A Multiplex PCR-Based Next Generation Sequencing-Panel to Identify Mutations for Targeted Therapy in Breast Cancer Circulating Tumor Cells
Abstract
:1. Introduction
2. Materials and Methods
2.1. Patients
2.2. Enrichment and Enumeration of CTCs
2.3. CTC Isolation
2.4. Whole-Genome Amplification
2.5. Multiplex PCR-Based Next Generation Sequencing
2.6. Validation Experiments with Spiked Cells
2.7. Statistical Analysis
3. Results
3.1. Development of the Assay
3.2. Validation of the Assay
3.3. Analysis of CTCs from Metastatic Breast Cancer Patients to Identify Targeted Therapies
4. Discussion
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Characteristics | Total | in % | |
---|---|---|---|
Patients | 13 | 100 | |
Age at blood draw | |||
Mean | 61.2 | ||
Median | 61 | ||
Range | 46–78 | ||
Tumor size | |||
pT1 | 4 | 30.8 | |
pT2–4 | 8 | 61.5 | |
na | 1 | 7.7 | |
Nodal status at time of diagnosis | |||
0 | 4 | 30.8 | |
1–3 | 7 | 53.8 | |
na | 2 | 15.4 | |
Metastasis status at time of diagnosis | |||
0 | 10 | 77.0 | |
1 | 2 | 15.4 | |
na | 1 | 7.7 | |
Histology | |||
NST | 8 | 61.5 | |
Invasive-lobular | 5 | 38.5 | |
Grading | |||
1–2 | 9 | 69.2 | |
3 | 2 | 15.4 | |
na | 2 | 23.1 | |
Subtype | |||
Luminal | 11 | 84.6 | |
Triple negative | 2 | 15.4 | |
CTC count (per 7.5 mL blood) | |||
Median | 94 | ||
Range | 6–15,340 |
Forward Primer | Reverse Primer | |
---|---|---|
PIK3CA Exon 5 | 5′ AAGACTCGGCAGCATCTCCAGCATTTCCACAGCTACACCA 3′ | 5′ GCGATCGTCACTGTTCTCCAGATGTTCTCCTAACCATCTGA 3′ |
PIK3CA Exon 10 | 5′ AAGACTCGGCAGCATCTCCAGGGAAAATGACAAAGAACAG 3′ | 5′ GCGATCGTCACTGTTCTCCAATTTTAGCACTTACCTGTGAC 3′ |
PIK3CA Exon 21 | 5′ AAGACTCGGCAGCATCTCCATTGATGACATTGCATACATTCG 3′ | 5′ GCGATCGTCACTGTTCTCCAGTGGAAGATCCAATCCATTT 3′ |
ESR1 Exon 5 | 5′ AAGACTCGGCAGCATCTCCATTGACCCTCCATGATCAGGT 3′ | 5′ GCGATCGTCACTGTTCTCCAGCTACTCCTAAGCTACAGCC 3′ |
ESR1 Exon 7 | 5′ AAGACTCGGCAGCATCTCCATCTCTCACTCTCTCTCTGCG 3′ | 5′ GCGATCGTCACTGTTCTCCAGATGTGGGAGAGGATGAGGA 3′ |
ESR1 Exon 8 | 5′ AAGACTCGGCAGCATCTCCAAGTAGTCCTTTCTGTGTCTTC 3′ | 5′ GCGATCGTCACTGTTCTCCAAATGCGATGAAGTAGAGCCC 3′ |
AKT1 Exon 3 | 5′ AAGACTCGGCAGCATCTCCAGTAGAGTGTGCGTGGCTC 3′ | 5′ GCGATCGTCACTGTTCTCCACCCCAAATCTGAATCCCGAG 3′ |
ERBB2 Exon 8 | 5′ AAGACTCGGCAGCATCTCCAGGCTACATGTTCCTGATCTCC 3′ | 5′ GCGATCGTCACTGTTCTCCAGGGTCTGAGGAAGGATAGGA 3′ |
ERBB2 Exon 18 | 5′ AAGACTCGGCAGCATCTCCAAAGTACACGATGCGGAGACT 3′ | 5′ GCGATCGTCACTGTTCTCCAACCTTCACCTTCCTCAGCTC 3′ |
ERBB2 Exon 19 | 5′ AAGACTCGGCAGCATCTCCAATCCTCCTCTTTCTGCCCAG 3′ | 5′ GCGATCGTCACTGTTCTCCAAGTCTAGGTTTGCGGGAGTC 3′ |
ERBB2 Exon 20 | 5′ AAGACTCGGCAGCATCTCCATGGTTTGTGATGGTTGGGAG 3′ | 5′ GCGATCGTCACTGTTCTCCAGACATGGTCTAAGAGGCAGC 3′ |
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Franken, A.; Rivandi, M.; Yang, L.; Jäger, B.; Krawczyk, N.; Honisch, E.; Niederacher, D.; Fehm, T.; Neubauer, H. A Multiplex PCR-Based Next Generation Sequencing-Panel to Identify Mutations for Targeted Therapy in Breast Cancer Circulating Tumor Cells. Appl. Sci. 2020, 10, 3364. https://doi.org/10.3390/app10103364
Franken A, Rivandi M, Yang L, Jäger B, Krawczyk N, Honisch E, Niederacher D, Fehm T, Neubauer H. A Multiplex PCR-Based Next Generation Sequencing-Panel to Identify Mutations for Targeted Therapy in Breast Cancer Circulating Tumor Cells. Applied Sciences. 2020; 10(10):3364. https://doi.org/10.3390/app10103364
Chicago/Turabian StyleFranken, André, Mahdi Rivandi, Liwen Yang, Bernadette Jäger, Natalia Krawczyk, Ellen Honisch, Dieter Niederacher, Tanja Fehm, and Hans Neubauer. 2020. "A Multiplex PCR-Based Next Generation Sequencing-Panel to Identify Mutations for Targeted Therapy in Breast Cancer Circulating Tumor Cells" Applied Sciences 10, no. 10: 3364. https://doi.org/10.3390/app10103364