Development and Validation of a Targeted ‘Liquid’ NGS Panel for Treatment Customization in Patients with Metastatic Colorectal Cancer
Abstract
:1. Introduction
2. Materials and Methods
2.1. Patient Enrollment and Data Collection
2.2. Sampling of Blood and Plasma Preparation
2.3. cfDNA Isolation and Quantitation
2.4. Library Preparation and Targeted NGS
2.5. NGS Data Analysis
2.6. BEAMing Digital PCR
2.7. Statistics
3. Results
3.1. Study Population
3.2. Development, Application and Evaluation of the NGS Assay
3.3. Validation of the NGS Plasma Assay by BEAMing dPCR
3.4. Concordance Analysis of RAS Testing in Tissue and Plasma
3.5. Association of Clinical Parameters with ctDNA
3.6. Cost and Turn-around Time of NGS Assay versus dPCR
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Patient Characteristics | n (%) |
---|---|
Gender | |
Male | 20 (67%) |
Female | 10 (33%) |
Age in years | |
Mean | 68 |
Range | 43–83 |
Primary tumor location | |
Right colon | 16 (53%) |
Left colon/Rectum | 14 (47%) |
First line chemotherapy | |
Oxaliplatin-based doublet | 21 (70%) |
Irinotecan-based doublet | 4 (13%) |
Other 1 | 5 (17%) |
First line targeted agent | |
Anti-angiogenic | 15 (50%) |
Anti-EGFR | 11 (37%) |
None | 4 (13%) |
Total | 30 (100%) |
Gene | Exon(s) | Codon Position | Number of Hotspots |
---|---|---|---|
KRAS | 2 | 12, 13 | 2 |
3 | 59, 61 | 2 | |
4 | 117, 146 | 2 | |
NRAS | 2 | 12, 13 | 2 |
3 | 59, 61 | 2 | |
4 | 117, 146 | 2 | |
MET | 14 | 1003 | 1 |
BRAF | 15 | 600 | 1 |
ERBB2 | 8 | 309, 310 | 2 |
19 | 740, 755, 767, 769 | 4 | |
20 | 773, 776, 777 | 3 | |
21 | 824, 842, 874 | 3 | |
EGFR | All exons (1–28) including 12 | 451, 464, 465, 467, 491, 492 | 6 |
Plasma ctDNA RAS Status by dPCR | |||||||
---|---|---|---|---|---|---|---|
RAS | Mutant | WT | Total | PPA | NPA | OPA | |
Plasma ctDNA RAS status by NGS | Mutant | 26 | 1 | 27 | 100 × 26/35 = 74.3% | 100 × 25/26 = 96.2% | 100 × 51/61 = 83.6% |
WT | 9 | 25 | 34 | ||||
Total | 35 | 26 | 61 |
Tissue RAS Status | |||||||
---|---|---|---|---|---|---|---|
RAS | Mutant | WT | Total | PPA | NPA | OPA | |
Plasma ctDNA RAS status by NGS | Mutant | 13 | 1 | 14 | 100 × 13/16 = 81.2% | 100 × 13/14 = 92.8% | 100 × 26/30 = 86.7% |
WT | 3 | 13 | 16 | ||||
Total | 16 | 14 | 30 |
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Kastrisiou, M.; Zarkavelis, G.; Kougioumtzi, A.; Sakaloglou, P.; Kostoulas, C.; Georgiou, I.; Batistatou, A.; Pentheroudakis, G.; Magklara, A. Development and Validation of a Targeted ‘Liquid’ NGS Panel for Treatment Customization in Patients with Metastatic Colorectal Cancer. Diagnostics 2021, 11, 2375. https://doi.org/10.3390/diagnostics11122375
Kastrisiou M, Zarkavelis G, Kougioumtzi A, Sakaloglou P, Kostoulas C, Georgiou I, Batistatou A, Pentheroudakis G, Magklara A. Development and Validation of a Targeted ‘Liquid’ NGS Panel for Treatment Customization in Patients with Metastatic Colorectal Cancer. Diagnostics. 2021; 11(12):2375. https://doi.org/10.3390/diagnostics11122375
Chicago/Turabian StyleKastrisiou, Myrto, George Zarkavelis, Anastasia Kougioumtzi, Prodromos Sakaloglou, Charilaos Kostoulas, Ioannis Georgiou, Anna Batistatou, George Pentheroudakis, and Angeliki Magklara. 2021. "Development and Validation of a Targeted ‘Liquid’ NGS Panel for Treatment Customization in Patients with Metastatic Colorectal Cancer" Diagnostics 11, no. 12: 2375. https://doi.org/10.3390/diagnostics11122375