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International audience; Aims: Recently, YAP1 fusion genes have been demonstrated in eccrine poroma and porocarcinoma, and the diagnostic use of YAP1 immunohistochemistry has been highlighted in this setting. In other organs, loss of YAP1 expression can reflect YAP1 rearrangement or transcriptional repression, notably through RB1 inactivation. In this context, our objective was to re-evaluate the performance of YAP1 immunohistochemistry for the diagnosis of poroma and porocarcinoma.Methods and results: The expression of the C-terminal part of the YAP1 protein was evaluated by immunohistochemistry in 543 cutaneous epithelial tumours, including 27 poromas, 14 porocarcinomas and 502 other cutaneous tumours. Tumours that showed a lack of expression of YAP1 were further investigated for Rb by immunohistochemistry and for fusion transcripts by real-time PCR (YAP1::MAML2 and YAP1::NUTM1). The absence of YAP1 expression was observed in 24 cases of poroma (89%), 10 porocarcinoma (72%), 162 Merkel cell carcinoma (98%), 14 squamous cell carcinoma (SCC) (15%), one trichoblastoma and one sebaceoma. Fusions of YAP1 were detected in only 16 cases of poroma (n = 66%), 10 porocarcinoma (71%) all lacking YAP1 expression, and in one sebaceoma. The loss of Rb expression was detected in all cases except one of YAP1-deficient SCC (n = 14), such tumours showing significant morphological overlap with porocarcinoma. In-vitro experiments in HaCat cells showed that RB1 knockdown resulted in repression of YAP1 protein expression.Conclusion: In addition to gene fusion, we report that transcriptional repression of YAP1 can be observed in skin tumours with RB1 inactivation, including MCC and a subset of SCC.

This file contains supplementary tables S1- S5. Table S1: siRNA and TaqMan probes; Table S2: Primers used for LATS2 gene sequencing; Table S3: Differential phosphorylation level between C2LN MPM subgroup and other MPM; Table S4: LATS2 mutations in MPM; Table S5: Differential gene expression between C2LN MPM subgroup and other MPM.

This file contains the new supplementary Material and Methods.

This file contains the legends for supplementary Figures S1-S7.

This file contains supplementary Figures S1-S7. Figure S1: YAP1 and WWTR1 gene expression; Figure S2: LATS2 and NF2 siRNA knockdown efficiency; Figure S3: Effect of LATS2/NF2 co-inactivation on MPM cell proliferation, migration and invasion; Figure S4: Analysis of Hippo signaling pathway activity and mTOR expression and phosphorylation in MPM; Figure S5: Activity of the Hippo signaling pathway in the C2LN MPM subgroup; Figure S6: Effect of mTOR inhibitors on MPM cell viability; Figure S7: Validation of a C2LN MPM subgroup biomarker.

Purpose: To better define malignant pleural mesothelioma (MPM) heterogeneity and identify molecular subtypes of MPM, we focus on the tumor suppressor gene LATS2, a member of the Hippo signaling pathway, which plays a key role in mesothelial carcinogenesis.Experimental Design: Sixty-one MPM primary cultures established in our laboratory were screened for mutations in LATS2. Gene inactivation was modeled using siRNAs. Gene and protein expressions were analyzed by quantitative RT-PCR, Western blot analysis, and reverse phase protein array. Cell proliferation, viability, apoptosis, mobility, and invasion were determined after siRNA knockdown or YAP (verteporfin), mTOR (rapamycin), and mTOR/PI3K/AKT (PF-04691502) inhibitor treatment.Results: The LATS2 gene was altered in 11% of MPM by point mutations and large exon deletions. Genetic data coupled with transcriptomic data allowed the identification of a new MPM molecular subgroup, C2LN, characterized by a co-occurring mutation in the LATS2 and NF2 genes in the same MPM. MPM patients of this subgroup presented a poor prognosis. Coinactivation of LATS2 and NF2 leads to loss of cell contact inhibition between MPM cells. Hippo signaling pathway activity, mTOR expression, and phosphorylation were altered in the C2LN MPM subgroup. MPMs of this new subgroup show higher sensitivity to PF-04691502 inhibitor. The MOK gene was identified as a potential biomarker of the C2LN MPM subgroup and PF-04691502 sensitivity.Conclusions: We identified a new MPM molecular subgroup that shares common genetic and transcriptomic characteristics. Our results made it possible to highlight a greater sensitivity to an anticancer compound for this MPM subgroup and to identify a specific potential biomarker. Clin Cancer Res; 23(12); 3191–202. ©2016 AACR.

MCC (Merkel cell carcinoma) is an aggressive neuroendocrine cutaneous neoplasm. Integration of the Merkel cell polyomavirus (MCPyV) is observed in about 80% of the cases, while the remaining 20% are related to UV exposure. Both MCPyV-positive and -negative MCCs-albeit by different mechanisms-are associated with RB1 inactivation leading to overexpression of SOX2, a major contributor to MCC biology. Moreover, although controversial, loss of RB1 expression seems to be restricted to MCPyV-negative cases.The aim of the present study was to assess the performances of RB1 loss and SOX2 expression detected by immunohistochemistry to determine MCPyV status and to diagnose MCC, respectively.Overall, 196 MCC tumors, 233 non-neuroendocrine skin neoplasms and 70 extra-cutaneous neuroendocrine carcinomas (NEC) were included. SOX2 and RB1 expressions were assessed by immunohistochemistry in a tissue micro-array. Diagnostic performances were determined using the likelihood ratio (LHR).RB1 expression loss was evidenced in 27% of the MCC cases, 12% of non-neuroendocrine skin tumors and 63% of extra-cutaneous NEC. Importantly, among MCC cases, RB1 loss was detected in all MCPyV(-) MCCs, while MCPyV( +) cases were consistently RB1-positive (p 0.001). SOX2 diffuse expression was observed in 92% of the MCC cases and almost never observed in non-neuroendocrine skin epithelial neoplasms (2%, p 0.0001, LHR + = 59). Furthermore, SOX2 diffuse staining was more frequently observed in MCCs than in extra-cutaneous NECs (30%, p 0.001, LHR + = 3.1).These results confirm RB1 as a robust predictor of MCC viral status and further suggest SOX2 to be a relevant diagnostic marker of MCC.

Diagnosis of osteocartilaginous pathologies depends on morphological examination and immunohistochemical and molecular biology analyses. Decalcification is required before tissue processing, but available protocols often lead to altered proteins and nucleic acids, and thus compromise the diagnosis. The objective of this study was to compare the effect of different methods of decalcification on histomolecular analyses required for diagnosis and to recommend an optimal protocol for processing these samples in routine practice. We prospectively submitted 35 tissue samples to different decalcification procedures with hydrochloric acid, formic acid, and EDTA, in short, overnight and long cycles for 1 to >10 cycles. Preservation of protein integrity was examined by immunohistochemistry, and quality of nucleic acids was estimated after extraction (DNA and RNA concentrations, 260/280 ratios, PCR cycle thresholds), analysis of DNA mutations (high-resolution melting) or amplifications (PCR, in situ hybridization), and detection of fusion transcripts (RT-PCR, in situ hybridization). Hydrochloric acid- and long-term formic acid-based decalcification induced false-negative results on immunohistochemistry and molecular analysis. EDTA and short-term formic acid-based decalcification (

Breast cancer remains the first cause of death in women worldwide even if the average 10-year survival rate is 83%. It remains difficult to accurately evaluate the risk of recurrence, and consequently the need of chemotherapy, in estrogen receptor (ER)-positive, HER2-negative early breast cancers, at the individual level, leading to overtreatment for some patients. In that context, multigene prognostic scores are proposed in combination with clinicopathological parameters, to guide adjuvant treatment decisions. In our study, patients diagnosed with ER-positive, Her2-negative breast cancer, analyzed with the 12-gene EndoPredictTM test are prospectively included in the national SiMosein registry. The decision for Endopredict testing is determined by an oncological tumor board in expert breast cancer centers. The test is locally performed (10 national laboratories, evaluated by a national quality control exchange program). Clinicopathological parameters are recorded and the treatment decision before and after the genomic test is requested. The aim of the study is to prospectively evaluate the real-life use of the EndopredictTM test and the impact of the recurrence risk on the adjuvant treatment decision. A total of 3153 patients were included between May 2016 and December 2018. Patients are 75.6% post-menopausal (median age: 57.55y) and are mostly diagnosed as invasive ductal carcinoma not otherwise specified (81.2%) with SBR grade 2 (74.02%) and positive Ki67 staining (>14%, 66.4%). Tumor sizes are pT1ab, pT1c or pT2 in respectively 12.1%, 54.7% and 31.4% of the cases. Lymph node involvement is negative (pN0) in 70.5% of the cases. However, 29.5% of cases are pN1-3 with 5.2% of pN1mi. All cases are ER-positive, HER2-negative early breast cancers and 74.8% are progesterone receptor (PR)-positive (PR>10%) and 76.43% of the cases are of luminal B-like subtype. The population evaluated in this prospective study is in accordance to the intermediate prognostic subpopulation of luminal breast cancers where genomic signatures could contribute to guide the adjuvant treatment decision. The risk of relapse at 10 years, as reflected by the EPclin score, is of 13.16% on average, with 55.18% of high risk (positive threshold 10%). Treatment decisions before and after the test were recorded for 599 patients. The decision remains unmodified after the test in 54.59% of the cases. Conversely, change in adjuvant treatment decision was made from hormone therapy (HT) to adjunction of chemotherapy (HT+CT) in 18.20% of the cases and from (HT+CT) to (HT) leading to chemotherapy withdrawn in 27.21%. Finally, the national SiMosein registry, evaluated the utility of the test. The use of EndopredictTM is mostly restricted to the intermediate prognostic subgroup of the early luminal breast subtype as requested by the French health authority (HAS). The test is routinely used in France and clinicians need mainly the test for treatment decision. The EPclin score leads to 45.41% of modification of treatment with a majority of avoidance of chemotherapy (59.93%). Citation Format: Jacqueline Lehmann-Che, Jennifer Wong, Lucie Tixier, Ludovic Lacroix, Magali Triki-Lacroix, Pierre-Jean Lamy, Agnes Garnier, Véronique Quillien, Florence Godey, Isabelle Soubeyran, Gaëtan MacGrogan, Laurent Arnould, Juliette Haudebourg, Anne Tallet, Linda Dainese, Caroline Hugonin, Frédérique Penault-Llorca. Simosein registry, the French national real-life prospective evaluation of the impact of Endopredict® (EPclin) on treatment decision in ER-positive, HER2-negative breast cancers [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-08-25.

International audience

International audience; Chronic stimulation by infectious pathogens or self-antigen glucosylsphingosine (GlcSph) can lead to monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM). Novel assays such as the multiplex infectious antigen microarray (MIAA) and GlcSph assays, permit identification of targets for >60% purified monoclonal immunoglobulins (Igs). Searching for additional targets, we selected 28 purified monoclonal Igs whose antigen was not represented on the MIAA and GlcSph assays; their specificity of recognition was then analyzed using microarrays consisting of 3760 B-cell epitopes from 196 pathogens. The peptide sequences PALTAVETG and PALTAAETG of the VP1 coat proteins of human poliovirus 1/3 and coxsackievirus B1/B3, respectively, were specifically recognized by 6/28 monoclonal Igs. Re-analysis of patient cohorts showed that purified monoclonal Igs from 10/155 MGUS/SM (6.5%) and 3/147 MM (2.0%) bound to the PALTAVETG or PALTAAETG epitopes. Altogether, PALTAV/AETG-initiated MGUS are not rare and few seem to evolve toward myeloma.

International audience; While virus-negative Merkel cell carcinoma (MCC) is characterized by high frequency of UV-induced mutations, expression of two viral oncoproteins is regarded as key mechanism driving Merkel cell polyomavirus (MCPyV)-positive MCC. The cells in which these molecular events initiate MCC oncogenesis have yet not been identified for both MCC subsets. A considerable proportion of virus-negative MCC is found in association with squamous cell carcinoma (SCC) suggesting (i) coincidental collision, (ii) one providing a niche for the other or (iii) one evolving from the other. Whole exome sequencing of four combined tumors consisting of SCC in situ and MCPyV-negative MCC demonstrated in all cases many mutations shared between SCC and MCC indicating a common ancestry and thereby a keratinocytic origin of these MCCs. Moreover, analyses of the combined cases as well as of pure SCC and MCC suggests that RB1 inactivation in SCC facilitates MCC development and that epigenetic changes may contribute to the SCC/MCC transition.

Nodular fasciitis, primary aneurysmal bone cyst, myositis ossificans, and their related lesions are benign tumors that share common histological features and a chromosomal rearrangement involving the ubiquitin-specific peptidase 6 (USP6) gene. The identification of an increasing number of new partners implicated in USP6 rearrangements demonstrates a complex tumorogenesis of this tumor spectrum. In this study on a series of 77 tumors (28 nodular fasciitis, 42 aneurysmal bone cysts, and 7 myositis ossificans) from the database of the French Sarcoma Group, we describe 7 new partners of the USP6 gene. For this purpose, rearrangements were first researched by multiplexed RT-qPCRs in the entire population. A targeted RNA sequencing was then used on samples selected according to a high USP6-transcription level expression estimated by RT-qPCR. Thanks to this multistep approach, besides the common USP6 fusions observed, we detected novel USP6 partners: PDLIM7 and MYL12A in nodular fasciitis and TPM4, DDX17, GTF2I, KLF3, and MEF2A in aneurysmal bone cysts. In order to try to bring to light the role played by the recently identified USP6 partners in this lesional spectrum, their functions are discussed. Taking into account that a traumatic participation has long been mentioned in the histogenesis of most of these lesions and because of their morphological resemblance to organizing granulation reparative tissue or callus, a focus is placed on their relationship with tissue remodeling and, to a lesser extent, with bone metabolism.

International audience; Merkel cell carcinoma (MCC), an aggressive neuroendocrine carcinoma of the skin, is to date the only human cancer known to be frequently caused by a polyomavirus. However, it is a matter of debate which cells are targeted by the Merkel cell polyomavirus (MCPyV) to give rise to the phenotypically multifaceted MCC cells. To assess the lineage of origin of MCPyV-positive MCC, genetic analysis of a very rare tumor combining benign trichoblastoma and MCPyV-positive MCC was conducted by massive parallel sequencing. Although MCPyV was found to be integrated only in the MCC part, six somatic mutations were shared by both tumor components. The mutational overlap between the trichoblastoma and MCPyV-positive MCC parts of the combined tumor implies that MCPyV integration occurred in an epithelial tumor cell before MCC development. Therefore, our report demonstrates that MCPyV-positive MCC can derive from the epithelial lineage.

Theranostic assays are based on single-gene testing, but the ability of next-generation sequencing (NGS) to interrogate numerous genetic alterations will progressively replace single-gene assays. Although NGS was evaluated to screen for theranostic mutations, its usefulness in clinical practice on large series of samples remains to be demonstrated. NGS performance was assessed following guidelines. TaqMan probes and NGS were compared for their ability to detect EGFR and KRAS mutations, and NGS mutation profiles were analyzed on a large series of non–small-cell lung cancers (n = 1343). The R2 correlation between expected and measured allelic ratio, using commercial samples, was >0.96. Mutation detection threshold was 2% for 10 ng of DNA input. κ Scores for TaqMan versus NGS were 0.99 (95% CI, 0.97–1.00) for EGFR and 0.98 (95% CI, 0.97–1.00) for KRAS after exclusion of rare EGFR (n = 40) and KRAS (n = 60) mutations. NGS identified 693 and 292 mutations in validated and potential oncogenic drivers, respectively. Significant associations were found between EGFR and PI3KCA or CTNNB1 and between KRAS and STK11. Potential oncogenic driver mutations or gene amplifications were more frequent in validated oncogenic driver nonmutated samples. This work is a proof of concept that targeted NGS is accessible in routine screening, including large screening, at reasonable cost. Clinical data should be collected and implemented in specific databases to make molecular data meaningful for direct patients' benefit.

Background: Unicentric mediastinal Castleman disease (CD) is a rare condition, poorly characterized due to the small number of cases and the absence of genomic study. We analyzed clinical, radiological, histological and genomic patterns associated with mediastinal CD in a substantial case series. Methods: We retrospectively reviewed cases of unicentric mediastinal CD managed in 2 French thoracic surgery departments between 1988 and 2012. Clinical, radiological, surgical and pathological data were recorded. On available FFPE blocks we performed mutation screening by next-generation-sequencing, using AmpliSeq™ Cancer Hotspot v2 (Life Technologies) and immunohistochemistry (IHC) (AKT-mTOR pathway). Results: Eleven patients were identified (mean age 41±15 years, sex-ratio 0.8, median follow-up 78 months). Surgical approach was thoracotomy (n=6), sternotomy (n=4), and VATS (n=1). Additional procedures included thymectomy in three cases, mediastinal lymphadenectomy in two cases, and bilobectomy in one case. One patient presented local relapse as a follicular dendritic cell sarcoma, leading to death 48 months after the first resection. Within 9 patients whose FFPE blocks were available, 2 mutations were found: VHL (p.F119L, 35%, n=1) and JAK3 (p.V718L, 53%, n=1). Phospho-AKT and phospho-mTOR stainings were negative in all cases, whereas phospho-S6RP staining was positive in eight cases, mainly in interfollicular cell cytoplasm. Conclusions: From this series of patients with unicentric mediastinal CD, we observed 2 cases of potential driver mutations and 8 cases of phospho-S6RP activation not related to AKT-mTOR. Larger studies are required to decipher more precisely the molecular abnormalities and potential therapeutic targets underlying this uncommon condition.

Aim Hepatocellular carcinoma (HCC) is a common outcome of chronic hepatitis C virus (HCV) infection and constitutes the main burden of this disease. The molecular mechanisms underlying the development of HCC are multiple and might involve certain microRNA (miR). Since discordant results have been reported concerning the detection of expression of miR-152 and miR-122 in HCC, our aim was to measure the levels of both miRs in serum and liver samples. Methods We analyzed miR-152 and miR-122 expression by RT-qPCR in a serum cohort from 14 HCV-infected patients who developed HCC, 20 HCV+ patients without HCC and 19 control patients. We also studied miR-152 and miR-122 in an independent tissue cohort from 11 normal livers, and from paired HCC and non-tumor adjacent livers of 11 HCV-infected patients and 12 non-infected patients. Results In serum samples, higher levels of miR-122 were found in non-HCC HCV+ compared to HCC HCV+ and control groups whereas miR-152 was detectable in a lower range in HCC HCV+ compared to non-HCC HCV+ and control groups. We found higher signals for miR-122 and miR-152 in non-tumor liver and HCC tissues compared to control tissues. HCC etiology had no detectable influence on miR-122 expression whereas miR-152 was increased in HCV+ tissue samples. Conclusions Detection of low values of circulating miR-152 is a potentially interesting marker of hepatocarcinogenesis in HCV+ patients, in contrast to miR-122, which varies according to hepatocyte damage.

Introduction Le carcinome a cellules de Merkel (CCM) est un carcinome neuroendocrine cutane rare et agressif. En 2008, Feng et coll. identifiaient l’integration du polyomavirus a cellules de Merkel (MCPyV) comme l’evenement oncogenique responsable de 80 % de ces tumeurs. Les cas de CCM depourvus de virus sont consideres comme des tumeurs induites par les UV. Ce second groupe est associe a un pronostic plus pejoratif. Objectif comparer les caracteristiques morphologiques et immunohistochimiques des CCM en fonction du statut viral des tumeurs. Materiel et methodes L’inclusion des cas etait realisee a partir d’une cohorte multicentrique de CCM. Le statut viral etait determine par PCR en temps reel. Au total, 80 CCM viro-positifs et de 21 cas viro-negatifs ont ete inclus. L’evaluation microscopique a ete realisee par deux pathologistes en aveugle du statut viral. Les criteres morphologiques suivants ont ete etudies : la forme du noyau, la presence d’un nucleole, la taille de la cellule, le volume cytoplasmique, la presence d’un cytoplasme clair, d’une architecture en rosettes, d’un epidermotropisme, d’un contingent d’une autre ligne de differenciation (tumeurs composites) ou l’association a une maladie de Bowen ou a une keratose actinique. L’expression des marqueurs immunohistochimiques suivants a egalement ete etudiee : ATOH1, CD99, cytokeratines (CK) 7, 8, 18 et 20, EMA, grand antigene T, neurofilament, PAX5, p53, SATB2, TDT et TTF- 1. Resultats L’evaluation microscopique ( Figure 1 ) a montre que des noyaux allonges, fusiformes etaient plus frequemment observes dans les tumeurs viro-negatives (p = 0,005). De plus, ces dernieres presentaient parfois un phenotype de type « carcinome neuroendocrine a grandes cellules » avec une plus grande taille cellulaire (p = 0,0026), un cytoplasme abondant (p = 10−7) et des nucleoles proeminents (p = 0,002). Un epidermotropisme, et l’association avec une maladie de Bowen/keratose actinique n’etaient jamais observes pour les tumeurs viro-positives. En immunohistochimie, les tumeurs viro-negatives montraient une frequente positivite de TTF1, de la CK7, une surexpression de p53, ainsi qu’une frequente negativite des neurofilaments. En revanche, une expression intense de MUC1, une expression en dot des cytokeratines 8, 18 et 20 ainsi que du CD99 ont ete identifies comme des caracteristiques des CCM viro-positifs. L’expression en dot du CD99 etait notamment associee a la presence du polyomavirus de Merkel (sensibilite = 81 %, specificite = 90 %, rapport de vraisemblance positif = 8,08). Conclusion Notre etude confirme l’existence de caracteristiques morphologiques et immunohistochimiques distinctes entre les CCM viro-positifs et viro-negatifs. De maniere interessante, notre etude a identifie le pattern d’expression en dot du CD99 comme un marqueur associe au statut viro-positif.

Introduction Dans le traitement du melanome metastatique, la sensibilite aux inhibiteurs du gene BRAF de mutations rares est parfois inconnue. Nous rapportons la premiere description d’une sensibilite aux BRAFi d’un melanome metastatique ayant la mutation pV600K601 delinsE, combinant la deletion de la valine (V) en position 600, de la lysine (K) en position 601, et l’insertion d’un acide aspartique (E). Observations Un homme de 46 ans etait pris en charge pour un melanome metastatique ganglionnaire inguino-iliaque, hepatique et cutane. Un sequencage Sanger etait realise sur la tumeur primitive et la metastase cutanee, permettant de caracteriser une mutation pV600_K601delinsE ( Fig. 1 ). La sensibilite aux BRAFi etant « non connue », le patient etait traite par ipilimumab nivolumab puis nivolumab. A M6, il presentait une reponse complete de la tumeur primitive, une reponse partielle hepatique et une progression des cibles ganglionnaires. L’immunotherapie etait maintenue, avec une stabilite des lesions hepatiques mais une progression ganglionnaire confirmee a M9 et M12 ( Fig. 2A ). L’immunotherapie etait relayee par une therapie ciblee (dafrafenib/trametinib). A S4, la TEP TDM objectivait une diminution du metabolisme et des dimensions des adenopathies ( Fig. 2B ). A M6, il existait une reponse complete hepatique et ganglionnaire iliaque, mais la persistance de l’adenopathie inguinale ( Fig. 2C ) que nous avons traitee par chirurgie. L’histologie montrait la persistance de cellules tumorales et la biologie moleculaire trouvait la mutation pV600_K601 delinsE du gene BRAF. A 6 mois de cette exerese le patient maintenait une reponse metabolique complete. Discussion Nous avons evalue la sensibilite de cette mutation aux therapies ciblees apres echec de l’immunotherapie. La cinetique rapide de reponse apres l’introduction des BRAFi/MEKi etait en faveur d’une reponse specifique a la therapie ciblee. La mutation pV600K601delinsE n’a jamais ete decrite a notre connaissance dans le melanome, mais rapportee dans les cancers papillaires de la thyroide. L’insertion d’un acide aspartique (E) apres deletion de la valine et de la lysine en position 600 et 601 est susceptible d’entrainer les memes consequences qu’une mutation V600E « classique » sur l’activite kinase de BRAF. Une mutation proche (pV600_K601delinsD) a ete rapportee chez un patient ayant un melanome metastatique sensible aux BRAFi. L’acide aspartique (E) et l’acide glutamique (D) etant similaires dans leurs proprietes biochimiques, il etait probable que le profil de reponse de notre patient soit similaire a celui decrit dans la litterature. Conclusion Nous rapportons la premiere description d’un melanome porteur de la mutation BRAF V600K601 delinsE avec une sensibilite a l’inhibiteur de BRAF.

Diagnosis of osteocartilaginous pathologies depends on morphological examination and immunohistochemical and molecular biology analyses. Decalcification is required before tissue processing, but available protocols often lead to altered proteins and nucleic acids, and thus compromise the diagnosis. The objective of this study was to compare the effect of different methods of decalcification on histomolecular analyses required for diagnosis and to recommend an optimal protocol for processing these samples in routine practice. We prospectively submitted 35 tissue samples to different decalcification procedures with hydrochloric acid, formic acid, and EDTA, in short, overnight and long cycles for 1 to10 cycles. Preservation of protein integrity was examined by immunohistochemistry, and quality of nucleic acids was estimated after extraction (DNA and RNA concentrations, 260/280 ratios, PCR cycle thresholds), analysis of DNA mutations (high-resolution melting) or amplifications (PCR, in situ hybridization), and detection of fusion transcripts (RT-PCR, in situ hybridization). Hydrochloric acid- and long-term formic acid-based decalcification induced false-negative results on immunohistochemistry and molecular analysis. EDTA and short-term formic acid-based decalcification (5 cycles of 6 h each) did not alter antigenicity and allowed for detection of gene mutations, amplifications or even fusion transcripts. EDTA showed superiority for in situ hybridization techniques. According to these results and our institutional experience, we propose recommendations for decalcification of bone samples, from biopsies to surgical specimens.

Supplementary information : https://static-content.springer.com/esm/art%3A10.1038%2Fs41379-019-0288-7/MediaObjects/41379_2019_288_MOESM1_ESM.pdf; International audience; In 2008, Feng et al. identified Merkel cell polyomavirus integration as the primary oncogenic event in ~80% of Merkel cell carcinoma cases. The remaining virus-negative Merkel cell carcinoma cases associated with a high mutational load are most likely caused by UV radiation. The current study aimed to compare the morphological and immunohistochemical features of 80 virus-positive and 21 virus-negative Merkel cell carcinoma cases. Microscopic evaluation revealed that elongated nuclei-similar to the spindle-shape variant of small cell lung cancer-were less frequent in Merkel cell polyomavirus-positive Merkel cell carcinoma compared to the virus-negative subset (p = 0.005). Moreover, virus-negative cases more frequently displayed a "large-cell neuroendocrine carcinoma" phenotype with larger cell size (p = 0.0026), abundant cytoplasm (p = 4×10-7) and prominent nucleoli (p = 0.002). Analysis of immunohistochemical data revealed frequent positivity for thyroid transcription factor 1 and cytokeratin 7, either absence or overexpression of p53, as well as frequent lack of neurofilament expression in virus-negative cases. By contrast, cytokeratin 8, 18 and 20 and a CD99 with a dot pattern as well as high EMA expression were identified as characteristic features of virus-positive Merkel cell carcinoma. In particular, the CD99 dot-like expression pattern was strongly associated with presence of the Merkel cell polyomavirus in Merkel cell carcinoma (sensitivity = 81%, specificity = 90%, positive likelihood ratio = 8.08). To conclude, virus-positive and -negative Merkel cell carcinoma are characterized by distinct morphological and immunohistochemical features, which implies a significant difference in tumor biology and behavior. Importantly, we identified the CD99 staining pattern as a marker indicating the virus status of this skin cancer.

Electronic supplementary material : https://static-content.springer.com/esm/art%3A10.1038%2Fs41379-018-0155-y/MediaObjects/41379_2018_155_MOESM1_ESM.docx; International audience; Merkel cell carcinoma is a rare neuroendocrine carcinoma of the skin mostly induced by Merkel cell polyomavirus integration. Cytokeratin 20 (CK20) positivity is currently used to distinguish Merkel cell carcinomas from other neuroendocrine carcinomas. However, this distinction may be challenging in CK20-negative cases and in cases without a primary skin tumor. The objectives of this study were first to evaluate the diagnostic accuracy of previously described markers for the diagnosis of Merkel cell carcinoma and second to validate these markers in the setting of difficult-to-diagnose Merkel cell carcinoma variants. In a preliminary set (n = 30), we assessed optimal immunohistochemical patterns (CK20, thyroid transcription factor 1 [TTF-1], atonal homolog 1 [ATOH1], neurofilament [NF], special AT-rich sequence-binding protein 2 [SATB2], paired box protein 5, terminal desoxynucleotidyl transferase, CD99, mucin 1, and Merkel cell polyomavirus-large T antigen) and Merkel cell polyomavirus load thresholds (real-time PCR). The diagnostic accuracy of each marker was then assessed in a validation set of 103 Merkel cell carcinomas (9 CK20-negative cases and 15 cases without a primary skin tumor) and 70 extracutaneous neuroendocrine carcinoma cases. The most discriminant markers for a diagnosis of Merkel cell carcinoma were SATB2, NF expression, and Merkel cell polyomavirus DNA detection (positive likelihood ratios: 36.6, 44.4, and 28.2, respectively). Regarding Merkel cell carcinoma variants, cases without a primary skin tumor retained a similar immunohistochemical profile and CK20-negative tumors displayed a different profile (decrease frequency of NF and SATB2 expression), but Merkel cell polyomavirus DNA remained detected (78% of cases by qPCR). Moreover, 8/9 (89%) CK20-negative Merkel cell carcinoma cases but only 3/61 (5%) CK20-negative extracutaneous neuroendocrine cases were positive for at least one of these markers. In conclusion, detection of SATB2 and NF expression and Merkel cell polyomavirus DNA helps distinguish between Merkel cell carcinoma classical and variant cases and extracutaneous neuroendocrine carcinomas.

International audience; INTRODUCTION: The transcriptional activity of the UGT1A1 gene is modulated by a variable number of repetitions of the dinucleotide (TA) within its promoter region. By comparison to the most common allele (TA)6 (UGT1A1*1), decreased activity is observed with increasing TA repetitions. The aim of this study was to determine whether the presence of the variant allele UGT1A1*28, harbouring seven TA repetitions, (TA)7, in the homozygous state, is associated with precancerous colonic lesions and/or with specific colorectal cancer characteristics. MATERIAL AND METHODS: All patients treated for colorectal cancer in a tertiary care centre, between January 2009 and December 2013, who had routine UGT1A1 genotyping for irinotecan dose-adjustment were included. Data were retrospectively collected. RESULTS: 292 patients were enrolled, including 23 UGT1A1*28/*28 homozygous (7.9%), 137 wild type homozygous (46.9%) and 132 heterozygous (45.2%). There were no significant differences in phenotypic colonic characteristics between homozygous and heterozygous patients carrying the UGT1A1*28 allele as compared to *1/*1 homozygous. Patients treated with aspirin were significantly more common in the UGT1A1*28/*28 homozygous group than in the other groups (7/23 (30.4%) compared to 22/269 (8.2%), p = 0.001). CONCLUSION: Dinucleotide polymorphism in the promoter region of the UGT1A1 gene is not associated with a specific colonic phenotype in patients with sporadic colorectal cancer.

International audience; Chondrosarcomas are rare primary malignant bone tumors that involve the head and neck region in 1% to 12% of cases. Central conventional chondrosarcoma is the most common subtype and is associated with isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) gene mutations in 50% to 60% of cases. We aimed to define the frequency of IDH1 and IDH2 gene mutations in a multicenter series of 88 cases of chondrosarcoma of the head and neck, including tumors involving the base of the skull (n = 30), the facial skeleton (n = 11), and the laryngeal and tracheal cartilages (n = 47). Petrous bone and cricoid cartilage were the most frequently involved sites for chondrosarcomas of the skull base and laryngotracheal tract (43.3% and 31.9%, respectively). Overall, 64.9% of craniofacial chondrosarcomas featured IDH mutations, with a high rate for skull base tumors (85.7%) but no IDH mutations in tumors of the facial skeleton. This different mutational profile could be related to the type of ossification, the bones of the base of the skull mainly resulting from endochondral ossification, and those of the face from intramembranous ossification. Conversely, mutation was infrequent in chondrosarcomas involving the laryngeal and tracheal cartilages (11.8% of 47 cases). Evaluation of IDH mutation status may be a useful diagnostic tool for bone tumors of the skull base, which are most often assessable with only small biopsy samples. The low rate of IDH mutations observed in laryngotracheal chondrosarcomas suggests a different mode of tumorigenesis needing further exploration.

Theranostic assays are based on single-gene testing, but the ability of next-generation sequencing (NGS) to interrogate numerous genetic alterations will progressively replace single-gene assays. Although NGS was evaluated to screen for theranostic mutations, its usefulness in clinical practice on large series of samples remains to be demonstrated. NGS performance was assessed following guidelines. TaqMan probes and NGS were compared for their ability to detect EGFR and KRAS mutations, and NGS mutation profiles were analyzed on a large series of non-small-cell lung cancers (n = 1343). The R

Introduction Le carcinome a cellules de Merkel (CCM) est un carcinome neuroendocrine cutane primitif, principalement induit par l’integration du polyomavirus de Merkel. L’expression de la cytokeratine 20 (CK20) est actuellement utilisee pour distinguer les CCM des carcinomes neuroendocrines d’autres organes. Cependant, cette distinction reste difficile dans les cas de CCM CK20-negatifs et dans les carcinomes a cellules de Merkel ganglionnaires sans primitif cutane. L’objectif de la presente etude est d’evaluer les performances diagnostiques des marqueurs precedemment decrits dans la litterature pour le diagnostic de CCM, notamment dans les cas de variantes difficiles a diagnostiquer (tumeurs CK20-negatives et CCM sans primitif cutane). Materiel et methodes Cent dix-huit cas de CCM (dont 9 MCC CK20-negatif et 15 MCC sans primitif cutane) ont ete compares a 85 cas de CNE extra-cutanes. Apres inclusion de l’ensemble des prelevements dans un tissu micro array, les marqueurs immunohistochimiques suivants ont ete realises : cytokeratine 20, TTF-1, ATOH1, neurofilament (NF), SATB2, PAX5, TdT, CD99, MUC1 et antigene Large T (CM2B4). L’evaluation a ete realisee en double insu. De plus, la presence d’ADN de MCPyV a ete recherchee par PCR quantitative. Apres la determination de seuils de positivite optimaux dans une premiere serie de validation (30 cas), les performances diagnostiques de chaque marqueur ont ete evaluees en utilisant un rapport positif de vraisemblance (seuil > 10). Dans les cas de CCM difficiles a diagnostiquer, la methode « CHi-squared Automatic Interaction Detector » (CHAID) a ete appliquee pour proposer un arbre de decision en utilisant une combinaison de marqueurs. Resultats SATB2, NF et la detection du Polyomavirus de Merkel apparaissent comme les marqueurs les plus robustes pour le diagnostic positif de MCC (rapports de vraisemblance : 36,6 ; 44,4 et 28,2, respectivement) ( Tableau 1 ). Alors que les CCM ganglionnaires sans primitif cutane avaient un phenotype prototypique de CCM, les cas CK20-negatifs presentaient frequemment une absence d’expression des marqueurs immunochimiques SATB2 et Neurofilament (37,5 et 44 % de cas positifs respectivement). Cependant le genome du Polyomavirus de Merkel etait frequemment present dans ces cas CK20-negatifs MCPyV (78 % par qPCR). Par consequent, dans cette derniere population, l’utilisation d’un arbre de decision combinant la detection des marqueurs immunohistochimiques SATB2, NF et la detection du Polyomavirus est proposee. Conclusion SATB2, le neurofilament et la detection du polyomavirus de Merkel permettent, en complement de la cytokeratine 20, de distinguer les CCM des carcinomes neuroendocrines extra-cutanes.

Introduction Les mutations activatrices V600E et V600K du proto-oncogene b-Raf sont identifiees en routine pour definir la sensibilite des melanomes stade IV ou stade III non operables a la bitherapie par BRAFi et MEKi. Cependant, les techniques de sequencage de nouvelle generation (next-gen sequencing [NGS]) permettent l’identification de mutations differentes du gene b-Raf et l’efficacite des therapies ciblees sur les neoplasies porteuses de ces mutations n’est pas toujours connue. Observation Un patient âge de 75 ans a ete opere d’un melanome ulcere, de 6 mm d’epaisseur du dos en aout 2016, puis d’un curage pour progression ganglionnaire avec rupture capsulaire en fevrier 2017. En novembre 2017, une TEP-TDM montrait une progression multimetastatique hepatique, osseuse et surrenale avec un etat general conserve (OMS = 0). Une mutation rare de l’exon 15 du gene b-Raf : c.1796_1797insCAC (p.T599_V600insT) etait identifiee en NGS puis validee en technique de Sanger. Une premiere ligne de traitement par anti-PD1 etait initiee. Au bout de 3 mois de traitement, l’etat general etait altere (OMS = 1) et la TEP-TDM trouvait une importante progression metastatique, notamment au niveau squelettique. Une deuxieme ligne par bitherapie par BRAFi et MEKi (dabrafenib et trametinib) etait alors initiee. Au bout de 6 semaines de traitement, une reponse partielle etait observee a la TEP-TDM. Cependant, une fievre grade III conduisait a changer de bitherapie pour vemurafenib et cometinib. La reevaluation suivante en avril 2018 montrait un maintien de la reponse partielle ( Fig. 1 et 2 ). Discussion Il s’agit du premier cas rapporte de traitement par BRAFi et MEKi d’un melanome metastatique possedant la mutation b-Raf p.T599_V600insT. Cette mutation rare consiste en l’addition d’un codon en position 1796, ce qui conduit a l’ajout d’une threonine en position 599. Cette mutation a egalement ete decrite dans des cas de carcinome thyroidien et chez une patiente atteinte de melanome metastatique, decedee avant que le traitement par BRAF inhibiteur n’ait pu etre instaure. Les donnees in silico et in vitro indiquent que certaines mutations rares ou complexes des codons 599 a 601 du gene b-Raf peuvent resulter en une activation constitutionnelle de la kinase b-Raf par le meme mecanisme que les mutations plus frequentes V600E et V600K. Conclusion Le traitement par une bitherapie BRAFi et MEKi est une option therapeutique valable chez les patients atteints de melanome metastatique possedant la mutation p.T599_V600insT du gene b-Raf. Chez les patients atteints de melanomes possedant des mutations rares du gene b-Raf, le potentiel activateur de la mutation in silico et in vitro doit etre etudie avant l’introduction d’un traitement par BRAF inhibiteur.

Summary Background Survival of metastatic colon cancer (mCC) patients has considerably improved with optimization of new drugs regimen. Inactivation of TP53 pathway by TP53 mutations is observed in nearly half of colorectal tumors. The impact of such mutations has been poorly studied in the metastatic setting. Methods The files of 254 mCC treated in a single institution at Saint-Louis hospital between January 1999 and April 2011 were retrospectively reviewed. Tissue samples for analysis of TP53 mutations were available for 68 patients, performed using FASAY. The prognostic value of TP53 status was evaluated by comparing progression free survival (PFS) and overall survival (OS) in the group of TP53-mutated and wild type patients. Results PFS was 6.9 months and OS 21.7 months in the whole population. There was no statistical difference in TP53- mutated and wild type groups in term of PFS (HR = 1.04; IC9 5% = 0.6–1.79) and OS (HR = 0.99; IC 95% = 0.53–1.55) whatever the chemotherapy regimen (oxaliplatin- or irinotecan-based). Only BRAF V600 mutation was demonstrated to be a poor prognostic factor for PFS and OS, and CEA level for OS. Conclusions Routine determination of TP53 mutations, even with a highly sensitive method, cannot be recommended to predict chemotherapy response in mCC.

All tumors have in common to reactivate a telomere maintenance mechanism to allow for unlimited proliferation. On the other hand, genetic instability found in some tumors can result from the loss of telomeres. Here, we measured telomere length in colorectal cancers (CRCs) using TRF (Telomere Restriction Fragment) analysis. Telomeric DNA content was also quantified as the ratio of total telomeric (TTAGGG) sequences over that of the invariable Alu sequences. In most of the 125 CRCs analyzed, there was a significant diminution in telomere length compared with that in control healthy tissue. Only 34 tumors exhibited no telomere erosion and, in some cases, a slight telomere lengthening. Telomere length did not correlate with age, gender, tumor stage, tumor localization or stage of tumor differentiation. In addition, while telomere length did not correlate with the presence of a mutation in BRAF (V-raf murine sarcoma viral oncogene homolog B), PIK3CA (phosphatidylinositol 3-kinase catalytic subunit), or MSI status, it was significantly associated with the occurrence of a mutation in KRAS. Interestingly, we found that the shorter the telomeres in healthy tissue of a patient, the larger an increase in telomere length in the tumor. Our study points to the existence of two types of CRCs based on telomere length and reveals that telomere length in healthy tissue might influence telomere maintenance mechanisms in the tumor.

Purpose: To better define malignant pleural mesothelioma (MPM) heterogeneity and identify molecular subtypes of MPM, we focus on the tumor suppressor gene LATS2, a member of the Hippo signaling pathway, which plays a key role in mesothelial carcinogenesis. Experimental Design: Sixty-one MPM primary cultures established in our laboratory were screened for mutations in LATS2. Gene inactivation was modeled using siRNAs. Gene and protein expressions were analyzed by quantitative RT-PCR, Western blot analysis, and reverse phase protein array. Cell proliferation, viability, apoptosis, mobility, and invasion were determined after siRNA knockdown or YAP (verteporfin), mTOR (rapamycin), and mTOR/PI3K/AKT (PF-04691502) inhibitor treatment. Results: The LATS2 gene was altered in 11% of MPM by point mutations and large exon deletions. Genetic data coupled with transcriptomic data allowed the identification of a new MPM molecular subgroup, C2LN, characterized by a co-occurring mutation in the LATS2 and NF2 genes in the same MPM. MPM patients of this subgroup presented a poor prognosis. Coinactivation of LATS2 and NF2 leads to loss of cell contact inhibition between MPM cells. Hippo signaling pathway activity, mTOR expression, and phosphorylation were altered in the C2LN MPM subgroup. MPMs of this new subgroup show higher sensitivity to PF-04691502 inhibitor. The MOK gene was identified as a potential biomarker of the C2LN MPM subgroup and PF-04691502 sensitivity. Conclusions: We identified a new MPM molecular subgroup that shares common genetic and transcriptomic characteristics. Our results made it possible to highlight a greater sensitivity to an anticancer compound for this MPM subgroup and to identify a specific potential biomarker. Clin Cancer Res; 23(12); 3191–202. ©2016 AACR.

Hepatocellular carcinoma (HCC) is a common outcome of chronic hepatitis C virus (HCV) infection and constitutes the main burden of this disease. The molecular mechanisms underlying the development of HCC are multiple and might involve certain microRNA (miR). As discordant results have been reported concerning the detection of expression of miR-152 and miR-122 in HCC, our aim was to measure the levels of both miRs in serum and liver samples.We analyzed miR-152 and miR-122 expression by reverse transcription-quantitative polymerase chain reaction in a serum cohort from 14 HCV-infected patients who developed HCC, 20 HCV+ patients without HCC, and 19 control patients. We also studied miR-152 and miR-122 in an independent tissue cohort from 11 normal livers, and from paired HCC and non-tumor adjacent livers of 11 HCV-infected patients and 12 non-infected patients.In serum samples, higher levels of miR-122 were found in non-HCC HCV+ compared to HCC HCV+ and control groups, whereas miR-152 was detectable in a lower range in HCC HCV+ compared to non-HCC HCV+ and control groups. We found higher signals for miR-122 and miR-152 in non-tumor liver and HCC tissues compared to control tissues. Hepatocellular carcinoma etiology had no detectable influence on miR-122 expression, whereas miR-152 was increased in HCV+ tissue samples.Detection of low values of circulating miR-152 is a potentially interesting marker of hepatocarcinogenesis in HCV+ patients, in contrast to miR-122, which varies according to hepatocyte damage.

Introduction Le carcinome a cellules de Merkel (CCM) est un carcinome viro-induit agressif avec une survie a 5 ans estimee a 40 %. Jusqu’a peu, le traitement des stades IV reposait sur une polychimiotherapie a base de sels de platine sans benefice demontre sur la survie. Malgre l’introduction recente de l’avelumab comme traitement de deuxieme ligne dans les MCC avances refractaires, le nombre eleve de patients non repondeurs incite au developpement de nouvelles therapeutiques. Une densite vasculaire intratumorale elevee est associee a une diminution de la survie dans le CCM. De plus, une expression intense du VEGF-A (vascular endothelial growth factor) au sein de la tumeur primitive est associee a un risque accru de metastase. L’hypothese de travail de la presente etude est que le VEGF-A pourrait constituer une cible therapeutique dans le CCM. Patients et methodes Afin de confirmer l’expression du VEGF-A au sein des CCM, 97 tumeurs incluses en paraffine, provenant d’une cohorte multicentrique, ont ete incluses dans un tissu micro array. Le niveau de VEGF-A intratumoral a ete evalue par immunohistochimie (score semi quantitatif) et les cas classes en deux categories (expression nulle/faible vs intense). Apres recueil des donnees cliniques et determination du statut viral par immunohistochimie et PCR en temps reel, les deux populations etaient comparees. Apres confirmation de l’expression du VEGF-A par la lignee tumorale de CCM Waga (PCR en temps reel et western blot), l’efficacite du bevacizumab, un anticorps anti-VEGF-A, a ete evalue dans un modele murin de CCM. Pour ce faire, les tumeurs ont ete generees par injection de cellules tumorales a des souris NOD/SCID (n = 16). Apres randomisation des animaux dans le groupe experimental (bevacizumab) ou dans le groupe temoin (PBS), le traitement ete administre trois fois par semaine. Le critere de jugement principal etait le poids tumoral a la fin de l’etude. Resultats Une expression du VEGF-A etait observee dans 95 % des tumeurs (n = 92/97). Un niveau eleve de VEGF-A (n = 38) etait significativement associee au statut viropositif (Fisher's exact test : p = 0,027). L’inhibition du VEGF-A par un anticorps monoclonal etait ensuite etait evaluee comme option therapeutique dans un modele murin. Les taux de croissance tumorale au cours de la procedure ( Fig. 1 ) etait significativement plus faibles dans le groupe experimental que dans le groupe temoin (test U de Mann-Whitney : p = 1,5 × 10−4). Apres sacrifice, les poids tumoraux etaient egalement plus faibles dans le groupe traite par bevacizumab (Mann-Whitney : p = 3 × 10−4). Conclusion Le VEGF-A est exprime par la majorite des CCM, et son blocage reduit la croissance tumorale in vivo. De ce fait, l’inhibition du VEGF-A par un anticorps monoclonal semble constituer une option therapeutique prometteuse dans le MCC metastatique, utilisable en alternative ou en combinaison avec les traitements actuels.

Background: Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells, which secrete >30g/L of monoclonal immunoglobulin (mc Ig). MM is always preceded by the usually asymptomatic stage called "monoclonal gammapathy of undetermined significance" (MGUS). Presently the detection of a mc Ig does not lead to analysis of its specificity of antigen (Ag) recognition. Yet certain infectious pathogens are associated with the development of B-cell malignancy: for instance, Epstein-Barr virus (EBV) and Burkitt lymphoma, Helicobacter pylori (H. pylori) and MALT lymphoma. Objective: Our aim was to determine the frequency of recognition by purified mc Ig from MGUS and MM patients, of infectious pathogens associated with chronic infection and B-cell malignancy. Methods: We used the "Multiplex Infectious Antigen Array" (MIAA)test set up by the team to study the specificity of purified mc IgG against 8 infectious pathogens: hepatitis C virus (HCV), EBV, H. pylori, varicella zoster virus (VZV), cytomegalovirus (CMV), herpesvirus simplex 1 (HSV-1), HSV-2, and Toxoplasma gondii. Second or third methods were used to verify the specificity of mc IgG (western blotting, ELISA, or another multiplex array). Sialylation of mc Ig, which reflects chronic inflammation, was also studied, via ELISAs and Western blotting, for 98 MGUS and MM patients and 43 controls. Results: The specificity of purified mc IgG was analyzed for 291 patients (60 MGUS, including 4 associated with a B-cell lymphoma; 108 MM; for the 123 other patients the diagnosis of MGUS or MM was not available). Purified mc IgG were found to be specific for one of the 8 infectious pathogens of the MIAA test for 17.5% of patients (51/291: 25 MGUS, 1 SMM, and 25 MM). Purified mc Ig specifically recognized EBV (n=27, including 8 MGUS, 1 SMM and 18 MM ; the Ag recognized were EBNA-1, 25 cases; and VCA, 2 cases); HCV (10 cases: 9 MGUS and 1 MM) ; HSV-1 (7 cases: 5 MGUS including 3 associated with a B-cell malignancy, and 2 MM) ; H. pylori (5 cases: 2 MGUS including 1 associated with a B-cell malignancy, and 3 MM); VZV (1 MM); and CMV (1 MGUS). The Ag most frequently recognized by purified mc IgG was EBV EBNA-1; MM patients with EBNA-1-specific mc IgG represented 10% of the MM in this series. Consistent with a pro-inflammatory effect, for 81.6% of MGUS and MM patients (80/98), the purified mc IgG was found to be hyposialylated. Moreover, purified polyclonal Ig were also hyposialylated for 37% of MM (compared to Conclusion: At least 6 infectious pathogens (EBV, HCV, H. pylori, HSV, VZV, CMV) may be involved in the pathogenesis of MGUS and MM with mc IgG. Importantly, EBV, HCV, H. pylori and HSV are known as oncogenic pathogens associated with solid cancer and/or B-cell malignancy. Finally, for 37% of the MM patients examined, both the polyclonal and mc Ig were found to be hyposialylated, which suggests that inflammation existed prior to clonality and MM. Disclosures Garderet: Takeda: Consultancy; Amgen: Consultancy; BMS: Consultancy, Honoraria; Novartis: Consultancy.

Tacrolimus (TAC) and cyclosporin (CsA) are commonly responsible for hypomagnesemia that predisposes in turn for hypertension, renal impairment and encephalopathy.The effects of TAC on Mg(2+)-homeostasis and of pre-existing Mg(2+)-deficiency on TAC immunosuppressive activity were compared to CsA in mice.Mg(2+) was quantified in plasma, erythrocytes, urine, feces, and femurs from mice treated with TAC 5mg/kg/day. Immunosuppression was assessed in splenocytes by mixed lymphocyte reaction, IL-2 quantification and CN activity determination.Plasma and urine Mg(2+) levels in TAC-treated mice were significantly lower from day 7 until day 21 (p0.05 versus control) and returned to control value at day 28. Mg(2+) levels were unchanged in erythrocyte, feces and femur. Inhibition of allogeneic proliferation, IL-2 production and CN activity were 68, 56 and 30% lower (p0.01) after 7 days of TAC-treatment, and 72, 68 and 51% lower (p0.01) after 7 days of CsA-treatment with a dose of 50mg/kg/day. Dietary-induced hypomagnesemia resulted in significant inhibition of CN activity (p0.01) without alteration of IL-2 production or allogeneic proliferation. However, it did not alter the effects observed with CsA- or TAC-treatment on allogeneic proliferation, IL-2 production and CN activity.By contrast with CsA, long-course TAC-treatment induced an early, but transient, and moderate hypomagnesemia without alteration of bone or erythrocyte stocks, intestinal absorption or renal function. Therefore, in clinical use, TAC should be preferred to CsA in patients with pathological or pharmacological conditions which favor Mg(2+)-deficiency. However, dietary-induced hypomagnesemia did not alter the immunosuppressive effects of TAC and CsA.