supplementary figure 1. supplementary figure 2 all patients characteristicn% median age of patients...
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ROC curve for CTC Summation Method
100% - Specificity%
Sen
siti
vity
%
0 50 1000
50
100
150
AUC-ROC= 0.9507 (95% CI: 0.9012 to 1.000)P<0.0001
Supplementary Figure 2
All patients
Characteristic N %
Median Age of patients
58 yrs
ER
Positive 40 69
Negative 18 31
PR
Positive 29 50
Negative 29 50
HER2
Positive 7 12
Negative 51 88
Status
Stable 18 31
Progression 40 69
Supplementary Table 1: Classification of patients based on receptor and disease status
Assay Blood volume per test CTC enrichment principle Detection method
CellSearch Method 7.5 mL Immunomagnetic using EpCAMVisual confirmation of CK+/DAPI+/CD45- cells using automated fluorescence imaging
CSV Method7.5 mL (for comparative analysis)
Depletion of CD45+ cells and positive selection of cell-surface vimentin positive cancer cells using 84-1 antibody
Visual confirmation of CSV+/Cancer specific marker using Confocal Imaging
Supplementary Table 2 Comparison between two detection techniques
Abbreviations: CSV= Cell-surface vimentin, CK= Cytokeratin,
K coefficient Degree of Agreement Agreement (%)
Comparison of Progressive population with the CellSearch CTC test
-0.017 Poor 45%
Comparison of Stable population with the CellSearch CTC test
0.440 Moderate 66.67%
Supplementary Table 3 Concordances between CSV and CellSearch detection techniques
Abbreviations: CSV= Cell-surface vimentin, CTC= Circulating tumor cells.
Triple Negative Luminal A Luminal B HER2 Type
CTCs<5 Stable Progression Stable Progression Stable Progression Stable Progression
CSV < 5 4 4 10 2 2 0 1 0
CellSearch < 5 2 8 10 12 2 1 1 0
CTCs≥ 5
CSV ≥ 5 0 8 1 22 0 3 0 1
CellSearch ≥ 5 2 4 1 12 0 1 0 1
CSV: Cell-surface vimentin; CTCs: Circulating tumor cells; ER: Estrogen receptor; PR: Progesterone receptor ; HER2: HER2/neu receptor; Triple negative: ER- PR- HER2-; Luminal A: ER/PR+ HER2- Luminal B: ER/PR+ HER2+, HER2 Type: ER/PR- HER2+
Supplementary Table 4: Receptor based classification for individual samples with stable/progression status as determined by both techniques
Triple Negative (n=16)
Sensitivity SpecificityPositive Predictive
ValueNegative Predictive
Value
CSV 66.67% 100% 100% 50%
CellSearch 33.34% 50% 66.67% 20%
Summation (Th 5) 83.33% 25% 76.92% 33.33%
Summation (Th 8) 83.33% 75% 90.91% 60%
Luminal A (n=35)
CSV 91.67% 90.91% 95.65% 83.33%
CellSearch 50% 90.91% 92.3% 45.45%
Summation (Th 5) 95.83% 72.73% 88.46% 88.89%
Summation (Th 8) 95.83% 81.82% 92% 90%
Supplementary Table 5: Analysis of Sensitivity, Specificity, Positive Predictive Value, and Negative Predictive Value based on receptor status of patients
CSV: Cell-surface vimentin Summation: sum of CTC counts from 84-1 and CellSearch methods. Triple negative: ER- PR- HER2-; Luminal A: ER/PR+ HER2- Th 5 indicates a threshold of 5 CTCs/ 7.5 mL as cutoff.Th 8 indicates a threshold of 8 CTCs/ 7.5 mL as cutoff.
n=20 Progression Stable
Nuclear EpCAM 13 1
No Nuclear EpCAM 2 4
Supplementary Table 6: Analysis of Nuclear EpCAM Status
Sensitivity Specificity Positive Predictive Value Negative Predictive Value
n=20 86.67% 80% 92.86% 66.67%
Supplementary Table 7: Sensitivity, Specificity, Positive prediction and Negative prediction values for Nuclear EpCAM method
Sample Age ER/PR/HER2 status Treatment Disease status 84-1 CellSearchS1 65 ER+/PR-/HER2+ Taxol Progression 20 1S3 42 ER+/PR+/HER2- letrozole Stable 0 0S4 48 ER+/PR+/HER2+ trastuzumab Stable 0 0S5 61 ER+/PR+/HER2- paclitaxel Stable 1 0
S7 78 ER+/PR+/HER2- everolimus, exemestane and zoledronic acid Progression 1 2S9 53 ER+/PR+/HER2+ Ixabepilone plus capecitabine, zoladex + AI Progression 32 74S10 44 ER-/PR-/HER2+ docetaxel, pertuzumab and trastuzumab Progression 12 44S11 27 ER-/PR-/HER2- Abraxane and bevacizumab Progression 2 22S12 72 ER+/PR+/HER2- tamoxifen Progression 0 10S13 58 ER+/PR+/HER2- Taxol Progression 5 69S14 67 ER+/PR+/HER2- letrozole Stable 0 0S15 61 ER-/PR-/HER2- Taxol and Avastin Stable 2 1S16 49 ER+/PR+/HER2- Afinitor and tamoxifen Progression 7 54S17 46 ER-/PR-/HER2- eribulin Progression 73 4S18 37 ER-/PR-/HER2- Taxol Stable 1 4
S19 69 ER+/PR-/HER2- everolimus, exemestane and zoledronic acid Stable 2 1S20 64 ER+/PR+/HER2- Taxol Stable 0 1S22 31 ER+/PR+/HER2+ Taxol Stable 3 4S23 53 ER+/PR-/HER2- capecitabine Progression 6 13S24 66 ER+/PR+/HER2- docetaxel and zoledronic Progression 8 11S26 58 ER-/PR-/HER2- eribulin Progression 1 3S27 48 ER-/PR-/HER2- gemcitabine and carboplatin Progression 17 1S28 36 ER+/PR+/HER2- Gemzar, carboplatin Progression 24 1S29 47 ER-/PR-/HER2- MK-2206 Stable 4 11S30 52 ER+/PR+/HER2- tamoxifen Stable 4 1S31 40 ER-/PR-/HER2- Taxol and Avastin Progression 48 1S33 42 ER+/PR-/HER2- abraxane plus bevacizumab Progression 6 34S35 67 ER+/PR+/HER2- everolimus Progression 8 1
S36 68 ER+/PR+/HER2- everolimus, exemestane and zoledronic acid Stable 0 9S38 51 ER+/PR+/HER2- Avastin and eribulin Progression 13 3S39 65 ER-/PR-/HER2- Taxol and Avastin Stable 0 5
S40 60 ER+/PR-/HER2- everolimus, exemestane and zoledronic acid Progression 15 1S41 57 ER-/PR-/HER2- Gemzar Progression 23 74S42 62 ER+/PR-/HER2- vinorelbine Progression 16 7S43 54 ER+/PR-/HER2+ Tamoxifen Progression 66 221S44 41 ER+/PR+/HER2- Abraxane Progression 6 3S45 56 ER-/PR-/HER2+ Pertuzumab, trastuzumab, docetaxel Stable 1 1S46 35 ER-/PR-/HER2- erlotinib plus pralatrexate Progression 60 1S47 69 ER+/PR-/HER2- everolimus and exemestane Progression 30 10S49 66 ER-/PR-/HER2- cisplatin and etoposide Progression 1 55S50 67 ER+/PR+/HER2- Arimidex Stable 0 0S52 67 ER+/PR+/HER2- letrozole Stable 9 0S53 65 ER+/PR-/HER2- anastrozole Progression 7 2S54 52 ER+/PR+/HER2- everolimus and exemestane Stable 0 0S55 63 ER-/PR-/HER2- capecitabine Progression 2 0S56 47 ER-/PR-/HER2- Avastin and eribulin Progression 148 369S57 59 ER+/PR+/HER2- Taxol and Avastin Progression 24 3S58 66 ER-/PR-/HER2- Abraxane Progression 96 2S59 41 ER+/PR+/HER2- eribulin Progression 6 2S60 59 ER+/PR+/HER2- Avastin and eribulin Progression 6 60S61 92 ER+/PR+/HER2- fluoxymesterone Progression 28 0S62 45 ER+/PR+/HER2- gemcitabine Progression 142 26S63 76 ER+/PR-/HER2- fulvestrant Stable 0 0S64 59 ER+/PR+/HER2- anastrozole Progression 6 21S65 67 ER+/PR+/HER2- everolimus and exemestane Progression 180 0S66 30 ER-/PR-/HER2- T-FAC Progression 12 0S67 58 ER+/PR-/HER2- Abraxane and Avastin Progression 30 124S68 40 ER+/PR+/HER2- exemestane, everolimus, and denosumab Progression 144 2
Supplementary Table 8: Clinical parameters, disease status and CTC counts from patients