Circulating extracellular vesicles provide valuable protein, but not DNA, biomarkers in metastatic breast cancer

Abstract Detection of cell‐free circulating tumour DNA (ctDNA) and cancer‐specific extracellular vesicles (EVs) in patient blood have been widely explored as non‐invasive biomarkers for cancer detection and disease follow up. However, most of the protocols used to isolate EVs co‐isolate other components and the actual value of EV‐associated markers remain unclear. To determine the optimal source of clinically‐relevant circulating biomarkers in breast cancer, we applied a size exclusion chromatography (SEC) procedure to analyse separately the content in nucleic acids of EV‐enriched and EV‐depleted fractions, in comparison to total plasma. Both cellular and mitochondrial DNA (cellDNA and mtDNA) were detected in EV‐rich and EV‐poor fractions. Analysing specific mutations identified from tumour tissues, we detected tumour‐specific cellular alleles in all SEC fractions. However, quantification of ctDNA from total plasma was more sensitive than from any SEC fractions. On the other hand, mtDNA was preferentially enriched in EV fractions from healthy donor, whereas cancer patients displayed more abundant mtDNA in total plasma, and equally distributed in all fractions. In contrast to nucleic acids, using a Multiplexed bead‐based EV‐analysis assay, we identified three surface proteins enriched in EVs from metastatic breast cancer plasma, suggesting that a small set of EV surface molecules could provide a disease signature. Our findings provide evidence that the detection of DNA within total circulating EVs does not add value as compared to the whole plasma, at least in the metastatic breast cancer patients used here. However, analysis of a subtype of EV‐associated proteins may reliably identify cancer patients. These non‐invasive biomarkers represent a promising tool for cancer diagnosis and real‐time monitoring of treatment efficacy and these results will impact the development of therapeutic approaches using EVs as targets or biomarkers of cancer

Gene- and exon-expression profiling reveals an extensive LPS-induced response in immune cells in patients with cirrhosis.

Lipopolysaccharide (LPS)-expressing bacteria cause severe inflammation in cirrhotic patients. The global gene response to LPS is unknown in cirrhotic immune cells.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Neutrophil extracellular traps formed during chemotherapy confer treatment resistance via TGF-β activation

International audienceMetastasis is the major cause of cancer death, and the development of therapy resistance is common. The tumor microenvironment can confer chemotherapy resistance (chemoresistance), but little is known about how specific host cells influence therapy outcome. We show that chemotherapy induces neutrophil recruitment and neutrophil extracellular trap (NET) formation, which reduces therapy response in mouse models of breast cancer lung metastasis. We reveal that chemotherapy-treated cancer cells secrete IL-1β, which in turn triggers NET formation. Two NET-associated proteins are required to induce chemoresistance: integrin-αvβ1, which traps latent TGF-β, and matrix metalloproteinase 9, which cleaves and activates the trapped latent TGF-β. TGF-β activation causes cancer cells to undergo epithelial-to-mesenchymal transition and correlates with chemoresistance. Our work demonstrates that NETs regulate the activities of neighboring cells by trapping and activating cytokines and suggests that chemoresistance in the metastatic setting can be reduced or prevented by targeting the IL-1β-NET-TGF-β axis

Microsatellite instability detection in breast cancer using drop-off droplet digital PCR

International audienceThe use of conventional methods (immunohistochemistry, pentaplex PCR) for detecting microsatellite instability (MSI), a predictive biomarker of immunotherapy efficacy, is debated for cancers with low MSI prevalence, such as breast cancer (BC). We developed two multiplex drop-off droplet digital PCR (ddPCR) assays targeting four microsatellites, initially identified from public BC whole-genome sequencing dataset. Performances of the assays were investigated and 352 tumor DNA and 28 circulating cell-free DNA from BC patients, with unknown MSI status were blindly screened. Cross-validation of ddPCR MSI status with other MSI detection methods was performed. We then monitored circulating tumor DNA (ctDNA) dynamics before and during pembrolizumab immunotherapy in one patient with MSI-high (MSI-H) metastatic BC. The assays showed high analytical specificity and sensitivity (limit of detection = 0.16%). Among N = 380 samples, seven (1.8%) were found as MSI-H by ddPCR with six of them confirmed by next-generation sequencing (NGS). Specificity was 100% in N = 133 microsatellite stable BC submitted to NGS. In the patient with MSI-H metastatic BC, ctDNA monitoring revealed an early decrease of microsatellite mutant allelic frequencies during immunotherapy. These results demonstrated MSI detection by ddPCR, a non-invasive, fast and cost-effective approach, allowing for large pre-screening of BC patients who may benefit from immunotherapy

Non-invasive multi-cancer diagnosis using DNA hypomethylation of LINE-1 retrotransposons

The detection of circulating tumor DNA, which allows non-invasive tumor molecular profiling and disease follow-up, promises optimal and individualized management of patients with cancer. However, detecting small fractions of tumor DNA released when the tumor burden is reduced remains a challenge. We implemented a new highly sensitive strategy to detect base-pair resolution methylation patterns from plasma DNA and assessed the potential of hypomethylation of LINE-1 retrotransposons as a non-invasive multi-cancer detection biomarker. Resulting machine learning-based classifiers showed powerful correct classification rates discriminating healthy and tumor plasmas from 6 types of cancers in two independent cohorts (AUC = 88% to 100%, N = 747). This should lead to the development of more efficient non-invasive diagnostic tests adapted to all cancer patients, based on the universality of these factors. One-Sentence Summary LINE-1 retrotransposons hypomethylation is a sensitive and specific biomarker to detect multiple forms of cancer non-invasively

Multimodal liquid biopsy for early monitoring and outcome prediction of chemotherapy in metastatic breast cancer

International audienceCirculating tumor cells (CTCs) and circulating tumor DNA (ctDNA) are two cancer-derived blood biomarkers that inform on patient prognosis and treatment efficacy in breast cancer. We prospectively evaluated the clinical validity of quantifying both CTCs (CellSearch) and ctDNA (targeted next-generation sequencing). Their combined value as prognostic and early monitoring markers was assessed in 198 HER2-negative metastatic breast cancer patients. All patients were included in the prospective multicenter UCBG study COMET (NCT01745757) and treated by first-line chemotherapy with weekly paclitaxel and bevacizumab. Blood samples were obtained at baseline and before the second cycle of chemotherapy. At baseline, CTCs and ctDNA were respectively detected in 72 and 74% of patients and were moderately correlated (Kendall’s τ = 0.3). Only 26 (13%) patients had neither detectable ctDNA nor CTCs. Variants were most frequently observed in TP53 and PIK3CA genes . KMT2C / MLL3 variants detected in ctDNA were significantly associated with a lower CTC count, while the opposite trend was seen with GATA3 alterations. Both CTC and ctDNA levels at baseline and after four weeks of treatment were correlated with survival. For progression-free and overall survival, the best multivariate prognostic model included tumor subtype (triple negative vs other), grade (grade 3 vs other), ctDNA variant allele frequency (VAF) at baseline (per 10% increase), and CTC count at four weeks (≥5CTC/7.5 mL). Overall, this study demonstrates that CTCs and ctDNA have nonoverlapping detection profiles and complementary prognostic values in metastatic breast cancer patients. A comprehensive liquid-biopsy approach may involve simultaneous detection of ctDNA and CTCs