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One-step nucleic acid amplification (OSNA) is a molecular procedure used intraoperatively for the detection of sentinel lymph node (SLN) metastases. The aim of the present study was to define a cut-off of cytokeratin (CK)19 mRNA copy number predictive of positive completion axillary lymph node dissection (ALND). The OSNA procedure was employed for SLN analysis in 812 patients with T1-T2 N0 breast cancer. A total of 197 patients with SLN metastases were retrospectively analyzed. A total of 40 patients (20%) had non-SLN metastases. Receiver operating characteristics curve analysis established a cut-off of 5,000 CK19 mRNA copy number with 75% sensitivity and 72% specificity. The positive and negative predictive values were 40.5 and 92%, respectively. Multivariate analysis showed that this cut-off and tumor localization in the outer or lower-outer quadrant of the breast were significantly associated with non-SNL involvement (P

International audience; This book is a comprehensive guide to the techniques, clinical applications, and benefits of the different forms of liquid biopsy employed in patients with a variety of tumor types, including lung, breast and colorectal cancer. Offering detailed explanations, it discusses the how changes in tumors can be tracked using these cutting-edge technologies, which enable the detection and analysis of diverse circulating biomarkers: tumor cells, tumor DNA, tumor RNA (free or in exosomes), and fluid biomarkers identifiable by means of targeted proteomics. The use of such advanced technologies is enabling us to tackle questions and problems in a way that was not possible just a few years ago. We now have at our disposal an effective means of overcoming the problem of intratumor heterogeneity, which has limited the value of conventional biopsy approaches. As a consequence, oncology practice is about to change radically, toward truly personalized precision medicine. This book provides both clinicians and researchers with a thorough and up-to-date overview of progress in the field.

published in Cancer Research,79(4 Suppl):OT1-03-02, February 2019; International audience; Activating ESR1 mutations have recently been reported as a key mechanism leading to Aromatase Inhibitor (AI) resistance. ESR1 mutations occur rarely in primary breast cancers. However, in large retrospective studies, ESR1 mutations occurred in up to 39% of Estrogen-Receptor(ER)-positive metastatic breast cancer resistant to AI. Numerous hotspot mutations have been identified, most of them affecting the ligand-binding domain (LBD) and leading to ligand-independent activation of the ER and to resistance to AI.Phosphatidylinositol 3-kinase (PI3K)/AKT pathway is involved in key Cellular Mechanisms and mutations in PIK3CA and AKT1 are frequently reported in breast cancer.In this study, we propose to use a capture-based Next Generation Sequencing (NGS) assay and to use the barcoding and polishing features in our analysis pipeline. This assay will be able to detect all mutations on AKT1, PIK3CA, ESR1 and other genes on circulating tumor DNA (ctDNA) extracted from blood samples of patients with breast cancer. We consider that this exon-screening strategy is relevant according to the recent knowledge.We plan to prospectively include women with advanced breast cancer about to begin standard-of-care first line endocrine therapy (ET). Patients will be required to have histologically confirmed ER-positive, HER2-negative breast cancer and documented loco-regionally advanced or metastatic disease, not amenable to surgery or radiation with curative intent. Patients with endocrine sensitive disease (no prior ET or relapse more than 12 months after completing adjuvant ET) as well as patients with endocrine resistant disease (relapse while on adjuvant ET or within 12 months of completing adjuvant ET) will be enrolled.ET can be prescribed alone or in combination with a targeted therapy. Nevertheless, we will recruit at least 25% of patients with exclusive ET in the endocrine sensitive group.Peripheral-blood samples, for analysis of ctDNA, will be obtained from participating patients at pre-specified time points: at start of ET to determine the baseline mutational status of ESR1, PIK3CA, AKT1 and other genes included in a panel of genes of interest in solid tumors, and then, at evaluation of response to therapy until disease progression or end of study.Patients will be followed for 36 months or until disease progression. Determination of progression will be done per local investigator.The primary objective is to describe the prevalence of activating ESR1 mutations affecting the LBD, using NGS, from the start of ET to progression or end of study. Secondary objectives include to describe the prevalence of ESR1 mutations affecting other domains, the prevalence of ESR1 mutations in patients with and without endocrine resistance at enrolment and the prevalence of PIK3CA and AKT1 mutations, to demonstrate that ESR1, PIK3CA and AKT1 mutations whatever their times of onset are predictors of progression free survival.As of June 2018, 8 sites were opened to recruitment and 18 pts were included; the target enrollment is 146. The trial is supported by AstraZeneca.

published in Journal of Clinical Oncology, 34(7-Suppl), 2016.; International audience; Background: Despite the great histological and molecular heterogeneity, the clinical management of EOC remains univocal. Additionally to conventional histological prognostic markers and oncogenetic investigations, molecular diagnostic was performed using NGS to identify "druggable" targets that could provide access to targeted therapy in high-grade EOC. Methods: This study was performed in 53 patients (pts) (mean age 58.9, range 25-87) with histologically proven high-grade EOC, mostly serous carcinomas (N = 45), 44 were treated by surgery, 9 received neo-adjuvant chemotherapy. BRCA1/2 germline mutations were screened in 19 pts with familial cancer history justifying oncogenetic investigations. P53 and PTEN expression was assessed on formalin fixed paraffin-embedded using immunohistochemistry. Somatic mutations of KRAS and NRAS (exons 2, 3 and 4), BRAF (exon 15), PIK3CA (exons 5, 10 and 21) and MET (exons 14, 16, 17, 18 and 19) were screened using NGS on DNA extracts from frozen tumor specimens taken at diagnosis. Sequences were aligned with reference sequences and variant calling was processed. At X1000 depth, NGS sensitivity was 1%. Mapping, sorting and indexing were performed twice using AVA, BWA and SAMtools. VarScan2 was finally used for variant calling. Results: With a median follow-up of 38.2 months (range 1.4-93.3), 24 pts are alive, 14 pts disease-free and15 progressed within 6 months after platinum-based therapy. All pts screened for BRCA mutations had serous EOC, mutations were detected in 7/19 pts. P53 overexpression was detected in 60% and PTEN loss in 41% of the pts. One KRAS (exon 2), 2 NRAS mutations (exon 3), 6 PIK3CA mutations (exon 5, 10 and 21) and 5 MET mutations (exons 14 and 18) were detected. All clear-cell EOC were mutated (N = 4). No mutation was found in endometrioid EOC (N = 3). All MET and NRAS mutations were found in serous EOC (N = 7). Conclusions: In high-grade EOC, NGS has adequate sensitivity and specificity to detect genetic abnormalities and provide molecular rationale for targeted therapies, potentially offering new therapeutic opportunities to the pts.

published in Journal of Clinical Oncology, 34(7-Suppl), 2016. Poster Session, Breast Cancer—HER2/ER. Abstract Number: 2571. Poster Board Number: #94; International audience; Background: Blockade of PI3K/AKT/mTOR pathway by E combined with T in HER2+ BC pts shows promising results. It has also been reported that T acted differently when used in PO setting with a lower implication of signaling blockade and a higher induction of ADCC when used alone in chemotherapy naive pts. Additionally, mTOR blockade has been experimentally reported to activate MAPK pathway through a feed-back loop effect. We present the results of a biological integrated approach including markers of PI3K/AKT/mTOR and MAPK pathways, proliferation and immune environment in HER-2 + PBC treated with T +/- E in PO setting. Methods: Eligible pts with HER2+ PBC (IHC 3+ or FISH/SISH+) amenable to surgery were randomized to receive T (loading dose 4 mg/kg, then 2 mg/kg/W), or T+E (10 mg/d) for a 6W PO treatment. All pts had baseline core needle biopsies, tissue collected at surgery and blood samples for biomarkers and pharmacokinetic analyses. FFPE samples were used for PIK3CA mutation status and IHC (pAKT, pS6K, eIF4E, LKB1, p4EBP1, Ki67, caspase 3, PTEN, CD8, CD68). Frozen samples were used for multiplex immunoanalyses of phosphorylated PI3K/AKT/mTOR and MAPKinase signaling proteins (p-AKT, p-GSK3, p-P70S6K, p-MEK1, p-ERK1/2, p-P90RSK). Results: FFPE and frozen tumor samples (n = 80), were obtained from 82 pts randomized from July 2008 to April 2012 (41 per arm). At baseline, only PIK3CA mutation status was predictive of clinical response, whatever the arm. No difference in the expression of phosphorylated signaling kinases was induced by exposure to T while T+E induced a significant activation of MAPKinase pathway. Between baseline and surgery we observed a down regulation of pAKT pS6K, LKB1, Ki67, a lower expression of PTEN and an increase in lymphocytes (CD8+) and macrophages (CD68+) infiltrates in both arms. Conclusions: These results confirm that in PO setting, T +/- E should mostly act via the activation immune response through tumor-associated lymphocytes and macrophages infiltrates. Rapid decrease in PTEN and LKB1 may be related to early cellular mechanism of resistance to HER2 inhibition. Clinical trial information: NCT00674414

International audience

Présentation Poster; International audience; Estrogen receptor alpha 36 (ERα36) is a variant of the canonical estrogen receptor alpha (ERα66), whose high expression level correlates with a poor survival prognosis for breast cancer patients. While our previous results indicated that a high ERα36 expression level correlates with hormone independent growth and enhanced metastatic potential (Chamard-Jovenin et al, 2015; doi: 10.1186/s12918-015-0178-7) the mechanisms of ERα36 expression onset and the consequences for the normal mammary gland are poorly documented. Therefore, we explored (i) the methylation status of ERα36 promoter in 60 mammary tumor samples and (ii) the consequences of a ERα36 overexpression in vitro in MCF-10A normal mammary epithelial cells and in vivo in a unique model of MMTV-ERα36 transgenic mouse strain wherein the human ERα36 mRNA was specifically expressed in the mammary gland. Our data indicate a marked correlation between promoter methylation and ERα36 expression. Concomitantly, ERα36 overexpression lowered proliferation rate but enhanced migration and survival of the MCF-10A cell line. By a combination of bioinformatics and computational analyses of microarray data, we identified hierarchical gene networks, downstream of ERα36. In vivo, human ERα36 expression led to duct epithelium thinning and E-cadherin expression loss in adult but not prepubescent mice. Here, we show that ERα36 expression could be driven, at least in part, by epigenetic mechanisms. Moreover, an enhanced expression of ERα36 is sufficient, by itself, to disrupt normal breast epithelial phenotype in vivo and in vitro through a dominant-positive effect on nongenomic estrogen signaling pathways. These results also suggest that, in the presence of endogenous estradiol, ERα36 overexpression in vivo contributes to alter mammary gland architecture which may support pre-neoplastic lesion and augment breast cancer risk.

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Présentation Poster; National audience

Présentation Poster; National audience

Présentation Poster; International audience; Background: Short-course preoperative radiotherapy (sc-preopRT) and long-course preoperative radiotherapy (lc-preopRT) followed by total mesorectal excision (TME) are worldwide standards of care in locally advanced T3–4 N0 or N1 rectal adenocarcinoma. It is now well established that the host immune system participates in the elimination of tumor cells and that significant tumor infiltration by T-cells (LT), such as CD8+, is associated with a better prognosis. In colorectal tumors, the infiltration of Treg FoxP3+ is also described as a prognostic factor associated with better survival. We aimed to investigate the impact of the immune response to preoperative RT on progression-free survival (PFS) and overall survival (OS) in rectal cancer managed with TME.Material and Methods: We analyzed data for 237 patients with rectal cancer who underwent TME between 1995 and 2007 after neo-adjuvant treatment with preoperative RT with or without CT in 3 French centers. The LYMPHOREC study was approved by the French national review boards and independent ethics committee (CPP, CCTIRS and the CNIL). Our primary objective was to assess the impact of the immune infiltration of the tumor or tumor site (in cases with complete response) by CD8+ and FoxP3+ lymphocytes after sc-preopRT or lc-preopRT with or without CT on progression-free survival (PFS) and overall survival (OS). Our secondary objectives were to assess changes in the quantities of these lymphocyte infiltrations with respect to the type of preoperative RT (with vs without chemotherapy) or the dose fractionation scheme (≤2Gy vs >2Gy/fraction). These second analyses were performed with 133 patients from whom one biopsy sample was collected. A biopsy-based pretherapeutic lymphocyte infiltration was thus evaluated.Results: In univariate analysis, TNM stage, the delay between surgery and RT, CD8+, FoxP3+ and the ratio CD8+/FoxP3+ were significantly correlated with survival outcomes while chemotherapy as a component of preoperative radiotherapy was not. In multivariate analysis, when adjusted for clinical and treatment-related variables, tumor infiltration by FoxP3 lymphocytes after treatment significantly correlated with PFS (p=0.007). Variations in the CD8/FoxP3 ratio inside the epithelial tissue from before to after preoperative RT correlated with PFS and OS (p=0.049 and p=0.024, respectively). Interestingly, the dose per fraction (

National audience

National audience

published in Journal of Clinical Oncology, 33(15-Suppl):11055, 2015. Poster Session, Tumor Biology. Abstract Number: 11055. Poster Board Number: #268; International audience; In most patients with mCRC who are being considered for anti-EGFR antibody therapy, RAS mutation testing i.e. KRAS and NRAS exon 2, 3 and 4, is routinely assessed using PCR-based assays only detecting major hotspot mutations of exon (ex) 2 (codon 12 and 13), 3 (codon 59 and 61) and 4 (codon 117 and 146). We performed deep sequencing of the entire exons using NGS as an alternative to detect additional rare mutations profiles with significant frequency of mutated allele (FMA). Methods: 188 formalin-fixed paraffin-embedded tumor samples from primary or metastatic lesions of patients (M/F sex ratio 1.27, mean age 69 years, range 32-90) with mCRC (150 colon, 38 rectum) were analyzed. DNA was extracted from macrodissected slides (mean tumor cell content 43.3%, range 5-80). Results: RAS mutation testing was routinely assessed using NGS in 177 mCRC samples. NGS could not be performed in 11 cases (6.2%) due to the insufficient quantity or quality of DNA. NGS sensitivity was 1% at X1000 depth. RAS mutations were found in 103 samples (62%) and relatively distributed as 69.9% KRAS ex2, 3.9% KRAS ex3, 14.6% KRAS ex4, 4.8% NRAS ex2, 1.0% NRAS ex3, 1.0% NRAS ex4 and 4.8% multiple mutations. Uncommon mutational profiles were detected in 10 cases (9.7%): 2 KRAS ex2 c.37G > T p.G13C single mutation with FMA > 30%, 5 silent mutations (4 with FMA > 25%), alone (n = 2) or combined with other rare mutations (n = 3) with lower but significant FMA ( > 1%), and 6 multiple mutation profiles among which 2 double hotspot mutation (KRAS ex2 c.34G > A p.G12S and NRAS ex3 c.181C > A p.Q61K, KRAS ex2 c.34G > A p.G12S and NRAS ex2 c.38G > T p.G13V), 1 secondary rare mutation associated with a KRAS ex2 c.35G > A p.G12D hotspot mutation, and 3 multiple mutations only with rare but potentially deleterious mutations located around the loops responsible for nucleotide (GTP) binding. In only 1 case, the FMA of the secondary mutations was < 1%. As a whole, 7 cases (6.8%) had RAS mutations out of hotspots. Conclusions: NGS proved accurate, sensitive and suitable for routine RAS genotyping in mCRC. It can detect uncommon RAS mutation profiles with significant FMA that can potentially impair the patient response to anti-EGFR antibody.

Présentation Poster; International audience; OBJECTIVES: In 2013, it was shown that mutations in KRAS exons 3 and 4, or NRAS exons 2 to 4 had a similar effect. The primary objective was to assess the practices in conducting RAS testing in 2014. The secondary objectives were to describe the evolution of the RAS testing prescription rates from 2011, the process and time required to obtain the results, and to analyze their impact on the therapeutic strategy. METHODS: FLASH-RAS is an observational retrospective French multicenter study. RESULTS: 375 mCRC patients diagnosed and initiating a 1st line treatment (L1) between March and June 2014 were analyzed. For 90.1% of the patients (IC95%= [87.1%; 93.2%]), a genotyping request for RAS biomarkers was made in L1, i.e. a significantly increased rate compared to 2011 (81.1% in 2011, p

International audience

International audience; KRAS mutation detection represents a crucial issue in metastatic colorectal cancer (mCRC). The optimization of KRAS mutation detection delay enabling rational prescription of first-line treatment in mCRC including anti-EGFR-targeted therapy requires robust and rapid molecular biology techniques. Routine analysis of mutations in codons 12 and 13 on 674 paraffin-embedded tissue specimens of mCRC has been performed for KRAS mutations detection using three molecular biology techniques, that is, high-resolution melting (HRM), polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP), and allelic discrimination PCR (TaqMan PCR). Discordant cases were assessed with COBAS 4800 KRAS CE-IVD assay. Among the 674 tumor specimens, 1.5% (10/674) had excessive DNA degradation and could not be analyzed. KRAS mutations were detected in 38.0% (256/674) of the analysable specimens (82.4% in codon 12 and 17.6% in codon 13). Among 613 specimens in whom all three techniques were used, 12 (2.0%) cases of discordance between the three techniques were observed. 83.3% (10/12) of the discordances were due to PCR-RFLP as confirmed by COBAS 4800 retrospective analysis. The three techniques were statistically comparable (κ > 0.9; P < 0.001). From these results, optimization of the routine procedure consisted of proceeding to systematic KRAS detection using HRM and TaqMan and PCR-RFLP in case of discordance and allowed significant decrease in delays. The results showed an excellent correlation between the three techniques. Using HRM and TaqMan warrants high-quality and rapid-routine KRAS mutation detection in paraffin-embedded tumor specimens. The new procedure allowed a significant decrease in delays for reporting results, enabling rational prescription of first-line-targeted therapy in mCRC.

International audience

International audience; Radiotherapy and surgery are the two main pillars of the locoregional treatment of cancer. The risk of second malignancy is better evaluated and constitutes a major issue regarding radioprotection of the patients. Among malignant disease observed in the surviving irradiated patients, the occurrence of sarcoma is a rare event but associated with a poor outcome since the 5 year overall survival is estimated at 10 to 35 %. The SARI protocol, written in 2011, included 120 patients and 240 controlled patients, irradiated in the same conditions but without sarcoma observed during the follow up. The main objective was to identify the clinical and biological factors associated with the occurrence of such a complication. The secondary objective was to identify the dosimetric characteristics of the treatment of the primary. Preliminary results will be presented during the 2016 meeting of the French radiation oncology society.; La radiothérapie constitue avec la chirurgie les deux piliers majeurs du traitement locorégional du cancer. Le risque de second cancer devient mieux évalué et représente un souci majeur en termes de radioprotection. Parmi les lésions malignes découvertes chez des patients traités, l’apparition d’un sarcome est un évènement rare mais de pronostic sombre puisque le taux de survie des patients est estimé entre 10 à 35 % à 5 ans. Le protocole SARI, conçu et écrit en 2011, a permis l’inclusion de 120 patients atteints d’un sarcome développé en territoire irradié appariés à 240 témoins, irradiés dans les mêmes conditions, mais sans avoir vu se développer la maladie. L’objectif principal de l’essai était de déterminer les facteurs de risque clinique et biologique prédictifs, de survenu d’un sarcome en territoire irradié. Les objectifs secondaires étaient de décrire les caractéristiques de la radiothérapie chez l’ensemble des patients ayant vu se développer un sarcome sur territoire irradié. Les résultats préliminaires seront présentés lors du congrès 2016 de la Société française de radiothérapie oncologie.

International audience

International audience; Aims. Trastuzumab, an antibody binding to epidermal growth factor receptor-2 (HER2), has been approved to treat HER2-positive breast cancer in different settings. This study aimed at evaluating the influence of tumour size on trastuzumab pharmacokinetics (PK) in non-metastatic breast cancer patients treated with short-term preoperative trastuzumab.Methods.Trastuzumab PK data were obtained from a multi-center, randomized and comparative study. This antibody was administered preoperatively to patients with localized HER2-positive breast cancer as a single 4 mg/kg loading dose followed by 5 weekly 2 mg/kg doses. Trastuzumab concentrations were measured repeatedly using an ELISA technique. Tumour size was evaluated at baseline using breast echography. Trastuzumab pharmacokinetics was studied using a population approach and a two-compartment model. The influence of tumour burden on trastuzumab pharmacokinetics was quantified as a covariate.Results.A total of 784 trastuzumab concentrations were available in the 79 eligible patients. Estimated parameters (interindiviual standard deviation) were: central volume of distribution = 2.1 L (23%), peripheral volume of distribution = 1.3 L (38%) intercompartment clearance = 0.36 L/day, with an elimination half-life of 11.8 days. Typical clearance was 0.22 L/day (19%) and its value was increased with tumour size: in patient with the highest tumour size, trastuzumab clearance was 50% [18%–92%] higher than in patients with the lowest tumour size.Conclusions.In non-metastatic breast cancer patients, trastuzumab clearance increases with tumour size. The elimination half-life of trastuzumab was shorter in the present population of patients than in metastatic breast cancer patients previously studied.

Présentation Poster; National audience; Introduction : De 2008 à 2013, la prescription des anticorps monoclonaux anti-EGFR (cetuximab et panitumumab) dans le cancer colorectal métastatique (CCRm) était soumise à la mise en évidence préalable de l’absence de mutation somatique de l’exon 2 du gène KRAS, dont l’impact sur l’efficacité thérapeutique avait été démontré. En 2013, l’identification d’autres mutations sur les exons 3, 4 du gène KRAS et 2, 3, 4 du gène NRAS exerçant un effet similaire, et la restriction de la prescription du cetuximab aux patients atteints de CCRm non porteurs d’une de ces mutations dites « mutations RAS » a permis d’affiner davantage la population susceptible de bénéficier de ce traitement.Objectifs et méthode : L’étude FLASH-RAS fait suite à l’étude FLASH-KRAS qui a permis de décrire les pratiques de prescription et de réalisation du test KRAS exon 2 en 2011. FLASH-RAS est une étude observationnelle rétrospective nationale multicentrique dont l’objectif principal était l’évaluation en 2014 des pratiques de prescription et de réalisation des tests RAS dans la prise en charge (PEC) du CCRm. Les objectifs secondaires étaient de décrire l’évolution des taux de prescription des tests RAS entre 2011 et 2014, de décrire le circuit et le délai d’obtention des résultats, d’analyser leur impact sur la stratégie thérapeutique.Résultats : 375 patients atteints d’un CCRm diagnostiqué après le 1er mars 2014 et pour lesquels un traitement de 1ère ligne métastatique a été instauré entre le 1er mars et le 30 juin 2014 ont été analysés. 90.1% (IC95%= [87.1% ; 93.2%]) d’entre eux ont bénéficié d’une demande de test RAS lors de leur PEC en 1ère ligne métastatique, un taux en augmentation significative par rapport à 2011 (81.1% dans FLASH-KRAS, p

Présentation Poster; National audience

International audience; Background: P38 kinases are members of the mitogen-activated protein kinase (MAPK) family. In breast cancers MAPK, as well as PI3 kinase-AKT pathway signaling proteins have major implication in molecular oncogenesis and are extensively investigated as putative targets for therapy. The present study reports the investigation of the expression of P38MAPK and its phosphorylated form (p-P38MAPK) in clinical specimens of invasive breast carcinomas in relation with estrogen receptor and HER2 expression, as well as MAPK and PI3K signaling phosphorylated proteins. Methods: The expression of P38MAPK and p-P38MAPK as well as p-AKT, p-GSK3β, p-S6 kinase, p-MEK1, p-ERK1/2 were semi-quantitatively assessed using multiplex bead immuno-assay. The analyses were performed retrospectively in frozen specimens from 46 invasive breast tumors classified according to estrogen receptor (ER) and HER2 status. Results: All specimens were taken at diagnosis and validated for tumor content >50%. Twenty-nine were ER+, 17 were HER2+, 10 were triple negative (TN) tumors. Analyses were performed in triplicate from total protein extracts and were achievable in all specimens. P38MAPK was found to be expressed in all tumor specimen and significantly (P=0.002) overexpressed in ER+ tumors. P38MAPK was lower in TN tumors than in all others. The median expression of phosphorylated-P38MAPK was also higher in ER+ than in ER- tumors and lower in TN tumors than in all others. HER2 status had no influence on P38MAPK and p-P38MAPK expression. No variation in the phosphorylation rate of P38MAPK was observed in relation with ER, HER2 or TN status. Significantly higher (P=0.0048) expression of p-AKT tumors was observed in HER2+ tumors. No significant difference in p-MEK1, p-GSK3β and p-S6K expression was evidenced in any other comparisons based on ER and HER2 expression subtypes. Conclusions: Investigation of the expression of multiple phosphorylated signaling proteins can be used as molecular biomarkers for personalized targeted therapy. In ER+ invasive breast cancer, the overexpression of P38MAPK could serve as biomarker for evaluation of P38MAPK inhibitors.

National audience; Le cancer tue chaque année 147 500 personnes en France. Le développement des thérapies personnalisées et plus récemment de la théranostique a complètement modifié l'approche biologique de cette maladie. Ainsi, aux marqueurs sériques déjà bien connus mais peu spécifiques, viennent s'ajouter de nombreux marqueurs tumoraux prédictifs de la résistance ou de la réponse à une thérapie ciblée. L'étude de ces biomarqueurs s'effectue par différentes approches techniques, relevant essentiellement de la biologie moléculaire. Ces déterminations biologiques sont réalisées en France au sein des 28 plateformes de génétique moléculaire des tumeurs labélisées par l'Institut national du cancer (INCa). Les techniques les plus utilisées pour ce type de diagnostic moléculaire reposent sur l'utilisation de prélèvements tumoraux fixés au formol et inclus en paraffine, ce qui rend les déterminations relativement difficiles, notamment lors de la réalisation de techniques comme la PCR en temps réel, le séquençage, l'immunohistochimie, la FISH ou la CISH. Cet article établit une synthèse sur les biomarqueurs validés en théranostique ainsi que les méthodes diagnostiques les plus utilisées, mais également sur les biomarqueurs candidats à venir et les évolutions proches comme l'utilisation de biopsies liquides.

National audience; Selon la législation relative à la biologie médicale du 16 janvier 2010, tous les laboratoires de biologie médicale doivent être accrédités selon la norme ISO 15189 pour au moins 50 % de leur activité avant fin 2016. Une des étapes critique en biologie moléculaire et plus précisément en génétique aussi bien somatique que constitutionnelle, consiste en l'extraction de l'ADN depuis un échantillon d'intérêt, qu'il soit solide ou liquide. L'ADN extrait doit ensuite répondre à un certain nombre de critères de qualité et également être en concentration suffisante pour permettre la réalisation des examens de biologie moléculaire comme la détection de mutations somatiques par exemple. Cet article décrit, à titre d'exemple, la validation de l'extraction et de la purification d'ADN par méthode d'extraction sur colonne, puis de son dosage quantitatif par méthode spectrophotométrique, selon la norme ISO 15189 et le guide technique d'accréditation de santé humaine du Cofrac SH-GTA-04.

National audience; Depuis le 16 janvier 2010, la législation relative à la biologie médicale impose la mise en place de l'accréditation selon la norme ISO 15189 dans tous les laboratoires de biologie médicale (LBM). Ainsi, tous les LBM doivent être accrédités pour au moins une partie de leur activité avant fin octobre 2013. Les examens de biologie moléculaire font partie intégrante de la biologie médicale et sont également concernés par l'accréditation. La validation des méthodes de biologie moléculaire est cependant rendue délicate pour des raisons à la fois liées aux types de méthodes utilisées, mais aussi par le type d'analytes qui sont généralement assez rares. Cet article décrit, à titre d'exemple, la validation de la détection qualitative des mutations du gène KRAS dans les cancers colorectaux métastatiques par technique de PCR TaqMan selon la norme ISO 15189 et le guide technique d'accréditation de santé humaine du Cofrac SH-GTA-04.

International audience; In high-grade gliomas, the identification of patients that could benefit from EGFR inhibitors remains a challenge, hindering the use of these agents. Using xenografts models, we evaluated the antitumor effect of the combined treatment "gefitinib + radiotherapy" and aimed to identify the profile of responsive tumors. Expression of phosphorylated proteins involved in the EGFR-dependent signaling pathways was analyzed in 10 glioma models. We focused on three models of anaplastic oligodendrogliomas (TCG2, TCG3 and TCG4) harboring high levels of phospho-EGFR, phospho-AKT and phospho-MEK1. They were treated with gefitinib (GEF 75 mg/kg/day x 5 days/week, for 2 weeks) and/or fractionated radiotherapy (RT: 5x2Gy/week for 2 weeks). Our results showed that GEF and/or RT induced significant tumor growth delays. However, only the TCG3 xenografts were highly responsive to the combination GEF+RT, with ∼50% of tumor cure. Phosphoproteins analysis five days after treatment onset demonstrated in TCG3 xenografts, but not in TCG2 model, that the EGFR-dependent pathways were inhibited after GEF treatment. Moreover, TCG3-bearing mice receiving GEF monotherapy exhibited a transient beneficial therapeutic response, rapidly followed by tumor regrowth, along with a major vascular remodeling. Taken together, our data evoked an "EGFR-addictive" behavior for TCG3 tumors. This study confirms that combination of gefitinib with fractionated irradiation could be a potent therapeutic strategy for anaplastic oligodendrogliomas harboring EGFR abnormalities but this treatment seems mainly beneficial for "EGFR-addictive" tumors. Unfortunately, neither the usual molecular markers (EGFR amplification, PTEN loss) nor the basal overexpression of phosphoproteins were useful to distinguish this responsive tumor. Evaluating the impact of TKIs on the EGFR-dependent pathways during the treatment might be more relevant, and requires further validation.

National audience

National audience

published in Journal of Clinical Oncology, vol. 30, 2012, abstract 103; International audience; Background: Colorectal cancer (CRC) is the 2nd most common cancer with more than one million new cases diagnosed every year. From 2006 to 2008, several studies showed the importance of the KRAS oncogene in the treatment of metastatic CRC (mCRC) and response to anti-EGFR therapies like cetuximab or panitumumab. KRAS is a small G protein which can bear activating mutations in 40% cases of mCRC. Numerous methods have been described for the detection of KRAS mutations in FFPE tissues. Direct sequencing is the gold standard but the most sensitive methods are real time PCR assays based. The new COBAS4800 KRAS developed by Roche Diagnostics is a CE IVD validated assay using real time PCR and TaqMelt technology. This assay is validated for the detection of 19 KRAS somatic mutations on exon 2 (codons 12/13) and exon 3 (codon 61) in specimen containing at least 5% of mutated tumoral cells. Methods: We compared COBAS with previously validated PCR TaqMan and PCR High Resolution Melting (HRM) assays. The PCR TaqMan and HRM are a real time PCR based assays. TaqMan can detect accurately 0.5 to 1% of mutated tumoral DNA on codon 12 (G12A, G12C, G12D, G12R, G12S and G12V) and on codon 13 (G13D). HRM can detect 5 to 10% of tumoral DNA on codon 12 and 13. 80 FFPE samples of colorectal tumors resections or biopsies have been collected and processed with the 3 assays. Results have been compared using kappa statistics. Results: On 80 samples, 69 were interpretable with COBAS and TaqMan and 62 with COBAS and HRM. No statistically significant difference between COBAS and TaqMan was found (k=0.942 ; p

National audience

National audience

published in Oncologie 13(9)S4; National audience

International audience; Our work aimed to identify the ability of the HNSCC cell line Cal 27 to induce angiogenesis, regarding PTEN expression and anti-EGFR treatment. Cal 27, a human HNSCC cell line expressing PTEN has been transfected by a PTEN-siRNA and exposed to cetuximab for 48 hours. The culture media were then collected to be used as conditionned media for HUVEC and aortic rings culture. Aortic rings sprouting and tubular structures from HUVEC have been investigated by microscopy and quantified by AngioQuant software. The conditioned media have been also characterized by an analysis of key factors involved in angiogenesis regulation using Proteome Profiler Array. The anti-angiogenic effect of cetuximab in HNSCC seems to result from a direct effect of the anti-EGFR on endothelial cells rather than an effect mediated by tumoral cells, despite a VEGF concentration significantly lower due to cetuximab exposure. PTEN is involved in the regulation of vasculature in HNSCC. But surprisingly, loss of PTEN expression in Cal 27 leads to a reduction of the tumoral cells ability to induce angiogenesis. This process is not linked to the VEGF concentration changes but is mediated by a global change in the molecular constitution of conditioned media.

International audience; Background: Keloids and hypertrophic scars (HSc) affect 4.5-16% of the population. Thus far, the different approaches of keloid treatment are not very efficient, with a 50% relapse rate and many ongoing researches are looking for simple, safe and more efficient therapeutic methods. Tacrolimus is an immunomodulator that could be useful in treating keloid. Objectives: The objective of this study is to evaluate the effectiveness of Tacrolimus in inhibiting HSc formation on rabbits' ears model and to check optical skin spectroscopy in tissue characterization. Methods: Our study was carried out on 20 New-Zealand female white rabbits. HSc were obtained by wounding rabbits' ear. These wounds were treated with intradermal injections of tacrolimus (0.2-0.5 mg/cm2) or a vehicule. The assessment of treatment efficacy was performed by clinical examinations, histological assay and skin spectrometry. Results: Tacrolimus did not induce general or local side-effects. The scar elevation index in treated subjects was half less than that of the untreated ones. Furthermore, dermal thickness and inflammatory cellular density were both significantly smaller for treated scars than for the control ones. In vivo optical skin spectroscopy can characterize hypertrophic and normal skin with high sensibility and specificity. Conclusion: Intradermal injection of tacrolimus at 0.5 mg/cm2 is an efficient way to prevent HSc in our experiment model and its tolerance is correct. Optical spectroscopy could be a good non-invasive tool to evaluate HSc treatment. These promising results might be proposed for patients suffering from keloid.

International audience; Introduction : Les kinases de signalisation des voies PI3K et MAPK, en aval des récepteurs à tyrosine kinase, ont une implication majeure dans la réponse aux thérapies ciblées. Cette étude a pour but de valider l'utilisation du Bio-Plex® phosphoprotein array pour explorer en situation clinique la fonctionnalité de ces voies de signalisation en aval des récepteurs à tyrosine kinase. Patients et méthodes : L'expression de phospho-EGFR et des phosphoprotéines de signalisation en aval du récepteur à l'EGF (AKT, P70S6 kinase, ERK1/2, GSK3β) a été évaluée par Bio-Plex® phos- phoprotein array à partir d'échantillons congelés de carci- nome canalaire infiltrant, issus de 49 tumeurs de patientes, prélevés lors du diagnostic et comparés aux résultats obtenus en western blot. Résultats : Dix-sept des 49 prélèvements surexprimaient HER2 (3+) selon les résultats obtenus en immunohistochimie. Dix tumeurs étaient triple-négatives. La validation de l'essai est basée sur les FDA Guidelines for Bioanalytical Method Validation. La régression linéaire (r de Pearson) et les repré- sentations de Bland-Altman mettent en évidence des corré- lations comprises entre 0,790 et 0,852 (p < 0,001) entre les résultats obtenus en Bio-Plex® phosphoprotein array et en western blot. Avec des limites de détection détermi- nées par un rapport signal sur bruit égal à 3, on observe dans les 49 échantillons tumoraux de grandes variations dans l'expression des phosphoprotéines, allant jusqu'à un facteur 1 000. Aucune variation significative entre l'expression de pERK1/2 et de pAKT, mesurées dans les tumeurs HER2 3+ et les non-HER2 3+, n'a été mise en évidence. Une expression plus basse de toutes les phosphoprotéines de signalisation a été observée dans les tumeurs triple-négatives (rapports des niveaux d'expression compris entre 1,48 et 3,12). Conclusions et perspectives Ces résultats montrent qu'avant initiation d'un traitement, la fonctionnalité des voies de signalisation PI3K/AKT et MAPK peut différer fortement d'un patient à l'autre et pour- rait probablement expliquer les disparités dans les réponses cliniques aux thérapies ciblées, même lorsqu'elles sont pres- crites en fonction des niveaux d'expression des récepteurs membranaires explorés par immunohistochimie. Ces résul- tats valident l'utilisation du Bio-Plex® phosphoprotein array dans des échantillons de tumeurs de taille compatible avec une situation de diagnostic clinique et justifient son investi- gation en vue de déterminer des marqueurs prédictifs de la réponse clinique aux thérapies ciblées. Cette étude a été soutenue par la Ligue contre le cancer.

The photodynamic therapy, more exactly named dynamic photochemotherapy, is a new approach based on the combined action of a photoactive drug or photosensitizer, light and oxygen. These compounds are non toxic without appropriated light exposure but produce, after irradiation, reactive species in presence of molecular oxygen, leading to target tissue destruction. This article describes first the photosensitization molecular mechanisms, then the photophysical and chemical properties requested to obtain a « good » photosensitizer. In order to propose photosensitizers really adapted to therapeutic applications, more efficient and selective new photoactive compounds are elaborated. At last, the main application fields of photodynamic therapy, the limits and the future of this treatment are developed., La thérapie photodynamique est une nouvelle approche, basée sur l'action sélective par la lumière de médicaments, dits photosensibilisateurs. Ces molécules, non toxiques en absence d'une lumière de longueur d'onde appropriée, génèrent, après irradiation lumineuse et en présence d'oxygène, des espèces très réactives entraînant la destruction du tissu cible. Cet article décrit tout d'abord les mécanismes moléculaires de photosensibilisation, puis les propriétés chimiques et photophysiques d'un « bon » photosensibilisateur. Une conception rationnelle de photosensibilisateurs adaptés aux applications thérapeutiques passe nécessairement par un effort d'élaboration de nouveaux composés photo-activables plus efficaces, donc plus sélectifs. Enfin, les principaux domaines d'application, notamment en cancérologie, ainsi que les freins et les perspectives de la thérapie photodynamique sont développés.

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