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Purpose: 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., Experimental Design: 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. The DIAMOND (Detection of Long Interspersed Nuclear Element Altered Methylation ON plasma DNA) method targets 30-40,000 young L1 scattered throughout the genome, covering about 100,000 CpG sites and is based on a reference-free analysis pipeline., Results: Resulting machine learning-based classifiers showed powerful correct classification rates discriminating healthy and tumor plasmas from 6 types of cancers (colorectal, breast, lung, ovarian, gastric cancers and uveal melanoma including localized stages) in two independent cohorts (AUC = 88% to 100%, N = 747). DIAMOND can also be used to perform copy number alterations (CNA) analysis which improves cancer detection., Conclusions: 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.

In a prospective study (NCT02866149), we assessed the efficacy of fulvestrant and everolimus in CDK4/6i pre-treated mBC patients and circulating tumor DNA (ctDNA) changes throughout therapy. Patients treated with fulvestrant and everolimus had their ctDNA assessed at baseline, after 3-5 weeks and at disease progression. Somatic mutations were identified in archived tumor tissues by targeted NGS and tracked in cell-free DNA by droplet digital PCR. ctDNA detection was then associated with clinicopathological characteristics and patients' progression-free survival (PFS), overall survival (OS) and best overall response (BOR). In the 57 included patients, median PFS and OS were 6.8 (95%CI [5.03-11.5]) and 38.2 (95%CI [30.0-not reached]) months, respectively. In 47 response-evaluable patients, BOR was a partial response or stable disease in 15 (31.9%) and 11 (23.4%) patients, respectively. Among patients with trackable somatic mutation and available plasma sample, N = 33/47 (70.2%) and N = 19/36 (52.8%) had ctDNA detected at baseline and at 3 weeks, respectively. ctDNA detection at baseline and PIK3CA mutation had an adverse prognostic impact on PFS and OS in multivariate analysis. This prospective cohort study documents the efficacy of fulvestrant and everolimus in CDK4/6i-pretreated ER + /HER2- mBC and highlights the clinical validity of early ctDNA changes as pharmacodynamic biomarker., (© 2024. The Author(s).)

Metastasis 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., Competing Interests: Declaration of interests M.E. is a member of the research advisory board for brensocatib for Insmed, Inc., a member of the scientific advisory board for Vividion Therapeutics, Inc., and a consultant for Protalix, Inc., and holds shares in Agios Pharmaceuticals, Inc. J.A., C.G., and A.M. have a patent related to this work (EP22306004)., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The 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., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Purpose: Blunt cerebrovascular injuries (BCVI) are a rare but serious complication after trauma. Among patients with BCVI, neurological status is altered in 30% of cases and the prognosis seems to be associated with ischemic complications. The aim of this study was to assess the long-term outcome of BCVI-associated ischemic events., Methods: This retrospective cohort study (2011-2017) included all patients admitted for severe trauma with identified BCVI in two level-1 trauma centers. Patients were considered to have a poor neurological outcome with a GOS-E between 2 and 5 and a good neurological outcome with GOS-E between 6 and 8. A multivariate logistic regression identified risk factors for poor neurological outcome at 1 year., Results: Of the 6,294 patients admitted in both trauma centers between 2011 and 2017, 81 patients presenting BCVI were identified (incidence of 1.3%). The median age was 35 years (24-44) with a median Injury Severity Score of 28 (17-41). 29 patients (50%) had a good neurological prognosis, while 25 patients (43%) had a poor neurological prognosis at 1 year. Ischemic stroke occurred in 11 patients (13.6%) within a median time of 2 days (2-2.5). No ischemic stroke occurred in the first year after ICU discharge in both groups. In our study, good prognosis at 1 year was not associated with ischemic complications [3 (10) vs 3 (12) p = 1]., Conclusion: Ischemic complications after BCVI are rare, occur within the first week and do not seem to impact independently the 1-year neurological prognosis., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany.)

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., Competing Interests: The authors have no disclosure., (© 2022 The Authors. Journal of Extracellular Biology published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Purpose: The aim of this work is to study a cohort of patients of ISS < 15 admitted to a TC, and to determine the number of patients that ultimately benefited from the skills and resources specific of a level 1 trauma center., Methods: Retrospective study from a prospective cohort of patients admitted to TC (Beaujon Hospital, APHP) for suspected severe trauma from January 2011 to December 2017. The main outcome criterion was the use of surgery or interventional radiology within the first 24 h after admission of patients with ISS < 15. The secondary outcomes were stratified into severe (mortality, resuscitation care, length of stay in intensive care units) and non-severe criteria (mild head injury, hospital discharge or transfer within 24 h)., Results: Of 3035 patients admitted during the study period, 1409 with an ISS < 15 were included, corresponding to a theoretical overtriage rate of 46.4%. Among these, 611 patients (43.4%) underwent emergency intervention within the first 24 h (586 surgical interventions, 19 direct transfers to the operating theater and 6 acts of interventional radiology), 238 (16.9%) of patients presented with severe and 531 (38%) with non-severe outcome criteria., Conclusion: This work demonstrates that in a cohort of patients classified as ISS < 15 admitted to a TC, a considerable amount of TC-specific resources are required, and patients present with severe outcome criteria despite being classified as overtriaged. These results suggest that triage of trauma patients should be based on resource use and clinical outcome rather than anatomic criteria., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

Circulating 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., (© 2021. The Author(s).)

Objective: To introduce the laparoscopic approach in liver transplant recipients., Summary of Background Data: Despite the increasingly frequent use of laparoscopy in living donor hepatectomy, the laparoscopic approach has never been reported in liver transplant recipients., Methods: A 52-year-old woman (body mass index: 18.5 kg/m) with neuroendocrine liver metastases of a digestive origin underwent hybrid liver transplantation by pure laparoscopic total hepatectomy and liver graft implantation using a preexisting midline incision. The hepatic pedicle vessels were dissected after division of the bile duct without a porto-caval shunt. Left lateral sectionectomy and early division of the common trunk allowed near completion of caval dissection with no prolonged inflow occlusion. The liver graft was reduced and latero-lateral caval anastomosis was performed., Results: Surgery lasted 400 minutes with 400 mL of blood loss. The anhepatic phase lasted 43 minutes. Warm ischemia time and cold ischemia times were 38 and 466 minutes, respectively. The postoperative course was uneventful., Conclusions: This case study suggests that the hybrid approach may be feasible and safe in selected recipients. The decision to use this surgical approach should be made in transplant centers with significant expertise in both laparoscopic liver and pancreatic surgery. Further reducing the size of the abdominal incision is the next step, which may be achieved with the development of vascular anastomoses devices.

Background: Transporting a severely injured patient directly to a trauma center (TC) is consensually considered optimal. Nevertheless, disagreement persists regarding the association between secondary transfer status and outcome. The aim of the study was to compare adjusted mortality between road traffic trauma patients directly or secondarily transported to a level 1 trauma center (TC) in an exclusive French trauma system with a physician staffed prehospital emergency medical system (EMS)., Methods: A retrospective cohort study was performed using 2015-2017 data from a regional trauma registry (Traumabase®), an administrative database on road-traffic accidents and prehospital-EMS records. Multivariate logistic regression models were computed to determine the role of the modality of admission on mortality and to identify factors associated with secondary transfer. The primary outcome was day-30 mortality. Results: During the study period, 121.955 victims of road-traffic accident were recorded among which 4412 trauma patients were admitted in the level 1 regional TCs, 4031 directly and 381 secondarily transferred from lower levels facilities. No significant association between all-cause 30-day mortality and the type of transport was observed (Odds ratio 0.80, 95% confidence interval (CI) [0.3-1.9]) when adjusted for potential confounders. Patients secondarily transferred were older, with low-energy mechanism and presented higher head and abdominal injury scores. Among all 947 death, 43 (4.5%) occurred in lower-level facilities. The population-based undertriage leading to death was 0.15%, 95%CI [0.12-0.19]., Conclusion: In an exclusive trauma system with physician staffed prehospital care, road-traffic victims secondarily transferred to a TC do not have an increased mortality when compared to directly transported patients., Competing Interests: The authors have declared that no competing interests exist.

Background: In nonmetastatic triple-negative breast cancer (TNBC) patients, we investigated whether circulating tumor DNA (ctDNA) detection can reflect the tumor response to neoadjuvant chemotherapy (NCT) and detect minimal residual disease after surgery., Methods: Ten milliliters of plasma were collected at 4 time points: before NCT; after 1 cycle; before surgery; after surgery. Customized droplet digital PCR (ddPCR) assays were used to track tumor protein p53 ( TP53 ) mutations previously characterized in tumor tissue by massively parallel sequencing (MPS)., Results: Forty-six patients with nonmetastatic TNBC were enrolled. TP53 mutations were identified in 40 of them. Customized ddPCR probes were validated for 38 patients, with excellent correlation with MPS ( r = 0.99), specificity (≥2 droplets/assay), and sensitivity (at least 0.1%). At baseline, ctDNA was detected in 27/36 patients (75%). Its detection was associated with mitotic index ( P = 0.003), tumor grade ( P = 0.003), and stage ( P = 0.03). During treatment, we observed a drop of ctDNA levels in all patients but 1. No patient had detectable ctDNA after surgery. The patient with rising ctDNA levels experienced tumor progression during NCT. Pathological complete response (16/38 patients) was not correlated with ctDNA detection at any time point. ctDNA positivity after 1 cycle of NCT was correlated with shorter disease-free ( P < 0.001) and overall ( P = 0.006) survival., Conclusions: Customized ctDNA detection by ddPCR achieved a 75% detection rate at baseline. During NCT, ctDNA levels decreased quickly and minimal residual disease was not detected after surgery. However, a slow decrease of ctDNA level during NCT was strongly associated with shorter survival., (© 2016 American Association for Clinical Chemistry.)

Background & Aims: Lipopolysaccharide (LPS)-expressing bacteria cause severe inflammation in cirrhotic patients. The global gene response to LPS is unknown in cirrhotic immune cells., Methods: Gene-expression profiling using Affymetrix Human Exon Array analyzed the expression of 14,851 genes in LPS-stimulated peripheral blood mononuclear cells (PBMCs) from 4 patients with cirrhosis and 4 healthy subjects. We performed validation studies using RT-qPCR in LPS-stimulated PBMCs from 52 patients and 9 healthy subjects and investigated the association of gene induction with mortality in 26 patients., Results: Gene-expression profiling of LPS-stimulated cirrhotic cells showed 509 upregulated genes and 1588 downregulated genes. In LPS-stimulated "healthy" cells, 952 genes were upregulated and 838 genes downregulated. The 741 LPS-regulated genes shared by cirrhotic and "healthy" cells were involved in cytokine production/activity and induction of "immune paralysis". Comparison of functions associated with the 1356 genes, specifically regulated by LPS in cirrhotic cells, to functions of the 1049 genes, specifically regulated in "healthy" cells, allowed to define a cirrhosis-specific phenotype. Unlike in "healthy" cells, LPS failed to induce an interferon-mediated program in cirrhotic cells. In cirrhotic PBMCs, LPS specifically induced certain molecules involved in apoptosis and downregulated molecules involved in endocytic trafficking. RT-qPCR experiments showed that LPS-stimulated cirrhotic PBMCs had an enhanced induction of certain proinflammatory cytokines and chemokines. In the prognosis study, higher ex vivo LPS-induction of the inflammatory genes IL6 and CXCL5 was a significant predictor of mortality., Conclusions: Our results show that LPS-stimulated cirrhotic PBMCs exhibit an extensive and often unexpected transcriptional response., (Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)