Carsten Hagenbeck

Circulating tumor cells in breast cancer

Detection of disseminated tumor cells (DTCs) in bone marrow and of circulating tumor cells (CTCs) in the blood has become a major focus of translational cancer research. DTC presence is a common phenomenon seen in 30-40% of primary breast cancer patients and is strongly associated with poor clinical outcome. Since bone marrow biopsy is an invasive procedure, evaluation of CTCs might become a desired alternative. Recent clinical trials have shown CTC detection to be a promising prognostic tool in both primary and metastatic setting. Evaluation of CTCs might be useful for therapy monitoring and their characterization might help to identify novel targets for biological therapies aimed at disrupting earliest steps of metastatic cascade.

The presence and prognostic impact of apoptotic and nonapoptotic disseminated tumor cells in the bone marrow of primary breast cancer patients after neoadjuvant chemotherapy

IntroductionNeoadjuvant systemic therapy of primary breast cancer (PBC) patients offers the possibility to monitor treatment response. However, patients might have metastatic relapse despite achieving a pathologic complete response (pCR). This indicates that local response to therapy must not be representative for systemic treatment efficacy. Therefore, the aim of this study was to compare local response with systemic tumor cell dissemination by determining the presence of disseminated tumor cells (DTCs), including apoptotic tumor cells, in the bone marrow (BM) of PBC patients after neoadjuvant chemotherapy (NACT).MethodsDTCs were detected by immunocytochemistry (pancytokeratin antibody A45-B/B3) and cytomorphology (DTC status). The presence of apoptotic tumor cells was determined by using the M30 antibody (M30 status). This antibody detects a neo-epitope that is expressed only during early apoptosis.ResultsBM aspirates from 400 PBC patients that had completed NACT were eligible for this study. Of these, 167 (42%) patients were DTC positive (DTC status). The M30 status was investigated in 308 patients. Apoptotic (M30-positive) tumor cells were detected in 89 (29%) of these. Whereas the DTC status was not correlated (P = 0.557) to local treatment response (that is, pCR or a clinical complete/partial response), the presence of M30-positive tumor cells was significantly higher in patients that responded to therapy (P = 0.026). Additionally, DTC-positive patients were at an increased risk for disease relapse (hazard ratio, 1.87; 95% CI, 1.11 to 3.15; P = 0.019).ConclusionThe presence of DTC is independent of therapy response of the primary tumor. As patients that are DTC positive after NACT have an unfavorable outcome, they might benefit from additional systemic treatment.

Circulating tumour cells in breast cancer

Evaluation of isolated tumour cells in bone marrow (BM) and peripheral blood has become a major focus of translational cancer research. The presence of disseminated tumour cells in BM is a common phenomenon observed in 30–40% of primary breast cancer patients and independently predicts reduced clinical outcome. The detection of circulating tumour cells (CTCs) in blood might become a desired alternative to the invasive and painful BM biopsy. Recent clinical trials confirmed the feasibility of CTC detection as a robust and reproducible parameter for prognostication in both adjuvant and metastatic setting. The characterisation of CTCs might become an important biomarker for therapy monitoring and help to identify specific targets for novel therapeutic strategies.

Isolation and Molecular Characterization of Amniotic Fluid-Derived Mesenchymal Stem Cells Obtained from Caesarean Sections

Human amniotic fluid cells are immune-privileged with low immunogenicity and anti-inflammatory properties. They are able to self-renew, are highly proliferative, and have a broad differentiation potential, making them amenable for cell-based therapies. Amniotic fluid (AF) is routinely obtained via amniocentesis and contains heterogeneous populations of foetal-derived progenitor cells including mesenchymal stem cells (MSCs). In this study, we isolated human MSCs from AF (AF-MSCs) obtained during Caesarean sections (C-sections) and characterized them. These AF-MSCs showed typical MSC characteristics such as morphology, in vitro differentiation potential, surface marker expression, and secreted factors. Besides vimentin and the stem cell marker CD133, subpopulations of AF-MSCs expressed pluripotency-associated markers such as SSEA4, c-Kit, TRA-1-60, and TRA-1-81. The secretome and related gene ontology (GO) terms underline their immune modulatory properties. Furthermore, transcriptome analyses revealed similarities with native foetal bone marrow-derived MSCs. Significant KEGG pathways as well as GO terms are mostly related to immune function, embryonic skeletal system, and TGFβ-signalling. An AF-MSC-enriched gene set included putative AF-MSC markers PSG5, EMX-2, and EVR-3. In essence, C-section-derived AF-MSCs can be routinely obtained and are amenable for personalized cell therapies and disease modelling.

The presence of human mesenchymal stem cells of renal origin in amniotic fluid increases with gestational time

BackgroundEstablished therapies for managing kidney dysfunction such as kidney dialysis and transplantation are limited due to the shortage of compatible donated organs and high costs. Stem cell-based therapies are currently under investigation as an alternative treatment option. As amniotic fluid is composed of fetal urine harboring mesenchymal stem cells (AF-MSCs), we hypothesized that third-trimester amniotic fluid could be a novel source of renal progenitor and differentiated cells.MethodsHuman third-trimester amniotic fluid cells (AFCs) were isolated and cultured in distinct media. These cells were characterized as renal progenitor cells with respect to cell morphology, cell surface marker expression, transcriptome and differentiation into chondrocytes, osteoblasts and adipocytes. To test for renal function, a comparative albumin endocytosis assay was performed using AF-MSCs and commercially available renal cells derived from kidney biopsies. Comparative transcriptome analyses of first, second and third trimester-derived AF-MSCs were conducted to monitor expression of renal-related genes.ResultsRegardless of the media used, AFCs showed expression of pluripotency-associated markers such as SSEA4, TRA-1-60, TRA-1-81 and C-Kit. They also express the mesenchymal marker Vimentin. Immunophenotyping confirmed that third-trimester AFCs are bona fide MSCs. AF-MSCs expressed the master renal progenitor markers SIX2 and CITED1, in addition to typical renal proteins such as PODXL, LHX1, BRN1 and PAX8. Albumin endocytosis assays demonstrated the functionality of AF-MSCs as renal cells. Additionally, upregulated expression of BMP7 and downregulation of WT1, CD133, SIX2 and C-Kit were observed upon activation of WNT signaling by treatment with the GSK-3 inhibitor CHIR99201. Transcriptome analysis and semiquantitative PCR revealed increasing expression levels of renal-specific genes (e.g., SALL1, HNF4B, SIX2) with gestational time. Moreover, AF-MSCs shared more genes with human kidney cells than with native MSCs and gene ontology terms revealed involvement of biological processes associated with kidney morphogenesis.ConclusionsThird-trimester amniotic fluid contains AF-MSCs of renal origin and this novel source of kidney progenitors may have enormous future potentials for disease modeling, renal repair and drug screening.

Use of Granulocyte-colony Stimulating Factor During Chemotherapy and Its Association With CA27.29 and Circulating Tumor Cells—Results From the SUCCESS A Trial

Background Little is known about the effect of granulocyte colony‐stimulating factor (G‐CSF) treatment during adjuvant chemotherapy on prognostic markers. The present study explored the association between G‐CSF and changes in cancer antigen (CA)27.29 and circulating tumor cell (CTC) levels during therapy. Patients and Methods A total of 3754 node‐positive or high‐risk node‐negative early‐stage breast cancer patients were treated within the SUCCESS‐A trial (simultaneous study of gemcitabine‐docetaxel combination adjuvant treatment, as well as extended bisphosphonate and surveillance‐trial). CA27.29 and CTCs were determined before the start and within 6 weeks after the end of chemotherapy. Results Overall, 1324 of the 2646 patients (50.0%) available for analysis had ≥ 1 G‐CSF applications during chemotherapy. G‐CSF application was significantly associated with CA27.29 status before and after chemotherapy (χ2 = 30.6, df = 3; P < .001), because 238 patients (18.0%) with G‐CSF treatment but only 146 (11.0%) without G‐CSF treatment switched from a negative CA27.29 status before to a positive CA27.29 status after chemotherapy. In addition, patients with G‐CSF application showed a significantly greater increase in CA27.29 levels after chemotherapy compared with patients without any G‐CSF application during chemotherapy (Mann‐Whitney U test; Z = −7.81, P < .001). No significant association was found between G‐CSF application and CTC status before or after chemotherapy (χ2 = 1.2, df = 3; P = .75). Conclusion Cautious interpretation is needed regarding elevated levels of MUC‐1–derived tumor markers such as CA27.29 shortly after adjuvant chemotherapy when G‐CSF has been given, because G‐CSF treatment was associated with increased CA27.29 levels after chemotherapy. Micro‐Abstract The present study examined the association between granulocyte colony‐stimulating factor (G‐CSF) and prognostic markers cancer antigen (CA)27.29 and circulating tumor cells (CTCs) in 2646 early‐stage breast cancer patients. Those with G‐CSF application showed a significantly greater increase in CA27.29 levels after chemotherapy than those without any G‐CSF application during chemotherapy, although no association with CTCs was found.

Abstract OT1-2-03: The DETECT-study concept: Treatment based on the phenotype of circulating tumor cells in HER2-negative metastatic breast cancer

Background: The prognostic impact of circulating tumor cells (CTC) in metastatic breast cancer (MBC) is well demonstrated. The role of CTCs in predicting specific treatment response and the importance of CTC phenotypes for therapeutic decisions will be investigated within the DETECT-study concept. Trial Design and eligibility criteria: The DETECT studies are prospective, multicenter, open-label clinical trials designed for patients with HER2-negative MBC and evidence of CTCs in the peripheral blood. DETECT III is a two-arm study for patients with HER2-positve CTCs, randomized to physician’s choice therapy (chemotherapy or endocrine treatment) with or without additional HER2-targeted treatment with lapatinib. DETECT IV combines tow single-arm studies aimed at patients with HER2-negative CTCs. Postmenopausal patients with hormone-receptor-positive MBC will be treated with the mTOR-inhibitor everolimus in combination with an endocrine therapy of physician’s choice (everolimus cohort), whereas patients with triple-negative or hormone-receptor-positive MBC and indication to chemotherapy will receive eribulin (eribulin cohort). Specific aims: The primary objective of the trials is to estimate the clinical efficacy of treatments, assessed by the CTC clearance rate for DETECT III and by progression-free survival (PFS) for DETECT IV. Methods: Prevalence of CTCs at various time points as well as the HER2 status of CTCs are assessed using the FDA-approved CellSearch System (Veridex, USA). After immunomagnetic enrichment with an anti-EpCam-antibody, cells were labelled with anti-CK8/18/19 and anti-CD45 antibodies to distinguish epithelial cells from leucocytes. A fluorescein conjugate antibody with anti-CK-Fluorescein Isothiocyanate (FITC) was used for HER2 phenotyping. The cut-off for CTC-positivity was 1 CTC and for HER2 1 CTC with strong HER2-staining (+++). Survival endpoints will be estimated using the Kaplan-Meier method. Present and target accrual: Overall, about 2000 patients with HER2-negative MBC will have to be screened for CTCs to be able to recruit 228 patients with HER2-positive CTCs for DETECT III (which started in February 2012), 400 patients with HER2-negative CTCs for DETECT IV- everolimus cohort (which started in December 2013) and 120 patients for DETECT IV- eribulin cohort (which will start in the second half of 2014). 907 patients have been recruited for CTC screening until June 2014. Perspectives: One screening for CTCs offers different treatment options for patients with HER2-negative MBC and evidence of CTCs within the DETECT-study concept. DETEC III is the first study to investigate a personalized targeted treatment based on the phenotype of CTCs. The addition of a HER2-targeted therapy in case of HER2-positive CTCs is innovative and in case of success will lead to new treatment strategies in MBC. DETECT IV complements DETECT III with regard to additional therapy indications. Citation Format: Bernadette AS Jaeger, Susanne Albrecht, Fabienne Schochter, Carola A Melcher, Carsten Hagenbeck, Thomas WP Friedl, Brigitte Rack, Volkmar Muller, Peter A Fasching, Wolfgang Janni, Tanja Fehm. The DETECT-study concept: Treatment based on the phenotype of circulating tumor cells in HER2-negative metastatic 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 OT1-2-03.

Abstract P4-01-08: Persistence of circulating tumor cells immediately after and two years after systemic adjuvant chemotherapy in patients with early breast cancer – Results of the German SUCCESS trials

Background There is growing evidence that circulating tumor cells (CTCs) have prognostic impact in patients (pts) with early breast cancer (EBC). In this study the persistence of CTCs immediately after and two years after chemotherapy (Ctx) was prospectively evaluated according to molecular subtypes within the German multicentre SUCCESS trials. Methods SUCCESS A and C were randomized Phase III studies including pts with node positive or high-risk node negative EBC. In each trial two different adjuvant Ctx regimen were compared: FEC-DOC (3 cycles of FEC followed by 3 cycles of Docetaxel) to FEC-DG (3 cycles of FEC followed by 3 cycles of Docetaxel/Gemcitabine) in SUCCESS A and in the SUCCESS C study FEC-DOC to an anthracycline-free Ctx regimen (6 cycles of Docetaxel/Cyclophosphamide). Both studies involved a second randomization after Ctx: 2 vs. 5 years of zoledronic acid treatment (SUCCESS A) or 2-years of an individualized lifestyle-intervention program vs. general lifestyle recommendations (SUCCESS C). Adequate endocrine treatment and treatment with trastuzumab as indicated were included in both trials. As part of the translational research program, 23ml of peripheral blood were drawn to isolate CTCs using the CellSearch System (Veridex, USA). After immunomagnetic enrichment with an anti-EpCam-antibody, cells were labelled with anti-CK8/18/19 and anti-CD45 antibodies to distinguish epithelial cells from leucocytes. The cut-off for CTC-positivity was ≥ 1 CTC. Molecular subtypes were defined as luminal-A-like (hormone-receptor positive, G1 or 2), luminal-B-like (hormone-receptor positive, G3), HER2-positive and triple-negative. Results CTC analyses were performed for 3344 blood samples collected immediately after Ctx and for 1352 blood samples two years after Ctx. After Ctx 17.5% (584/3344) of the pts were CTC-positive (range 1 – 124 CTCs), and two years after Ctx the positivity rate for CTCs was 17.2% (233/1352, range 1-99). CTC positivity as assessed immediately after Ctx differed significantly among molecular subtypes (chi-square test, p Two years after Ctx CTC-positivity did not differ significantly among molecular subtypes (chi-square test, p = 0.463). CTC-positivity rates were 15.7% (96/613) for luminal-A-like tumors, 19.1% (49/256) for luminal-B-like tumors, 17.2% (51/296) for HER2-positive tumors, and 19.8% (37/187) for triple-negative tumors. Conclusions The data of this study confirm previous findings that CTCs may persist after standard adjuvant therapy. Immediately after Ctx CTCs seem to be more frequent in pts with HER2-positive tumors as compared to other molecular subtypes, while two years after Ctx no differences in CTC positivity among molecular subtypes were detected. These results might indicate good efficacy of HER2-targeted therapies on CTCs. Citation Format: Bernadette AS Jaeger, Ulrich Andergassen, Julia K Neugebauer, Marianna Alunni-Fabbroni, Carola A Melcher, Carsten Hagenbeck, Susanne Albrecht, Ralf Lorenz, Thomas Decker, Georg Heinrich, Tanja Fehm, Andreas Schneeweiss, Matthias W Beckmann, Klaus Pantel, Klaus Friese, Peter A Fasching, Thomas WP Friedl, Wolfgang Janni, Brigitte K Rack. Persistence of circulating tumor cells immediately after and two years after systemic adjuvant chemotherapy in patients with early breast cancer – Results of the German SUCCESS trials [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 P4-01-08.

Abstract OT1-1-11: DETECT III - A multicenter, randomized, phase III trial to compare standard therapy alone versus standard therapy plus lapatinib in patients with initially HER2-negative metastatic breast cancer and HER2-positive circulating tumor cells

Background: In metastatic breast cancer (MBC) patients, HER2 status may be subject to change and it has been shown that 20-30% of initially HER2-negative patients have HER2-positive metastases. The discordance of the HER2 status between primary tumor and circulating tumor cells (CTCs) may be an important factor affecting the response to HER2-targeted treatments; however, it is unclear if therapy based on the HER2 status of CTC offers a clinical benefit for patients. Trial Design: DETECT III is a recruiting prospective, multicenter, open-label, two-arm, phase-III study for patients with HER2-negative MBC. Patients with HER2-positve CTCs are randomized to receive either a standard therapy of the physician9s choice alone or a standard therapy plus additional HER2-targeted treatment with Lapatinib. Main Eligibility criteria: - Metastatic breast cancer; - HER2-negative primary tumor tissue and/or biopsies from metastatic sites; - Evidence of at least 1 HER2-positive CTC out of 7.5 ml blood; - Indication for systemic treatment - ≥1 lesion evaluable according to RECIST Specific aims: The objective of the DETECT III trial is to estimate the clinical efficacy of standard therapy plus lapatinib as assessed by progression free survival (PFS) in patients with initially HER2-negative MBC and HER2-positive CTCs. Additional research will be performed on CTC dynamic and characteristics. Present accrual and target accrual: The DETECT III – trial started February 2012 and has analyzed so far (2013-06-06) 506 patients. We found CTCs in 336 (66.4%) patients (median 7 CTCs, range 1-35078). 67 of these patients (19.9%) had at least one HER2 positive CTC. Estimated patient enrollment is 228. Methods: The primary endpoint progression-free survival will be estimated using the Kaplan-Meier method. Prevalence and number of CTCs at various time points as well as the HER2 status of CTCs will be determined using the FDA-approved CellSearch System (Veridex, USA). In addition, different measures of CTC dynamics and their value for evaluating therapy efficacy or as a prognostic tool will be examined in detail by explorative data analyses. Perspectives: The DETECT III-Trail is the first study where treatment is based on phenotypic characteristics of CTCs. If this trial succeeds in proving efficacy of lapatinib in patients with initially HER2-negative primary tumor but HER2-positive CTCs, it may lead to a new strategy for the treatment of metastatic breast cancer. Contact information: nikolaus.de-gregorio@uniklinik-ulm.de. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr OT1-1-11.

Abstract OT2-6-10: DETECT IV – A multicenter, single arm, phase II study evaluating the efficacy of everolimus in combination with endocrine therapy in patients with HER2-negative, hormone-receptor positive metastatic breast cancer and HER2-negative circulating tumor cells (CTCs)

BACKGROUND: The monitoring of therapy efficacy in metastatic breast cancer (MBC) is a very important component of treatment. Several studies have indicated that the determination of prevalence and number of circulating tumor cells (CTCs) at various times during treatment may be an effective tool for assessing treatment efficacy. Even if the prognostic value of CTCs in MBC is well understood, the role of CTC prevalence and - in particular - CTC phenotype in predicting treatment response requires further investigation. While the recently started DETECT III-trial is designed to evaluate the effect of HER2-targeted therapy in patients with HER2-negative MBC and HER2-postive CTCs, DETECT IV aims to assess the effect of an endocrine treatment in combination with the mTOR-inhibitor everolimus for patients with HER2-negative CTCs. SPECIFIC AIMS/TRIAL DESIGN: DETECT IV is a prospective, multicenter, open-label, single arm phase II study aimed at postmenopausal patients with hormone-receptor positive, HER2-negative metastatic breast cancer. Patients with HER2-negative CTCs will be treated with Everolimus in combination with an endocrine therapy according to the physician9s choice (letrozole, anastrozole, exemestan or tamoxifen). The primary objective of the trial is to estimate the clinical efficacy of everolimus in combination with endocrine therapy as assessed by progression-free survival (PFS) in the targeted patient population. Additional research will be done on CTC dynamics and characteristics (e.g. PI3K-mutation-analysis). This will provide a better understanding of prognostic and predictive value of the CTCs. This clinical trial is one more step into a more personalized therapy for metastatic breast cancer and will provide further information on CTCs and their informative value during the therapy. ELIGIBILITY CRITERIA: In this trial postmenopausal female patients suffering from hormone-receptor positive, HER2-negative metastasizing breast cancer with HER2-negative circulating tumor cells (CTC) and indication for standard anticancer therapy will be included. STATISTICAL METHODS/TARGET ACCRUAL: DETECT IV will start in September 2013. The estimated number of patients is 400. Therefore, 2000 patients need to be screened for HER2-negative CTCs. This screening is combined with the DETECT III - trail, which started February 2012. The primary endpoint progression-free survival will be estimated using the Kaplan-Meier method. Prevalence and number of CTCs at various time points will be determined using the FDA-approved CellSearch System (Veridex, USA). In addition, different measures of CTC dynamics and their value for evaluating therapy efficacy or as a prognostic tool will be examined in detail by explorative data analyses. Contact information: fabienne.schochter@uniklinik-ulm.de. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr OT2-6-10.