Maren Bredemeier

Circulating U2 Small Nuclear RNA Fragments as a Novel Diagnostic Tool for Patients with Epithelial Ovarian Cancer

BACKGROUND Ovarian cancer is the leading cause of death among malignancies in women. Despite advances in treatment, >50% of patients relapse. For disease monitoring, the identification of a blood-based biomarker would be of prime interest. In this regard, noncoding RNAs, such as microRNA (miRNA) or small nuclear RNA (snRNA), have been suggested as biomarkers for noninvasive cancer diagnosis. In the present study, we sought to identify differentially expressed miRNA/snRNA in sera of ovarian cancer patients and investigate their potential to aid in therapy monitoring. METHODS miRNA/snRNA abundance was investigated in serum (n = 10) by microarray analysis and validated in an extended serum set (n = 119) by reverse-transcription quantitative PCR. RESULTS Abundance of U2-1 snRNA fragment (RNU2-1f) was significantly increased in sera of ovarian cancer patients (P < 0.0001) and paralleled International Federation of Gynecology and Obstetrics stage as well as residual tumor burden after surgery (P < 0.0001 and P = 0.011, respectively). Moreover, for patients with suboptimal debulking, preoperative RNU2-1f concentration was associated with radiographic response after chemotherapy and with platinum resistance (P = 0.0088 and P = 0.0015, respectively). Interestingly, according to the RNU2-1f abundance dynamics, persistent RNU2-1f positivity before surgery and after chemotherapy identified a subgroup of patients with high risk of recurrence and poor prognosis. CONCLUSIONS This is the first report to suggest that a circulating snRNA can serve as an auxiliary diagnostic tool for monitoring tumor dynamics in ovarian cancer. Our results provide a rationale to further investigate whether this high-risk patient group may benefit from additional therapies that are directly applied after chemotherapy.

Establishment of a multimarker qPCR panel for the molecular characterization of circulating tumor cells in blood samples of metastatic breast cancer patients during the course of palliative treatment

Background Circulating tumor cells (CTC) are discussed to be an ideal surrogate marker for individualized treatment in metastatic breast cancer (MBC) since metastatic tissue is often difficult to obtain for repeated analysis. We established a nine gene qPCR panel to characterize the heterogeneous CTC population in MBC patients including epithelial CTC, their receptors (EPCAM, ERBB2, ERBB3, EGFR) CTC in Epithelial-Mesenchymal-Transition [(EMT); PIK3CA, AKT2), stem cell-like CTC (ALDH1) as well as resistant CTC (ERCC1, AURKA] to identify individual therapeutic targets. Results At TP0, at least one marker was detected in 84%, at TP1 in 74% and at TP2 in 79% of the patients, respectively. The expression of ERBB2, ERBB3 and ERCC1 alone or in combination with AURKA was significantly associated with therapy failure. ERBB2 + CTC were only detected in patients not receiving ERBB2 targeted therapies which correlated with no response. Furthermore, patients responding at TP2 had a significantly prolonged overall-survival than patients never responding (p = 0.0090). Patients and Methods 2 × 5 ml blood of 62 MBC patients was collected at the time of disease progression (TP0) and at two clinical staging time points (TP1 and TP2) after 8–12 weeks of chemo-, hormone or antibody therapy for the detection of CTC (AdnaTest EMT-2/StemCell Select™, QIAGEN Hannover GmbH, Germany). After pre-amplification, multiplex qPCR was performed. Establishment was performed using various cancer cell lines. PTPRC (Protein tyrosine phosphatase receptor type C) and GAPDH served as controls. Conclusions Monitoring MBC patients using a multimarker qPCR panel for the characterization of CTC might help to treat patients accordingly in the future.

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.

Abstract 372: Expression profiling of circulating tumor cells: A prognostic and predictive biomarker in metastatic breast cancer

Background: Circulating tumor cells (CTCs) are discussed to be an ideal surrogate marker to monitor disease progression in metastatic breast cancer (MBC) since response to therapy can only be assessed retrospectively after a therapy strategy has already failed. Here we established a new profiling method to characterize the heterogeneous CTC population and investigated if it is possible to predict treatment response based on expression of 46 genes in CTCs of MBC patients. Materials and Methods: 2×5 ml blood of 45 MBC patients was collected at the time of disease progression (T0) and at two consecutive clinical staging (T1 and T2) after 8-12 weeks of chemo-, hormone or antibody therapy for the detection of CTCs applying positive immunomagnetic selection targeting EpCAM, EGFR and HER2 using the AdnaTest EMT-2/Stem Cell Select (AdnaGen GmbH, Germany). Patients were classified into responders and non-responders at the time of clinical staging according to RECIST criteria. PCR assays targeting 46 selected transcript comprising breast cancer, stem cell, EMT, and references markers were designed and extensively optimized for a workflow based on pre-amplification and high throughput profiling. Each sample was profiled in duplicates with the full set of markers including also ValidPrime to correct for genomic background and InterPlate Calibrator to even out variations between runs. The entire workflow was validated to establish excellent technical reproducibility from sample collection through extraction, pre-amplification and analysis. Measured data were analyzed using parametric as well as non-parametric statistics with GenEx and SAS. qPCR as well as technical reads were normalized using the average expression of the reference genes B2M and ActB. Results: A number of genes, including ADAM17, CD24L4, EPCAM, KRT19, MTOR, HER2, TOP2A, and CD44 were differentially expressed in MBC patients (n = 45) as compared to healthy controls (n = 20). A group of non-responders could be identified based on gene expression. Interestingly, expression of ADAM17 (tumor necrosis factor-α-converting enzyme) differed significantly when responders were compared with non-responders at T1 (p = 0.000567). Conclusion: It is possible to distinguish MBC patients from healthy controls based on the expression of the genes investigated. Preliminary results indicate that ADAM17 is a key marker, distinguishing responders from non-responders. For more detailed analysis, it is desirable to build up a larger patient cohort in order to correlate gene expression profiles of CTC enriched samples to a given therapy for individualized treatment. Citation Format: Maren Bredemeier, Mikael Kubista, Robert Sjoback, Marie Jendrichova, Eva Rohlova, Vednula Novosadova, Katarina Kolostova, Siegfried Hauch, Bahriye Aktas, Mitra Tewes, Rainer Kimmig, Sabine Kasimir-Bauer. Expression profiling of circulating tumor cells: A prognostic and predictive biomarker in metastatic breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 372. doi:10.1158/1538-7445.AM2015-372

Abstract 3066: Establishment of a new method for the selection and detection of circulating tumor cells in metastatic breast cancer patients

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Circulating tumor cells (CTCs) are discussed to be an ideal surrogate marker for individualized treatment options in metastatic breast cancer (MBC) since metastatic tissue may be difficult to obtain for repeated analysis. Here we established a new method for selection and detection of the heterogeneous CTC population using immunomagnetic enrichment followed by multi-marker profiling of genes related to different CTC phenotypes. Materials and Methods: Establishment of a nine gene qPCR panel was performed using various cancer cell lines for the markers EpCAM (epithelial); PI3K, AKT2 [epithelial-mesenchymal-transition (EMT)]; ALDH1 (stem cell); ERCC1, Aurora kinase (resistance markers); HER2, HER3, EGFR (receptors); CD45 (leucocyte control) and GAPDH (housekeeping gene) as well as the synthetic EpCAM fragment as an internal reference. 2x5 ml blood of 20 MBC patients was collected at the time of disease progression (T0) and at clinical staging (T1) after 8-12 weeks of chemo-, hormone or antibody therapy for the detection of CTCs applying a new positive immunomagnetic selection using anti-EpCAM, anti-HER2 and anti-EGFR coated magnetic beads (AdnaTest EMT-2/Stem Cell Select, AdnaGen AG Germany). cDNA was gene specifically pre-amplified using the TATAA Multiplex Grand Master Mix according to in-house designed assays. qPCR was performed using Bio-Rad iTaq Universal Supermix SYBR Green Mix. The cutoff was calculated for each gene separately in a way that the false positive rate in all healthy donors (n=18) was lower than 10% (specificity >90%). Subsequently, delta delta Ct was calculated as Ct (cutoff)-Ct(sample) - [Ct(CD45 cutoff)-Ct(CD45sample)]. Results were correlated with clinical response to a given therapy. Results: At T0, at least one of the studied markers was detected in 19/20 patients and at T1 in 16/20 patients, respectively. The distribution of the markers across all patients was highly variable at both time points. However, EpCAM, Aurora kinase as well as EGFR and HER3 were observed most frequently. Interestingly, some patients expressed only one CTC-subtype. Clinically, we observed an increased frequency for epithelial, EMT, stem cell and resistance markers in therapy resistant patients as compared to responders. Whereas EMT and stem cell like CTCs were only detected in the resistant group, CTCs still detectable in the responder group mainly represented an epithelial phenotype showing HER2, HER3 or EGFR expression. Interestingly, some of these persisting CTCs in responders were also positive for ERCC1 and Aurora kinase. Conclusion: We successfully established a new method for the detection of the heterogeneous CTC population which might help to match the right therapy to the right patient in the future. Despite these promising preliminary findings, the qPCR panel has to be further optimized and needs to be verified in a larger patient population. Citation Format: Maren Bredemeier, Bahriye Aktas, Jenny Wagner, Doreen Schellbach, Rainer Kimmig, Sabine Kasimir-Bauer. Establishment of a new method for the selection and detection of circulating tumor cells in metastatic breast cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3066. doi:10.1158/1538-7445.AM2014-3066

Abstract 3076: A fully automated q-PCR-based circulating tumor cell analysis using the Alere TM q-Analyzer test platform

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Circulating Tumor Cell (CTC) analysis has emerged as a promising new diagnostic field for cancer patients towards the estimation of risk for metastatic relapse and metastatic progression providing unique information for therapeutic strategies. The reliable clinical utility of CTCs, however, relies on the standardization of assays used for the detection of CTCs. Here, we report a fully integrated and robust system for the analysis of distinct molecular marker expression profiles of CTCs in blood samples of metastatic breast cancer (MBC) patients. Material and Methods: The CTC test platform consists of the portable q-Analyzer instrument and a disposable test-specific cartridge accommodating all required reagents for the processing of lysates of enriched CTCs from whole blood samples, thus removing subjectivity from the test process by eliminating any dependence on the test environment. Due to the full automation of the actual process, handling is limited to the loading of 100µl of enriched cell lysate obtained from CTC Alere™ AdnaTest BreastCancerSelect onto the test cartridge and insertion into the instrument. Released mRNA is selectively captured to a solid phase and subjected to reverse transcription providing a template for target-specific amplification and real-time quantification of the tumor-associated transcripts EPCAM, MUC1, HER2, ESR1 and PGR. Real-time monitoring is achieved by utilizing the Competitor Monitored Amplification (CMA) combining the benefits of quantitative real-time PCR and microarray analysis for the analysis of expression profiles of multiple genes in a single reaction sample. The implementation of an additional control, assessing the level of contaminating leucocytes present in the enriched sample, provides true quality monitor for the purity of the sample and means to substantially improve the test specificity. Results: Spiking two and five T47D cells into 5 ml blood of 44 healthy donors and evaluating 83 non-spiked healthy donor blood samples, analytical sensitivity revealed 100% recovery and 95% specificity, respectively. The duplicated inter-assay coefficient of variation was <2.5% for each individual transcript. The overall agreement for detecting CTCs between the manual Alere™ AdnaTest BreastCancerDetect and this procedure proved to be 93% with high corresponding individual concordance for MUC1 (κ=1), EPCAM (κ=1), PGR (κ=0.97) and a lower agreement for HER2 (κ=0.46) and ESR1 (κ=0.74). Data were confirmed in 10 MBC patients and more samples are currently evaluated. Conclusion: The Alere™ q CTC Breast Test presents a robust and sensitive test platform, addressing the growing need for standardization which is a prerequisite for multicentric evaluations. We believe that our approach ultimately will pave the way of CTC markers into clinical practice. Moreover, the test facilitates comparisons and improvements of different CTC enrichment methods. Citation Format: Stephan Hubold, Ivan Loncarevic, Jana Thiele, Maren Bredemeier, Heidi Klemm, Heike Klabunde, Danny Michel, Rainer Kimmig, Siegfried Hauch, Bahriye Aktas, Eugen Ermantraut, Sabine Kasimir-Bauer. A fully automated q-PCR-based circulating tumor cell analysis using the Alere TM q-Analyzer test platform. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3076. doi:10.1158/1538-7445.AM2014-3076

Abstract P1-04-13: The role of excision repair cross-complementation 1 enzyme (ERCC1)-positive circulating tumor cells in primary and metastatic breast cancer

Background: Multi-drug resistance to chemotherapeutic agents, as well as resistance to radiation therapy is a major cause of treatment failure in breast cancer. The expression of the excision repair cross-complementation 1 (ERCC1) enzyme in tumor tissue has been associated with resistance to platinum based chemotherapy but also with radiation therapy in different cancer diseases. However, studies on ERCC1 in breast cancer are limited. Furthermore, after primary tumor resection, there is no validated clinical parameter to predict the sensitivity to radio/chemotherapy. Circulating Tumor Cells (CTCs), as potential surrogate markers for minimal residual disease, would be an ideal ‘surrogate tissue’ to identify such biomarkers. Here we evaluated ERCC1 expression in CTCs of primary and metastatic breast cancer patients as a potential biomarker for radio/chemoresistance and correlated these findings with the presence of HER2-positive and stemness like CTCs (slCTCs), including cells being able to perform epithelial-mesenchymal transition (EMT). Patients and Methods: 2 × 5 ml blood from primary (n = 110) and metastatic (n = 56) breast cancer patients were analyzed for CTCs with the AdnaTest BreastCancer (AdnaGen AG, Hannover, Germany) for the detection of EpCAM, MUC-1, HER-2, and beta-Actin transcripts. The recovered c-DNA was additionally tested for the presence of a) ERCC1 b) slCTCs using the AdnaTest TumorStemCell for the expression of ALDH1 (both using single-plex RT-PCR) and c) CTCs in EMT applying the AdnaTest EMT (multiplex RT-PCR for TWIST, AKT2, PI3K). The analysis of PCR products was performed by capillary electrophoresis on the Agilent Bioanalyzer 2100. Results: In primary breast cancer, the overall detection rate for CTCs was 27% (30/110 patients) with the expression rate of 83% for HER2. ERCC1 was detected in 51/110 patients (46%), slCTCs in 27/110 patients (25%) and CTCs in EMT in 47/110 patients (43%), respectively. ERCC1 expression was significantly associated with the presence of HER2-positive CTCs (p = 0.001), slCTCs (p = 0.014) and CTCs in EMT (p = 4×10E-8), respectively. In metastatic breast cancer, the positivity rate for CTCs was 36% (20/56 patients) with the expression rate of 40% for HER2. ERCC1 was detected in 15/56 patients (27%), slCTCs in 16/56 patients (29%) and CTCs in EMT in 12/56 patients (22%), respectively. ERCC1 expression was significantly associated with the presence of HER2-positive CTCs (p = 0.0019), slCTCs (p = 0.00014) and CTCs in EMT (p = 0.00037), respectively. Conclusion: The tumor stem cell phenotype and EMT are widely discussed to be correlated with increasing cell survival and chemotherapy resistance including increased DNA repair capacity characterized by the overexpression of genes like ERCC1. Using CTCs as a potential surrogate marker for radio/chemoresistance, we could show a significant correlation of the ERCC1 profile and CTCs displaying tumor stem cell or EMT characteristics but also a HER2-positive phenotype. Thus, the presence as well as the molecular profile of CTCs might become an important tool to predict radio/chemoresistance in breast cancer patients. The clinical relevance on prognosis and therapy response has to be evaluated in a prospective trial. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-04-13.

Abstract 1466: Establishment of a multimarker gene panel for the characterization of circulating tumor cells in metastatic breast cancer.

Background: Improved treatment strategies for metastatic breast cancer (MBC) patients are urgently needed. Since metastatic tissue may be difficult to obtain for repeated analysis, circulating tumor cells (CTC) would be an ideal surrogate tissue to identify prognostic and predictive factors that will help to select the optimal therapeutic strategy for each individual patient. Assuming that the population of CTC contains epithelial-like, EMT (Epithelial-Mesenchymal-Transition)-like and stem cell-like cells, we established a multi-marker qPCR for the characterization of these cells.Materials and Methods: Establishment of a 16 gene qPCR panel was performed using various epithelial cancer cell lines for the markers: EpCAM, MUC1 (epithelial); PI3K, PTEN, TWIST, mTOR, KRAS, AKT2 (EMT); ALDH1, CD44, CD24L4 (stem cell); ER, PR, HER2 and EGFR (receptors) and CD45 as a leucocyte control. The prostate cancer cell line LNCAP, expressing most of these genes, was used for spiking experiments. 10 ml blood of eight healthy donors (HDs), five HDs spiked with 10 LNCAP cells and 25 MBC patients were selected for CTC using AdnaTest BreastCancerSelect (AdnaGen AG) resulting in cDNA1. Subsequently, the same sample (with removed target cells) was processed again using the same procedure resulting in cDNA2. cDNA1 and cDNA2 were gene specifically preamplified using TaqMan PreAmp Master Mix according to in house designed assays. qPCR was performed using Bio-Rad SYBR Green Mix. If the CD45 deltaCt was > zero, deltaCt value of a given gene was calculated as the difference between Ct (cDNA2) and Ct(cDNA1). A gene with a deltaCt > zero was considered positive.Results: When HDs were tested for all genes, no false positive findings were observed except for AKT2, CD24L4, CD44 (n=3 cases). All of the genes except for TWIST, ER and PR could be positively detected in samples spiked with 10 LNCAP cells. In patient samples, at least one of all studied markers was detected in 21/25 (84%) of the patients. The distribution of the markers across all patients was highly variable. However, PI3K expression was observed most frequently (n=8/21 patients), followed by the expression of CD44 (n=6/21 patients), HER2 (n=5/21 patients) and TWIST, KRAS, mTOR and EpCAM (4/21 patients), respectively. No expression was observed for MUC1, PR and CD45. In general, EMT- and stem cell-like CTC were predominantly detected. HER2 positive and epithelial-like CTC as well as CTC expressing ER, PR and EGFR were observed less frequently. Interestingly, some patients expressed only one CTC-subtype.Conclusion: Multi-gene expression profiling improves the characterization of CTC of an individual patient. Furthermore, it was possible to classify individual patient samples into CTC subtypes. Despite these promising preliminary findings, the method has to be further optimized and needs to be verified in a larger patient population. Citation Format: Maren Bredemeier, Bahriye Aktas, Rainer Kimmig, Sabine Kasimir-Bauer. Establishment of a multimarker gene panel for the characterization of circulating tumor cells in metastatic breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1466. doi:10.1158/1538-7445.AM2013-1466