P4-07-09: Automated Quantitative Assessment of HER2 Expression of Circulating Tumor Cells (CTC) in Metastatic Breast Cancer (IC 2006–04 Study)

Background Beyond quantitative CTC analysis for outcome assessment, their qualitative analysis could predict drug response and resistance. Classification of CTC and quantification of treatment targets on CTC can be subjective as they are morphologically heterogeneous. Current HER2 fluorescence assessment methods rely on a visual comparison between CTC images and external controls (cells lines) and are poorly reproducible. We report an automated classification and quantification of HER2 expression on CTC in a large cohort of metastatic breast cancer (MBC) patients (pts). Materials and Methods: CTC enumeration and HER2 assessment was performed with the CellSearch® system in pts with MBC included in the prospective IC 2006–04 study (Pierga, Ann Oncol 2011), before first line chemotherapy. The FITC conjugated antibody CB11 was used to identify HER2. For quantitative assessment of HER2 the breast cancer lines SKBR3 and MCF7 with known HER2 status were spiked in blood of normal donors and processed with the CellSearch system. Digital images of Cytokeratin-PE, DAPI, CD45-APC, and HER2−FITC from these samples were stored. An algorithm, previously developed in prostate cancer (Ligthart, AACR 2011), was applied on these images to automate identification of CTC. The fluorescence of the HER2 FITC channel for each CTC as well as the CD45+, DAPI+ leukocytes contained within each sample were also quantified using this algorithm. HER2 fluorescence levels of CTC were compared to HER2 status of status of pts. Results: Among 267 pts included in this study, 103 had at least 1 CTC/7.5 ml and HER2 fluorescence assessment. In these pts, correlation analysis between automated (aCTC) and manual (mCTC) CTC counts showed an excellent correlation (R2 =0.978). HER2 fluorescence of CTC, varied greatly within and between pts, whereas leukocyte fluorescence was more homogeneous. The vast majority of CTC exhibited less fluorescence than SKBR3 cells, even in HER2+ pts. Primary tumor was HER2+ and HER2− in 36 and 58 pts respectively. Percentage of HER2+ CTC was higher in pts with primary HER2+ status. Although a rare event, the presence within a sample of at least 1 CTC whose HER2 expression is superior to SKBR3 cells was also strongly associated with HER2+ primary tumor (N=15/36 vs. 3/58; p Conclusion: This study is the first to assess HER2 staining intensity in each CTC isolated from a large number of MBC pts. This was made possible by the aCTC count that was highly correlated with the mCTC count in our cohort and permitted HER2 quantification. Our study shows that HER2 staining is highly heterogeneous among CTC within each pt. These findings demonstrate the feasibility of real time quantitative assessment of treatment targets on CTC and opens a path towards personalized treatment. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-07-09.