Microarray Approach Combined with ddPCR: An Useful Pipeline for the Detection and Quantification of Circulating Tumour DNA Mutations
Abstract
:1. Introduction
2. Material and Methods
2.1. Sample Collection and Processing
2.2. Microarray
2.2.1. PCR Conditions
2.2.2. Microarray Preparation, Hybridization, Image Scanning and Data Analysis
2.3. Droplet Digital PCR (ddPCR)
3. Results
Microarray and ddPCR Analyses
4. Discussion
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Sample ID | Tissue Biopsy | Sidedness of the Primary Tumour | Number and Location of Metastatic Sites | First-Line Treatment |
---|---|---|---|---|
1 | KRAS G12D | descending colon | 2 (liver, lung) | FOLFOX + BEVA |
2 | BRAF V600E | ascending colon | 3 (stomach, peritoneum, lymph nodes) | Not applicable: performance status deterioration |
3 | wt | sigmoid colon | 1 (lung) | FOLFOX + PANI |
4 | wt | sigmoid colon | 2 (liver, lymph nodes) | FOLFOXIRI + PANI |
5 | wt | sigmoid colon | 1 (liver) | FOLFOX + PANI |
6 | wt | rectum | 2 (lung, liver) | FOLFOX + BEVA |
7 | wt | splenic flexure, sigmoid colon-rectum | 1 (liver) | FOLFOX + PANITUMUMAB |
8 | wt | descending colon | 1 (peritoneum) | FOLFIRI + CETUXIMAB |
9 | wt | ascending colon | 2 (lung, liver) | CAPECITABINA |
10 | wt | rectum | 1 (lung) | FOLFIRI + CETUXIMAB |
11 | wt | sigmoid colon | 2 (liver, peritoneum) | FOLFOX + CETUXIMAB |
12 | wt | transverse colon | 1 (liver) | FOLFOX + PANI |
13 | KRAS A146T | descending colon | 3 (liver, peritoneum, lymph nodes) | XELOX + BEVA |
14 | KRAS G13D | sigmoid colon | 2 (liver, lymph nodes) | FOLFOX + BEVA |
15 | wt | descending colon | 1 (liver) | FOLFOX + CETUXIMAB |
16 | KRAS A146T | cecum | 2 (right iliac bone, muscles) | XELOX+BEVA |
17 | wt | cecum, rectum | 1 (peritoneum) | FOLFOX + PANITUMUMAB |
18 | KRAS G12D | hepatic flexure | 1 (liver) | FOLFOX+BEVACIZUMAB |
19 | KRAS G12S | descending colon | 1 (liver) | FOLFOX+BEVACIZUMAB |
20 | KRAS G12D | rectum | 1 (liver) | FOLFOX+BEVACIZUMAB |
Sample ID | Tissue Biopsy | Liquid Biopsy | |
---|---|---|---|
Microarray | ddPCR Mutated Allele % | ||
1 | KRAS G12D | KRAS G12D + G12R | KRAS G12D = 0.033 KRAS G12R = 0.95% |
2 | BRAF V600E | BRAF V600E | BRAF V600E = 15% |
3 | wt | wt | - |
4 | wt | wt | - |
5 | wt | wt | - |
6 | wt | wt | - |
7 | wt | KRAS G12C | KRAS G12C = 32.3% |
8 | wt | wt | - |
9 | wt | wt | - |
10 | wt | wt | - |
11 | wt | wt | - |
12 | wt | wt | - |
13 | KRAS A146T | KRAS A146T | KRAS A146T = 20% |
14 | KRAS G13D | KRAS G13D | KRAS G13D = 1.3% |
15 | wt | wt | - |
16 | KRAS A146T | KRAS A146T | KRAS A146T = 77.5% |
17 | wt | wt | - |
18 | KRAS G12D | KRAS G12D | KRAS G12D = 65.3% |
19 | KRAS G12S | KRAS G12S | KRAS G12S = 17.7% |
20 | KRAS G12D | KRAS G12D | KRAS G12D = 3.7% |
Sample ID | Microarray | ddPCR Mutated Allele % | Clinical Evaluation RECIST Classification |
---|---|---|---|
Pz 1 T0 Pz 1 T1 Pz 1 T2 | G12D + G12R G12D wt | G12D = 0.033%; G12R = 0.95% G12D = 0.4 %; G12R = 0.22% G12D = 0%; G12R = 0.25% | >> PRSD |
Pz 3 T0 Pz 3 T1 Pz 3 T2 | wt wt wt | - - - | >>PR CR |
Pz 4 T0 Pz 4 T1 Pz 4 T2 | wt wt wt | - - - | >>PR CR |
Pz 5 T0 Pz 5 T1 | wt wt | - - | SD |
Pz 7 T0 Pz 7 T1 Pz 7 T2 | G12C G12C wt | G12C = 32.3% G12C = 0.17% G12C = 0% | >>PR CR |
Pz 10 T0 Pz 10 T1 | wt wt | - - | SD |
Pz 11 T0 Pz 11 T1 | wt wt | - - | SD |
Pz 13 T0 Pz 13 T1 Pz 13 T2 | A146T wt A146T | A146T = 20% A146T = 1% A146T = 3.3% | >>PR PD |
Pz 14 T0 Pz 14 T2 | G13D G13D | G13D = 1.3% G13D = 0.22% | >>PR |
Pz 17 T0 Pz 17 T1 | wt wt | - - | SD |
Pz 19 T0 Pz 19 T1 | G12S G12S | G12S = 17.7% G12S = 14% | PD |
Pz 20 T0 Pz 20 T1 | G12D G12D | G12D = 3.7% G12D = 0.3% | PR |
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Galbiati, S.; Damin, F.; Ferraro, L.; Soriani, N.; Burgio, V.; Ronzoni, M.; Gianni, L.; Ferrari, M.; Chiari, M. Microarray Approach Combined with ddPCR: An Useful Pipeline for the Detection and Quantification of Circulating Tumour DNA Mutations. Cells 2019, 8, 769. https://doi.org/10.3390/cells8080769
Galbiati S, Damin F, Ferraro L, Soriani N, Burgio V, Ronzoni M, Gianni L, Ferrari M, Chiari M. Microarray Approach Combined with ddPCR: An Useful Pipeline for the Detection and Quantification of Circulating Tumour DNA Mutations. Cells. 2019; 8(8):769. https://doi.org/10.3390/cells8080769
Chicago/Turabian StyleGalbiati, Silvia, Francesco Damin, Lucia Ferraro, Nadia Soriani, Valentina Burgio, Monica Ronzoni, Luca Gianni, Maurizio Ferrari, and Marcella Chiari. 2019. "Microarray Approach Combined with ddPCR: An Useful Pipeline for the Detection and Quantification of Circulating Tumour DNA Mutations" Cells 8, no. 8: 769. https://doi.org/10.3390/cells8080769