Research Article: HER2-Positive Circulating Tumor Cells in Breast Cancer

Date Published: January 10, 2011

Publisher: Public Library of Science

Author(s): Michail Ignatiadis, Françoise Rothé, Carole Chaboteaux, Virginie Durbecq, Ghizlane Rouas, Carmen Criscitiello, Jessica Metallo, Naima Kheddoumi, Sandeep K. Singhal, Stefan Michiels, Isabelle Veys, José Rossari, Denis Larsimont, Birgit Carly, Marta Pestrin, Silvia Bessi, Frédéric Buxant, Fabienne Liebens, Martine Piccart, Christos Sotiriou, Syed A. Aziz.

Abstract: Circulating Tumor Cells (CTCs) detection and phenotyping are currently evaluated in Breast Cancer (BC). Tumor cell dissemination has been suggested to occur early in BC progression. To interrogate dissemination in BC, we studied CTCs and HER2 expression on CTCs across the spectrum of BC staging.

Spiking experiments with 6 BC cell lines were performed and blood samples from healthy women and women with BC were analyzed for HER2-positive CTCs using the CellSearch®.

Based on BC cell lines experiments, HER2-positive CTCs were defined as CTCs with HER2 immunofluoresence intensity that was at least 2.5 times higher than the background. No HER2-positive CTC was detected in 42 women without BC (95% confidence interval (CI) 0–8.4%) whereas 4.1% (95%CI 1.4–11.4%) of 73 patients with ductal/lobular carcinoma in situ (DCIS/LCIS) had 1 HER2-positive CTC/22.5 mL, 7.9%, (95%CI 4.1–14.9%) of 101 women with non metastatic (M0) BC had ≥1 HER2-positive CTC/22.5 mL (median 1 cell, range 1–3 cells) and 35.9% (95%CI 22.7–51.9%) of 39 patients with metastatic BC had ≥1 HER2-positive CTC/7.5 mL (median 1.5 cells, range 1–42 cells). In CTC-positive women with DCIS/LCIS or M0 BC, HER2-positive CTCs were more commonly detected in HER2-positive (5 of 5 women) than HER2-negative BC (5 of 12 women) (p = 0.03).

HER2-positive CTCs were detected in DCIS/LCIS or M0 BC irrespective of the primary tumor HER2 status. Nevertheless, their presence was more common in women with HER2-positive disease. Monitoring of HER2 expression on CTCs might be useful in trials with anti-HER2 therapies.

Partial Text: Minimal residual disease (MRD) after primary surgery is thought to contribute to disease relapse in early breast cancer (BC) [1]; [2]. The presence of peripheral blood Circulating Tumor Cells (CTCs) is considered as a surrogate marker for the evaluation of MRD. CTCs are epithelial tumor cells detected in the peripheral blood of patients with solid tumors using mainly cytometric/antibody-based and molecular approaches [1]; [2]. Currently, CellSearch® is the only technology for CTC detection that has been cleared by the Food and Drug administration (FDA) as an aid in the monitoring of patients with metastatic breast, colorectal and prostate cancers. The detection of ≥5 CTCs/7.5 mL of peripheral blood before starting a new treatment in metastatic BC was associated with worse clinical outcome [3]. Data for CTC detection and phenotyping in early BC by CellSearch® are only now emerging. In the German Success trial, ≥1 CTC/23 mL of peripheral blood were detected in 20% and 21% of approximately 1500 patients with early BC before or after adjuvant chemotherapy, respectively [Rack B, et al. 2010 J Clin Oncol 28:7s; suppl; abstr 1003]. Two multicenter neoadjuvant studies (Remagus II and GeparQuattro) and two single center studies have used CellSearch® for CTC detection in early BC [4]–[7]. In the Remagus II study, detection of ≥1 CTC/7.5 mL before, after neoadjuvant chemotherapy or at both time points was associated with worse distant metastasis free survival (DMFS) and overall survival (OS) at a median follow-up of 36 months [8]. In the GeparQuattro trial, HER2 overexpressing CTCs were observed in 14 of 58 CTC-positive cases (24.1%) including 8 patients with HER2-negative primary tumors and 3 patients after neoadjuvant trastuzumab. Several groups have reported the detection of bone marrow HER2-positive DTCs or peripheral blood HER2-positive CTCs in BC using different detection methods [9]–[15].

In the present study ≥1 CTC/22.5 mL of blood were detected in 7.1% of women without BC, 8.2% of women with DCIS/LCIS and 11.9% of women with M0 BC. Pierga et al. detected ≥1 CTC/7.5 mL in 23% of 97 patients before the administration of neoadjuvant chemotherapy and in 17% of 86 patients after the administration of neoadjuvant chemotherapy [4]. Riedthorf et al. detected ≥1 CTC/7.5 mL in 21.6% of 213 patients before neoadjuvant treatment (NT) and in 10.6% of 207 patients after NT [5]. Sandri et al. detected one to three CTCs/30 mL in approximately 30% of 56 patients with localized BC before surgery [7]. Lang et al. detected ≥1 CTC/30 mL of blood in 38% of 92 patients [6]. In the SUCCESS study, ≥1 CTC/23 mL of peripheral blood were detected in 20% and 21% of patients before or after adjuvant chemotherapy, respectively in a cohort of approximately 1500 patients with early BC [Rack B, et al. 2010 J Clin Oncol 28:7s; suppl; abstr 1003]. In the above studies, blood draws were performed before primary surgery with the exception of the SUCCESS study in which blood draws were performed 3 weeks after surgery and at the end of chemotherapy. The lower CTC detection rates in M0 BC in our series could be due: (1) to the fact that blood draw was not performed before primary surgery but instead 3 weeks after surgery up to 5 years from the diagnosis, (2) a step of ficoll enrichment was performed which is known to lead to cell loss [25], (3) only cells meeting the stringent criteria for CTC definition by CellSearch® were considered positive after an independent blind review by Veridex. Although a direct comparison between different studies cannot be performed, the CTC detection rate when analyzing 22.5 mL of blood by CellSearch® after a modified ficoll procedure in our study was not higher than the CTC detection in 7.5 mL of blood by Pierga et al [4] and Riedthorf et al [5]. Our results question the utility of applying a modified ficoll procedure, before CellSearch®, which may additionally increase complexity and raise issues of reproducibility among laboratories.