HER2 Status Of 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.

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.

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 & 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<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.

Real-time HER2 status detected on circulating tumor cells predicts different outcomes of anti-HER2 therapy in histologically HER2-positive metastatic breast cancer patients.

BackgroundThis study was initiated to investigate the difference in HER2 status between tumor tissue and circulating tumor cells (CTCs), as well as the predictive value of CTC HER2 status for predicting the outcomes of anti-HER2 therapy in histologically HER2-positive metastatic breast cancer (MBC) patients.MethodsHER2 expression on CTCs was detected using a CellSearch system within 7 days before a new line of anti-HER2 therapy was begun. According to the criterion proposed in our previous report, patients were defined as CTC HER2-positive or -negative. After close follow-up, the correlation between CTC HER2 status and the outcome of the treatment was evaluated by statistical analysis.ResultsCTCs were detected in 57.4 % (58/101) of the patients. Notably, 62.1 % (36/58) of these patients had an inconsistent HER2 status between their tissue and CTCs. The discordant rate may correlate with the time interval between histological and CTC HER2 testing and is more likely to occur in the subgroup of patients with an interval of > 1 year than in those with an interval < 1 year (70.7 % vs. 41.2 %, P = 0.043). For PFS, positive HER2 status on CTCs was shown to be a valuable predictor, both in univariate (HR = 0.321, 95%CI, 0.156–0.62, P = 0.0011) and multivariate (HR = 0.383, 95%CI, 0.166–0.831, P = 0.019) Cox regression analysis. Meanwhile, Kaplan-Meier survival curves revealed that the median PFS of CTC HER2-positive patients was significantly longer than CTC HER2-negative ones (8.5 vs. 3.5 months, P < 0.001).ConclusionsHER2 status on CTCs was different from that of tumor tissues and predicted a different outcome of the patients’ anti-HER2 therapy. This difference may be correlated with the time interval between tissue and CTC HER2 testing, indicating the necessity of real-time HER2 analysis for histologically HER2-positive MBC patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2578-5) contains supplementary material, which is available to authorized users.

Abstract P2-02-03: HER2 expression on circulating tumor cells before adjuvant chemotherapy and during follow-up in patients with HER2-negative early breast cancer

Abstract Background: Detection of circulating tumor cells (CTCs) before adjuvant chemotherapy as well as during follow-up is related to poor prognosis in early breast cancer. The presence of HER2-positive CTCs has been also associated with significantly decreased disease-free and overall survival. We hereby assessed the HER2 status of CTCs in HER2-negative patients with early breast cancer, before the initiation of adjuvant chemotherapy and during follow-up. Methods: Double staining immunofluorescent experiments using pancytokeratin (A45-B/B3) and HER2 antibodies were performed in peripheral blood mononuclear cells (PBMCs) of patients. Patients were evaluated before adjuvant therapy (n=103) and during follow-up (n=290) at intervals ranging from 6 months to 20 years post-chemotherapy. Results: CTCs were detected in 28 (27.2%) out of 103 patients pre-chemotherapy and in 94 (32.4%) out of 290 during follow-up. The mean CTC count detected at ≤2 years of follow-up was not significantly different compared to baseline [3.4 CTCs (range 1-13) vs 2.9 (range 1-12), respectively]. However, this value showed a significant decrease from 2-5 to ≥10 years of follow-up (4.2, range 1-11 vs. 1.8, range 1-4). Among CTC-positive patients, CTCs expressing HER2 were detected in 21 (75%) and in 73 (77.7%) patients pre-chemotherapy and during follow-up, respectively. The mean proportion of HER2-positive CTCs per patient was 73.5% pre-chemotherapy and 67% at follow-up. This proportion showed a significant increase at follow-up time points ≤2, 2-5 and 5-10 years [52% (p=0.0027), 65.6% (p=0.0476) and 69% (p=0.0440), respectively] compared to ≥10 years (89.2%). Conclusions: These data demonstrate that CTCs persist during follow-up in a significant percentage of early breast cancer patients. Similarly, HER2 positive CTCs are detected in a significant proportion of patients both at baseline and during follow-up. Mean CTC count appears to decrease after 10 years, whereas the population of HER2 positive CTCs seems to increase during follow-up. These results suggest that targeting HER2-positive CTCs may have implications for the prevention of late relapses. Citation Format: Markomanolaki H, Mavroudis D, Kallergi G, Vetsika E-K, Stratigos M, Makrigiannaki K, Georgoulias V, Agelaki S. HER2 expression on circulating tumor cells before adjuvant chemotherapy and during follow-up in patients with HER2-negative early breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-03.

Abstract OT1-2-03: The DETECT-study concept: Treatment based on the phenotype of circulating tumor cells in HER2-negative metastatic breast cancer

Abstract Background: The prognostic impact of circulating tumor cells (CTC) in metastatic breast cancer (MBC) is well demonstrated. The role of CTCs in predicting specific treatment response and the importance of CTC phenotypes for therapeutic decisions will be investigated within the DETECT-study concept. Trial Design and eligibility criteria: The DETECT studies are prospective, multicenter, open-label clinical trials designed for patients with HER2-negative MBC and evidence of CTCs in the peripheral blood. DETECT III is a two-arm study for patients with HER2-positve CTCs, randomized to physician’s choice therapy (chemotherapy or endocrine treatment) with or without additional HER2-targeted treatment with lapatinib. DETECT IV combines tow single-arm studies aimed at patients with HER2-negative CTCs. Postmenopausal patients with hormone-receptor-positive MBC will be treated with the mTOR-inhibitor everolimus in combination with an endocrine therapy of physician’s choice (everolimus cohort), whereas patients with triple-negative or hormone-receptor-positive MBC and indication to chemotherapy will receive eribulin (eribulin cohort). Specific aims: The primary objective of the trials is to estimate the clinical efficacy of treatments, assessed by the CTC clearance rate for DETECT III and by progression-free survival (PFS) for DETECT IV. Methods: Prevalence of CTCs at various time points as well as the HER2 status of CTCs are assessed using the FDA-approved CellSearch System (Veridex, USA). After immunomagnetic enrichment with an anti-EpCam-antibody, cells were labelled with anti-CK8/18/19 and anti-CD45 antibodies to distinguish epithelial cells from leucocytes. A fluorescein conjugate antibody with anti-CK-Fluorescein Isothiocyanate (FITC) was used for HER2 phenotyping. The cut-off for CTC-positivity was 1 CTC and for HER2 1 CTC with strong HER2-staining (+++). Survival endpoints will be estimated using the Kaplan-Meier method. Present and target accrual: Overall, about 2000 patients with HER2-negative MBC will have to be screened for CTCs to be able to recruit 228 patients with HER2-positive CTCs for DETECT III (which started in February 2012), 400 patients with HER2-negative CTCs for DETECT IV- everolimus cohort (which started in December 2013) and 120 patients for DETECT IV- eribulin cohort (which will start in the second half of 2014). 907 patients have been recruited for CTC screening until June 2014. Perspectives: One screening for CTCs offers different treatment options for patients with HER2-negative MBC and evidence of CTCs within the DETECT-study concept. DETEC III is the first study to investigate a personalized targeted treatment based on the phenotype of CTCs. The addition of a HER2-targeted therapy in case of HER2-positive CTCs is innovative and in case of success will lead to new treatment strategies in MBC. DETECT IV complements DETECT III with regard to additional therapy indications. Citation Format: Bernadette AS Jaeger, Susanne Albrecht, Fabienne Schochter, Carola A Melcher, Carsten Hagenbeck, Thomas WP Friedl, Brigitte Rack, Volkmar Müller, Peter A Fasching, Wolfgang Janni, Tanja Fehm. The DETECT-study concept: Treatment based on the phenotype of circulating tumor cells in HER2-negative metastatic breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr OT1-2-03.

Abstract OT1-1-04: HER2 status of circulating tumor cells in HER2-positive metastatic breast cancer patients: A valuable biomarker in anti-HER2 therapy

Abstract Background: In the earlier study which we have conducted, we observed that: among the patients who were histologically positive for HER2 and received anti-HER2 therapy, only those with HER2-positive circulating tumor cells (CTCs) have benefited. We also demonstrated that CTC enumeration with a modified cut-off is a valuable prognostic tool for HER2-positive MBC patients, and formulated the criterion for evaluating CTCs HER2 status. Although the number of cases was not adequate enough so as to make the persuasion of the statistical analysis limited, it was still a valuable innovative study worthy of further exploring. Purpose: we conducted a prospective study in order to find out whether HER2 expression in circulating tumor cells (CTCs) influenced anti-HER2 therapy in those HER2-positive metastatic breast cancer (MBC) patients. Trial design Patients: All the patients randomly enrolled in the trial were diagnosed with metastatic breast cancer, and the metastasis of all eligible subjects can be measured or evaluated, the Eastern Cooperative Oncology Group (ECOG) performance status score is 0 to 3. Each patient had a pathology report confirmed that this was a HER2-positve patient by the means of an immunohistochemistry (IHC) score or a fluorescent in situ hybridization (FISH) ratio, the researcher was also informed of the histological type, nodal status, estrogen receptor (ER), progesterone receptor (PgR) statuses. All of the patients would receive a new line or a new cycle of systematic therapy which must include the anti-HER2 therapy. The blood sample (10 ml) was drawn with an interval of &amp;lt;7days before the initiating of the new treatment. All the therapeutic regimens were made without knowing the CTC results and according to the National Comprehensive Cancer Network (NCCN) clinical practice guidelines (Breast Cancer V.2.2010). Disease status was evaluated by Response Evaluation Criteria in Solid Tumors (RECIST). Detection method: CTC isolation, enumeration and characterization were performed by using the CellSearch technology. HER2 expression intensity in CTC was given a score of 0, 1+, 2+, or 3+, according to the trial we have conducted previously, the CTC HER2 positive criterion was defined as &amp;gt;30% of CTCs over-expressing HER2 (3+). Statistical method: PFS was measured as the time between the baseline CTC assessment and the documentation of disease progression or death. Patients who were alive without progression at the time of analysis, PFS was calculated by the most recent follow-up evaluations. Kaplan-Meier survival curves were generated based on the CTC levels at baseline and the HER2 status of CTC, the curves were compared using the log-rank test. Present accrual and target accrual: 174 eligible patients were enrolled during September 2010 to April 2013, 76 patients were detected with CTC31, 50 patients received a new line or a new cycle of anti-HER2 therapy within 7days. Among the 50 patients, 40 patients received Trastuzumab and others received Lapatinib. With the ongoing of the trial, we’ll continue following up with PFS and other data of the patients. Types of anti-HER2 therapyAnti-HER2 therapyNTrastuzumab40Lapatinib10Total50 Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr OT1-1-04.

Abstract OT1-1-11: DETECT III - A multicenter, randomized, phase III trial 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

Abstract Background: In metastatic breast cancer (MBC) patients, HER2 status may be subject to change and it has been shown that 20-30% of initially HER2-negative patients have HER2-positive metastases. The discordance of the HER2 status between primary tumor and circulating tumor cells (CTCs) may be an important factor affecting the response to HER2-targeted treatments; however, it is unclear if therapy based on the HER2 status of CTC offers a clinical benefit for patients. Trial Design: DETECT III is a recruiting prospective, multicenter, open-label, two-arm, phase-III study for patients with HER2-negative MBC. Patients with HER2-positve CTCs are randomized to receive either a standard therapy of the physician's choice alone or a standard therapy plus additional HER2-targeted treatment with Lapatinib. Main Eligibility criteria: - Metastatic breast cancer; - HER2-negative primary tumor tissue and/or biopsies from metastatic sites; - Evidence of at least 1 HER2-positive CTC out of 7.5 ml blood; - Indication for systemic treatment - ≥1 lesion evaluable according to RECIST Specific aims: The objective of the DETECT III trial is to estimate the clinical efficacy of standard therapy plus lapatinib as assessed by progression free survival (PFS) in patients with initially HER2-negative MBC and HER2-positive CTCs. Additional research will be performed on CTC dynamic and characteristics. Present accrual and target accrual: The DETECT III – trial started February 2012 and has analyzed so far (2013-06-06) 506 patients. We found CTCs in 336 (66.4%) patients (median 7 CTCs, range 1-35078). 67 of these patients (19.9%) had at least one HER2 positive CTC. Estimated patient enrollment is 228. Methods: The primary endpoint progression-free survival will be estimated using the Kaplan-Meier method. Prevalence and number of CTCs at various time points as well as the HER2 status of CTCs will be determined using the FDA-approved CellSearch System (Veridex, USA). In addition, different measures of CTC dynamics and their value for evaluating therapy efficacy or as a prognostic tool will be examined in detail by explorative data analyses. Perspectives: The DETECT III-Trail is the first study where treatment is based on phenotypic characteristics of CTCs. If this trial succeeds in proving efficacy of lapatinib in patients with initially HER2-negative primary tumor but HER2-positive CTCs, it may lead to a new strategy for the treatment of metastatic breast cancer. Contact information: nikolaus.de-gregorio@uniklinik-ulm.de. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr OT1-1-11.

Persistence of HER2 overexpression on circulating tumor cells in patients after systemic treatment for HER2-positive breast cancer: Follow-up results of the German Success B trial.

11043 Background: The discordance between HER2-expression on circulating tumor cells (CTC) in peripheral blood and the primary tumor has already been shown by our study group for early breast cancer patients with HER2-positive tumors. Here, we compare the results to CTC prevalence and HER2-status of CTC after adjuvant chemotherapy. Methods: The SUCCESS B trial compares FEC-Docetaxel vs. FEC-Docetaxel-Gemcitabine and HER2-targeted therapy as adjuvant treatment for patients with early, HER2-positive, node positive or high risk node negative primary breast cancer. We prospectively analyzed 23ml peripheral blood before and after chemotherapy. CTC and HER2-status were assessed with the CellSearchSystem (Veridex, USA). After immunomagnetic enrichment with an anti-Epcam-antibody, cells were labeled with anti-CK 8/18/19, anti-CD45 antibodies as well as a fluorescein conjugate antibody for HER2-phenotyping. Cutoff for CTC positivity was ≥ 1 CTC. HER-positivity of CTC was assigned if at least one CTC showed strong HER2 staining (3+). Results: CTCs and their HER2-status both before and after chemotherapy were available for 392 patients. In 179 (45.7%) patients no CTC were detected before and after chemotherapy. CTC status changed from positive before to negative after chemotherapy in 104 (26.5%) patients and from negative before to positive after chemotherapy in 69 (17.6%) patients, while 40 (10.2%) patients had a consistently positive CTC status. Patients were significantly more likely to change their CTC status from positive to negative than from negative to positive (p = 0.01). Of the 40 patients with CTC both before and after chemotherapy, 14 (35%) patients had HER2-positive CTC before and after therapy, and 9 (22%) patients had HER2-negative CTC at both time points. 7 (18%) patients had HER2-positive CTC before but not after chemotherapy, while 10 (25%) patients showed the reverse pattern (p = 0.63). Conclusions: Cytotoxic treatment does not seem to influence the HER2-status on CTC. Follow-up data within the Success B trial will analyze the relevance of the HER2-expression of CTC to predict the efficacy of targeted treatment.

Biomarq: A novel approach to automated HER2-analysis of circulating tumor cells (CTCs).

638 Background: Targeted treatment approaches directed against CTCs might improve the outcome of metastatic breast cancer patients. Accurate and reproducible assessment of the CTC phenotype is a requirement to use CTCs as treatment targets. The aim of this study was to test the suitability of the automated BioMarQ System (Veridex, USA) – an enhancement of the FDA-approved CellSearch System (Veridex USA) for CTC detection– for assessing the Her2-status of CTCs based on fluorescence and cell-morphology data. Methods: Data on classification of the Her2-status into four groups (0, +, ++, +++) obtained by visual inspection of CTCs after immunocytochemical staining with the CellSearch System, and of BioMarQ-generated automatic measures on fluorescence and cell morphology via image clips generated from raw images were available for 329 CTCs from 17 metastatic breast cancer patients. A discriminant analysis was performed to test whether linear combinations of the fluorescence and/or cell morphology measures allow a separation of CTCs into four groups according to the visual classification of their Her2-status. Results: The highest concordance with the visual classification was obtained by a set of three discriminant functions based on the five variables mean-, maximum- and integrated fluorescence intensity, cell area (µm2), and coefficient of variation for the pixel fluorescence values within a cell. All five variables differed significantly among the 17 patients (Kruskal-Wallis-test, all p &lt; 0.001). Based on the three discriminant functions, 74.5% of all cases were correctly assigned to the four visually obtained categories (6 out of 8 CTCs classified as +++; 57 out of 80 as ++; 17 out of 65 as +, 165 out of 176 classified as 0). Conclusions: Our preliminary analysis shows that discriminant functions derived from measures on fluorescence and cell morphology generated by BioMarQ correctly assigned the Her2-status of CTCs to the four visually obtained categories in 75% of all cases. Further analysis based on a larger number of Her2-positive CTCs, and cross validation by FISH-analysis will provide deeper insight into the suitability of BioMarQ for objectively assessing the Her2-status of CTCs.

Discordance in HER2 gene amplification in circulating and disseminated tumor cells in patients with operable breast cancer

Human epidermal growth factor receptor 2 (HER2) gene amplification in circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) might be useful for modifying Herceptin therapy in breast cancer. In the process of investigating the utility of a microfluidic platform for detecting HER2 gene amplification in these cells, we observed novel results on discordance of HER2 status. Peripheral blood (8.5 mL) and bone marrow (BM) (7.5–10 mL) were collected prospectively from patients with clinical stages I–IV breast cancer. Mononuclear cells were recovered, stained with cytokeratin (CK), CD45, and DAPI, and processed through microfluidic channels for fluorescence in situ hybridization (FISH). A ratio of HER2:CEP17 >2 in any CK+/CD45 or CK−/CD45 cell was regarded as positive for HER2 gene amplification. Peripheral blood from 95 patients and BM from 78 patients were studied. We found CK+/CD45−/DAPI+ CTCs in 27.3% of patients. We evaluated HER2 gene amplification by FISH in 88 blood and 78 BM specimens and found HER2+ CTCs in 1 of 9 (11.1%) and HER2+ DTCs (27.2%) in 3 of 11 patients with HER2+ primary tumor. Among patients with a HER2− primary tumor, 5 of 79 had HER2+ CTCs (6.3%) and 14 of 67 had HER2+ DTCs (20.8%). The overall rate of discordance in HER2 status was 15% between primary tumor and CTCs and 28.2% between primary tumor and DTCs. HER2 was amplified in CTCs and DTCs in a portion of both HER2+ and HER2− primary tumors. HER2 discordance was more frequent for DTCs. The clinical implications of evaluating HER2 status in CTCs and DTCs in breast cancer needs to be established in prospective clinical trials. The cell enrichment and extraction microfluidic technology provides a sensitive platform for evaluation of HER2 gene amplification in CTCs and DTCs.

Abstract OT1-1-10: DETECT III - 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 but with HER2-positive circulating tumorcells

Abstract Background: Despite a HER2-negative primary tumor approximately 20–30% of patients develop HER2-positive metastases (Zidan et al. 2005; Tewes et al. 2009). As previously described in the DETECT I trial (Fehm et al. 2010) determination of HER2 status on circulating tumor cells (CTCs) is one option for re-evaluating HER2-status in the metastatic setting. Currently it is unclear if HER2-targeted therapy based on the assessment of HER2-status of CTCs reveals a clinical benefit. Trial design: DETECT III is a randomized, open-label, two arm phase III study comparing standard treatment alone vs. standard treatment plus HER2-targeted therapy with lapatinib in HER2-negative metastatic breast cancer patients with HER2-positive CTCs. Choices of chemotherapy and endocrine therapy include: docetaxel, paclitaxel, capecitabine, vinorelbine, non pegylated liposomal doxorubicin, letrozole, exemestane and anastrozole. Main eligibility criteria: 1. metastatic breast cancer with HER2-negative primary tumor tissue and/or biopsies from metastatic sites or locoregional recurrences2. evidence of ≥ 1 HER2-positive CTC3. ≥ 1 evaluable metastatic lesion according to RECIST4. Tumor evaluation within 6 weeks before randomization Specific aims: Objective: The objective of the trial is to prove the clinical efficacy of lapatinib in patients with metastasizing breast cancer who exhibit HER2-positive circulating tumor cells (CTC) although the primary tumor tissue and/or biopsies from metastatic sites were investigated for HER2 status and showed HER2-negativity. Primary endpoint: Progression free survival Secondary endpoints: Overall response rateClinical benefit rateOverall survivalDynamic of CTCQuality of lifeSafety and tolerability of lapatinib Statistical methods: The primary endpoint will be analyzed by Kaplan-Meier method using the logrank test in order to compare the PFS distributions of the two arms. Efficacy, toxicity and other event rates are calculated, providing confidence intervals. In case of comparison between patient groups, these rates will be analyzed by Fisher's exact test or test. The Kaplan Meier analysis for all event related data will be carried out overall for the whole patient population. Furthermore a Cox regression analysis will be done using the following covariates Hormone receptor status (positive/negative)Number of prior chemotherapy lines for metastatic diseasePrior endocrine therapy for metastatic diseaseEndocrine treatment vs. cytotoxic treatmentOne metastatic site vs. multiple metastatic sitesBone metastases vs. no bone involvementPerformance status ECOG Score (0/&amp;gt; 0) Present accrual and target accrual: As only half of the patients with HER2-negative metastatic breast cancer show CTC-positivity and of those approximately 32% will exhibit HER2-positive CTC (Fehm et al. 2010), screening of about 1420 patients is required to enroll 228 patients. First patient was screened in February 2012. As of July 27th 2012 117 patients were screened and 21 were found to have HER2-positive CTCs Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr OT1-1-10.

O2-034 - Detection and HER2 Expression of Circulating Tumor Cells in Advanced Gastric Cancer Patients

ABSTRACT Purpose This study was aimed at detecting and HER2-expressiong circulating tumor cells (CTC) in peripheral blood of chemo-naive or chemo-resistant patients with advanced gastric cancer. Materials and methods All patients were enrolled using institutional review board-approved protocols at the Cancer Institute Hospital in the Japanese Foundation for Cancer Research and provided informed consent. The study population consisted of patients of aged 18 years or older with histologically proven advanced gastric cancer. A total of 140 patients with advanced gastric cancer were enrolled into a prospective study. We used CellSearch system for the detection and HER2 expression for CTC. Results We detected more than or equal to 1 CTC/7.5 ml in 80 of 140 patients (57.1%). HER2-positive CTC were observed in 19 of 80 CTC-positive patients (23.8%), including six patients with HSR2-negative primary tumors. Twenty-one patients with HER2-positive primary tumors administered trastuzumab in combination with chemotherapy (Xeloda + cisplatin; 15, weekly paclitaxel; 5). We detected more than or equal to 1 CTC/7.5 ml at the baseline in 11 of 20 patients with trastuzumab treatment. HER2-positive CTCs were observed in 6 of 11 CTC-positive patients (54.5%). Patients with HER2-positive CTC at the baseline were shorter PFS than those with HER2-negative CTC at the baseline. Conclusion HER2 expression on CTC was associated with chemoresistance. Information on the HER2 status of CTC might be helpful for stratification of HER2-directed therapies.

Clinical role of detecting circulating tumor cells

Using ultrasensitive molecular or immunological/cytometric approaches, identification of circulating tumor cells (CTC) thus far undetectable at primary diagnosis even by high resolution imaging technologies is feasible. Results from several clinical studies revealed that in breast cancer patients with metastatic disease positivity for CTC is associated with worse prognosis. Thus, information obtained from CTC research might support clinicians in assessing individual prognosis, stratifying patients at risk to systemic adjuvant anti-cancer therapies, early response evaluation and monitoring therapeutic efficacy. At present, the CellSearchTM system combining automated capturing of CTC, immunostaining and image analysis is the only standardized technology approved by the FDA for the detection of CTC in patients with metastatic breast, colorectal and prostate cancer [1–3]. However, evidence has emerged that most currently used CTC detection methods still lack sensitivity or specificity to find all CTC especially those that have lost epithelial features, e.g. in the course of epithelial-mesenchymal transition [4–7]. Therefore special emphasis is placed to the development of innovative new strategies with the capacity to track additional CTC and to identify those with the potential to initiate metastasis. In this context, discovery of approaches enabling further phenotypical and molecular characterisation of CTC, e.g., analysis of single cell whole genomes, next generation sequencing and gene expression analysis on CTC is pivotal particularly in view of the development of new personalized targeting therapies. It is also worth mentioning that detection and characterization of CTC within translational research projects has entered clinical trials also enrolling patients with early breast cancer. First promising results on CTC detection especially within treatment regimens including targeted therapy, e.g., trastuzumab directed against HER2 in breast cancer patients receiving neoadjuvant or adjuvant therapy have been published recently [8–11]. Furthermore, characterization of the HER2 status of CTC might help identifying additional patients profiting from an anti-HER2 treatment and getting insights into therapy-induced selection of CTC.

Detection and HER2 expression of circulating tumor cells in advanced gastric cancer patients.

10541 Background: This study was aimed at detecting and HER2-expressiong circulating tumor cells (CTC) in peripheral blood of chemo-naïve or chemo-resistant patients with advanced gastric cancer. Methods: All patients were enrolled using institutional review board-approved protocols at the Cancer Institute Hospital in the Japanese Foundation for Cancer Research and provided informed consent. The study population consisted of patients of age 18 years or older with histologically proven advanced gastric cancer. A total of 140 patients with advanced gastric cancer were enrolled into a prospective study. We used CellSearch system for detection and HER2 expression for CTC. Results: We detected ≥1 CTC/7.5ml in 80 of 140 patients (57.1%). HER2-positive CTC were observed in 19 of 80 CTC-positive patients (23.8%), including 6 patients with HSR2-negative primary tumors. 21 patients with HER2-positive primary tumors administered trastuzumab in combination with chemotherapy (Xeloda+cisplatin; 15, weekly Paclitaxel; 5). We detected ≥1 CTC/7.5ml at the baseline in 11 of 20 patients with trastuzumab treatment. HER2-positive CTCs were observed in 6 of 11 CTC-positive patients (54.5%). Patients with HER2-positive CTC at the baseline were shorter PFS than those with HER2-negative CTC at the baseline. Conclusions: HER2 expression on CTC was associated with chemo-resistance. Information on the HER2 status of CTC might be helpful for stratification of HER2-directed therapies.

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

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

DETECT III: A multicenter, randomized, phase III study to compare standard therapy alone versus standard therapy plus lapatinib in patients (pts) with initially HER2-negative metastatic breast cancer but with HER2-positive circulating tumor cells (CTC).

TPS1146 Background: HER2 status may change over the course of disease in breast cancer pts. Approx. 20-30% of pts with initially HER2-negative breast cancer have HER2-positive metastasis (Zidan et al. 2005; Tewes et al. 2009). Determining HER2 status on CTC is one option to re-evaluate HER2 status at the time metastasis is diagnosed. Currently it is unclear if HER2-targeted therapy based on the assessment of HER2 status of CTC reveals a clinical benefit. Methods: This is a randomized, open-label, two arm phase III study to investigate the clinical efficacy of lapatinib, as a HER2-targeted therapy in initially HER2-negative metastatic breast cancer pts with HER2-positive CTC at the time of distant disease. As only half of the pts with HER2-negative metastatic breast cancer show CTC-positivity and of those approx. 32% will exhibit HER2-positive CTC (Fehm et al. 2010), screening of about 1420 pts is required to enroll 228 pts. Main inclusion criteria: metastatic breast cancer with HER2-negative primary tumor tissue and/or biopsies from metastatic sites or locoregional recurrences, evidence of ≥1 HER2-positive CTC and ≥1 measurable metastatic lesion according to RECIST. Eligible pts will be randomized 1:1 to receive standard treatment vs. standard treatment plus lapatinib. Standard chemo- or endocrine therapy must be approved in combination with lapatinib or been investigated in prior clinical trials. Primary endpoint is progression free survival. Secondary endpoints include overall response rate, clinical benefit rate, overall survival and dynamic of CTC. The DETECT III trial is one of the first trials where treatment is based on phenotypic characteristics of CTC. If this trial succeeds in proving efficacy of lapatinib in pts with initially HER2-negative metastatic breast cancer but HER2-positive CTC, this will establish a new strategy in the treatment of metastatic breast cancer.

Abstract 1217: Detection of HER2 expression and phosphorylation in breast cancer CTC samples by CEER™

Abstract Survival rates of metastatic breast cancers (mBCAs) are considerably low. Often, tumor cells at the primary site may not reflect the profile of the tumor cell population in recurrent disease. Circulating tumor cells (CTCs) isolated from peripheral blood offer a non-invasive disease monitoring modality. Identification of reliable molecular markers within (CTCs) from patients with recurrent disease may further improve breast cancer survival. Hence, we have developed a multiplexed immunoassay for monitoring activated HER2 protein and their expression levels using a multiplexed immuno-microarray platform. Collaborative Enzyme Enhanced Reactive-immunoassay (CEER™) was utilized to analyze HER2 profiles in CTCs isolated from 76 BCA patients (stages III to IV) with HER2 negative primary disease. The CEER technology utilizes the formation of a unique immuno-complex requiring co-localization of two detector antibodies around capture antibodies immobilized on immuno-array to profile HER2 protein expression and activation. The collaboration between two channeling-enzymes conjugated on two detection antibodies in proximity, enables the profiling of the target proteins with extreme sensitivity and specificity. Approximately 25% of HER2 negative BCA patients in this cohort showed varying levels of HER2 activation in CTCs isolated from the recurrent disease. About 8% of patients with HER2 activation in CTCs also showed significant HER2 over-expression. This suggests mechanisms of HER2 activation either through formation of receptor heterodimer (in samples without HER2 over-expression) or homodimer (in samples with HER2 overexpression) formation. The distinct HER2 discordance between primary tumor and recurrent disease demonstrates an urgent need for routine monitoring of HER2 status in CTCs found in mBCA patients. Incidence of HER2 alterations in CTCs should be considered in selecting effective treatment regimens for BCA patients with relapsed disease. Furthermore, CEER can be used for profiling other druggable targets for guiding effective clinical strategies including rational targeted agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1217. doi:1538-7445.AM2012-1217

Prognostic Role and HER2 Expression of Circulating Tumor Cells in Peripheral Blood of Patients Prior to Radical Cystectomy: A Prospective Study

BackgroundPreliminary research has suggested the potential prognostic value of circulating tumor cells (CTC) in patients with advanced nonmetastatic urothelial carcinoma of the bladder (UCB). ObjectiveProspectively analyze the clinical relevance and human epidermal growth factor receptor 2 (HER2) expression of CTC in patients with clinically nonmetastatic UCB. Design, setting, and participantsBlood samples from 100 consecutive UCB patients treated with radical cystectomy (RC) were investigated for the presence (CellSearch system) of CTC and their HER2 expression status (immunohistochemistry). HER2 expression of the corresponding primary tumors and lymph node metastasis were analyzed using fluorescence in situ hybridization. InterventionBlood samples were taken preoperatively. Patients underwent RC with lymphadenectomy. MeasurementsOutcomes were assessed according to CTC status. HER2 expression of CTC was compared with that of the corresponding primary tumor and lymph node metastasis. Results and limitationsCTC were detected in 23 of 100 patients (23%) with nonmetastatic UCB (median: 1; range: 1–100). Presence, number, and HER2 status of CTC were not associated with clinicopathologic features. CTC-positive patients had significantly higher risks of disease recurrence and cancer-specific and overall mortality (p values: ≤0.001). After adjusting for effects of standard clinicopathologic features, CTC positivity remained an independent predictor for all end points (hazard ratios: 4.6, 5.2, and 3.5, respectively; p values ≤0.003). HER2 was strongly positive in CTC from 3 of 22 patients (14%). There was discordance between HER2 expression on CTC and HER2 gene amplification status of the primary tumors in 23% of cases but concordance between CTC, primary tumors, and lymph node metastases in all CTC-positive cases (100%). The study was limited by its sample size. ConclusionsPreoperative CTC are already detectable in almost a quarter of patients with clinically nonmetastatic UCB treated with RC and were a powerful predictor of early disease recurrence and cancer-specific and overall mortality. Thus CTC may serve as an indication for multimodal therapy. Molecular characterization of CTC may serve as a liquid biopsy to guide individual targeted therapy in future clinical trials.

OT1-02-06: DETECT III – 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.

Abstract Background In breast cancer patients, HER2 status may change over the course of the disease. Approximately 20–30 % of initially HER2−negative patients have HER2−positive metastasis (Zidan et al. 2005, Tewes et al. 2009). Re-evaluation of HER2 status on metastatic tissue is warranted, but not always possible, especially during the course of therapy. Determining HER2 status on circulating tumor cells is one option for re-evaluating HER2 status at the time metastasis is diagnosed as described in our previous study DETECT I (Fehm et al. 2010). However, at present it is unclear if therapy based on the HER2 status of CTC offers a clinical benefit for patients. Therefore, the study DETECT III aims to assess whether lapatinib, as one of the HER2−targeted therapies, in initially HER2−negative breast cancer patients with HER2−positive CTC is effective at the time of distant disease. Trial Design DETECT III is a prospective, multicenter, randomized, open-label, two arm phase III study. As only half of the patients with HER2−negative MBC will be CTC-positive and approx. 32% will exhibit HER2−positive CTCs a screening of about 1420 patients will be needed. Approx. 228 patients will be enrolled in the study and randomized 1:1 to one of the following regimens Arm A (n=114): Standard Treatment, Arm B (n=114): Standard Treatment plus Lapatinib. Planned duration of individual study participation Max. duration of randomized treatment period: 12 months • Max. follow-up period: 24 months Main eligibility criteria 1. Metastatic breast cancer 2. HER2−negative primary tumor tissue and/or biopsies from metastatic sites or locoregional recurrences 3. Evidence of HER2−positive CTCs 4. Indication for a standard chemo- or endocrine therapy whose combination with lapatinib is either approved or has been investigated in prior clinical trials 5. Tumor evaluation within 6 weeks before randomization 6. ≥ 1 lesion that can be accurately measured according to RECIST 7. LVEF ≥ 50 Specific aims Objective The objective of the trial is to prove the clinical efficacy of lapatinib in patients with metastasizing breast cancer who exhibit HER2−positive circulating tumor cells (CTC) although the primary tumor tissue and/or biopsies from metastatic sites were investigated for HER2 status and showed HER2−negativity. Primary endpoint: Progression free survival Secondary endpoints: Overall response rate Clinical benefit rate Overall survival Dynamic of CTC Quality of life Safety and tolerability of lapatinib Statistical methods The primary endpoint will be analyzed by Kaplan-Meier method using the logrank test in order to compare the PFS distributions of the two arms. Efficacy, toxicity and other event rates are calculated, providing confidence intervals. In case of comparison between patient groups, these rates will be analyzed by Fisher's exact test or x2 test. The Kaplan Meier analysis for all event related data will be carried out overall for the whole patient population. Furthermore a Cox regression analysis will be done using the following covariates Hormone receptor status (positive/negative) Number of prior chemotherapy lines for metastatic disease Prior endocrine therapy for metastatic disease Endocrine treatment vs. cytotoxic treatment One metastatic site vs. multiple metastatic sites Bone metastases vs. no bone involvement Performance status (0 / &amp;gt; 0) Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr OT1-02-06.