A Novel Workflow to Enrich and Isolate Patient-Matched EpCAMhigh and EpCAMlow/negative CTCs Enables the Comparative Characterization of the PIK3CA Status in Metastatic Breast Cancer
Abstract
:1. Introduction
2. Results
2.1. A Novel Workflow to Enrich and Isolate Patient-Matched EpCAMhigh and EpCAMlow/negative CTCs
2.2. Validation of Immunostaining on Cytospins
2.3. Validation of Tumor Cell Enrichment via Parsortix™ System
2.4. Establishment of in Situ Staining of Captured Tumour Cells
2.5. Processing of MBC Clinical Samples: Enrichment, Detection and Isolation of EpCAMhigh and EpCAMlow/negative Cells
2.6. Whole Genome Amplification of Single Isolated Cells
2.7. Mutational Analysis of PIK3CA Exons 9 and 20 in both CTC-Subpopulations
3. Discussion
4. Materials and Methods
4.1. Cell Lines and Culture Conditions
4.2. Patient Material
4.3. Enrichment and Enumeration of EpCAMhigh CTCs via CellSearch®
4.4. Enrichment of EpCAMlow/negative Cells with Parsortix™ System
4.5. Detection and Isolation of EpCAMhigh and EpCAMlow/negative Cells via CellCelector™
4.5.1. Selection Criteria
4.5.2. Cell Isolation Parameters
4.6. Whole Genome Amplification of Isolated CTCs
4.7. Sanger Sequencing of the PIK3CA Exons 9 and 20 on CTCs
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
CTC | Circulating Tumor Cell |
EpCAM | Epithelial Cell Adhesion Molecule |
FDA | Food and Drug Administration |
EMT | Epithelial to Mesenchymal Transition |
MBC | Metastatic Breast Cancer |
PI3K | Phosphatidylinositol 3-Kinase |
PIK3CA | Phosphatidylinositol 3-Kinase Catalytic subunit Alpha |
WGA | Whole Genome Amplification |
WT | Wild-type |
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# | Patient ID | EpCAMhigh CTCs | EpCAMlow/negative CTCs | ||||
---|---|---|---|---|---|---|---|
Sorted CTCs | High Integrity WGA Products | Low Integrity WGA Products | Sorted CTCs | High Integrity WGA Products | Low INTEGRITY WGA Products | ||
1 | I | 5 | 0 | 5 | 19 | 0 | 19 |
2 | V | 1 | 0 | 1 | 2 | 0 | 2 |
3 | VI | 11 | 5 | 6 | 24 | 3 | 21 |
4 | VIII | 8 | 0 | 8 | 12 | 0 | 12 |
5 | IX | 26 | 11 | 15 | 16 | 2 | 14 |
6 | X | 13 | 4 | 9 | 5 | 0 | 5 |
7 | XI | 7 | 3 | 4 | 6 | 0 | 6 |
8 | XVI | 9 | 1 | 8 | 27 | 1 | 26 |
9 | XXIV (2) | 6 | 1 | 5 | 4 | 0 | 4 |
10 | XXXV | 8 | 2 | 6 | 18 | 3 | 15 |
11 | XLI | 2 | 1 | 1 | 5 | 1 | 4 |
12 | XLVI | 7 | 2 | 5 | 4 | 0 | 4 |
13 | XLVII | 4 | 0 | 4 | 3 | 1 | 2 |
107 | 30 | 77 | 145 | 11 | 134 | ||
28% | 72% | 8% | 92% |
EpCAMhigh CTCs | |||||
# | Patient ID | PIK3CA Exon 9 Mutational Analysis | PIK3CA Exon 20 Mutational Analysis | ||
Sequenced CTCs | Mutational Status | Sequenced CTCs | Mutational Status | ||
1 | I | 2 | WT | 1 | WT |
2 | VI | 5 | WT | 2 | WT |
3 | IX | 13 | WT | 11 | 2: p.H1047L (c.CAT > CTT); 9: WT |
4 | X | 7 | WT | 5 | 2: p.H1047R (c.CAT > CGT); 3: WT |
5 | XI | 1 | WT | 3 | WT |
6 | XVI | 3 | WT | 3 | WT |
7 | XXXV | 1 | WT | 3 | WT |
8 | XXXVI | 1 | WT | 1 | WT |
9 | XLVII | 3 | WT | 3 | WT |
10 | XLVII | 2 | WT | 2 | 1: p.H1047L (c.CAT > CTT); 1: WT |
38 | 34 | ||||
EpCAMlow/negative CTCs | |||||
# | Patient ID | PIK3CA Exon 9 Mutational Analysis | PIK3CA Exon 20 Mutational Analysis | ||
Sequenced CTCs | Mutational Status | Sequenced CTCs | Mutational Status | ||
1 | I | 10 | 1: p.E545K (c.CAG > AAG); 9: WT | 7 | WT |
2 | VI | 7 | WT | 7 | WT |
3 | IX | 5 | 1: p.E545K (c.CAG > AAG); 4: WT | 3 | WT |
4 | X | 2 | WT | 1 | WT |
5 | XI | 1 | WT | 1 | WT |
6 | XVI | 0 | n.d. | 2 | WT |
7 | XXXV | 8 | WT | 8 | WT |
8 | XXXVI | 3 | WT | 3 | WT |
9 | XLVII | 1 | WT | 1 | WT |
10 | XLVII | 2 | WT | 2 | WT |
39 | 35 |
PIK3CA Exon | Primer Name | Sequence (5′→3′) | Primer Length (bp) |
---|---|---|---|
9 | forward | CATCCGATGTACCTGATTGAACTGCATGCAGACAAAGAACAGCTCAAAGCAA | 52 |
reverse | CATTCCTTAGATAGCTCGGAAGTCCATTGCATTTTAGCACTTACCTGTGAC | 52 | |
20 | forward | CATCCGATGTACCTGATTGAACTGCATGCATTGATGACATTGCATACATTCG | 52 |
reverse | CATTCCTTAGATAGCTCGGAAGTCCATTGCGTGGAAGATCCAATCCATTT | 50 | |
Sequencing | forward | TCCGATGTACCTGATTGAAC | 20 |
reverse | TTCCTTAGATAGCTCGGAAG | 20 |
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Lampignano, R.; Yang, L.; Neumann, M.H.D.; Franken, A.; Fehm, T.; Niederacher, D.; Neubauer, H. A Novel Workflow to Enrich and Isolate Patient-Matched EpCAMhigh and EpCAMlow/negative CTCs Enables the Comparative Characterization of the PIK3CA Status in Metastatic Breast Cancer. Int. J. Mol. Sci. 2017, 18, 1885. https://doi.org/10.3390/ijms18091885
Lampignano R, Yang L, Neumann MHD, Franken A, Fehm T, Niederacher D, Neubauer H. A Novel Workflow to Enrich and Isolate Patient-Matched EpCAMhigh and EpCAMlow/negative CTCs Enables the Comparative Characterization of the PIK3CA Status in Metastatic Breast Cancer. International Journal of Molecular Sciences. 2017; 18(9):1885. https://doi.org/10.3390/ijms18091885
Chicago/Turabian StyleLampignano, Rita, Liwen Yang, Martin H. D. Neumann, André Franken, Tanja Fehm, Dieter Niederacher, and Hans Neubauer. 2017. "A Novel Workflow to Enrich and Isolate Patient-Matched EpCAMhigh and EpCAMlow/negative CTCs Enables the Comparative Characterization of the PIK3CA Status in Metastatic Breast Cancer" International Journal of Molecular Sciences 18, no. 9: 1885. https://doi.org/10.3390/ijms18091885