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. 2020 Feb 24;9(2):522.
doi: 10.3390/cells9020522.

Detection of MET Alterations Using Cell Free DNA and Circulating Tumor Cells from Cancer Patients

Patricia Mondelo-Macía  1 , Carmela Rodríguez-López  2   3 , Laura Valiña  4   5 , Santiago Aguín  2   3 , Luis León-Mateos  2   3 , Jorge García-González  2   3   6 , Alicia Abalo  1 , Oscar Rapado-González  1   7 , Mercedes Suárez-Cunqueiro  3   6   7 , Angel Díaz-Lagares  6   8 , Teresa Curiel  3 , Silvia Calabuig-Fariñas  6   9   10   11 , Aitor Azkárate  5   12 , Antònia Obrador-Hevia  5   13 , Ihab Abdulkader  14 , Laura Muinelo-Romay  1   6 , Roberto Diaz-Peña  1   15 , Rafael López-López  1   2   6
Affiliations

Detection of MET Alterations Using Cell Free DNA and Circulating Tumor Cells from Cancer Patients

Patricia Mondelo-Macía et al. Cells. .

Abstract

MET alterations may provide a potential biomarker to evaluate patients who will benefit from treatment with MET inhibitors. Therefore, the purpose of the present study is to investigate the utility of a liquid biopsy-based strategy to assess MET alterations in cancer patients. We analyzed MET amplification in circulating free DNA (cfDNA) from 174 patients with cancer and 49 healthy controls and demonstrated the accuracy of the analysis to detect its alteration in patients. Importantly, a significant correlation between cfDNA concentration and MET copy number (CN) in cancer patients (r = 0.57, p <10-10) was determined. Furthermore, we evaluated two approaches to detect the presence of MET on circulating tumor cells (CTCs), using the CellSearch® and Parsortix systems and monitored patients under anti-EGFR treatment (n = 30) combining both cfDNA and CTCs analyses. This follow-up provides evidence for the potential of MET CN assessment when patients develop resistance to anti-EGFR therapy and a significant association between the presence of CTCs MET+ and the Overall Survival (OS) in head and neck cancer patients (P = 0.05; HR = 6.66). In conclusion, we develop specific and noninvasive assays to monitor MET status in cfDNA/CTCs and demonstrate the utility of plasma MET CN determination as a biomarker for monitoring the appearance of resistance to anti-EGFR therapy.

Keywords: MET amplification; MET copy number; MET protein expression; circulating free DNA (cfDNA); circulating tumor cells (CTCs); targeted therapy.

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Conflict of interest statement

Rafael López-López reports grants and personal fees from Roche, Merck, AstraZeneca, Bayer, Pharmamar, Leo, and personal fees and non-financial support from Bristol-Myers Squibb and Novartis, outside of the submitted work. Jorge Garcia-González reports personal fees from Novartis, Bristol-Myers Squibb, MSD Oncology, Roche/Genentech, Lilly, Boehringer Ingelheim, AstraZeneca, Pierre Fabre, Leo Pharma, and Travel, Accommodations and Expenses from Bristol-Myers Squibb, MSD Oncology, Roche/Genentech, and Lilly. Luis León-Mateos reports personal fees from Astra Zeneca, Boehringer, MSD, Sanofi, Novartis, Roche, and Bristol-Myers outside of the submitted work.

Figures

Figure 1
Figure 1
MET CN analysis. (A) Scatterplot representing correlation between MET CN in cancer cell lines determined by ddPCR versus single-nucleotide polymorphism (SNP) array (n = 8) using Pearson’s correlation; (B) Plasma MET CN detected in healthy controls (n = 49), non-metastatic patients (n = 34), and metastatic patients (n = 140) using the Mann–Whitney–Wilcoxon U-Test.
Figure 2
Figure 2
MET CN analysis in circulating free DNA (cfDNA) from metastatic cancer patients. (A) Correlation between cfDNA levels and plasma MET CN in all metastatic cancer patients (n = 140); (B) Correlation between cfDNA levels and plasma MET CN in lung and head and neck cancer patients (n = 30).
Figure 2
Figure 2
MET CN analysis in circulating free DNA (cfDNA) from metastatic cancer patients. (A) Correlation between cfDNA levels and plasma MET CN in all metastatic cancer patients (n = 140); (B) Correlation between cfDNA levels and plasma MET CN in lung and head and neck cancer patients (n = 30).
Figure 3
Figure 3
Comparison of MET CN status in tissue and cfDNA. (A) Distribution of MET 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) Representative example of a negative case for MET amplification obtained in a NSCLC patient by FISH; and (C) Representative example of a positive case for MET amplification obtained in a NSCLC patient by FISH.
Figure 4
Figure 4
Percentage of spiked tumor cancer cells captured using CellSearch® and Parsortix systems. Evaluation of the enrichment capacity of CellSearch® 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. p-value < 5 × 10−3, in all comparisons between CellSearch® and Parsortix System in each cell line.
Figure 5
Figure 5
Detection of MET expression using tumor cancer cells with the CellSearch® and Parsortix systems (A and 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).
Figure 5
Figure 5
Detection of MET expression using tumor cancer cells with the CellSearch® and Parsortix systems (A and 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).
Figure 6
Figure 6
CTCs enumeration and MET expression in blood samples evaluated by the CellSearch® (upper panel) and Parsortix (down panel) systems. Distribution of MET scores in CTCs from patients with NSCLC (A) and head and neck cancer (B).
Figure 7
Figure 7
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+).
Figure 8
Figure 8
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 MET CN for patient id60 are shown.
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