HER-2 expression in CTCs in each of the 132 patients.

<p>Patients are sorted according to the percentage of HER-2+ CTCs using a threshold of (>0). HER-2 negative CTCs are colored in green, dim HER-2 in blue (0 < HER-2 < 100) and bright HER-2 (≥100) CTCs red.</p

Number of manually scored HER-2+ cells versus number of automatically scored HER-2+ cells using a threshold of >0.

<p>Samples of 132 patients were investigated.</p

Comparisons of HER-2 assessment by different sites using ACCEPT versus CellTracks Analyzer II^® (Menarini Silicon Biosystems Inc) visualization.

<p>Indicated are the percentages of scored CTCs where all, five, four or only three out of six investigators agreed on the HER-2 status. In the case of 3 agree indifferent means that three investigators vote for HER-2 positive and the other three for HER-2 negative.</p

Expression of Cytokeratin and HER-2 on 4084 CTCs in 132 breast cancer patients.

<p>HER-2 negative CTCs are colored in green, dim HER-2 (0 < HER-2 < 100) in blue and bright HER-2 (≥100) in red.</p

Sample Visualizer of ACCEPT.

<p>In the scatter plots 3 of the 158 objects are depicted blue and the corresponding thumbnail images are highlighted. In this example ‘Marker1’ represents signals for HER-2. The corresponding HER-2 images are shown below the Sample Visualizer, in the right image the red line indicates the boundary detected by ACCEPT of the identified CTC and the number indicates the median value of the HER-2 staining within this boundary. Size bar in overlay: 6.4μm.</p

Cytokeratin and HER-2 mean intensities of cells in breast cancer cell lines MDA-MB 231, MDA-MB 453 and SKBR-3.

<p>Panel A, 373 MDA-MB 231 cells (magenta), 496 MDA-MB 453 cells (cyan), 361 SKBR-3 cells (orange). Average number of HER-2 antigens included for each cell line. Panel B classification of the MDA-MB 231, MDA-MB 453 and SKBR-3 into 428 negative HER-2 (green), 462 dim HER-2 (blue) and 340 bright HER-2 (red) expressing cells identified by cluster analysis.</p

Additional file 1: Figure S1. of Methylation patterns in serum DNA for early identification of disseminated breast cancer

Samples from the SUCCESS trial analyzed within this study. Figure S2. Samples from the UKCTOCS cohort analyzed within this study (nested case/control setting). Figure S3. Absolute pattern counts for all patterns detected in the region of marker EFC#93 in Serum Set 1 samples. Figure S4. Pattern frequency of EFC#93 serum DNAme in two prospectively independently collected cohorts. Figure S5. DNA amount per mL serum in the prospectively collected serum (Set 1 and 2), SUCCESS cohort, and UKCTOCS cohort. Figure S6. Pattern frequency for EFC#93 measured in SUCCESS serum set samples from women with no, 1–4 or ≥ 5 CTCs in the matched blood sample before (A) or after (B) chemotherapy. Figure S7. Impact of the presence (+ve, ≥ 1 cancer cell in blood sample) or absence (-ve) of CTCs on patient outcome. Figure S8. Impact of the presence (+ve, EFC#93 pattern frequency ≥ 0.00008) or absence (-ve) of serum DNA methylation in CTC + ve (≥1 cancer cell in pre-chemotherapy blood sample) or absence CTC-ve patients. Figure S9. Relapse-free and overall survival according to samples taken after chemotherapy. Figure S10. Relapse-free and overall survival according to samples taken after chemotherapy. Figure S11. Average serum DNA amount correlates with average UK temperature. Figure S12. Average serum DNA fragment size correlates with average UK temperature. Figure S13. Correlation of DNA fragment size and DNA amount. Figure S14. Overall survival of women whose samples were taken before and after chemotherapy and before anti-hormonal treatment in hormone receptor-negative and -positive SUCCESS participants. Figure S15. Overall survival of women whose samples were taken before and after chemotherapy and before anti-hormonal treatment in hormone receptor-negative and -positive SUCCESS participants. (PDF 2123 kb

Additional file 3: Table S1. of Methylation patterns in serum DNA for early identification of disseminated breast cancer

Coordinates and primers used to amplify the identified target regions using bisulfite sequencing. (PDF 578 kb