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Introduction: Progressive advanced non-small cell lung cancer (NSCLC) accounts for about 80–85% of all lung cancers. Approximately 10–50% of patients with NSCLC harbor targetable activating mutations, such as in-frame deletions in Exon 19 (Ex19del) of EGFR. Currently, for patients with advanced NSCLC, testing for sensitizing mutations in EGFR is mandatory prior to the administration of tyrosine kinase inhibitors. Patients and Methods: Plasma was collected from patients with NSCLC. We carried out targeted NGS using the Plasma-SeqSensei™ SOLID CANCER IVD kit on cfDNA (circulating free DNA). Clinical concordance for plasma detection of known oncogenic drivers was reported. In a subset of cases, validation was carried out using an orthogonal OncoBEAMTM EGFR V2 assay, as well as with our custom validated NGS assay. Somatic alterations were filtered, removing somatic mutations attributable to clonal hematopoiesis for our custom validated NGS assay. Results: In the plasma samples, driver targetable mutations were studied, with a mutant allele frequency (MAF) ranging from 0.00% (negative detection) to 82.25%, using the targeted next-generation sequencing Plasma-SeqSensei™ SOLID CANCER IVD Kit. In comparison with the OncoBEAMTM EGFR V2 kit, the EGFR concordance is 89.16% (based on the common genomic regions). The sensitivity and specificity rates based on the genomic regions (EGFR exons 18, 19, 20, and 21) were 84.62% and 94.67%. Furthermore, the observed clinical genomic discordances were present in 25% of the samples: 5% in those linked to the lower of coverage of the OncoBEAMTM EGFR V2 kit, 7% in those induced by the sensitivity limit on the EGFR with the Plasma-SeqSensei™ SOLID CANCER IVD Kit, and 13% in the samples linked to the larger KRAS, PIK3CA, BRAF coverage of the Plasma-SeqSensei™ SOLID CANCER IVD kit. Most of these somatic alterations were cross validated in our orthogonal custom validated NGS assay, used in the routine management of patients. The concordance is 82.19% in the common genomic regions (EGFR exons 18, 19, 20, 21; KRAS exons 2, 3, 4; BRAF exons 11, 15; and PIK3CA exons 10, 21). The sensitivity and specificity rates were 89.38% and 76.12%, respectively. The 32% of genomic discordances were composed of 5% caused by the limit of coverage of the Plasma-SeqSensei™ SOLID CANCER IVD kit, 11% induced by the sensitivity limit of our custom validated NGS assay, and 16% linked to the additional oncodriver analysis, which is only covered by our custom validated NGS assay. Conclusions: The Plasma-SeqSensei™ SOLID CANCER IVD kit resulted in de novo detection of targetable oncogenic drivers and resistance alterations, with a high sensitivity and accuracy for low and high cfDNA inputs. Thus, this assay is a sensitive, robust, and accurate test.

The isolation of circulating tumor cells (CTCs) directly from blood, as a liquid biopsy, could lead to a paradigm shift in cancer clinical care by providing an earlier diagnosis, a more accurate prognosis, and personalized treatment. Nevertheless, CTC-specific challenges, including their rarity and heterogeneity, have hampered the wider use of CTCs in clinical studies. Microfluidic-based isolation technologies have emerged as promising tools to circumvent these limitations but still fail to meet the constraints of high purity and short processing time required to ensure compatibility with clinical follow-up. In this study, we developed an immunomagnetic-based microfluidic device, the MagPure chip, to achieve the negative selection of CTCs through the depletion of white blood cells (WBCs) and provide highly purified samples for subsequent analysis. We demonstrate that the MagPure chip depletes all magnetically labeled WBCs (85% of WBCs were successfully labeled) and ensures a CTC recovery rate of 81%. In addition, we show its compatibility with conventional biological studies, including 2D and 3D cell culture, as well as phenotypic and genotypic analyses. Finally, we successfully implemented a two-step separation workflow for whole blood processing by combining a size-based pre-enrichment system (ClearCell FX1®) with the MagPure chip as a subsequent purification step. The total workflow led to high throughput (7.5 mL blood in less than 4 h) and high purity (947 WBCs per mL remaining, 99.99% depletion rate), thus enabling us to quantify CTC heterogeneity in size and tumor marker expression level. This tumor-marker-free liquid biopsy workflow could be used in a clinical context to assess phenotype aggressiveness and the prognosis rate.

Summary We report a case of metastatic pancreatic neuroendocrine carcinoma associated with paraneoplastic Cushing’s syndrome, successively treated with five lines of treatment (platin-etoposide, LV5FU2-dacarbazine, FOLFIRINOX, pembrolizumab, and paclitaxel) and anti-secretory treatment. Circulating-free DNA (cfDNA) was analysed at each morphological evaluation starting from the second-line treatment. cfDNA changes were well correlated with the disease course, and cfDNA may be used as a predictive marker and/or as an early marker of response. In addition, the absolute count of atypical cells was elevated upon disease progression. Learning points cfDNA changes were well correlated with the Cushing’s syndrome course and with the tumour burden changes assessed by laboratory markers and by RECIST criteria. cfDNA analysis was used to determine the pharmacogenetic patterns of the present patient. An elevated number of atypical circulating cells was noticed upon disease progression.

Patient-Derived Xenografts (PDXs) in the Chorioallantoic Membrane (CAM) are a representative model for studying human tumors. Circulating Tumor Cells (CTCs) are involved in cancer dissemination and treatment resistance mechanisms. To facilitate research and deep analysis of these few cells, significant efforts were made to expand them. We evaluated here whether the isolation of fresh CTCs from patients with metastatic cancers could provide a reliable tumor model after a CAM xenograft. We enrolled 35 patients, with breast, prostate, or lung metastatic cancers. We performed microfluidic-based CTC enrichment. After 48–72 h of culture, the CTCs were engrafted onto the CAM of embryonated chicken eggs at day 9 of embryonic development (EDD9). The tumors were resected 9 days after engraftment and histopathological, immunochemical, and genomic analyses were performed. We obtained in ovo tumors for 61% of the patients. Dedifferentiated small tumors with spindle-shaped cells were observed. The epithelial-to-mesenchymal transition of CTCs could explain this phenotype. Beyond the feasibility of NGS in this model, we have highlighted a genomic concordance between the in ovo tumor and the original patient’s tumor for constitutional polymorphism and somatic alteration in one patient. Alu DNA sequences were detected in the chicken embryo’s distant organs, supporting the idea of dedifferentiated cells with aggressive behavior. To our knowledge, we performed the first chicken CAM CTC-derived xenografts with NGS analysis and evidence of CTC dissemination in the chicken embryo.

Introduction: Treatment options of lung cancer (LC) comprise radiotherapy, and/or combined treatment approaches, including chemotherapy, immunotherapy and targeted therapies based on the tumoral molecular profile. Following curative-intent first-line therapies, clinical surveillance involves serial CT imaging. However, such surveillance can detect only macroscopic disease recurrence and is frequently inconclusive. NGS has been utilized to help identify and monitor treatment plans. Nucleosomes, complexes of DNA and histones proteins, are released during cell death into blood circulation. Trimethylation of lysine 27 on histone H3 (H3K27Me3), catalyzed by enhancer of zeste homolog 2 (EZH2), is a crucial epigenetic process in tumorigenesis. We investigated if H3K27Me3-nucleosome concentration could be a biomarker for molecular residual disease (MRD). Patients and Methods: Plasmas were retrospectively collected from patients with advanced LC during treatment (CIRCAN’s cohort, n= 200) and from healthy donors (n=100). We carried out standard targeted NGS on paired plasmas. Samples were divided in two sub-groups based on genetical results: ctDNA negative (n=120) or positive (n=80) for presence of somatic alterations. Concentration of circulating H3K27Me3-nucleosome was measured using chemiluminescent Nu.Q® immunoassay (Belgian Volition SRL, Belgium). Results: Significantly elevated concentrations of H3K27Me3-nucleosomes were found in LC plasmas during the follow-up of patients compared to healthy donors (median 14.9 ng/ml vs 6.15 ng/ml, respectively, p Conclusions: High levels of Nu.Q® H3K27Me3 could allow physicians to detect MRD in LC patients following treatment with curative intent. This could be achieved by monitoring testing of patients at defined intervals of treatment and recovery, alongside imaging, to incorporate analyses of evolving molecular landscapes during treatment. In this setting, the H3K27Me3-nucleosome quantification, to complete the molecular exploration of cfDNA is highly encouraging, especially in advanced NSCLC, where re-tissue biopsies are impractical, expensive, and may cause undue harm. H3K27Me3-nucleosome quantification may also be useful in patient identification for specific treatments such as EZH2 inhibitor, but this requires investigation. Citation Format: Emmanuel Grolleau, Julie Candiracci, Arnaud Gauthier, Gaelle Lescuyer, David Barthelemy, Christine Haon, Florence Geiguer, Margaux Raffin, Nathalie Hardat, Julie Balandier, Rémi Rabeuf, Anne-Sophie Wozny, Guillaume Rommelaere, Claire Rodriguez-Lafrasse, Fabien Subtil, Sébastien Couraud, Marielle Herzog, Lea Payen-Gay. Circulating H3K27 nucleosomes to monitor lung cancer patients during treatment, a universal biomarker quantifying the molecular residual disease (MRD) in plasma samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2163.

Introduction: NSCLC accounts for about 80-85% of all lung cancers. Approximately 10-50% of patients with NSCLC harbor EGFR activating mutations, such as in-frame deletions in exon 19 (Ex19del). Currently, for patients with advanced NSCLC, testing for sensitizing mutations in EGFR is mandatory prior to the administration of anti-EGFR inhibitors such as Osimertinib. The CAM xenograft offers a rich environment in nutrients and embryonic growth factors, favorable for aggressive tumor development, with its highly vascularized membrane and natural immunodeficiency at engraftment (at EDD9). Methods: HCC827 Ex19del cells have been engrafted in ovo, and treated with various doses of osimertinib for seven days (4 treatments). The xenografts were collected and immunohistopathological, transcriptional and genetic analyses were carried out. Based on human transcriptional data, supervised multivariate discrimination between conditions and ontology analysis was performed through GSEA. Results: The xenograft growth was inversely dependent on the exposure dose of osimertinib, with a reduction in tumor weight of 58% for 10 µM (corresponding to 8.3 µg/Kg, T1/2 =4hr). The histological analysis showed epithelial tumor cells composed of solid sheets. The quantity of tumor cells was significantly lower in the treated group, with an area of tumor necrosis. The immunohistochemistry of tumor cells confirmed the human pulmonary epithelial origin, with TTF1 positive in treated and control groups. As expected, the ontology analysis of transcriptomic findings highlighted a downregulation of the EGFR pathway and its downstream effectors. Furthermore, the transcriptomic responses associated with chemotaxis, immune cell recruitment, and angiogenesis were dampened in the presence of osimertinib. This suggests that therapeutic efficacy was not uniquely guided through cell-autonomous mechanisms, but also takes place at the tissue level. We confirmed this in an independent experiment of in ovo HCC827 engraftment, showing a statically significant reduction of 15% in the number of vessels surrounding the xenograft in the osimertinib-treated condition. This demonstrates the advantage of using the in ovo model to decipher the regulations associated with therapeutics in a complex tissue context. Conclusions: For the first time, we have observed that osimertinib modulates angiogenesis and chemotaxis, which apparently reduces immune system recruitment and immune checkpoint responses. The ontologic analysis of transcriptomic findings in the CAM model strongly support the clinical observations of the pharmacological tumoral response to targeted therapy. The fact that immune infiltration was reduced in osimertinib-treated tumors may partially explain the lower response to immunotherapy regimens in osimertinib resistance contexts. Citation Format: David Barthelemy, Arnaud Vigneron, Xavier Rousset, Jerome Guitton, Emmanuel Grolleau, Margaux Raffin, Julie Balandier, Gaelle Lescuyer, Florence Geiguer, Sebastien Couraud, Jean Vaillet, Nazim Benzerdjeb, Lea Payen-Gay. Pharmacological mechanisms of osimertinib in advanced non-small lung cancer bearing the deletion exon 19 of EGFR In an original chicken chorioallantoic membrane (CAM) Model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4866.

Background: Noninvasive genotyping using plasma cell-free DNA (cfDNA) has the potential to obviate the need for some invasive biopsies in cancer patients. We sought to validate an ultra-deep plasma Next-Generation Sequencing (NGS) assay for patients with Non-Small-Cell Lung Cancers (NSCLC) for detecting Minimal Residual Disease (MRD) following curative-intent NSCLC treatment or EGFR detection at an early stage. Patients and Methods: We carried out targeted NGS using the Plasma-SeqSensei™ Solid Cancer IVD Kit on cfDNA extracted from plasma. Somatic alterations were filtered, removing somatic mutations attributable to clonal hematopoiesis. Sensitivity and specificity for plasma detection of known oncogenic drivers were calculated. In a subset of cases, validation was carried out using an orthogonal OncoBEAMTM EGFR V2 assay, as well as with a custom-validated NGS assay. Results: In comparison with the OncoBEAMTM EGFR V2 kit, we observed a 75% concordance with the Plasma-SeqSensei™ Solid Cancer IVD Kit. The 25% genomic discordances were respectively observed in 5%, 7% and 13% linked to the limit of coverage of the OncoBEAMTM EGFR V2 kit, the limit of EGFR sensitivity of the Plasma-SeqSensei™ Solid Cancer IVD Kit and the larger KRAS, PIK3CA, BRAF coverage of the Plasma-SeqSensei™ Solid Cancer IVD Kit. In comparison with the custom-validated NGS assay, we observed a 68% concordance with the Plasma-SeqSensei™ Solid Cancer IVD Kit. The 32% genomic discordances were respectively observed in 5%, 11% and 16% linked to the limit of coverage of the Plasma-SeqSensei™ Solid Cancer IVD Kit, the limit of covered sensitivity of the custom-validated NGS assay and the additional oncodrivers analysis only covered by the custom-validated NGS assay. Benefitting from the higher coverage from targetable BRAF, KRAS, PIK3CA targetable alterations, the plasma targeted NGS Plasma-SeqSensei™ Solid Cancer IVD Kit showed 13% additional discordances with the Plasma OncoBEAMTM EGFR V2 kit. Most of these additional somatic alterations were cross-validated in our orthogonal custom-validated NGS assay, used in the routine management of patients. The custom-validated NGS assay sensitivity is determined for MAF at 1%, explaining the limit of sensitivity with the Plasma-SeqSensei™ Solid Cancer IVD Kit. Conclusions: Plasma-SeqSensei™ Solid Cancer IVD Kit resulted in de novo detection of targetable oncogenic drivers and resistance mechanisms in patients with NSCLC, including when tissue biopsies were inadequate for genotyping with high sensitivity and accuracy for low and high cfDNA inputs. Citation Format: David Barthelemy, Emmanuel Grolleau, Gaelle Lescuyer, Florence Geiguer, Arnaud Gauthier, Julie Balandier, Margaux Raffin, Claire Bardel, Bruno Bouyssounouse, Claire Rodriguez-Lafrasse, Sébastien Couraud, Anne-Sophie Wozny, Lea Payen-Gay. Paired comparison of routine molecular screening of patient samples with advanced non-small cell lung cancer in circulating cell-free DNA using OncoBEAMTM EGFR V2, targeted next-generation sequencing Plasma-SeqSensei™ Solid Cancer IVD Kit and custom-validated NGS assay. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6691.

Magnetophoresis-based microfluidic devices offer simple and reliable manipulation of micro-scale objects and provide a large panel of applications, from selective trapping to high-throughput sorting. However, the fabrication and integration of micro-scale magnets in microsystems involve complex and expensive processes. Here we report on an inexpensive and easy-to-handle fabrication process of micrometer-scale permanent magnets, based on the self-organization of NdFeB particles in a polymer matrix (polydimethylsiloxane, PDMS). A study of the inner structure by X-ray tomography revealed a chain-like organization of the particles leading to an array of hard magnetic microstructures with a mean diameter of 4 µm. The magnetic performance of the self-assembled micro-magnets was first estimated by COMSOL simulations. The micro-magnets were then integrated into a microfluidic device where they act as micro-traps. The magnetic forces exerted by the micro-magnets on superparamagnetic beads were measured by colloidal probe atomic force microscopy (AFM) and in operando in the microfluidic system. Forces as high as several nanonewtons were reached. Adding an external millimeter-sized magnet allowed target magnetization and the interaction range to be increased. Then, the integrated micro-magnets were used to study the magnetophoretic trapping efficiency of magnetic beads, providing efficiencies of 100% at 0.5 mL/h and 75% at 1 mL/h. Finally, the micro-magnets were implemented for cell sorting by performing white blood cell depletion.

Background and Objectives The use of ultra-sensitive diagnostic tests to detect clinically actionable somatic alterations within the gene encoding the epidermal growth factor receptor (EGFR) within circulating cell-free DNA is an important first step in determining the eligibility of patients with non-small cell lung cancer to receive tyrosine kinase inhibitors. Methods We present the clinical validation (accuracy, sensitivity, and specificity) of a highly sensitive OncoBEAMTM EGFR V2 test, which we compare to a custom next-generation sequencing assay, for the treatment of patients with non-small cell lung cancer with EGFR tyrosine kinase inhibitor therapies. The OncoBEAMTM digital-polymerase chain reaction method detects 36 different EGFR alterations in circulating cell-free DNA, whereas the next-generation sequencing assay covers major solid tumor oncodrivers. Of the 540 samples analyzed with the OncoBEAMTM EGFR V2 test, 42.4% of patients had undergone molecular testing at diagnosis (N = 229/540) and 57.7% of patients during disease progression (N = 311/540). Results The sensitivity and specificity were measured for this BEAMing assay. The number of mutant beads and mutant allelic fraction were measured for each EGFR alteration and the level of detection was established at 0.1% for a median of 2861 genome equivalent (GE) in each reaction using HD780 horizon control DNA, as well as by an internal quality reference standard. Approximately 10%, 27%, and 63% of the 540 samples contained < 1500 GE, a range of 1500–3000 GE, and > 3000 GE, which corresponded to a maximal assay sensitivity of 2.0%, 0.5–0.1%, and 0.1–0.05% mutant allelic fraction, respectively. In a routine hospital setting, 11.4% of non-small cell lung cancer tumors were positive at diagnosis for EGFR alterations, while 43.7% samples harbored EGFR mutations at progression, among which 40.3% expressed EGFR resistance mutations after first-line tyrosine kinase inhibitor treatment with first- and second-generation drugs. Conclusions The OncoBEAMTM EGFR V2 is a sensitive, robust, and accurate assay that delivers reproducible results. Next-generation sequencing and BEAMing technologies act complementarily in the routine molecular screening. We show that using a next-generation sequencing assay, despite its lower sensitivity, enables the identification of rare EGFR alterations or resistance mechanisms (mutation, deletion, insertion, and copy number variation) to orient first- and second-line treatments. Supplementary Information The online version contains supplementary material available at 10.1007/s40291-021-00515-9.

International audience; Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1-10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.

The materials present the good practices of Total E&P Qatar to make its crisis management and emergency response effective: the way it is organized and the way the people involved are behaving. To protect its assets, TEPQ has developed its own Affiliate Emergency Response Plan. In case of emergency response, TEPQ sets different group of people structured in cells. Each cell has its own role in the emergency response, depending on the situation. The cross-functionality between each cell for the application of the emergency response plans is warranted by a central Emergency Response Cell. Real cases confirmed the effectiveness of the TEPQ emergency response organization. Nevertheless it highlighted the need of having the involved people more comfortable with their role and responsibilities. For this purpose two main actions were taken: The drills and exercises frequency of the cells have been increased;The number of people likely to be involved was decreased to better train them. As a summary, less people but better trained with more experience. For the trainings, TEPQ has set-up training matrices, called ComPass, defining the trainings or awareness mandatory for each position. All the employees and contractors involved in the emergency response are namely identified in the ComPass matrix of their site and their training status is followed. For the experience, each person likely to be involved in one of the emergency response cell participates to weekly tests, monthly table top, three-monthly exercises and yearly large scale exercises.