Franziska Meier-Stiegen

Molecular profiling of single circulating tumor cells with diagnostic intention

Several hundred clinical trials currently explore the role of circulating tumor cell (CTC) analysis for therapy decisions, but assays are lacking for comprehensive molecular characterization of CTCs with diagnostic precision. We therefore combined a workflow for enrichment and isolation of pure CTCs with a non‐random whole genome amplification method for single cells and applied it to 510 single CTCs and 189 leukocytes of 66 CTC‐positive breast cancer patients. We defined a genome integrity index (GII) to identify single cells suited for molecular characterization by different molecular assays, such as diagnostic profiling of point mutations, gene amplifications and whole genomes of single cells. The reliability of > 90% for successful molecular analysis of high‐quality clinical samples selected by the GII enabled assessing the molecular heterogeneity of single CTCs of metastatic breast cancer patients. We readily identified genomic disparity of potentially high relevance between primary tumors and CTCs. Microheterogeneity analysis among individual CTCs uncovered pre‐existing cells resistant to ERBB2‐targeted therapies suggesting ongoing microevolution at late‐stage disease whose exploration may provide essential information for personalized treatment decisions and shed light into mechanisms of acquired drug resistance.

Genomic High-Resolution Profiling of Single CKpos/CD45neg Flow-Sorting Purified Circulating Tumor Cells from Patients with Metastatic Breast Cancer

BACKGROUND Circulating tumor cells (CTCs) are promising surrogate markers for systemic disease, and their molecular characterization might be relevant to guide more individualized cancer therapies. To enable fast and efficient purification of individual CTCs, we developed a work flow from CellSearch(TM) cartridges enabling high-resolution genomic profiling on the single-cell level. METHODS Single CTCs were sorted from 40 CellSearch samples from patients with metastatic breast cancer using a MoFlo XDP cell sorter. Genomes of sorted single cells were amplified using an adapter-linker PCR. Amplification products were analyzed by array-based comparative genomic hybridization, a gene-specific quantitative PCR (qPCR) assay for cyclin D1 (CCND1) locus amplification, and genomic sequencing to screen for mutations in exons 1, 9, and 20 of the phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) gene and exons 5, 7, and 8 of the tumor protein p53 (TP53) gene. RESULTS One common flow-sorting protocol was appropriate for 90% of the analyzed CellSearch cartridges, and the detected CTC numbers correlated positively with those originally detected with the CellSearch system (R(2) = 0.9257). Whole genome amplification was successful in 72.9% of the sorted single CTCs. Over 95% of the cells displayed chromosomal aberrations typical for metastatic breast cancers, and amplifications at the CCND1 locus were validated by qPCR. Aberrant CTCs from 2 patients harbored mutations in exon 20 of the PIK3CA gene. CONCLUSIONS This work flow enabled effective CTC isolation and provided insights into genomic alterations of CTCs in metastatic breast cancer. This approach might facilitate further molecular characterization of rare CTCs to increase understanding of their biology and as a basis for their molecular screening in the clinical setting.

Expression of Stem Cell and Epithelial-Mesenchymal Transition Markers in Circulating Tumor Cells of Breast Cancer Patients

Evaluation and characterization of circulating tumor cells (CTCs) have become a major focus of translational cancer research. Presence of CTCs predicts worse clinical outcome in early and metastatic breast cancer. Whether all cells from the primary tumor have potential to disseminate and form subsequent metastasis remains unclear. As part of the metastatic cascade, tumor cells lose their cell-to-cell adhesion and undergo epithelial-mesenchymal transition (EMT) in order to enter blood circulation. During EMT epithelial antigens are downregulated; thus, such tumor cells might elude classical epithelial marker-based detection. Several researchers postulated that some CTCs express stem cell-like phenotype; this might lead to chemoresistance and enhanced metastatic potential of such cells. In the present review, we discuss current data on EMT and stem cell markers in CTCs of breast cancer and their clinical significance.

Analysing the mutational status of PIK3CA in circulating tumor cells from metastatic breast cancer patients

The frequently altered phosphatidylinositol‐3‐kinase (PI3K)/Akt signaling pathway is involved in the regulation of cellular processes required for breast carcinogenesis. The aim of the project was to develop a method to identify hotspot mutations in the PIK3CA gene in circulating tumor cells (CTCs) of metastatic breast cancer (metBC) patients.

Therapeutic intervention based on circulating tumor cell phenotype in metastatic breast cancer: concept of the DETECT study program

AbstractPurpose The aim of the ongoing DETECT study program is to evaluate therapeutic intervention based on phenotypes of circulating tumor cells (CTC) in patients with metastatic breast cancer (MBC). Currently (as of July 2015) more than half of the projected about 2000 patients with MBC have already been screened for CTC.MethodsWomen with HER2-negative primary tumor and presence of CTC are recruited into different DETECT trials according to the HER2-phenotype of CTC. Patients with HER2-positive CTC are randomized to treatment with physicians’ choice therapy (standard chemo- or endocrine therapy) with or without additional HER2-targeted therapy with lapatinib in the DETECT III trial. In DETECT IVa, postmenopausal patients with hormone-receptor positive primary cancer and HER2-negative CTC receive everolimus and standard endocrine therapy. For women with HER2-negative CTC and triple negative MBC or hormone-receptor positive tumor and indication for chemotherapy, a treatment with eribulin is offered (DETECT IVb). The clinical efficacy is investigated by CTC-Clearance and progression-free survival (PFS). The DETECT V/CHEVENDO trial extends the DETECT study program for women with HER2-positive and hormone-receptor positive MBC. The primary objective of this trial is to compare safety and quality of life (QoL) as assessed by the occurrence of adverse events in patients treated with dual (trastuzumab plus pertuzumab) HER2-targeted therapy plus either endocrine or chemotherapy. The translational research projects of the DETECT study program focus on further molecular characterization of CTC and evaluation of markers for their suitability to predict treatment response and to facilitate the development of more personalized treatment options.

Dormancy in breast cancer

Tumor dormancy describes a prolonged quiescent state in which tumor cells are present, but disease progression is not yet clinically apparent. Breast cancer is especially known for long asymptomatic periods, up to 25 years, with no evidence of the disease, followed by a relapse. Factors that determine the cells decision to enter a dormant state and that control its duration remain unclear. In recent years, considerable progress has been made in understanding how tumor cells circulating in the blood interact and extravasate into secondary sites and which factors might determine whether these cells survive, remain dormant, or become macrometastases. The mechanisms of tumor cell dormancy are still not clear. Two different hypotheses are currently discussed: tumor cells persist either by completely withdrawing from the cell cycle or by continuing to proliferate at a slow rate that is counterbalanced by cell death. Because dormant disseminated tumor cells may be the founders of metastasis, one hypothesis is that dormant tumor cells, or at least a fraction of them, share stem cell-like characteristics that may be responsible for their long half-lives and their suggested resistance to standard chemotherapy. Therefore, knowledge of the biology of tumor cell dormancy may be the basis from which to develop innovative targeted therapies to control or eliminate this tumor cell fraction. In this review, we discuss biological mechanisms and clinical implications of tumor dormancy in breast cancer patients.

Circulating tumor cells in breast cancer—current status and perspectives

The phenomenon of tumor cell dissemination through the blood stream has been known since the 19th century. Circulating tumor cells (CTCs) may be detected in peripheral blood of patients with breast cancer and may serve as a surrogate marker for minimal residual disease. Prognostic relevance of CTCs has already been demonstrated in early and metastatic breast cancer and commercially available detection systems are currently employed in various clinical trials. Since peripheral blood is an easily accessible compartment, serial reevaluation of CTCs is possible and may contribute to better therapy monitoring. Another potential of CTCs lies in the characterization of tumor cells. Expression profiles may differ between CTCs and primary tumor, which may result in different responses to treatment. Assessment of molecular features of CTCs may be an important step for the optimization of adjuvant and metastatic systemic therapy.

Abstract PD6-6: A pooled analysis of the prognostic relevance of circulating tumor cells in early breast cancer

Background: While there is unequivocal evidence regarding the prognostic relevance of circulating tumor cells (CTCs) in peripheral blood of patients (pts.) with metastatic breast cancer (BC), less data is available for the prognostic relevance at time of primary diagnosis. Methods: We conducted a pooled analysis with individual data of 3172 pts with non-metastatic (Stage I-III) BC from five academic institutions. Prevalence and number of CTCs in the peripheral blood were assessed at time of primary diagnosis using the FDA-approved CellSearch System (Veridex, USA). Patient outcomes were analyzed using univariate log-rank tests and multivariate Cox regressions. The median follow-up time was 61 months. Results: At least one CTC was detected in 20.2% of the pts. The presence of CTCs was associated with larger tumors, more axillary lymph node metastases, and higher histological tumor grade (all p < 0.005). The presence of CTCs was significantly related to poor progression-free (PFS, log-rank test, p < 0.001, hazard ratio [HR] 2.0) and overall survival (OS, p < 0.001, HR 2.6). Multivariate Cox regressions, including tumor size, nodal status, histological tumor grade, hormone receptor status and HER2 status, and CTC prevalence confirmed that the presence of CTCs was an independent prognostic factor for both poor PFS (HR 1.8) and OS (HR 2.1, both p < 0.001). In the univariate subgroup analysis concerning OS, the HR for pts. with positive HRS was 3.1 (p< 0.0001), and 2.0 (p = 0.001) for pts. with a negative HRS. The prognostic relevance of CTC status was similar for patients with HER2 negative and HER2 positive tumors (HR 2.6 and 2.5, respectively). The HR in CTC-positive pts. with HRS positive/HER2 positive tumors was 3.4 (p = 0.003). For patients with more than 3 CTCs, the hazard ratio for death was increased to 5.2 (1.5 - 2.8) in univariate and to 4.0 (2.6 - 6.2) in multivariate analysis. View this table: Cox Regression Analysis for Overall Survival Conclusions: In patients with early breast cancer, the presence of CTCs in peripheral blood is an independent predictor of poor progression-free and overall survival. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD6-6.

Discordance in Human Epidermal Growth Factor Receptor 2 (HER2) Phenotype Between Primary Tumor and Circulating Tumor Cells in Women With HER2-Negative Metastatic Breast Cancer

PurposeDiscordance in human epidermal growth factor receptor 2 (HER2) status between primary tumor and metastases might have important implications for treatment response and therapy decisions. Here, we evaluate both the frequency of circulating tumor cells (CTCs) and the factors predicting HER2 discordance between primary tumor and CTCs as a potential surrogate for tumor biology and tumor heterogeneity in patients with metastatic breast cancer.Patients and MethodsThe number of CTCs in 7.5 mL of peripheral blood and HER2 status were evaluated in 1,123 women with HER2-negative metastatic breast cancer. HER2 discordance was defined as the presence of at least one CTC with a strong immunocytochemical HER2 staining intensity. Factors predicting discordance in HER2 phenotype were assessed using multivariable logistic regression.ResultsOverall, 711 (63.3%) of 1,123 screened patients were positive for CTCs (≥ one CTC). Discordance in HER2 phenotype between primary tumor and CTCs was observed in 134 patients (18....

Abstract OT1-02-04: The DETECT V-study – Comparison of dual HER2-targeted therapy with trastuzumab plus pertuzumab in combination with chemo- or endocrine therapy in patients with HER2-positive and hormone-receptor positive metastatic breast cancer

Background: Maintenance of quality of life (QoL) is one of the main aims of treatment of incurable diseases such as metastatic breast cancer (MBC). In patients with HER2-positive MBC, taxan-based chemotherapy in combination with dual HER2 targeted therapy with trastuzumab and pertuzumab has shown promising efficacy results in terms of prolonged survival. However, cytostatic treatment is often accompanied by adverse events of grade 3 or higher, seriously impacting the patients9 QoL. In patients with HER2-positive and hormone-receptor positive MBC, the combination of trastuzumab with aromatase inhibitors was shown to be a safe and effective treatment option. The synergistic combination of dual HER2-targeted therapy with trastuzumab and pertuzumab plus endocrine therapy might offer an even better treatment option for these patients. DETECT V is the first prospective randomized phase III clinical trial comparing the safety and efficacy of the dual HER2-targeted therapy in combination with either endocrine therapy or chemotherapy. Trial design and eligibility criteria: Women with HER2-positive and hormone-receptor positive MBC with first to third line therapy are 1:1 randomized either to a dual HER2-targeted therapy with Pertuzumab and Trastuzumab plus endocrine therapy or to the dual HER2-targeted therapy plus chemotherapy. Specific aims: The primary objective of this study is to compare the safety and QoL in both arms, as assessed by the occurrence of AEs during the treatment period. We developed a modified adverse event score - including all adverse events grade 3 or higher, except neutropenia, which is included only if rated grade 4, and alopecia, rash, hand-foot-syndrome and peripheral neuropathy which are included if rated grade 2 or higher – in order to better reflect the clinical, physiological and psychological impact of AEs on patients9 QoL. Key secondary endpoint, besides the efficacy endpoints progression free survival (PFS) and overall survival, is to compare quality-adjusted survival (QAS), as measured using the quality-adjusted time without symptoms and toxicity (Q-TWiST) method, between both treatment arms. QAS as measured using the Q-TWiST method provides a single metric value that is a composite measure of quantity of survival time and quality of survival as assessed by the patients themselves. Q-TWiST analyses account for possible trade-offs between quantity and quality of life (e.g. prolonged time to progression at the cost of higher toxicity, which adversely affects QoL), and provide an excellent tool to evaluate whether two treatment options differ with regard to the overall perceived value to the patients. Translational research projects focus on Circulating Tumor Cell(CTC)-enumeration (the presence of CTCs is not obligatory in DETECT V), prognostic role of CTC dynamics, and the assessment of marker expression on CTCs in order to calculate an endocrine responsiveness score which will be evaluated regarding its suitability to predict treatment success. Contact: For further information on the DETECT V study please contact www.detect-studien.de or studienzentrale.ufk@uniklinik-ulm.de. Citation Format: Polasik A, Schramm A, Friedl TWP, Rack B, Trapp E, Tzschaschel M, Fasching PA, Taran F-A, Hartkopf A, Schneeweiss A, Muller V, Aktas B, Pantel K, Meier-Stiegen F, Wimberger P, Janni W, Fehm T. The DETECT V-study – Comparison of dual HER2-targeted therapy with trastuzumab plus pertuzumab in combination with chemo- or endocrine therapy in patients with HER2-positive and hormone-receptor positive metastatic breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr OT1-02-04.