Circulating Tumor Cells Positive Patients Research Articles

Abstract 599: Investigation of PD-L1 expression in circulating tumor cells isolated using the Parsortix system in metastatic lung and breast cancer patients

Abstract Programmed death-ligand 1 (PD-L1) allows cancer cells to evade the host immune response when upregulated. PD-L1 antagonists have been shown to be effective immunotherapies. The FDA approved the PD-L1 inhibitor nivolumab for treatment of previously treated, non-responding metastatic non-small cell lung cancer (NSCLC) patients, independent of the PD-L1 status identified in tumour tissue, likely due to the controversial value of PD-L1 detection on tissue biopsy that may be out-of-date. Measurement of PD-L1 expression in circulating tumor cells (CTCs) may enable repeat testing to provide up-to-date PD-L1 status and the potential to monitor patients on these therapies. Whilst the epitope-dependent CELLSEARCH® system is the only technology FDA-cleared for the enumeration of CTCs, its application is limited to epithelial CTCs and has limited sensitivity in NSCLC. We developed a research use only assay for the characterisation of PD-L1 expression on CTCs isolated using the Parsortix® system, a label-independent microfluidic device that harvests CTCs of all phenotypes (epithelial and/or mesenchymal) based on size and compressibility. Peripheral blood (8 – 10mL) was drawn into K2EDTA tubes from 17 healthy volunteers, 17 metastatic breast cancer (MBC) patients and 18 metastatic NSCLC patients and processed on Parsortix systems within 24 hours. Harvested CTCs were cytospun and immunofluorescently stained for detection of cytokeratins and PD-L1. Slides were imaged using a BioView AllegroPlus imaging system.No CTCs were identified in the healthy volunteers, highlighting the specificity of the assay. CTCs (≥1) were identified in 70% of the MBC patients and 55% of the NSCLC patients. Importantly, the positivity rate observed in NSCLC patients was 2-fold higher than that in previously described studies using the CELLSEARCH system. A mean of 9 CTCs/tube of blood were identified in the CTC positive MBC patients, with a range of 1 – 128 CTCs. A mean of 11 CTCs/tube of blood were identified in the CTC positive NSCLC patients, with a range of 1 – 23 CTCs. CTC clusters, consisting of 3 – 45 cells per cluster, were observed in both NSCLC and MBC patients. High heterogeneity of PD-L1 expression was observed. The CTC positive patients were classified into three groups: patients with CTCs positive for both cytokeratins (CK+) and PD-L1 (36% in MBC and 40% in NSCLC), patients with a mixed population of CK+, PD-L1+/- CTCs (27% in MBC and 40% in NSCLC) and patients with CK+, PD-L1- CTCs only (36% in MBC and 20% in NSCLC).The optimized assay for detection of PD-L1 expression on CTCs allowed for the identification of PD-L1 positive and PD-L1 negative CTCs in a significant proportion of the NSCLC and MBC patients studied. The ability to isolate significant numbers of PD-L1 positive CTCs in blood lays the groundwork for development of dynamic PD-L1 monitoring to support personalized patient management. Citation Format: Mariacristina Ciccioli, Amy Davis, Ofure Alenkhe, Anne-Sophie Pailhes-Jimenez. Investigation of PD-L1 expression in circulating tumor cells isolated using the Parsortix system in metastatic lung and breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 599.

Abstract 768: Predictive role of circulating tumor cells in women with ovarian cancer

Abstract Background: “Liquid biopsy”, where body fluids is screened for biomarkers is gathering substantial research. We aimed to examine women with suspected ovarian cancer for the presence of circulating tumor cells (CTCs) and study its role in prediction of chemoresistance and survival. Methods: Magnetic powder labeled monoclonal antibodies for epithelial cell adhesion molecule (EpCAM), Mucin 1 cell surface associated ( MUC1) ,mucin 16, cell surface associated MUC16 or CA125 were prepared according to the manufacturer's protocol. Expression of three ovarian cancer related genes were detected in CTCs using multiplex reverse transcriptase-polymerase chain reaction (Multiplex RT-PCR). CTCs and serum CA125 were measured in 100 patients with suspected ovarian cancer. Correlations with clinicopathological parameters and treatment were analyzed. Results: CTCs were detected in 18/70 (25.7%) of women with malignancy compared to 0/30 (0.0%) in those with benign gynecologic diseases (p=0.001). The sensitivity and specificity of the CTC test for predicting a malignant histology in pelvic masses was 27.7% (95% CI :16.3%,37.7%) and 100% (95% CI: 85.8%, 100%), respectively. The number of CTCs correlated with stage of ovarian cancer (p=0.030). The presence of EpCAM+CTC at primary diagnosis in ovarian cancer was found to be an independent predictor of a poor progression free survival (PFS) (HR,3.3; 95% CI, 1.3-8.4; p=0.010), overall survival (OS) (HR, 2.6; 95% CI,1.1-5.6; p=0.019) and resistance to chemotherapy (OR 8.6; 95% CI, 1.8-43.7; p=0.009). Conclusion: Expression of EpCAM in CTC positive patients predicts chemotherapy resistance and prognosis, could aid clinicians in individualizing therapy, saving the patient from unnecessary side effects and systemic toxicity of platinum-based chemotherapy. Also, EpCAM expression can be a strong basis for further investigating anti-EpCAM targeted therapies in ovarian cancer. Citation Format: Swarnima Singh, Uday Kumar. Predictive role of circulating tumor cells in women with ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 768.

ARTO trial (NCT03449719), a randomized phase II trial enrolling oligometastatic castration-resistant prostate cancer patients treated with first-line abiraterone acetate with or without stereotactic body radiation therapy: Preliminary results comprehensive of biochemical outcomes and circulating tumor cells analysis.

118 Background: Androgen Receptor Targeted Agents (ARTA) represent one of the main treatment options for metastatic castrate resistant prostate cancer (mCRPC). Addition of stereotactic radiation therapy (SBRT) to ablate metastatic foci may improve clinical outcomes in oligometastatic setting. ARTO trial (NCT03449719) is a randomized phase II trial testing the benefit of upfront SBRT on all sites of metastatic disease in oligo-mCRPC patients undergoing I line therapy with Abiraterone Acetate (AA). In this preliminary analysis, we report results after 6 months of follow up, together with an exploratory analysis of androgen receptor splice variants (ARV7/ARFL) Prostate Specific Antigen (PSA) and Prostate Specific Membrane Antigen (PSMA) expression on Circulating Tumor Cells (CTCs) detected in this cohort of patients. Methods: 31 patients affected by oligo-mCRPC (defined as < 3 non-visceral metastatic lesions) were randomized to receive I line AA therapy with or without SBRT on all metastatic sites. Baseline blood samples to detect CTCs and evaluate their ARV7, ARFL, PSA and PSMA expression were taken before AA treatment start. Assessments comprehensive of clinical examination and serum PSA were performed every three months. Toxicity was assessed by the Common Terminology Criteria for Adverse Events toxicity scale (CTCAE v.4.03). Results: Thirteen and 18 patients were enrolled in the treatment and control arm, respectively. Nineteen metastatic lesions were treated with SBRT in the treatment arm. At 6 months, complete response (defined as a serum PSA level < 0.2 ng/dl) and biochemical response (defined as a PSA reduction > 50% if compared to baseline) were achieved in 6 vs 4 and in 10 vs 8 patients in the treatment vs control arm, respectively. One patient in the treatment arm died for other causes, 1 biochemical progression occurred in the control arm. No adverse events occurred in both arms of treatment. CTCs analysis was available for 10 patients, out of whom 4 were found positive for CTCs (1 and 3 from the treatment and control arm, respectively). ARV7 and ARFL were expressed in 1 patient from the control arm, PSMA was expressed in all CTC positive patients, PSA was expressed in 2 patients from the control and one from the treatment arm. Conclusions: SBRT+AA in oligo-mCRPC patients was safe and yielded promising biochemical results. CTCs detection in this selected cohort of oligo-mCRPC was lower if compared to historical data of unselected mCRPC patients. Data about ARV7, ARFL, PSA and PSMA expression represent an interesting snapshot of biomarker arrangement in this setting. Clinical trial information: NCT03449719.

Cell by cell immuno- and cancer marker profiling of non-small cell lung cancer tissue: Checkpoint marker expression on CD103+, CD4+ T-cells predicts circulating tumor cells

Non-small cell lung cancer (NSCLC) has a poor prognosis. Targeted therapy and immunotherapy in recent years has significantly improved NSCLC patient outcome. In this study, we employed cell-by-cell immune and cancer marker profiling of the primary tumor cells to investigate possible signatures that might predict the presence or absence of circulating tumor cells (CTCs). We performed a comprehensive study on 10 NSCLC patient tissue samples with paired blood samples. The solid tissue biopsy samples were dissociated into single cells by non-enzymatic tissue homogenization and stained with a total 25 immune, cancer markers and DNA content dye and analyzed with high-parameter flow cytometry. CTCs were isolated and analyzed from the paired peripheral blood. We investigated a total of 74 biomarkers for their correlation with CTC number. Strong correlations were observed between CTC number and the frequency of immune checkpoint marker expressing lymphocytes (CTLA-4, LAG3, TIM3, PD-1), within the CD103+CD4+ T lymphocyte subset. CTC number is also correlated with the frequency of PD-L1 expressing cancer cells and cancer cell DNA content. In contrast, CTC number inversely correlated to the frequency of CD44+E-cadherin− cancer cells. Unsupervised clustering analysis based on the biomarker analysis separated the CTC negative patients from the CTC positive patients. Profiling multiple immune and cancer markers on cancer samples with multi-parametric flow cytometry allowed us to obtain protein expression information at the single cell level. Clustering analysis of the proteomic data revealed a signature driven by checkpoint marker expression on CD103+CD4+ T cells that could potentially be predictive of CTCs and targets of therapy.

Correlation of circulating tumor enumeration and immune checkpoint marker expression on CD103+, CD4+ T cells in non-small cell lung cancer tissue.

109 Background: Non-small cell lung cancer (NSCLC) has a poor prognosis as most patients are at advanced stage when diagnosed. Targeted therapy and immunotherapy in recent years has significantly improved NSCLC patient outcome. In this study, we employed cell-by-cell immune and cancer marker profiling of the primary tumor cells to investigate possible signatures that might predict the presence or absence of circulating tumor cells (CTCs). Methods: We performed a comprehensive study on 10 NSCLC patient tissue samples with paired blood samples. The solid tissue biopsy samples were dissociated into single cells by non-enzymatic tissue homogenization. The single cell suspensions were stained with a total 25 immune, cancer markers and a DNA content dye and analyzed with advanced, high-parameter flow cytometry. CTCs were isolated and analyzed from the paired peripheral blood. Results: Out of the 26 unique cell markers stained, we investigated a total of 72 biomarkers for their correlation with CTC number. Strong correlations were observed between CTC number and the frequency of immune checkpoint marker expressing lymphocytes, especially with the immune checkpoint marker expressing CD103+CD4+ T lymphocytes. CTC number is also correlated with the frequency of PD-L1 expressing cancer cells and cancer cell DNA content. In contrast, CTC number inversely correlated to the frequency of CD44+E-cadherin- cancer cells. Unsupervised clustering analysis based on the biomarker analysis separated the CTC negative patients from the CTC positive patients. Conclusions: Profiling multiple immune and cancer markers on cancer samples with multi-parametric flow cytometry allowed us to obtain protein expression information at the single cell level. Clustering analysis of the proteomic data revealed a signature driven by checkpoint marker expression on CD103+CD4+ T cells that could potentially be predictive of CTCs.

Abstract P3-01-04: DETECT V – Expression analysis of human epidermal growth factor receptor 2 and estrogen receptor on circulating tumor cells of metastatic breast cancer patients

Abstract Introduction: The prognostic relevance of Circulating Tumor Cells (CTCs) in patients with metastatic breast cancer (MBC) has been shown in multiple clinical trials. Beside their quantification it is of particular interest to analyze the predictive value of these cells and investigate if therapeutic decisions can be based on the phenotype of CTCs. The DETECT study concept evaluates this possible implication of CTCs. The DETECT V study compares endocrine therapy vs. chemotherapy, both combined with Trastuzumab and Pertuzumab in MBC patients with a HER2-positive (Human Epidermal Growth Factor Receptor 2), hormone receptor-positive tumor. Translational projects to identify potential predictive markers are associated to the study program. The expression of predictive markers HER2 and estrogen receptor (ER) on CTCs is analyzed to establish and validate an “Endocrine Responsiveness Score” (ERS). The ERS is aiming to estimate the potential benefit of endocrine therapy. Methods: Quantification and characterization of CTCs was performed using Cell Search® CXC Kit. The staining protocol was validated using cell lines with known expression of ER and HER2. CTCs were quantified at randomization, after six weeks and at the end of treatment in two samples. Staining intensities of both markers were specified. Results: Staining was successfully established. ER staining intensity was specified as negative or positive; HER2 staining intensity as negative, weak, moderate or strong. Detection and characterization of CTCs was analyzed for the first 30 patients. At time of randomization 17 of 26 samples were CTC positive with ≥1 CTC in one sample (HER2 and ER), 6/25 in both samples. After six weeks 8 of 26 patients contained ≥1 CTC in one sample and 5 of 26 in both samples. 11 of 15 CTC positive patients at time of randomization were CTC negative in the subsequent sample after six weeks. The cumulative frequency of ER- and HER2-positivity regarding CTCs detected in all samples was analyzed. 13% of all CTCs detected were ER positive (14/115). The percentage of HER2 3+ CTCs decreased from 27% at time of randomization (25/92) to 0% after six weeks (0/87). Analyzing the distribution of the expression of ER and HER2 in all CTCs of one sample, 10 of 19 samples containing ≥2 CTC showed heterogenic intensity of HER2-specific immunofluorescence. 8 of 14 samples containing ≥2 CTC showed heterogenic intensity of ER-specific immunofluorescence, respectively. Conclusion: The implementation of “Endocrine Responsiveness Score” ERS aims to predict the potential benefit of endocrine therapy in patients with a HER2-positive, hormone receptor-positive tumor. Descriptive analysis of first patient samples has shown to detect heterogenic expression of HER2 and ER in CTCs of patients participating in the DETECT V study. Citation Format: Fehm T, Meier-Stiegen F, Riethdorf S, Rack B, Taran F-A, Pantel K, Müller V, Janni W, Huober J. DETECT V – Expression analysis of human epidermal growth factor receptor 2 and estrogen receptor on circulating tumor cells of metastatic breast cancer patients [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-01-04.

Analysis of Circulating Tumor Cells in Ovarian Cancer and Their Clinical Value as a Biomarker

Background/Aims: Monitoring the appearance and progression of tumors are important for improving the survival rate of patients with ovarian cancer. This study aims to examine circulating tumor cells (CTCs) in epithelial ovarian cancer (EOC) patients to evaluate their clinical significance in comparison to the existing biomarker CA125. Methods: Immuomagnetic bead screening, targeting epithelial antigens on ovarian cancer cells, combined with multiplex reverse transcriptase-polymerase chain reaction (Multiplex RT-PCR) was used to detect CTCs in 211 samples of peripheral blood (5 ml) from 109 EOC patients. CTCs and CA125 were measured in serial from 153 blood and 153 serum samples from 51 patients and correlations with treatment were analyzed. Immunohistochemistry was used to detect the expression of tumor-associated proteins in tumor tissues and compared with gene expression in CTCs from patients. Results: CTCs were detected in 90% (98/109) of newly diagnosed patients. In newly diagnosed patients, the number of CTCs was correlated with stage (p=0.034). Patients with stage IA-IB disease had a CTC positive rate of 93% (13/14), much higher than the CA125 positive rate of only 64% (9/14) for the same patients. The numbers of CTCs changed with treatment, and the expression of EpCAM (p=0.003) and HER2 (p=0.035) in CTCs was correlated with resistance to chemotherapy. Expression of EpCAM in CTCs before treatment was also correlated with overall survival (OS) (p=0.041). Conclusion: Detection of CTCs allows early diagnose and expression of EpCAM in CTC positive patients predicts prognosis and should be helpful for monitoring treatment.

Using the New CellCollector to Capture Circulating Tumor Cells from Blood in Different Groups of Pulmonary Disease: A Cohort Study

Circulating tumor cells (CTCs) are promising biomarkers for clinical application. Cancer screening with Low-Dose Computed Tomography (LDCT) and CTC detections in pulmonary nodule patients has never been reported. The aim of this study was to explore the effectiveness of the combined methods to screen lung cancer. Out of 8313 volunteers screened by LDCT, 32 ground-glass nodules (GGNs) patients and 19 healthy volunteers were randomly selected. Meanwhile, 15 lung cancer patients also enrolled. CellCollector, a new CTC capturing device, was applied for CTCs detection. In GGNs group, five CTC positive patients with six CTCs were identified, 15.6% were positive (range, 1–2). In lung cancer group, 73.3% of the analyzed CellCollector cells were positive (range, 1–7) and no “CTC-like” events were detected in healthy group. All CTCs detected from GGNs group were isolated from the CellCollector functional domain and determined by whole genomic amplification for next-generation sequencing(NGS) analysis. NGS data showed that three cancer-related genes contained mutations in five CTC positive patients, including KIT, SMARCB1 and TP53 genes. In four patients, 16 mutation genes existed. Therefore, LDCT combined with CTC analysis by an in vivo device in high-risk pulmonary nodule patients was a promising way to screen early stage lung cancer.

Association between microRNAs expression in primary breast cancer and presence of circulating tumor cells (CTC) in peripheral blood.

e23022 Background: CTCs play major role in tumor dissemination and progression and represent one of the key component of metastatic cascade. MicroRNAs (miRs) are involved in regulation in several biological processes in cancer including invasion, epithelial-mesenchymal transition (EMT) and disease progression. The aim of this study was to identify miRs in primary tumor associated with presence of CTCs in peripheral blood (PB) in non-metastatic breast cancer patients. Methods: This translational study included 79 patients with primary breast cancer for whom fresh frozen tumor tissue and status of CTCs in peripheral blood were available. CTCs were detected before surgery by qRT-PCR assay for expression of epithelial (CK19) or epithelial-mesenchymal transition (EMT) genes (TWIST1, SNAIL1, SLUG, ZEB1). Total RNA was extracted from fresh frozen primary tumor and the expression profiles were obtained using Human microRNA Microarray v21.0 (Agilent Technologies). Results: We analyzed 48 (60.8%) tumor samples from patients with presence of CTCs in PB and 31 (39.2%) tumors with non-detectable CTCs. From CTCs positive patients, in 20 (41.7%) of them epithelial CTCs (EP_CTC) were detected while in 28 (38.3%) CTCs with EMT (CTC_EMT) phenotype were present. We identified 178 miRs that were expressed at significantly different levels (FDR < 0.05) in tumors with presence of any type of CTCs in PB compared to tumors with non-detectable CTCs. We also identified 174 and 137 miRs (33 overlapping) that were expressed at significantly different levels in tumors with EP_CTCs and CTC_EMT, respectively, compared to tumors with non-detectable CTCs. Overlapping miRs with highest different levels in expression (FDR < 0.01) were miR-3137, miR-3138, miR-3168, miR-605-5p, miR-6165 and miR-6790-5p. Conclusions: We identified for the first time miRs expressed in primary tumor associated with CTCs in peripheral blood in breast cancer patients. Moreover, we identified miRs specifically associated with various subpopulations of CTCs. We suppose, that these miRs could be involved in tumor dissemination and might lead to identification of new therapeutic targets. Study was supported by APVV-14-0327.

Abstract P1-01-03: Dynamics of circulating tumor cells during the course of chemotherapy and prognostic relevance across molecular subtypes in high-risk early breast cancer patients – Results from the adjuvant SUCCESS A trial

Abstract Background: The presence of circulating tumor cells (CTCs) before chemotherapy is known to be associated with reduced disease free survival (DFS) and overall survival (OS) in early breast cancer (EBC). In addition, recent findings suggest that CTCs persisting after adjuvant chemotherapy indicate poor prognosis. In an explorative analysis of the SUCCESS A trial, we evaluated the prognostic relevance of changes in CTC counts during the course of adjuvant chemotherapy across molecular subtypes to assess whether the prognostic role of persisting CTCs varies according to tumor biology. Methods: The SUCCESS A trial is a phase III study, where patients with high-risk EBC (stage pN1-3 or pT2-4 or grade 3 or age ≤ 35 or hormone-receptor negative) were randomized to adjuvant chemotherapy with 3 cycles of epirubicin-fluorouracil-cyclophosphamide followed by either 3 cycles of docetaxel or 3 cycles of gemcitabine-docetaxel. CTC enumeration was performed before and after chemotherapy using the FDA-approved CellSearch® System (Janssen Diagnostics, LLC), and CTC positivity was defined as ≥ 1 CTC in 23 ml blood. Molecular subtypes were defined as luminal A like (hormone-receptor positive, grading 1 or 2), luminal B like (hormone-receptor positive, grading 3), triple-negative or HER2-positive. Patient outcome in terms of DFS and OS was analyzed using univariate log-rank tests and Cox regression models (median follow-up time 65.2 months). Results: Data on both molecular subtypes and CTC status before and after chemotherapy were available for 1485 (39.6%) of 3754 patients randomized. This cohort contained 577 (38.9%) luminal A like, 236 (15.9%) luminal B like, 379 (25.5%) HER2-positive and 293 (19.7%) triple negative tumors. Overall, 917 (61.8%) patients were CTC negative before and after chemotherapy (neg/neg), 260 (17.5%) patients had a negative CTC status before and a positive CTC status after chemotherapy (neg/pos), 229 (15.4%) patients converted from positive to negative CTC status (pos/neg), and 79 (5.3%) patients were positive for CTCs at both time points (pos/pos). There were significant differences in DFS and OS among these four groups in patients with luminal A like tumors (log rank test, both p < 0.003) and patients with luminal B like tumors (log rank test, both p < 0.001). In both patients with luminal A like or luminal B like tumors, persistently CTC positive patients had the worst outcome (relative to persistently CTC-negative patients) in terms of DFS and OS. In contrast to luminal-like tumors, no significant differences with regard to DFS or OS were found among the four groups (neg/neg, neg/pos, pos/neg, pos/pos) in patients with HER2-positive or triple-negative tumors (log rank test, all p > 0.13). Conclusion: The presence of CTCs both before and after adjuvant chemotherapy was associated with poor survival in luminal A like and luminal B like tumors, but not in HER2-positive or triple-negative tumors. Further research is needed to evaluate the effect of chemotherapy on CTC prevalence in different molecular subtypes of EBC. Citation Format: Tzschaschel MLJ, Rack B, Andergassen U, Friedl TWP, Schneeweiss A, Mueller V, Tanja F, Pantel K, Gade J, Lorenz R, Rezai M, Tesch H, Soeling U, Polasik A, Alunni-Fabbroni M, Trapp EK, Mahner S, Schindlbeck C, Lichtenegger W, Beckmann MW, Fasching PA, Janni W. Dynamics of circulating tumor cells during the course of chemotherapy and prognostic relevance across molecular subtypes in high-risk early breast cancer patients – Results from the adjuvant SUCCESS A trial [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 P1-01-03.

P1.07-031 Clinical Evaluation of Folate Receptor-Positive Circulating Tumor Cells Detection in Patients with Small Cell Lung Cancer

Small cell lung cancer (SCLC) is distinguished by extremely high numbers of circulating tumor cells (CTCs) in comparison to other malignancies; however, the role of CTCs in evaluating chemotherapy effect of SCLC is to be further clarified. The purpose of this study was to investigate the predictive and prognostic role of folate receptor–positive CTCs in unresectable SCLC. In this single-center prospective study, blood samples for folate receptor–positive CTCs analysis were obtained from 80 patients with chemotherapy-naive unresectable SCLC at baseline, after two cycles of chemotherapy, and on disease progression. All patients received chemotherapy with EC or EP regimen for at least two cycles. CTCs number at baseline, after chemotherapy and changes with chemotherapy were evaluated as predictive factors for chemotherapy effect, along with clinical characteristics. Of all 80 patients, CTCs was detected at baseline as positive (CTCs>8.7 FU/3mL) in 67 patients, with the percentage of 83.8%, which was not associated with age, sex, smoking status or disease stage. In 72 evaluable patients, the disease control rate was 83.9% (52/62) and 50% (5/10) in CTCs positive and negative patients respectively (P=0.004). In CTCs positive patients, those harboring low levels of folate receptor (8.7<CTCs<14, n=29) were associated with longer PFS (275 days, 95%CI:82-468) than those with high expression levels (CTCs ≥14, n=38)(PFS: 210days, 95% CI:159-261; P=0.0458). Reduction in CTCs number after two cycles of chemotherapy was associated with partial response (P=0.049) but not with stable disease (P=0.47). The results of a multivariate analysis showed that the baseline CTCs level was an independent prognostic factor for survival time (HR=2.19; P=0.024). CTCs number at baseline could be used as a useful prognostic biomarker for SCLC. Reduction in CTCs number with chemotherapy could predict better chemotherapy effect of SCLC.

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

Abstract 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 &amp;lt; 0.001) and DFS (HR 2.28, 95% CI 1.48 – 3.50, p &amp;lt; 0.001). The prognostic value of CTC status 2 years after chemotherapy was independent from hormone- and HER2-receptor status. Overall, 719 (65.2%) patients were CTC negative before and 2 years after chemotherapy, while 157 (14.2%) had a negative CTC status before and a positive CTC status 2 years after chemotherapy. 180 (16.3%) patients converted from positive to negative CTC status and 47 (4.3%) patients were persistently positive for CTCs. There were significant differences in OS and DFS among the four patient groups (log rank tests, both p &amp;lt; 0.001) and persistently CTC positive patients had the worst outcome in terms of OS and DFS. 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 patients' 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, Schneeweiß 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.

Abstract 374: Expression of the autophagosomal marker LC3 on circulating tumor cells (CTCs) of patients with early and metastatic breast cancer (BC)

Abstract Background: Autophagy is a protein and organelle degradation pathway involved in the regulation of cancer development and progression. Recent evidence suggests that autophagy serves as a cell survival mechanism under stressed conditions and it is considered to be involved in determining the response of cancer cells to anticancer therapy. Microtubule-associated protein 1 light chain 3 (LC3) has been identified as a marker for monitoring autophagy. CTCs, which have been linked to tumor progression in BC, may employ autophagy associated mechanisms to survive. Here, we investigated the expression of LC3B on CTCs isolated from patients with BC. Methods: The expression of LC3B was first assessed by immunofluorescence using MCF7 treated with tamoxifen as a model system. LC3B expression was then evaluated in 22 and 25 CTC positive patients with early and metastatic BC before the initiation of adjuvant and first-line chemotherapy, respectively. Metastatic patients were also assessed before the administration of the second treatment cycle. Cytospins of MCF7 cells and of peripheral blood mononuclear cells (PBMCs) were stained with a monoclonal A45-B/B3 anti-cytokeratin (CK) antibody and an LC3B anti-rabbit antibody. LC3B density was measured using the automated fluorescent microscope ARIOL system. The cutoff value for high and low LC3B expression, was set at the median value of the integrated optical density (IOD). Results: Among 142 CTCs identified in patients with early BC, 55 (38%) presented high LC3B expression. A total of 176 CTCs were detected in metastatic patients before the initiation of first-line chemotherapy and 99 (59%) presented high LC3B expression (p = 0.0003 compared with early BC patients). After the first treatment cycle, 62 CTCs were detected with 37 (60%) of them demonstrating high LC3B expression. Nine metastatic patients remained CTC-positive both before and after the first cycle of chemotherapy. In these patients, the proportion of cells presenting high LC3B expression was 40% and 75%, pre- and post- chemotherapy, respectively (p = 0.0049). Conclusions: The autophagosome marker LC3B is expressed in CTCs from patients with BC. High LC3B expression in CTCs was more commonly observed in metastatic, whereas low LC3B expression prevailed in early disease. Moreover, LC3B expression on CTCs was significantly increased after the administration of chemotherapy. The above observations suggest that metastatic progression in breast cancer could be linked to LC3B expression. Moreover the evaluation of LC3B on CTCs merits further investigation regarding its association with chemotherapy efficacy. Citation Format: Maria Spiliotaki, Chrysa Koukorava, Alexios Matikas, Vassilis Georgoulias, Dimitrios Mavroudis, Sofia Agelaki. Expression of the autophagosomal marker LC3 on circulating tumor cells (CTCs) of patients with early and metastatic breast cancer (BC). [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 374. doi:10.1158/1538-7445.AM2015-374

402P - Circulating Tumor Cells Analysed By Scanning Fluorescence Microscopy in the Xena Trial

ABSTRACT Aim: The hypothesis is that circulating tumor cells (CTC) can be used as an indicator for treatment response in metastatic breast cancer patients. CTC results from the first 38 patients in a monitoring study are presented. Methods: CTC are tested as a monitoring marker for treatment response in the clinical trial XeNa. Metastatic or locally advanced HER2 negative breast cancer patients are treated with a combined therapy with Xeloda and Navelbine Oral vs standard of care treatment. We aim to include 120 breast cancer patients in a randomized two-armed study with equal numbers in each arm. Blood samples before treatment and approximately four weeks after first treatment will be analyzed for CTC by a scanning fluorescence microscope (CytoTrack®). Details regarding XeNa can be found at clinicaltrials.org, ClinicalTrials.gov Identifier: NCT01941771. The endpoints of the study includes overall survival, clinical response according to RECIST criteria and clinical response according to CTC count. The null hypothesis is that there is no difference in outcome for patients with CTC compared to patients without CTC. It is estimated that with a power of 0.80, a total of 80 patients are required to reject the null hypothesis with a significance level of 0.05. CTC analysis is performed by the scanning fluorescence microscope CytoTrack. Results: In 38 consecutive patients, 17 were found positive for CTC prior to treatment. Follow up samples were CTC positive in twelve of the original CTC positive patients and CTC negative in five of the original CTC positive patients. Twentyfour patients had no CTC prior to or following treatment. The study is ongoing, so outcome for the patients are not yet known. No of patients Median prior Range prior Median Post Range post No CTC 24 0 0 0 0 CTC unchanged 12 17 2-36 0 0 CTC decreased 5 4 2-77 4 2-49 Conclusions: CTC analysis is regarded as a promising marker for monitoring treatment effect in metastatic breast cancer. The utility of CTC in monitoring treatment response and residual disease in the current study will become more clear when the study is completed and the patient outcome is known. Disclosure: All authors have declared no conflicts of interest.

Toll-like receptor (TLR) expression of immune system cells from metastatic breast cancer patients with circulating tumor cells

The risk posed by breast cancer represents a complex interaction among factors affecting tumor immunity of the host. Toll-like receptors (TLRs) are members of the innate immune system and generally function to attract host immune cells upon activation. However, the good intentions of TLRs are sometimes not transferred to positive long-term effects, due to their involvement in exacerbating inflammatory effects and even contributing to continued inflammation. Chronic inflammatory states are considered to favor an increased predisposition to cancer, with continuous activation of inflammatory cytokines and other hallmarks of inflammation exerting a deleterious effect. Circulating tumor cells (CTCs) are neoplastic cells present in the peripheral blood circulation that have been found to be an indicator of disease progression and long-term survival. In the present study, we examined the expression of TLRs on dendritic cells, which play a major role in eliciting anti-tumor immunity, in metastatic breast cancer patients with CTCs. Flow cytometric data showed significant differences between circulating tumor cell (CTC) positive patients and CTC negative patients in their expression of TLR2 by CD8 positive cytotoxic T cells and TLR2, TLR4, TLR3, and TLR8 by CD11c positive dendritic cells (p<0.05). Expression of TLR2, TLR4, and TLR8 was increased in CTC positive patients, whereas TLR3 expression was decreased in the dendritic cell population.

Abstract P2-01-02: Circulating Tumor Cells (CTC) may Express HER2/neu in Patients With Early HER2/neu Negative Breast Cancer – Results of the German SUCCESS C Trial

Abstract Background: There is growing evidence that the HER2/neu-status of distant metastases or minimal residual disease in blood and bone marrow may differ from the primary tumor in patients with breast cancer. The HER2/neu-status of CTCs was prospectively evaluated in patients with HER2/neu negative primary breast cancer randomized into the German multicenter SUCCESS C study. Methods: The SUCCESS C trial is a randomized Phase III study comparing FEC-Docetaxel (FEC-Doc) vs. Docetaxel-Cyclophosphamid (DC) as well as 2 years of a lifestyle-intervention in patients with early, HER2/neu negative, node positive or high-risk node negative primary breast cancer. As part of the translational research program, 23ml peripheral blood were drawn after adjuvant chemotherapy. In 505 samples, the prevalence of CTCs and their HER2/neu-status were assessed 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. A fluorescein conjugate antibody with anti-CK-Fluorescein Isothiocyanate (FITC) was used for HER2/neu phenotyping. The cut-off for CTC-positivity was ≥ 1 CTC and for HER2/neu ≥ 1 CTC with strong HER2/neu-staining (+++). Results: 26,9% of pts (n = 136) were positive for CTCs (mean 1.78; range 1–7; median = 1). The number of detected CTC was distributed as follows: 1 CTC (n = 76; 55.9%), 2 CTCs (n = 35; 25.7%), 3 CTCs (n = 13; 9.6%), 4 CTCs (n = 7; 5.2%) and ≥ 5 CTCs (n = 5; 3.7%). HER2/neu staning of CTCs was not detectable or weak in 26.5% (n = 36) and 4.4% (n = 6) of CTC positive patients respectively and therefore categorized as HER2/neu negative. In 32.4% of the CTC-positive patients (n = 44), we detected moderate and in 36.8% (n = 50) strong HER2/neu-staining of ≥ 1 CTC per sample. No association was found between CTCs or the HER2/neu-status of CTCs with tumor size, histopathological grading, hormone receptor status or axillary lymph node involvement. Conclusions: The data of this trial confirm previous findings that patients with HER2/neu negative primary breast cancer can show HER2/neu positive minimal residual disease. These results underline the importance of frequent HER2/neu determination during follow up and disease progression. Survival data within the Success C trial will give further insight into the tumor biology of HER2/neu negative disease. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-01-02.

HER2 expression on circulating tumor cells (CTC) in patients with early HER2-positive breast cancer: Results of the German SUCCESS B trial.

10540 Background: Recent reports showed a discrepancy in the Her2-status of metastases or minimal residual disease in blood and bone marrow compared to the primary tumor in patients with breast cancer. The Her2-status of CTC was prospectively evaluated in the German multicenter SUCCESS B study. Methods: The SUCCESS B trial is a randomized Phase III study comparing FEC-Docetaxel (Doc) vs. FEC-Docetaxel-Gemcitabine (Doc-G) as well as Her2 targeted therapy with Trastuzumab as adjuvant treatment in patients with early, Her2 positive, node positive or high risk node negative primary breast cancer. As part of the translational research program, 23ml of peripheral blood were drawn before adjuvant chemotherapy. In 638 samples CTC and Her2-status were assessed using the CellSearch System (Veridex, USA). After immunomagnetic enrichment with an anti-Epcam-antibody, cells were labeled with anti-CK8/18/19, anti-CD45 antibodies as well as a fluorescein conjugate antibody for Her2 phenotyping. Cutoff for CTC-positivity was ≥1 CTC and for Her2 ≥1 CTC with strong Her2-staining (+++). Results: 40.2% of pts (n=257) were positive for CTC (mean 4.52; range 0-1689). The number of detected CTC was distributed as follows: 1 CTC (n=112; 43.6%), 2 CTC (n=65; 25.3%), 3 CTC (n=36; 14.0%), 4 CTC (n=12; 4.7%) and ≥5 CTC (n=31; 12.1%). Her2 status on CTC was negative or weak in 12.5% (n=32) and 8.9% (n=23) of CTC positive patients respectively and therefore categorized as Her2 negative. In 21.4% (n=55) we detected moderate and in 57.2% (n=147) strong Her2-staining of ≥1 CTC per sample. Therefore 57.2% of CTC-positive samples were diagnosed as Her2 positive and 21.4% as questionable. No association was found between the detection of CTC or the Her2-status on CTC with tumor size, histopathological grading, hormone receptor status or axillary lymph node involvement. Conclusions: The data of this trial confirms the frequent discordance of Her2 expression on CTC compared to the primary tumor. Survival data within the Success B trial will give further insight into the tumor biology of Her2 positive disease and the role of the Her2-status on CTC to predict treatment efficacy.

38P Evolution of Estrogen Receptor (ER) and ErbB2 Expression Between Primary Tumor and Circulating Tumor Cells (CTC) of Metastatic Breast Cancer Patients

ABSTRACT Several trials have shown the prognostic relevance of circulating tumor cells (CTC) in early and metastatic breast cancers. CTC, and consequently the metastatic cells, may have a very different receptor status than the related primary tumors. Nonetheless hormone therapy and anti-ErbB2 therapy are prescribed according to the hormone status (ER/PR protein expression) and ErbB2 expression of the initial tumor. Consequently, it is of the highest relevance to analyze ER and ErbB2 expression in the CTC. We tested the CTC presence (CK positivity) and ER and ErbB2 expressions on blood samples (cytospins) collected from 21 metastatic breast cancer patients. A triple fluorescence protocol was used to visualize CK cytokeratin (8/18, 8/19) in green (DyeLight488 conjugate), ER in red (cy3 conjugate) and ErbB2 in blue (AMCA conjugate). We were able to identify more than one third CTC positive patients with the possibility of dramatic changes between primary tumor status and CTC status. One patient which had an ER-positive and erbB2-negative primary tumor exhibited simultaneously the 4 possibilities of receptor expression (ER-pos/erbB2-neg plus ER-pos/erbB2-pos, ER-neg/erbB2-neg and ER-neg/erbB2-pos). The ER-negative and erbB2-negative CTC may direct towards tamoxifen and traztuzumab resistances and have to be identified among other ER-positive and ErbB2 positive cells. We demonstrated that the individual assessment of ER and ErbB2 expression in each CTC outlines the heterogeneity of the CTC and the extreme changes of their receptor status compared to the primary tumor status. Large multicentric cohorts of breast cancer patients treated with adjuvant therapy should now be analyzed for both ER and ErbB2 status of the CTC and of the primary tumors. Such analysis should lead to optimal individualized treatments. Disclosure All authors have declared no conflicts of interest.

37P HER2 Tumor Heterogeneity and Discrepancies in HER2 Status Between Primary Tumor and Corresponding Circulating Tumor Cells in Metastatic Breast Cancer Patients

ABSTRACT Introduction Presence of circulating tumor cells (CTCs) in peripheral blood of patients with metastatic breast cancer (MBC) represents an independent prognostic factor. Discrepancies in HER2 status between primary breast tumors and CTCs have been reported. Indeed, 5-38% of patients with HER2 negative (HER2-) primary breast cancers show HER2 overexpression in corresponding CTCs at time of metastatic disease. However, the biological explanation of such discrepancies is still matter of debate. Sparse reports indicate that 5-30% of breast cancers show HER2 heterogeneity. We hypothesize that heterogeneity of HER2 expression within HER2- primary tumors might explain the presence of HER2 positive (HER2+) CTCs. Methods We analyzed 14 CTC positive (≥ 2 CTCs/7.5ml blood) MBC patients whose primary tumors were HER2- according to the adjuvant trastuzumab trials criteria. CTCs were evaluated for HER2 expression by immunofluorescence (CellSearch™ System, Veridex LLC) and defined positive if ≥ 50% cells stained for HER2. According to these criteria, seven of the patients had discordant HER2+ CTCs (cases), while seven had concordant HER2- CTCs (controls). For both cases and controls, we evaluated HER2 status by immunohistochemistry on additional tumor-bearing blocks, either from the primary tumor or from synchronous metastatic axillary lymphnodes (average of 3 blocks/patient). HER2 heterogeneity was defined as presence of incomplete or complete membrane staining in Results Five of the seven (71%) discordant cases showed HER2 heterogeneity. Heterogeneity was present either in the additional primary tumor blocks (40%) or in the metastatic axillary lymphnodes (40%) or in both (20%). Intriguingly, none of the concordant cases (controls) showed HER2 heterogeneity. Conclusion Here we show that areas of HER2 expression in HER2- tumors are associated with the presence of HER2+ CTCs at time of metastatic disease, suggesting that HER2+ clones might be selected during cancer progression. Trials investigating whether patients with HER2 heterogeneous tumors might benefit from HER2 targeted therapies are warranted. Disclosure A. Di Leo: Dr. Di Leo received research grants from GSK and honoraria as a speaker at satellite symposia or as a consultant from Roche and GSK. All other authors have declared no conflicts of interest.

Abstract 2386: Triple fluorescence analysis of estrogen receptor alpha (ER) and ErbB2 status of circulating tumor cells (CTC) in breast cancer: from cell models to metastatic patients

Abstract Purpose: Hormone therapy and anti-ErbB2 (HER-2) therapy are prescribed according to the hormone status (ER/PR protein expression) and ErbB2 expression of the initial tumor. Several trials have shown the prognostic relevance of circulating tumor cells (CTC) in early and metastatic breast cancers. As it appears that the CTC, and consequently the metastatic cells, may have a very different receptor status than the related primary tumors, it is of the highest relevance to analyze ER and ErbB2 expression of the CTC. Experimental procedures: We developed a triple fluorescence technique to visualize simultaneously cytokeratin (8/18, 8/19), ER and ErbB2 on each individual cell. Using this protocol, we analyzed ER and ErbB2 expression in various breast cancer cell lines (MCF-7, T47D, Cama-1, ZR75, SK-Br-3, HCC 3153, MDA-MB-231), and compared ER expression in the different cell lines using also immunostaining. We were then able to test the CTC presence (CK positivity) and ER and ErbB2 expressions on blood samples (cytospins) collected from 15 metastatic breast cancer patients. Results: The 2-step triple fluorescence protocol was optimized to visualize CK in green (DyLight488 conjugate), ER in red (cy3 conjugate) and ErbB2 in blue (AMCA conjugate). Using in parallel this triple fluorescence protocol and the reference immunostaining technique, we could analyze that ER-positive cell lines express very different levels of ER and within one cell line, cells can express low, moderate and/or high levels of ER. The identification of cells that express a very low level of ER is compulsory as the CTC from one unique patient can potentially express differently ER. Consequently, ER-positivity and -negativity have to be clearly defined. CTC analysis from patient blood samples was then performed with an individual assessment, for each single isolated cell, of cytokeratin (epithelial marker that peripheral blood cells should not express) and of ER and ErbB2 expressions. We were able to identify, among the CTC positive patients, changes between primary tumor status and CTC status, e.g. ER-positive primary tumors with both ER-positive and ER-negative CTC. Those ER-negative CTC may direct towards tamoxifen resistance and have to be identified among other ER-positive cells, similarly as any ErbB2 negative CTC. Conclusion: We demonstrated that triple immunofluorescence is very relevant, first to identify CTC, and then to allow an individual assessment of ER and ErbB2 expression, at the protein level. Moreover, the approach we developed allows to characterize a population of CTC with a very low expression of ER. Large multicentric cohorts of breast cancer patients treated with adjuvant therapy should now be analyzed for both ER and ErbB2 status of the CTC, compared to the primary tumor profile. The expected results should lead to optimal individualized treatments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2386. doi:1538-7445.AM2012-2386