Circulating Tumor Cells HER2 Research Articles

Early drop of circulating tumor cells (CTC) and increase of circulating endothelial cells (CEC) during neoadjuvant chemotherapy (CT) combined with bevacizumab in HER2-negative inflammatory breast cancer (IBC) in multicenter phase II trial BEVERLY 1.

10510 Background: CTC detection in blood is an independent prognostic factor in primary breast cancer with a detection rate from 20 to 25% (Pierga CCR 2008). Changes in the CTC HER2 status compared to primary tumor have been reported. Predictive value of CEC for response to anti-angiogenic agents is debated. Methods: CTC and CEC were detected in 7.5 ml and 4 ml of blood respectively in the neoadjuvant setting of HER2 negative IBC (T4d) patients (pts) enrolled in the phase II multicenter trial BEVERLY 1, evaluating bevacizumab (15mg/kg q3w) in combination with sequential neoadjuvant CT of 4 cycles of FEC followed by 4 cycles of Docetaxel. The CellSearch System, combining EpCAM immunomagnetic selection followed by anti-cytokeratin (A45B/B3) and anti-HER2 staining for CTC and CD146 IMS and CD105 staining for CEC. Results: From 12/08 to 09/10, 101 pts were included and 92 were evaluable for CTC and CEC. At baseline, 37 pts out of 92 had ≥ 1 detectable CTC (40%, 95%CI 30-50%). No correlation was found between ...

Circulating Tumor Cells in Breast Cancer Patients: An Evolving Role in Patient Prognosis and Disease Progression

In this paper, we examine the role of circulating tumor cells (CTCs) in breast cancer. CTCs are tumor cells present in the peripheral blood. They are found in many different carcinomas but are not present in patients with benign disease. Recent advances in theories regarding metastasis support the role of early release of tumor cells in the neoplastic process. Furthermore, it has been found that phenotypic variation exists between the primary tumor and CTCs. Of particular interest is the incongruency found between primary tumor and CTC HER2 status in both metastatic and early breast cancer. Overall, CTCs have been shown to be a poor prognostic marker in metastatic breast cancer. CTCs in early breast cancer are not as well studied, however, several studies suggest that the presence of CTCs in early breast cancer may also suggest a poorer prognosis. Studies are currently underway looking at the use of CTC level monitoring in order to guide changes in therapy.

Abstract S6-6: High Independent Prognostic and Predictive Value of Circulating Tumor Cells in a Large Prospective Multicenter Trial Including Serum Tumor Markers in First Line Chemotherapy Metastatic Breast Patients

Abstract Background: Circulating tumor cells (CTCs) in peripheral blood and decrease during treatment has been reported as an independent prognostic factor in metastatic breast cancer (MBC) (Cristofanilli M., NEJM 2004). Data comparing CTC with circulating tumor markers are limited. We conducted a multicenter prospective study to validate these markers in a large series of patients receiving specifically first line chemotherapy (CT) for MBC including assessment of CA15.3, CEA and LDH. Methods: CTC were measured in 7.5ml of whole blood at baseline, before cycle 2, at clinical and/or radiological evaluation (C3-C4) and at tumor progression. Analysis was performed using the CellSearch™ System, combining EpCAM immunomagnetic selection (IMS) followed by anti-cytokeratin (A45B/B3). Double labeling for HER2 was performed in a subgroup of patients. Tumor markers were assessed locally with ELISA kit and high if above normal. Results: From 06/07 to 9/09, 267 patients with measurable or evaluable disease starting first line CT for MBC were included in five French Cancer Centers. Median follow-up was 16 months, 57 deaths and 160 progressions were observed. Median age was 57 years, 39 patients (15%) had HER2+ tumor and received trastuzumab, 55 (21%) had triple negative tumor, 125 received bevacizumab associated with CT, 260 were evaluable for CTC levels at baseline, 170 (65%) had ≥1CTC and 115 (44%) ≥5CTC. CTC HER2 status was assessed in 113 patients at baseline and was positive in 64% of the cases in at least one cell. CA15.3 was high in159/247 (64%), CEA in 109/212 (51%) and LDH in 99/220 (45%). CTC were highly correlated with tumor markers, tumor burden, performans status (PS), number of metastatic sites, liver metastasis) but were independent of tumor biology (HER2 status, Triple Negative BC (TNBC), grade). At first evaluation (C3-C4), 110/225 patients (49%) had objective response. At univariate analysis, high CTC level with a threshold of 1 or 5 at base line, before C2 (3-4w) and at radiological evaluation (C3-C4, 9-12w) was predictive of poor progression-free survival (PFS) and overall survival (OS) (p<10-5). Baseline CA15.3, CEA and LDH were prognostic for PFS and LDH only for OS. At multivariate analysis, TNBC, PS ≥2, CTC ≥5, and high CEA were prognostic for PFS and TNBC, high LDH, CTC ≥5 and PS ≥2 for OS. Multivariate analyses including CTC and tumor markers variations during treatment are ongoing. Conclusions: This is the largest prospective series validating the prognostic value of CTC in first line chemotherapy MBC independently from serum tumor marker for PFS and OS. Persistence of a high level of CTC before second cycle of chemotherapy is a strong and early predictive marker of poor PFS, before radiological evaluation. A prospective multicenter randomized trial (CIRCE01) to assess the use of CTC to modify treatment after second line chemotherapy is ongoing. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr S6-6.

Changes of HER2 Status in Circulating Tumor Cells Compared With the Primary Tumor During Treatment for Advanced Breast Cancer

Background HER2/ neu status of tumor cells at metastatic sites in patients with advanced disease may differ from that of the primary tumor. Assessing the presence of target antigens on circulating tumor cells (CTCs) might affect treatment choice. Patients and Methods From June 2007 to October 2008, we collected 23 mL of blood from each of the 76 consecutive patients before and during chemotherapy to determine CTC numbers and HER2 overexpression. CTCs were isolated with the CellSearch System® (Veridex, LLC; Raritan, NJ) and fluorescently stained with the Epithelial Cell Kit®. Tumor Phenotyping Reagent® was used to investigate HER2/ neu overexpression. Results Concordance of HER2 status between the primary tumor and CTCs was 86% (49 out of 57 patients) at baseline and 82% (50 out of 61 patients) in the treatment samples. HER2 overexpression in CTCs was acquired in 8 out of 45 patients (18%) and lost in 3 out of 16 patients (19%) during a treatment containing trastuzumab. The overall discordance rate between the primary tumor and CTCs was 18% (11 out of 61 patients). Patients with HER2 overexpression in CTCs had poorer progression-free survival compared with those without CTCs or with HER2− CTCs (log-rank P =.036). Conclusion Information on the presence or absence of HER2 overexpression can be obtained in CTCs. Larger trials are needed to evaluate the activity of HER2-targeted therapy in patients with acquired HER2 overexpression in CTCs.

Prospective multicentric trial evaluating prognostic and predictive value of circulating tumor cells in first-line chemotherapy metastatic breast patients.

10544 Background: Detection of five or more circulating tumor cells (CTCs) per 7.5 ml in peripheral blood has been reported as an independent prognostic factor in metastatic breast cancer (MBC). A decrease in CTC during treatment could be associated with a better outcome (Cristofanilli, NEJM 2004). We conducted a multicentric prospective study to validate this marker in a large series of patients receiving specifically first line chemotherapy for MBC. Methods: CTC were measured in 7.5 ml of whole blood at baseline, before cycle 2, at clinical and/or radiological evaluation (C3-C4) and at tumor progression. Analysis was performed using the CellSearch System, combining EpCAM immunomagnetic selection (IMS) followed by anti-cytokeratin (A45B/B3) staining for CTC. Double labeling for HER2 was performed in a subgroup of patients. Results: From 06/07 to 9/09, 271 patients with measurable or evaluable disease were included in five French Cancer Centers. 261 were evaluable for CTC levels at baseline, before first cycle of chemotherapy. At baseline, median number of CTC was 2 (range 0-16 885). After one cycle of chemotherapy, median CTC number was 1 (range: 0-202). Incidence of CTC during treatment is shown in the Table. CTC HER2 status was assessed in 117 patients at baseline and was positive in 62% of the cases in at least one cell. Correlation with other clinical and biological parameters as serum marker (CA15.3) is ongoing. 18 months median follow-up will be reached in April 2010. Conclusions: We observed a clear decrease of CTC level after only one cycle of chemotherapy. These findings support the role of CTC enumeration as an adjunct to standard methods of monitoring disease status in MBC. However, about one-third of the patients with MBC had undetectable CTC level during first line treatment and even at tumor progression, suggesting a need for more sensitive methods of detection. Baseline C2 C3-C4 Progression ≥ 1 CTC 169 (65%) 77 (37%) 59 (28%) 20 (46%) ≥ 5 CTC 113 (43%) 38 (18%) 25 (12%) 13 (30%) Number of patients 261 209 210 43 Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Veridex Veridex

Multiple biomarker expression in circulating tumor cells (CTCs) from locally advanced/inflammatory (LA/IBC) and metastatic breast cancer (MBC) patients (pts)

1092 Background: Numeration of CTCs from MBC pts is predictive of outcome. Quantitative changes in CTC-s are currently tested for their potential to monitor therapy (Rx). Biomarker characterization of CTCs may be a useful adjunctive guide for Rx selection. Methods: At the COHCC between May 1, 2008 and December 31, 2008, consecutively treated pts with LABC/IBC, or with newly diagnosed/progressing MBC were accrued. Blood samples (20–30 mL) were procured prior to initiating neoadjuvant (neo)Rx (LABC and IBC patients) or systemic Rx (MBC), and were sent to PARC for analysis. A novel high-speed scanning instrument located CTCs from cytokeratin (CK) labeling enabling high resolution images to be selectively acquired using digital microscopy. From these images, CTCs were identified by CK, DAPI (nuclear marker) and CD45, and protein expression levels were determined for HER-2, estrogen receptor (ER), Excision repair cross-complementation group 1 (ERCC1), and EGFR. Cell lines with expression of each marker were used for normalization of the cell intensities, and a scoring system was used to account for relative number and expression levels of markers on the CTCs. Results: Twenty-seven pts with LABC, 4 pts with IBC, and 11 pts with MBC were enrolled. We have observed CTCs prior to initiating neoRx in all pts with IBC relative to 39% of all LABC/IBC cases, and in 57% of pts (n:14) with HER-2+ primary BC versus 24% (n = 17) with HER-2- BC . ER status, size, or grade did not predict for CTC detection. Numeration of CTCs was seen in 45% of pts with MBC. Expression of EGFR and ERCC1 was detected in 3 of 4, and 2 of 4 tested CTC samples from MBC cases. Expression of HER-2 and ER was observed on 1 of 3 and 3 of 4 CTC samples; there was discrepancy between the CTC expression profile and HER-2 and ER status of the primary BC in one case each. Conclusions: Detecting multiple markers in CTCs from pts with MBC is feasible, and similar testing in LABC/IBC patients is needed. Such multiplex testing may allow for more personalized Rx for pts with LABC/IBC and MBC. No significant financial relationships to disclose.

Prognostic Value of the Molecular Detection of Circulating Tumor Cells Using a Multimarker Reverse Transcription-PCR Assay for Cytokeratin 19, Mammaglobin A, and HER2 in Early Breast Cancer

To investigate the prognostic value of the molecular detection of circulating tumor cells (CTCs) using three markers [cytokeratin 19 (CK19), mammaglobin A (MGB1), and HER2] in early breast cancer. CK19mRNA+, MGB1mRNA+, and HER2mRNA+ cells were detected using real-time (CK19) and nested (MGB1 and HER2) reverse transcription-PCR in the peripheral blood of 175 women with stage I to III breast cancer before the initiation of adjuvant chemotherapy. The detection of CTCs was correlated with clinical outcome. In 10 patients, immunofluorescence staining experiments were done to investigate the coexpression of cytokeratin, MGB1, and HER2 in CTCs. CK19mRNA+, MGB1mRNA+, and HER2mRNA+ cells were detected in 41.1%, 8%, and 28.6% of the 175 patients, respectively. Patients had one of the following molecular profiles: CK19mRNA+/MGB1mRNA+/HER2mRNA+ (n = 8), CK19mRNA+/MGB1mRNA+/HER2mRNA- (n = 1), CK19mRNA+/MGB1mRNA-/HER2mRNA+ (n = 42), CK19mRNA+/MGB1mRNA-/HER2mRNA- (n = 21), CK19mRNA-/MGB1mRNA+/HER2mRNA- (n = 5), and CK19mRNA-/MGB1mRNA-/HER2mRNA- (n = 98). Double-immunofluorescence experiments confirmed the following CTC phenotypes: CK+/MGB1+, CK+/MGB1-, CK-/MGB1+, CK+/HER2+, CK+/HER2-, MGB1+/HER2-, and MGB1+/HER2+. In univariate analysis, the detection of CK19mRNA+, MGB1mRNA+, and HER2mRNA+ cells was associated with shorter disease-free survival (DFS; P < 0.001, P = 0.001, and P < 0.001, respectively), whereas the detection of CK19mRNA+ and MGB1mRNA+ cells was associated with worse overall survival (P = 0.044 and 0.034, respectively). In multivariate analysis, estrogen receptor-negative tumors and the detection of CK19mRNA+ and MGB1mRNA+ cells were independently associated with worse DFS. The detection of peripheral blood CK19mRNA+ and MGB1mRNA+ cells before adjuvant chemotherapy predicts poor DFS in women with early breast cancer.