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Figure S5 shows the effects of the combination of streptozotocin with mTOR inhibitors on the size of intrahepatic tumor nodules.

Streptozotocin-based chemotherapy is the first-line chemotherapy recommended for advanced pancreatic neuroendocrine tumors (pNETs), whereas targeted therapies, including mTOR inhibitors, are available in second-line treatment. Unfortunately, objective response rates to both treatments are limited. Because mTOR pathway activation, commonly observed in pNETs, has been reported as one of the major mechanisms accounting for chemoresistance, we investigated the potential benefit of mTOR inhibition combined with streptozotocin treatment in a subset of pNETs, namely insulinomas. To evaluate the potential of mTOR inhibition in combination with streptozotocin, we selected four different inhibitors acting at various levels of the pathway (everolimus: inhibition of mTORC1; MK-2206: inhibition of AKT; BKM120: inhibition of PI3K, mTORC1, and mTORC2; and BEZ235: inhibition of mTORC1 and mTORC2). Effects on cell viability and apoptosis were assessed in insulinoma cell lines INS-1E (rat) and MIN6 (mouse) in vitro and were confirmed in vivo by using a mouse model of hepatic tumor dissemination after intrasplenic xenograft. In vitro, all four combinations display synergistic effects. These combinations lead to heterogeneous mTOR pathway inhibition, in agreement with their respective target, and increased apoptosis. In vivo, tumor growth in the liver was significantly inhibited by combining streptozotocin with everolimus (P = 0.0014), BKM120 (P = 0.0092), or BEZ235 (P = 0.008) as compared to each agent alone. These results suggest that targeting the mTOR pathway in combination with streptozotocin could be of potential benefit for insulinomas and pNET patients and thus support further clinical investigations. Mol Cancer Ther; 17(1); 60–72. ©2017 AACR.

This file gathers all supplementary data (material and methods, supplementary figures and table)

Table S1 provides results from the whole-genome analysis of the INS-1E cell line.

Nutrient availability is a key determinant of tumor cell behavior. While nutrient-rich conditions favor proliferation and tumor growth, scarcity, and particularly glutamine starvation, promotes cell dedifferentiation and chemoresistance. Here, linking ribosome biogenesis plasticity with tumor cell fate, we uncover that the amino acid sensor GCN2 represses the expression of the precursor of ribosomal RNA, 47S, under metabolic stress. We show that blockade of GCN2 triggers cell death by an irremediable nucleolar stress and subsequent TP53-mediated apoptosis in patient-derived models of colon adenocarcinoma (COAD). In nutrient-rich conditions, GCN2 activity supports cell proliferation through the transcription stimulation of 47S rRNA, independently of the canonical ISR axis. However, impairment of GCN2 activity prevents nuclear translocation of the methionyl tRNA synthetase (MetRS) underlying the generation of a nucleolar stress, mTORC1 inhibition and autophagy induction. Inhibition of the GCN2-MetRS axis drastically improves the cytotoxicity of RNA pol I inhibitors, including the first-line chemotherapy oxaliplatin, on patient-derived COAD tumoroids. Our data thus reveal that GCN2 differentially controls the ribosome biogenesis according the nutritional context. Furthermore, pharmacological co-inhibition of the two GCN2 branches and the RNA pol I activity may represent a valuable strategy for elimination of proliferative and metabolically-stressed COAD cell.

The glucose-requiring hexosamine biosynthetic pathway (HBP), which produces UDP-N-acetylglucosamine for glycosylation reactions, promotes lung adenocarcinoma (LUAD) progression. However, lung tumor cells often reside in low-nutrient microenvironments, and whether the HBP is involved in the adaptation of LUAD to nutrient stress is unknown. Here, we show that the HBP and the coat complex II (COPII) play a key role in cell survival during glucose shortage. HBP up-regulation withstood low glucose-induced production of proteins bearing truncated N-glycans, in the endoplasmic reticulum. This function for the HBP, alongside COPII up-regulation, rescued cell surface expression of a subset of glycoproteins. Those included the epidermal growth factor receptor (EGFR), allowing an EGFR-dependent cell survival under low glucose in anchorage-independent growth. Accordingly, high expression of the HBP rate-limiting enzyme GFAT1 was associated with wild-type EGFR activation in LUAD patient samples. Notably, HBP and COPII up-regulation distinguished LUAD from the lung squamous-cell carcinoma subtype, thus uncovering adaptive mechanisms of LUAD to their harsh microenvironment.

International audience; Genetic alterations in non-small cell lung cancers (NSCLC) stimulate the generation of energy and biomass to promote tumor development. However, the efficacy of the translation process is finely regulated by stress sensors, themselves often controlled by nutrient availability and chemotoxic agents. Yet, the crosstalk between therapeutic treatment and glucose availability on cell mass generation remains understudied. Herein, we investigated the impact of pemetrexed (PEM) treatment, a first-line agent for NSCLC, on protein synthesis, depending on high or low glucose availability. PEM treatment drastically repressed cell mass and translation when glucose was abundant. Surprisingly, inhibition of protein synthesis caused by low glucose levels was partially dampened upon co-treatment with PEM. Moreover, PEM counteracted the elevation of the endoplasmic reticulum stress (ERS) signal produced upon low glucose availability, providing a molecular explanation for the differential impact of the drug on translation according to glucose levels. Collectively, these data indicate that the ERS constitutes a molecular crosstalk between microenvironmental stressors, contributing to translation reprogramming and proteostasis plasticity.

Detection of molecular alterations in lung cancer bone metastasis (LCBM) is particularly difficult when decalcification procedure is needed. The Idylla™ real-time (RT)-PCR is compared to the routine method used in our laboratory, which combines next generation and Sanger sequencing, for the detection of EGFR mutations in LCBM.LCBM subjected to EDTA or formic acid decalcification were analysed for EGFR mutational status using two methods: first, the Ion Torrent Ampliseq next generation sequencing (NGS) assay +/- Sanger sequencing was used prospectively; then, the fully-automated, RT-PCR based molecular testing system Idylla™ EGFR Mutation Test was applied retrospectively.Out of the 34 LCBM assayed, 14 (41.2%) were unsuitable for NGS analysis and five remained unsuitable after additional Sanger EGFR sequencing (5/34, 14.7%). Using Idylla™, valid results were observed for 33/34 samples (97.1%). The concordance between the NGS +/- Sanger sequencing method and the RT-PCR method was 89.7% (26/29), one false positive EGFR S768I mutation and two false negative results were observed using Idylla™; one of these false negative cases was diagnosed by Sanger sequencing with a rare exon 19 EGFR mutation not covered by the Idylla™ EGFR Mutation Test design.Detection of EGFR mutations in decalcified LCBM is challenging using NGS, more than half of samples showing invalid results. Alternative methods should thus be preferred to spare clinical samples and decrease delay. The Idylla™ EGFR Mutation Test shows a good performance on decalcified bone samples and could be used as a first step. In case of negative results, a sequencing approach is mandatory to check the presence of rare EGFR mutations sensitive to EGFR tyrosine kinase inhibitors. Keywords: Lung carcinoma, EGFR mutation, Bone metastases, Decalcification, Tyrosine kinase inhibitor

Highlights • Molecular status determination following decalcification procedures is challenging. • The Idylla™ EGFR assay demonstrates good performance on decalcified bone samples. • The choice of EGFR assay should be adapted to patient and sample specificities., Detection of molecular alterations in lung cancer bone metastasis (LCBM) is particularly difficult when decalcification procedure is needed. The Idylla™ real-time (RT)-PCR is compared to the routine method used in our laboratory, which combines next generation and Sanger sequencing, for the detection of EGFR mutations in LCBM. LCBM subjected to EDTA or formic acid decalcification were analysed for EGFR mutational status using two methods: first, the Ion Torrent Ampliseq next generation sequencing (NGS) assay +/- Sanger sequencing was used prospectively; then, the fully-automated, RT-PCR based molecular testing system Idylla™ EGFR Mutation Test was applied retrospectively. Out of the 34 LCBM assayed, 14 (41.2%) were unsuitable for NGS analysis and five remained unsuitable after additional Sanger EGFR sequencing (5/34, 14.7%). Using Idylla™, valid results were observed for 33/34 samples (97.1%). The concordance between the NGS +/- Sanger sequencing method and the RT-PCR method was 89.7% (26/29), one false positive EGFR S768I mutation and two false negative results were observed using Idylla™; one of these false negative cases was diagnosed by Sanger sequencing with a rare exon 19 EGFR mutation not covered by the Idylla™ EGFR Mutation Test design. Detection of EGFR mutations in decalcified LCBM is challenging using NGS, more than half of samples showing invalid results. Alternative methods should thus be preferred to spare clinical samples and decrease delay. The Idylla™ EGFR Mutation Test shows a good performance on decalcified bone samples and could be used as a first step. In case of negative results, a sequencing approach is mandatory to check the presence of rare EGFR mutations sensitive to EGFR tyrosine kinase inhibitors.

Streptozotocin-based chemotherapy is the first-line chemotherapy recommended for advanced pancreatic neuroendocrine tumors (pNETs), whereas targeted therapies, including mTOR inhibitors, are available in second-line treatment. Unfortunately, objective response rates to both treatments are limited. Because mTOR pathway activation, commonly observed in pNETs, has been reported as one of the major mechanisms accounting for chemoresistance, we investigated the potential benefit of mTOR inhibition combined with streptozotocin treatment in a subset of pNETs, namely insulinomas. To evaluate the potential of mTOR inhibition in combination with streptozotocin, we selected four different inhibitors acting at various levels of the pathway (everolimus: inhibition of mTORC1; MK-2206: inhibition of AKT; BKM120: inhibition of PI3K, mTORC1, and mTORC2; and BEZ235: inhibition of mTORC1 and mTORC2). Effects on cell viability and apoptosis were assessed in insulinoma cell lines INS-1E (rat) and MIN6 (mouse) in vitro and were confirmed in vivo by using a mouse model of hepatic tumor dissemination after intrasplenic xenograft. In vitro, all four combinations display synergistic effects. These combinations lead to heterogeneous mTOR pathway inhibition, in agreement with their respective target, and increased apoptosis. In vivo, tumor growth in the liver was significantly inhibited by combining streptozotocin with everolimus (P = 0.0014), BKM120 (P = 0.0092), or BEZ235 (P = 0.008) as compared to each agent alone. These results suggest that targeting the mTOR pathway in combination with streptozotocin could be of potential benefit for insulinomas and pNET patients and thus support further clinical investigations. Mol Cancer Ther; 17(1); 60–72. ©2017 AACR.

Genomic duplications and fusion involving BRAF and KIAA1549 that create fusion proteins with constitutive B-RAF kinase activity are a hallmark of pilocytic astrocytomas (PAs). The detection of KIAA1549–BRAF fusion transcripts is of paramount importance to classify these tumors and to identify patients who could benefit from BRAF inhibitors. In a clinical setting, the available material for molecular analysis from these pediatric tumors is often limited to formalin-fixed paraffin-embedded (FFPE) tissue. The aim of the present study was to develop a new method to detect the three most frequent KIAA1549–BRAF fusion transcripts, 15–9, 16–11, and 16–9, where numbers refer to the exons fused together, using a FFPE-compatible multiplex quantitative reverse transcription polymerase chain reaction (qRT-PCR). We compared performance of the assay to a reference singleplex method on a collection of 46 FFPE PAs. The results showed that both methods are comparable. The multiplex method had an overall 97% sensitivity and 100% specificity compared to the singleplex method, and agreement between the two techniques was almost perfect (Cohen’s kappa: 0.97). There was no evidence of a significant difference between the qRT-PCR efficiencies of the multiplex technique and of the singleplex assay for all fusion transcripts and for GAPDH, the latter used as a reference gene. The multiplex method consumed four times less complementary DNA (cDNA), cost less, and required half the hands-on technical time. The results show that it could be beneficial to implement the multiplex method in a clinical setting, where samples presenting low quantity of degraded RNA are not unusual.

Streptozotocin-based chemotherapy is the first-line chemotherapy recommended for advanced pancreatic neuroendocrine tumors (pNETs), whereas targeted therapies, including mTOR inhibitors, are available in second-line treatment. Unfortunately, objective response rates to both treatments are limited. Because mTOR pathway activation, commonly observed in pNETs, has been reported as one of the major mechanisms accounting for chemoresistance, we investigated the potential benefit of mTOR inhibition combined with streptozotocin treatment in a subset of pNETs, namely insulinomas. To evaluate the potential of mTOR inhibition in combination with streptozotocin, we selected four different inhibitors acting at various levels of the pathway (everolimus: inhibition of mTORC1; MK-2206: inhibition of AKT; BKM120: inhibition of PI3K, mTORC1, and mTORC2; and BEZ235: inhibition of mTORC1 and mTORC2). Effects on cell viability and apoptosis were assessed in insulinoma cell lines INS-1E (rat) and MIN6 (mouse)

We thank the Dr. J.D. Minna (University of Texas Southwestern Medical Center, Dallas, USA) for the HBEC 3KT-RL cell line. We are grateful to Dr. D. Ron (Institute of Metabolic Science, Cambridge, UK) for GCN2 deficient Mouse Embryonic Fibroblasts and for the F2vE-PERK construct. The authors thank the Dr. B. Manship for language editing and manuscript corrections. C. Chaveroux. is the recipient of the program for fundamental and clinical research on cancer from the "Fondation de France". S. Manie is supported by the foundation "Association pour la Recherche sur le Cancer" (PJA20131200334), and by the "Programme INCA" (INCA_7981); The hexosamine biosynthetic pathway (HBP) is a nutrient-sensing metabolic pathway that produces the activated amino sugar UDP-N-acetylglucosamine, a critical substrate for protein glycosylation. Despite its biological significance, little is known about the regulation of HBP flux during nutrient limitation. Here, we report that amino acid or glucose shortage increase GFAT1 production, the first and rate-limiting enzyme of the HBP. GFAT1 is a transcriptional target of the activating transcription factor 4 (ATF4) induced by the GCN2-eIF2 alpha signalling pathway. The increased production of GFAT1 stimulates HBP flux and results in an increase in O-linked beta-N-acetylglucosamine protein modifications. Taken together, these findings demonstrate that ATF4 provides a link between nutritional stress and the HBP for the regulation of the O-GlcNAcylation-dependent cellular signalling

Context: Detection of thyroid cancer among benign nodules on fine-needle aspiration biopsies (FNAB), which presently relies on cytological examination, is expected to be improved by new diagnostic tests set up from genomic data. Objective: The aim of the study was to use a set of genes discriminating benign from malignant tumors, on the basis of their expression levels, to build tumor classifiers and evaluate their capacity to predict malignancy on FNAB. Design: We analyzed the level of expression of 200 potentially informative genes in 56 thyroid tissue samples (benign or malignant tumors and paired normal tissue) using nylon macroarrays. Gene expression data were subjected to a weighted voting algorithm to generate tumor classifiers. The performances of the classifiers were evaluated on a series of 26 sham FNAB, i.e. FNAB carried out on thyroid nodules after surgical resection. Results: A series of 19 genes with a similar expression in follicular adenomas and normal tissue and discriminating follicular adenomas+normal tissue from the following: 1) follicular thyroid carcinomas (FTCs), 2) papillary thyroid carcinomas (PTCs), or 3) both FTCs and PTCs. These were used to generate four classifiers, the FTCs, PTCs, common (FTC+PTCs), and global classifiers. In 23 of the 26 sham FNAB, the four classifiers yielded a diagnosis in agreement with the diagnosis of the pathologist used as reference; in the three other cases, the correct diagnosis was given by three of four classifiers. Conclusions: We developed a procedure of molecular diagnosis of benign vs. malignant tumors applicable to the material collected by FNAB. The molecular test complied with a preclinical validation stage; it must be now evaluated on ultrasound-guided FNAB in a large-scale prospective study.

SLC5A8, proposed as a thyroid apical iodide transporter, was recently defined as a Na+-coupled transporter of short-chain fatty acid. To document the expression pattern of SLC5A8 in the thyroid, we analyzed the regulation of its expression in normal human thyrocytes in culture and in tissues with distinct functional activity. To determine whether SLC5A8 expression is altered in all thyroid carcinomas or only in particular subtypes, we investigated the level of its expression in a series of 50 hypofunctioning tumors. SLC5A8 expression was studied at the transcript level and compared with that of SLC26A4 or Pendrin and SLC5A5 or Na+/iodide symporter. SLC5A8 expression, unlike that of SLC5A5 and SLC26A4, was not regulated by TSH in normal human thyrocytes in culture and was not related to the functional state of thyroid tissue; toxic adenomas and adjacent resting tissues exhibited the same SLC5A8 transcript content. SLC5A8 expression was selectively down-regulated (40-fold) in papillary thyroid carcinomas of classical form (PTC-cf.). Methylation-specific PCR analyses showed that SLC5A8 was methylated in 90% of PTC-cf. and in about 20% of other papillary thyroid carcinomas. In a series of 52 PTC-cf., a low SLC5A8 expression was highly significantly associated with the presence of BRAF T1796A mutation. These data identify a relationship between the methylation-associated silencing of the tumor-suppressor gene SLC5A8 and the T1796A point mutation of the BRAF gene in the PTC-cf. subtype of thyroid carcinomas.

Exposure of phosphatidylserine (PS) on the outer leaflet of the plasma membrane is a key feature of apoptosis. As the signals underlying these phenomena are unknown, it is generally assumed that PS exposure is a consequence of caspase activation, another hallmark of apoptosis. In this study we investigated the role of caspases in PS externalization during apoptosis of activated PBL triggered by drugs (etoposide, staurosporine), CD95 engagement, or IL-2 withdrawal. Anti-CD95 mAb induces a rapid activation of caspases, followed by PS exposure and mitochondrial transmembrane potential (ΔΨm) disruption. In contrast, etoposide (ETO), staurosporine (STS), or IL-2 withdrawal triggers concomitant caspase activation, PS exposure, and ΔΨm disruption. Such kinetics suggest that PS exposure could be independent of caspase activation. As expected, in activated PBL treated by anti-CD95 mAb, the pan-caspase inhibitor Cbz-Val-Ala-Asp(OMe)-fluoromethylketone and the caspase-8 inhibitor Cbz-Leu-Glu-Thr-Asp(OMe)-fluoromethylketone, but not the caspase-9 inhibitor Cbz-Leu-Glu-His-Asp(OMe)-fluoromethylketone, inhibit PS externalization and ΔΨm disruption. Surprisingly, during apoptosis induced by ETO, STS, or IL-2 withdrawal, none of those caspase inhibitors prevents PS externalization or ΔΨm disruption, whereas they all inhibit DNA fragmentation as well as the morphological features of nuclear apoptosis. In Jurkat and H9 T cell lines, as opposed to activated PBL, PS exposure is inhibited by Cbz-Val-Ala-Asp(OMe)-fluoromethylketone during apoptosis induced by CD95 engagement, ETO, or STS. Thus, caspase-independent PS exposure occurs in primary T cells during apoptosis induced by stimuli that do not trigger death receptors.

e23056 Background: A number of RAS mutations confer resistance to anti-EGFR therapies routinely used in the treatment of colon cancer. The objective of this study was to evaluate the pertinence of analyzing circulating-free plasma DNA (cfDNA) as an indicator of the mutational status of a tumor, in order to use liquid biopsies instead of invasive and painful tumor biopsies during tumor progression. Methods: A cohort of 24 lung and 25 colon cancer patients was constituted in the Hospices Civils of Lyon. Liquid biopsy plasma samples were collected at diagnosis (colon cancer) and during tumor progression (lung cancer) for the purpose of the current study. KRAS and NRAS somatic alterations were quantified using three different technologies: the Droplet Digital polymerase chain reaction (ddPCR) from BioRad, the BEAMing Digital PCR from Sysmex Inostics, and the NGS NextSeq 500 by Illumina with the Accel-Amplicon 56G Oncology Panel from SWIFT BIOSCIENCES. Results: We observed a high level of sensitivity and specificity with the BEAMing technology, which provided us with excellent matches, around 96% and 73%, between solid and liquid biopsies taken at diagnosis (colon cancer) or during tumor progression (lung cancer), respectively. Indeed, when examining cfDNA from patients displaying one of the KRAS or NRAS mutations, 11 of the 13 mutations were confirmed using this technology, whereas only 5-6 matched the initial NGS status, using the two other technologies. The detection threshold was estimated at 1% for samples containing at least 0.8 ngctDNA/µL for the multiplex screening ddPCR from BioRad and for the 56G Oncology Panel from SWIFT BIOSCIENCES. The threshold was lower, at 0.03%, in samples containing only 0.25 ng ctDNA/µL for the BEAMing technology, which includes a PCR pre-amplification step. Conclusions: The advantage of the Illumina NGS technology is the larger coverage of longer gene regions, and thus the detection of more genetic mutations. Finally, the BEAMing technology enabled us to follow the appearance and disappearance of somatic alterations, with a very high level of sensitivity.

Background/Aims: While the range of therapeutic options for well-differentiated gastroenteropancreatic neuroendocrine tumors has recently increased with the emergence of targeted therapies, such as mTOR inhibitors, there is no recent progress in the treatment of poorly differentiated neuroendocrine carcinomas (PDNECs). Since PDNECs have been shown to strongly express mTOR pathway components, the aim of the present study was to assess the antitumor effect of the mTOR inhibitor everolimus in preclinical models of PDNECs. Methods: The expression of mTOR pathway components and their response to everolimus were assessed in two neuroendocrine cell lines: STC-1 and GluTag. A xenograft model of intrahepatic dissemination in the nude mouse, based on the intrasplenic injection of either STC-1 and GluTag tumor cells, was used. Animals were started on everolimus treatment 3 days after injection. The effects of treatment on tumor growth, proliferative capacities, apoptosis and in situ expression of mTOR pathway components were assessed. Results: The expression of mTOR pathway components was comparable in STC-1 and GluTag cells and in human PDNECs and could be inhibited in vitro by everolimus. In vivo, the tumor volume of STC-1 and GluTag xenografts was significantly reduced in treated animals (6.05 ± 1.84% as compared to 21.76 ± 3.88% in controls). Everolimus treatment also induced a significant decrease in Ki67 index and in the phosphorylation levels of the two major effectors of mTOR, p70S6K and 4E-BP1. Conclusion: Our experimental data suggest that mTOR inhibition could be considered a therapeutic option for high-grade gastroenteropancreatic neuroendocrine tumors.

International audience; The efficacy of anti-TNF-α therapies highlights the role of TNF-α in the pathogenesis of rheumatoid arthritis (RA). However, the mechanism of action of these agents is poorly understood at the molecular level. The aim of this study was to characterize the effects of anti-TNF-α treatment on the global gene expression profile in peripheral blood mononuclear cells (PBMCs) of responder RA patients. Changes in gene expression were determined using oligonucleotide microarrays (25,341 genes) in PBMCs obtained before and after 12 wk of treatment with either etanercept or adalimumab from responder RA patients. Two hundred fifty-one genes displayed significant changes (false discovery rate < 0.1%) in expression level (178 upregulations with mean fold change = 1.5 and 73 downregulations with mean fold change = -1.50) after 12 wk of treatment. Importantly, the expression of several genes, including those coding for the calcium binding proteins S100A12 and A8, CD14 antigen, Selectin P, or ribosomal protein L39, reported to be upregulated in RA patients, were found to be decreased after anti-TNF-α treatment. Globally, inflammation, immune response, apoptosis, protein synthesis, and mitochondrial oxido-reduction were the most affected pathways in response to anti-TNF-α treatment. The obtained gene expression signature in PBMCs provides new information to better understand the mechanisms of action of anti-TNF-α treatment in RA patients.

Contact hypersensitivity is one of the most common skin diseases and its pharmacological control is an important clinical issue. We investigated the control of contact hypersensitivity by immunosuppressive drugs administered during sensitization or challenge. Mycophenolate mofetil, methotrexate and 5-fluorouracil completely inhibited contact hypersensitivity when administered during sensitization whereas they did not decrease inflammatory reaction when administered during challenge. Conversely, mitoxantrone, and cyclophosphamide, given as a single injection at the time of sensitization or challenge, completely inhibited the reaction, a property associated with T and B cell depletion. The data indicate that antimetabolites which are cell cycle dependent inhibit clonal expansion and subsequent differentiation of cytotoxic CD8+ T cells. Their lack of effect at the time of challenge indicates that T cell proliferation is not required for the expression of effector or regulatory T cell activation. Conversely lymphoablative drugs can inactivate or destroy differentiated cytotoxic T cells with rapid kinetics.

To determine the clinical usefulness of measuring antistratum corneum (ASC) and antifilaggrin autoantibodies (AFA) to discriminate between rheumatoid arthritis (RA) and other rheumatic or autoimmune diseases, using an indirect immunofluorescence (IIF) assay, along with a complementary immunoblotting technique (IB) when IIF detection of ASC was negative.Sera from 346 patients were studied: 189 sera from patients with RA seen in the same clinic, 92 from patients with non-RA rheumatic diseases, 24 from nonrheumatic autoimmune diseases, and 41 from healthy blood donors. ASC and AFA were detected using IIF and IB, respectively.ASC detection using IIF showed a specificity of 97.5% for RA with 44.4% sensitivity. When both IIF and IB techniques were used, sensitivity for RA increased significantly (up to 53.4%; p0.01) with no decrease in specificity (p0.01).These data confirm the usefulness of 2 different techniques performed simultaneously for detecting ASC/AFA, and the usefulness of these biological markers for discriminating between RA and other rheumatic diseases in clinical practice.

V600 mutations of BRAF are present in 40% of cutaneous melanoma and are currently targeted for therapy by specific inhibitors (vemurafenib, dabrafenib). While BRAFV600 mutations are mostly heterozygous, the impact of BRAF mutated allelic specific imbalance (MASI) on the sensitivity to BRAF inhibitors and on clinical outcome has not been evaluated so far. Here we compared cell proliferation, tumor growth and vemurafenib sensitivity of heterozygous versus homozygous BRAF V600E mutated melanoma cell lines. We also performed a retrospective study evaluating the impact on overall survival and drug response of BRAF V600E mutated melanoma patients with or without MASI. Methods: We conducted proliferation and cytotoxity assays to compare cell growth and sensitivity to vemurafenib and trametinib in heterozygous, versus homozygous BRAF V600 mutated human melanoma cell lines. Their in vivo tumorigenesis and sensitivity to vemurafenib was also evaluated upon engraftment in SCID mice. A retrospective series of 179 patients with metastatic melanoma harboring a BRAF V600 mutations were analyzed and early first line treatment response was compared between patients with or without MASI. Results: Our results showed that heterozygous BRAF V600 mutated melanoma cell lines proliferate less rapidly than homozygous cell lines in vitro (cell proliferation counts after 7 days range from 3.6 x106 to 7.7x106 cells for homozygote cells, and from 4.8x104 to 5.5x105 for heterozygous cell lines) and in vivo (palpable tumors appeared on average 7 days after inoculation for homozygous cells but after 39 days for heterozygous cells). Heterozygous cell lines are more resistant to BRAF inhibitors either in vitro or in vivo (IC50 of vemurafenib ranged from 0,1 to 0,198 µM for homozygous cells and from 8,62 to 22,65 µM for heterozygous cell lines). We have also shown that patients with BRAF V600 mutated tumors with MASI may have initially a poorer prognosis (median overall survival 733 days IC95% [381; 1762] versus 1011 days IC95%[879;2969] p=0.236) but tend to have better responses to systemic drugs than patients without MASI (percentage of primary resistance 23% versus 34%; p=0.56). In conclusion: This translational study shows that a MASI of BRAF in melanoma is associated with a higher cell proliferation and tumorigenic potential in vitro and in vivo. This mutational status is also associated with a better response to BRAF inhibitor in vitro and in mice xenografts. Clinical relevance of these data should be more thoroughly evaluated on larger and more homogeneous series of patients however our preliminary data show a trend toward a clinical-experimental correlation with similar yet still statistically insignificant results. If confirmed, our data suggest that study of the MASI of BRAF mutation could serve as a predictive marker of patient response to vemurafenib. The role of paradoxical activation of the MAPK signaling pathway in cells without MASI of BRAF needs to be further explored. Citation Format: Amelie Boespflug, Luc Thomas, Pascal Rousset, Charles Dumontet, Julie Caramel, Carole Ferraro-Peyret, Pierre Paul Bringuier, Brigitte Balme, Stephane Dalle. BRAF V600 mutated allelic specific imbalance (MASI) impact on sensitivity to vemurafenib. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5602. doi:10.1158/1538-7445.AM2014-5602

Treatment of rheumatoid arthritis (RA) with infliximab (Remicade®) has been associated with the induction of antinuclear autoantibodies (ANA) and anti-double-stranded DNA (anti-dsDNA) autoantibodies. In the present study we investigated the humoral immune response induced by infliximab against organ-specific or non-organ-specific antigens not only in RA patients but also in patients with ankylosing spondylitis (AS) during a two-year followup. The association between the presence of autoantibodies and clinical manifestations was then examined. The occurrence of the various autoantibodies was analyzed in 24 RA and 15 AS patients all treated with infliximab and in 30 RA patients receiving methotrexate but not infliximab, using the appropriate methods of detection. Infliximab led to a significant induction of ANA and anti-dsDNA autoantibodies in 86.7% and 57% of RA patients and in 85% and 31% of AS patients, respectively. The incidence of antiphospholipid (aPL) autoantibodies was significantly higher in both RA patients (21%) and AS patients (27%) than in the control group. Most anti-dsDNA and aPL autoantibodies were of IgM isotype and were not associated with infusion side effects, lupus-like manifestations or infectious disease. No other autoantibodies were shown to be induced by the treatment. Our results confirmed the occurrence of ANA and anti-dsDNA autoantibodies and demonstrated that the induction of ANA, anti-dsDNA and aPL autoantibodies is related to infliximab treatment in both RA and AS, with no significant relationship to clinical manifestations.

Gastroenteropancreatic neuroendocrine neoplasms (GEP NEN) are defined as rare neoplastic lesions developing from neuroendocrine cells. Therapeutic options available for GEP NEN are scarce, although targeted therapies such as mTOR or tyrosine-kinase inhibitors provide new opportunities. However, tumor cells develop resistances to these treatments, which reduce their effectiveness. To date, no biomarker is available to select patients responding to these treatments or to anticipate the development of resistances. Identifying new therapeutic targets and understanding mechanisms of resistance are therefore a relevant issue in the treatment of GEP NEN.We found that GEP NEN cells induce the Unfolded Protein Response (UPR) when subjected to hypoxia or glucose depletion, and that PERK pathway promotes cell survival. Modulation of the UPR thanks to UPR inhibitors or inducers decreases tumor growth in a murine model of metastatic dissemination of GEP NEN. Moreover, the mTOR inhibitor rapamycin preferentially induces PERK arm of the UPR, thereby promoting survival of rapamycin-treated cells. These results show the interest in targeting the UPR in the treatment of NNE GEP. In addition, we here suggest a new resistance mechanism to mTOR inhibitors involving PERK pathway. If these results are confirmed in vivo and ex vivo, the combination of mTOR inhibitor and PERK inhibitor could overcome mTOR inhibitors resistances in GEP NEN, Les néoplasmes neuroendocrines gastro-entéro-pancréatiques (NNE GEP) représentent un groupe de tumeurs rares se développant à partir des cellules neuroendocrines de l'organisme. L'arsenal thérapeutique disponible aujourd'hui pour les NNE GEP reste faible, même s'il s'est étoffé au cours de ces dix dernières années avec l'arrivée des thérapies ciblées (inhibiteurs de mTOR et de tyrosine-kinase). Cependant, ces traitements présentent des résistances qui conditionnent leur efficacité et aucun biomarqueur permettant de sélectionner les patients répondeurs à ces traitements ou d'anticiper le développement de résistances n'est connu. Identifier de nouvelles cibles thérapeutiques et comprendre les mécanismes de résistance est donc un enjeu dans le traitement des NNE GEP. Nos travaux montrent que les cellules de NNE GEP soumises à l'hypoxie ou la déplétion en glucose activent l'Unfolded Protein Response (UPR) et que la voie PERK favorise la survie cellulaire. De plus, la modulation de la réponse UPR (via des inhibiteurs ou inducteurs de l'UPR) diminue la croissance tumorale dans un modèle murin de dissémination métastatique de NNE GEP. Nous avons également découvert qu'un inhibiteur de mTOR, la rapamycine, permet d'activer préférentiellement la voie PERK de l'UPR, favorisant la survie des cellules traitées par la rapamycine. Ces résultats montrent l'intérêt de cibler la réponse UPR dans le traitement des NNE GEP. De plus, nous suggérons la mise en place d'un mécanisme de résistance aux inhibiteurs de mTOR impliquant la voie PERK. Si ces résultats se confirment in vivo et ex vivo, l'association d'un inhibiteur de mTOR et d'un inhibiteur de PERK pourrait palier aux phénomènes de résistance rencontrés avec les inhibiteurs de mTOR dans les NNE GEP