International study on inter-reader variability for circulating tumor cells in breast cancer.

INTRODUCTION: Circulating tumor cells (CTCs) have been studied in breast cancer with the CellSearch® system. Given the low CTC counts in non-metastatic breast cancer, it is important to evaluate the inter-reader agreement. METHODS: CellSearch® images (N = 272) of either CTCs or white blood cells or artifacts from 109 non-metastatic (M0) and 22 metastatic (M1) breast cancer patients from reported studies were sent to 22 readers from 15 academic laboratories and 8 readers from two Veridex laboratories. Each image was scored as No CTC vs CTC HER2- vs CTC HER2+. The 8 Veridex readers were summarized to a Veridex Consensus (VC) to compare each academic reader using % agreement and kappa (κ) statistics. Agreement was compared according to disease stage and CTC counts using the Wilcoxon signed rank test. RESULTS: For CTC definition (No CTC vs CTC), the median agreement between academic readers and VC was 92% (range 69 to 97%) with a median κ of 0.83 (range 0.37 to 0.93). Lower agreement was observed in images from M0 (median 91%, range 70 to 96%) compared to M1 (median 98%, range 64 to 100%) patients (P < 0.001) and from M0 and <3CTCs (median 87%, range 66 to 95%) compared to M0 and ≥3CTCs samples (median 95%, range 77 to 99%), (P < 0.001). For CTC HER2 expression (HER2- vs HER2+), the median agreement was 87% (range 51 to 95%) with a median κ of 0.74 (range 0.25 to 0.90). CONCLUSIONS: The inter-reader agreement for CTC definition was high. Reduced agreement was observed in M0 patients with low CTC counts. Continuous training and independent image review are required.

HER2-positive circulating tumor cells in breast cancer.

PURPOSE: 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. METHODS: 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®. RESULTS: Based on BC cell lines experiments, HER2-positive CTCs were defined as CTCs with HER2 immunofluorescence 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). CONCLUSION: 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.

Knockout mice reveal a role for P2Y6 receptor in macrophages, endothelial cells, and vascular smooth muscle cells.

P2Y receptors are G-protein-coupled receptors activated by extracellular nucleotides. The P2Y(6) receptor is selectively activated by UDP, and its transcript has been detected in numerous organs, including the spleen, thymus, intestine, blood leukocytes, and aorta. To investigate the biological functions of this receptor, we generated P2Y(6)-null mice by gene targeting. The P2Y(6) knockout (KO) mice are viable and are not distinguishable from the wild-type (WT) mice in terms of growth or fertility. In thioglycollate-elicited macrophages, the production of inositol phosphate in response to UDP stimulation was lost, indicating that P2Y(6) is the unique UDP-responsive receptor expressed by mouse macrophages. Furthermore, the amount of interleukin-6 and macrophage-inflammatory protein-2, but not tumor necrosis factor-alpha, released in response to lipopolysaccharide stimulation was significantly enhanced in the presence of UDP, and this effect was lost in the P2Y(6) KO macrophages. The endothelium-dependent relaxation of the aorta by UDP was abolished in KO P2Y(6) mice. The contractile effect of UDP on the aorta, observed when endothelial nitric-oxide synthase is blocked, was also abolished in P2Y(6)-null mice. In conclusion, we generated P2Y(6)-deficient mice and have shown that these mice have a defective response to UDP in macrophages, endothelial cells, and vascular smooth muscle cells. These observations might be relevant to several physiopathological conditions such as atherosclerosis or hypertension.