Julia Neugebauer

The German SUCCESS C Study - The First European Lifestyle Study on Breast Cancer.

Cohort trials have shown evidence that obesity and a low level of physical activity are not only associated with a higher risk of developing breast cancer, but also with an increased risk for recurrence and reduced survival in breast cancer patients. The SUCCESS C study is the first European trial to evaluate the effect of an intensive lifestyle intervention program on disease-free survival in women with early breast cancer and to examine the predictive value of selected biomarker candidates. A total of 3,547 women with early-stage, Her2/neu-negative breast cancer will be included. The first randomization will compare disease-free survival in patients treated with either 3 cycles of FEC (epirubicine, fluorouracil, cyclophosphamide), followed by 3 cycles of docetaxel or 6 cycles of docetaxel-cyclophosphamide, and thus assess the role of anthracycline-free chemotherapy. The second randomization compares disease-free survival in patients with a body mass index of 24–40 kg/m2 receiving either a telephone-based individualized lifestyle intervention program aiming at moderate weight loss or general recommendations for a healthy lifestyle alone. In addition, the study will evaluate the predictive role of cancer-associated and obesity-related biomarkers for the prediction of disease recurrence and survival. This SUCCESS C trial will provide valuable information on the effects of a lifestyle intervention program on the prognosis of early breast cancer patients.

Pooled analysis of the prognostic relevance of progesterone receptor status in five German cohort studies

The progesterone receptor (PR) has been increasingly well described as an important mediator of the pathogenesis and progression of breast cancer. The aim of this study was to assess the role of PR status as a prognostic factor in addition to other well-established prognostic factors. Data from five independent German breast cancer centers were pooled. A total of 7,965 breast cancer patients were included for whom information about their PR status was known, as well as other patient and tumor characteristics commonly used as prognostic factors. Cox proportional hazards models were built to compare the predictive value of PR status in addition to age at diagnosis, tumor size, nodal status, grading, and estrogen receptor (ER) status. PR status significantly increased the accuracy of prognostic predictions with regard to overall survival, distant disease-free survival, and local recurrence-free survival. There were differences with regard to its prognostic value relative to subgroups such as nodal status, ER status, and grading. The prognostic value of PR status was greatest in patients with a positive nodal status, negative ER status, and low grading. The PR-status adds prognostic value in addition to ER status and should not be omitted from clinical routine testing. The significantly greater prognostic value in node-positive and high-grade tumors suggests a greater role in the progression of advanced and aggressive tumors.

Detection of Tumor Cell-Specific mRNA in the Peripheral Blood of Patients with Breast Cancer—Evaluation of Several Markers with Real-Time Reverse Transcription-PCR

It is widely known that cells from epithelial tumors, e.g., breast cancer, detach from their primary tissue and enter blood circulation. We show that the presence of circulating tumor cells (CTCs) in samples of patients with primary and metastatic breast cancer can be detected with an array of selected tumor-marker-genes by reverse transcription real-time PCR. The focus of the presented work is on detecting differences in gene expression between healthy individuals and adjuvant and metastatic breast cancer patients, not an accurate quantification of these differences. Therefore, total RNA was isolated from blood samples of healthy donors and patients with primary or metastatic breast cancer after enrichment of mononuclear cells by density gradient centrifugation. After reverse transcription real-time PCR was carried out with a set of marker genes (BCSP, CK8, Her2, MGL, CK18, CK19). B2M and GAPDH were used as reference genes. Blood samples from patients with metastatic disease revealed increased cytokine gene levels in comparison to normal blood samples. Detection of a single gene was not sufficient to detect CTCs by reverse transcription real-time PCR. Markers used here were selected based on a recent study detecting cancer cells on different protein levels. The combination of such a marker array leads to higher and more specific discovery rates, predominantly in metastatic patients. Identification of CTCs by PCR methods may lead to better diagnosis and prognosis and could help to choose an adequate therapy.

Estrogen Receptor and HER2 Status on Disseminated Tumor Cells and Primary Tumor in Patients with Early Breast Cancer

BACKGROUND: We evaluated both estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) status on disseminated tumor cells (DTCs) in the bone marrow of 54 patients with early breast cancer and compared these with the corresponding primary tumor (PT). MATERIALS AND METHODS: Bone marrow aspirates were obtained at the time of first surgery, and ER and HER2 status on DTCs was assessed simultaneously by immunocytochemistry using a triple fluorescence staining method. RESULTS: The median number of DTCs was 13 (range 1-95). The concordance rate between ER status on DTC and PT was 74%. Patients with an ER-positive PT were significantly more likely to have at least one ER-positive DTC (34 out of 42) than patients with an ER-negative PT (6 out of 12; P = .031). Thirty-nine (93%) of the 42 patients with ER-positive PT had at least one ER-negative DTC. The concordance rate between HER2 status on DTC and PT was 52%. The probability of having at least one HER2-positive DTC was not related to the HER2 status of the PT (P = 0.56). Twenty-two (46%) of the 48 patients with a HER2-negative PT had at least one HER2-positive DTC. All the six patients with a HER2-positive PT had at least one HER2-negative DTC. CONCLUSION: Taken together, our study confirms that ER and/or HER2 status may differ between DTC and PT. This discordance could be important for patients lacking ER or HER2 expression on the PT but showing ER-positive or HER2-positive DTC because they might benefit from an endocrine and/or HER2-targeted therapy.

Abstract S2-03: Persistence of circulating tumor cells in high risk early breast cancer patients during follow-up care suggests poor prognosis – Results from the adjuvant SUCCESS A trial

Background: Recent data suggest that circulating tumor cells (CTCs) are of prognostic relevance in early as well as metastatic breast cancer (BC). While persisting CTCs immediately after chemotherapy are known to indicate poor prognosis, there is a lack of data regarding the prognostic role of CTCs assessed during long-term follow-up care. Hence the prognostic value of CTCs two years after chemotherapy was analyzed. Methods: The SUCCESS A trial is a randomized, open-label, 2x2 factorial design Phase III study in high-risk breast cancer patients (≥N0 or T2–T4 or grade 3 or age ≤ 35 or hormone-receptor negative). Patients were first randomized to adjuvant chemotherapy treatment with 3 cycles of epirubicin-fluorouracil-cyclophosphamide followed by either 3 cycles of docetaxel or 3 cycles of gemcitabine-docetaxel. In addition, patients were randomized to 2 vs. 5 years of zoledronate treatment. Presence of CTCs was assessed using the FDA-approved CellSearch System (Janssen Diagnostics, LLC). CTC positivity was defined as ≥ 1 CTC in 7.5 ml whole blood. To investigate if CTC status 2 years after chemotherapy is of prognostic relevance independent from CTC status before chemotherapy and to evaluate the prognostic relevance of changed CTC status, only patients with data on CTC status before and 2 years after chemotherapy were included. Patient outcomes in terms of overall survival (OS) and disease-free survival (DFS) were analyzed by univariate log-rank tests and multivariate Cox regressions adjusted for age, menopausal status, tumor stage, nodal stage, grading, histological type, hormone receptor status and HER2 status. Survival time was measured beginning with the date of follow-up CTC assessment two years after chemotherapy. Results: Data on CTC status before and 2 years after chemotherapy were available for 1103 (29.4 %) of 3754 randomized patients. The CTC status 2 years after chemotherapy was positive in 204 (18.5%) patients. The median follow-up time was 37 months. Multivariate Cox regressions including CTC status before chemotherapy showed significant independent prognostic role for CTC status 2 years after chemotherapy on OS (hazard ratio (HR) 3.95, 95% confidence interval (CI) 2.13 – 7.32, p Conclusion: The presence of CTCs two years after chemotherapy analyzed during routine breast cancer follow-up care was associated with decreased survival. According to these results, persisting CTCs during long term follow-up independently predict patients9 outcome and may serve as surveillance marker. Citation Format: Janni W, Rack B, Fasching P, Haeberle L, Friedl T, Tesch H, Lorenz R, Neugebauer J, Koch J, Jaeger B, Fehm T, Mueller V, Schneeweis A, Lichtenegger W, Beckmann M, Scholz C, Pantel K, Trapp E. Persistence of circulating tumor cells in high risk early breast cancer patients during follow-up care suggests poor prognosis – Results from the adjuvant SUCCESS A trial. [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 S2-03.

Comparison of HER2 Expression in Primary Tumor and Disseminated Tumor Cells in the Bone Marrow of Breast Cancer Patients.

Objective: The aim of this study was to measure the human epidermal growth factor receptor 2 (HER2) status of disseminated tumor cells (DTCs) from bone marrow (BM) aspirates and to assess correspondence or discrepancy with the primary tumor. Methods: DTCs were isolated from the BM of 156 breast cancer patients. Cytokeratin-positive DTCs were further analyzed by the chromogenic in situ hybridization method to detect HER2 gene amplification. Results: A significant correlation (p = 0.021) was found between the HER2 status of DTCs and the primary tumors. Sixty-one (68.5%) patients had a corresponding status. However, a shift of phenotype between primary tumor and DTCs was found in the remaining patients. Conclusion: This study showed a significant grade of discordance of the HER2 status between primary tumors and DTCs in the BM of a relevant subgroup of patients. Detection of HER2 amplification on DTCs could therefore help to better stratify patients for a more tailored therapy, since they would benefit from a HER2-targeted therapy.

Abstract P4-01-16: Detection of EMT, anoikis and stem cell markers in metastatic breast cancer patients under different lines of treatment

Background: Metastasis are thought to be induced by occult spreading of tumor cells already during the early phases of the disease. Circulating Tumor Cells (CTCs) are regarded as precursors of distant metastasis, while detaching from the primary tumor and originating micrometastases in distant organs. Recent evidences pointed to CTC heterogeneity, showing that CTCs can present different phenotypes. Goal of this study was to identify in metastatic breast cancer (mBC) patients CTCs with EMT features and to further characterize them with respect to cellular heterogeneity. Methods: This prospective ongoing study included mBC patients (n=12) with a median age of 58,5 years (range: 35-78 years), enrolled in a time frame of 7 months while undergoing therapy. The majority of patients were estrogen and/or progesterone receptor positive (11/12) and HER2 non-amplified (10/12). Patients had metastatic lesions in viscera as well as bone (6/12), only bone (2/12), only viscera (2/12), only viscera in combination with locoregional recurrence (1/12), or visceral and bone metastases in combination with locoregional recurrence (1/12). Current therapy was endocrine therapy (4/12), chemotherapy (3/12), chemotherapy and HER2-targeted therapy (2/12), HER2-targeted monotherapy (1/12), antiangiogenic therapy (1/12), or surgical therapy only (1/12). Blood samples, withdrawn at any time point during treatment, were depleted of EpCAM+ cells and CD45+ white blood cells (EpCAM/CD45 depleted fraction) (Mego et al., Int J Cancer 2012;130(4):808-816) and expression of epithelial markers (EpCAM, E-Cadherin, Cytokeratin 8,18,19), mesenchymal markers (n-Cadherin, Vimentin), EMT-inducing factors (Twist1, Snail1, SLUG, Zeb1 and FoxC2), anoikis markers (TrkB1, Bcl2) and stem cell markers (CD24, CD44, CD133) were analyzed by qRT-PCR. CTC counting with the CellSearchTM system (Veridex, Raritan NJ) was run in parallel. Healthy donors (n=10) were included in the study as negative controls. Results: The data collected so far showed that 50% of the patients were positive for CTCs in the EpCAM+ fraction as detected with the CellSearchTM system, while 33% were still CTC positive in the EpCAM/CD45 depleted fraction. 50% of the patients, found CTC negative with CellSearchTM, were nevertheless positive for the epithelial and EMT markers in the EpCAM/CD45 depleted fraction (EpCAM 25%, E-Cadherin 25%, CK8 16,6%, CK18 16,6%, CK19 16,6%, SLUG 8,3%, Zeb1 25%, Twist1 8,3%, Vimentin 58,3%). N-cadherin, FoxC2, Snail1, TrkB1, CD24, CD44 and CD133 were never detectable. Conclusions: These preliminary results suggest that mBC patients undergoing different lines of therapy present heterogeneous CTCs. 50% of the patients with undetectable EpCAM+ CTCs, were found CTC positive in the EpCAM/CD45 depleted fraction. mBC patients might be insensitive to treatment due to a selection of resistant CTCs subpopulations with different phenotypes. Additional patients with the same clinical characteristics will be analyzed in the next 6 coming months and updated results will be included. Citation Format: Elisabeth K Trapp, Brigitte Rack, Leonie Majunke, Julian Koch, Simone Hofmann, Thomas WP Friedl, Julia Neugebauer, Julia Juckstock, Bernadette Jager, Jens Huober, Wolfgang Janni, Marianna Alunni-Fabbroni. Detection of EMT, anoikis and stem cell markers in metastatic breast cancer patients under different lines of treatment [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 P4-01-16.

Abstract P4-11-21: Prevalence of circulating tumor cells (CTCs) after five years of zoledronate treatment in the adjuvant SUCCESS-A study

Aim: The prognostic value of CTCs at primary diagnosis has recently been confirmed by the SUCCESS A Study (Rack et al. JNCI 2014). Key questions on the role of adjuvant bisphosphonate treatment, including patient populations deriving benefit and optimal timing/scheduling of therapy are still controversial. Aim of this study was therefore to evaluate CTCs in the context of two different zoledronate regimens. Methods: The SUCCESS A trial is a large, randomized, open-label, 2x2 factorial design Phase III study in patients with high risk breast cancer (stage N1 or T2–T4 or grade 3 or age ≤ 35 or hormone-receptor negative). Patients were first randomized to adjuvant chemotherapy treatment with 3 cycles of Epirubicin-Fluorouracil-Cyclophosphamide (FEC) followed by either 3 cycles of Docetaxel or 3 cycles of Gemcitabine-Docetaxel. In addition, patients were randomized to 2 years vs. 5 years of zoledronate treatment. CTC status 5 years after primary diagnosis was assessed using the FDA-approved CellSearch System (Veridex, USA), and CTC positivity was defined as ≥ 1 CTC. We studied the influence of zoledronate treatment duration on CTC prevalence at 5 years in addition to well-known patient and tumour characteristics using a multiple logistic regression analysis on the ITT population. Results: Data on CTC status at 5 years after primary diagnosis were available for 728 (19.4%) out of 3754 randomized patients. 310 patients had been randomized to 2 years of zoledronate treatment and 418 patients had been randomized to 5 years of zoledronate treatment. In these patients a difference in CTC positivity after 5 years could not be shown between patients randomized to 2 or 5 years of zoledronate (p = 0.13, Wald test). The adjusted odds ratio for 2 years vs 5 years was 0.65 (95%CI: 0.37 to 1.13). Conclusions: The final survival analysis of the SUCCESS A trial will give further insight, whether CTCs can be used as early predictive marker for the efficacy of adjuvant zoledronate treatment. Citation Format: Brigitte Rack, Peter A Fasching, Lothar Haberle, Thomas Friedl, Mahdi Rezai, Jorn Hilfrich, Hans Tesch, Georg Heinrich, Helmut Forstbauer, Julia Neugebauer, Elisabeth Trapp, Susanne Albrecht, Bernadette Jager, Tanja Fehm, Volkmar Muller, Andreas Schneeweiss, Klaus Friese, Werner Lichtenegger, Matthias W Beckmann, Wolfgang Janni. Prevalence of circulating tumor cells (CTCs) after five years of zoledronate treatment in the adjuvant SUCCESS-A study [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 P4-11-21.

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.

E8. Clinical applications of circulating tumour cells

Introduction The clinical potential of detecting minimal residual disease (MRD) in primary and metastatic breast cancer stretches far beyond its mere prognostic relevance, and may contribute to individualising systemic treatment. The role of MRD in the development of tailored treatment may comprise the following aspects: • detection of MRD could serve as an early tool for evaluating treatment efficacy, leading to a more individualised therapeutic approach; • detection of MRD could be implemented in everyday patient care to select patients with an increased risk for recurrence of their disease, and who might benefit from a change in treatment, or where there might be a need for additional therapeutic interventions; • MRD could serve as a source to understand tumour biology in MRD well after the completion of primary treatment; • phenotyping MRD might lead to a more individualised and thus targeted treatment based on the phenotype of persistent MRD. Thus, the significance of MRD may reach far beyond the potential of established prognostic parameters at the time of primary diagnosis of breast cancer.