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1. CD63-GPC1-Positive Exosomes Coupled with CA19-9 Offer Good Diagnostic Potential for Resectable Pancreatic Ductal Adenocarcinoma

Author

Aurélie Bedel, Camille Buscail, O Degrandi, Laurence Chiche, Christophe Laurent, Charline Caumont, Séverine Verdon, Alain Brisson, Alexandre Chauvet, François Moreau-Gaudry, Véronique Vendrely, Marion Marty, Pascaline Quincy, Isabelle Lamrissi, Etienne Buscail, Isabelle Moranvillier, Sandrine Dabernat, Biothérapies des maladies génétiques et cancers, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), CHU Bordeaux [Bordeaux], Equipe 3: EREN- Equipe de Recherche en Epidémiologie Nutritionnelle (CRESS - U1153), Université Paris 13 (UP13)-Institut National de la Recherche Agronomique (INRA)-Conservatoire National des Arts et Métiers [CNAM] (CNAM)-Centre de Recherche Épidémiologie et Statistique Sorbonne Paris Cité (CRESS (U1153 / UMR_A_1125 / UMR_S_1153)), Université Paris Diderot - Paris 7 (UPD7)-Université Sorbonne Paris Cité (USPC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de la Recherche Agronomique (INRA)-Université Paris Diderot - Paris 7 (UPD7)-Université Sorbonne Paris Cité (USPC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de la Recherche Agronomique (INRA), Université de Bordeaux (UB), Chimie et Biologie des Membranes et des Nanoobjets (CBMN), École Nationale d'Ingénieurs des Travaux Agricoles - Bordeaux (ENITAB)-Institut de Chimie du CNRS (INC)-Université de Bordeaux (UB)-Centre National de la Recherche Scientifique (CNRS), Patrick Brou de Lauriere Fondation, Celgene company, Groupement Interregional de Recherche Clinique et d'Innovation Sud-Ouest OutreMer (GIRCI-SOOM), Admin, Oskar, AUDOUX, Michèle, Université Paris 13 (UP13)-Institut National de la Recherche Agronomique (INRA)-Conservatoire National des Arts et Métiers [CNAM] (CNAM), HESAM Université - Communauté d'universités et d'établissements Hautes écoles Sorbonne Arts et métiers université (HESAM)-HESAM Université - Communauté d'universités et d'établissements Hautes écoles Sorbonne Arts et métiers université (HESAM)-Centre de Recherche Épidémiologie et Statistique Sorbonne Paris Cité (CRESS (U1153 / UMR_A_1125 / UMR_S_1153)), Institut National de la Recherche Agronomique (INRA)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Université Sorbonne Paris Cité (USPC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de la Recherche Agronomique (INRA)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Université Sorbonne Paris Cité (USPC)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université de Bordeaux (UB)-École Nationale d'Ingénieurs des Travaux Agricoles - Bordeaux (ENITAB)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Inserm U1035, Biotherapies des Maladies Genetiques et Cancers, Univ Bordeaux, CHU de Bordeaux, Pole de Biologie et Pathologie, and Université de Bordeaux (UB)-CHU Bordeaux [Bordeaux]-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
0301 basic medicine, Cancer Research, medicine.medical_specialty, Original article, [SDV]Life Sciences [q-bio], [SDV.CAN]Life Sciences [q-bio]/Cancer, Exosome, Gastroenterology, lcsh:RC254-282, Flow cytometry, 03 medical and health sciences, 0302 clinical medicine, [SDV.CAN] Life Sciences [q-bio]/Cancer, Internal medicine, Medicine, Liquid biopsy, Prospective cohort study, [CHIM.MATE] Chemical Sciences/Material chemistry, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, medicine.diagnostic_test, CD63, business.industry, Cancer, [CHIM.MATE]Chemical Sciences/Material chemistry, medicine.disease, lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens, Microvesicles, 3. Good health, [SDV] Life Sciences [q-bio], 030104 developmental biology, Oncology, 030220 oncology & carcinogenesis, CA19-9, business, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract

Tumor-released extracellular vesicles (EVs) contain tumor-specific cargo distinguishing them from healthy EVs, and making them eligible as circulating biomarkers. Glypican 1 (GPC1)-positive exosome relevance as liquid biopsy elements is still debated. We carried out a prospective study to quantify GPC1-positive exosomes in sera from pancreatic ductal adenocarcinoma (PDAC) patients undergoing up-front surgery, as compared to controls including patients without cancer history and patients displaying pancreatic preneoplasic lesions. Sera were enriched in EVs, and exosomes were pulled down with anti-CD63 coupled magnetic beads. GPC1-positive bead percentages determined by flow cytometry were significantly higher in PDAC than in the control group. Diagnosis accuracy reached 78% (sensitivity 64% and specificity 90%), when results from peripheral and portal blood were combined. In association with echo-guided-ultrasound-fine-needle-aspiration (EUS-FNA) negative predictive value was 80% as compared to 33% for EUS-FNA only. This approach is clinically relevant as a companion test to the already available diagnostic tools, since patients with GPC1-positive exosomes in peripheral blood showed decreased tumor free survival.

Published
2019

2. High Clinical Value of Liquid Biopsy to Detect Circulating Tumor Cells and Tumor Exosomes in Pancreatic Ductal Adenocarcinoma Patients Eligible for Up-Front Surgery

Author

Pascaline Quincy, Laurence Chiche, Marion Marty, Aurélie Bedel, François Moreau-Gaudry, Isabelle Lamrissi, Isabelle Moranvillier, Alexandre Chauvet, Charline Caumont, Sandrine Dabernat, Camille Buscail, O Degrandi, Etienne Buscail, Christophe Laurent, Catherine Alix-Panabières, Véronique Vendrely, Séverine Verdon, Thomas Cauvin, Biothérapies des maladies génétiques et cancers, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), CHU Bordeaux [Bordeaux], Aide à la Décision pour une Médecine Personnalisé - Laboratoire de Biostatistique, Epidémiologie et Recherche Clinique - EA 2415 (AIDMP), Université Montpellier 1 (UM1)-Université de Montpellier (UM), Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier), Université de Bordeaux (UB), Hôpital Bretonneau, Centre Hospitalier Régional Universitaire de Tours (CHRU Tours), Equipe 3: EREN- Equipe de Recherche en Epidémiologie Nutritionnelle (CRESS - U1153), Université Paris 13 (UP13)-Institut National de la Recherche Agronomique (INRA)-Conservatoire National des Arts et Métiers [CNAM] (CNAM)-Centre de Recherche Épidémiologie et Statistique Sorbonne Paris Cité (CRESS (U1153 / UMR_A_1125 / UMR_S_1153)), Institut National de la Recherche Agronomique (INRA)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Université Sorbonne Paris Cité (USPC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de la Recherche Agronomique (INRA)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Université Sorbonne Paris Cité (USPC)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de médecine moléculaire de Rangueil (I2MR), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-IFR150-Institut National de la Santé et de la Recherche Médicale (INSERM), Service de Chirurgie Viscérale et Digestive [CHU Caen], CHU Caen, Normandie Université (NU)-Tumorothèque de Caen Basse-Normandie (TCBN)-Normandie Université (NU)-Tumorothèque de Caen Basse-Normandie (TCBN), Salvy-Córdoba, Nathalie, Université Paris 13 (UP13)-Institut National de la Recherche Agronomique (INRA)-Conservatoire National des Arts et Métiers [CNAM] (CNAM), HESAM Université - Communauté d'universités et d'établissements Hautes écoles Sorbonne Arts et métiers université (HESAM)-HESAM Université - Communauté d'universités et d'établissements Hautes écoles Sorbonne Arts et métiers université (HESAM)-Centre de Recherche Épidémiologie et Statistique Sorbonne Paris Cité (CRESS (U1153 / UMR_A_1125 / UMR_S_1153)), and Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées- Institut Fédératif de Recherche Bio-médicale Institution (IFR150)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
0301 basic medicine, Cancer Research, medicine.medical_specialty, endocrine system diseases, pancreatic cancer, [SDV.CAN]Life Sciences [q-bio]/Cancer, exosomes, circulating tumor cells, medicine.disease_cause, lcsh:RC254-282, Exosome, Article, Flow cytometry, 03 medical and health sciences, 0302 clinical medicine, Circulating tumor cell, [SDV.CAN] Life Sciences [q-bio]/Cancer, Pancreatic cancer, Medicine, Digital polymerase chain reaction, Liquid biopsy, medicine.diagnostic_test, liquid biopsy, business.industry, lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens, medicine.disease, digestive system diseases, 3. Good health, Surgery, Clinical trial, 030104 developmental biology, Oncology, 030220 oncology & carcinogenesis, KRAS, business
Abstract

Purpose: Expediting the diagnosis of pancreatic ductal adenocarcinoma (PDAC) would benefit care management, especially for the start of treatments requiring histological evidence. This study evaluated the combined diagnostic performance of circulating biomarkers obtained by peripheral and portal blood liquid biopsy in patients with resectable PDAC. Experimental design: Liquid biopsies were performed in a prospective translational clinical trial (PANC-CTC #NCT03032913) including 22 patients with resectable PDAC and 28 noncancer controls from February to November 2017. Circulating tumor cells (CTCs) were detected using the CellSearch&reg, method or after RosetteSep&reg, enrichment combined with CRISPR/Cas9-improved KRAS mutant alleles quantification by droplet digital PCR. CD63 bead-coupled Glypican-1 (GPC1)-positive exosomes were quantified by flow cytometry. Results: Liquid biopsies were positive in 7/22 (32%), 13/22 (59%), and 14/22 (64%) patients with CellSearch&reg, or RosetteSep&reg, based CTC detection or GPC1-positive exosomes, respectively, in peripheral and/or portal blood. Liquid biopsy performance was improved in portal blood only with CellSearch&reg, reaching 45% of PDAC identification (5/11) versus 10% (2/22) in peripheral blood. Importantly, combining CTC and GPC1-positive-exosome detection displayed 100% of sensitivity and 80% of specificity, with a negative predictive value of 100%. High levels of GPC1+-exosomes and/or CTC presence were significantly correlated with progression-free survival and with overall survival when CTC clusters were found. Conclusion: This study is the first to evaluate combined CTC and exosome detection to diagnose resectable pancreatic cancers. Liquid biopsy combining several biomarkers could provide a rapid, reliable, noninvasive decision-making tool in early, potentially curable pancreatic cancer. Moreover, the prognostic value could select patients eligible for neoadjuvant treatment before surgery. This exploratory study deserves further validation.

Published
2019

3. Assessment of BRAFV600E mutation in pulmonary Langerhans cell histiocytosis in tissue biopsies and bronchoalveolar lavages by droplet digital polymerase chain reaction

Author

Gaël Dournes, Marion Marty, Hugues Begueret, Camille Brochet, Elodie Blanchard, Jean-Philippe Merlio, Séverine Verdon, Charline Caumont, Thomas Bandres, Audrey Gros, and Clémence Pierry

Subjects
0301 basic medicine, Pathology, medicine.medical_specialty, Biology, medicine.disease_cause, Pulmonary Langerhans cell histiocytosis, Pathology and Forensic Medicine, law.invention, 03 medical and health sciences, 0302 clinical medicine, law, medicine, Digital polymerase chain reaction, In patient, Molecular Biology, Polymerase chain reaction, Mutation, medicine.diagnostic_test, Cell Biology, General Medicine, Molecular biology, BRAF V600E, 030104 developmental biology, Bronchoalveolar lavage, 030220 oncology & carcinogenesis, Immunohistochemistry
Abstract

The neoplastic nature of pulmonary Langerhans cell histiocytosis (PLCH) is still debated. As the detection of BRAF V600E and MAP2K1 mutations in patients with PCLH is now considered for such assessment, the aim of our study was to evaluate digital droplet polymerase chain reaction (ddPCR) in PCLH diagnosis. We retrospectively analyzed BRAFV600E detection in a cohort of 42 PCLH tissues and 18 bronchoalveolar lavages (BALs) by ddPCR, immunohistochemistry, high-resolution melting PCR (HRM), and next-generation sequencing (NGS). The presence of BRAFV600E mutation was assessed by at least two concordant techniques to further evaluate specificity and sensitivity of each method. The BRAF V600E mutation prevalence was detected in 18 out of 41 cases by ddPCR, 10 out of 36 cases by HRM PCR, and 16 out of 31 cases by NGS. BRAFV600E immunohistochemistry sensitivity was 94%, and specificity was 79%. HRM PCR sensitivity was only 59%, and specificity was 100%. NGS sensitivity and specificity were 100% for interpretable cases (n = 31), but in 11 cases, this technique was non-contributive. The analysis of BAL samples by ddPCR revealed a BRAFV600E mutation both in tissue and in BAL samples in one patient, a wild-type status both in tissue and in BAL samples in two patients, and a wild-type BRAF status in BAL and a BRAFV600E mutation in tissue samples in four patients. The study supports the usefulness of ddPCR for BRAF status assessment in either tissue or BAL samples to increase the accuracy of PLCH diagnosis.

Published
2017

4. Assessment of BRAF

Author

Clémence, Pierry, Charline, Caumont, Elodie, Blanchard, Camille, Brochet, Gael, Dournes, Audrey, Gros, Thomas, Bandres, Séverine, Verdon, Marion, Marty, Hugues, Bégueret, and Jean-Philippe, Merlio

Subjects
Adult, Genetic Markers, Male, Proto-Oncogene Proteins B-raf, Biopsy, DNA Mutational Analysis, High-Throughput Nucleotide Sequencing, Middle Aged, Bronchoalveolar Lavage, Immunohistochemistry, Polymerase Chain Reaction, Sensitivity and Specificity, Histiocytosis, Langerhans-Cell, Mutation, Humans, Female, Bronchoalveolar Lavage Fluid, Lung, Retrospective Studies
Abstract

The neoplastic nature of pulmonary Langerhans cell histiocytosis (PLCH) is still debated. As the detection of BRAF

Published
2017

5. The Detection of Double Mutations in KRAS Depends on the Mutation-Detection Assay Used

Author

Isabelle Hostein, Jean-Christophe Sabourin, Nicolas Faur, Aude Lamy, Séverine Verdon, Isabelle Soubeyran, and Charlotte Primois

Subjects
Colorectal cancer, medicine.drug_class, Clinical Biochemistry, Viral Oncogene, Adenocarcinoma, Biology, medicine.disease_cause, Monoclonal antibody, Polymerase Chain Reaction, Proto-Oncogene Proteins p21(ras), Proto-Oncogene Proteins, medicine, Humans, Panitumumab, Epidermal growth factor receptor, Codon, neoplasms, Mutation, Cetuximab, Biochemistry (medical), Exons, medicine.disease, Molecular biology, ras Proteins, biology.protein, KRAS, Colorectal Neoplasms, medicine.drug
Abstract

To the Editor: Epidermal growth factor receptor is frequently overexpressed in colorectal cancer, and targeted therapies have been developed to block this receptor. Monoclonal antibodies such as cetuximab and panitumumab have demonstrated effectiveness in increasing the survival rates for some metastatic patients. Patients with mutations in codon 12 or 13 of the KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) gene are not responsive to this therapy, however, and tumors from patients are systematically screened for somatic KRAS mutations before treatment. Several techniques have been developed to screen for KRAS mutations, and the differences in these techniques with respect to diagnostic sensitivity, diagnostic specificity, and efficiency for mutation detection have been described (1,–,4). The main potential weakness of direct sequencing is its low diagnostic sensitivity (approximately 20%). Other techniques, such as TaqMan® PCR–based assays and SNaPshot® (Applied Biosystems), have diagnostic sensitivities between 1% and 10%. In this study, we analyzed 1970 colorectal carcinoma tumors for KRAS mutations by direct sequencing. We detected 10 cases …

Published
2011

6. CD63-GPC1-Positive Exosomes Coupled with CA19-9 Offer Good Diagnostic Potential for Resectable Pancreatic Ductal Adenocarcinoma

Author

Etienne Buscail, Alexandre Chauvet, Pascaline Quincy, Olivier Degrandi, Camille Buscail, Isabelle Lamrissi, Isabelle Moranvillier, Charline Caumont, Severine Verdon, Alain Brisson, Marion Marty, Laurence Chiche, Christophe Laurent, Veronique Vendrely, François Moreau-Gaudry, Aurelie Bedel, and Sandrine Dabernat

Subjects
Neoplasms. Tumors. Oncology. Including cancer and carcinogens, RC254-282
Abstract

Tumor-released extracellular vesicles (EVs) contain tumor-specific cargo distinguishing them from healthy EVs, and making them eligible as circulating biomarkers. Glypican 1 (GPC1)-positive exosome relevance as liquid biopsy elements is still debated. We carried out a prospective study to quantify GPC1-positive exosomes in sera from pancreatic ductal adenocarcinoma (PDAC) patients undergoing up-front surgery, as compared to controls including patients without cancer history and patients displaying pancreatic preneoplasic lesions. Sera were enriched in EVs, and exosomes were pulled down with anti-CD63 coupled magnetic beads. GPC1-positive bead percentages determined by flow cytometry were significantly higher in PDAC than in the control group. Diagnosis accuracy reached 78% (sensitivity 64% and specificity 90%), when results from peripheral and portal blood were combined. In association with echo-guided-ultrasound-fine-needle-aspiration (EUS-FNA) negative predictive value was 80% as compared to 33% for EUS-FNA only. This approach is clinically relevant as a companion test to the already available diagnostic tools, since patients with GPC1-positive exosomes in peripheral blood showed decreased tumor free survival.

Published
2019
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7. Tumor homogeneity between primary and metastatic sites for BRAF status in metastatic melanoma determined by immunohistochemical and molecular testing.

Author

Lucile Boursault, Véronique Haddad, Béatrice Vergier, David Cappellen, Severine Verdon, Jean-Pierre Bellocq, Thomas Jouary, and Jean-Philippe Merlio

Subjects
Medicine, Science
Abstract

BRAF inhibitors have demonstrated improvement of overall survival in patients with metastatic melanoma and BRAF(V600) mutations. In order to evaluate BRAF tumor heterogeneity between primary and metastatic site, we have evaluated the performance of immunohistochemistry (IHC) with an anti-BRAF(V600E) antibody in both localization by comparison with high resolution melting analysis followed by Sanger sequencing in a parallel blinded study. A total of 230 samples distributed as primary melanoma (n = 88) and different types of metastatic samples (n = 142) were studied in 99 patients with advanced or metastatic melanoma (stage III or IV). The prevalence of each BRAF mutation was c.1799T>A, BRAF(V600E) (45.2%), c.1799_1800TG>AA, BRAF(V600E2) (3.0%), c.1798_1799GT>AA, BRAF(V600K) (3.0%), c.1801 A>G, BRAF(K601E) (1.3%), c.1789_1790CT>TC, BRAF(L597S) (0.4%), c.1780G>A, BRAF(D594N) (0.9%) respectively. IHC was positive in 109/112 samples harboring BRAF(V600E/E2) mutations and negative in other cases. The cytoplasmic staining was either strongly positive in tumor cells of BRAF(V600E) mutated cases. It appeared strong brown, different from the vesicular grey cytoplasmic pigmentation of melanophages. Concordance between the two techniques was 96.4%. Sensitivity of IHC for detecting the BRAF(V600E/E2) mutations was 97.3%, while specificity was 100%. Both our IHC and molecular study demonstrated homogeneity between primary and metastatic sites for BRAF status in melanoma. This study also provides evidence that IHC may be a cost-effective first-line method for BRAF(V600E) detection. Thereafter, molecular techniques should be used in negative, ambiguous or non-contributive cases.

Published
2013
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