Siegfried Hauch

Stem cell and epithelial-mesenchymal transition markers are frequently overexpressed in circulating tumor cells of metastatic breast cancer patients.

IntroductionThe persistence of circulating tumor cells (CTC) in breast cancer patients might be associated with stem cell like tumor cells which have been suggested to be the active source of metastatic spread in primary tumors. Furthermore, these cells also may undergo phenotypic changes, known as epithelial-mesenchymal transition (EMT), which allows them to travel to the site of metastasis formation without getting affected by conventional treatment. Here we evaluated 226 blood samples of 39 metastatic breast cancer patients during a follow-up of palliative chemo-, antibody – or hormonal therapy for the expression of the stem cell marker ALDH1 and markers for EMT and correlated these findings with the presence of CTC and response to therapy.Methods2 × 5 ml blood was analyzed for CTC with the AdnaTest BreastCancer (AdnaGen AG) for the detection of EpCAM, MUC-1 and HER2 transcripts. The recovered c-DNA was additionally multiplex tested for three EMT markers [Twist1, Akt2, PI3Kα] and separately for the tumor stem-cell markers ALDH1. The identification of EMT markers was considered positive if at least one marker was detected in the sample.Results97% of 30 healthy donor samples investigated were negative for EMT and 95% for ALDH1 transcripts. CTC were detected in 69/226 (31%) cancer samples. In the CTC (+) group, 62% were positive for at least one of the EMT markers and 69% for ALDH1, respectively. In the CTC (-) group the percentages were 7% and 14%, respectively. In non-responders, EMT and ALDH1 expression was found in 62% and 44% of patients, in responders the rates were 10% and 5%, respectively.ConclusionsOur data indicate that a major proportion of CTC of metastatic breast cancer patients shows EMT and tumor stem cell characteristics. Further studies are needed to prove whether these markers might serve as an indicator for therapy resistant tumor cell populations and, therefore, an inferior prognosis.

ERCC1-Positive Circulating Tumor Cells in the Blood of Ovarian Cancer Patients as a Predictive Biomarker for Platinum Resistance

BACKGROUND Platinum resistance constitutes one of the most recognized clinical challenges for ovarian cancer. Notably, the detection of the primary tumor-based excision repair cross-complementation group 1 (ERCC1) protein by immunohistochemistry was recently shown to be inaccurate for the prediction of platinum resistance. On the basis of the previous finding that circulating tumor cells (CTC) in the blood of ovarian cancer patients are prognostically significant, and given our hypothesis that the negative prognostic impact of CTC may arise from a cellular phenotype associated with platinum resistance, we asked whether expression of the excision repair cross-complementation group 1 (ERCC1) gene in the form of the ERCC1 transcript in CTC may be a suitable blood-based biomarker for platinum resistance. METHODS The presence of CTC was analyzed by immunomagnetic CTC enrichment (n = 143 patients) targeting the epithelial epitopes epithelial cell adhesion molecule (EPCAM) (also known as GA733-2) and mucin 1, cell surface associated (MUC1), followed by multiplex reverse-transcription PCR to detect the transcripts EPCAM, MUC1, and mucin 16, cell surface associated (MUC16) (also known as CA125), including ERCC1 transcripts in a separate approach. ERCC1 expression in primary tumors was comparatively assessed by immunohistochemistry, using the antibody 8F1. RESULTS At primary diagnosis, the presence of CTC was observed in 14% of patients and constituted an independent predictor of overall survival (OS) (P = 0.041). ERCC1-positive CTC (ERCC1(+)CTC) were observed in 8% of patients and constituted an independent predictor, not only for OS but also for progression-free survival (PFS) (P = 0.026 and P = 0.009, respectively). More interestingly, we discovered the presence of ERCC1(+)CTC at primary diagnosis to be likewise an independent predictor of platinum resistance (P = 0.010), whereas ERCC1 expression in corresponding primary tumor tissue predicted neither platinum resistance nor prognosis. CONCLUSIONS The presence of ERCC1(+)CTC can serve as a blood-based diagnostic biomarker for predicting platinum resistance at primary diagnosis of ovarian cancer.

ERCC1-expressing circulating tumor cells as a potential diagnostic tool for monitoring response to platinum-based chemotherapy and for predicting post-therapeutic outcome of ovarian cancer

Background We recently showed that the presence of ERCC1+CTCs is an independent predictive biomarker for platinum-resistance and poor prognosis of ovarian cancer. The goal of our current research was to determine how the auxiliary assessment of ERCC1-transcripts influences overall CTC-detection rate. We extended this investigation from an initially predictive setting to paired pre- and post-therapeutic blood analysis in order to see, whether ERCC1+CTCs dynamics mirror response to chemotherapy. Methods 65 Paired blood samples (10ml) of primary ovarian cancer patients at primary diagnosis and after chemotherapy were studied for CTCs with the AdnaTest Ovarian Cancer (QIAGEN Hannover GmbH). We analyzed the tumor-associated transcripts EpCAM, MUC-1 and CA-125. ERCC1-transcripts were investigated in a separate approach by singleplex RT-PCR. RESULTS Auxiliary assessment of ERCC1-transcripts enhanced the overall CTC-detection rate up to 17%. ERCC1+CTCs (defined as positive for one of the AdnaTest markers plus ERCC1-positivity) were detected in 15% of patients at primary diagnosis and in 12% after chemotherapy. The presence of ERCC1+CTCs after chemotherapy correlated with platinum-resistance (P=0.01), reduced PFS (P=0.0293) and OS (P=0.0008) and their persistence indicated poor post-therapeutic outcome (PFS: P=0.005; OS: P=0.0058). Interestingly, the assessment of ERCC1-transcripts alone was sufficient for the detection of prognostic relevant ERCC1-expressing CTCs. Conclusion Auxiliary assessment of ERCC1-transcripts expands the phenotypic spectrum of CTC detection and defines an additional overlapping fraction of ERCC1-expressing CTCs, which are potentially selected by platinum-based chemotherapy. Specifically, we suggest that ERCC1+CTCs could additionally be useful as a surrogate for monitoring platinum-based chemotherapy and to assess the post-therapeutic outcome of ovarian cancer.

EMT-like circulating tumor cells in ovarian cancer patients are enriched by platinum-based chemotherapy

Background Assuming that tumor cell dissemination requires a shift to a mesenchymal phenotype, we analyzed the incidence of epithelial-to-mesenchymal-transition (EMT)-like circulating tumor cells (CTCs) in ovarian cancer patients and inquired, how their molecular phenotypes respond to platinum-based chemotherapy and influence outcome. Results Before surgery, overall detection rate for epithelial CTCs was 18%. EMT-like CTCs were more frequently observed (30%) and were mutually exclusive to epithelial CTCs in the majority of patients (82%). After chemotherapy, EMT-like CTCs increased up to 52%, accompanied by the “de novo” emergence of PI3Kα+/Twist+ EMT-like CTCs. Before surgery, PI3K+ EMT-like CTCs in combination with epithelial CTCs indicated decreased OS (p = 0.02) and FIGO I-III patients with residual tumor burden after surgery were more likely to be positive for EMT-like CTCs after chemotherapy (p = 0.02). In the latter group, epithelial CTCs alone significantly correlated with decreased PFS and OS (p = 0.02, p = 0.002), supported by an additional inclusion of PI3K+ CTCs (OS, p = 0.001). Materials and Methods Blood samples of 91 ovarian cancer patients before surgery and 31 matched samples after adjuvant chemotherapy were evaluated for CTCs with the AdnaTest ovarian cancer and EMT-1, analyzing the epithelial-associated transcripts EpCAM, Muc-1 and CA125 and the EMT-associated transcripts PI3Kα, Akt-2 and Twist. Conclusions Platinum-based chemotherapy seems to select for EMT-like CTCs in ovarian cancer patients and provokes a shift towards PI3Kα and Twist expressing CTCs, which may reflect clonal tumor evolution towards therapy resistance. It has to be determined, whether this CTC subgroup may serve as a biomarker to identify patients at high risk.

Gene Expression Signatures in Circulating Tumor Cells Correlate with Response to Therapy in Metastatic Breast Cancer

BACKGROUND Circulating tumor cells (CTCs) are thought to be an ideal surrogate marker to monitor disease progression in metastatic breast cancer (MBC). We investigated the prediction of treatment response in CTCs of MBC patients on the basis of the expression of 46 genes. METHODS From 45 MBC patients and 20 healthy donors (HD), 2 × 5 mL of blood was collected at the time of disease progression (TP0) and at 2 consecutive clinical staging time points (TP1 and TP2) to proceed with the AdnaTest EMT-2/StemCellSelectTM (QIAGEN). Patients were grouped into (a) responder (R) and non-responder (NR) at TP1 and (b) overall responder (OR) and overall non-responder (ONR) at TP2. A 46-gene PCR assay was used for preamplification and high-throughput gene expression profiling. Data were analyzed by use of GenEx (MultiD) and SAS. RESULTS The CTC positivity was defined by the four-gene signature (EPCAM, KRT19, MUC1, ERBB2 positivity). Fourteen genes were identified as significantly differentially expressed between CTC+ and CTC- patients (KRT19, FLT1, EGFR, EPCAM, GZMM, PGR, CD24, KIT, PLAU, ALDH1A1, CTSD, MKI67, TWIST1, and ERBB2). KRT19 was highly expressed in CTC+ patients and ADAM17 in the NR at TP1. A significant differential expression of 4 genes (KRT19, EPCAM, CDH1, and SCGB2A2) was observed between OR and ONR when stratifying the samples into CTC+ or CTC-. CONCLUSIONS ADAM17 could be a key marker in distinguishing R from NR, and KRT19 was powerful in identifying CTCs.

Comparison of the PI3KCA pathway in circulating tumor cells and corresponding tumor tissue of patients with metastatic breast cancer

The aim of the present study was to compare the phosphatidylinositol 3-kinase (PI3KCA)-AKT serine/threonine kinase (AKT) pathway in circulating tumor cells (CTCs) and corresponding cancerous tissues. Stemness-like circulating tumor cells (slCTCs) and CTCs in epithelial-mesenchymal transition (EMT) have been implicated as the active source of metastatic spread in breast cancer (BC). In this regard, the PI3KCA-AKT signaling pathway was demonstrated to be implicated in and to be frequently mutated in BC. The present study compared this pathway in slCTCs/CTCs in EMT and the corresponding tumor tissues of 90 metastatic BC patients (pts). slCTCs and CTCs in EMT were isolated using the AdnaTest EMT-1/StemCell for the detection of aldehyde dehydrogenase 1 family member A1 (ALDH1) (singleplex PCR) and PI3KCA, AKT2 and twist family bHLH transcription factor 1 (multiplex PCR). Tumor tissue was investigated for PI3KCA hotspot mutations using Sanger sequencing of genomic DNA from micro-dissected formalin-fixed paraffin-embedded tissue, and for the expression of ALDH1 and phosphorylated AKT (pAKT), and phosphatase and tensin homolog (PTEN) loss, by immunohistochemistry. slCTCs were identified in 23% of pts (21/90 pts) and CTCs in EMT in 56% (50/90 pts) of pts. pAKT and ALDH1 positivity in tumor tissue was identified in 47 and 9% of cases, respectively, and a PTEN loss was observed in 18% of pts. A significant association was detected between pAKT expression in cancerous tissue and AKT2 expression in CTCs (P=0.037). PI3KCA mutations were detected in 32% of pts, most frequently on exons 21 (55%) and 10 (45%). Pts with PI3KCA mutations in tumor tissue had a significantly longer overall survival than pts with wild-type PI3KCA expression (P=0.007). Similar results were obtained for pts with aberrant PI3KCA signaling in CTCs and/or aberrant signaling in cancerous tissue (P=0.009). Therapy-resistant CTCs, potentially derived from the primary tumor or metastatic tissue, may be eliminated with specific PI3K pathway inhibitors, alone or in combination, to improve the prognosis of metastatic BC pts.

Stem cell and epithelial-mesenchymal transition markers on circulating tumor cells in patients with metastatic breast cancer.

Abstract #107 Background: Stem cell like tumor cells have been suggested to be the active source of metastatic spread in primary tumors. Furthermore, these cells may undergo phenotypic changes, known as epithelial-mesenchymal transition (EMT), which allows them to travel to the site of metastasis formation without getting affected by conventional treatment. Assuming that metastasis requires a dissemination of tumor stem cells or tumor cells showing EMT, it was the purpose of this study to identify these cells among circulating tumor cells (CTC) in blood samples of 28 metastatic breast cancer patients.
 Materials and Methods: 5 ml blood was analyzed for EpCAM, MUC-1 and HER-2 transcripts with the AdnaTest BreastCancer (AdnaGen AG). The recovered c-DNA was additionally multiplex tested for three EMT markers [Twist, Akt2, PI3K (TAP)] and separately for the tumor stem-cell marker ALDH1. The analytical sensitivity was determined by the detection of a low number of target cells (5 IGROV cells spiked into 5 ml blood of healthy donors) using the AdnaTest BreastCancer procedure. The identification of EMT markers was considered positive if at least one marker was detected in the sample. Healthy donor samples without spiked tumor cells were used to determine the specificity of the test. The resulting PCR fragments were separated and quantified using the Agilent Bioanalyzer 2100.
 Results: Applying an amplicon cut-off value of 0.2 ng/μl for Akt2, 0.15 ng/μl for Twist, 0.25 ng/μl for PI3K and 0.15 ng/μl for ALDH1, 97% of 30 healthy donor samples investigated were negative for TAP and 95% for ALDH1 transcripts. The spiking experiments revealed 80% recovery of the IGROV cells. CTC were detected in 12/28 (43%) breast cancer samples. All samples were further examined for tumor stem cell or TAP markers. In the CTC (+) group 50% were positive for at least one of the TAP markers and 42% for ALDH1. In the CTC (-) group the percentages were 19% and 12%, respectively. The expression of TAP and/or stem cell markers in CTC was compared with the clinical follow up results. In CTC (+) patients diagnosed as non-responders the expression of TAP and ALDH1 was found in 5/12 (40%) samples. In the CTC (+) responder group only one sample expressed TAP and no positive case was found for ALDH1. In the CTC (-) non-responder group 1/14 samples was positive for TAP and ALDH1. 2/14 (14%) of CTC (-) responders were positive for TAP and 1/14 for ALDH1, respectively.
 Conclusion: A major proportion of CTC found in the blood of metastatic breast cancer patients shows EMT and tumor stem cell characteristics supporting the hypothesis that these markers are an indicator for therapy resistant tumor cell populations and, therefore, for an inferior prognosis. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 107.

RNA Profiles of Circulating Tumor Cells and Extracellular Vesicles for Therapy Stratification of Metastatic Breast Cancer Patients

BACKGROUND Liquid biopsies are discussed to provide surrogate markers for therapy stratification and monitoring. We compared messenger RNA (mRNA) profiles of circulating tumor cells (CTCs) and extracellular vesicles (EVs) in patients with metastatic breast cancer (MBC) to estimate their utility in therapy management. METHODS Blood was collected from 35 hormone receptor-positive/HER2-negative patients with MBC at the time of disease progression and at 2 consecutive staging time points. CTCs were isolated from 5 mL of blood by positive immunomagnetic selection, and EVs from 4 mL of plasma by a membrane affinity-based procedure. mRNA was reverse transcribed, preamplified, and analyzed for 18 genes by multimarker quantitative polymerase chain reaction (qPCR) assays. RNA profiles were normalized to healthy donor controls (n = 20), and results were correlated with therapy outcome. RESULTS There were great differences in mRNA profiles of EVs and CTCs, with only 5% (21/403) of positive signals identical in both fractions. Transcripts involved in the PI3K signaling pathway were frequently overexpressed in CTCs, and AURKA, PARP1, and SRC signals appeared more often in EVs. Of all patients, 40% and 34% showed ERBB2 and ERBB3 signals, respectively, in CTCs, which was significantly associated with disease progression (P = 0.007). Whereas MTOR signals in CTCs significantly correlated with response (P = 0.046), signals in EVs indicated therapy failure (P = 0.011). The presence of AURKA signals in EVs seemed to be a marker for the indication of unsuccessful treatment of bone metastasis. CONCLUSIONS These results emphasize the potential of CTCs and EVs for therapy monitoring and the need for critical evaluation of the implementation of any liquid biopsy in clinical practice.

Abstract 3777: RNA profiles of circulating tumor cells and extracellular vesicles for therapy stratification of metastatic breast cancer patients

Background: Blood analytes, as liquid biopsies, are discussed to be surrogate markers for therapy stratification of metastatic breast cancer (MBC) patients. Repeated analysis is enabled by the minimal invasive nature of blood draw. Analysis of RNA enclosed in circulating tumor cells (CTCs) or extracellular vesicles (EVs) may be sensitive enough to detect disease progression earlier than contemporary visual staging methods. A prediction of the ideal therapy strategy via characterization of CTCs or EVs would be even more beneficial. Here we compare RNA profiles of CTCs and EVs in MBC patients to get insight into their feasibility for therapy stratification. Patients and methods: Blood was collected from 10 MBC patients at the time of disease progression (T0) and at two consecutive clinical staging time points (T1 and T2) during therapy. Two cohorts were separated according to RECIST criteria a) Overall Responder showed response at T1 and T2 and b) Late Non-Responder displayed stable disease or partial remission at T1, but showed progressive disease at T2. CTCs were isolated from 5 ml blood by positive immunomagnetic selection targeting EpCAM, EGFR and HER2 (AdnaTest EMT2/StemCell Select TM, QIAGEN, Germany). EVs were isolated from 4 ml pre-filtered plasma by affinity-based binding to a spin column (exoRNeasy, QIAGEN, Germany). mRNA bound to Oligo-dT beads was purified and reverse transcribed (AdnaTest EMT2/StemCell Detect TM, QIAGEN, Germany). Pre-amplified cDNA was analysed by a multimarker qPCR (AdnaPanel TNBC, QIAGEN, Germany). RNA profiles of 18 genes (including AKT2, ALK, AR, AURKA, BRCA1, cKIT, cMET, EGFR, ERCC1, HER2, HER3, KRT5, mTOR, NOTCH1, PARP1, PI3K, SRC1, GAPDH) were normalized by data of healthy donors (n=20) and CD45 served as leukocyte control in the CTC preparation. Results: In general, data analysis showed great differences in RNA profiles of EVs and CTCs. However, PI3K and SRC1 signals were found in similar frequencies in the EV and CTC fraction. HER2, HER3, cKIT or cMET signals significantly correlated with disease progression by analysis of either CTCs or EVs (p=0.001). In EVs, BRCA1 signals positively indicated response, while AR and KRT5 signals were related to a negative response. No marker was exclusively found in CTCs to correlate to the therapy-response course. In CTCs and EVs, however, similar signal courses for PI3K and cMET were found across all time points, revealing PI3K as potential positive response marker and cMET as potential negative response marker in both blood analytes. Conclusions: Expression profiling in CTCs as well as in EVs is enabled by the described workflows. Preliminary data indicated great differences in RNA profiles of EVs and CTCs. The amount of included patients is continuously increased to validate the preliminary results obtained until now. Citation Format: Corinna Keup, Siegfried Hauch, Linda Plappert, Markus Sprenger-Haussels, Pawel Mach, Mitra Tewes, Bahriye Aktas, Hans-Christian Kolberg, Rainer Kimmig, Sabine Kasimir-Bauer. RNA profiles of circulating tumor cells and extracellular vesicles for therapy stratification of metastatic breast cancer patients [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 3777. doi:10.1158/1538-7445.AM2017-3777

Abstract P6-08-39: Influence of lifestyle factors and tumor cell dissemination in 632 early breast cancer patients

Introduction: Influence of lifestyle behaviour in risk of developing breast cancer is supported by several lines. Data from 632 early breast cancer (EBC) patients were collected to evaluate the influence of lifestyle factors in progression free survival (PFS) and overall survival (OS). Results of disseminated tumor cell (DTC) in bone marrow and circulating tumor cells (CTC) in blood were available as well. A complete pathological data set and medical history were obtained. It was the purpose of the present study to correlate these data to compare the findings. Methods: We evaluated 629 bone marrow samples and 606 blood samples from EBC patients treatet between 2004 to 2010 at the time of first diagnosis. All samples underwent immunomagnetic enrichment using the AdnaTest BreastCancerSelect (AdnaGen AG, Germany) within 4 hours after blood withdrawal followed by RNA isolation and subsequent gene expression analysis by reverse transcription and Multiplex-PCR in separated tumor cells using the AdnaTest BreastCancerDetect. CTCs were analyzed for the three breast cancer associated markers: GA733-2, Muc-1, Her-2 and actin as an internal PCR control. BM aspirates were analyzed for DTCs by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3. Lifestyle data icluding menopausal status, BMI, usage of Metformin, hormone replacement drugs, beta blockers and Bisphosphonates were collected by accessing the patient files. Histological data of the primary tumor were available for each patient. Results: The overall detection rate for CTCs was 15.41% (88/571 patients) and for DTC was 38.5% (242/628 patients), respectively. The mean BMI of 428 patients was 26.4 in 81/574 premenopausal, 426/574 postmenopausal and 67/574 perimenopausal patients. Medical history of smoking (128/394 patients), using hormone replacement therapy (92/354 patients), alcohol consumption (68/378 patients), having allergies (188/419), using Metformin (22/389 patients), taking beta blockers (83/392 patients) and Bisphosphonates (201/526 patients) were compared to PFS and OS as well as the histological data of the primary tumor. Conclusion: Lifestyle factors seems to influence the outcome in our cohort of EBC patients as shown in previous studies. Final data and results regarding to tumor cell dissemination compared to lifestyle behaviour will be available for the SABCS 2014. Citation Format: Bahriye Aktas, Anna Frackenpohl, Siegfried Hauch, Johann Kraus, Hans Armin Kestler, Rainer Klaus Kimmig, Sabine Kasimir-Bauer. Influence of lifestyle factors and tumor cell dissemination in 632 early breast cancer patients [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 P6-08-39.