Machine Learning-Assisted Evaluation of Circulating DNA Quantitative Analysis for Cancer Screening.

While the utility of circulating cell-free DNA (cfDNA) in cancer screening and early detection have recently been investigated by testing genetic and epigenetic alterations, here, an original approach by examining cfDNA quantitative and structural features is developed. First, the potential of cfDNA quantitative and structural parameters is independently demonstrated in cell culture, murine, and human plasma models. Subsequently, these variables are evaluated in a large retrospective cohort of 289 healthy individuals and 983 patients with various cancer types; after age resampling, this evaluation is done independently and the variables are combined using a machine learning approach. Implementation of a decision tree prediction model for the detection and classification of healthy and cancer patients shows unprecedented performance for 0, I, and II colorectal cancer stages (specificity, 0.89 and sensitivity, 0.72). Consequently, the methodological proof of concept of using both quantitative and structural biomarkers, and classification with a machine learning method are highlighted, as an efficient strategy for cancer screening. It is foreseen that the classification rate may even be improved by the addition of such biomarkers to fragmentomics, methylation, or the detection of genetic alterations. The optimization of such a multianalyte strategy with this machine learning method is therefore warranted.

Blood contains circulating cell-free respiratory competent mitochondria.

Mitochondria are considered as the power-generating units of the cell due to their key role in energy metabolism and cell signaling. However, mitochondrial components could be found in the extracellular space, as fragments or encapsulated in vesicles. In addition, this intact organelle has been recently reported to be released by platelets exclusively in specific conditions. Here, we demonstrate for the first time, that blood preparation with resting platelets, contains whole functional mitochondria in normal physiological state. Likewise, we show, that normal and tumor cultured cells are able to secrete their mitochondria. Using serial centrifugation or filtration followed by polymerase chain reaction-based methods, and Whole Genome Sequencing, we detect extracellular full-length mitochondrial DNA in particles over 0.22 µm holding specific mitochondrial membrane proteins. We identify these particles as intact cell-free mitochondria using fluorescence-activated cell sorting analysis, fluorescence microscopy, and transmission electron microscopy. Oxygen consumption analysis revealed that these mitochondria are respiratory competent. In view of previously described mitochondrial potential in intercellular transfer, this discovery could greatly widen the scope of cell-cell communication biology. Further steps should be developed to investigate the potential role of mitochondria as a signaling organelle outside the cell and to determine whether these circulating units could be relevant for early detection and prognosis of various diseases.

Hypoxia differently modulates the release of mitochondrial and nuclear DNA.

BACKGROUND: We investigated the influence of hypoxia on the concentration of mitochondrial and nuclear cell-free DNA (McfDNA and NcfDNA, respectively). METHOD: By an ultra-sensitive quantitative PCR-based assay, McfDNA and NcfDNA were measured in the supernatants of different colorectal cell lines, and in the plasma of C57/Bl6 mice engrafted with TC1 tumour cells, in normoxic or hypoxic conditions. RESULTS: Our data when setting cell culture conditions highlighted the higher stability of McfDNA as compared to NcfDNA and revealed that cancer cells released amounts of nuclear DNA equivalent to the mass of a chromosome over a 6-h duration of incubation. In cell model, hypoxia induced a great increase in NcfDNA and McfDNA concentrations within the first 24 h. After this period, cfDNA total concentrations remained stable in hypoxia consecutive to a decrease of nuclear DNA release, and noteworthy, to a complete inhibition of daily mitochondrial DNA release. In TC1-engrafted mice submitted to intermittent hypoxia, plasma NcfDNA levels are much higher than in mice bred in normoxia, unlike plasma McfDNA concentration that is not impacted by hypoxia. CONCLUSION: This study suggests that hypoxia negatively modulates nuclear and, particularly, mitochondrial DNA releases in long-term hypoxia, and revealed that the underlying mechanisms are differently regulated.

Quantifying circulating cell-free DNA in humans.

To our knowledge, this is the first comprehensive study on the influence of several pre-analytical and demographic parameters that could be a source of variability in the quantification of nuclear and mitochondrial circulating DNA (NcirDNA and McirDNA). We report data from a total of 222 subjects, 104 healthy individuals and 118 metastatic colorectal cancer (mCRC) patients. Approximately 50,000 and 3,000-fold more mitochondrial than nuclear genome copies were found in the plasma of healthy individuals and mCRC patients, respectively. In healthy individuals, NcirDNA concentration was statistically influenced by age (p = 0.009) and gender (p = 0.048). Multivariate analysis with logistic regression specified that age over 47 years-old was predictive to have higher NcirDNA concentration (OR = 2.41; p = 0.033). McirDNA concentration was independent of age and gender in healthy individuals. In mCRC patients, NcirDNA and McirDNA levels were independent of age, gender, delay between food intake and blood collection, and plasma aspect, either with univariate or multivariate analysis. Nonetheless, ad hoc study suggested that menopause and blood collection time might have tendency to influence cirDNA quantification. In addition, high significant statistical differences were found between mCRC patients and healthy individuals for NcirDNA (p < 0.0001), McirDNA (p < 0.0001) and McirDNA/NcirDNA ratio (p < 0.0001). NcirDNA and McirDNA levels do not vary in the same way with regards to cancer vs healthy status, pre-analytical and demographic factors.

Inhibition of DDR1-BCR signalling by nilotinib as a new therapeutic strategy for metastatic colorectal cancer.

The clinical management of metastatic colorectal cancer (mCRC) faces major challenges. Here, we show that nilotinib, a clinically approved drug for chronic myeloid leukaemia, strongly inhibits human CRC cell invasion in vitro and reduces their metastatic potential in intrasplenic tumour mouse models. Nilotinib acts by inhibiting the kinase activity of DDR1, a receptor tyrosine kinase for collagens, which we identified as a RAS-independent inducer of CRC metastasis. Using quantitative phosphoproteomics, we identified BCR as a new DDR1 substrate and demonstrated that nilotinib prevents DDR1-mediated BCR phosphorylation on Tyr177, which is important for maintaining ß-catenin transcriptional activity necessary for tumour cell invasion. DDR1 kinase inhibition also reduced the invasion of patient-derived metastatic and circulating CRC cell lines. Collectively, our results indicate that the targeting DDR1 kinase activity with nilotinib may be beneficial for patients with mCRC.