HER2-positive Circulating Tumor Cells Research Articles

Abstract PO3-16-02: Trastuzumab and taxane chemotherapy in the first-line in MBC patients with a HER2-negative primary tumor and HER2-positive circulating tumor cells: a phase II trial

Abstract Purpose: HER2 overexpressing circulating tumor cells (CTCs) are observed in up to 20% of HER2-negative metastatic breast cancer patients. Since targeted anti-HER2 therapy has drastically increased the outcome of patients with HER2 positive breast cancer, we hypothesized that patients with HER2 overexpressing CTCs might benefit from the addition of trastuzumab to chemotherapy. Methods: In this single-arm, phase II trial, HER2 negative patients with HER2-positive CTCs received trastuzumab as addition to first-line treatment with taxane chemotherapy (trastuzumab group). Patients with detectable CTCs but without HER2 overexpression and that received taxane chemotherapy only, were used as internal control group (taxane monotherapy group). The primary outcome measure was progression-free rate at 6 months (PFR6). The study was powered to reach a PFR6 of 80%; in this case this strategy was considered worth further investigation. In November 2022, the study was terminated early due to slow patient accrual. Results: 63 HER2 negative patients with metastatic disease were screened, of which 8 patients had HER2-positive CTCs and were treated in the first line with trastuzumab in addition a taxane. Patients receiving taxane monotherapy were used as a control group (n = 27). The median number of CTCs was 15 (range 1 – 131) in patients with HER2-positive CTCs, compared to median 5 (range 1-1047) in the control group. PFR6 was 50% in the trastuzumab group and 64% in the taxane monotherapy group, with no significant difference in median PFS (8 versus 9 months, p=0.51). Excluding the patients that had no measurable CTCs, there was a borderline significant difference between median CTC count in the patients that did and that did not have HER2 positive CTCs (p=0.05). We confirmed a significant higher CTC-count in samples that had at least one HER2-postive CTC in an independent patient set (n=233, p< 0.001). Conclusions: Although this study was performed in a limited number of patients, no clinical benefit of trastuzumab was observed. Firstly, we conclude that our strategy was not feasible for clinical implementation due to high numbers needed to screen. And, second, due to the strong correlation between CTC numbers and detected CTC HER2 positivity, the prognostic value of the CTC numbers precludes judgement of any predictive value of the HER2 status of CTCs. Citation Format: Noortje Verschoor, Manouk Bos, Inge de Kruijff, Ngoc M Van, Jaco Kraan, Jan Drooger, Johanna Zuetenhorst, Saskia M Wilting, Stefan Sleijfer, Agnes Jager, John WM Martens. Trastuzumab and taxane chemotherapy in the first-line in MBC patients with a HER2-negative primary tumor and HER2-positive circulating tumor cells: a phase II trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-16-02.

Trastuzumab and first-line taxane chemotherapy in metastatic breast cancer patients with a HER2-negative tumor and HER2-positive circulating tumor cells: a phase II trial

PurposeHER2 overexpressing circulating tumor cells (CTCs) are observed in up to 25% of HER2-negative metastatic breast cancer patients. Since targeted anti-HER2 therapy has drastically improved clinical outcomes of patients with HER2-positive breast cancer, we hypothesized that patients with HER2 overexpressing CTCs might benefit from the addition of trastuzumab to chemotherapy.MethodsIn this single-arm, phase II trial, patients with HER2-positive CTCs received trastuzumab as addition to first-line treatment with taxane chemotherapy. Patients with detectable CTCs but without HER2 overexpression that received taxane chemotherapy only, were used as control group. The primary outcome measure was progression-free rate at 6 months (PFR6), with a target of 80%. In November 2022, the study was terminated early due to slow patient accrual.Results63 patients were screened, of which eight patients had HER2-positive CTCs and were treated with trastuzumab. The median number of CTCs was 15 per 7.5 ml of blood (range 1–131) in patients with HER2-positive CTCs, compared to median 5 (range 1–1047) in the control group. PFR6 was 50% in the trastuzumab group and 54% in the taxane monotherapy group, with no significant difference in median PFS (8 versus 9 months, p = 0.51).ConclusionNo clinical benefit of trastuzumab was observed, although this study was performed in a limited number of patients. Additionally, we observed a strong correlation between the number of evaluable CTCs and the presence of HER2-positive CTCs. We argue that randomized studies investigating agents that are proven to be solely effective in the HER2-positive patient group in patients with HER2-positive CTCs and HER2-negative tissue are currently infeasible. Several factors contribute to this impracticality, including the need for more stringent thresholds, and the rapidly evolving landscape of cancer treatments.

A highly sensitive nanobiosensor based on aptamer-conjugated graphene-decorated rhodium nanoparticles for detection of HER2-positive circulating tumor cells

AbstractAssessment of human epidermal growth factor receptor-2 (HER2) tumor marker status is an impressive factor in screening, diagnosing and monitoring breast cancer (BC). The electrochemical biosensor is a revolutionary method in cancer diagnosis, which is used in this research to detect HER2+circulating tumor cells. The electrochemical activity, size, shape, and morphology of the synthesized nanomaterials were analyzed. The hybrid nanocomposite established by the coupling of reduced graphene oxide nanosheets (rGONs) and rhodium nanoparticles (Rh-NPs) on the surface of graphite electrode resulted in improved surface area, electrochemical activity, and biocompatibility. The graphite electrode-based aptasensor (g-aptasensor) demonstrated exceptional performance against HER2-overexpressed SKBR3 cancer cells, with a linear dynamic range of 5.0 to 10.0 × 104 cells/mL, an analytical limit of detection (LOD) as low as 1.0 cell/mL, and a limit of quantification (LOQ) of 3.0 cells/mL. The G-rich DNA aptamers can fold into an intermolecular G-quadruplex, which specifically bind to the target molecule. Consequently, the advantages of this highly efficient nanocomposite platform include broad dynamic range, high specificity, selectivity, stability, reproducibility, and low cost. These characteristics indicate that the fabricated nanobiosensor has a high potential for use in detecting and monitoring HER2 level for the care of BC patients and clinical diagnosis.

Her2 Expression in Circulating Tumor Cells Is Associated with Poor Outcomes in Patients with Metastatic Castration-Resistant Prostate Cancer.

HER2-dependent signaling may support the development of metastatic castration-resistant prostate cancer (mCRPC) by activating androgen receptor signaling through ligand-independent mechanisms. From 41 mCRPC patients (including 31 treated with Androgen Receptor Signaling Inhibitors [ARSI]), Circulating Tumor Cells (CTCs) were prospectively enriched with AdnaTest platform and analyzed with a multiplexed assay for HER2 and AR-V7 mRNA expression. Then, we evaluated the impact of HER2 expression on PSA-response, Progression Free Survival (PFS) and Overall Survival (OS). HER2 expression was detected in CTCs of 26 patients (63%). Although PSA response was similar regardless of HER2 status, patients with HER2 positive CTCs had shorter PSA-PFS (median: 6.2 months versus 13.0 months, p = 0.034) and radiological-PFS (6.8 months versus 25.6 months, p = 0.022) than patients without HER2 expression. HER2 expression was also associated with a shorter OS (22.7 months versus not reached, p = 0.05). In patients treated with ARSI, multivariate analyses revealed that the prognostic impact of HER2 status on PSA-PFS was independent of AR-V7 expression and of the detection of CTCs by an AdnaTest. We showed for the first time the poor prognostic value of HER2 expression in CTCs from patients with mCRPC. The therapeutic interest of targeting this actionable pathway remains to be explored.

MXene-based cytosensor for the detection of HER2-positive cancer cells using CoFe2O4@Ag magnetic nanohybrids conjugated to the HB5 aptamer

MXenes are a new class of conductive two-dimensional material which have received growing attention in biosensing for their significant surface area and unique surface chemistry. Here, gold electrodes were modified with MXene nanosheets of about 2 nm thickness and 1.5 μm lateral size for the electrochemical detection of tumor cells. An HB5 aptamer with high selectivity for HER-2 positive cells was immobilized on the MXene layers via electrostatic interactions. To minimize electrode biofouling with blood matrix, magnetic separation of HER-2 positive circulating tumor cells was carried out using CoFe2O4@Ag magnetic nanohybrids bonded to the HB5. The formation of sandwich-like structures between the magnetically captured cells and the functionalized MXene electrodes effectively shields the electron transfer of a redox probe, enabling quantitative cell detection using the change in current. This label-free MXene-based cytosensor platform yielded a wide linear range of 102-106 cells/mL, low detection limit of 47 cells/mL, and good sensitivity and selectivity in the detection of HER2-posetive cells in blood samples. The presented aptacytosensor demonstrates the great potential of using CoFe2O4@Ag magnetic nanohybrids and MXenes to monitor cancer progression via circulating tumor cells in blood at low cost.

Clinical significance of circulating tumor cells in the response to trastuzumab for HER2-negative metastatic gastric cancer.

The prognosis of metastatic gastric cancer has improved due to trastuzumab in patients with HER2 positive. Circulating tumor cells (CTCs) have been examined as a prognostic predictor in gastric cancer. The clinical advantage of trastuzumab was examined in gastric cancer patients with HER2-negative tumor tissues and HER2-positive CTCs. A total of 105 patients with metastatic or recurrence gastric cancer were enrolled. All patients were examined HER2 expression in CTC using the CellSearch system in blood specimens. CTCs were detected in 65 of 105 patients (61.9%) and 61 patients were divided into three groups: Group A (n = 27), histological HER2-positive; Group B (n = 17), histological HER2-negative and HER2-positive CTCs; and Group C (n = 17), HER2-negative on histology and CTCs. Patients received capecitabine plus cisplatin. Groups A and B were additionally treated by trastuzumab. There was no relationship between tumor tissues and CTCs in HER2 expression. Even if group B had no histological HER2 expression, group B showed a good prognosis as same as group A, and group C had a significantly worse overall survival than groups A and B. The multivariate analysis demonstrated that HER2-expression on CTCs was an independent prognostic factor for both overall and progression-free survival. The present results indicate the potential clinical utility of trastuzumab combined chemotherapy in patients with HER2-positive CTCs even if they are histologically HER2-negative.

Abstract PD3-12: Efficacy of the tyrosine kinase inhibitor lapatinib in the treatment of patients with HER2-negative metastatic breast cancer and HER2-positive circulating tumor cells - results from the randomized phase III DETECT III trial

Abstract Background: It is well-known that tumor biology may change during the course of the disease due to clonal evolution, and such changes might have important implications for response to targeted treatments. Circulating tumor cells (CTCs) could serve as a real-time liquid biopsy to detect changes in tumor biology. It has been demonstrated that patients with HER2-negative metastatic breast cancer (MBC) may have discordant, HER2-positive CTCs in the peripheral blood. However, up to now there is no randomized clinical trial investigating whether treatment decisions based on CTC phenotype provide benefits in terms of improved outcome. The aim of the DETECT III study is to investigate whether patients with initially HER2-negative MBC and HER2-positive CTCs benefit from HER2-targeted therapy with the tyrosine kinase inhibitor lapatinib. In addition, the significance of CTCs as an early predictive marker for response to therapy will be analyzed. Methods: The randomized phase III DETECT III trial (NCT01619111) compares lapatinib in combination with standard therapy versus standard therapy alone in patients with initially HER2-negative MBC and HER2-positive CTCs. Efficacy of lapatinib treatment is evaluated by CTC clearance rate, progression-free survival (PFS) and overall survival (OS). In addition, we investigate the association between CTC results and both PFS and OS to assess the utility of CTCs as an early predictive marker for treatment response. CTC enumeration and phenotyping was performed using the CellSearch® technology (Menarini Silicon Biosystems; Bologna, Italy). Survival data are analyzed using log rank tests, univariable and adjusted multivariable cox regressions. Results: First results on CTC clearance rates, PFS and OS of 105 prospectively randomized patients will be presented. Conclusion: This first randomized clinical trial in breast cancer patients with treatment decisions being based on the phenotype of CTCs will show whether patients with HER2 negative MBC and HER2 positive CTCs benefit from additional HER2-targeted therapy with lapatinib. This finding might be increasingly important as novel HER2-targeted drugs become available. Citation Format: Tanja Fehm, Volkmar Mueller, Maggie Banys-Paluchowski, Peter A Fasching, Thomas WP Friedl, Andreas Hartkopf, Jens Huober, Christian Loehberg, Brigitte Rack, Sabine Riethdorf, Andreas Schneeweiss, Diethelm Wallwiener, Franziska Meier-Stiegen, Oliver Hoffmann, Lothar Müller, Pauline Wimberger, Eugen Ruckhaeberle, Jens Blohmer, Wolfgang Janni. Efficacy of the tyrosine kinase inhibitor lapatinib in the treatment of patients with HER2-negative metastatic breast cancer and HER2-positive circulating tumor cells - results from the randomized phase III DETECT III trial [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD3-12.

Abstract PS2-11: Ctc-her2+ a novel subset in stage IVaggressive: Molecular correlations, outcome and clinical characteristics in metastatic breast cancer

Abstract Background The enumeration of Circulating Tumor Cells (CTCs) in the peripheral blood of patients (pts) with metastatic breast cancer (MBC) showed prognostic value independent of clinic-pathological characteristics and proposed as a new tool for Stage IV stratification. Molecular characterization of CTCs with analysis of standard HER2 and estrogen-receptor (ER) status provides additional predictive value. The presence of HER2 positive CTCs (CTCs-HER2+) has been documented in pts with HER2 negative MBC and suggestive of tumor heterogeneity. The molecular features associated with the detection of these cells and the clinical significance of CTCs-HER2+ remains of unclear clinical significance. The lack of validated prospective trials limits the use of this immunophenotypic characterization as a prognostic and predictive factor. We conducted an analysis of a large cohort of patients with MBC evaluated for CTCs enumeration (CTCs+), molecular analysis of CTCs (CTCs-HER2+) and ctDNA. Methods The IRB-approved study prospectively analyzed blood samples of pts with MBC enrolled before starting a new line of therapy. Samples were collected from pts treated at Northwestern University (Chicago, IL) between 2016 and 2020. ctDNA was analyzed using the Guardant360 NGS assay (Guardant Health) with a specific focus on the detection of ERBB2 mutations. CTCs were enumerated through CellSearch™ (Menarini Silicon Biosystems, Bologna, Italy), and characterized for HER2 expression using the CellSearch CXC Kit. HER2+ CTCs were divided in 4 different categories (0,1+,2+,3+), leaning on fluorescence intensity, as previously reported by Riethdorf et al. Pts with <5 CTCs (stage IV indolent ) were excluded from the analysis. The cHER2 ratio, defined as the sum of 2+ CTCs and 3+ CTCs divided by the total number of HER2+ CTCs was used to split the remaining pts in 2 different subgroups: cHER2 ratio> 0.75 (cHER2high), cHER2 ratio<0.75 (cHER2low). Pts' characteristics and genomic alterations were compared between these two subgroups and a long-rank test was performed to analyze the outcome (progression-free survival, PFS). Results A total of 247 pts with MBC about to start new therapy were enrolled. 136 patients (55%) demonstrated CTCs count ≥ 5 (stage IV aggressive). Of these, 111 pts had a blood sample bearing HER2+ CTCs. 88 pts were classified as cHER2low, while 32 were allocated in the second subgroup (cHER2high). The clinic subtype distribution of the two groups was as follows: 65% ER+, 19% HER2+ and 16% TNBC among cHER2low, compared to 55% ER+, 38% HER2+ and 7% TNBC among cHER2high pts. The HER2+ subtype was significantly more represented in the cHER2high group (p<0.05), along with ERBB2 mutations (p<0.05). We observed a total of 89 events (progression to treatment), 67 and 22 respectively. The median PFS was 87 days in the cHER2low and 209 days among cHER2high pts (log Rank test, p<0.001). Conclusion This study shows a significant association between the cHER2 ratio and outcome in pts with Stage IV aggressive disease, suggesting a potential predictive/prognostic role of this biomarker among MBC pts bearing CTC-HER2+. cHER2high pts, in which blood was collected a prevalent number of CTC-HER2+ 2+ and 3+, were more likely to have a HER2 FISH amplification by tissue biopsy and ERBB2 gene alteration by ctDNA. These findings, taken together, suggest a potential role of these subsets of CTC-HER2+ in the characterization of the HER2+ metastatic disease. The cHER2low is intrinsically characterized by a higher percentage of CTC-HER2+ 1+. This CTC-HER2+ subset of cells is commonly detected among all subtypes but, more frequently in ER+ disease and their significance remains unclear. Exploring the underlying biological nature of the CTC-HER2+ 1+ could allow to better understand the role these cells in the natural evolution of the MBC. Citation Format: Paolo D'Amico, Qiang Zhang, Lorenzo Gerratana, Youbin Zhang, Ami Shah, Carolina Reduzzi, Andrew Davis, Saya Jacob, Firas Wehbe, Amir Behdad, Giuseppe Curigliano, Massimo Cristofanilli. Ctc-her2+ a novel subset in stage IVaggressive: Molecular correlations, outcome and clinical characteristics in metastatic breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS2-11.

HER2-targeted therapy influences CTC status in metastatic breast cancer

PurposeAs an independent, negative-prognostic biomarker for progression-free survival (PFS) and overall survival (OS), circulating tumor cells (CTCs) constitute a promising component for developing a liquid biopsy for patients with metastatic breast cancer (MBC). The effects of HER2-targeted therapy such as trastuzumab, pertuzumab, T-DM1, and lapatinib on CTC status and longitudinal enumeration were assessed in this trial.MethodsCTC status of 264 patients with MBC was analyzed prior to and after 4 weeks of a new line of palliative systemic therapy. CTCs were assessed using CellSearch®. Three groups were compared: patients with HER2-positive MBC receiving ongoing HER2-targeted therapy (n = 28), patients with de novo HER2-positive MBC and no HER2-targeted therapy in the last 12 months prior to enrollment and start of HER2-targeted therapy (n = 15), and patients with HER2-nonamplified disease and no HER2-targeted therapy (n = 212).ResultsPositive CTC status (≥ 5 CTC/7.5 ml blood) at enrollment was observed in the 3 groups for 17.9, 46.7, and 46.2% (p = 0.02) of patients, respectively. At least one CTC/7.5 ml was seen in 28.6, 53.3, and 67.0% (p < 0.001) of these patients. Furthermore, 3.6, 40.0, and 3.3% (p < 0.001) of the patients had at least one HER2-positive CTC. After 4 weeks of therapy 7.1, 0.0, and 31.1% (p = 0.001) of patients had still a positive CTC status (≥ 5 CTC/7.5 ml blood). At least one CTC/7.5 ml was still observed in 25.0, 20.0, and 50.5% (p = 0.004) of the patients. Furthermore, 7.1, 0.0, and 1.9% (p = 0.187) had at least one HER2-positive CTC. After 3 months of therapy, 35.7, 20.0, and 28.3% (p = 0.536) showed disease progression.ConclusionsHER2-targeted therapy seems to reduce the overall CTC count in patients with MBC. This should be taken into account when CTC status is used as an indicator for aggressive or indolent metastatic tumor disease.

Evaluation of HER2 expression in urothelial carcinoma cells as a biomarker for circulating tumor cells.

Detection of circulating tumor cells (CTC) by techniques based on epithelial cell adhesion molecule (EpCAM) is suboptimal in urothelial carcinoma (UC). As HER2 is thought to be broadly expressed in UC, we explored its utility for CTC detection. HER2 and EpCAM expression was analyzed in 18 UC cell lines (UCCs) by qRT-PCR, western blot and fluorescence-activated cell scanning (FACS) and compared to the strongly HER2-expressing breast cancer cell line SKBR3 and other controls. HER2 expression in UC patient tissues was measured by qRT PCR and correlated with data on survival and risk for metastasis. UCCs with high EpCAM and variable HER2 expression were used for spike-in experiments in the CellSearch system. Twenty-one blood samples from 13 metastatic UC patients were analyzed for HER2-positive CTCs with CellSearch. HER2 mRNA and protein were broadly expressed in UCC, with some heterogeneity, but at least 10-fold lower than in the HER-2+ SKBR3 cells. Variations were unrelated to cellular phenotype or clinicopathological characteristics. EpCAM expression was essentially restricted to UCCs with epitheloid phenotypes. Heterogeneity of EpCAM and HER2 expression was observed also in spike-in experiments. The 7 of 21 blood samples from 6 of 13 patients were enumerated as CTC positive via EpCAM, but only one sample stained weakly positive (1+) for HER2. Detection rate of CTCs by EpCAM in UC is poor, even in metastatic patients. Because of its widespread expression, particularly in patients with high risk of metastasis, detection of HER2 could improve identification of UC CTCs, which is why combined detection using antibodies for EpCAM and HER2 may be beneficial.

Abstract OT3-18-01: Detect III/IV study trial - The multicenter study program in patients with HER2-negative MBC and circulating tumor cells

Abstract Background: The collaborative DETECT study program represents a large program on metastatic breast cancer (MBC) worldwide. The main objective of the DETECT trial is to evaluate the efficacy of individualized breast cancer treatment based on the presence and phenotype setting of circulating tumor cells (CTCs). Thus, the DETECT study program is one of the first clinical trials translating the role of CTC enumeration/phenotyping directly into treatment intervention within different breast cancer subtypes. Trial design: The DETECT-III trial is a multicenter, randomized, phase III study comparing standard therapy alone versus standard therapy plus lapatinib in patients with initially HER2-negative MBC and HER2-positive CTCs. Patients with HER2-negative MBC and only HER2-negative CTCs can be included within the DETECT-IV trial, a prospective, multicenter, open-label, phase II study including patients with HER2-negative MBC. Within the DETECT-IV study setting postmenopausal patients with hormone-receptor positive MBC are treated with the CDK 4/6 inhibitor ribociclib and endocrine therapy, while women with triple negative MBC or a hormone-receptor positive tumor and indication for chemotherapy will receive eribulin. Specific aims: The DETECT study program offers various up-to-date treatment options, generating a wealth of clinical data including long-term follow-up data, evaluated in the controlled setting of a single large clinical trial program. The primary endpoint of the DETECT III trial is the comparison of efficacy as assessed using CTC clearance rate between patients receiving standard anticancer therapy with lapatinib and patients receiving standard anticancer therapy alone. The secondary objective of this trial is to assess the level of compliance to study procedures comparing the efficacy of lapatinib between given treatment groups. The primary objectives of the DETECT- IV trial are to evaluate CTC clearance rate for the ribociclib cohort and progression-free survival (defined as time interval from date of recruitment until progressive disease) for the eribulin cohort. The main focal point of the extensive collaborative translational oncology research projects is to apply innovative biomarkers and assays focusing on molecular characteristics of CTCs. This “biological status” of CTCs may provide new information about their potential function in terms of a repeatedly performed liquid biopsy to assess the patients’ response to therapy. Citation Format: Sabrina Krause, Thomas WP Friedl, Peter A Fasching, Andreas Schneeweiss, Volkmar Müller, Sabine Riethdorf, Klaus Pantel, Florin-Andrei Taran, Arkadius Polasik, Marie Tzschaschel, Amelie de Gregorio, Franziska Meier-Stiegen, Jens Huober, Wolfgang Janni, Tanja Fehm. Detect III/IV study trial - The multicenter study program in patients with HER2-negative MBC and circulating tumor cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr OT3-18-01.

Nuclear-Biased DUSP6 Expression is Associated with Cancer Spreading Including Brain Metastasis in Triple-Negative Breast Cancer.

DUSP6 is a dual-specificity phosphatase (DUSP) involved in breast cancer progression, recurrence, and metastasis. DUSP6 is predominantly cytoplasmic in HER2+ primary breast cancer cells, but the expression and subcellular localization of DUSPs, especially DUSP6, in HER2-positive circulating tumor cells (CTCs) is unknown. Here we used the DEPArray system to identify and isolate CTCs from metastatic triple negative breast cancer (TNBC) patients and performed single-cell NanoString analysis to quantify cancer pathway gene expression in HER2-positive and HER2-negative CTC populations. All TNBC patients contained HER2-positive CTCs. HER2-positive CTCs were associated with increased ERK1/ERK2 expression, which are direct DUSP6 targets. DUSP6 protein expression was predominantly nuclear in breast CTCs and the brain metastases but not pleura or lung metastases of TNBC patients. Therefore, nuclear DUSP6 may play a role in the association with cancer spreading in TNBC patients, including brain metastasis.

The Potential Clinical Implications of Circulating Tumor Cells and Circulating Tumor Microemboli in Gastric Cancer.

Gastric adenocarcinoma (GAC) is the third deadliest malignant neoplasm worldwide, mostly because of late disease diagnosis, low chemotherapy response rates, and an overall lack of tumor biology understanding. Therefore, tools for prognosis and prediction of treatment response are needed. Quantification of circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) and their expression of biomarkers has potential clinical relevance. Our aim was to evaluate CTCs and CTM and their expression of HER2 and plakoglobin in patients with nonmetastatic GAC, correlating the findings to clinicopathological data. CTC enrichment was performed with isolation by size of epithelial tumor cells, and the analysis was performed with immunocytochemistry and microscopy. Two collections were made: one at diagnosis (55 samples before neoadjuvant treatment) and one after surgery and before adjuvant therapy (33 samples). A high detection rate of CTCs (90%) was observed at baseline. We evaluated HER2 expression in 45/55 biopsy samples and in 42/55 CTC samples, with an overlap of 36 subjects. Besides the good agreement observed for HER2 expression in primary tumors and paired CTCs for 36 cases (69.4%; κ = 0.272), the analysis of HER2 in CTCs showed higher positivity (43%) compared with primary tumors (11%); 3/5 patients with disease progression had HER2-negative primary tumors but HER2-positive CTCs. A significant CTC count drop in follow-up was seen for CTC-HER2-positive cases (4.45 to 1.0 CTCs per mL) compared with CTC-HER2-negative cases (2.6 to 1.0 CTCs per mL). The same was observed for CTC-plakoglobin-positive cases (2.9 to 1.25 CTCs per mL). CTC analysis, including their levels, plakoglobin, and HER2 expression, appears to be a promising tool in the understanding the biology and prognosis of GAC. The analysis of circulating tumor cell levels from the blood of patients with gastric adenocarcinoma, before and after neoadjuvant treatment, is useful to better understand the behavior of the disease as well as the patients more likely to respond to treatment.

Abstract P3-01-19: HER2-positive circulating tumor cells (CTCs) in advanced breast cancer (BC): A feature independent of BC subtype

Abstract Introduction: HER2 overexpression is observed on CTCs in advanced BC (ABC), but their significance is not known. We aimed to describe clinical, pathologic, and molecular associations with HER2 overexpression on CTCs in ABC patients (pts). Methods: We conducted a retrospective analysis of data from ABC pts treated at Thomas Jefferson University and Northwestern University who had evaluation of CTCs and circulating tumor DNA (ctDNA). CTCs were enumerated with the CellSearch immunomagnetic kit (Menarini Silicon Biosystems), HER2 expression on CTCs was evaluated using the CellSearch CXC Kit, and ctDNA was analyzed using the Guardant360 NGS assay (Guardant Health). Associations with the presence of HER2+ CTCs were explored through univariate and multivariate logistic regression. Kruskal-Wallis testing evaluating HER2+ CTCs as a continuous variable was also conducted to confirm consistency of findings. Time to development of HER2+ CTCs was evaluated using Cox proportional hazards regression analysis. Results: Baseline CTCs were evaluated in 209 pts (10% stage III, 90% stage IV) of whom 41% had no detectable CTCs, 23% had 1-4 CTCs, and 36% had &amp;gt;5 CTCs (stage IV aggressive). Twelve percent had CTC clusters. At least 1 HER2+ CTC was seen in 33% of pts at baseline draw. Of 39 patients with HER2+ BC, only 18% had HER2+ CTCs. Of patients with HER2+ CTCs, 55% had hormone receptor positive BC, 28% had triple negative BC, and 18% had HER2+ BC. On univariate logistic analysis, BC subtype or HER2 status was not associated with the presence of HER2+ CTCs. IBC pts represented 52% of pts and were less likely to have HER2+ CTCs (OR 0.40 95% CI 0.19-0.84). Bone metastases were associated with an increased likelihood of HER2+ CTCs (OR 2.46, 95% CI 1.12-5.38); however, other sites of metastases and number of metastatic sites were not correlated with HER2+ CTCs. Aggressive disease features including &amp;gt;5 CTCs and presence of CTC clusters were strongly associated with HER2+ CTCs (OR 15.72, 95% CI 6.89-35.8 and 8.97, 95% CI 3.23-24.89, respectively). Of 168 pts with ctDNA analysis, ERRB2 aberrations were seen in 22% of pts and were significantly associated with HER2+ CTCs (OR of 3.74, 95% CI 1.45-9.63). On multivariate analysis, the associations with &amp;gt;5 CTCs and ERBB2 alterations in ctDNA remained statistically significant. The associations of HER2+ CTCs with bone disease, &amp;gt;5 CTCs, CTC clusters, and ERBB2 alterations in ctDNA, and the inverse relationship with IBC were consistent when HER2+ CTCs were evaluated as a continuous variable with Kruskal-Wallis testing. Among pts without HER2+ CTCs at baseline, the time to detection of HER2+ CTCs correlated with the presence of bone metastases (HR 3.40, 95% CI 1.14-10.19), &amp;gt;5 CTCs (3.77, 95% CI 1.33-10.70), and visceral disease (HR 3.00, 95% CI 1.07-8.44). Conclusions: HER2+ CTCs are common in ABC, independent of HER2 status of the tumor, and, in fact, common in the luminal BC. HER2+ CTCs were also strongly associated with CTC characteristics of aggressive disease with poor survival (CTCs clusters and &amp;gt;5 CTCs) and ERBB2 aberrations in ctDNA. Further studies will be investigating the role of HER2+ CTCs in endocrine resistance and the potential of anti-HER2 therapy in this unique CTC-defined setting. Citation Format: Shah AN, Gerratana L, Davis AA, Zhang Q, Zhang Y, Rossi G, Wang C, Strickland K, Yang H, Flaum L, Abu-Khalaf M, Behdad A, Ye Z, Platanias L, Gradishar WJ, Cristofanilli M. HER2-positive circulating tumor cells (CTCs) in advanced breast cancer (BC): A feature independent of BC subtype [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 P3-01-19.

Abstract P3-01-08: HER2-negative metastatic breast cancer with HER2-positive circulating tumor cells (CTCs): A new CTC-defined HER2-positive subgroup

Abstract Introduction: CTCs can overexpress HER2 discordant from tumor HER2 expression. We aimed to describe characteristics of a CTC-defined group of pts with metastatic breast cancer (MBC) that is tumor HER2- and CTC HER2+ (HER2 tumor- CTC+). Methods: We retrospectively analyzed data from pts treated at Northwestern University who had serial evaluation of CTCs and circulating tumor DNA (ctDNA). We included pts with pathologically confirmed HER2- MBC and HER2+ CTCs. CTCs were enumerated with the CellSearch immunomagnetic kit (Menarini Silicon Biosystems), HER2 expression on CTCs was determined using the CellSearch CXC Kit in 7.5 cc whole blood, and ctDNA was analyzed using the Guardant360 NGS assay (Guardant Health). Results: Among 98 pts with HER2- MBC and CTC analysis, 46 (47%) had at least 1 HER2+ CTC. In this cohort the median age was 53. At initial BC diagnosis, 80% had early stage or locally advanced BC and 20% had de-novo metastatic disease. Baseline histology was 65% ductal, 20% lobular, 2% mixed ductal and lobular, and 13% unknown. Pathology of metastatic tumor was hormone receptor positive (HR+)/HER2- in 78% and triple negative in 22%. Detailed HER2 immunohistochemistry (IHC) and FISH results from metastases were available from 63% of pts of whom 72% had an IHC score of 0 or 1 and 28% had an IHC score of 2 with negative FISH testing. The median time from the most recent pathologic metastatic tumor assessment to the detection of a HER2+ CTC was 6.5 mo. Twenty-two pts had simultaneous (within 8 weeks) HER2- tumor confirmation and HER2+ CTC detection. The median lines of endocrine therapy (ET) for MBC prior to detection of HER2+ CTCs was 1 (range 0-5, 41% no ET, 17% 1 line, 41% &amp;gt;2 lines). Pts received a median of 2 (range 0-10) prior systemic therapies for MBC prior to detection of HER2+ CTCs, (20% 0 lines, 41% 1-3 lines, and 39% &amp;gt;4 lines). Among these 46 pts, CTCs were analyzed longitudinally in 104 samples, with HER2+ CTCs detected in 77 samples. Number of HER2+ CTCs at initial detection ranged from &amp;lt;5 in 24%, 5-50 in 43%, and &amp;gt;50 in 33%, with a median of 11.5 HER2+ CTCs. CTC clusters were noted in 37% of pts. At initial detection the proportion of CTCs that were HER2+ was 0-25% in 13% of pts, 26-50% in 46% of pts, and 51-100% in 41% of pts. Seven pts had ERBB2 aberrations in ctDNA. Of 12 pts with tumor sequencing, 2 had ERBB2 mutations, 1 had ERBB3 amplification, and 1 had overexpression of ERBB3 RNA. After detection of HER2+ CTCs, 18 pts received HER2 directed therapy (with chemotherapy in 13 pts, with endocrine therapy in 4 pts, and as monotherapy in 1 pt). Imaging demonstrated a partial response or stable disease in 9 pts (clinical benefit rate 50%), including in 1 pt with trastuzumab monotherapy, progressive disease in 8 pts, and not evaluated in 1 pt. Conclusions: HER2+ CTCs are frequently detected simultaneously or soon after HER2- tumor assessment in MBC. Within this newly defined subgroup, the several responses seen with HER2 targeted therapy serve as a proof of concept that HER2 tumor- CTC+ patients can benefit from HER2 targeted therapy. Future studies are needed to determine a clinically relevant threshold for HER2+ CTCs to guide further study of HER2 therapy combinations in HER2 tumor- CTC+ pts. Citation Format: Shah AN, Gerratana L, Zhang Q, Davis AA, Zhang Y, Flaum L, Behdad A, Platanias L, Gradishar WJ, Cristofanilli M. HER2-negative metastatic breast cancer with HER2-positive circulating tumor cells (CTCs): A new CTC-defined HER2-positive subgroup [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 P3-01-08.

Abstract P5-17-03: How is inflammatory breast cancer (IBC) different? Integration of clinico-pathological features and circulating tumor cells (CTCs)-based biomarkers for disease and prognostic assessment

Abstract Background: Since IBC is rare and burdened by a particularly unfavorable prognosis, biomarkers able to enhance diagnosis and risk assessment are of pivotal importance and a current unmet need. The aim of this study is to integrate standard clinico-pathological features with CTCs-based biomarkers for a more objective and detailed characterization of IBC. Methods: This study analyzed retrospectively 251 Advanced Breast Cancer (BC) patients (pts) longitudinally characterized for CTCs and CTCs-based biomarkers at Thomas Jefferson University (Philadephia, PA) and Northwestern University (Chicago. IL). CTCs were enumerated through the CellSearch system (Menarini Silicon Biosystems), and characterized for HER2 expression using the CellSearch CXC Kit. Pts were defined as stage IV aggressive based on the previously reported ≥5 CTCs cut-off (Davis et al. 2018). Associations between clinical features, CTC-derived biomarkers and IBC were tested through uni and multivariate logistic regression. Survival was tested though log-rank test. Results: Within the analyzed cases, 46% were diagnosed with IBC and among them, 38% was stage IV aggressive. CTC clusters (CTC_CL) were detectable in 12.5% of pts and HER2 positive CTCs (HER2_CTC) in 29.5%. Notably, IBC patients (pts) had a significantly lower CTC count with respect to non-IBC (median 2.5 vs 0 respectively for non-IBC and IBC; P=0.019). BC subtype (HER2 positive BC: OR 2.97; Triple negative BC: OR 2.13), liver and bone involvement (liver: OR 0.46; bone involvement: OR 0.31) were the only significant clinico-pathological features associated with IBC at univariate logistic regression. Interestingly, a marginal significance was observed for soft tissue involvement (OR 1.65, 95%CI 0.95 - 2.87, P=0.07). Stage IV aggressive and presence of HER2_CTC at baseline were moreover inversely associated with IBC. The multivariate model confirmed the significant association between IBC and HER2 positive BC subtype (OR 2.64, 95%CI 1.08 - 6.48, P=0.034), absence of bone involvement (OR 0.31, 95%CI 0.14 - 0.68, P=0.003) and absence of HER2_CTC (OR 0.38, 95%CI 0.15 - 0.98, P=0.045). The baseline detection of CTC_CL was a strong predictor of prognosis for OS in IBC pts (median OS (mOS) 7.6 months (mts) vs not reached (NR) respectively for detectable vs non-detectable CTC_CL; P&amp;lt;0.0001), while a trend was observed for HER2_CTC (mOS 9.9 mts vs NR respectively for detectable vs non-detectable HER2_CTC; P&amp;lt;0.082). Pts negative for CTC_CL at baseline had higher odds of developing CTC_CL in later time-points if stage IV aggressive (OR 12.27, 95%CI 2.10 - 71.57, P=0.005). Despite no baseline factors were significantly associated with the onset of HER2_CTC in later time-points, a trend (P=0.05) was observed for patients without lymph node involvement (OR: 5) and with bone involvement (OR: 4.3). Conclusion: HER2_CTC and in particular CTC_CL are promising prognostic predictors in IBC. Stage IV aggressive IBC pts could benefit from a longitudinal CTCs assessment, being more prone to develop CTC_CL and therefore at higher risk of rapid disease progression. Probably due to the tropism for soft tissue, IBC is characterized by a lower number of HER2_CTC. Citation Format: Gerratana L, Zhang Q, Wang C, Shah A, Davis AA, Ye Z, Zhang Y, Abu-Khalaf M, Flaum L, Strickland K, Rossi G, Behdad A, Gradishar W, Platanias L, Yang H, Cristofanilli M. How is inflammatory breast cancer (IBC) different? Integration of clinico-pathological features and circulating tumor cells (CTCs)-based biomarkers for disease and prognostic assessment [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 P5-17-03.

Abstract P5-17-02: Dissecting the biology of inflammatory breast cancer (BC) through cell free DNA and a circulating tumor cells (CTC)-derived signature

Abstract Background: The biological characteristics conferring Inflammatory BC's (IBC) distinctive and aggressive clinical features are currently not fully clarified. The aim of this study is to dissect IBC's biology through the integration of DNA and CTC-based circulating biomarkers. Methods: This study retrospectively analyzed 251 Advanced BC (ABC) patients (pts) treated and longitudinally characterized for CTCs and circulating tumor DNA (ctDNA) at Thomas Jefferson University (Philadephia, PA) and Northwestern University (Chicago, IL). CTCs were enumerated through CellSearch (Menarini Silicon Biosystems), and characterized for HER2 expression using the CellSearch CXC Kit, while ctDNA was analyzed using the Guardant360 NGS assay (Guardant Health) and its percentage (%ctDNA) was classified based on the previously reported cut-off of 5.7% (Gerratana et al 2018). A subset of 117 pts was further characterized for circulating cell-free DNA (ccfDNA) through Qubit® dsDNA HS quantitation Assay (Thermo Fisher Scientific) and quantitative real-time PCR assay for ALU DNA repeats on chromosome 1.Associations between clinical characteristics, CTCs-derived biomarkers and IBC were explored through Fisher's exact test; survival was tested though Cox regression and log-rank test. Results: Of the total 251 pts, 115 were diagnosed with IBC. Among the 117 patients characterized for ccfDNA, 70 had IBC. Median ccfDNA was 1.59 for IBC (IQR 1.02-3.19) and 2.37 for non-IBC (nIBC) (IQR 1.13-3.52), P=0.27. Consistent results were observed for %ctDNA levels (median value: 2 vs 1.6). The impact on OS of ccfDNA after log transformation was significant for the total population (HR 1.73 95%CI: 1.11-2.69) but not in IBC pts (HR 1.40 95%CI: 0.84-2.34). On the other hand, ctDNA high pts had a significantly worse OS (nIBC: HR 5.34 95%CI: 1.70-18.81 P=0.004; IBC: HR 4.05 95%CI: 1.91-8.58 P&amp;lt; 0.001). In the ctDNA high subgroup no differences in total number of CTCs were observed between IBC and nIBC, while significantly lower CTCs were observed in ctDNA low IBC pts (P=0.0097). The ctDNA low IBC subgroup had a higher incidence of HER2 positive BC (P=0.003) and a significantly lower incidence of CTCs clusters (P=0.006), HER2 positive CTCs (P=0.041). Notably, no associations were observed with stage at baseline, number of metastatic sites, liver, lung and visceral involvement. On the other hand, the ctDNA_high IBC subgroup was characterized by a lower incidence in liver, bone and visceral involvement (P=0.017, P=0.014 and P=0.03 respectively) and a marginally high incidence in soft tissue involvement (0.084). Moreover, IBC diagnosis conferred a significantly worse prognosis only in the ctDNA low subgroup (OS at 12 months nIBC: 100% vs IBC: 70%; P=0.049), while no differences were observed in the ctDNA_high subgroup (OS at 12 months nIBC: 29% vs IBC: 26%; P=0.767). Conclusion: ctDNA is able to stratify BC according to aggressiveness independently from the sites and type of metastases, both in the IBC and nIBC subgroups. IBC has a distinctive CTCs/ctDNA-based signature, in particular ctDNAlow pts have a lower incidence of HER2 positive CTCs and CTC clusters. This signature is probably due to predominant lymphatic metastatic spread and aggressive phenotype. Citation Format: Gerratana L, Zhang Q, Wang C, Shah A, Davis AA, Ye Z, Zhang Y, Abu-Khalaf M, Flaum L, Strickland K, Rossi G, Behdad A, Gradishar W, Platanias L, Yang H, Cristofanilli M. Dissecting the biology of inflammatory breast cancer (BC) through cell free DNA and a circulating tumor cells (CTC)-derived signature [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 P5-17-02.

Abstract OT1-10-01: DETECT III/IV study trial – The multicenter study program in patients with HER2-negative metastatic breast cancer and circulating tumor cells

Abstract Background: The collaborative DETECT study program represents one of the largest study program on metastatic breast cancer worldwide. The main objective of the DETECT trial is to evaluate the efficacy of individualized breast cancer treatment based on the presence and phenotype setting of circulating tumor cells (CTCs). Thus, the DETECT study program is one of the first clinical trial translating the role of CTC enumeration/phenotyping directly into treatment intervention within different breast cancer subtypes. Trial design: The DETECT III trial is a multicenter, randomized, phase III study to compare standard therapy alone versus standard therapy plus lapatinib in patients with initially HER2-negative metastatic breast cancer and HER2-positive circulating tumor cells. Patients with persisting HER2-negative circulating tumor cells (CTCs) can be included within the DETECT IV trial, a prospective, multicenter, open-label, phase II study including patients with HER2-negative metastatic breast cancer. Within the DETECT IV study setting postmenopausal patients with hormone-receptor positive metastatic breast cancer are treated with everolimus or ribociclib and endocrine therapy, while women with triple negative metastatic breast cancer or a hormone-receptor positive tumor and indication for chemotherapy receive eribulin. Specific aims: The DETECT study program comprises all breast cancer subtypes and therefore offers various up-to-date treatment options, generating a wealth of clinical data including long-term follow-up data, evaluated in a controlled setting of a single large clinical trial. The primary endpoint of the DETECT III trial is the comparison of patients receiving standard anticancer therapy with lapatinib and patients receiving standard anticancer therapy alone, with regard to the CTC clearance rate. The secondary objective of this trial is to assess the level of compliance to study procedures comparing the efficacy of lapatinib between given treatment groups (Progression free survival, overall response rate and dynamic of CTCs). Primary objective of the DETECT IV trial is to evaluate CTC clearance rate within the everolimus/ribociclib cohort and additionally assess progression-free survival defined as time interval from date of recruitment until progressive disease within the eribulin cohort. The main focal point of the extensive collaborative translational oncology research projects is to apply innovative biomarkers and assays focusing on molecular characteristics of CTCs. This “biological status” may give new information about CTCs potential function as liquid biopsy in order to determine their relevance for therapy prediction. Citation Format: Krause S, Friedl T, Romashova T, Fasching PA, Schneeweiss A, Müller V, Taran F-A, Arkadius P, Marie T, De Gregorio A, Meier-Stiegen F, Huober J, Janni W, Fehm T. DETECT III/IV study trial – The multicenter study program in patients with HER2-negative metastatic breast cancer and circulating tumor cells [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 OT1-10-01.

Detection of HER2-positive Circulating Tumor Cells Using the LiquidBiopsy System in Breast Cancer

BackgroundMost previous studies of circulating tumor cells (CTCs) are based on the CellSearch platform, but CellSearch has a number of limitations. This study aimed to use the LiquidBiopsy system and immunofluorescence to test the human epidermal growth factor receptor 2 (HER2) status of CTCs in patients with breast cancer. Materials and MethodsThe LiquidBiopsy system was used to detect HER2-positive (HER2+) cells in whole blood by microfluidic immunomagnetic bead screening and immunofluorescence assay, according to the manufacturer;s instructions. HER2 expression on CTCs was assessed using the Ariol system, calibrated through spiking experiments of 100 cells (BT474, SKBR3, A431, and MDA-MB-231) and 2.5 × 107 white blood cells/mL from healthy donors. Seventy-one patients with breast cancer and 107 non-cancer donors consented to provide blood. ResultsBased on breast cancer cell lines experiments, HER2+ CTCs were defined as CTCs with HER2 immunofluorescence intensity ≥ 3.5 times higher than the CD45 immunofluorescence intensity (100% sensitivity and 99.9% specificity). Among the 71 patients with breast cancer, 31 (43.7%) had HER2+ tumor. Among the HER2+ patients, 41.9% (13/31) were found to be HER2+ based on CTC ≥ 1, and 25.8% (8/31) were positive based on CTC ≥ 3. In HER2-negative patients by pathologic examination, 1 (2.5%) patient was found to have ≥ 3 HER2+ CTCs, whereas 15 (37.5%) patients had ≥ 1 HER2+ CTC. HER2+ CTCs were detected at all stages, even in early breast cancer, but the detection rate was higher in metastatic breast cancer. ConclusionThis proof-of-concept study strongly suggests that HER2+ CTCs can be detected using the LiquidBiopsy system.

Abstract P2-01-02: Heterogeneity and variability of human epidermal growth factor receptor 2 (HER2) expression on circulating tumor cells (CTC) in HER2 negative metastatic breast cancer patients treated with first line weekly paclitaxel and bevacizumab in a prospective cohort from the French Breast Cancer InterGroup Unicancer (UCBG): COMET study

Abstract Background: It has been reported in women with advanced estrogen-receptor (ER)-positive/(HER2)-negative breast cancer the acquisition of a HER2-positive CTC subpopulation during therapy (Jordan NV Nature 2016). The clinical significance of acquired HER2 heterogeneity during the evolution of metastatic breast cancer is unknown. We report here the analysis of HER2 status of CTC before and after one cycle of treatment in HER2 negative metastatic breast cancer patients treated with first line weekly paclitaxel and bevacizumab. Patients &amp; methods:The French cohort COMET is a prospective study including first line HER2 negative patients (pts) receiving weekly paclitaxel and bevacizumab according to EMA approved combination. The aim of this cohort is to evaluate clinical, biological and radiological parameters associated with pts outcome. We confirmed previously the outcome of patients with high CTC count at base line and after one cycle of treatment (Bidard et al, Lancet Oncol 2014). We present here the analysis on 203 pts evaluated for the expression of HER2 on CTC using the FDA cleared CellSearch method. The HER2 expression of CTCs (CB11 clone) was categorized (class 0–3) as described by Riethdorf et al.CCR2010. Results: At base line, 144 out of 203 pts had at least one detectable CTC (71%), (median 4, and range 1- 30,000). Among them, 104 (72%) had one or more HER2 positive CTC (1-21,484). In 25 patients with HER2 2+ primary tumor with FISH or CISH non amplified, the incidence of CTC HER2+ cases (13/25, 52%) was similar than in pts with HER2 0 or HER2 1+ (51%) primary tumor. In each case, 3 to 100% of detectable CTC could be HER2+ stained (median 50% of CTC). Only 12 cases (8% of all CTC cases) had 2+ HER2 staining score on CTC and none 3+. After one cycle of treatment, the number of pts with detectable CTC dropped to 64, including 42 with HER2+ CTC (65%). Out of these cases, 14 were 3+ or 2+ HER2 score (22% of CTC+ cases). This was a significant increase compared to baseline (8%) (p&amp;lt;0.001), including 6 cases with 100% of HER2+ CTC. To note, 7 patients without HER2+ CTC at baseline, had detectable HER2+ CTC after one cycle of treatment. With a median follow-up of 2 years, correlation of CTC variations with pts outcome is planned. Conclusion: HER2 staining on CTC was heterogeneous with HER2 positive and negative subpopulations in the same patient with primary HER2 negative breast cancer. We observe a variability of HER2 CTC status with an increased intensity or appearance of immunostaining in few cases during treatment. We hypothesize that these phenotypes changes within patient-derived circulating tumor cells could contribute to progression of breast cancer and acquisition of drug resistance. Citation Format: Pierga J-Y, Proudon C, Tredan O, Decraene C, Dubot C, Lorgis V, Jacot W, Goncalves A, Debled M, Levy C, Ferrero J-M, Jouannaud C, Luporsi E, Mouret-Reynier M-A, Dalenc F, Lemonnier J, Berger F, Bidard F-C. Heterogeneity and variability of human epidermal growth factor receptor 2 (HER2) expression on circulating tumor cells (CTC) in HER2 negative metastatic breast cancer patients treated with first line weekly paclitaxel and bevacizumab in a prospective cohort from the French Breast Cancer InterGroup Unicancer (UCBG): COMET study [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-01-02.