Ulrich Pachmann

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.

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

BackgroundLung 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.Methods30 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®) for quantification of these cells.ResultsIn 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.ConclusionWe 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.

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.

Abstract P4-15-20: Insulin-like growth factor receptor-1 (IGF-1R) expression is highly correlated with HER2 amplification on circulating epithelial tumor cells (CETCs) in breast cancer - this may be the reason for resistance to trastuzumab

Background: HER2 status has been the most extensively studied biomarker in circulating epithelial tumor cells of breast cancer patients. HER2 status can change during recurrence of disease and HER2 overexpression is a mandatory requirement to administer anti-HER2-directed drugs. Nevertheless, 70% of patients with HER2-positive breast cancers develop intrinsic or secondary resistance to trastuzumab. This resistance has been associated with the activation of an alternative signalling pathway such as the insulin-like growth factor (IGF) pathway. Therefore we investigated the expression of IGF-IR on the CETCs in addition to HER2 amplification in breast cancer patients to identify patients who might benefit from a combined targeted therapy against HER2 and IGF-IR. Methods: CETCs were determined from blood of 30 breast cancer patients. The number of vital CETCs and the expression of IGF-IR were investigated using the maintrac® approach. Fluorescence in situ hybridisation was used for analysis of HER2 amplification in CETCs. Results: CETCs could be detected in all breast cancer patients. The number of CETCs ranged from 4 to 163 in 100 µl of cell suspension. IGF-IR expression on the surface of CETCs was detected in all patients. Setting a cut-off 30% positive cells as HER2 positive in 75% of patients HER2 positive CETCs were observed irrespective of the status in the primary tumor. In contrast, only 6% of patients changed their HER2 status from positive tissue to negative CETCs. A statistically high correlation was found between the percentage of IGF-IR positive and HER2 positive CETCs. A statistically significant association was found between IGF-IR expression or HER2 amplification and ER/PR receptor status. The higher frequency of HER2 amplified circulating tumor cells might be due to the difference in preparation steps between tissue and blood borne cells and one of the reasons for trastuzumab resistance and for the response of some HER2 negative patients to trastuzumab. Re-evaluation of HER2 status in CETCs could be a valid strategy with potential clinical applications. Conclusion: Our results demonstrate a parallel expression of IGF-IR and HER2 amplification in CETCs. IGF-IR may be involved in the development of resistance to trastuzumab and may be an important potential therapeutic target in breast cancers with HER2 positive circulating tumor cells. Combining targeting of IGF-IR and HER2 may be a rational approach to improve response to trastuzumab in the sub-group of CETCs that express both, HER2 and IGF-IR. Citation Format: Dorothea Zimon, Monika Pizon, Ulrich Pachmann, Katharina Pachmann. Insulin-like growth factor receptor-1 (IGF-1R) expression is highly correlated with HER2 amplification on circulating epithelial tumor cells (CETCs) in breast cancer - this may be the reason for resistance to trastuzumab [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 P4-15-20.

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

Abstract 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.

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 patients 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.

Chemosensitivity Testing of Circulating Epithelial Cells (CETC) in Breast Cancer Patients and Correlation to Clinical Outcome.

In spite of ample prognostic markers available in breast cancer, still a considerable proportion of patients with good prognostic markers suffers relapse whereas patients with poor prognostic markers may remain disease free. It would, therefore, be desirable to control, at the individual patient level, whether the applied therapy is effective. Our previous work indicates, that in cancer patients most of the epithelial cells circulating in peripheral blood (CETC) are part of the tumor and that the response of these cells reflects the response of the tumor to the applied therapies.Therefore, these cells were used to assay chemosensitivity in short time cultures analyzing the percent of cell killing during short time incubation and monitoring the decrease or increase in numbers of these cells during treatment with the respective agents providing a unique tool for therapy surveillance. Patients were prospectively analysed for the number of CETC before each new combination of chemotherapy. 1ml of blood was drawn into EDTA vials, red blood cells lysed and the white blood cell pellet stained with FITC-labelled anti-Epcam. Green fluorescent cells were detected by image analysis and dead cells excluded due to red PI fluorescence. Activity of individual compounds was determined using three different concentrations of each compound at 3h, 6h and 12hs and displayed as % cell killing. The in vitro results were then compared to in vivo reduction of CETCs and to the reduction of a marker lesion.215 patients have been investigated so far. Cell killing was dose and time dependent. The highest killing rates were observed with Epirubicin and Taxanes, agents which are known for their high activity in breast cancer. Less than 20% killing activity was termed marginal activity. In vitro sensitivitity was highly significantly predictive of in vivo CETC reduction. In some cases an increasing resistance could be shown to develop during repeated cycles of the same combination therapy. CETC reduction was correlated with a prolonged progression free survival.Thus this approach can in the future be used to test in advance the sensitivity of the circulating tumor cells to chemotherapy and at the same time monitor the current response of the cells to therapy in vivo in order to optimize and individualize therapy. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2044.

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-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<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.

Abstract 4955: PD-L1 expression on circulating epithelial tumor cells (CETCs) correlates with the presence of metastasis in breast cancer patients and differs from PD-L2 expression

Background: Strategies to improve the efficacy of the immune system against malignant tumors represent a major innovation focusing on the programmed cell death-1 receptor (PD-1), with its two ligands PD-L1 and PD-L2. The expression of PD-L1 has been evaluated in a number of different tumor types and can be used as a predictive biomarker for PD-1/PD-L1 checkpoint inhibitor treatment response. PD-L2 has not received as much attention and its role in modulating tumor immunity is less clear. We used a non-invasive, real-time liquid biopsy to better characterize PD-L1/-L2 expression on circulating epithelial tumor cells (CETCs) in breast cancer patients. Methods: CETCs were determined from blood of 72 patients suffering from breast cancer. The number of vital CETCs and their 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 cancer patients whereas only 82% patients had PD-L2 positive CETCs. 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 Conclusion: Breast cancer patients have detectable CETCs with a high frequency of PD-L1 which correlates with progression of cancer disease. PD-L1 seems to be a major factor in immune evasion and may be a promising target of anticancer therapies. Monitoring the frequency of PD-L1 positive CETCs could reflect individual patient’s response to an anti-PD-1/PD-L1 therapy. Citation Format: Monika Pizon, Dorothea Schott, Ulrich Pachmann, Katharina Pachmann. PD-L1 expression on circulating epithelial tumor cells (CETCs) correlates with the presence of metastasis in breast cancer patients and differs from PD-L2 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4955. doi:10.1158/1538-7445.AM2017-4955