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

Detection and characterisation of disseminated tumour cells in bone marrow of breast cancer patients by immunostaining of Her-2 and MUC-1 in combination with Thomsen-Friedenreich (CD176)

Disseminated tumour cells (DTCs) in the bone marrow derive from many primary tumours, such as breast cancer. Their mere existence hints to present or future metastasis and implicates a worse prognosis for the patient. DTCs may possess different characteristics in comparison to the primary tumour due to events like Epithelial-Mesenchymal-Transition. Therefore these cells might be able to survive chemotherapy and cause relapses of the disease at a later point. We aimed to detect and further characterise DTCs by an immunostaining approach with three different antigen markers (Her-2, MUC-1 and TF, also known as CD176). For that reason, bone marrow of 41 breast cancer patients was obtained during surgery; DTCs were enriched by density gradient centrifugation and cytospins were prepared. After fixation, immunofluorescent double-stainings were carried out with antibodies against CD176 in combination with HER-2 or MUC-1. Cells co-expressing two antigens were found in all staining combinations (Her-2 and CD176: 46.14%; MUC-1 and CD176: 18.15% of all cases). Cells that stained for a single antigen only were also found (Her-2: 36.86%; MUC-1: 34.45%; CD176: 29.65% of all cases). Significant correlations between the stainings of all markers could be shown (p<0,001). In conclusion, Thomsen-Friedenreich Antigen (TF, CD176) is a promising marker in combination with the established marker Her-2 and other markers like MUC-1. These results may serve as a basis for future DTC detection routines and help to individualize medical treatment, reducing side effects and increasing the efficiency of the therapy