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SSc is an auto-immune disease characterized by life-threatening manifestations such as lung fibrosis or pulmonary arterial hypertension. Symptoms with a detrimental impact on quality of life are also reported and sicca syndrome (xerostomia, xeropthalmia) is present in up to 80% of patients with SSc. Sicca syndrome can occur in the absence of overlap with Sjögren’s disease and recent studies highlight that fibrosis of minor and major salivary glands, directly linked to the pathogenesis of SSc, could be a major contributor of xerostomia in SSc. This narrative review provides an overview of the clinical presentation, diagnostic strategies, management and future perspectives on sicca syndrome in patients with SSc.

International audience

Purpose:Follicular lymphoma arises from a germinal center B-cell proliferation supported by a bidirectional crosstalk with tumor microenvironment, in particular with follicular helper T cells (Tfh). We explored the relation that exists between the differentiation arrest of follicular lymphoma cells and loss-of-function of CREBBP acetyltransferase.Experimental Design: The study used human primary cells obtained from either follicular lymphoma tumors characterized for somatic mutations, or inflamed tonsils for normal germinal center B cells. Transcriptome and functional analyses were done to decipher the B- and T-cell crosstalk. Responses were assessed by flow cytometry and molecular biology including ChIP-qPCR approaches.Results:Conversely to normal B cells, follicular lymphoma cells are unable to upregulate the transcription repressor, PRDM1, required for plasma cell differentiation. This defect occurs although the follicular lymphoma microenvironment is enriched in the potent inducer of PRDM1 and IL21, highly produced by Tfhs. In follicular lymphoma carrying CREBBP loss-of-function mutations, we found a lack of IL21-mediated PRDM1 response associated with an abnormal increased enrichment of the BCL6 protein repressor in PRDM1 gene. Moreover, in these follicular lymphoma cells, pan-HDAC inhibitor, vorinostat, restored their PRDM1 response to IL21 by lowering BCL6 bound to PRDM1. This finding was reinforced by our exploration of patients with follicular lymphoma treated with another pan-HDAC inhibitor. Patients showed an increase of plasma cell identity genes, mainly PRDM1 and XBP1, which underline the progression of follicular lymphoma B cells in the differentiation process.Conclusions:Our data uncover a new mechanism by which pan-HDAC inhibitors may act positively to treat patients with follicular lymphoma through the induction of the expression of plasma cell genes.

Figure S1 : (A) The 1769-genes signature corresponding to CB/CC transition and CB response to IL-21/CD40L co-stimulation and that are differentially expressed in FL compared to CC and CB were explored using Ingenuity Pathway Analysis (IPA). IPA enrichment analysis showed that these genes are mainly involved in CD40L signaling and IPA diagram highlight activated pathway including NF-KappaB (Nuclear Factor-KappaB), MAPK (Mitogen-Activated Protein Kinase) and STAT3 (Signal Transducers and Activators of Transcription-3). This observation suggests that FL-tumor cells can respond to the Il- 21/CD40L signaling. (B) Modulation of gene expression in CB compared to FL during CB/CC transition related to CD40L+IL-21 stimulation was explored by geneset enrichment assays (GSEA) using signatures available on MSigDB: M8493 for CD40 up-regulated genes, M2827 for IL-21 up-regulated genes, M4552 for up-regulated genes in plasma cells. (C) BLIMP-1 immnunostaining performed on multiple myeloma (1), reactive lymph node (2) and follicular lymphoma (3) gives strong, weak (very few positive cells) and negative signal respectively, using the PRDM1 antibody (3H2-E8) from Thermo Scientific. Figure S2: Burkitt lymphoma-derived L3055 cell line characteristics. Figure S3: Purity and viability of FL B-cell fractions. Figure S4: Visualization of gating strategy of a representative sample. Figure S5 : BCL6 binding analysis on Intron 3 of PRDM1 gene. Figure S6: Detection of PRDM1 mRNA and BLIMP1 protein in unstimulated or treated L3055 cells. Figure S7 : FL B cell number for each fraction and percentage of cell viability in each cell culture condition (N=6). Figure S8 : Uncropped original images of immunoblots. Table S1 : Top-20 pathways associated with 804-upregulated (upper panel) or 967-downregulated genes (lower panel). Table S2: Clinical data and somatic mutations for epigenetic genes - CREBBP, EP300, EZH2, KMT2D & MEF2B - in the 20 FLs of the cohort. Table S3: Available clinical data and somatic mutations found in genes designed in the lymphopanel for the 4 patients with a refractory or multiple relapsing FL tested for the oral administration of the HDACi treatment. Table S5: List of primers used for quantitative RQ-PCR and QChIP-PCR

SummaryDiffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL) are the two most common B-cell lymphomas and are characterized by a dynamic crosstalk between tumor B cells and a heterogeneous tumor-supportive microenvironment, including immune, endothelial, and stromal components. Although their impact on the pathogenesis and prognosis of B-cell lymphoma has been acknowledged for years, tumor-associated macrophages (TAM) have not been extensively explored in DLBCL and FL. Herein, we investigate mononuclear phagocytes (MNP) heterogeneity at the single cell level and their potential co-regulation with the stromal and endothelial compartments in B-cell lymphoma lymph nodes compared to reactive secondary lymphoid organs, using a combination of mass cytometry, single cell RNA sequencing, andin silicoapproaches. We reveal a co-regulation between TAM and blood endothelial cells (BEC) in lymphoma. Moreover, we identify a specific interaction between Annexin A1 (ANXA1)-expressing BEC and formyl-peptide receptors (FPR1/2)-expressing monocytes/macrophages in DLBCL, which we confirmin situby multiplex immunofluorescence and imaging mass cytometry. This crosstalk is associated to an immunosuppressive tumor microenvironment and an adverse prognosis in DLBCL.

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The chemokine CXCL13 controls the normal organization of secondary lymphoid tissues and the neogenesis of ectopic lymphoid structures in nonlymphoid organs, particularly the lungs. The progression and severity of idiopathic pulmonary fibrosis (IPF), a fatal and irreversible interstitial lung disease, is predicted by the circulating blood concentrations of CXCL13. Although CXCL13 is produced by pulmonary tissues, it has not been determined which cells are involved. This study examines CXCL13 production by lung tissue macrophages from patients with IPF and the signaling pathways controlling CXCL13 gene expression in human alveolar macrophages (AM) and monocyte-derived macrophages (MoDM). CXCL13 is found in CD68- and CD206-positive AM from patients with IPF, and the CXCL13 gene is induced in these macrophages and MoDM when they are stimulated with LPS. We found that TNF-α and IL-10 control optimal CXCL13 gene expression in MoDM and possibly in AM by activating the NF-κB and JAK/STAT pathways, respectively. We also found that blood TNF-α and CXCL13 concentrations are significantly correlated in patients with IPF, suggesting that TNF-α contributes to CXCL13 production in humans. In conclusion, the results of this study demonstrate that AM from patients with IPF produces CXCL13 and that the NF-κB and JAK/STAT pathways are required to induce the expression of this major chemokine.

The differentiation of B cells into plasmablasts (PBs) and then plasma cells (PCs) is associated with extensive cell reprogramming and new cell functions. By using specific inhibition strategies (including a novel morpholino RNA antisense approach), we found that early, sustained upregulation of the proviral integrations of Moloney virus 2 (PIM2) kinase is a pivotal event during human B-cell in vitro differentiation and then continues in mature normal and malignant PCs in the bone marrow. In particular, PIM2 sustained the G1/S transition by acting on CDC25A and p27Kip1 and hindering caspase 3–driven apoptosis through BAD phosphorylation and cytoplasmic stabilization of p21Cip1. In PCs, interleukin-6 triggered PIM2 expression, resulting in antiapoptotic effects on which malignant PCs were particularly dependent. In multiple myeloma, pan-PIM and myeloid cell leukemia-1 (MCL1) inhibitors displayed synergistic activity. Our results highlight a cell-autonomous function that links kinase activity to the newly acquired secretion ability of the PBs and the adaptability observed in both normal and malignant PCs. These findings should finally prompt the reconsideration of PIM2 as a therapeutic target in multiple myeloma.

The cIMPACT-NOW Update 7 has replaced the WHO nosology of “ependymoma, RELA fusion positive” by “Supratentorial-ependymoma, C11orf95-fusion positive”. This modification reinforces the idea that supratentorial-ependymomas exhibiting fusion that implicates the C11orf95 (now called ZFTA) gene with or without the RELA gene, represent the same histomolecular entity. A hot off the press molecular study has identified distinct clusters of the DNA methylation class of ZFTA fusion-positive tumors. Interestingly, clusters 2 and 4 comprised tumors of different morphologies, with various ZFTA fusions without involvement of RELA. In this paper, we present a detailed series of thirteen cases of non-RELA ZFTA-fused supratentorial tumors with extensive clinical, radiological, histopathological, immunohistochemical, genetic and epigenetic (DNA methylation profiling) characterization. Contrary to the age of onset and MRI aspects similar to RELA fusion-positive EPN, we noted significant histopathological heterogeneity (pleomorphic xanthoastrocytoma-like, astroblastoma-like, ependymoma-like, and even sarcoma-like patterns) in this cohort. Immunophenotypically, these NFκB immunonegative tumors expressed GFAP variably, but EMA constantly and L1CAM frequently. Different gene partners were fused with ZFTA: NCOA1/2, MAML2 and for the first time MN1. These tumors had epigenetic homologies within the DNA methylation class of ependymomas-RELA and were classified as satellite clusters 2 and 4. Cluster 2 (n = 9) corresponded to tumors with classic ependymal histological features (n = 4) but also had astroblastic features (n = 5). Various types of ZFTA fusions were associated with cluster 2, but as in the original report, ZFTA:MAML2 fusion was frequent. Cluster 4 was enriched with sarcoma-like tumors. Moreover, we reported a novel anatomy of three ZFTA:NCOA1/2 fusions with only 1 ZFTA zinc finger domain in the putative fusion protein, whereas all previously reported non-RELA ZFTA fusions have 4 ZFTA zinc fingers. All three cases presented a sarcoma-like morphology. This genotype/phenotype association requires further studies for confirmation. Our series is the first to extensively characterize this new subset of supratentorial ZFTA-fused ependymomas and highlights the usefulness of ZFTA FISH analysis to confirm the existence of a rearrangement without RELA abnormality.

International audience

Choriocarcinoma is a highly malignant neoplasm resulting from the malignant transformation of proliferating trophoblastic cells and the molecular mechanisms leading to this transformation remain to be characterized. We report here the first case of a female germline TP53 mutation carrier who developed, as a first tumour, a lung choriocarcinoma, 6 months after a normal delivery. Molecular analyses established the gestational origin of the choriocarcinoma and showed, within the tumour, the presence of the germline mutant TP53 allele and loss of the wild-type allele. Resistance to methotrexate chemotherapy led to perform a surgical resection of the tumour. In agreement with the permissive role of TP53 mutations to oncogenic events, this report strongly suggests that TP53 mutations may promote malignant transformation of proliferating trophoblastic cells. Therefore, female TP53 mutation carriers may have an increased risk of developing gestational choriocarcinoma and might benefit from β-hCG level monitoring after pregnancy.

Nintedanib and pirfenidone have been approved for the treatment of idiopathic pulmonary fibrosis (IPF). Both drugs were shown to reduce but not to stop disease progression. Hence the medical need is still high for new effective anti-fibrotic molecules. Arsenic trioxide (ATO), a safe and effective drug used in cancer therapy, decreased pulmonary fibrosis induced by bleomycin in mice. In order to specify the anti-fibrotic properties of ATO, we investigated its effects on main functions of human lung fibroblasts (HLFs). IPF and control HLFs were derived from lung biopsies isolated from patients with IPF or without intertitial lung disease. After pre-treatment with ATO (0.01-1 μM) and stimulation with PDGF-BB (50 ng/ml) or TGF-β1 (1 ng/ml), proliferation, migration and differentiation of HLFs were assessed as well as the cell stress response triggered by ATO. The metalloid did not affect cell viability but it significantly decreased, in a concentration-dependent manner, the proliferation and migration of control and IPF HLFs induced by PDGF-BB. ATO also prevented the up-regulation of α-SMA, Collagen-1 and p-SMAD3 protein levels in TGF-β1-stimulated HLFs. The metalloid effects were associated with stabilization of the transcription factor NRF2 and induction of the antioxidant proteins NQO1 and HO-1 that emphases the stress response developed by ATO-treated HLFs. In conclusions, our results demonstrate that, in vitro, ATO concentrations, in the range of arsenic plasmatic levels measured in patients treated with standard dosing, counteracts the detrimental functions of IPF HLFs. ATO may thus be taken into consideration as a new therapeutic option for IPF treatment.

Purpose: Follicular lymphoma arises from a germinal center B-cell proliferation supported by a bidirectional crosstalk with tumor microenvironment, in particular with follicular helper T cells (Tfh). We explored the relation that exists between the differentiation arrest of follicular lymphoma cells and loss-of-function of CREBBP acetyltransferase. Experimental Design: The study used human primary cells obtained from either follicular lymphoma tumors characterized for somatic mutations, or inflamed tonsils for normal germinal center B cells. Transcriptome and functional analyses were done to decipher the B- and T-cell crosstalk. Responses were assessed by flow cytometry and molecular biology including ChIP-qPCR approaches. Results: Conversely to normal B cells, follicular lymphoma cells are unable to upregulate the transcription repressor, PRDM1, required for plasma cell differentiation. This defect occurs although the follicular lymphoma microenvironment is enriched in the potent inducer of PRDM1 and IL21, highly produced by Tfhs. In follicular lymphoma carrying CREBBP loss-of-function mutations, we found a lack of IL21-mediated PRDM1 response associated with an abnormal increased enrichment of the BCL6 protein repressor in PRDM1 gene. Moreover, in these follicular lymphoma cells, pan-HDAC inhibitor, vorinostat, restored their PRDM1 response to IL21 by lowering BCL6 bound to PRDM1. This finding was reinforced by our exploration of patients with follicular lymphoma treated with another pan-HDAC inhibitor. Patients showed an increase of plasma cell identity genes, mainly PRDM1 and XBP1, which underline the progression of follicular lymphoma B cells in the differentiation process. Conclusions: Our data uncover a new mechanism by which pan-HDAC inhibitors may act positively to treat patients with follicular lymphoma through the induction of the expression of plasma cell genes.

Myeloid sarcoma (MS) are extramedullary tumor masses consisting of a proliferation of myeloid immature cells that efface the underlying tissue architecture.[1] It may develop de novo or concurrentl...

Follicular lymphoma (FL) arises from a germinal center (GC) B cell proliferation supported by a bidirectional crosstalk with tumor microenvironment, in particular with follicular helper T cells (Tfh). Besides BCL2 translocation hallmark, tumor B cells progressively acquire loss-of-function alterations within chromatin organization factor genes. Notably, frequent inactivating mutations in CREBBP acetyltransferase are found in B-cell lymphoma and drive deep epigenetic and transcriptional profile modifications. Our transcriptional analysis on highly purified cell fractions revealed that, conversely to normal GC B cells (centroblasts and centrocytes), primary FL B cells are unable to upregulate the transcription repressor PRDM1 that is required for plasma cell differentiation and maintenance. This defect happens although FL tumor environment is enriched in the potent inducer of PRDM1, IL-21 that we found highly produced by CD4+ Tfhs. In 20 different FLs perfectly characterized at clinical and genetic level, we analyzed their FL cells obtained after purification from the initial tumor. Globally, we found a significant lack of IL-21-mediated PRDM1 response while our control cell line showed a positive increase of PRDM1 expression. We analyzed some of these primary FL tumor cells by qPCR-ChIP to assess BCL6 protein binding at intron 3 of the PRDM1 gene. We found that the lack of PRDM1 response to IL-21 was associated with an abnormal increased enrichment of the BCL6 protein repressor in PRDM1 gene. Our data indicated this was especially the case for FLs with nonfunctional CREBBP. We next explored 6 FLs, cells were treated in vitro with vorinostat, a pan-HDAC inhibitor. We found that for FL B cells carrying CREBBP loss-of-function mutations, vorinostat restored partially their PRDM1 response to IL-21 by lowering BCL6 bound to PRDM1. Finally, we wanted to assess this drug effect in the context of FL patients treated with a pan-HDACi. We found 4 patients involved in a phase I/II clinical at the Gustave Roussy Institute in Villejuif and treated with abexinostat, a new and off-label pan-HDAC inhibitor. All the patients presented a progressive FL disease, were heavily pretreated and their tumor were characterized by genomic DNA sequencing. One patient reached a stable complete remission, the drug was discontinued after 4 years of treatment. Among the three other patients, one had an objective 28-month response to the drug before relapse, one a stable disease for seven months while the last one did not response to the drug. For these 3 patients we've got fine needle tumor biopsy when patients re-progressed. Re-biopsies showed an increased expression of plasma cell-identity genes, mainly PRDM1 and XBP1, which underline the progression of FL B cells in the process of differentiation. Altogether, our data uncover a new mechanism by which pan-HDAC inhibitors may act positively to treat FL patients. Disclosures Ribrag: BMS: Consultancy, Honoraria, Other: travel; argenX: Research Funding; Servier: Consultancy, Honoraria; epizyme: Consultancy, Honoraria; NanoString Technologies: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Amgen: Research Funding; Roche: Honoraria, Other: travel; Incyte Corporation: Consultancy; pharmamar: Other: travel; Infinity: Consultancy, Honoraria; MSD: Honoraria.

Peripheral T-cell lymphomas (PTCL) comprise usually aggressive neoplasms of mature T cells with a heterogeneous molecular background. Over the past years, several genomic studies stemming from different research groups have shown evidence that mutation-induced activation of the T-cell receptor (TCR

L’avenement des therapies ciblees a considerablement ameliore la prise en charge des patients atteints de cancer bronchopulmonaire non a petites cellules (CBNPC). A cote des anomalies bien connues de ALK et de l’EGFR, les rearrangements de RET comptent pour 2 % des adenocarcinomes pulmonaires. Dans ce travail, nous avons etudie par hybridation in situ en fluorescence (FISH), le statut RET de 714 prelevements de CBNPC non epidermoides negatifs pour les mutations KRAS/EGFR/BRAF/ERBB2 et ne presentant pas de remaniements des genes ALK et ROS1 afin de determiner la prevalence et les caracteristiques clinico-pathologiques des patients RET-positifs. Ces analyses ont ete realisees en utilisant la sonde ZytoLight® SPEC RET Dual Color Break Apart Probe (Zytovision). Un rearrangement de RET a ete mis en evidence dans 18 prelevements sur 714 (2,52 %). L’âge moyen des patients RET-positifs etait de 67,7 ans. Parmi ces patients, 10/18 (56 %) etaient des femmes. Au niveau histologique, les tumeurs avec un rearrangement de RET presentaient une architecture solide (n = 10), acinaire (n = 4), papillaire (n = 1) et lepidique (n = 1). L’association decrite dans la litterature du rearrangement de RET avec le sexe feminin et l’architecture solide a ete retrouvee dans notre etude. Sur le plan therapeutique, 7/18 patients ont ete traites par une chimiotherapie a base de pemetrexed et seulement 2 ont pu beneficier d’inhibiteurs de tyrosine kinase (ITK) anti-RET non valides dans le CBNPC (vandetanib et sunitinib). Notre etude montre que le rearrangement de RET se produit chez un nombre significatif de patients. Des ITK anti-RET comme le cabozantinib ou le vandetanib disposant d’une AMM dans d’autres pathologies pourraient etre utilises chez ces patients. La recherche en routine du rearrangement de RET dans les CBNPC non epidermoides pan-negatifs pour les marqueurs precites apparait donc souhaitable.

Nous exposons ici le cas d’un lymphome intra-vasculaire diagnostique sur biopsies duodenales realisees dans le cadre du bilan d’une alteration de l’etat general et de douleurs epigastriques evoluant depuis 3 mois chez un homme de 77 ans. Cette entite tres rare, appartient au sous-groupe des lymphomes B diffus a grandes cellules dans la classification OMS 2008. Il se caracterise par une localisation quasi exclusive des cellules lymphomateuses dans les capillaires et les vaisseaux de petit calibre. Le pronostic de cette hemopathie generalement diagnostiquee post mortem reste de nos jours tres pejoratif, notamment du fait du retard diagnostic en lien avec le caractere peu specifique des symptomes. L’originalite de notre cas tient a son diagnostic precoce realise sur biopsies duodenales dans un contexte de douleurs epigastriques sans anomalie endoscopique, revelant une atteinte viscerale disseminee confirmee par TEP scanner et myelogramme. L’atteinte ganglionnaire et l’infiltration medullaire permettent egalement de discuter le sous-type rarissime dit « asiatique ».

International audience; Intrahepatic cholangiocarcinoma (ICC) is the second most common type of primary cancer in the liver. ICC is an aggressive cancer with poor prognosis and limited therapeutic strategies. The identification of new drug targets and prognostic biomarkers is an important clinical challenge for ICC. The presence of an abundant stroma is a histological hallmark of ICC. Given the well-established role of the stromal compartment in the progression of cancer diseases, we hypothesized that relevant biomarkers could be identified by analyzing the stroma of ICC. By combining laser capture microdissection and gene expression profiling, we demonstrate that ICC stromal cells exhibit dramatic genomic changes. We identified a signature of 1,073 nonredundant genes that significantly discriminate the tumor stroma from nontumor fibrous tissue. Functional analysis of differentially expressed genes demonstrated that up-regulated genes in the stroma of ICC were related to cell cycle, extracellular matrix, and transforming growth factor beta (TGFβ) pathways. Tissue microarray analysis using an independent cohort of 40 ICC patients validated at a protein level the increased expression of collagen 4A1/COL4A1, laminin gamma 2/LAMC2, osteopontin/SPP1, KIAA0101, and TGFβ2 genes in the stroma of ICC. Statistical analysis of clinical and pathological features demonstrated that the expression of osteopontin, TGFβ2, and laminin in the stroma of ICC was significantly correlated with overall patient survival. More important, multivariate analysis demonstrated that the stromal expression of osteopontin was an independent prognostic marker for overall and disease-free survival. CONCLUSION: The study identifies clinically relevant genomic alterations in the stroma of ICC, including candidate biomarkers for prognosis, supporting the idea that tumor stroma is an important factor for ICC onset and progression.

Lymphoedema, myelodysplasia, fibrosis and pulmonary alveolar proteinosis complicating a novel GATA2 mutation http://ow.ly/RHci303h8ks

International audience; Background - Lung cancer represents the leading cause of cancer-related death worldwide. Despite great advances in lung cancer management with the recent emergence of molecular targeted therapies for non-squamous non-small-cell lung cancer, no dramatic improvements have been achieved in lung squamous cell carcinoma (SCC). Mutations in discoidin domain receptor 2 (DDR2) gene were recently identified as promising molecular targets in this histology. The aim of this study is to describe the DDR2 mutational landscape of lung SCC and investigate the associated clinical factors. Methods - Next-generation sequencing of the DDR2 gene was performed on 271 samples of lung SCC. Patients followed in our institution from January 2011 to August 2014 were retrospectively selected for data collection. Other driver gene alterations (EGFR, KRAS, BRAF, HER2, and PI3KCA) were analyzed using pyrosequencing. Results - A total of 11 patients harboring a DDR2 mutation was detected among the 271 sequenced lung SCC samples (4%). We describe 10 unreported mutations, comprising a novel DDR2 exon 7 splice mutant. DDR2 mutations were not mutually exclusive with other driver gene alterations. One hundred thirty-six patients were included for clinical comparison and logistic regression analysis. No difference was detected between DDR2-mutant and DDR2 wild-type lung SCC regarding clinical characteristics or survival. Conclusion - DDR2 mutations were observed in 4% of cases of lung SCC of European descent. DDR2-mutated tumors can exhibit other driver gene alterations. No clinical characteristics were significantly associated with DDR2 mutation.

Objet Les systemes de capture et d’analyse d’image permettent une analyse par FISH semi-automatisee des coupes de tissus fixes par le formol et inclus en paraffine. Le service de cytogenetique et biologie cellulaire du CHU de Rennes est equipe du systeme BIOVIEW® - Encore, pouvant scanner 200 lames par serie avec une camera haute resolution. Methodes Nous rapportons l’etude de 47 cas de lymphomes. L’analyse histologique correspondait a 42 lymphomes B diffus a grandes cellules pour lesquels un rearrangement des genes BCL2, BCL6 et MYC a ete recherche ainsi que 5 lymphomes folliculaires analyses pour les genes BCL2 et BCL6 avec les sondes VYSIS® LSI BA correspondantes. Resultats Une analyse semi-automatique a ete possible pour 73 % (99/136) des lames avec des resultats sans ambiguite (rearrangement de BCL2 [n = 9], BCL6 [n = 8], MYC [n = 8], non rearranges [n = 63]) dans 89 % (88/99) et douteux dans 11 % (11/99). Pour ces derniers, l’analyse manuelle au microscope a permis a chaque fois de confirmer le resultat initialement suspecte (rearrangement de BCL2 [n = 1], BCL6 [n = 2], MYC [n = 3], non rearranges [n = 5]). Les resultats etaient non contributifs pour 27 % (37/136) des preparations. Une lecture manuelle au microscope a permis un rendu de resultat dans 35 % (13/37) des cas alors que les autres (24/37) restaient non interpretables. Le temps necessaire pour l’analyse semi-automatisee d’une preparation (12 ± 7 min) etait inferieur a celui de l’analyse manuelle (23 ± 5 min). Conclusion Ces resultats montrent l’interet de l’utilisation en routine du systeme BIOVIEW® - Encore pour l’analyse par FISH des lymphomes. Outre le gain de temps et l’analyse ciblee des zones d’interet, ce systeme permet l’archivage automatique et standardise des images. Son utilisation systematique est en cours pour les autres activites du service (tumeurs solides, onco-hematologie, cytogenetique constitutionnelle/prenatale) ainsi qu’en recherche pour l’etude de l’heterogeneite tumorale.

National audience; Introduction Les adénocarcinomes pulmonaires peuvent être causés par des mutations des gènes EGFR (10 %) ou KRAS (30 %), et des translocations du gène ALK (4 %). La détection de ces oncogènes rend les patients éligibles à une thérapie ciblée par inhibiteurs de tyrosine kinases (TKI). Récemment, le gène RET qui code pour un récepteur à tyrosine kinase apparenté à ALK, a été décrit dans 2 % des adénocarcinomes pulmonaires. Matériel et méthode Au cours de l’année 2014, 951 prélèvements d’adénocarcinomes pulmonaires ont été adressés à la plateforme de biologie moléculaire des cancers de Rennes. Les prélèvements négatifs pour les mutations KRAS/EGFR et ne présentant pas de remaniement du gène ALK ont été testés par hybridation in situ en fluorescence pour évaluer leur statut RET. Les remaniements de RET ont été analysés avec des sondes Break-Appart (Zytovision). Le prélèvement était considéré réarrangé lorsque plus de 15 % des cellules tumorales présentaient une séparation des signaux ou un signal 3′ isolé. Résultats Sur 951 adénocarcinomes, 199 ont été testés pour un réarrangement de RET. Un réarrangement de RET a été mis en évidence dans 7 prélèvements (3,7 %). Le ratio homme/femme était de 33 % pour les cas RET-positifs, contre 63 % dans la cohorte. L’âge moyen était de 71 ans pour les RET-positifs contre 65 ans dans la cohorte. Sur le versant anatomopathologique, 5 des 7 cas positifs présentaient une composante solide et deux une composante papillaire. Discussion Dans cette étude, 4 % des adénocarcinomes négatifs pour les oncogènes recherchés selon les recommandations de l’INCa se sont avérés RET-positifs. Récemment, deux cas de réponse de patients RET-positifs à des TKI (cabozantinib et vandétanib) ont été rapportés. Ainsi, la détection du remaniement de RET pourrait permettre l’inclusion de patients dans des essais cliniques évaluant l’efficacité de ces TKI

Les rearrangements du gene ALK representent 5 a 7 % de l’ensemble des adenocarcinomes bronchiques. La comparaison des performances de l’hybridation in situ en fluorescence (FISH) et de l’immunohistochimie (IHC) dans l’identification de patients ALK-positifs a conduit a proposer l’IHC comme methode de prescreening et la FISH en confirmation des cas positifs et douteux. Cependant, plusieurs etudes a large echelle retrouvent des discordances entre ces deux techniques. Dans cette etude, nous avons reanalyse 36 cas pour lesquels un diagnostic FISH/IHC discordant avait ete rendu. Une nouvelle analyse des IHC avec le clone 5A4 a ete realisee par les pathologistes ayant effectue le diagnostic initial. Un nouvelle IHC a ete realisee en utilisant le kit Ventana D5F3 CDx Assay recemment valide par la FDA pour identifier les patients eligibles au crizotinib et l’influence d’une variation interindividuelle du scoring a ete evaluee en comparant l’analyse de plusieurs pathologistes. Nos resultats montrent une augmentation du seuil de positivite utilise pour l’analyse 5A4. Le nombre de cas scores 1+ se reduit rapprochant les resultats de l’IHC de ceux de la FISH. En corollaire, 4 cas declares initialement positifs deviennent negatifs et discordants par rapport a la FISH. Les resultats obtenus avec le D5F3 montrent un profil plus proche de la FISH : 5 cas FISH-positifs sont scores negatifs avec le 5A4 mais positifs avec le D5F3. Enfin, la comparaison entre les 3 pathologistes impliques dans l’analyse D5F3 montre une heterogeneite du seuil de positivite utilise. En conclusion, cette etude souligne le travail qui reste a accomplir pour standardiser l’analyse IHC sur l’ensemble du territoire. La validation d’EEQ et la participation a des comparaisons inter laboratoires apparaissent necessaires pour homogeneiser les pratiques et modalites de l’analyse afin de garantir une identification exhaustive des patients susceptibles de beneficier des inhibiteurs de tyrosine kinase ciblant ALK.