Search

Summary Targetable kinase fusions are extremely rare ( We searched for NTRK1, NTRK2, NTRK3, ALK, ROS1, BRAF, RET, and NRG1 kinase fusions in CRCs using methods easy-to-implement in pathology laboratories: immunohistochemistry (IHC), fluorescent in situ hybridization (FISH), and fully automated real-time PCR targeted analyses. RNA-sequencing analyses were used for confirmation. Among 84 selected MLH1 deficient (IHC) CRCs cases, MLH1loss MSI-High wild-type CRCs consisted first in 19 cases after Idylla™ analyses and finally in 18 cases (21%) after RNA-sequencing (detection of one additional KRASG12D mutation). FISH (and when relevant, IHC) analyses concluded in 5 NTRK1, 3 NTRK3, 1 ALK, 2 BRAF, and 2 RET FISH positive tumors. ALK and NTRK1 rearranged tumors were IHC positive, but pan-TRK IHC was negative in the 3 NTRK3 FISH positive tumors. RNA-sequencing analyses confirmed 12 of 13 fusions with only one false positive RET FISH result. Finally, 12/18 (67%) of MLH1loss MSI-High wild-type CRCs contained targetable kinase fusions. Our study demonstrates the feasibility, but also the cost-effectiveness, of a multistep but rapid diagnostic strategy based on nonsequencing methods to identify rare and targetable kinase fusions in patients with advanced CRCs, as well as the high prevalence of these kinase fusions in MLH1loss MSI-High wild-type CRCs. Nevertheless, confirmatory RNA-sequencing analyses are necessary in case of low FISH positive nuclei percentage to rule out FISH false-positive results.

AimsWe aimed to evaluate the performances of the Idylla GeneFusion Assay (IGFA) designed to detect, in a single, rapid and fully automated assay,ALK,ROS1,RET,NTRK1,NTRK2andNTRK3gene fusions andMETexon 14 skipping in cancer samples.MethodsBased on a set of tumours enriched in cases with gene fusions, we applied the IGFA to tumour areas of various sizes and tumour cell contents. IGFA results were compared with those obtained with other methods (immunohistochemistry, fluorescent in situ hybridisation, DNA and RNA next-generation sequencing).ResultsWe selected 68 tumours: 49 cases with known gene fusions (8ALK, 8ROS1, 5RET, 7NTRK1, 3NTRK2and 6NTRK3ones) orMETexon 14 skipping mutations (12 cases) and 19 cases with no fusion and noMETmutation. We performed 128 IGFA tests on distinct tissue areas. The global sensitivity and specificity of the IGFA were, respectively, 62.82% and 99.2% with variations between molecular targets and tissue areas. Of note, 72.5% sensitivity and 98.79% specificity were obtained in 37 tissue areas fulfilling the manufacturer’s recommendations (ie, at least 10% of tumour cells in at least 20 mm² of tissue area). The rate of non-conclusive results was higher in small samples with low percentages of tumour cells.ConclusionsThe IGFA could contribute to the rapid detection of targetable gene fusions and mutations, especially in context of rapidly growing cancers requiring urgent therapeutic choices.

As a major cancer hallmark, there is a sustained interest in understanding the telomerase contribution to carcinogenesis in order to therapeutically target this enzyme. This is particularly relevant in primary cutaneous T-cell lymphomas (CTCL), a malignancy showing telomerase dysregulation with few investigative data available. In CTCL, we examined the mechanisms involved in telomerase transcriptional activation and activity regulation. We analyzed 94 CTCL patients from a Franco-Portuguese cohort, as well as 8 cell lines, in comparison to 101 healthy controls. Our results showed that not only polymorphisms (SNPs) located at the promoter of human telomerase reverse transcriptase (hTERT) gene (rs2735940 and rs2853672) but also an SNP located within the coding region (rs2853676) could influence CTCL occurrence. Furthermore, our results sustained that the post-transcriptional regulation of hTERT contributes to CTCL lymphomagenesis. Indeed, CTCL cells present a different pattern of hTERT spliced transcripts distribution from the controls, mostly marked by an increase in the hTERT β+ variants proportion. This increase seems to be associated with CTCL development and progression. Through hTERT splicing transcriptome modulation with shRNAs, we observed that the decrease in the α-β+ transcript induced a decrease in the cell proliferation and tumorigenic capacities of T-MF cells in vitro. Taken together, our data highlight the major role of post-transcriptional mechanisms regulating telomerase non canonical functions in CTCL and suggest a new potential role for the α-β+ hTERT transcript variant.

Background Diffuse Midline Glioma, H3K27M-mutant (DMG) is a rare, highly aggressive pediatric tumor affecting the brainstem, and is one of the deadliest cancers. Currently available treatment options such as chemotherapy and radiotherapy do only modestly prolong survival. In this pathology, H3K27 mutations deregulate Polycomb Repressive Complex 2 (PRC2), including enzymatic activity of EZH2, which is therefore under investigation as a therapeutic target. Methods We used a chemical EZH2 inhibitor, GSK126, small interfering RNAs, and a CRISPR/Cas9 knockout approaches in a series of DMG tumor cell lines to investigate metabolic treatment responses by proteomic analysis. A combination strategy was elaborated and studied in primary and established DMG cells, spheroid 3D cultures, and in vivo in a chick chorio-allantoic membrane DMG assay and an orthotopic intracranial DMG mouse model. Results GSK126 shows significant (P < .05–.001) inhibitory effects in in vitro cell proliferation assays and induces apoptosis. Chemical targeting of EZH2 induced expression of proteins implicated in cholesterol metabolism. Low-dose GSK126 treatment together with statins revealed strong growth inhibition in combinatorial treatments, but not in single treatments, both in DMG cells in vitro, in DMG spheroid cultures, and in chick and mouse in vivo models (P < .05). All statistical tests were two-sided. Conclusions Our results reveal an unexpected GSK126-inducible sensitivity to cholesterol biosynthesis inhibitors in highly aggressive pediatric glioma that warrants further evaluation as treatment strategy. This combinatorial therapy should have few side effects because of the low doses used to achieve significant anti-tumor activity.

PURPOSE Emerging evidence suggests a correlation between the tumor mutational burden (TMB) and the response to programmed cell death-1 protein (PD-1) monotherapy across multiple cancer types. In skin cancers, as high TMB is mostly because of ultraviolet (UV) exposure, we hypothesized a correlation between the primary melanoma cutaneous location according to sun exposure and response to anti–PD-1 monotherapy. METHODS The aim of this study was to analyze, in advanced melanoma, the relationship between TMB, locations according to sun exposure, and response to PD-1 inhibitors. We conducted a prospective multicentric analysis, by sequencing the most recent metastatic sample before PD-1 inhibitors using FoundationOne assay. RESULTS One hundred two patients were included, with TMB available for 94 cases. In univariate and multivariate linear regression, TMB was significantly associated with sun-exposed areas of the primary melanoma location and with age (coefficients of the association with log-TMB: non-UV location, –1.05; chronic sun-exposed area, 1.12; P value for the location, < 10–5; age, 0.021 per year, P value for age, .002). Molecular UV signature present on the metastatic site was associated with higher TMB (P = .003). Melanomas bearing a high TMB had a higher probability of response to PD-1 inhibitors compared with melanomas with a low TMB, with a dose-dependent effect following an exponential curve and a negative odds ratio of 0.40 (95% CI, 0.20 to 0.72, P = .004) between log-TMB and 6-month progression. CONCLUSION Cumulative sun exposure related to skin location and molecular UV signature present on the metastatic site appear to be relevant biomarkers directly linked to TMB. Because TMB is not yet available to all for routine clinical use, the location of the primary melanoma in a sun-exposed area may play an important role in clinical decisions regarding therapeutic choice., The location of the primary melanoma in a sun-exposed area can help choosing first-line advanced melanoma treatment.

Cutaneous T-cell lymphoma (CTCL) such as Sézary syndrome or mycosis fungoides corresponds to an abnormal infiltration of T lymphocytes in the skin. CTCL cells have a heterogeneous phenotype and express cell adhesion molecules such as cutaneous lymphocyte antigen (CLA) supporting skin homing. The use of a mAb (HECA-452) against CLA significantly decreased transendothelial migration and survival of CTCL cells from patient samples and My-La cell line. The decrease of CLA expression by inhibition of its maturation enzyme, ST3 β-galactoside α-2,3-sialyltransferase 4, also impaired CTCL cell migration, proliferation, and survival. We confirmed in vivo that treatment with anti-CLA mAb decreased the tumorigenicity as well as dissemination of CTCL cells in different tissues compared with the control group. Our findings provide evidence of the involvement of CLA in CTCL cell migration and survival, supporting that CLA inhibition could represent an actionable therapy in patients with CTCL.

Somatic mutations in isocitrate dehydrogenase1 (IDH1) gene1-3 are common in Ollier disease and in several neoplasms including cholangiocarcinoma. We report the case of a woman with Ollier disease with chondrosarcomas and an anaplastic astrocytoma. A mutation in IDH1 was found causing R132H at the protein level in both tumors. At the age of 40, the patient presented an acute abdominal pain.

Glioblastomas are frequent malignant brain tumours with a very poor prognosis and a need for new and efficient therapeutic strategies. With the approval of anti-TRK targeted therapies to treat patients with advanced NTRK-rearranged cancers, independent of the type of cancer, potential new treatment opportunities are available for the 0.5-5% of patients with NTRK-rearranged glioblastomas. Identification of these rare NTRK-rearranged glioblastomas requires efficient diagnostic tools and strategies which are evaluated in this study. We compared the results of NTRK1, NTRK2 and NTRK3 fluorescent in situ hybridisation (FISH) assays to those of pan-TRK immunohistochemistry (IHC) using two EPR17341 and A7H6R clones in a set of 196 patients with glioblastomas. Cases with at least 15% of positive nuclei using FISH analyses were further analysed using RNA sequencing. Above the 15% threshold, seven positive glioblastomas (3.57%) were identified by FISH assays (4 NTRK1, 3 NTRK2, no NTRK3). NTRK rearrangements were confirmed by RNA sequencing analyses in four cases [1 LMNA-NTRK1, 1 PRKAR2A-NTRK2, 1 SPECC1L-NTRK2 and 1 NACC2-NTRK2 fusions, i.e., 4/196 (2%) of NTRK-rearranged tumours in our series] but no rearrangement was detected in three samples with less than 30% of positive tumour nuclei as determined by NTRK1 FISH. Pan-TRK immunostaining showed major discrepancies when using either the EPR17341 or the A7H6R clones for the following criteria: main intensity, H-Score based scoring and homogeneity/heterogeneity of staining (Kappa values

Introduction Le pronostic du melanome metastatique a beaucoup evolue ces dernieres annees avec le developpement des therapies ciblees et des inhibiteurs de check-points. Nous rapportons une reponse clinique remarquable aux anti-MEK chez une patiente presentant une fusion RAF1. Observations Il s’agit d’une patiente de 65 ans presentant un melanome metastatique avec des localisations secondaires ganglionnaires, sous-cutanees et ileales. Les mutations BRAF, NRAS et KIT etaient negatives et un essai therapeutique ouvert de phase 3 associant un anti-PD-L1 (atezoluzimab 840 mg j1–j15) et un anti-MEK (cobimetinib, 60 mg par jour j1–j21) chez les patients non mutes BRAF etait propose en premiere ligne. A j10 du debut du traitement, une regression quasi complete des nodules sous-cutanes etait constatee. A j15, la patiente etait hospitalisee pour une pneumopathie interstitielle de grade 4 attribuee a l’atezoluzimab d’evolution favorable mais entrainant la sortie de l’essai therapeutique. Elle etait ensuite traitee en 2e et 3e ligne par anti-PD1 et par chimiotherapie avec une progression clinique notable. Devant l’absence d’alternative therapeutique, un screening moleculaire large FoundationOne etait realise. Une fusion de RAF1 etait mise en evidence. Un traitement par anti-MEK (trametinib) a demi-dose etait debute sous surveillance stricte notamment sur le plan pulmonaire. La reponse clinique et scanographique apres deux mois de traitement etait spectaculaire avec une remission partielle (−35 % selon les criteres RECIST) de sa maladie avec un recul de 3 mois. La tolerance du traitement etait excellente sans recidive de la pneumopathie interstitielle. Discussion La reponse clinique rapide des cibles sous-cutanees sous l’association anti-PD-L1 et anti-MEK nous faisait deja suspecter une sensibilite aux anti-MEK. Le programme Experience FoundationOne® (partenariat CHU-Roche®) propose une analyse par sequencage nouvelle generation (NGS) grâce a un panel de 315 genes et de 28 introns, ceci afin d’etablir un profil genomique de la tumeur. Ce large panel peut etre d’un grand interet pour la recherche de nouvelles cibles therapeutiques notamment en cas d’echec des traitements standards. La fusion du gene RAF1 (ou CRAF) est un evenement rare aboutissant a une hyperactivite de la proteine kinase Raf1. Il en resulte une activation constitutionnelle de la voie des MAP-kinases. Sur le plan moleculaire, les fusions impliquant le gene BRAF comme celles impliquant le gene CRAF aboutissent a une activation de la voie des MAP-kinases et constituent donc des cibles therapeutiques. Conclusion Nous decrivons le troisieme cas de fusion du gene RAF1 permettant une efficacite surprenante des anti-MEK sur le melanome metastatique. La voie des MAP-kinases est une cible therapeutique de predilection dans le traitement des melanomes. Le developpement de panels larges de screening moleculaire permet d’augmenter l’arsenal therapeutique et la survie des patients atteints de melanome metastatique.

// Pierre Melard 1, 2, * , Yamina Idrissi 1, * Laetitia Andrique 1, 3 , Sandrine Poglio 1 , Martina Prochazkova-Carlotti 1 , Sabine Berhouet 3 , Cecile Boucher 3 , Elodie Laharanne 1, 3 , Edith Chevret 1 , Anne Pham-Ledard 1, 4 , Andrea Carla De Souza Goes 1, 5 , Veronique Guyonnet-Duperat 6 , Alice Bibeyran 6 , Francois Moreau-Gaudry 6, 7 , Beatrice Vergier 1, 2 , Marie Beylot-Barry 1, 4 , Jean-Philippe Merlio 1, 3 , David Cappellen 1, 3 1 Institut National de la Sante et de la Recherche Medicale (Inserm) U1053, Universitaire de Bordeaux, F-33076 Bordeaux, France 2 Service de Pathologie, Centre Hospitalier Universitaire de Bordeaux, Hopital Haut-Leveque, F-33604 Pessac, France 3 Service de Biologie des Tumeurs-Tumorotheque, Centre Hospitalier Universitaire de Bordeaux, Hopital Haut-Leveque, F-33604 Pessac, France 4 Service de Dermatologie, Centre Hospitalier Universitaire de Bordeaux, Hopital Saint-Andre, F-33000 Bordeaux, France 5 Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro, CEP 20550-013 Rio de Janeiro, Brazil 6 Plateforme de Vectorologie, Unite Mixte de Services (UMS TBM-Core), Centre National de la Recherche Scientifique (CNRS)- Institut National de la Sante et de la Recherche Medicale (Inserm)-Universitaire de Bordeaux, F-33076 Bordeaux, France 7 Biotherapies des Maladies Genetiques et Cancers, Institut National de la Sante et de la Recherche Medicale (Inserm), U1035, Universitaire de Bordeaux, F-33076 Bordeaux, France * These authors have contributed equally to this work Correspondence to: David Cappellen, email: david.cappellen@u-bordeaux.fr Jean-Philippe Merlio, email: jp.merlio@u-bordeaux.fr Keywords: T-cell lymphomas, DUSP22, silencing, mutations, tumor suppressor function Received: April 28, 2016 Accepted: August 31, 2016 Published: September 10, 2016 ABSTRACT Monoallelic 6p25.3 rearrangements associated with DUSP22 (Dual Specificity Phosphatase 22 ) gene silencing have been reported in CD30+ peripheral T-cell lymphomas (PTCL), mostly with anaplastic morphology and of cutaneous origin. However, the mechanism of second allele silencing and the putative tumor suppressor function of DUSP22 have not been investigated so far. Here, we show that the presence, in most individuals, of an inactive paralog hampers genetic and epigenetic evaluation of the DUSP22 gene. Identification of DUSP22 -specific single-nucleotide polymorphisms haplotypes and fluorescence in situ hybridization and epigenetic characterization of the paralog status led us to develop a comprehensive strategy enabling reliable identification of DUSP22 alterations. We showed that one cutaneous anaplastic large T-cell lymphomas (cALCL) case with monoallelic 6p25.3 rearrangement and DUSP22 silencing harbored exon 1 somatic mutations associated with second allele inactivation. Another cALCL case carried an intron 1 somatic splice site mutation with predicted deleterious exon skipping effect. Other tested PTCL cases with 6p25.3 rearrangement exhibited neither mutation nor deletion nor methylation accounting for silencing of the non-rearranged DUSP22 allele, thus inactivated by a so far unknown mechanism. We also characterized the expression status of four DUSP22 splice variants and found that they are all silenced in cALCL cases with 6p25.3 breakpoints. We finally showed that restoring expression of the physiologically predominant isoform in DUSP22-deficient malignant T cells inhibits cellular expansion by stimulating apoptosis and impairs soft agar clonogenicity and tumorigenicity. This study therefore shows that DUSP22 behaves as a tumor suppressor gene in PTCL.

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We report here an exceptional pattern of atypical lentiginous melanocytic proliferation within an adenoma, leading to focal lamina propria infiltration and pulmonary metastasis, which was considered as primary colonic mucosal melanoma (MM) in a Caucasian patient. Such case illustrates the diagnosis criteria required to differentiate primary MM from colonic metastasis of melanoma, including the absence of past history of other primary melanoma, a unique colonic and abdominal lesion with predominant features of in situ lentiginous MM and a very focal and unique invasive area without other digestive tract or abdominal localization. This tumor displayed a KIT exon 11 mutation leading to a unique combination of p.I571M and p.D572G deleterious amino acid changes. Such pattern also favors the diagnosis as KIT appears as a master oncogenic player in MM oncogenesis.

Introduction Le syndrome de Sezary (SS) est un lymphome T cutane caracterise par un clone T dominant circulant et une infiltration cutanee associee a une erythrodermie et un prurit. Les mecanismes controlant la migration entre les compartiments sanguin et cutane sont mal connus, expliquant le manque de traitements efficaces. Des etudes anterieures nous ont amenees a etudier l’expression et la fonction du chimiorecepteur CXCR4 dans le SS. Materiel et methodes Sur une cohorte de 21 patients atteints de SS, nous avons quantifie l’expression de CXCR4 a la surface des cellules de Sezary circulantes par cytometrie multicouleur, et compare ces resultats aux donnees cliniques des patients. Les proprietes migratoires des cellules en reponse au chimioattractant de CXCR4 (SDF-1) ont ete etudiees in vitro, en presence d’un antagoniste de CXCR4 (AMD3100) ou d’un anticorps (Ac) anti-CXCR4. Le role de CXCR4 dans la survie des cellules de Sezary a ete explore par test d’apoptose-necrose apres traitement des cellules par AMD3100 ou Ac anti-CXCR4. Enfin, nous avons construit un shARN inhibant l’expression de CXCR4 afin de transduire des cellules de Sezary de patients et de confirmer son role fonctionnel in vivo apres injection chez la souris. Resultats La majorite des cellules de Sezary des 21 patients exprimaient fortement CXCR4 (pourcentage median de cellules CXCR4+ = 96,9 ± 9,6 %), et ce de facon homogene entre les patients. Le niveau d’expression de CXCR4 etait independant des principaux facteurs pronostiques de la maladie. L’AMD3100 et l’Ac anti-CXCR4 bloquaient significativement la migration des cellules de Sezary en reponse a SDF-1 (taux d’inhibition respectifs = 90,6 ± 5,6 % et 60,8 ± 16,4 %, p Discussion Le role fonctionnel de CXCR4 que nous decrivons sur les cellules de patients n’a pas ete valide avec la lignee HUT78 (SS). Ceci suggere que CXCR4 pourrait avoir des conformations differentes selon les cellules ce qui affecterait sa fonction. La validation de ces resultats dans un modele de xenogreffe de cellules de Sezary modifiees avec un shARN inhibant l’expression de CXCR4 permettra de determiner si son inhibition est une strategie therapeutique impactant la survie et/ou la dissemination des cellules tumorales. Conclusion La forte proportion de cellules CXCR4+ dans le SS et la preuve fonctionnelle de son implication dans la migration cellulaire et la survie des cellules in vitro font de CXCR4 une cible therapeutique potentielle.

Telomere erosion may be counteracted by telomerase. Here we explored telomere length (TL) and telomerase activity (TA) in primary cutaneous T-cell lymphoma (CTCL) by using quantitative polymerase chain reaction and interphase quantitative fluorescence in situ hybridization assays. Samples from patients with Sézary syndrome (SS), transformed mycosis fungoides (T-MF), and cutaneous anaplastic large cell lymphoma were studied in parallel with corresponding cell lines to evaluate the relevance of TL and TA as target candidates for diagnostic and therapeutic purposes. Compared with controls, short telomeres were observed in aggressive CTCL subtypes such as SS and T-MF and were restricted to neoplastic cells in SS. While no genomic alteration of the hTERT (human telomerase catalytic subunit) locus was observed in patients' tumor cells, TA was detected. To understand the role of telomerase in CTCL, we manipulated its expression in CTCL cell lines. Telomerase inhibition rapidly impeded in vitro cell proliferation and led to cell death, while telomerase overexpression stimulated in vitro proliferation and clonogenicity properties and favored tumor development in immunodeficient mice. Our data indicate that, besides maintenance of TL, telomerase exerts additional functions in CTCL. Therefore, targeting these functions might represent an attractive therapeutic strategy, especially in aggressive CTCL.

Neurofibromatosis 1 (NF1) is one of the most common genetic disorders and is caused by mutations in the NF1 gene. NF1 gene mutational analysis presents a considerable challenge because of its large size, existence of highly homologous pseudogenes located throughout the human genome, absence of mutational hotspots, and diversity of mutations types, including deep intronic splicing mutations. We aimed to evaluate the use of hybridization capture-based next-generation sequencing to screen coding and noncoding NF1 regions. Hybridization capture-based next-generation sequencing, with genomic DNA as starting material, was used to sequence the whole NF1 gene (exons and introns) from 11 unrelated individuals and 1 relative, who all had NF1. All of them met the NF1 clinical diagnostic criteria. We showed a mutation detection rate of 91% (10 out of 11). We identified eight recurrent and two novel mutations, which were all confirmed by Sanger methodology. In the Sanger sequencing confirmation, we also included another three relatives with NF1. Splicing alterations accounted for 50% of the mutations. One of them was caused by a deep intronic mutation (c.1260 + 1604A > G). Frameshift truncation and missense mutations corresponded to 30% and 20% of the pathogenic variants, respectively. In conclusion, we show the use of a simple and fast approach to screen, at once, the entire NF1 gene (exons and introns) for different types of pathogenic variations, including the deep intronic splicing mutations.

Introduction L’immunotherapie est une avancee majeure dans le traitement du melanome metastatique mais la recherche de biomarqueurs predictifs de son efficacite est necessaire. Une charge mutationnelle elevee serait un marqueur de bonne reponse aux anti-PD-1. Le programme Experience FoundationOne® (partenariat CHU-Roche®) propose un sequencage de nouvelle generation par NGS avec un panel de 315 genes et de 28 introns, afin de determiner le profil genomique de la tumeur. Nous rapportons l’analyse de la charge mutationnelle (tumor mutational burden [TMB], exprimee en nombre de mutations par megabase d’ADN sequence) et la reponse a l’immunotherapie de patients inclus dans 2 services hospitalo-universitaires. Materiel et methodes Etude prospective de la TMB et de la reponse aux immunotherapies dans 32 cas de melanomes sequences par NGS avec le test FoundationOne® a partir d’un prelevement inclus en paraffine. La TMB etait consideree comme basse entre 1 et 5 mutations par megabase (Muts/Mb), intermediaire entre 6 et 19 et eleve a partir de 20 Muts/Mb. Les cas selectionnes etaient prioritairement ceux non mutes pour les cibles theranostiques habituelles (BRAF, NRAS et KIT), afin d’identifier de nouvelles possibilites therapeutiques. Resultats Les melanomes de 32 patients âges de 24 a 85 ans ont ete inclus dans le programme d’octobre 2017 a mai 2018. Trois sequencages (9 %) etaient non contributifs du fait de la mauvaise qualite de l’ADN extrait. Parmi les 8 patients avec une TMB elevee (28 % de la cohorte analysable), 5 recevaient un traitement par anti-PD-1 avec une reponse partielle ou complete pour 4 (80 %) (TMB [25–60]) et une stabilite pour un (TMB = 20 Muts/Mb). Tous les melanomes choroidiens (n = 3) avaient une TMB basse et n’ont repondu ni aux anti-PD-1 ni a l’anti-CTLA-4. Un patient avec une TMB basse (melanome acrolentigineux ; 2 Muts/Mb) a eu une reponse spectaculaire et immediate a l’ipilimumab apres avoir progresse sous anti-PD-1 et radiotherapie ( Tableau 1 ). Discussion Nos donnees confirment que la TMB est tres variable dans le melanome et sont en accord avec Goodman et al. qui ont analyse retrospectivement la TMB de 1638 patients et montre une association etroite et independante entre reponse a l’immunotherapie et TMB elevee versus intermediaire et basse (58 % vs 20 %, p = 0,0001). Dans notre cohorte un seul patient avec une TMB basse a presente une reponse spectaculaire a l’ipilimumab. Il avait recu prealablement une radiotherapie sur une masse ganglionnaire axillaire qui pourrait avoir favorise de cette reponse. Conclusion L’analyse de la TMB a permis de mettre en evidence des profils differents chez nos patients avec des reponses differentes a l’immunotherapie. Dans le contexte actuel ou le choix therapeutique de la 1re ligne est primordial en particulier chez les patients BRAF sauvage, l’analyse de la TMB pourrait etre un indicateur dans la decision du clinicien, a developper en pratique courante.

MYCN activation, mainly by gene amplification, is one of the most frequent molecular events in neuroblastoma (NB) oncogenesis, and is associated with increased malignancy and decreased neuronal differentiation propensity. The frequency of concomitant loss of heterozygosity at the 1p36.3 locus, which harbours the p53 anti-oncogene homologue TP73, indicates that MYCN and p73 alterations may cooperate in the pathogenesis of NB. We have previously shown that p73 isoforms are deregulated in NB tumours and that TAp73 co-operates synergistically with p53 for apoptosis of NB cells, whereas DeltaNp73 activates the expression of neuronal differentiation genes such as BTG2. Herein, using both ectopic expression and RNA interference-mediated silencing of p73 in MYCN amplified NB cells, we show that p73alpha isoforms inhibit MYCN expression at both transcript and protein levels, in spite of transactivator effects on MYCN promoter. To explain this paradox, we found that TAp73alpha exerts negative post-transcriptional effects leading to reduced MYCN mRNA stability. RNA immunoprecipitation experiments suggest that this dominant inhibitory post-transcriptional effect could be due to an interaction between the p73 protein and MYCN mRNA, a hypothesis also raised for the regulation of certain genes by the p53 protein.

Gastric MALT lymphoma (GML) can be induced by Helicobacter pylori infection in BALB/c mice thymectomised at day 3 post-birth (d3Tx). This represented a unique opportunity to investigate the inflammatory process involved in the recruitment, proliferation and structuration of lymphoid infiltrates in the gastric mucosa of mice developing GML. Complementary molecular and proteomic approaches demonstrated that Th1 and Th2 cytokines were upregulated, along with activators/regulators of the lymphoid response and numerous chemokines. Interleukin-4, interferon γ, lymphotoxin-α and -β were significantly upregulated and correlated with the inflammatory scores for all the d3Tx mice. GML lesions in d3Tx mice infected with H. pylori were associated with the presence of the inflammatory response. The dysregulation of numerous members of the tumour necrosis factor superfamily was also evident and suggests that they could play an important role in GML pathology, especially in light of their ability to promote and control lymphocyte proliferation.

Summary Background Transformed mycosis fungoides (TMF) large cells may express CD30 antigen, and because of this, the differential diagnosis between CD30-rich TMF and primary cutaneous anaplastic large-cell lymphoma (cALCL) may be difficult, and especially in distinguishing cALCL associated with MF vs. CD30-rich TMF. Objectives To find clinical, histological and molecular diagnostic features useful for differential diagnosis between cALCL and CD30-rich TMF. To analyse and compare the prognostic value of clinical and pathological factors in these two diseases. Material and methods We conducted a retrospective study (1999–2012) of 32 patients with cALCL and 34 with CD30-rich TMF, seen in reference centres of the French Study Group of Cutaneous Lymphoma. Clinical, histological and molecular features were analysed and compared to determine their diagnostic and prognostic value. Results Comparison of the two groups showed that age ˃ 60 years, ≥ 5 skin lesions, early progression, absence of spontaneous regression and trunk involvement were significantly associated with the diagnosis of TMF. Abnormal T-cell phenotype and perforin expression were significantly more frequent in cALCL (both P

The developmental and oncogenic roles of MYC proteins are well established, but the transcriptional targets mediating their functions remain elusive. Using small interfering RNA-mediated knockdown in breast and cervix carcinoma cell lines, which overexpress c-MYC, we show that c-MYC independently controls metabolism and cell proliferation, and can, depending on the cells, promote or inhibit migration. We identified new c-MYC target genes in these cell lines, and show that selective regulation of some targets correlates with the phenotypic responses of these different cell lines to c-MYC depletion. Notably, we show that a positive regulation of the WNT signalling pathway contributes to c-MYC pro-mitogenic effects in breast and cervix carcinoma cells. We also show that repression of CCL5/RANTES accounts for c-MYC anti-migratory effects in specific breast cancer cells. Our combined genomic and phenotypic analysis indicates that c-MYC functions are cellular-context-dependent and that selectively regulated genes are responsible for its differential properties.

To examine early events in osteoblast differentiation, we analyzed the expression of about 9,400 genes in the murine MC3T3 cell line, whose robust differentiation was documented cytochemically and molecularly. The cells were stimulated for 1 and 3 days with the osteogenic stimulus containing bone morphogenic protein 2. Total RNA was extracted and analyzed by Affymetrix GeneChip oligonucleotide arrays. A regulated expression of 394 known genes and 295 expressed sequence tags was detected. The sensitivity and reliability of detection by microarrays was shown by confirming the expression pattern for 20 genes by radioactive quantitative reverse transcription-PCR. Functional classification of regulated genes was performed, defining the groups of regulated growth factors, receptors, and transcription factors. The most interesting finding was concomitant activation of transforming growth factor-β, Wnt, and Notch signaling pathways, confirmed by strong up-regulation of their target genes by PCR. The transforming growth factor-β pathway is activated by stimulated production of the growth factor itself, while the exact mechanism of Wnt and Notch activation remains elusive. We showed that bone morphogenic protein 2 stimulated expression of Hey1, a direct Notch target gene, in mouse MC3T3 and C2C12 cells, in human mesenchymal cells, and in mouse calvaria. Small interfering RNA-mediated inhibition of Hey1 induction led to an increase in osteoblast matrix mineralization, suggesting that Hey1 is a negative regulator of osteoblast maturation. This negative regulation is apparently achieved via interaction with Runx2: Hey1 completely abrogated Runx2 transcriptional activity. These findings identify the Notch-Hey1 pathway as a negative regulator of osteoblast differentiation/maturation, which is a completely novel aspect of osteogenesis and could point to possible new targets for bone anabolic agents.

BCL-2/JH rearrangement has been proposed as a biomarker for evaluating the genotoxicity of occupational and environmental exposures. Available data on time-related modification of this rearrangement in peripheral blood lymphocytes in unexposed healthy individuals is scarce. We investigated the characteristics of BCL-2/JH rearrangements in 33 adults unexposed to genotoxins at 2 seasonal time points: winter and spring. BCL-2/JH rearrangement was detected in 79% of individuals (detection limit = 8.48 × 10−8). Its frequency ranged from

L’equipe de A.L. Feldman et la notre avons mis en evidence des rearrangements du locus 6p25.3 dans environ 20 % des lymphomes T anaplasiques, notamment cutanes (LTAc). Combinant des approches de cytogenetique et biologie moleculaire, nous avons, parmi les genes candidats presents dans la region des points de cassures en 6p25.3, identifie un nouveau gene candidat, dual specificity phosphatase 22 (DUSP22), de fonction peu connue jusqu’alors. Nous avons mis en evidence, du fait d’epissages et promoteurs alternatifs, de differents transcrits codant des isoformes proteiques distinctes. Les transcrits majoritaires dans les cellules normales sont reprimes dans les tumeurs presentant des alterations du locus 6p25.3. Des transcrits normalement minoritaires sont, au contraire, occasionnellement surexprimes dans les tumeurs. Nous avons ensuite montre, en modulant leur expression dans des lignees cellulaires au moyen de vecteurs lentiviraux d’expression ou d’ARN interference, que les isoformes inactivees dans les tumeurs ont des proprietes suppressives de tumeurs, inhibant la croissance en agar semi-solide et la tumorigenicite dans les souris immuno-deficientes. A l’oppose, celles qui sont surexprimees dans certaines tumeurs exercent des activites pro-oncogeniques. Nos cas de LTAc avec extinction de DUSP22 presentant des rearrangements mono-alleliques du 6p25.3, nous avons recherche les mecanismes responsables de l’inactivation du second allele du gene. Nous avons alors mis a jour, en accord avec les predictions bioinformatiques de Genovese et al., l’existence d’un paralogue du gene DUSP22, localise en 16p11.2, present chez 90 % des individus, methyle ou delete au niveau de la region promotrice, et transcriptionnellement inactif. L’existence de ce paralogue a un impact majeur sur les analyses cytogenetiques et moleculaires de DUSP22. Nous avons ainsi developpe des strategies permettant une etude objective des alterations genetiques (translocations, deletions, mutations) et epigenetiques (methylation) de ce gene, dont l’application a l’analyse des lymphomes T cutanes sera presentee.

Cytokines macrophage colony stimulating factor (M-CSF) and the receptor activator of NFkappaB ligand (RANKL) induce differentiation of bone marrow hematopoietic precursor cells into bone-resorbing osteoclasts without the requirement for stromal cells of mesenchymal origin. We used this recently described mouse cell system and oligonucleotide microarrays representing about 9,400 different genes to analyze gene expression in hematopoietic cells undergoing differentiation to osteoclasts. The ability of microarrays to detect the genes of interest was validated by showing expression and expected regulation of several osteoclast marker genes. In total 750 known transcripts were up-regulated by > or =2-fold, and 91% of them at an early time in culture, suggesting that almost the whole differentiation program is defined already in pre-osteoclasts. As expected, M-CSF alone induced the receptor for RANKL (RANK), but also, unexpectedly, other RANK/NFkappaB pathway components (TRAF2A, PI3-kinase, MEKK3, RIPK1), providing a molecular explanation for the synergy of M-CSF and RANKL. Furthermore, interleukins, interferons, and their receptors (IL-1alpha, IL-18, IFN-beta, IL-11Ralpha2, IL-6/11R gp130, IFNgammaR) were induced by M-CSF. Although interleukins are thought to regulate osteoclasts via modulation of M-CSF and RANKL expression in stromal cells, we showed that a mix of IL-1, IL-6, and IL-11 directly increased the activity of osteoclasts by 8.5-fold. RANKL induced about 70 novel target genes, including chemokines and growth factors (RANTES (regulated on activation, normal T cell expressed and secreted), PDGFalpha, IGF1), histamine, and alpha1A-adrenergic receptors, and three waves of distinct receptors, transcription factors, and signaling molecules. In conclusion, M-CSF induced genes necessary for a direct response to RANKL and interleukins, while RANKL directed a three-stage differentiation program and induced genes for interaction with osteoblasts and immune and nerve cells. Thus, global gene expression suggests a more dynamic role of osteoclasts in bone physiology than previously anticipated.

The p15 gene which encodes a cyclin-dependent kinase inhibitor, is located in the 9p21 chromosomal region that is frequently deleted in human bladder transitional cell carcinomas (TCCs). The aim of the present paper is to study the potential involvement of the p15 gene in the evolution of TCCs. p15 mRNA expression was investigated by semi-quantitative RT-PCR in a series of 75 TCCs, 13 bladder cell lines and 6 normal bladder urothelia by semi-quantitative RT-PCR. p15 was expressed in the normal urothelium but p15 mRNA levels were significantly decreased in 66% of the superficial (Ta-T1) TCCs (P = 0.0015). In contrast, in muscle-invasive (T2-T4) TCCs, p15 expression differed widely between samples. p16 mRNA levels were also studied and there was no correlation between p15 and p16 mRNA levels, thus indicating that the two genes were regulated independently. Lower p15 expression in superficial tumours did not reflect a switch from quiescence to proliferative activity as normal proliferative urothelial controls did not present decreased p15 mRNA levels relative to quiescent normal urothelia. We further investigated the mechanisms underlying p15 down regulation. Homozygous deletions of the p15 gene, also involving the contiguous p16 gene, were observed in 42% of the TCCs with decreased p15 expression. No hypermethylation at multiple methylation-sensitive restriction sites in the 5′-CpG island of p15 was encountered in the remaining tumours. Our data suggest that decreased expression of p15 may be an important step in early neoplastic transformation of the urothelium and that a mechanism other than homozygous deletions or hypermethylation, may be involved in p15 down regulation. © 2001 Cancer Research Campaign http://www.bjcancer.com

Loss of heterozygosity (LOH) on chromosome 10 has been observed in several human cancers including glioblastomas, meningiomas, melanomas and endometrial and prostate carcinomas. We have investigated the incidence of LOH on chromosome 10 in 36 human transitional cell carcinomas (TCCs) of the bladder, three upper urinary tract TCCs and one lymph node metastasis, using a panel of 27 highly polymorphic markers spanning 10p (short arm) and 10q (long arm). Fourteen bladder tumours (39%), the three upper urinary tract tumours and the lymph node metastasis showed LOH for at least one locus on chromosome 10. Remarkably, LOH on chromosome 10 was observed mainly in muscle-invasive (P = 0.01) and high grade tumours (P = 0.03). For five tumours and the lymph node metastasis, LOH was found at all informative loci, indicating monosomy or isodisomy of chromosome 10. The deletion mapping of the tumours with partial loss delineated two minimal regions of loss on chromosome 10q. One region, the most telomeric, was bounded by markers D10S214 and D10S169 and the other, the most proximal, was bounded by markers D10S222 and D10S531. Our results demonstrate that chromosome 10q LOH is common in muscle-invasive bladder cancers and that two potential tumour suppressor loci, at 10q24.1-q24.3 and 10q26.1-q26.2, may contribute to the malignant progression of these tumours. Localization of the smallest common regions of loss in bladder tumours provides a starting point for the identification of the genes involved.

An experimental model of advanced human neuroblastoma, IGR-N-91, which is able to disseminate in the nude mouse, has been described. The present study was designed to ascertain which cell population from the IGR-N-91 primary tumour actually disseminates throughout the animals. In s.c. IGR-N-91 tumour xenografts, 3 areas, called pearly, vascularized and haemorrhagic, depending on the presence of blood vessels and haemorrhagic suffusions, were consistently observed and independently resected. Molecular analysis of tumour materials revealed a significant increase inMYCNandmaxgene transcript levels in the haemorrhagic area, as compared with the pearly and vascularized areas. Given the growth kinetics observed bothin vitroandin vivo,and the DNA flow-cytometry profiles of tumour cells obtained from the haemorrhagic area, this transcriptional increase did not appear to be associated with enhanced proliferation. In this area of the tumours, multidrug-resistance-related genes,i.e., MDRI, MRP, GST-πandtopoisomerase IIαwere activated concomitantly withMYCNandmaxgenes. The same observations were made, except for thetopoisomerase-IIαgene, when sub-lines derived from metastases were compared with that derived from the primary tumour. These data demonstrate that over-expression of several genes determining the multi-drug-resistance phenotype precedes the metastatic spread of IGR-N-91 NB tumour cells in the nude mouse. Data also suggest that the cell sub-population exhibiting this pleiotropic over-expression within the primary tumour undergoes selection during metastatic dissemination.Int. J. Cancer,70:000–000. © 1997 Wiley-Liss, Inc.

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

WNT5A, a member of the WNT family of secreted lipid-modified glycoproteins, is a critical regulator of a host of developmental processes, including limb formation, lung morphogenesis, intestinal elongation and mammary gland development. Altered WNT5A expression has been associated with a number of cancers. Interestingly, in certain types of cancers, such as hematological malignancies and colorectal carcinoma, WNT5A is inactivated and exerts a tumor suppressive function, while in other cancers, such as melanoma and gastric carcinoma, WNT5A is overexpressed and promotes tumor progression. The mechanism by which WNT5A achieves these distinct activities in cancers is poorly understood. Here, we provide evidence that the WNT5A gene produces two protein isoforms, WNT5A-long (WNT5A-L) and WNT5A-short (WNT5A-S). Amino-terminal sequencing and a WNT5A-L specific antibody demonstrate that the mature and secreted isoforms are distinct, with WNT5A-L carrying an additional 18 N-terminal amino acids. Biochemical analysis indicates that both purified proteins are similar with respect to their stability, hydrophobicity and WNT/β-catenin signaling activity. Nonetheless, modulation of these two WNT5A isoforms, either through ectopic expression or knockdown, demonstrates that they exert distinct activities in cancer cell lines: while WNT5A-L inhibits proliferation of tumor cell lines, WNT5A-S promotes their growth. Finally, we show that expression of these two WNT5A isoforms is altered in breast and cervix carcinomas, as well as in the most aggressive neuroblastoma tumors. In these cancers, WNT5A-L is frequently down-regulated, whereas WNT5A-S is found overexpressed in a significant fraction of tumors. Altogether, our study provides evidence that the distinct activities of WNT5A in cancer can be attributed to the production of two WNT5A isoforms.

Primary cutaneous large B-cell lymphoma, leg type has been individualized from nodal diffuse large B-cell lymphoma. The objective of this study was to screen primary cutaneous large B-cell lymphoma, leg type for genetic alterations recently described in nodal diffuse large B-cell lymphoma. Skin biopsies from 23 patients were analyzed for IRF4, BCL2, BCL6, and MYC expression. FISH testing was performed for BCL2, BCL6, MYC with separation probes and for CDKN2A and PRDM1/BLIMP1 deletion. Multiple sequential FISH analyses with up to six probes were performed to define samples with multiple cytogenetic alterations. MYD88 mutations were studied by Sanger sequencing. All cases but one displayed at least one genetic alteration (96%). Nine patients exhibited a single genetic mutation and 12 combined several alterations (52%). We observed a split for BCL2, BCL6, or MYC in 1/23, 6/23, and 3/23 of cases, respectively. No double-hit lymphoma was observed. CDKN2A deletion was detected by FISH in only 5/23 cases. BLIMP1 and/or 6q deletion was observed at a higher rate in 10/20 of cases. No correlation between rearrangement and immunohistochemical expression was found for BCL2 or MYC. FISH tracking of sequential hybridizations showed that several alterations were carried by the same nuclei. The p.L265P MYD88 mutation was found in 11/18 (61%) of cases. Contrary to most cutaneous lymphomas that rarely harbor primary genetic alteration of their nodal histological equivalent, primary cutaneous large B-cell lymphoma, leg type seems to be a 'cutaneous counterpart' of activated B-cell-like diffuse large B-cell lymphoma with a similar cytogenetic profile and a high rate of MYD88 oncogenic L265P mutation. This also suggests a common lymphomagenesis with NF-κB activation, strong IRF4 expression and terminal B-cell differentiation blockage. Our data support the use of therapies targeting NF-κB, as most patients displayed disease progression and resistance to conventional therapies.

Objectives Recent studies have shown that telomere length was significantly reduced in placentas collected at delivery from pregnancies complicated by intrauterine growth restriction secondary to placental insufficiency. Placental telomere length measurement during ongoing pregnancies complicated by intrauterine growth restriction has never been reported. This was the main objective of our study. Methods In our center, late chorionic villus samplings were performed between 18 and 37 weeks of amenorrhea in 24 subjects with severe intrauterine growth restriction (cases) and in 28 subjects with other indications for prenatal diagnosis (controls). Placental insufficiency was assessed by histo-pathological examination. Relative measurement of telomere length was carried out prospectively by quantitative Fluorescent In Situ Hybridization using fluorescent Peptide Nucleic Acid probes on interphase nuclei obtained from long-term cultured villi and with an automated epifluorescent microscope. A quantitative Polymerase Chain Reaction technique was performed to confirm the quantitative Fluorescent In Situ Hybridization results. The number of copies of gene loci encoding the RNA template (hTERC) and the catalytic subunit (hTERT) of the enzyme complex telomerase were also estimated in these placentas by Fluorescent In Situ Hybridization. Results Mean fluorescence intensity of telomere probes estimated by quantitative Fluorescent In Situ Hybridization was significantly less for cases compared to controls (p

Abbreviations: ABC, activated B cell; DLBCL, diffuse large B-cell lymphoma; GC, germinal center; PCBCL, primary cutaneous B-cell lymphoma; PCFCL, primary cutaneous follicle center lymphoma; PCDLBCL-LT, primary cutaneous diffuse large B-cell lymphoma, leg type; PCMZL, primary cutaneous marginal zone lymphoma

International audience; Macrosomia, obesity, macrocephaly, and ocular abnormalities syndrome (MOMO syndrome) has been reported in only four patients to date. In these sporadic cases, no chromosomal or molecular abnormality has been identified thus far. Here, we report on the clinical, cytogenetic, and molecular findings in a child of healthy consanguineous parents suffering from MOMO syndrome. Conventional karyotyping revealed an inherited homozygous balanced reciprocal translocation (16;20)(q21;p11.2). Uniparental disomy testing showed bi-parental inheritance for both derivative chromosomes 16 and 20. The patient's oligonucleotide array-comparative genomic hybridization profile revealed no abnormality. From the homozygous balanced reciprocal translocation (16;20)(q21;p11.2), a positional cloning strategy, designed to narrow 16q21 and 20p11.2 breakpoints, revealed the disruption of a novel gene located at 20p11.23. This gene is now named LINC00237, according to the HUGO (Human Genome Organization) nomenclature. The gene apparently leads to the production of a non-coding RNA. We established that LINC00237 was expressed in lymphocytes of control individuals while normal transcripts were absent in lymphocytes of our MOMO patient. LINC00237 was not ubiquitously expressed in control tissues, but it was notably highly expressed in the brain. Our results suggested autosomal recessive inheritance of MOMO syndrome. LINC00237 could play a role in the pathogenesis of this syndrome and could provide new insights into hyperphagia-related obesity and intellectual disability.

Dans 70 a 90 % des cellules tumorales humaines, une activite telomerase est detectee alors que dans les tissus avoisinants non tumoraux, l’activite de la telomerase est faible voire nulle. La telomerase et la longueur des telomeres ont ete proposees comme des biomarqueurs des cellules tumorales. Ces biomarqueurs ont ete etudiee dans les tumeurs solides [1] et hematologiques [2] . En revanche dans les lymphomes T cutanes primitifs (LTCP) les donnees sont rares. Dans une premiere etude [3] , nous avons evalue l’impact de la longueur des telomeres et de l’activite de la telomerase dans la lymphomagenese des LTCP. Nous avons montre que les LTCP agressifs possedaient des telomeres courts par rapport aux LTCP indolents et aux cellules saines, que dans les cellules de LTCP une activite telomerase etait presente, faisant de ces tumeurs des candidates potentielles a un traitement cible anti-telomerase. Pour mieux comprendre la fonction de la telomerase dans ces cellules, nous avons module l’expression de la telomerase, via sa sous-unite catalytique hTERT, a l’aide de constructions lentivirales dans des lignees cellulaires de LTCP. Nous avons montre que l’inhibition de la telomerase se traduisait par une diminution brutale de la proliferation cellulaire et conduisait a la mort cellulaire. En revanche, la surexpression de la telomerase est associee a une augmentation de la proliferation cellulaire, a une augmentation des capacites clonogene et tumorigene des cellules infectees aussi bien in vitro (test de soft agar) que in vivo (xenogreffe chez la souris). Ces dernieres observations laissent supposer qu’au-dela de son role de maintenance des telomeres dans les cellules de LTCP, la telomerase exerce d’autres fonctions, qualifiees de non-canoniques, car independantes de ses fonctions sur les telomeres. La connaissance de ses fonctions est indispensable avant d’envisager d’utiliser la telomerase comme cible therapeutique dans les LTCP.

P53 is a tumor suppressor protein critical for genome integrity. Although its control at the protein level is well known, the transcriptional regulation of the TP53 gene is still unclear. We have analyzed the organization of the TP53 gene domain using DNA arrays in several breast cancer and control cell lines. We have found that in the control breast epithelial cell line, HB2, the TP53 gene is positioned within a relatively small DNA domain, encompassing 50 kb, delimited by two nuclear matrix attachment sites. Interestingly, this domain structure was found to be radically different in the studied breast cancer cell lines, MCF7, T47D, MDA-MB-231, and BT474, in which the domain size was increased and TP53 transcription was decreased. We propose a model in which the organization of the TP53 gene domain correlates with the transcriptional status of TP53 and neighboring genes. J. Cell. Biochem. 112: 2072–2081, 2011. © 2011 Wiley-Liss, Inc.

International audience; Neuroblastoma (NB), the most frequent solid tumor of early childhood, is diagnosed as a disseminated disease in >60% of cases, and several lines of evidence support the resistance to apoptosis as a prerequisite for NB progression. We show that autocrine production of netrin-1, a multifunctional laminin-related molecule, conveys a selective advantage in tumor growth and dissemination in aggressive NB, as it blocks the proapoptotic activity of the UNC5H netrin-1 dependence receptors. We show that such netrin-1 up-regulation is a potential marker for poor prognosis in stage 4S and, more generally, in NB stage 4 diagnosed infants. Moreover, we propose that interference with the netrin-1 autocrine loop in malignant neuroblasts could represent an alternative therapeutic strategy, as disruption of this loop triggers in vitro NB cell death and inhibits NB metastasis in avian and mouse models.

Introduction La mutation activatrice du gene MYD88 L265P est un evenement frequent dans le lymphome cutane B diffus a grandes cellules de type jambe (LCBDGC-TJ), rapporte jusque dans 69 % des cas. Les particularites cliniques et evolutives associees a cette mutation n’ont pas ete etudiees notamment sa valeur pronostique, ce qui fait l’objet de ce travail. Patients et methodes Soixante et un patients de la cohorte SURVIELEG avec du materiel biologique exploitable pour l’analyse moleculaire ont ete identifies, provenant de 19 centres francais du Groupe francais d’Etude des lymphomes cutanes, diagnostiques entre 1988 et 2010. La recherche de la mutation MYD88 L265P etait effectuee sur l’ADN tumoral extrait a partir des biopsies cutanees par une technique de PCR en temps reel, avec detection allele specifique par sondes type TaqMan®. Les donnees cliniques au diagnostic, le traitement realise et l’evolution etaient recueillies. Les caracteristiques des patients au diagnostic et l’evolution etaient comparees selon le statut du gene MYD88. L’analyse statistique multivariee etait ajustee sur l’âge. Resultats Les resultats moleculaires etaient interpretables pour 58 patients (95 %). Il s’agissait de 34 femmes et 24 hommes, d’âge median 79 ans. Il existait une localisation a la jambe dans 44 (76 %) des cas. Vingt (34 %) etait classes T1, 26 (45 %) T2 et 12 (21 %) T3. Cinquante-neuf pour cent avaient recu un traitement comprenant du rituximab associe a une polychimiotherapie. La mutation MYD88 L265P etait presente dans 34 cas (59 %), plus frequemment chez des sujets âges (p = 0,006) et etait associee a une atteinte du membre inferieur (p = 0,008). Il n’y avait pas de difference significative en terme de traitement recu entre les 2 groupes. La presence de la mutation MYD88 etait associee a une survie specifique plus courte en analyse univariee (p = 0,03). L’analyse multivariee ajustee sur l’âge identifiait la mutation MYD88 (p = 0,04) et l’existence de tumeurs multiples (p = 0,03) comme facteurs pronostiques independants. Discussion Cette etude confirme la forte prevalence de la mutation MYD88 L265P au sein de ce lymphome, et montre son association avec des facteurs cliniques lies a cette maladie que sont la localisation a la jambe et un âge avance. De plus, cette etude identifie pour la premiere fois cette mutation comme un facteur pronostique pejoratif. Conclusion L’impact clinique demontre ici de la mutation MYD88, activatrice de la voie NF-kB, pousse a explorer la biologie de ces lymphomes en vue de proposer des therapies ciblees et personnalisees en cas de rechute ou de resistance apres un traitement conventionnel.

Importance The activating mutation of MYD88 L265P is a frequent feature of primary cutaneous diffuse large B-cell lymphoma, leg-type (PCLBCL-LT), reported in up to 69% of the cases. Whether patients with MYD88 mutation display specific clinical and evolutive features has not been evaluated. Objective To identify the clinical characteristics associated with MYD88 mutation, confirm its high prevalence, and evaluate its effect on prognosis in patients with PCLBCL-LT. Design, Setting, and Participants A retrospective multicenter study was conducted using the medical records of patients from dermatology departments belonging to the French Study Group for Cutaneous Lymphomas. Sixty-one patients with a diagnosis of PCLBCL-LT made between 1988 and 2010 who were available for molecular study were included. Of these, 58 patients displaying interpretable results constituted the study group. Median follow-up was 33 months, and 39 patients (67%) were monitored until death. Main Outcomes and Measures Clinical features (age, sex, number of skin lesions, tumor stage, and location as leg vs elsewhere), MYD88 mutation (allele-specific TaqMan polymerase chain reaction assay), treatment regimen, and outcome were recorded. Baseline characteristics and outcome were compared according to the status of MYD88. Results The median age of the patients was 79 years, and 59% were female. Skin lesions were located on the leg in 76% of the cases. Thirty-four of 58 patients (59%) harbored the MYD88 L265P mutation. Patients had similar clinical characteristics at presentation regardless of their MYD88 status, except that those harboring the MYD88 mutation were older (P = .006) and had more frequent involvement of the leg (P = .008). Patients harboring the MYD88 mutation had 3- and 5-year–specific survival rates of 65.7% and 60.2% vs 85.4% and 71.7% in patients with the wild-type allele. The MYD88 mutation was significantly associated with shorter disease-specific survival in univariate (P = .03) and multivariate (odds ratio, 3.01; 95% CI, 1.03-8.78; P = .04) analysis. There was no significant difference between the groups in their treatment regimens. Considering overall survival, in univariate (P = .002) and multivariate (odds ratio, 2.94; 95% CI, 1.18-7.30; P = .02) analysis, MYD88 L265P mutation was an independent adverse prognostic factor. Conclusions and Relevance This study confirms the high prevalence of MYD88 L265P mutation in PCLBCL-LT and shows its association with shorter survival. The clinical effect of MYD88 mutation activating the nuclear factor-κB pathway supports the use of targeted therapies at the time of relapse after conventional therapies.