Circulating Tumor Cells HER2 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.

Liquid biopsy identification of ERBB2 amplified and HER2 expressing metastatic breast cancer: Comparison and combination of cell and cell-free platforms.

3061 Background: Liquid biopsies are a non-invasive diagnostic approach for detecting circulating tumor cells (CTCs) or circulating tumor DNA (ctDNA) that may provide clinically actionable information for treatment decisions for metastatic breast cancer (MBC) patients when a conventional biopsy is otherwise infeasible. Here we report a comprehensive liquid biopsy platform including: 1) quantitative immunofluorescent HER2 and ER protein expression in CTCs (ctcIF), 2) determination of ERBB2 amplification and the number of Large-scale State transitions (LST) by single-cells CTC genomics (ctcDNA) and 3) alterations in plasma ctDNA. Methods: Blood samples from 457 progressing metastatic breast cancer patients and 25 healthy donors (HDs) as controls were collected for cell & cell-free DNA analysis. After plasma isolation, nucleated cells were plated, & slides were bio-banked for Immunofluorescent staining & subsequent imaging. CTCs were identified using Epic Sciences digital imaging & machine learning algorithms, and individual CTCs were sequenced for genomic quantification of LSTs and Copy Number Variants (CNV). Bio-banked plasma was analyzed to detect ctDNA alterations with high clinical relevance (Class IA). Results: Within this cohort of 457 MBC patients, the presence of CTCs, ctcDNA (LST+) and ctDNA alterations were detected in 98%, 58%, and 42%, respectively, while no CTCs and no ctDNA alterations were detected in the HD cohort, suggesting high specificity. ctcDNA genomics was more sensitive than ctDNA in detecting ERBB2amp in MBC patients (16%, and 2%, respectively). A mean variant allele frequency (VAF) of < 25%, which is the threshold for detecting a two-fold ERBB2 amplification by ctDNA, was present in 20% and 80% of ERBB2amp MBC detected by the ctDNA and ctcDNA platform, respectively, suggesting that the ctcDNA platform can identify the majority of ERBB2amp among patients with a low ctDNA fraction. HER2exp or ERexp expression by ctcIF were detected in 67% and 67% of MBC patients, respectively. A liquid biopsy classification of HER2 status by combining the three platforms (ctcIF, ctcDNA, and ctDNA) identified that in the tested population, 16% were ERBB2amp, 52% were HER2 expressing (HER2exp and ERBB2nonamp), and 27% were HER2neg (HER2- and ERBB2nonamp). To identify MBC patients that may potentially change treatment as a result of the test we identified a small cohort of MBC patients (n=46) with HER2- disease and tissue biopsy IHC=0, 74% of these patients had HER2exp CTC consistent with HER2 low expression which may aid therapeutic decisions in later line MBC. Conclusions: Here we report a comprehensive liquid biopsy profile combining ctcIF, ctcDNA, and ctDNA platforms with high sensitivity and specificity in determining clinically actionable HER2 and ER biomarker status that may impact therapeutic decision-making in MBC patients.

Abstract P5-06-03: Analyzing the results of liquid biopsy in the identification of ERBB2 amplified and HER2 expressing metastatic breast cancer: comparison and combination of cell and cell-free platforms

Abstract Background Liquid biopsies are a non-invasive diagnostic approach for detecting circulating tumor cells (CTCs) or circulating tumor DNA (ctDNA) that may provide clinically actionable information for treatment decisions for metastatic breast cancer (MBC) patients when a conventional biopsy is otherwise infeasible. In addition, the development of quantitative, reproducible, and more sensitive HER2 assays is expected to enable the identification of patients with HER2-low MBC that may benefit from novel HER2-targeted therapies. Here we report a comprehensive liquid biopsy platform including immunofluorescent HER2 and ER quantitative protein expression in CTCs (ctcIF) coupled with the determination of ERBB2 amplification and the number of Large-scale State Transitions (LST) by single-cell CTC genomics (ctcDNA), and ctDNA alterations in plasma. Methods Blood samples were collected for cell-based and cell-free analysis from 62 patients with documented MBC and from 24 blood donors (HD) with no known cancer history. After isolation, nucleated cells were plated, and slides and plasma were bio-banked. CTCs were identified using Epic Sciences digital imaging and machine learning algorithms, and ctcIF enables quantitation of HER2 and ER protein expression. ctcDNA was analyzed by low-pass whole-genome sequencing (WGS), allowing detection of ERBB2 amplification (ERBB2amp) and quantification of large-scale state transitions (LST+) in individual CTCs. cfDNA from plasma was analyzed using a validated NGS panel (56 genes of interest) to detect ctDNA alterations. Results Within this cohort of 62 MBC patients, the presence of CTCs, ctcDNA (LST+) and ctDNA alterations were detected in 87%, 70%, and 59%, respectively, while no CTCs and no ctDNA alterations were detected in the HD cohort, suggesting high specificity. ctcDNA genomics was more sensitive than ctDNA in detecting ERBB2amp in MBC patients (11%, and 2%, respectively). A variant allele frequency (VAF) of > 40%, which is required for detecting a two-fold ERBB2 amplification by ctDNA, was not present among 86% and 0% of MBC detected by the ctcDNA and ctDNA platforms, respectively, suggesting that the ctcDNA platform can identify ERBB2amp among patients with a low ctDNA fraction. HER2+ or ER+ expression by ctcIF were detected in 37% and 58% of MBC patients, respectively. At the cellular level, across 62 patients, CTC with detectable ERBB2amp by ctcDNA had a higher median expression of HER2 protein by ctcIF compared to CTC with ERBB2nonamp (1772 MFI vs 122.5 MFI, respectively; p< 0.001). Similarly, at the patient level, among patients with circulating ERBB2amp, HER2 protein was detected by ctcIF in 100% of MBC patients (p< 0.001), suggesting a very high positive correlation between the presence of ctcDNA genomic ERBB2amp and ctcIF HER2 protein expression. A liquid biopsy classification of HER2 status by combining the three platforms (ctcIF, ctcDNA, and ctDNA) identified that among MBC, 11% were ERBB2amp, 26% were HER2 expressing (HER2+ and ERBB2nonamp), and 60% were HER2neg (HER2- and ERBB2nonamp). Combination models of the three individual platforms (ctcIF, ctcDNA, and ctDNA) were able to provide potentially clinically actionable biomarker data (LST+ CTC, ERBB2amp CTC, HER2+ CTC, ER+ CTC and 1A+ SNVs) to 79% of MBC patients while retaining 100% specificity. Conclusions Here we reported a comprehensive liquid biopsy profile combining ctcIF, ctcDNA, and ctDNA platforms with high sensitivity and specificity in determining clinically actionable HER2 and ER biomarker status that may impact therapeutic decision-making in late MBC patients. The comprehensive liquid biopsy platform’s combined utility can aid biomarker profiling of MBC among often biologically heterogeneous tumor sites of metastatic disease and those inaccessible by conventional tissue biopsy. Citation Format: Giuseppe Di Caro, Ernest T. Lam, Megan M. Slade, Anna Lundberg, Martin Blankfard, Nilesh Dharajiya, Alisa Tubbs, Rick Wenstrup, Lee Schwartzberg. Analyzing the results of liquid biopsy in the identification of ERBB2 amplified and HER2 expressing metastatic breast cancer: comparison and combination of cell and cell-free platforms [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-06-03.

Heterogeneity of HER2 Expression in Circulating Tumor Cells of Patients with Breast Cancer Brain Metastases and Impact on Brain Disease Control.

Simple SummaryResults from a previous study suggested that the number of circulating tumor cells (CTC) might have a role as a biomarker of early distant brain failure in patients with breast cancer brain metastases (BCBM). However, it remains largely underexplored whether heterogeneous HER2 expression in CTC may have a prognostic implication. We evaluated the status of HER2 expression in CTC before and after radiotherapy/radiosurgery for BCBM and observed that the presence of HER2 expression in any moment was associated with longer distant brain failure-free survival, irrespective of the primary immunophenotype of the breast tumor. This finding suggests that the status of HER2 expression in CTC has the potential to improve the treatment selection for patients with BCBM.HER2 expression switching in circulating tumor cells (CTC) in breast cancer is dynamic and may have prognostic and predictive clinical implications. In this study, we evaluated the association between the expression of HER2 in the CTC of patients with breast cancer brain metastases (BCBM) and brain disease control. An exploratory analysis of a prospective assessment of CTC before (CTC1) and after (CTC2) stereotactic radiotherapy/radiosurgery (SRT) for BCBM in 39 women was performed. Distant brain failure-free survival (DBFFS), the primary endpoint, and overall survival (OS) were estimated. After a median follow-up of 16.6 months, there were 15 patients with distant brain failure and 16 deaths. The median DBFFS and OS were 15.3 and 19.5 months, respectively. The median DBFFS was 10 months in patients without HER2 expressed in CTC and was not reached in patients with HER2 in CTC (p = 0.012). The median OS was 17 months in patients without HER2 in CTC and was not reached in patients with HER2 in CTC (p = 0.104). On the multivariate analysis, DBFFS was superior in patients who were primary immunophenotype (PIP) HER2-positive (HR 0.128, 95% CI 0.025–0.534; p = 0.013). The expression of HER2 in CTC was associated with a longer DBFFS, and the switching of HER2 expression between the PIP and CTC may have an impact on prognosis and treatment selection for BCBM.

Evaluation of Liquid Biopsy in Patients with HER2-Positive Breast Cancer.

Breast cancer is one of the common malignant tumors, and liquid biopsy has become a hot spot for clinical testing. To clarify the detection effect of liquid biopsy in breast cancer, we collected peripheral blood of HER2-positive (human epidermal growth factor receptor 2-positive) patients. Circulating tumor cells (CTCs) were isolated and analyzed. HER2 expression on CTCs was detected. The results showed that in the 198 HER2-positive samples, the CTC detection rate was 79.8% (158/198), and the mean number of CTCs was 21, ranging from 1 to 63/7.5 mL peripheral blood. Only 41.1% (65/158) of patients had histology and CTC HER2 status consistent with the remaining 58.9% (93/158) of patients, although their histological HER2 was positive, and CTC HER2 was negative. Our study confirmed the value of CTC HER2 real-time status testing in HER2-positive breast cancer patients. The inconsistency in HER2 status between CTCs and histology may be related to the time interval between CTCs and histological HER2 detection, suggesting that real-time HER2 detection is necessary for histological HER2-positive patients.

Heterogeneity of HER2 expression in circulating tumor cells of patients with breast cancer brain metastases and impact on brain disease control.

2029 Background: The HER2 expression switching in circulating tumor cells (CTC) of breast cancer is dynamic and may have prognostic and predictive clinical implications. This study aims to analyse the association between expression of HER2 in CTC of patients with breast cancer brain metastases (BCBM) and brain disease control. Methods: Exploratory analysis of a prospective assessment (NCT02941536) of CTC before (CTC1) and 4–5 weeks after (CTC2) stereotactic radiotherapy/radiosurgery (SRT). CTC were isolated and quantified by a method of isolation by size of tumors and analyzed by immunocytochemistry to evaluate the expression of HER2. Distant brain failure-free survival (DBFFS), the primary endpoint, and overall survival (OS) were estimated by Kaplan-Meier estimator. Log-rank tests were applied in order to compare the survival curves. For multivariate analysis of prognostic factors that affected DBFFS and OS, the Cox proportional model was adjusted. Results: The median age at SRT was 54 (34-70), the diagnosis-specific graded prognostic assessment (DS-GPA) was 1–2 in 17.5% and 2.5–4 in 82.5% and the primary immunophenotype (PIP) was HER2-enriched in 51%, luminal B (LB) in 31% and triple negative (TN) in 18% of the total of 39 patients. CTC were detected in all 39 patients before SRT and the median CTC1 was 2 CTC/mL. After SRT, CTC were detected in 34 of 35 patients (4 deaths between CTC1 and CTC2) and the median CTC2 was 2.33 CTC/mL. HER2 was expressed in CTC1 and/or CTC2 in 9 patients, of which only 2 patients had PIP HER2-enriched. After a median follow-up of 16.6 months, there were 15 patients with distant brain failure and 16 deaths. The median DBFFS and OS were 15.3 and 19.5 months, respectively. Median DBFFS was 7 months in patients with PIP TN and was not reached in PIP LB and HER2-enriched (p = 0.036); 14 months in patients with DS-GPA 1-2 and 7 months with DS-GPA 2.5-4 (p = 0.017); 10 months in patients without HER2 expressed in CTC and not reached in patients with HER2 expressed in CTC (p = 0.012). Median OS was 4.8 months in patients with PIP TN and was not reached in PIP LB and HER2-enriched (p = 0.0026); 19.54 months in patients with DS-GPA 1-2 and 7.6 months with DS-GPA 2.5-4 (p = 0.00088); 17 months in patients without HER2 expressed in CTC and not reached in patients with HER2 expressed in CTC (p = 0.104). On multivariate analysis, DBFFS was superior in patients with PIP HER2-enriched (HR 0.128, 95% CI 0.025–0.534; p = 0.013) and OS was superior in patients with PIP HER2-enriched (HR 0.073, 95% CI 0.018-0.288; p < 0.0001) and LB (HR 0.224, 95% CI 0.062–0.816; p = 0.023). The status of expression of HER2 in CTC was not included in Cox model for DBFFS due to lack of events in patients with HER2 expressed in CTC. Conclusions: The expression of HER2 in CTC was associated with a longer DBFFS and the switching of HER2 expression between PIP and CTC may have impact on prognosis and treatment selection of BCBM.

Correlation between different levels of HER2 expression in circulating tumor cells (cHER2 ratio) and metastatic behavior in stageIVaggressive breast cancer.

3036 Background: The presence of HER2 expressing (HER2+) circulating tumor cells (CTCs) occurs often in metastatic breast cancer (MBC) patients (pts). We have previously showed that the ratio among CTCs expressing high level of HER2 and the total number of HER2+ CTCs (circulating HER2 ratio, cHer2 ratio) has a prognostic role in MBC patients. Here we further investigate the role of the cHER2 ratio in the process of metastatic spread. Methods: Under IRB-approved study we prospectively analyzed blood samples of patients 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. 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 3 different categories (1+,2+,3+) leaning on fluorescence intensity. 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 cohorts: cHER2 ratio high (> 0.75) (cHER2high) and cHER2 ratio low (≤0.75) (cHER2low). The frequency of each metastatic site (i.e. liver, lung, central nervous system, bone, lymph nodes, skin/soft tissue, serosa) and the total number of different sites involved (1-7, ≤2 and >2 sites) were compared among the two sub-populations and analyzed through Fisher exact test. Results: Out of 98 pts enrolled, 77 were classified as cHER2low and 21 as cHER2high. We observed a higher frequency of oligometastatic pts (≤2 sites involved) in the cHER2high cohort (16, 76%), compared to only 29 (37%) in the cHER2low (p<0.005). Moreover, the cHER2 ratio was associated with a tropism toward specific sites of disease spread with higher incidence of liver, lung and lymph nodes metastases in the cHER2low cohort (p<0.05). No other statistical associations were observed in respect of specific organ tropism. The frequency of involvement for each metastatic site among the two cohorts are reported in the table. Conclusions: Measuring CTCs enumeration and HER2 expression we identified two cohorts, cHER2high and cHER2low, associated with distinct patterns of metastatic spread. The cHER2low pts were correlated to multiple sites of metastatic involvement, with particular tropism toward liver, lung and lymph nodes. These results confirm the prognostic role of the cHER2 ratio, suggesting a peculiar biological meaning of the HER2+ 1+ CTCs.[Table: see text]

Functional Characterization of Circulating Tumor Cells (CTCs) from Metastatic ER+/HER2- Breast Cancer Reveals Dependence on HER2 and FOXM1 for Endocrine Therapy Resistance and Tumor Cell Survival: Implications for Treatment of ER+/HER2- Breast Cancer.

Simple SummaryAcquired endocrine resistance and late recurrence in patients with ER+/HER2− breast cancer are complex and not fully understood. Here, we evaluated mechanisms of acquired resistance in circulating tumor cells (CTCs) from an ER+/HER2− breast cancer patient who initially responded but later progressed under endocrine treatment. We found a switch from ERα-dependent to HER2-dependent and ERα-independent expression of FOXM1, which may enable disseminated ER+/HER2− cells to re-initiate tumor cell growth and metastasis formation in the presence of endocrine treatment. We found that NFkB signaling sustains HER2 and FOXM1 expression in CTCs in the presence of ERα inhibitors suggesting that NFkB and FOXM1 might be an efficient therapeutic approach to prevent late recurrence and to treat endocrine resistance. Collectively our data show that CTCs from patients with endocrine resistance allow mechanisms of acquired endocrine resistance to be delineated, and can be used to test potential drug regimens for combatting resistance.Mechanisms of acquired endocrine resistance and late recurrence in patients with ER+/HER2− breast cancer are complex and not fully understood. Here, we evaluated mechanisms of acquired resistance in circulating tumor cells (CTCs) from an ER+/HER2− breast cancer patient who initially responded but later progressed under endocrine treatment. We found a switch from ERα-dependent to HER2-dependent and ERα-independent expression of FOXM1, which may enable disseminated ER+/HER2− cells to re-initiate tumor cell growth and metastasis formation in the presence of endocrine treatment. Our results also suggest a role for HER2 in resistance, even in ER+ breast cancer cells that have neither HER2 amplification nor activating HER2 mutations. We found that NFkB signaling sustains HER2 and FOXM1 expression in CTCs in the presence of ERα inhibitors. Inhibition of NFkB signaling blocked expression of HER2 and FOXM1 in the CTCs, and induced apoptosis. Thus, targeting of NFkB and FOXM1 might be an efficient therapeutic approach to prevent late recurrence and to treat endocrine resistance. Collectively our data show that CTCs from patients with endocrine resistance allow mechanisms of acquired endocrine resistance to be delineated, and can be used to test potential drug regimens for combatting resistance.

Abstract PS2-06: The detection and enumeration of circulating tumor cells (CTCs) and circulating endothelial cells (CECs) in metastatic breast cancer

Abstract Introduction: Circulating tumor cells (CTCs) are the roots of metastasis which is the main cause for death in metastatic breast cancer (MBC). CTCs enumeration is strongly prognostic in advanced disease and can stratify patients in two distinct disease, Stage IV aggressive and Stage IV indolent. In the former disease, the detection of CTC clusters and HER2-expression increase prognostic and predictive value. The metastatic cascade is a complex, regulated process involving immune cells and endothelial cells for progression and neoangiogenesis. Circulating endothelial cells (CECs) from the inner wall of blood vessels are shed into the blood stream during formation of blood vessels which is considered a sensitive marker of endothelial damage in pathological conditions such as cancer. CECs have been also studied as a biomarker for tumor progression and monitoring anti-angiogenic therapeutic effects in MBC. We evaluated the concomitant detection of CTCs and CECs in MBC patients, along with expression of HER2 in CTCs that may offer an interesting clue to elucidate the metastasis mechanisms. Methods: Whole blood samples (7.5ml/each) were collected from 14 stage IV MBC patients before systemic therapy. CTCs enumeration was performed in FDA approved CELLTRACKS System (Menarini) by using CTC Kit contains specific antibodies targeting the EpCAM for capturing CTCs, anti-CK-PE (for epithelial cells), DAPI (for nucleus), anti-CD45-APC (for leukocytes), and anti-HER-2/neu-FLU. The CTCs were classified as CK+, EpCAM+, DAPI+ and CD45-. Meanwhile, the same patients’ blood samples (4.0ml/each) were processed for CEC analyzed by using CEC kit which immunomagnetically captures CD146+ cells, and then stains the cells for CD105-PE (specific for protein endolgin), CD45-APC, nucleus-DAPI. The positive CECs were classified as CD 146+, CD105+, DAPI+ and CD45-. The associations between CTCs, HER2 expression and CECs were evaluated. Results: The average age of patients was 53.1. Subtypes of Luminal, HER2 positive and TNBC were 64.2% 7.2% and 28.6% respectively. Distant metastasis were found in 13 out of 14 patients, including bone (7), liver (5), Lymph nodes (5) and Pleura (2). CTCs were found positive (≥5, Stage IV aggressive) in 5 patients (range: 5-47, mean=24), and HER expression was identified in all 5 of these cases with a range of numbers between 1 and 7 (mean=4.2). The ratios of HER+ CTC/total ratios were 8.51%, 17.95%, 20%, 30.77%, and 33.33%. HER2 expression were defined officially in our lab according to the percentile of positive HER2 CTCs/Total CTCs and the expression intensity as - (<20%), + (20-39%), ++ (40-59%) and +++ (≥60%) respectively. There were 9 patients (%) were identified as CTCs negative (<5, Stage IV indolent) with the mean=1, and HER+ CTCs were found in only 2 patients with Stage IV indolent. Meanwhile, CECs were found in all 14 patients with a range of numbers between 4 to 115. There were an average of 33 CECs in Stage IV aggressive disease, compared to 53 CECs in Stage IV indolent. The average of CECs were 53.44, 12 and 37.25 in Luminal, HER2 positive and TNBC groups respectively. On the other hand, patients with HER2+ CTCs had an average of 50 CECs which is significantly higher than average of 41 CECs in patients without HER2+ CTCs. Moreover, there were average of 94.5, 56 and 40.22 CECs were found in groups when HER2 expression was ++/+++, above + and - respectively. The results demonstrated that although CTC enumeration have a reverse correlation with CECs numbers, HER2 expression in CTCs was significantly related with high CECs numbers. Conclusions: Our data provides the first evidence of potential association between CTCs and CECs in metastatic breast cancer. The association between HER2 expression and CECs offers a potential new insight to mechanism connections between CECs and disease metastasis in MBC. Citation Format: Qiang Zhang, Paolo D'Amico, Jeannine Donahue, Lorenzo Gerratana, Andrew A. Davis, Saya Liz Jacob, Zheng Cai, Elena Vagia, Wenan Qiang, Ami N. Shah, Katy Kerby, Lisa Flaum, Youbin Zhang, Firas Wehbe, Amir Behdad, William Gradishar, Leonidas Platanias, Massimo Cristofanilli. The detection and enumeration of circulating tumor cells (CTCs) and circulating endothelial cells (CECs) 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 PS2-06.

Abstract PD8-03: Clinico-pathological and molecular features in young women with metastatic breast cancer

Abstract Introduction: Advanced breast cancer can be more aggressive and portend a worse prognosis in the younger population (age < 50). While several studies have outlined clinico-pathologic features and molecular features of primary disease in young women with breast cancer, data in the metastatic setting remain scarce. Thus, the goal of this study was to explore the underlying clinic-pathologic and molecular profile of young women with metastatic breast cancer. Methods: We conducted a retrospective analysis of 138 females with metastatic breast cancer treated at Northwestern Medicine who provided consent for serial evaluation of circulating biomarkers. Patient were divided into two cohorts based on age at the time of metastasis, namely premenopausal (defined as age < 50) and postmenopausal (age ≥ 50). CellSearch™ immunomagnetic kit (Menarini Silicon Biosystems) was utilized to enumerate circulating tumor cells (CTCs), and the CellSearch™ CXC Kit (in 7.5 cc whole blood) characterized CTC HER2 expression. Circulating tumor DNA (ctDNA) was sequenced using the Guardant360 next-generation sequencing (NGS) assay (Guardant Health). When available, tissue samples from the primary and metastatic site(s) were sequenced using FoundationOne and/or Tempus xT NGS assays. Associations were drawn using Pearson’s χ2 test, independent samples T-tests, and multivariate logistic regression. Results: Of the 138 women, 54 (39%) were premenopausal with median age of 42 (range: 28-49), and 84 (61%) were postmenopausal with median age of 57 (range: 50-81). For the premenopausal cohort, 96% had invasive ductal carcinoma, 2% invasive lobular carcinoma and 2% mixed/unknown, compared to postmenopausal with 74%, 19% and 7% respectively, p=0.003. No statistically significant association was observed based on disease subtype (HR+/HER2-, HR+/HER2+, HR-/HER2+, TNBC), correspondingly stratified as 46%, 17%, 9%, 28% (premenopausal) and 57%, 10%, 13% 20% (postmenopausal). In total, 39% of the premenopausal and 40% of the postmenopausal patients had inflammatory breast cancer (IBC). Among patients initially diagnosed with non-metastatic disease, time (years) to metastasis was 2.76 (95% CI 2.11 to 3.41) for the premenopausal and 6.08 (95% CI 4.89 to 7.27) for the postmenopausal cohort, p=0.0001. Statistically significant associations were found when comparing the NGS datasets, derived from serial collection of ctDNA +/- tissue samples. Specifically, the premenopausal group had a higher incidence of GATA3 (11 vs 6 cases; p=0.017) alterations, but a lower incidence of NF1 (1 vs 18; p<0.001) and RB1 (1 vs 9; p=0.049) alterations. Most common gene alterations included TP53 (67%), PIK3CA (45%), ERBB2 (26%), GATA3 (26%), MYC (24%), FGFR1 (19%) and ESR1 (17%) for the younger cohort, versus TP53(67%), PIK3CA (39%), ESR1 (27%), NF1 (27%), MYC (23%), ERBB2 (23%) and FGFR1 (20%) for the postmenopausal cohort. There were no statistically significant differences between the cohorts in terms of total number of ctDNA alterations at baseline draw (median[IQR]: 3[1-7] premenopausal group; 5[2-7] postmenopausal group), presence of ≥5 CTCs (54%; 46% of total cases, respectively), occurrence of CTC clusters (26%; 24%), or HER2+ CTC expression (44%; 45%). Conclusion: Our data reveal that premenopausal women diagnosed with metastatic breast cancer had a more rapid progression to metastasis and differ from their postmenopausal counterparts in both their pathologic profile (almost exclusively invasive ductal carcinoma) and molecular profile (notably, gene alteration frequencies of NF1, RB1 and GATA3), which could have significant implications in developing targeted treatment paradigms for younger women. Additionally, CTC prevalence in the metastatic setting differs from earlier stage breast cancer data showing a higher proportion of ≥5 CTCs in postmenopausal patients. Citation Format: Kristen Carroll, Ami M Shah, Lorenzo Gerratana, Chenyu Lin, Andrew A Davis, Qiang Zhang, Youbin Zhang, Lisa Flaum, Amir Behdad, Leonidas C Platanias, William J Gradishar, Massimo Cristofanilli. Clinico-pathological and molecular features in young women with metastatic breast cancer [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 PD8-03.

Abstract 408: Expression of CCR5 associated with HER2 in circulating tumor cells (CTCs) is a novel biomarker for patients with metastatic breast cancer (MBC)

Abstract Introduction: The Chemokine C-C motif ligand 5 (CCL5) and its receptor 5 (CCR5) play a significant role in solid tumors, particularly triple negative breast cancer (TNBC) and HER2+ subtypes with prognostic implications. CCL5-CCR5 axis was reported to govern cancer stem cells expansion and play key role in MBC progression. Our group recently reported that overexpression of HER2 was associated with CTC-clusters which caused poor prognosis of patients with MBC. Herein, we first reported the expression of CCR5 in CTCs of MBC patients, and described the correlation between CCR5 and HER2 expression in CTCs. Methods: Whole blood samples (7.5ml/each) were collected from stage III/IV MBC patients before systemic therapy. CTCs enumeration was performed in FDA approved CELLTRACKS ANALYZERII® System (Menarini) by using CTC Kit contains antibodies targeting the Epithelial Cell Adhesion Molecule (EpCAM) antigen for capturing CTCs, and immunofluorescent staining reagents including anti-CK-PE (specific for epithelial cells), DAPI (for nucleus), anti-CD45-APC (specific for leukocytes), and anti-HER-2/neu-FLU. The CTCs were classified as CK+, EpCAM+, DAPI+ and CD45-. After confirming CTCs were positive by CellSearch system, CCR5 expression were evaluated by using CXC kit for multiple staining includes anti-CCR5-PE (R&D Systems), Anti-CK-FITC, DAPI and Anti-CD45-APC. The correlation between HER2 expression and CCR5 expression was analyzed by Kruskal-Wallis test was used for statistics. Results: CTCs were found positive (≥5) in all seven MBC patients with a range of numbers between 124 and 442, and HER expression was identified in 6 out 7 cases (between 57 and 149 total cells). The ratios of HER2+ CTCs/total CTCs were varied between 28.5% and 88.1%. Meanwhile, CCR5 expression were found in 3 out 7 patients (Group 1), with CCR5+ CTCs/ total CTCs ratios were 4.45%, 43.1% and 59.1% respectively. There were 4 patients without CCR5 expression in CTCs (Group 2). The average HER2+ CTCs was 127.6 with HER2+ CTC/Total CTC as average of 59.4% in Group 1, which were significantly higher in compared with the corresponding numbers as 80.3 and 29.97% respectively in Group 2, which indicated that upregulation of CCR5 was positively associated with high level of HER2 expression in CTCs. Conclusions: Our data provides the first evidence of strong expression of CCR5 in CTCs of MBC as potential new marker. The significant correlation between overexpression of HER2 in CTCs and high level of CCR5 indicated that CCR5 may contributes to more aggressive MBC subtypes with HER2 expression, which promotes carcinogenesis and metastasis at least partly by maintaining and increasing cancer stem cells leading to increased invasion. We conclude that the further understanding of the molecular interactions of CCR5 and HER2 in CTCs will be important to elucidate the mechanism of metastasis of MBC and predict prognosis. Citation Format: Qiang Zhang, Lorezo Gerratana, Ami N. Shah, Andrew A. Davis, Lisa Flaum, Youbin Zhang, Richard G. Pestell, Firas Wehbe, Amir Behdad, Leonidas Platanias, William Gradishar, Massimo Cristofanilli. Expression of CCR5 associated with HER2 in circulating tumor cells (CTCs) is a novel biomarker for patients with metastatic breast cancer (MBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 408.

Abstract 429: Increased chromatin heterogeneity in circulating tumor cells (CTCs) is associated with high levels of HER2 expression in metastatic breast cancer (MBC)

Abstract Introduction: Molecular and genomic characterization of CTCs may help to understand MBC prognosis and predict treatment benefit. We previously reported that overexpression of HER2 in CTCs is associated with detection of clusters, more aggressive clinical features and prognosis in MBC (2018 AACR #5195). Herein, we report a new finding that a widespread variation on chromatin distribution in CTCs is associated with HER2 expression in MBC which may indicate more aggressive tumor. Methods: Whole blood sample (7.5ml/each) was collected from stage III/IV BCa patients before therapy. CTC enumeration were performed in FDA approved CELLTRACKS ANALYZERII® System (Menarini) by targeting the Epithelial Cell Adhesion Molecule antigen for capturing CTCs. After confirming CTCs were positive by CellSearch system, live CTCs enrichment was performed by using Parsortix system (ANGLE) utilizes microfluidic based 10μm separation cassettes. The captured CTCs were stained by Anti-CK-PE (specific for epithelial cells), DAPI (for nucleus), anti-CD45-APC (specific for leukocytes), and anti-HER2-FITC. CTCs chromatin packing density was scanned by Partial Wave Spectroscopic (PWS) microscopy which is a label-free spectroscopic microscopy method that resolves structures in cells and quantify the cell nucleus heterogeneity of chromatin packing density scaling between 20-350 nm, or from the kb-pair to 10 Mb-pair range. For each nuclei, the nanoscale heterogeneity of chromatin packing was analyzed as nuclear statistical parameter standard deviation Σ (sigma, RMS). Kruskal-Wallis test was used for statistics. Results: We identified 400 CTCs by CellSearch system, including 115 HER2+ CTCs and 285 HER2- CTCs. The live CTCs were sorted by Parsortix system and were proceeded with multiple staining and scanning by PWS microscopy. CTCs were classified based on morphology and correct phenotype as CK+, DAPI+ and CD45-. There were 97 acquired nuclei that were scanned and evaluated successfully, including 17 HER2+ CTCs, 12 HER2- CTCs and 80 non-CTC cells. According to the images, stronger nanoscopic variations at each pixel in internal structure represents higher chromatin distribution heterogeneity quantified by RMS. The average RMS was 0.05125 in CTC cells which is significantly higher than the non-CTC population (RMS=0.0381, p<0.01). Moreover, the HER2+ CTCs demonstrated the highest nuclear sigma (RMS=0.05366) among all the subgroups when compared to HER2- CTCs (RMS=0.04546, p<0.01). Conclusion: This is the first report of chromatin heterogeneity in CTCs, and the association between HER2 expression and high variations of chromatin distribution in MBC patients. This novel property of CTCs describing nanoscale heterogeneity of chromatin packing requires further evaluation and validation but, it may offer a novel dimension in the understanding of the metastatic process. Citation Format: Qiang Zhang, Lorenzo Gerratana, Ami N. Shah, Andrew Adam Davis, Lisa Flaum, Xiang Zhou, Youbin Zhang, Vadim Backman, Amir Behdad, Firas Wehbe, William Gradishar, Leonidas Platanias, Massimo Cristofanilli. Increased chromatin heterogeneity in circulating tumor cells (CTCs) is associated with high levels of HER2 expression in metastatic breast cancer (MBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 429.

Abstract 457: HER2, AR protein expression and chromosomal instability in circulating tumor cells (CTCs) of metastatic breast cancer (MBC) patients (pts)

Abstract Background: Upregulation of HER2 and AR protein expression, and increase in chromosomal instability, are mechanisms of acquired resistance to endocrine therapy, and are being investigated as treatment-guiding biomarkers. However, measurement of these biomarkers requires metastatic biopsies, which are costly, invasive, and prone to under-sampling which precludes their utility to guide treatment in late stage metastatic pts. A CTC-based test could expand the clinical utility. Using the Epic Sciences platform, MBC blood samples were characterized for CTC prevalence, HER2 and AR expression on CTCs, and CTC chromosomal instability at time of disease progression. Material and methods: Blood samples were acquired from 127 total pts with HR+/HER2-, HR-/HER2+ and TNBC metastatic breast cancer pts. 114 samples were analyzed for HER2 and 110 were analyzed for AR using the Epic CTC Platform. Single-cell whole genome sequencing was performed on 157 CTCs from 19 pts to assess large scale transitions (a surrogate of chromosome instability) and gene copy number alterations. Results: 87/114 (76.3%) of pts had detectable CTCs, with a median of 1.1 CTC/mL. 17/114 (14.9%) had at least one HER2(+) CTC, 21/110 (19.1%) had at least one AR(+) CTC, and 10/110 (9%) had both AR(+) and HER2(+) CTCs detected in technical replicates. HER2 expression on individual CTCs showed distinctive cytoplasmic membrane staining, and AR expression on individual CTCs showed frequent nuclear localization. The majority of HER2+ and AR+ CTCs had high cytokeratin expression. Most pt samples showed heterogeneous expression of these markers at disease progression indicating subclonal sensitivity to targeted therapies. CTCs with larger nuclear area and higher N/C ratio had higher frequency of chromosomal instability. ERBB2, FGFR1 gain, PTEN, CDH1, BRCA1, BRCA2 loss were frequently observed and the prevalence were associated with CTC phenotypes. Conclusions: CTCs are detected in most MBC pts upon disease progression. CTC based biomarkers assessing HER2, AR and chromosomal instability status could guide therapy selection. The results warrant prospective evaluation of these biomarkers on MBC pts’ CTCs as a resistance marker for targeted therapies. Citation Format: Priscilla Ontiveros, Connie Landaverde, Maren K. Levin, Sarah Hippely, Mark Landers, Yipeng Wang, Ryan Dittamore, Joyce A. O'Shaughnessy. HER2, AR protein expression and chromosomal instability in circulating tumor cells (CTCs) of metastatic breast cancer (MBC) patients (pts) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 457.

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 P3-01-04: 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.

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% >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% >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 <5 in 24%, 5-50 in 43%, and >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 P3-01-07: Enabling HER2 and androgen receptor (AR) protein expression and localization in circulating tumor cells (CTCs) of ER(+/-)HER2(-) metastatic breast cancer (MBC) patients (pts)

Abstract Background: Upregulation of HER2 and AR are mechanisms of acquired resistance to endocrine therapy, and are being investigated as treatment-guiding biomarkers. However, measurement of these proteins and their localization requires metastatic biopsies, which are costly, invasive, and prone to under-sampling which limits their utility to guide treatment in late stage metastatic patients. A CTC-based test could expand the clinical utility of these biomarkers. Here we utilized the Epic Sciences CTC platform for CTC detection and characterization. MBC blood samples were characterized for CTC prevalence, HER2 and AR expression at time of disease progression. Material and methods: HER2 and AR expression levels were determined based on model cell lines. A total of 72 blood samples were acquired from ER(+/-)/HER2(-) patients (by standard tissue pathology) at disease progression. 72 samples were analyzed for HER2 and 64 were analyzed for AR using the Epic Platform. Single-cell whole genome sequencing was performed to assess clonality and inter-patient heterogeneity of CTCs detected. Results: 55/72 (76.4%) of patients had CTCs detected across two slides. 13/72 (18.1%) had at least one HER2(+) CTC, 14/64 (21.9%) had at least one AR(+) CTC, and 7/64 (10.9%) had at both AR(+) and HER2(+) CTCs detected on replicate slides. HER2 expression on individual CTCs showed distinctive cytoplasmic membrane staining, and AR expression on individual CTCs showed frequent nuclear localization. Most patient samples showed heterogeneous expression of these markers at disease progression indicating subclonal sensitivity to targeted therapies. Subsequently, these cells will be individually sequenced to better determine the clonality of resistance. Conclusions: CTCs are detected in most MBC pts upon disease progression, with expression of known endocrine therapy resistance markers, HER2 and AR, observed that CTCs could guide subsequent therapy selection. Prospective evaluation of HER2 and AR on MBC pts' CTCs as predictive biomarkers of benefit from inhibitors of these proteins is needed. Citation Format: Ontiveros P, Landaverde C, Graf R, Levin MK, Hippely S, Wang Y, Landers M, Dittamore R, O'Shaughnessy JA. Enabling HER2 and androgen receptor (AR) protein expression and localization in circulating tumor cells (CTCs) of ER(+/-)HER2(-) metastatic breast cancer (MBC) patients (pts) [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 P3-01-07.

Absence of HER2 Expression of Circulating Tumor Cells in Patients with Non-Metastatic Esophageal Cancer.

The clinical significance of circulating tumor cells (CTC) in non-metastatic esophageal cancer (EC) remains controversial and the cellular and molecular characteristics of CTCs are poorly understood. Especially the frequency and oncological impact of HER2 expression in CTCs in patients with EC have not been evaluated until now. In this single-center, prospective study, peripheral blood samples were obtained preoperatively from 45 patients who were diagnosed with resectable EC. CTC detection and HER2 expression were performed using the CellSearch System. Data were correlated with clinicopathological parameters and patient outcomes. The study included 13 patients with squamous cell carcinomas (SCC) and 32 patients with adenocarcinomas (AC). HER2 gene amplification in the primary tumor was detected in 9.1% of patients. One or more CTCs were detected in 15.6% (SCC 1/13; AC 6/32) of the patients. None of the detected CTCs showed HER2 expression. Patients with CTCs showed significantly shorter relapse-free (p<0.001) and overall survival (p=0.015) than CTC-negative patients. This is the first study analyzing HER2 expression and the clinical significance of CTCs in patients with non-metastatic EC using an automated immunomagnetic detection system. HER2 expression in CTCs is very rare in patients with non-metastatic EC and seems to have a low clinical and oncological impact.

Abstract 5195: Increased circulating tumor cell (CTC) clusters are associated with significantly higher levels of HER2 expression and metastasis in stage III/IV breast cancer

Abstract Introduction: Molecular and genomic characterization of CTCs may help to understand breast cancer prognosis and predict treatment benefit, especially for metastatic or recurrent disease. Overexpression of HER2 protein has been associated with rapid cell division and the metastasis and prognosis of advanced breast cancer in the clinic. Herein, we report a correlation between the detection of CTC clusters and HER2 overexpression in otherwise biomarker negative disease. Methods: 146 whole blood samples (7.5ml/each) were collected from 95 patients with stage III/IV breast cancer at baseline, before and after systemic therapy. All samples were transported and stored at 15-30 °C in EDTA tubes. CTC enrichment and enumeration were performed in FDA approved semi-automated fluorescence CELLTRACKS ANALYZERII® System (Janssen Diagnostics) by using CELLSEARCH® CXC Kit (Cell Search) contains ferrofluid-based capture reagent particles with a magnetic core surrounded by a polymeric layer coated with antibodies targeting the Epithelial Cell Adhesion Molecule (EpCAM) antigen for capturing CTCs. Furthermore, it includes immunofluorescent staining reagents including Anti-CK-PE which is specific for the intracellular protein cytokeratin in epithelial cells, DAPI stains the cell nucleus, anti-CD45-APC is specific for leukocytes, and anti-HER-2/neu-FLU is specific for HER-2/neu antigen. The CTCs were classified based on morphology and correct phenotype as CK+, EpCAM+, DAPI+ and CD45-. Database of CTCs was generated and linked with clinical database. Kruskal-Wallis test was used for statistics. Results: Of the 146 samples analyzed, 80 samples contained CTCs, among which 54 cases did not form clusters (Group 1), 11 samples had 1-5 clusters (Group 2), and 15 had &amp;gt;5 clusters (Group 3). The average ratio of CTCs/ total EpCAM+ was directly correlated with the number of clusters and were reported as 15.36%, 25.32% and 43.65% for the 3 cohorts respectively (P&amp;lt;0.01). The highest expression of HER-2 was found in Group 3 (66.67% samples are positive, and 120 positive cells/each sample). This value was significantly higher than Group 2 (27.27% samples are positive, 24.33 positive cells/each sample) and Group 1 (20.37% samples are positive, 6.18 positive cells/each sample) (P&amp;lt;0.001). The results indicated that high level of CTCs clusters strongly associated with over expression of HER2 in CTCs. Conclusion: We demonstrate that patients with detectable CTCs clusters in comparison with CTCs with no or low numbers of clusters, high levels of CTCs clusters (&amp;gt; 5 clusters/7.5ml blood) is a strong indicator for more aggressive of CTCs with high level of HER2 expression, which tend to be more aggressive in metastasis and worse prognosis than HER2-negative breast cancers. Along with and cancer stage and HER2 status, CTCs clusters helps determine your treatment options in clinic. Citation Format: Qiang Zhang, Youbin Zhang, Lisa Flaum, Lorenzo Gerratana, William Gradishar, Leonidas Platanias, Massimo Cristofanilli. Increased circulating tumor cell (CTC) clusters are associated with significantly higher levels of HER2 expression and metastasis in stage III/IV breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5195.

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.