P4-06-04: Detection of HER2 Gene Amplification in Circulating Tumor Cells and Disseminated Tumor Cells by Fluorescence In Situ Hybridization Using OncoCEE™.

Abstract

BACKGROUND: The status of HER2 gene amplification in circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) might provide useful information for monitoring response to trastuzumab therapy, and may provide a basis for consideration of trastuzumab in patients with HER2 negative primary tumors who have HER2 positive CTCs and/or DTCs. The majority of techniques utilized for detection of minimal residual disease are limited in their ability to allow detailed phenotypic and genotypic evaluation of the cells. We report the utility of a microfluidic platform (OncoCEE™,Biocept, San Diego) for detecting HER2 gene amplification in CTCs and DTCs in patients with non-metastatic breast cancer. METHODS: Peripheral blood (10ml) and bone marrow (BM) (1-2ml) were collected from patients with clinical stage I-III breast cancer in acid citrate dextrose solution (BD, Franklin Lakes, NJ) and anti-clumping reagent (OncoCEE-Sure™). Mononuclear cells were recovered using a Percoll density gradient method, incubated with a mixture of 10 primary capture antibodies (Abs), introduced into CEE™ microchannels, stained with fluorescent anti cytokeratin (CK) and CD45 abs and finally processed for fluorescence in situ hybridization (FISH) using probes specific to centromere 17 (spectrum green) and HER2 (spectrum arrange). The ratio of HER2:CEP17 >2.2 in any CK+/CD45- and CK-/CD45- cell was regarded as positive for HER2 gene amplification. RESULTS: Peripheral blood and/or BM from 78 patients (65 BM; 70 blood; 57 matched blood and BM) with T1NO (39), T1N1 (8), T2N0 (12), T2N1 (2), T2N2 (1), T2N3 (3), T3N0 (2), T3N1 (2), T3N2 (1), T4N0(5), T4N1 (3), with HER2+ (n=12) and HER2− (n=58) primary invasive breast tumors were studied. The 12 patients with HER2+ primary tumors had HER2+ DTCs in 3/12 (25%) and HER2+ CTCs in 1/9 (11%) cases respectively. HER2+ DTCs and HER2+ CTCs occurred in 12/55 (24%) and in 4/63 (6%) of the patients with HER2− primary breast tumors. HER2+ CTCs and DTCs occurred simultaneously in only 2 patients and in either blood (3) or BM (13) in the remaining patients. CONCLUSION: 1. The cell enrichment and extraction microfluidic technology (OncoCEE™) provides a sensitive platform for evaluation of HER2 gene amplification of CTCs and DTCs. 2. HER2+ primary tumors were associated with either HER2+ CTCs or DTCs in 25% of the patients. 3. HER2+ CTCs or DTCs occurred in 28% of patients with HER2−primary tumor. 4. Discordant HER2 status was contributed mainly by HER2+ DTCs occurring in HER2 - primary tumors. 5. The clinical significance of evaluating the status of HER2 gene amplification in CTCs and DTCs in the management of patients with breast cancer needs to be evaluated prospectively in larger clinical trials. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-06-04.