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The diagnosis of well-differentiated adenocarcinoma on bile aspiration is a well-known challenge. This study was aimed at improving the diagnostic performance and providing a biliary cytology learning atlas.This single-center, retrospective study included 135 cases of informative biliary samples collected between 2009 and 2018 that were classified as benign, atypical, or malignant. A double assessment was performed by a novice and a cytopathologist experienced in biliary cytology to establish the specificities, sensitivities, and inter- and intraobserver κ index agreements of 24 cytological criteria, which were illustrated in a learning atlas.A multivariate logistic regression was used to assess whether the most specific and reproducible criteria were associated with malignancy. A scoring system was statistically determined: 6 points were attributed in the presence of a 3-dimensional (3D) cluster, anisonucleosis, and a nuclear to cytoplasmic (N:C) ratio0.5, whereas 4 points were given in the presence of an enhanced nuclear membrane. A score higher than 10 points resulted in a malignancy diagnosis with 96% sensitivity and 97% specificity.A diagnostic tree of malignancy based on 4 criteria, together with a multidisciplinary approach, allows the diagnosis of adenocarcinoma with a specificity of 100% and a sensitivity of 88% or 72% depending on the presence of a single malignant cell or the presence of 3 combined criteria (a 3D cluster, anisonucleosis, and an N:C ratio0.5). It comes with a learning atlas useful for cytopathologist training and accuracy in this uncommon cytology.

Diffuse large B‐cell lymphoma (DLBCL) is a clinically heterogeneous entity, in which the first‐line treatment currently consists of an immuno‐chemotherapy regimen (R‐CHOP). However, around 30% of patients will not respond or will relapse. Overexpression of c‐MYC or p53 is frequently found in DLBCL, but an association with prognosis remains controversial, as for other biomarkers previously linked with DLBCL aggressivity (CD5, CD23, or BCL2). The aim of this study was to explore the expression of these biomarkers and their correlation with outcome, clinical, or pathological features in a DLBCL cohort. Immunohistochemical (c‐MYC, p53, BCL2, CD5, and CD23), morphological (‘starry‐sky’ pattern [SSP]), targeted gene panel sequencing by next‐generation sequencing (NGS), and fluorescence in situ hybridisation analyses were performed on tissue microarray blocks for a retrospective cohort of 94 R‐CHOP‐treated de novo DLBCL. In univariate analyses, p53 overexpression (p53high) was associated with unfavourable outcome (p = 0.04) and with c‐MYC overexpression (p = 0.01), whereas c‐MYC overexpression was linked with an SSP (p = 0.004), but only tended towards an inferior prognosis (p = 0.06). Presence of a starry‐sky morphology was found to be correlated with better survival in p53high DLBCL (p = 0.03) and/or c‐MYC‐positive DLBCL (p = 0.002). Furthermore, NGS data revealed that these three variables were associated with somatic mutations (PIM1, TNFRSF14, FOXO1, and B2M) involved in B‐cell proliferation, survival, metabolism, and immune signalling. Taken together, these results show that the SSP pattern seems to be a protective factor in high‐risk DLBCL subgroups and highlight cell death as a built‐in failsafe mechanism to control tumour growth.

NUT carcinoma of the thorax is a rare and very aggressive tumor, whose definition is based on the demonstration of a nuclear protein in testis (NUTM1; also known as NUT) gene fusion on 15q14 with different partners from the bromodomain-containing proteins gene family. This fusion results in an activation of MYC oncoprotein responsible for the tumor's aggressivity. NUT carcinoma arises preferentially in young adults, presenting a large thoracic mass frequently associated with lymph nodes, bone or pleural metastases. At histology, this tumor is often poorly differentiated, mainly composed of sheets of small cells with scant cytoplasm, a round nucleus with a central nucleolus. Focal areas of squamous differentiation can be observed. Mitoses and necrosis are frequent, as well as neutrophilic infiltrate. The diagnosis is based on the detection of NUT protein expression by immunohistochemistry using the rabbit monoclonal antibody C52B1 in more than 50% of the tumor nuclei. This technique offers 87% sensitivity and nearly 100% specificity with reference to FISH or RT-PCR, which confirm the NUTM1 rearrangement. The differential diagnoses include basaloid carcinoma of the lung, small cell carcinoma, thymic carcinoma (basaloid variant), SMARCA4_deficient thoracic sarcoma, other NUTM1 rearranged undifferentiated tumors, small round cell tumors, non-Hodgkin lymphoma/leukemia, and melanoma. The prognosis of NUT carcinoma remains very poor, with a median survival of 6.7 months, and 1- and 2-year overall survival rates of 30% and 19%, respectively. NUT carcinoma is often refractory to conventional chemotherapy, but ifosfamide-based regimens or BET inhibitors could represent promising therapies.

Simple Summary CYCLON is a nuclear protein, which has been associated with disease progression and treatment resistance in DLBCL, the most common form of aggressive B-cell lymphoma, but also represents a predictive factor of refractory disease and relapse for immuno-chemotherapy-treated DLBCL patients. The molecular mechanisms related to this unstructured protein remain largely uncharacterized. Here, we performed a mass-spectrometry-based identification of the CYCLON protein interactome that suggested it could exert nucleolar functions related to cell proliferation. Among the CYCLON oncogenic network, we performed an immunohistochemical evaluation of the multi-functional nucleolar protein NPM1 in a DLBCL cohort and showed that CYCLON/NPM1 concomitant expression delineates a poor prognosis subgroup of patients. Multivariate survival analyses demonstrated that specific sub-cellular localizations of CYCLON and NPM1 represent independent novel predictors specifically associated with refractory DLBCL. Abstract R-CHOP immuno-chemotherapy significantly improved clinical management of diffuse large B-cell lymphoma (DLBCL). However, 30–40% of DLBCL patients still present a refractory disease or relapse. Most of the prognostic markers identified to date fail to accurately stratify high-risk DLBCL patients. We have previously shown that the nuclear protein CYCLON is associated with DLBCL disease progression and resistance to anti-CD20 immunotherapy in preclinical models. We also recently reported that it also represents a potent predictor of refractory disease and relapse in a retrospective DLBCL cohort. However, only sparse data are available to predict the potential biological role of CYCLON and how it might exert its adverse effects on lymphoma cells. Here, we characterized the protein interaction network of CYCLON, connecting this protein to the nucleolus, RNA processing, MYC signaling and cell cycle progression. Among this network, NPM1, a nucleolar multi-functional protein frequently deregulated in cancer, emerged as another potential target related to treatment resistance in DLBCL. Immunohistochemistry evaluation of CYCLON and NPM1 revealed that their co-expression is strongly related to inferior prognosis in DLBCL. More specifically, alternative sub-cellular localizations of the proteins (extra-nucleolar CYCLON and pan-cellular NPM1) represent independent predictive factors specifically associated to R-CHOP refractory DLBCL patients, which could allow them to be orientated towards risk-adapted or novel targeted therapies.

Large cell neuroendocrine carcinoma (LCNECs) and small cell lung carcinomas (SCLCs) are high-grade neuroendocrine carcinomas of the lung with very aggressive behavior and poor prognosis. Their histological classification as well as their therapeutic management has not changed much in recent years, but genomic and transcriptomic analyses have revealed different molecular subtypes raising hopes for more personalized treatment. Indeed, four subtypes of SCLCs have been recently described, SCLC-A driven by the master gene ASCL1, SCLC-N driven by NEUROD1, SCLC-Y by YAP1 and SCLC-P by POU2F3. Whereas SCLC standard of care is based on concurrent chemoradiation for limited stages and on chemotherapy alone or chemotherapy combined with anti-PD-L1 checkpoint inhibitors for extensive stage SCLC, SCLC-A variants could benefit from DLL3 or BCL2 inhibitors, and SCLC-N variants from Aurora kinase inhibitors combined with chemotherapy, or PI3K/mTOR or HSP90 inhibitors. In addition, a new SCLC variant (SCLC-IM) with high-expression of immune checkpoints has been also reported, which could benefit from immunotherapies. PARP inhibitors also gave promising results in combination with chemotherapy in a subset of SCLCs. Regarding LCNECs, they represent a heterogeneous group of tumors, some of them exhibiting mutations also found in SCLC but with a pattern of expression of NSCLC, while others harbor mutations also found in NSCLC but with a pattern of expression of SCLC, questioning their clinical management as NSCLCs or SCLCs. Overall, we are probably entering a new area, which, if personalized treatments are effective, will also lead to the implementation in practice of molecular testing or biomarkers detection for the selection of patients who can benefit from them.

Background High-grade B-cell lymphoma with rearrangements of MYC and BCL2 and/or BCL6 is an aggressive mature B-cell neoplasm, whereas B-lymphoblastic lymphoma is immature cell proliferation, with a frequent positivity for terminal deoxynucleotidyl transferase. The transformation of a low-grade follicular lymphoma into a lymphoblastic neoplasm expressing terminal deoxynucleotidyl transferase is a very rare event. Case presentation A 55-year-old Caucasian man was followed for a grade 1–2 follicular lymphoma carrying a t(14;18) IGH/BCL2+ and was initially treated with R-CHOP. The follicular lymphoma presented two relapses. In the third relapse, the patient had multiple lymphadenopathy and ascites, which motivated a retroperitoneal biopsy and an ascitic tap. These samples were analyzed by histological, cytological, flow cytometric, cytogenetic, and molecular assessments. The patient died of a multiple organ dysfunction syndrome 2 weeks after his third relapse. The biopsy revealed a diffuse proliferation made up of two types of tumor cells: centroblasts (Bcl-6-positive) and immature cells (terminal deoxynucleotidyl transferase-positive). Flow cytometric analysis confirmed the immature phenotype, with an expression of terminal deoxynucleotidyl transferase, combined with a loss of membrane immunoglobulins. The cytogenetic analysis performed on the ascites revealed a clonal evolution characterized by a t(8;22)(q24;q11) MYC+ translocation not previously detected in follicular lymphoma. Fluorescence in situ hybridization confirmed the double rearrangement of the BCL2 and MYC genes. Polymerase chain reactions and sequencing were used to study the clonal relationship between follicular lymphoma and the secondary tumors. The IGVH gene rearrangement revealed a unique clonal rearrangement involving an IGVH4–59 subset in all three specimens. Conclusion These findings suggest a clonal relationship between the two types of lymphoma cells. Furthermore, they support the transformation of an acute follicular lymphoma into a composite lymphoma combining a high-grade B-cell lymphoma and a lymphoblastic neoplasm expressing terminal deoxynucleotidyl transferase. This case report highlights the possible transformation of follicular lymphoma into a highly aggressive and immature proliferation.

La proteine S100A8 est deregulee dans de nombreux types de cancer. Dans les leucemies aigues myeloides (LAM), son expression intracellulaire est associee a un pronostic defavorable. Bien qu’elle puisse etre secretee et qu’elle exerce une activite d’alarmine, le role extracellulaire de la proteine S100A8 dans la niche hematopoietique reste meconnu. Nous avons mesure la proteine S100A8 par technique ELISA chez 78 plasmas medullaires, dont 50 LAM. La concentration en S100A8 ([S100A8]) est significativement plus elevee chez les patients atteints de LAM que chez les patients sains ou presentant des etats pre-leucemiques (syndrome myeloproliferatif, syndrome myelodysplasique). Dans les LAM, nous montrons que la [S100A8] est significativement associee a la leucocytose. Fait interessant, la [S100A8] est fortement correlee avec le pourcentage de monocytes mais negativement correlee avec le pourcentage de blastes medullaires. Ce lien entre la [S100A8] et monocytes a ete confirme par la correlation avec le pourcentage de cellules CD36+ CD64+ CD14+ determine par immunophenotypage. De plus, la [S100A8] est plus elevee dans la leucemie aigue monocytaire et myelomonocytaire (LAM4/5) que dans les autres sous-types de FAB. En analyse multivariee, la [S100A8] est principalement exprimee lorsque les blastes exprimaient des marqueurs monocytaires CD11c+, CD4+, CD3- ou CD117-. Pour confirmer l’origine monocytaire de la proteine S100A8, nous avons mesure par cytometrie en flux la proteine S100A8 intracellulaire dans les blastes, les lymphocytes, les PNN, les promonocytes et les monocytes de patients atteints de LAM. Les blastes CD34+ expriment peu de S100A8 intracellulaire. La proteine S100A8 est significativement plus exprimee par les monocytes et les promonocytes, confirmant par une seconde approche l’origine monocytaire de la S100A8 dans les LAM. Pour finir, nous avons etudie l’impact pronostique de la [S100A8] qui semble associee a une diminution de la survie globale dans les LAM4/M5. En conclusion, nos travaux caracterisent la secretion de S100A8 dans les plasmas medullaires et son role dans la leucemogenese.

International audience; Objectives: Current understanding of the histological features of the vocal folds is still insufficient to make the link to their vibromechanical performance. In particular, the 3D microscale rearrangement of the loaded tissues is still to be explored. Thus, the aim of this work is to characterize the 3D histological specificities of human vocal folds' fibrous networks and their straininduced microstructure evolutions under tensile loading, at the scale of the muscular, collagen and elastin microfiber bundles.

The use of complementary and alternative medicine at least once during or after cancer treatment has increased over the past years from an estimated 25% in the 1970s and 1980s to more than 32% in the 1990s and to 49% after 2000. The risk of herb-drug interaction is therefore increasingly recognized as a public health problem. To the best of our knowledge, we report here the first case of interaction between ginger and anticancer drug, with serious consequences for the patient. There is an urgent need regarding complementary and alternative medicine: Both clinicians and patients should be aware of the potential interactions between herbs and prescribed drugs.

Purpose The survival benefit with adjuvant chemotherapy for patients with resected stage II-III non–small-cell lung cancer (NSCLC) is modest. Efforts to develop prognostic or predictive biomarkers in these patients have not yielded clinically useful tests. We report findings from the Lung Adjuvant Cisplatin Evaluation (LACE)-Bio-II study, in which we analyzed next-generation sequencing and long-term outcomes data from > 900 patients with early-stage NSCLC treated prospectively in adjuvant landmark clinical trials. We used a targeted gene panel to assess the prognostic and predictive effect of mutations in individual genes, DNA repair pathways, and tumor mutation burden (TMB). Methods A total of 908 unmatched, formalin-fixed, paraffin-embedded, resected lung cancer tumor specimens were sequenced using a targeted panel of 1,538 genes. Stringent filtering criteria were applied to exclude germline variants and artifacts related to formalin fixation. Disease-free survival, overall survival, and lung cancer—specific survival (LCSS) were assessed in Cox models stratified by trial and adjusted for treatment, age, sex, performance score, histology, type of surgery, and stage. Results Nonsynonymous mutations were identified in 1,515 genes in 908 tumor samples. High nonsynonymous TMB (> 8 mutations/Mb) was prognostic for favorable outcomes (ie, overall survival, disease-free survival, and LCSS) in patients with resected NSCLC. LCSS benefit with adjuvant chemotherapy was more pronounced in patients with low nonsynonymous TMBs (≤ 4 mutations/Mb). Presence of mutations in DNA repair pathways, tumor-infiltrating lymphocytes, TP53 alteration subtype, and intratumor heterogeneity was neither prognostic nor predictive. Statistically significant effect of mutations in individual genes was difficult to determine due to high false-discovery rates. Conclusion High nonsynonymous TMB was associated with a better prognosis in patients with resected NSCLC. In addition, the benefit of adjuvant chemotherapy on LCSS was more pronounced in patients with low nonsynonymous TMBs. Studies are warranted to confirm these findings.

International audience

Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-31849-w.; International audience; Human vocal folds possess outstanding abilities to endure large, reversible deformations and to vibrate up to more than thousand cycles per second. This unique performance mainly results from their complex specific 3D and multiscale structure, which is very difficult to investigate experimentally and still presents challenges using either confocal microscopy, MRI or X-ray microtomography in absorption mode. To circumvent these difficulties, we used high-resolution synchrotron X-ray microtomography with phase retrieval and report the first ex vivo 3D images of human vocal-fold tissues at multiple scales. Various relevant descriptors of structure were extracted from the images: geometry of vocal folds at rest or in a stretched phonatory-like position, shape and size of their layered fibrous architectures, orientation, shape and size of the muscle fibres as well as the set of collagen and elastin fibre bundles constituting these layers. The developed methodology opens a promising insight into voice biomechanics, which will allow further assessment of the micromechanics of the vocal folds and their vibratory properties. This will then provide valuable guidelines for the design of new mimetic biomaterials for the next generation of artificial larynges.

Pulmonary large-cell neuroendocrine carcinomas (LCNECs) have similarities with other lung cancers, but their precise relationship has remained unclear. Here we perform a comprehensive genomic (n = 60) and transcriptomic (n = 69) analysis of 75 LCNECs and identify two molecular subgroups: “type I LCNECs” with bi-allelic TP53 and STK11/KEAP1 alterations (37%), and “type II LCNECs” enriched for bi-allelic inactivation of TP53 and RB1 (42%). Despite sharing genomic alterations with adenocarcinomas and squamous cell carcinomas, no transcriptional relationship was found; instead LCNECs form distinct transcriptional subgroups with closest similarity to SCLC. While type I LCNECs and SCLCs exhibit a neuroendocrine profile with ASCL1high/DLL3high/NOTCHlow, type II LCNECs bear TP53 and RB1 alterations and differ from most SCLC tumors with reduced neuroendocrine markers, a pattern of ASCL1low/DLL3low/NOTCHhigh, and an upregulation of immune-related pathways. In conclusion, LCNECs comprise two molecularly defined subgroups, and distinguishing them from SCLC may allow stratified targeted treatment of high-grade neuroendocrine lung tumors., The molecular nature of large-cell neuroendocrine lung carcinomas (LCNEC) has remained unclear. Here, the authors show LCNECs represent a distinct transcriptional subgroup among lung cancers and comprise two molecular subgroups, type I (TP53 and STK11/KEAP1 alterations) and type II (TP53 and RB1 inactivation).

Objectives The aim of the present study was firstly to assess in a clinical setting the yields of an amplicon-based parallel RNA sequencing (RNA-seq) assay for ALK fusion transcript variants detection in comparison with immunohistochemistry (IHC) and fluorescent in-situ hybridization (FISH) in a selected population of ALK-positive and ALK-negative non-small cell lung cancer (NSCLC) cases, and secondly to evaluate the impact of the ALK variant on crizotinib efficacy. Materials and methods The cohort used for the assessment of the RNA-seq assay comprised 53 samples initially diagnosed as being ALK-positive based on the results obtained by IHC and/or FISH, and 23 ALK-negative samples. A distinction was made between ‘truly’ IHC/FISH positive or ‘truly’ IHC/FISH negative samples, and those for which the IHC and/or FISH were equivocal (IHC) or borderline-positive (FISH). Results On the overall population, RNA-seq sensitivity (Se) and specificity (Spe) were of 80% and 100%, respectively when IHC and FISH were combined. For the 31 ‘truly positive’ samples, Se and Spe of 100% were reached. An ALK status could be assigned by RNA-seq in 10/10 of the equivocal and/or borderline-positive IHC/FISH cases, 2/7 IHC/FISH discordant cases. When crizotinib efficacy was evaluated according to the type of ALK variant, better clinical outcomes were observed in crizotinib-treated patients with EML4-ALK v1/v2/others variants compared to v3a/b variants. Conclusion RNA-seq detects ALK rearrangements with a high sensitivity and specificity using only 10 ng of RNA. It appears to be a promising rescue technique for non-clear-cut IHC/FISH cases and also offers a unique opportunity to identify ALK fusion variants and evaluate their predictive value for ALK inhibitors efficacy.

International audience; Since the last decades, the development of fundamental knowledge on mechanics and multi-physics phenomena driving biological systems has enabled the outstanding improvement of biomaterials able to mimic the mechanical behaviour of human soft tissues. Vocal-fold tissues are very challenging to replace as they possess specific abilities to endure large reversible deformations and to vibrate up to more than thousand cycles per second. Such unique performances result from complex physical and biomechanical features which remain poorly understood so far. This is mainly ascribed to a lack of knowledge of their unique macro-and microstructures, very difficult to investigate experimentally [1,2]. The 3D characterization of each constitutive element at the micrometric scale is a current challenge using confocal microscopy (limited depth of field), micro-IRM (limited spatial resolution) or X-ray microtomography in absorption mode (low sensitivity to distinguish fresh biological components owning similar absorptive properties). The present work aims (i) at characterizing the 3D microscale arrangement of the vocal-fold tissue on excised anatomical samples by means of X-ray synchrotron phase-contrast microtomography; (ii) at studying the relationship between 3D microstructure and vocal-fold biomechanical behaviour using a multiscale modeling approach. Regarding the experimental part, 7 human larynges have been scanned at mean (12.3μm) and high-resolution (0.65μm), using X-ray microtomographs of the ESRF's ID19 beamline. Histological colorations have allowed a comparative analysis of the acquired images using optical microscopy. A morphological and histological database will be presented, quantifying the 3D geometries, orientations and densities of vocal-fold fibrous structure. In agreement with the histological analyses, the striated-cells network of inferior thyroarytenoid muscle (vocalis) has been differentiated from the fibrous network of lamina propria (type-I and type-III collagen fibers, elastin fibers). Regarding the theoretical part, a recent 2D micro-mechanical model of the vocal folds [3] has been improved to account for the 3D fibrous networks of the tissue. The impact of several microstructural parameters (fiber-bundles initial diameter, waviness and orientation) on the vibro-mechanical response of the tissue has been evidenced.

International audience

Présentation pdf diffusée en ligne, figures publiées dans la photothèque du CNRS - donnant suite à Leroux et al., CNRS Le Journal, 2017http://www.cnrs.fr/insis/recherche/docs-evenements/HommeElastique_LucieBailly.pdf; National audience

Background Complete resection of non–small-cell lung cancer (NSCLC) offers the potential for cure after surgery and adjuvant chemotherapy. Patients may not benefit and may experience severe toxicity. There are no validated molecular tools to allow better patient selection. Materials and Methods The LACE-Bio (LACE [Lung Adjuvant Cisplatin Evaluation]) project includes 4 trials (International Adjuvant Lung Cancer Trial [IALT], Adjuvant Navelbine International Trialist Association [ANITA], JBR10, and Cancer and Leukemia Group B (CALGB)-9633). Immunohistochemistry biomarkers shown in one trial to have a prognostic/predictive effect on overall survival were tested. Results The majority of the promising biomarkers could not be validated; the prognostic effect of tumor infiltrating lymphocytes and β-tubulin was confirmed. Potential causes include tissue fixation, storage, the use of tissue microarrays, and varying reagent/antibody batches. Conclusions Immunohistochemistry assays from single trials may be misleading and require validation before being used for patient selection. LACE-Bio-2 is evaluating potential genomic biomarkers that may allow more precise selection of patients with NSCLC for adjuvant chemotherapy in NSCLC.

Background ALK (anaplastic lymphoma kinase) gene rearrangement is a novel oncogenic driver in non-small cell lung cancer (NSCLC) against which a selective inhibitor, namely crizotinib, is effective. Fluorescence in situ hybridization (FISH) is considered the reference method in selecting patients with ALK-positive tumors for treatment with crizotinib. Case report We report the case of a 42-year-old non-smoking woman with an advanced pulmonary ALK FISH-negative adenocarcinoma characterized by strong immunohistochemical expression of ALK fusion protein. The patient received targeted therapy with crizotinib in compassionate use and experienced a long-lasting clinical response. Conclusion FISH testing should not be considered the only method to select patients for therapy with ALK inhibitors and the use of multiple ALK-detecting techniques could be helpful in screening ALK-positive patients more appropriately.

International audience; Malignant pleural mesothelioma (MPM) is an aggressive tumor with no effective therapy. However PD-L1/PD-1 immunity checkpoint therapies gave encouraging results; TLR3 is a programmed death factor, which triggering up-regulates PD-L1. As PD-1/PD-L1 blocking antibodies could restore antitumor immune responses alone or in combination with TLR3 agonists, we investigated PD-L1/PD-1 and TLR3 expressions in MPM to select patients for immunotherapy. Sixty-eight pleural surgical specimens, including 58 MPM (epithelioid, n = 34; biphasic, n = 11; sarcomatoid, n = 13) and 10 benign lesions, were studied. PD-L1 expression was assessed using E1L3N and SP142 clones in tumor cells (TCs) and in tumor-infiltrating lymphocytes (TILs) (positivity threshold of 1%), and compared with overall survival. PD-1, CD3 and CD8 expression by TILs, and TLR3 expression by TCs were analyzed concomitantly. PD-L1 was more expressed by sarcomatoid subtype than by other MPM (62% versus 23% and 9% for E1L3N; 38% versus 11% for SP142) (P = .01 and .04, respectively). Specificity and sensitivity of E1L3N and SP142 were of 53% and 98%, and 90% and 86%, respectively. PD-L1 expression by TILs and TCs correlated for SP142 (P = .023), and PD-L1 SP142 expression by TCs was associated with shorter overall survival (P = .016). TLR3 was expressed in most MPM, but weakly in sarcomatoid MPM. We confirm by comparing two commercially available antibodies that PD-L1 expression is higher in sarcomatoid MPM and correlates with a shorter survival. Whereas TLR3 agonists could be tested in MPM expressing TLR3, the sarcomatoid subtype could benefit from anti-PD-L1/PD-1 therapies alone or in combination

International audience; Invasive mucinous lung adenocarcinoma (IMA) is a rare subtype of lung adenocarcinoma with no effective treatment option in advanced disease. KRAS mutations occur in 28–87% of the cases. NRG1 fusions were recently discovered in KRAS-negative IMA cases and otherwise negative for known driver oncogenes and could represent an attractive therapeutic target. Published data suggest that NRG1 fusions occur essentially in nonsmoking Asian women. From an IMA cohort of 25 French patients of known ethnicity, driver oncogenes EGFR, KRAS, BRAF, ERBB2 mutations, and ALK and ROS1 rearrangements presence were analyzed. In the IMA samples remaining negative for these driver oncogenes, an NRG1 rearrangement detection was performed by FISH. A driver oncogene was identified in 14/25 IMA, namely 12 KRAS mutations (48%), one ROS1 rearrangement (4%), and one ALK rearrangement (4%). The detection of NRG1 rearrangement by FISH was conducted in the 11 pan-negative IMA. One sample was NRG1FISH-positive and 100% of the tumor nuclei analyzed were positive. This NRG1-positive patient was a 61-year-old nonsmoking woman of Vietnamese ethnicity and was the sole patient of Asian ethnicity of the cohort. She died 6 months after the diagnosis with a pulmonary multifocal disease. NRG1FISH detection should be considered in patients with IMA pan-negative for known driver oncogenes. These results might suggest that NRG1 fusion is more frequent in IMA from Asian patient. Larger studies are needed.

Le cancer du poumon est la premiere cause de deces par cancer dans le monde. La majorite des cancers pulmonaires correspond a des carcinomes non a petites cellules, representes essentiellement par des adenocarcinomes ou des carcinomes malpighiens. Leur pronostic reste redoutable car les cancers du poumon sont souvent diagnostiques a un stade tardif, metastatique. Cependant, la decouverte des mutations du gene EGFR dans un sous-type d’adenocarcinomes, conferant a ces tumeurs une sensibilite majeure aux inhibiteurs de tyrosine-kinase (ITK), erlotinib et gefitinib, a suscite de nouveaux espoirs quant a l’identification de nouvelles anomalies genetiques comme autant de cibles therapeutiques potentielles.

Introduction: In 2011, the French National Cancer Institute recommended ALK -fluorescence in situ hybridization (FISH) testing in all EGFR / KRAS -negative adenocarcinomas by all the hospital molecular genetics platforms of cancers; however, this technique remains time and cost consuming and not suitable for a large-scale screening, in contrast to immunohistochemistry (IHC). Methods: To evaluate IHC as a prescreening tool, 441 specimens, including small biopsies and surgical specimens, were analyzed prospectively on the Grenoble molecular genetics platform. EGFR and KRAS mutation analyses and ALK IHC, using the 5A4 mAb on an automated staining module, were performed on all specimens; 100 were tested by both ALK IHC and FISH (break-apart probe). Results: Twenty-seven cases out of 441 were strongly positive (3+ intensity in more than 60% of cells) with ALK mAb, two additional cases exhibited a faint staining (1+) in less than 30% of the cells. Among the 100 cases analyzed by IHC and FISH, 19 were not interpretable by FISH, but 21 were positive with both techniques. Sensitivity and specificity of IHC when compared with FISH were 95 and 100%, respectively. Eleven patients were included in crizotinib trials. Among the 352 analyzable specimens for mutations, 7% were EGFR and 29% were KRAS mutated. Conclusions: Our IHC protocol, using a commercially available antibody and an amplification step on an automated staining module, led to intense cytoplasmic staining in 6.5% of the adenocarcinomas screened. Our results favor ALK IHC prescreening on a daily routine on surgical specimens and on small biopsies before FISH testing.

A series of A-ring variously methoxylated 4-(3-hydroxy-4-methoxyphenyl)coumarins related to combretastatin A-4 was prepared by cross-coupling reactions. Cytotoxicity studies indicated a potent activity against HBL100 cell line. Substitution patterns on A-ring had only a slight effect on antiproliferative activity. For most cytotoxic compounds, the activity as potential modulators of P-gp and BCRP efflux pumps was evaluated. The results show that compounds 2 and 7 were able to restore mitoxantrone accumulation (BCRP) at concentrations similar to that of cyclosporine A. Compound 7 was the most efficient to reverse P-gp activity. All compounds were found to potently inhibit in vitro microtubule formation via a substoichiometric mode of action for the most part. Compounds 1 and 2 were found to have an apparent affinity binding constant similar to that of combretastatin A-4, i.e., 1 × 10 (6) M(-1). The molecular modeling of coumarin derivatives was performed on the basis of the molecular structure of 7, as determined by single-crystal X-ray crystallography. The calculations suggested that the presence of a methoxy group out of the plane of the chromenone moiety is an important steric hindrance factor embedding the accessibility of those molecules inside the binding pocket on tubulin.

Objectif Les proprietes vibromecaniques remarquables du pli vocal, mal comprises a ce jour, sont liees a sa structure multicouches et a l’architecture de ses reseaux fibreux [1] . La caracterisation 3D de chaque element constitutif aux echelles micrometriques est un challenge en microscopie confocale (profondeur de champ limitee), en micro-IRM (resolution spatiale limitee) et en microtomographie a rayons X (imagerie par absorption pas assez sensible pour visualiser des tissus aux proprietes d’absorption similaires). Cette etude a pour but de caracteriser pour la premiere fois, sur pieces anatomiques excisees, les arrangements 3D de la microstructure du pli vocal grâce au rayonnement X synchrotron et au mode d’imagerie de contraste de phase [2] . Methodes Sept larynx humains ont ete scannes grâce aux microtomographes a rayons X a moyenne (12,3 μm) et haute resolution (0,65 μm) de la ligne ID19 de l’ESRF. Diverses conditions de conservation des tissus (non fixes, formoles, immerges dans l’ethanol) et de placement geometrique (au repos, etire) ont ete testees. Des colorations histologiques ont permis une analyse comparative des resultats en microscopie optique. Resultats Une base de donnees morphologiques et histologiques a ainsi ete elaboree, quantifiant les geometries 3D, orientations et densites de la structure fibreuse du pli vocal. En accord avec les coupes histologiques, les reseaux 3D des cellules striees au sein du muscle thyro-arytenoidien (vocalis) ont pu etre differenciees des reseaux fibreux de la lamina propria (fibres de collagene de Type I et III, fibres d’elastine). Conclusion Cette base de donnees multi-echelles comble un manque reel en biomecanique de la voix. Les developpements experimentaux (biomecaniques, optiques et anatomiques) et premiers resultats ouvrent des perspectives prometteuses pour une meilleure comprehension des micromecanismes impliques en phonation.

Anaplastic lymphoma kinase (ALK) gene rearrangements in lung adenocarcinoma result in kinase activity targetable by crizotinib. Although fluorescence in situ hybridisation (FISH) is the reference diagnostic technique, immunohistochemistry (IHC) could be useful for pre-screening.Diagnostic yields of ALK IHC, FISH and quantitative reverse transcriptase PCR performed in 14 French pathology/molecular genetics platforms were compared. 547 lung adenocarcinoma specimens were analysed using 5A4 and D5F3 antibodies, two break-apart FISH probes and TaqMan kits. Clinicopathological data were recorded.140 tumours were ALK rearranged (FISH with ≥15% of rearranged cells) and 400 were ALK FISH negative (ALK patients were young (p=0.003), mostly females (p=0.007) and light/nonsmokers (p20%. Variants were undetected in 36% of ALK tumours.Discordances predominated with FISH ranging from 10% to 20% of rearranged cells and were centre dependent. IHC remains a reliable pre-screening method for ALK rearrangement detection.

Renal cell carcinomas (RCCs) have notoriously been shown to be refractory to traditional therapies including radiation and cytokine therapies. The use of molecularly targeted therapies against mTOR, VEGF, and other angiogenic factors has significantly improved the standards of care of this disease. Yet, improvements are still required as many of the current therapies are limited by acquired resistance. However, the recent development of molecular targeted therapies involving kinase inhibitors has changed the clinical management of RCCs. Protein kinase CK2 is critical for the activation of multiple pro-survival signaling pathways, and its catalytic activity is invariably elevated in various types of tumors. However, the precise role of CK2 has never been addressed in RCCs. In this study, we have analyzed the activity of CK2 and the expression of its subunits in a small cohort of RCC tumors. This analysis revealed, in the majority of tumor samples, an upregulation of the CK2 catalytic subunits that was not correlated with mRNA abundance in the majority of tumor samples. Moreover, relative levels of the three CK2 subunits varied significantly between tumor samples, and a positive correlation was observed between low CK2β expression and an upregulation of the ZEB2 mesenchymal marker in a subset of tumor samples. Using the CK2 inhibitor CX-4945 to downregulate the CK2 catalytic activity in 786-O cells as a model of VHL-deficient renal cancer cell line, we showed that CK2 represents a potential promising therapeutic target in RCCs.

Contexte Les rearrangements du gene ALK dans les adenocarcinomes pulmonaires (ADK) induisent une activation constitutive de la kinase ALK, ciblable par le crizotinib [1] , [2] . Bien que la FISH (hybridation in situ fluorescente) soit la technique de reference pour le diagnostic de ces rearrangements, l’immunohistochimie (IHC) pourrait etre utile en tant qu’outil de pre-screening. But de l’etude Comparer les techniques d’IHC, FISH et RT-qPCR ALK realisees par 14 plateformes de genetique moleculaire des cancers (PGMC). Materiel et methodes Pour cette etude methodologique, 547 ADK, enrichis en prelevements chirurgicaux et en cas positifs, ont ete analyses, en utilisant les anticorps 5A4 et D5F3 (IHC), 2 sondes break-apart (FISH), et des kits TaqMan (RT-qPCR), et les donnees clinicopathologiques des patients renseignees. Resultats Au total, 140 tumeurs presentaient un rearrangement du gene ALK par FISH (≥ 15 % de cellules rearrangees) et 400 etaient negatives ( 20 %. Aucun variant de transcription n’a ete detecte dans 36 % des cas ALK-positifs. Conclusion L’analyse des resultats a mis en evidence que les discordances IHC/FISH sont plus frequentes pour les cas « borderline » (FISH entre 10 et 20 %), et qu’elles sont PGMC-dependantes. L’IHC est donc un outil de prescreening fiable pour la detection des rearrangements du gene ALK.

�� This essay examines the popular 1960s fi lms Mary Poppins (Stevenson 1964) and The Sound of Music (Wise 1965) which, despite their foreign settings and stars and their historical timeframes, are seen in the light of their relevance to social and political concerns of the United States in the 1960s. The fi lms set about addressing anxieties surrounding masculinity, motherhood, domestic gender roles, and the constitution of the family that were current at the time. The fi lms are seen as a response to the burgeoning women’s movement of the mid-1960s and rising levels of recognition of women’s changing place in society, as well as what was considered men’s loosening grip on patriarchal power in the family. In both fi lms, the character of the nanny, a threshold fi gure and family interloper, is the person responsible for reinstalling the father, whose domestic role as head of the intact, patriotic family was thought to be in jeopardy, by ensuring that his familial relations are modernized.

This paper reconsiders the use of the term “victim” in feminist theory to attempt to find common ground for the intersection and interconnection of Western and indigenous feminisms. The role of the victim in the discourse of victimology, a branch of criminology, is assessed and applied to the work of Rajeswari Sunder Rajan and Lata Mani who both examine the construction of women's subjectivity in the practice of “sati” in India.

Introduction As metastatic pulmonary adenocarcinomas are routinely investigated for EGFR, KRAS, and ALK mutations/rearrangement, adequacy of CT-guided trans-thoracic needle biopsies (TTNB) needs to be evaluated in respect with the 2011 ATS/ERS/IASLC guidelines. Methods Two series of consecutive TTNB with 18-gauge needles performed before and after the publication of the ATS/ERS/IASLC guidelines, were retrospectively compared regarding their adequacy for histological sub-typing and EGFR/KRAS mutations and ALK rearrangement testing; the first series included 43 TTNB collected from January 2010 to February 2011, and the second one 48 TTNB collected from March 2011 to December 2012. Results 28 women and 63 men were included; the 2 groups were comparable in age, in mean size of lesions (32.5 mm), and distance of the lesion from the pleura. By comparing the first to the second series, the number of biopsies increased from 1.6 to 1.85, their mean length increased from 10.9 to 12.5 mm, and the mean number of stainings (TTF1, P63, CK5-6, mucins) per biopsy decreased from 2.6 to 1. Mean tumor cell percentage was 42%, mean total DNA extracted increased from 2.7 to 3.8 μg. In the first series, 85% of TTNB allowed EGFR exons 19 and 21 and KRAS mutations pyrosequencing and 72% additional EGFR exons 18 and 20 mutation analyses, versus 98% and 92% in the second. Conclusions With respect to ATS/ERS/IASLC guidelines, radiologists, biologists and pathologists have improved their practice; accordingly, CT-guided TTNB enable a precise histological sub-typing and provide sufficient DNA amount for genetic analyses.

International audience; Epigenetic perturbations are increasingly described in cancer cells where they are thought to contribute to deregulated gene expression and genome instability. Here, we report the first evidence that a distinct category of chromosomal translocations observed in human tumours--those targeting 1q12 satellite DNA--can directly mediate such perturbations by promoting the formation of aberrant heterochromatic foci (aHCF). By detailed investigations of a 1q12 translocation to chromosome 2p, in a case of human B cell lymphoma, aberrant aHCF were shown to be localized to the nuclear periphery and to arise as a consequence of long range 'pairing' between the translocated 1q12 and chromosome 2 centromeric regions. Remarkably, adjacent 2p sequences showed increased levels of repressive histone modifications, including H4K20me3 and H3K9me3, and were bound by HP1. aHCF were associated to aberrant spatial localization and deregulated expression of a novel 2p gene (GMCL1) that was found to have prognostic impact in diffuse large B cell lymphoma. Thus constitutive heterochromatin rearrangements can contribute to tumourigenesis by perturbing gene expression via long range epigenetic mechanisms.

Over the past decades, in spite of intensive search, no significant increase in the survival of patients with glioblastoma has been obtained. The role of the blood-brain barrier (BBB) and especially the activity of efflux pumps belonging to the ATP Binding Cassette (ABC) family may, in part, explain this defect., info:eu-repo/semantics/published

8065 Background: Molecular alterations of crizotinib (crz) targets (ALK, MET, ROS1) are found in a wide range of malignancies. To avoid uncontrolled off-label use and allow for a nationwide safe access to crz for patients (pts) with an ALK, MET or ROS1 positive (+) tumor, the French National Cancer Institute (INCa) launched the AcSe program, funding both access to tumor molecular diagnosis and an exploratory multi-tumor 2-stage design phase II trial. We report the preliminary results of the ROS1+ NSCLC cohort. Methods: ROS1 status was determined in 28 regional INCa molecular genetic centers by break-apart FISH assays. Patients with ROS1 rearrangements, progressing after at least one standard treatment (including a platinum-based doublet, unless pts were considered as unfit for chemotherapy) were proposed to receive crz 250 mg BID. Responses were centrally assessed using RECIST v1.1. The objective response rate (ORR) and disease control rate (DCR) were assessed every 8 weeks. Results: From Aug. 5, 2013 to ...

8100 Background: ALK rearrangements occur in nearly 5% of NSCLC and lead to a permanent ALK protein activation, targeted by a small molecule, the crizotinib. To date, FISH (Fluorescent In situ Hybridization) is considered as the gold standard to identify ALK abnormalities, but dual testing and pre-screening by immunohistochemistry have been proposed. Methods: The purpose of the study was to compare immunohistochemistry (IHC) using 5A4 and D5F3 Abs, with FISH and quantitative RT-PCR in a series of 500 surgical specimens, collected within one year from 15 French Thoracic Pathology Departments and INCa genetic platforms. Our study was deliberately enriched in ALK positive cases and clinicopathological data were recorded. Results: Among the 459 cases included to date, 340 were both FISH and IHC ALK negative, and 85 were ALK FISH and IHC positive. Fifteen cases were FISH neg/IHC pos, but with low staining scores; 12 cases were FISH pos/IHC neg, most provided by two centers. Seven cases were non interpretable by FISH, but 5 were ALK IHC positive. Regarding RTqPCR, nearly 50% of ALK positive cases presented a variant 1, 30% a variant 3a/b, and less than 5%, variants 2 or 7; 20%were negative or non interpretable. Discordant cases will be further discussed according to the crizotinib response. ALK positive patients were more frequently women (65 vs 42%) and younger than ALK negative patients (mean age 59 vs 64yrs); 72% were non or light smokers, whereas 75% of ALK negative patients were smokers (mean of 41PY). Histologically, most ALK positive and negative tumors presented a solid or acinar predominant architecture and were P63 negative. However, ALK positive tumors were more frequently TTF1 positive (91 vs 76%). Conclusions: 5A4 or D5F3 immunohistochemistry is a reliable and easy technique for routine diagnosis of ALK abnormalities, while FISH and RT-qPCR still dependent on pre-analytic conditions and technical expertise. However, in case of a suggestive clinical presentation, double testing with FISH remains the safer testing option as false negative IHC cases exist.

8099 Background: After ALK (anaplastic lymphoma kinase) rearrangements, ROS1 (c-ros oncogene 1, located at 6q22) translocations were recently shown to define a new molecular subgroup in non-small cell lung cancers (NSCLC). These translocations, activating a proto-oncogene with a tyrosine kinase domain, are also targeted by the MET and ALK crizotinib. ROS1 rearrangements are found in only 1.2-1.7% NSCLCs, but seemingly only in triple negative (EGFR and KRAS non-mutated and ALK non-rearranged) adenocarcinomas and preferentially in non- or light smokers. Methods: 60 triple negative non- or light-smoking patients with lung adenocarcinoma were screened for ROS1 rearrangements by fluorescent in situ hybridization (FISH). Two commercially available break-apart FISH probe kits containing a mixture of two probes hybridizing on each side of the ROS1 locus, one located in a centromeric (3’) position, and the other one in a telomeric (5’) position relative to the ROS1 gene were used. ROS1 fusions were therefore detected without any preconception on the fusion partner, as multiple fusion partners have been described. At least 100 nuclei were screened, and a tumor was considered positive if it contained more than 15% rearranged nuclei. Results: Forty-nine of the 60 tumors were interpretable. Four (8.2%) of theses 49 tumors were ROS1 rearranged by FISH. Only one of the positive cases showed a “classical” split rearrangement pattern (one fusion, one green and one red signal in more than 15% of nuclei), whereas the three other tumors showed a variant pattern, with a deletion of the telomeric probe, located in a 5’ position relative to the ROS1 gene locus, indicating the presence of a fusion partner upstream of the ROS1 gene. The four positive cases were all at an advanced stage (3 or 4), but did not display any common architectural feature. Conclusions: The prevalence of ROS1 positivity in our selected population was far superior (8.2%) to that of a non-selected NSCLC population, suggesting that this type of screening may be more effective (faster and more cost-worthy) to detect ROS1 positive tumors.