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Warawdekar et al. CTCs from patients with metastatic breast cancer

Table 2: Tricolor flow cytometry for enumeration of CTC as well as control samples showed Ct values lower
done as described in Methods (complements Figure 3) than 30 for GAPDH. From the 17 patient samples, 14
Number of dual positive cells (82%) gave Ct values below 36. Of the 14, 9 (64%)
Patient number
[EpCAM+ CK+] CTCs were found to be positive for two marker genes, 10
1 25 (71.4%) for EpCAM, 9 (64%) for CK19, and 6 (43%) for
2 36 Muc-1. There were two samples that were positive for
3 85 all the four markers, with a high copy number recorded
4 60 for Her2. Her2 expression levels were comparable to
5 15 protein levels obtained by IHC, which was reported as
6 15
7 27 3+. In the 15 control samples, 4 recorded Ct values
8 21 that could be converted to copies/mL and these values
9 50 were below 1.
11 7
13 1 Imaging CTCs
14 3 In blood samples from patients, where CTCs were
15 4 higher, captured cells were subjected to morphological
16 8
17 15 characterization and examined for presence of a
20 19
22 6
For 5 patients only
RT-PCR no flow
Normal individual
number Number of dual positive cells [EpCAM + CK]
1 8
2 4
3 14
4 7
5 7
6 3
7 8
9 0
10 1
17 2
18 6
19 7
20 2
For 6 normal individuals only RT-PCR done
Flow cytometry not
done
One sample lost
during enrichment
CTC: circulating tumor cell; RT-PCR: reverse transcription
polymerase chain reaction; CK: cytokeratin

[22]
Figure 4: Standard curve for Aerts et al. method for T47-D cell
Figure 3: Tri color flow cytometry for CTC enumeration. CK+/ line. (A) Log number of EpCAM-positive cells versus Delta Ct
EpCAM+ dual-positive cells represent CTCs seen in positive (EpCAM-GAPDH); (B) log number of CK-19-positive cells versus
enriched fraction. The number of dual-positive cells in patient and Delta Ct (CK-19-GAPDH); (C) log number of MUC-1-positive cells
normal healthy individuals is shown. CTC: circulating tumor cell versus Delta Ct (MUC-1-GAPDH)
30 Journal of Cancer Metastasis and Treatment ¦ Volume 3 ¦ February 23, 2017