Detection of MET Alterations Using Cell Free DNA and Circulating Tumor Cells from Cancer Patients
<p><span class="html-italic">MET</span> CN analysis. (<b>A</b>) Scatterplot representing correlation between <span class="html-italic">MET</span> CN in cancer cell lines determined by ddPCR versus single-nucleotide polymorphism (SNP) array (<span class="html-italic">n</span> = 8) using Pearson’s correlation; (<b>B</b>) Plasma <span class="html-italic">MET</span> CN detected in healthy controls (<span class="html-italic">n</span> = 49), non-metastatic patients (<span class="html-italic">n</span> = 34), and metastatic patients (<span class="html-italic">n</span> = 140) using the Mann–Whitney–Wilcoxon U-Test.</p> "> Figure 2
<p><span class="html-italic">MET</span> CN analysis in circulating free DNA (cfDNA) from metastatic cancer patients. (<b>A</b>) Correlation between cfDNA levels and plasma <span class="html-italic">MET</span> CN in all metastatic cancer patients (<span class="html-italic">n</span> = 140); (<b>B</b>) Correlation between cfDNA levels and plasma <span class="html-italic">MET</span> CN in lung and head and neck cancer patients (<span class="html-italic">n</span> = 30).</p> "> Figure 2 Cont.
<p><span class="html-italic">MET</span> CN analysis in circulating free DNA (cfDNA) from metastatic cancer patients. (<b>A</b>) Correlation between cfDNA levels and plasma <span class="html-italic">MET</span> CN in all metastatic cancer patients (<span class="html-italic">n</span> = 140); (<b>B</b>) Correlation between cfDNA levels and plasma <span class="html-italic">MET</span> CN in lung and head and neck cancer patients (<span class="html-italic">n</span> = 30).</p> "> Figure 3
<p>Comparison of <span class="html-italic">MET</span> CN status in tissue and cfDNA. (<b>A</b>) Distribution of <span class="html-italic">MET</span> CN measured by ddPCR and fluorescence in situ hybridization (FISH) (the point larger indicates the discordant value, whereas the horizontal and vertical dotted lines indicate cut-off points of ddPCR and FISH, respectively); (<b>B</b>) Representative example of a negative case for <span class="html-italic">MET</span> amplification obtained in a NSCLC patient by FISH; and (<b>C</b>) Representative example of a positive case for <span class="html-italic">MET</span> amplification obtained in a NSCLC patient by FISH.</p> "> Figure 4
<p>Percentage of spiked tumor cancer cells captured using CellSearch<sup>®</sup> and Parsortix systems. Evaluation of the enrichment capacity of CellSearch<sup>®</sup> and Parsortix systems, using healthy blood spiked with LNCaP, NCI-N87, Hs746T, AU565, SNU-5, and C32 cancer cell lines. LNCaP, NCI-N87, SNU-5, and AU565 express Epithelial cell adhesion molecule (EpCAM) while Hs746T and C32 express low levels or do not express EpCAM, respectively. <span class="html-italic">p</span>-value < 5 × 10<sup>−3</sup>, in all comparisons between CellSearch<sup>®</sup> and Parsortix System in each cell line.</p> "> Figure 5
<p>Detection of MET expression using tumor cancer cells with the CellSearch<sup>®</sup> and Parsortix systems (<b>A</b> and <b>B</b>, respectively). Representative images of MET expression scored on score 0 (cell line LNCaP), 1 (cell line AU565), 2 (cell line Hs746T), and 3 (cell line SNU-5).</p> "> Figure 5 Cont.
<p>Detection of MET expression using tumor cancer cells with the CellSearch<sup>®</sup> and Parsortix systems (<b>A</b> and <b>B</b>, respectively). Representative images of MET expression scored on score 0 (cell line LNCaP), 1 (cell line AU565), 2 (cell line Hs746T), and 3 (cell line SNU-5).</p> "> Figure 6
<p>CTCs enumeration and MET expression in blood samples evaluated by the CellSearch<sup>®</sup> (upper panel) and Parsortix (down panel) systems. Distribution of MET scores in CTCs from patients with NSCLC (<b>A</b>) and head and neck cancer (<b>B</b>).</p> "> Figure 7
<p>Prognostic value to predict Overall Survival (OS) of CTCs enumeration and MET expression in head and neck cancer patients starting with anti-EGFR treatment. CTCs MET-positive ≥1: CTCs with high MET expression (scores 2+ or 3+); CTCs MET-positive <1: CTC with low MET expression (score 1+).</p> "> Figure 8
<p>Timeline for the clinical course of patient id60. The blue and yellow bars represent the treatments time frame, and the red drops indicate blood collection time points. Percent mutant allelic frequency (L858R and T790M) and <span class="html-italic">MET</span> CN for patient id60 are shown.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Lines
2.2. Patients and Samples
2.3. Circulating Free DNA Isolation from Plasma Samples
2.4. Droplet Digital PCR to Detect MET CN
2.5. Fluorescent In Situ Hybridization (FISH) Analysis
2.6. Spiked Experiments
2.7. Analysis of MET Expression on CTCs Isolated Using Cell Search®
2.8. Analysis of MET Expression on CTCs Isolated Using Parsortix System
2.9. Statistical Analysis
3. Results
3.1. Accuracy of ddPCR to Detect MET CN Alterations
3.2. MET CN Assessment in cfDNA from Cancer Patients and Healthy Controls
3.3. MET Amplification Concordance between cfDNA and Tissue Samples
3.4. MET Expression Analysis on CTCs
3.5. MET Amplification and CTCs Expression in Patients under Anti-EGFR Treatment
4. Discussion
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Features | All | Number of Lines of Treatment | Number of Metastatic Sites | ||
---|---|---|---|---|---|
≤1 | >1 | ≤2 | >2 | ||
Gender | N * (%) MET CN (Mean ± SD) | N (%) MET CN (Mean ± SD) | N (%) MET CN (Mean ± SD) | N (%) MET CN (Mean ± SD) | N (%) MET CN (Mean ± SD) |
Male | 83 (59.29%) 2.47 ± 0.40 | 48 (34.29%) 2.45 ± 0.36 | 31 (22.14%) 2.47 ± 0.46 | 36 (25.71%) 2.47 ± 0.45 | 44 (31.43%) 2.45 ± 0.34 |
Female | 57 (40.71%) 2.59 ± 1.40 | 28 (20%) 2.42 ± 0.30 | 27 (19.29%) 2.81 ± 2.04 | 25 (17.86%) 2.35 ± 0.35 | 28 (20%) 2.87 ± 1.98 |
Tumor type | |||||
NSCLC | 77 (55%) 2.52 ± 1.21 | 34 (24.29%) 2.43 ± 0.29 | 36 (25.71%) 2.64 ± 1.75 | 28 (20%) 2.36 ± 0.31 | 42 (30%) 2.66 ± 1.61 |
Head and neck | 30 (21.43%) 2.43 ± 0.37 | 28 (20%) 2.42 ± 0.38 | 1 (0.71%) 2.5 | 26 (16.43%) 2.40 ± 0.39 | 6 (4.29%) 2.52 ± 0.30 |
Colon | 8 (5.71%) 2.65 ± 0.81 | 1 (0.71%) 2.52 | 6 (4.29%) 2.52 ± 0.87 | 2 (1.43%) 2.92 ± 1.65 | 5 (3.57%) 2.36 ± 0.38 |
Melanoma | 6 (4.29%) 2.40 ± 0.49 | 4 (2.86%) 2.42 ± 0.50 | 2 (1.43%) 2.36 ± 0.68 | 3 (2.14%) 2.17 ± 0.12 | 3 (2.14%) 2.62 ± 0.66 |
Others ** | 19 (13.38%) 2.63 ± 0. | 6 (4.29%) 2.63 ± 0.29 | 11 (7.86%) 2.68 ± 0.29 | 4 (2.86%) 2.85 ± 0.19 | 12 (8.57%) 2.60 ± 0.29 |
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Mondelo-Macía, P.; Rodríguez-López, C.; Valiña, L.; Aguín, S.; León-Mateos, L.; García-González, J.; Abalo, A.; Rapado-González, O.; Suárez-Cunqueiro, M.; Díaz-Lagares, A.; et al. Detection of MET Alterations Using Cell Free DNA and Circulating Tumor Cells from Cancer Patients. Cells 2020, 9, 522. https://doi.org/10.3390/cells9020522
Mondelo-Macía P, Rodríguez-López C, Valiña L, Aguín S, León-Mateos L, García-González J, Abalo A, Rapado-González O, Suárez-Cunqueiro M, Díaz-Lagares A, et al. Detection of MET Alterations Using Cell Free DNA and Circulating Tumor Cells from Cancer Patients. Cells. 2020; 9(2):522. https://doi.org/10.3390/cells9020522
Chicago/Turabian StyleMondelo-Macía, Patricia, Carmela Rodríguez-López, Laura Valiña, Santiago Aguín, Luis León-Mateos, Jorge García-González, Alicia Abalo, Oscar Rapado-González, Mercedes Suárez-Cunqueiro, Angel Díaz-Lagares, and et al. 2020. "Detection of MET Alterations Using Cell Free DNA and Circulating Tumor Cells from Cancer Patients" Cells 9, no. 2: 522. https://doi.org/10.3390/cells9020522