodies of the IgM isotype has been reported, and AITP is partially mediated by antibodies of both isotypes, IgM and IgG. Using a technique of quantitative immunoblotting of immunoglobulins on self-tissues, followed by multiparametric statistical analysis of the data, we here demonstrate that patients with IgM- and IgG-mediated AITP are readily discriminated from patients with IgM-mediated AITP as well as from patients with IgG-mediated AITP at the basis of self-reactive antibody repertoires of isotypes IgM and IgG toward non-platelet antigens of human origin. Our data suggest that, in view of the important physiological functions of self-reactive antibody repertoires, human AITP mediated by both immunoglobulin isotypes IgG and IgM may be an independent disease entity. The role of autoantibody isotype for the pathophysiology of AITP might currently be underestimated, and diagnostic and therapeutic procedures in AITP might profit from considering autoantibody isotype more carefully.

Standardized quantification of circulating peripheral tumor cells from lung and breast cancer.

Detection and quantitation of circulating tumor cells from solid epithelial tumors could become a valuable tool for therapy monitoring if the procedure can be standardized. In the present work we assessed the influence of pre-analytical handling, storage and white blood cell isolation on analysis of a population of spiked tumor cell-line cells and intrinsically present epithelial cells in the peripheral blood of breast and lung cancer patients and the sensitivity of their detection. Sucrose density separation did not enrich epithelial cells, and even depleted them, leading to a gross underestimation of their numbers (3/13 positive, between 2.9 and 50 cells/mL) in comparison to red blood cell lysis (13/13 positive, between 77,200 and 800 cells/mL). Short-term storage of whole blood samples for up to 7 days had little influence on the number of epithelial cells recovered. The effectiveness of magnetic bead enrichment was dependent on the number of relevant cells and the volume used for enrichment. Red blood cell lysis and fluorochrome-labeled antibody staining in a no-wash procedure with subsequent laser scanning cytometry allowed the detection of circulating epithelial cells in 92% of breast and lung cancer patients. Two examples of how this method can be applied for the longitudinal analysis in individual patients are shown, with an increase in numbers preceding relapse and a decrease paralleling tumor reduction. The proposed simple and easy method allows close monitoring, which may help in real-time analysis of the response of solid tumors, especially their systemic component, to therapy and hopefully will contribute to more individually tailored therapy.

Increase in number of circulating disseminated epithelial cells after surgery for non-small cell lung cancer monitored by MAINTRAC(R) is a predictor for relapse: A preliminary report.

BACKGROUND: Lung cancer still remains one of the most commonly occurring solid tumors and even in stage Ia, surgery fails in 30% of patients who develop distant metastases. It is hypothesized that these must have developed from occult circulating tumor cells present at the time of surgery, or before. The aim of the present study was to detect such cells in the peripheral blood and to monitor these cells following surgery. METHODS: 30 patients treated for lung cancer with surgery were monitored for circulating epithelial cells (CEC) by taking peripheral blood samples before, 2 weeks and 5 months after surgery and/or radiotherapy (RT) chemotherapy (CT) or combined RT/CT using magnetic bead enrichment and laser scanning cytometry (MAINTRAC(R)) for quantification of these cells. RESULTS: In 86% of the patients CEC were detected before surgery and in 100% at 2 weeks and 5 months after surgery. In the control group, which consisted of 100 normal donors without cancer, 97 % were negative for CEC. A significantly higher number of CEC was found preoperatively in patients with squamous cell carcinoma than in those with adenocarcinoma. In correlation to the extent of parenchymal manipulation 2 weeks after surgery, an increase in numbers of CEC was observed with limited resections (18/21) whereas pneumonectomy led to a decrease (5/8) of CEC, 2 weeks after surgery. The third analysis done 5 months after surgery identified 3 groups of patients. In the group of 5 patients who received neo- or adjuvant chemo/radiotherapy there was evidence that monitoring of CEC can evaluate the effects of therapy. Another group of 7 patients who underwent surgery only showed a decrease of CEC and no signs of relapse. A third group of 11 patients who had surgery only, showed an increase of CEC (4 with an initial decrease after surgery and 7 with continuous increase). In the group with a continuous increase during the following 24 months, 2 early relapses in patients with stage Ia adenocarcinoma were observed. The increase of CEC preceded clinical detection by six months. CONCLUSION: We consider, therefore, that patients with adenocarcinoma and a continuous increase of CEC after complete resection for lung cancer are at an increased risk of early relapse.