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The prospective multicenter COMET trial followed a cohort of 306 consecutive metastatic breast cancer patients receiving bevacizumab and paclitaxel as first-line chemotherapy. This study was intended to identify and validate reliable biomarkers to better predict bevacizumab treatment outcomes and allow for a more personalized use of this antiangiogenic agent. To that end, we aimed to establish risk scores for survival prognosis dichotomization based on classic clinico-pathological criteria combined or not with single nucleotide polymorphisms (SNPs). The genomic DNA of 306 patients was extracted and a panel of 13 SNPs, covering seven genes previously documented to be potentially involved in drug response, were analyzed by means of high-throughput genotyping. In receiver operating characteristic (ROC) analyses, the hazard model based on a triple-negative cancer phenotype variable, combined with specific SNPs in VEGFA (rs833061), VEGFR1 (rs9582036) and VEGFR2 (rs1870377), had the highest predictive value. The overall survival hazard ratio of patients assigned to the poor prognosis group based on this model was 3.21 (95% CI (2.33&ndash, 4.42), p &lt, 0.001). We propose that combining this pharmacogenetic approach with classical clinico-pathological characteristics could markedly improve clinical decision-making for breast cancer patients receiving bevacizumab-based therapy.

Hyperprogressive disease (HPD), an unexpected acceleration of tumor growth kinetics, is described in cancer patients treated with anti-PD-1/anti-PD-L1 agents. Here, our aim was to take into consideration the host and explore whether single nucleotide polymorphisms (SNPs) in key genes involved in immune response might predispose to HPD. DNA was extracted from blood-samples from 98 patients treated under CPI monotherapy. Four candidate genes (PD-1, PD-L1, IDO1 and VEGFR2) and 15 potential SNPs were selected. The TGKR (ratio of the slope of tumor growth before treatment and the slope of tumor growth on treatment) was calculated. Hyperprogression was defined as a TGKR≥2. TGKR calculation was feasible for 80 patients (82%). HPD was observed for 11 patients (14%) and was associated with shorter overall survival (P = 0.003). In univariate analysis, HPD was significantly associated with age ≥70 y (P = 0.025), immune-related toxicity (P = 0.016), VEGFR2 rs1870377 A/T or A/A (P = 0.005), PD-L1 rs2282055 G/T or G/G (P = 0.024) and PD-L1 rs2227981 G/A or A/A (P = 0.024). Multivariate analysis confirmed the correlation between HPD and age ≥70 y (P = 0.006), VEGFR2 rs1870377 A/T or A/A (P = 0.007) and PD-L1 rs2282055 G/T or G/G (P = 0.018). Immunogenetics could become integral predictive factors for CPI-based immunotherapy.

Background Checkpoint inhibitors bring marked benefits but only in a minority of patients and may also be associated with severe adverse events. Treatment outcome still cannot be faithfully predicted. The following study hypothesized that host genetics could be applied as predictive biomarkers for checkpoint inhibitor response and immune-related adverse events. We conducted a study based on germinal polymorphisms from genes coding for proteins involved in immune regulation. Methods Germinal DNA was obtained from advanced cancer patients treated with anti-PD-1/PD-L1 checkpoint inhibitors. DNA was genotyped using a custom panel of 166 single nucleotide polymorphisms covering 86 preselected immunogenetic-related genes. Computational analysis using a GTEX portal was made to determine potential expression Quantitative Trait Loci in tissues. Results Ninety-four consecutive patients were included. Objective response rate (complete or partial response) was significantly correlated to tumor microenvironment-related SNPs concerning CCL2, NOS3, IL1RN, IL12B, CXCR3 and IL6R genes. Toxicity were linked to target-related gene SNPs including UNG, IFNW1, CTLA4, PD-L1 and IFNL4 genes. The Area Under the ROC curve (AUC) was 0.81 (95% CI: 0.72–0.9) for response and 0.89 (95% CI: 0.76–1.00) for toxicity. In silico functionality exploring pointed rs4845618 (IL6R), rs10964859 (IFNW1) and rs3087243 (CTLA4) as potentially impacting gene expression. Conclusion These results strongly support a role for distinct immunogenetic-related gene SNPs able to predict efficacy and safety of anti-PD1/PD-L1 therapies. The results highlight the existence of patient-specific, germinal biomarkers able predict response to checkpoint inhibitor efficacy and, possibly, to predict treatment-related adverse events.

Introduction The systematic assessment of anti-programmed cell death ligand 1 (PD-L1) expression by immunohistochemistry (IHC) in lung adenocarcinomas is becoming standard practice. However, the assessment of PD-L1 expression on small tissue specimens needs to be evaluated and the association with other features more thoroughly analyzed. Methods This retrospective single center study evaluated the immunohistochemical expression of the SP263 anti-PD-L1 antibody on tissue microarrays (TMA) of 152 surgically resected lung adenocarcinomas, using a 25% positivity threshold. The positive cases and 50 randomly chosen negative cases in tissue microarray (TMA) were reassessed on whole tissue sections. The results were correlated to clinical, histopathological and to molecular data obtained through the screening of 214 mutations in 26 genes (LungCarta panel, Agena Biosciences). Results Among 152 primary lung adenocarcinomas, 19 cases (13%) showed PD-L1 expression. The agreement between TMA and whole tissue sections was 89%, specificity was 97%. PD-L1 expression was correlated to RAS mutations (p = .04), RAS/TP53 co-mutations (p = .01) and to the solid or acinar subtype (p = .048). Conclusions With the SP263 PD-L1 antibody, small samples appear as a reliable means to evaluate the PD-L1 status in lung adenocarcinoma. The association between PD-L1 expression and RAS/TP53 mutations may have clinical relevance to predict the efficacy of PD-1/PD-L1 immune checkpoints inhibitors.

Background The purpose of this study was to investigate the effects of combining the phosphotidylinositol-3-kinase (PI3K) inhibitor buparlisib (BKM)120 with the anti-epidermal growth factor receptor (EGFR) agent cetuximab and radiotherapy (RT) on an orthotopic model of head and neck squamous cell carcinoma (HNSCC). Methods We evaluated the antitumor efficacy of BKM120, cetuximab, and RT, administered alone or in combination, using the human PIK3CA-mutated HNSCC cell line, CAL33, injected into the floor of the mouth of nude mice. Results Compared with control, the BKM120-cetuximab and the BKM120-cetuximab-RT combinations led to the highest tumor inhibition (p < .001). The highest inhibitory effect of treatments on cell proliferation, mitogen-activated protein kinase (MAPK) and PI3K/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathways were found with the BKM120-cetuximab association. The association of BKM120 and cetuximab with RT inhibited RT-induced activation of the MAPK pathway. Conclusion These results can serve as a preclinical rationale for innovative treatments combining PI3K inhibition with anti-EGFR therapies and irradiation in patients with HNSCC. © 2016 Wiley Periodicals, Inc. Head Neck, 2016

Background: Although bevacizumab (BVZ) is no longer unanimously recommended in the management of breast cancer (BRCA), few patients have shown objective benefit under BVZ treatment. We thus hypothesized that individual characteristics (germinal polymorphisms, SNPs) might help to subgroup patients drawing benefit from this therapy. We herein report confirmation of a preliminary pharmacogenetic ancillary study (ASCO 2017, abstract #1079) in the prospective COMET trial conducted on metastatic BRCA patients receiving first-line BVZ associated with paclitaxel. Both the number of patients (203 to 306) and median follow-up (24 to 50 months) were markedly increased. Methods: Patients were enrolled in the COMET prospective cohort study from 2012 to 2015. Germinal DNA was analyzed by high-throughput genotyping in 306 patients using an 18 SNPs panel covering 7 objectively preselected genes (VEGFA, VEGFR1, VEGFR2, CYP450, CYP2C8, ABCB1, IL8). Dominant and recessive models were investigated to test possible associations between SNPs and Progression-Free Survival (PFS) and Overall Survival (OS) with a median follow-up of 50 months (CI95%: 47-57). In multivariate analysis, association between clinical characteristics, SNPs and PFS or OS was expressed as a hazard ratio (HR) with a 95% confidence interval, which was estimated using a Cox proportional-hazards model. P values ≤ 0.05 were considered statistically significant. Computational analysis using a GTEX portal was used to determine potential eQTL (expression Quantitative Trait Loci) in tissues. Results: Median age was 55.5 (28-80), 80% ductal carcinoma, with a majority (57%) of histological grade equal to I or II. In multivariate Cox analysis, histological grade III and rs833061 C/C (VEGF-A) were associated with shorter PFS (HR= 1.7, 95%CI [1.3-2.1], p Conclusions: Using an easy-to-perform, low-cost genotyping test, germinal DNA identified predictors for BVZ-based treatment outcome in metastatic BRCA patients. These results open up the possibility of reconsidering BVZ treatment in a defined subgroup of BRCA patients. Citation Format: Gerard Milano, Jocelyn Gal, Patrick Brest, Nathalie Ebran, Laurence Llorca, Coraline Dubot, Gilles Romieu, Isabelle Desmoulins, Etienne Brain, Anthony Goncalves, Jean-Marc Ferrero, Paul Cottu, Marc Debled, Olivier Tredan, Emmanuel Chamorey, Marco Carlo Merlano, Jerôme Lemonnier. VEGFA and VEGFR2 SNPs identify a subgroup of breast cancer patients with favorable outcome under Bevacizumab-based therapy - A message from COMET, a French Unicancer multicentric study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4291.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Objectives/Hypothesis Recent preclinical and clinical studies on head and neck squamous cell carcinoma (HNSCC) revealed synergistic effects when combining anti-EGFR agents with conventional chemotherapeutic drugs. Activation of the PI3-kinase/AKT/mTOR signaling pathway has been identified as an important mechanism implicated in tumor progression and resistance to EGFR inhibitors. The aim of this study was to investigate the effects of combining the mTOR inhibitor temsirolimus (Tem) with the anti-EGFR agent cetuximab (Cet) and conventional chemotherapeutic drugs (cisplatin and fluorouracil (C/F)) on an orthotopic model of HNSCC. Study Design Preclinical in vivo study. Methods We evaluated the anti-tumor efficacy (measured tumor volume) of Tem, Cet, and C/F, administered alone or in combination. Investigations were performed using a human HNSCC cell line, CAL33, injected into the mouth floor of nude mice. Results As compared with the control, the combination of Tem and Cet led to the highest tumor inhibition and induced almost complete tumor growth arrest (P = 0.001). Tem significantly enhanced the impact of the Cet-C/F combination on tumor growth (P < 0.001). The highest inhibitory effects of treatments on cell proliferation (Ki67 labeling), MAPK (pP42/44 labeling), and PI3K/AKT/mTOR (pS6R labeling) signaling pathways were found with the Tem-Cet association. Conclusion In this orthotopic HNSCC model, the combination of Tem with Cet produced synergistic effects on tumor growth. These results were corroborated by a strong inhibition of both MAPK and PI3K-mTOR signaling pathways. Level of Evidence N/A. Laryngoscope, 126:E156–E163, 2016

Background Checkpoint inhibitors (CPIs) benefit only a proportion of patients and may be associated with severe adverse events (AEs) which cannot be predicted. We hypothesized that the host genetics could be used as predictive biomarkers for CPI response and AE prediction. Therefore, we conducted a study based on single nucleotide polymorphisms (SNPs) from genes affiliated with immune response and tumor-microenvironment interaction. Methods Germinal DNA was obtained from advanced cancer patients treated with anti PD-1/PD-L1 CPIs in the Centre Antoine Lacassagne (Nice, France) from July 2012 to January 2018. DNA was genotyped on the MassARRAY system (Agena Bioscience®) using a custom panel of 166 SNPs covering 86 preselected immunogenetic-related genes (Minor allele frequency MAF>0.05 in Caucasians). All tested SNPs were in Hardy-Weinberg equilibrium. Univariate analysis was performed to select the significant SNPs (p 0.5 being considered as good prediction). Computational analysis using a GTEX portal was used to determine potential eQTL (expression Quantitative Trait Loci) in tissues. Results 94 patients were identified, with median age 68 (32-85), 67% male, with a majority (51%) having advanced non-small cell lung cancer. Median follow-up was 16.3 months (95% CI: 12.5-18.3). Overall response rate (ORR) was observed in 49/94 (54%) of patients, with adverse events (grade 3-4) observed in 15/94 (16%) of patients. ORR was significantly predicted by tumor microenvironment related gene polymorphisms (CCL2, NOS3, IL1RN, IL12B, CXCR3, IL6R). In contrast, grade 3-4 AEs were linked to target-related gene SNPs (UNG, IFNW1, CTLA-4, PD-L1, IFNL4). The predictive (c)-index was 0.81 (95% CI: 0.72-0.9) for response and 0.89 (95% CI: 0.76-1.00) for toxicity. In silico functionality exploring (GTEX portal) pointed IL6R (rs4845618) and CTLA4 (rs3087243) as impacting gene expression. Conclusion Our data strongly support the role of distinct SNPs in immunogenetic related genes to predict efficacy and safety of anti PD1/PD-L1 therapies. These data support the notion that patient-specific, germinal biomarkers may supplement tumor-specific biomarkers in predicting response to CPI therapy, and that additional germinal biomarkers may predict grade 3-4 AEs. Citation Format: Sadal REFAE, Jocelyn GAL, Nathalie EBRAN, Josiane OTTO, Delphine BORCHIELLINI, Frederic Peyrade, Emmanuel CHAMOREY, Patrick Brest, Gerard Alain Milano, Esma SAADA-BOUZID. Germinal immunogenetics predicts treatment outcome for PD1 PD-L1 checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1370.

Introduction Single nucleotide polymorphisms (SNPs) of DNA repair and apoptosis genes have been associated with outcome in head and neck squamous cell carcinoma (HNSCC) patients receiving radiotherapy (RT). Our goal was to conduct a candidate gene study in HNSCC patients receiving RT or chemoRT. Methods 122 non-resectable HNSCC patients undergoing RT (N = 38) or chemoRT (N = 84) between 1992 and 2006 were retrospectively analyzed. ERCC1 Lys259Thr (rs735482), ERCC2 Lys751Gln (rs13181), ERCC5 His46His C > T (rs1047768), XRCC1 Arg399Gln (rs25487), TP53 Arg72Pro (rs1042522) and MDM2 309T > G (rs2279744) were analyzed on tumor DNA. SNP profile was considered to assess RT-related toxicity. Results All 120 evaluable patients experienced RT-related toxicity at any time. Among them, 83% had G3-4 acute side-effects during RT, mainly dysphagia, mucositis, epithelitis and/or xerostomia (DMEX). 28/105 patients (27%) had early G3-4 toxicity up to 3 months after the end of RT. 29/96 patients (30%) had G3-4 late toxicity thereafter. The presence of G allele of MDM2 or Thr allele of ERCC1 was associated with a significantly higher risk of acute and/or early DMEX toxicity. The MDM2 309GG genotype was linked to a higher risk of acute G3-4 dermatitis. The ERCC5 TT genotype was associated with more frequent G3-4 late cervical skin fibrosis or xerostomia. Pro allele of TP53 72 was associated with a higher risk of G3-4 osteoradionecrosis. Conclusion Relevant SNPs in DNA repair (ERCC1 and ERCC5) and apoptosis (MDM2 and TP53) genes might influence the severity of radiation-related side-effects in HNSCC patients. Prospective clinical SNP-based validation studies are needed on these bases.

Background: Hyperprogressive disease (HPD) has been described in advanced solid tumors patients treated with anti PD1 anti PDL1 monotherapy (Champiat, Clin Can Res 2016; Saada-Bouzid, Ann Oncol, 2017). Our aim was to explore whether host constitutional variations in the nucleotide sequence of genes involved in immune response might be associated with the occurrence of HPD in patients treated with anti PD-(L)1. Methods : From April to August 2017, 98 patients were treated in the Centre Antoine Lacassagne (Nice, France) with anti PD-(L)1. Potential SNPs for four candidate genes with a minor allele frequency≥ 5% in caucasians were selected according to genome browser data-base: PD1 (rs10204525, rs11568821, rs2227981), PD-L1 (rs2282055, rs2297136, rs2297137, rs4143815, rs10815225), IDO1 (rs3739319, rs3808606, rs373931, rs9657182) and VEGR2 (rs2305948, rs1870377, rs2071559). High through-output genotyping was done by MassARRAY (AGENA). HPD was defined as a TGKR (ratio of the slope of tumor growth before treatment and the slope of tumor growth on treatment) ≥ 2. Results: Patients were treated for head and neck squamous cell carcinoma (16/98), non-small cell lung cancer (48/98), melanoma (13/98), renal cell carcinoma (14/98) and 7 others. Median age was 66 years old, 33 were male. All tested SNPs were in Hardy-Weinberg equilibrium. Responses were associated with G3-G4 toxicities (10% vs 42%, p=0.001). TGKR was assessable in 80 patients. HPD was observed in 11/80 (14 %) patients. HPD was significantly associated with age≥70y (6% vs 25%, p=0.02) and genotype for PD1 rs 2227981 (8/32=35% G/G vs 2/43=5% A/G or A/A, p=0.02), PDL1 rs 2282055 (1/37=3% T/T vs 10/43=23% T/G or G/G,p=0.02) and VEGFR2 rs1870377 (2/48=5% T/T vs 9/32=28% T/A or A/A, p=0.005). Multivariate analysis confirmed the association between HPD and VEGFR2 rs1870377 T/A or A/A (OR=15.3, p=0.007), PDL1 rs 2282055 T/G or G/G (OR=18.7, p=0.01) and age≥70y (OR=14.4, p=0.006).). Combining the three alleles at risk for HPD gave an OR =12.4 (p=0.001). Conclusion: In patients treated with anti PD-(L)1, HPD was independently associated with older age and host related gene variations. Host related immunogenetics could become an integrative part of predictive factor for immunotherapy outcome. Citation Format: Sadal Refae, Nathalie Ebran, Jocelyn Gal, Josiane Otto, Damien Giacchero, Delphine Borchiellini, Joel Guigay, Frederique Peyrade, Gerard Milano, Esma Saada. Host immunogenetics and hyperprogression under PD1/PD-L1 checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4548.

3034Background: In the context of the checkpoint inhibitors (CPI) era and the strong demand for identification of predictive biomarkers, the role of the host must be considered. We developed a cust...

The epidermis of fish is covered with a layer of mucus, which contributes to the defence of the species against parasites, bacteria and fungi. We have previously extracted glycoproteins from various mucus samples from fish and have shown that they present pore-forming activities well correlated with strong antibacterial properties [Ebran, Julien, Orange, Saglio, Lemaitre and Molle(2000) Biochim. Biophys. Acta 1467, 271-280]. The present study focuses on the 65 kDa glycoprotein, Tr65, from the rainbow trout (Oncorhynchus mykiss, formerly Salmo gairdneri).Enzymatic digestion of Tr65 yielded a fragment pattern with strong homology with that of trout type II cytokeratin. Sequence analysis of the cDNA clone obtained by PCR confirmed this homology. We thus constructed a plasmid to overproduce the recombinant Tr65. We extracted and purified this recombinant Tr65, using it for multichannel and single-channel experiments in azolectin bilayers. Our results with recombinant Tr65 confirmed the pore-forming properties already shown with native antibacterial Tr65. These findings offer new insights into the function of keratin proteins present in various mucosal surfaces of animals and human beings.The epidermis of fish is covered with a layer of mucus, which contributes to the defence of the species against parasites, bacteria and fungi. We have previously extracted glycoproteins from various mucus samples from fish and have shown that they present pore-forming activities well correlated with strong antibacterial properties [Ebran, Julien, Orange, Saglio, Lemaitre and Molle(2000) Biochim. Biophys. Acta 1467, 271-280]. The present study focuses on the 65 kDa glycoprotein, Tr65, from the rainbow trout (Oncorhynchus mykiss, formerly Salmo gairdneri).Enzymatic digestion of Tr65 yielded a fragment pattern with strong homology with that of trout type II cytokeratin. Sequence analysis of the cDNA clone obtained by PCR confirmed this homology. We thus constructed a plasmid to overproduce the recombinant Tr65. We extracted and purified this recombinant Tr65, using it for multichannel and single-channel experiments in azolectin bilayers. Our results with recombinant Tr65 confirmed the pore-forming properties already shown with native antibacterial Tr65. These findings offer new insights into the function of keratin proteins present in various mucosal surfaces of animals and human beings.

Recent preclinical and clinical studies on head and neck squamous cell carcinoma (HNSCC) revealed synergistic effects when combining anti-EGFR agents with conventional chemotherapeutic drugs. Activation of the PI3-kinase/AKT/mTOR signaling pathway has been identified as an important mechanism implicated in tumor progression and resistance to EGFR inhibitors. The aim of this study was to investigate the effects of combining the mTOR inhibitor temsirolimus (Tem) with the anti-EGFR agent cetuximab (Cet) and conventional chemotherapeutic drugs (cisplatin and fluorouracil (C/F)) on an orthotopic model of HNSCC.Preclinical in vivo study.We evaluated the anti-tumor efficacy (measured tumor volume) of Tem, Cet, and C/F, administered alone or in combination. Investigations were performed using a human HNSCC cell line, CAL33, injected into the mouth floor of nude mice.As compared with the control, the combination of Tem and Cet led to the highest tumor inhibition and induced almost complete tumor growth arrest (P = 0.001). Tem significantly enhanced the impact of the Cet-C/F combination on tumor growth (P0.001). The highest inhibitory effects of treatments on cell proliferation (Ki67 labeling), MAPK (pP42/44 labeling), and PI3K/AKT/mTOR (pS6R labeling) signaling pathways were found with the Tem-Cet association.In this orthotopic HNSCC model, the combination of Tem with Cet produced synergistic effects on tumor growth. These results were corroborated by a strong inhibition of both MAPK and PI3K-mTOR signaling pathways.N/A.

Chromosomal translocations are common genetic abnormalities found in both leukemias and solid tumors. While much has been learned about the effects of specific translocations on cell proliferation, much less is known about what causes these chromosome rearrangements. This article describes the development and use of a system that genetically selects for rare translocation events using the yeast Saccharomyces cerevisiae. A translocation YAC was created that contains the breakpoint cluster region from the human MLL gene, a gene frequently involved in translocations in leukemia patients, flanked by positive and negative selection markers. A translocation between the YAC and a yeast chromosome, whose breakpoint falls within the MLL DNA, physically separates the markers and forms the basis for the selection. When RAD52 is deleted, essentially all of the selected and screened cells contain simple translocations. The detectable translocation rates are the same in haploids and diploids, although the mechanisms involved and true translocation rates may be distinct. A unique double-strand break induced within the MLL sequences increases the number of detectable translocation events 100- to 1000-fold. This novel system provides a tractable assay for answering basic mechanistic questions about the development of chromosomal translocations.

Among several biological functions, the epidermal mucus of fish may play an important role in host defense, particularly in the prevention of colonization by parasites, bacteria and fungi. In previous work, two hydrophobic proteins of 27 and 31 kDa were isolated from carp mucus. This study identified a strong antibacterial activity (0.16–0.18 μ M) well correlated with pore-forming properties. Here this work was extended to other fish species, four fresh water fish and one sea water fish. After a first step of purification, water-soluble and hydrophobic material were separated, and both fractions were analyzed by SDS-PAGE and capillary electrophoresis. Only the hydrophobic component induced pore-forming activity, when reconstituted in planar lipid bilayers. This pore-forming activity was well correlated to a strong antibacterial activity against several bacteria strains. These results suggest that fish secrete antibacterial proteins able to permeabilize the membrane of the target cell and thus act as a defense barrier.

We present a 10-year-old girl with a pure Bednar tumor (pigmented dermatofibrosarcoma protuberans) of the right shoulder. Cytogenetic analysis demonstrated 47 chromosomes with 2 copies of a derivative chromosome 22, der(22)t(17;22)(q22;q13). Fluorescence in situ hybridization (FISH) analysis demonstrated the COL1A1-PDGFB fusion on both der(22) chromosomes. By RT-PCR and sequencing, we observed a fusion of the COL1A1 exon 41 with PDGFB exon 2. This pure pediatric Bednar tumor in a child, like childhood dermatofibrosarcoma protuberans, had a linear structural abnormality rather than a ring chromosome that is more commonly encountered in adult Bednar and dermatofibrosarcoma protuberans tumors. The underlying molecular abnormality in this pediatric Bednar tumor is the same as in dermatofibrosarcoma protuberans.

The PI3-kinase (PI3K)/AKT/mTOR signaling pathway is of particular interest in head and neck squamous cell carcinoma (HNSCC). Its activation has been identified as an important mechanism implicated in tumor progression and resistance to epidermal growth factor receptor (EGFR) inhibitors. The anti-EGFR monoclonal antibody cetuximab (Cet), radiotherapy (RT) and their combination are widely used in the therapeutic management of patients with HNSCC. Inhibition of the PI3K/AKT/mTOR signaling pathway has been shown to potentialize the effects of anti-EGFR agents and RT. We conducted a preliminary in vitro study demonstrating synergistic effects of Cet followed by the PI3K inhibitor BKM120 (Buparlisib) in both PI3KCA wild-type and mutated cells. The aim of the present study was to investigate the effects of combining BKM120 with Cet and RT on an orthotopic xenograft model of HNSCC. In the present study we have used an orthotopic (floor of the mouth) model of human HNSCC to measure the effects of a treatment including BKM120 (40 mg/kg, 5 days/week, p.o.), Cet (2,5 mg/kg/day, 1 day/week, i.p.) and RT (6 Gy/dose, 3 days/week), administered alone or in combination. Investigations were performed using the human PI3KCA-mutated (H1047R) HNSCC cell line, CAL33, injected into the mouth floor of nude mice. The tumor growth was followed by IVIS imagery on days 3, 7 and 12 and tumor size was checked with a caliper ruler on day 13 after animal euthanasia. As compared with the control, the BKM120-Cet and the BKM120-Cet-RT combinations led to the highest tumor inhibition and induced almost complete tumor growth arrest (p < 0.001). The addition of BKM120 and Cet to RT significantly enhanced the impact of RT alone on tumor growth (p < 0.001). The highest inhibitory effects of treatments on cell proliferation (Ki67 labelling), MAPK (pERK labelling) and PI3K/AKT/mTOR (pS6R labelling) signaling pathways were found with the BKM120-Cet association. RT alone (p = 0.10) tended to activate the MAPK pathway but the association of BKM120 and Cet to RT inhibited the RT-induced activation of the MAPK pathway (p = 0.03). In conclusion, in this orthotopic HNSCC model, the combination of BKM120 with Cet and RT produced synergistic effects on tumor growth. These results could serve as a strong preclinical basis for innovative treatments combining PI3K inhibition with anti-EGFR therapies and RT in patients with HNSCC. Citation Format: Gérard Milano, Alexandre Bozec, Nathalie Ebran. Combination of PI3K targeting with cetuximab and irradiation: a preclinical study on an orthotopic xenograft model of head and neck cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1282.

International audience; There is now substantial evidence that imatinib may affect immune responses, especially those mediated by T lymphocytes. Fas (CD95/Apo-1), a cell death receptor, is a key regulator of the immune system. We have explored the consequences of treatment on the Fas system in chronic myeloid leukemia patients treated with imatinib. In comparison with healthy controls, we found not only a mild blood lymphopenia but also impairment of phytohemagglutinin activation in CD4Fas and CD8Fas lymphocytes of imatinib-treated patients. Moreover, these lymphocyte populations were more sensitive to FasL-induced cell death in relation to an increase in Fas expression at the cell surface. Taken together, these results reveal the role of Fas receptor in the lymphopenia observed in patients treated with imatinib, with potential deleterious consequences on antileukemic responses against this immunogenic hematological malignancy.

International audience

International audience; Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma and rarely occurs in adults. There are six main subtypes, each histologically, clinically, and cytogenetically distinct. Embryonal RMS is characterized by chromosomal gains, usually not associated with any consistent structural anomaly. We describe here a case of embryonal RMS in a 19-year-old female patient. The conventional cytogenetic analysis showed a t(4;22)(q35;q12) translocation as the sole cytogenetic change. Complementary fluorescence in situ hybridization analysis showed that the translocation breakpoints were located in the EWSR1 gene at 22q12 and the region of the DUX4 and FSHMD1A at 4q35. This constitutes a novel example of the high frequency of EWSR1 rearrangements in various types of sarcomas as well as of its ability to fuse with a large variety of partner genes. Because DUX4 is involved in myogenic differentiation and cell-cycle control, the striated muscle differentiation observed in the present case might be a direct consequence of the alteration of the DUX4 region generated by the t(4;22). The involvement of the DUX4 region might represent the genetic hallmark of a novel subclass of small round cell tumors.

Lipomas are frequently characterized by rearrangements resulting in the fusion of the HMGA2 gene (12q14.3) with a variety of partners. Chromosome band 9p22 rearrangements occur in about 1% of lipomas. We report here the molecular cytogenetic analysis of five cases of lipoma with a 9p22 aberration, including the first cytogenetic analysis of a colonic lipoma. Three out of the five cases showed a rearrangement of NFIB at 9p22.3. The NFIB rearrangement involved a fusion with HMGA2 in two cases. We have identified an in-frame fusion of the first three exons of HMGA2 with exon 6 of MSRB3 (12q14.3) and exons 8 and 9 of NFIB by using 3'RACE-PCR in a case of superficial lipoma. In a case of retroperitoneal lipoma we found a fusion of HMGA2 with NFIB by fluorescence in situ hybridization analysis. The colonic lipoma was characterized by a t(9;16;19)(p22;q21;q13) with a rearrangement of NFIB and no rearrangement of HMGA2. NFIB belongs to the nuclear factor I transcription family. It has been previously shown to be fused with HMGA2 in one case of lipoma and to be a recurrent partner of HMGA2 in pleormorphic adenoma of salivary glands. We here demonstrate that NFIB can also be rearranged independently from HMGA2, indicating a potentially important role in lipoma pathobiology. Our findings suggest that the rearrangement of NFIB might be associated with deep-seated lipomas, such as retroperitoneal or gastro-intestinal lipomas. (c) 2008 Wiley-Liss, Inc.

Background The diagnosis of dermatofibrosarcoma protuberans (DFSP) in childhood is often difficult because of the deceptive appearance of the lesions. Little is known about congenital DFSP, the frequency of which is probably underestimated because the initial lesion may pass unnoticed. Observations We studied 9 DFSP congenital cases (8 plaques and 1 nodule) initially suspected to be benign lesions. The first biopsies or excisions were performed after a delay of 5½ months to 15 years. All cases were CD34+. Histologic patterns were similar to the DFSP adult classic pattern in 4 cases. One case was a Bednar tumor. The histologic diagnosis of the 4 remaining cases was difficult. The collagen, type I, α 1–platelet-derived growth factor β fusion gene (COL1A1-PDGFB) was detected by means of reverse transcriptase–polymerase chain reaction or fluorescence in situ hybridization. Conclusions All cases of congenital DFSP were difficult to identify clinically. The diagnosis was suspected by means of histologic and immunohistochemical evaluation and was confirmed using molecular analyses. This study illustrates the difficulties and pitfalls of the recognition of congenital DFSP and emphasizes the value of immunohistochemical study with anti-CD34 and complementary molecular analysis for all cutaneous spindle cell tumors and plaques in neonates and infants.

In fish, a layer of mucus covers the external body surface contributing therefore, among other important biological functions, to the defense system of fish. The prevention of colonization by aquatic parasites, bacteria and fungi is mediated both by immune system compounds (IgM, lysozyme, etc.) and by antibacterial peptides and polypeptides. We have recently shown that only the hydrophobic components of crude epidermal mucus of fresh water and sea water fish exhibit strong pore-forming properties, which were well correlated with antibacterial activity [N. Ebran, S. Julien, N. Orange, P. Saglio, C. Lemaitre, G. Molle, Comp. Biochem. Physiol. 122 (1999)]. Here, we have isolated novel glycosylated proteins from the hydrophobic supernatant of tench (Tinca tinca), eel (Anguilla anguilla) and rainbow trout (Oncorhynchus mykiss) mucus. The study of their secondary structure was performed by circular dichroism and revealed structures in random coil and alpha-helix in the same proportions. When reconstituted in planar lipid bilayer, they induced the formation of ion channels. This pore-forming activity was well correlated with a strong antibacterial activity (minimal inhibitory concentration < 1 microM for the three proteins) against both gram-negative and gram-positive bacteria. Our results suggest that fish secrete antibacterial glycoproteins able to kill bacteria by forming large pores (several hundreds to thousands of pS) in the target membrane.

Background: Preclinical and clinical studies on head and neck squamous cell carcinoma (HNSCC) demonstrated synergistic anti-tumoral effects when combining anti-EGFR agents with conventional chemotherapeutic drugs such as cisplatin (C) and 5-fluorouracil (F). In a previous experimental study, our group pointed out an enhancement of PIK3CA signaling (pAKT) in tumors re-growing after cetuximab-based treatment (Bozec et al. Br J Cancer 2007). This context justifies investigations on mTOR inhibitors combined with cetuximab (Cetux) in HNSCC. Methods: The effects on tumor growth of treatments combining an mTOR inhibitor (Temsirolimus (Tem)), an anti-EGFR (Cetux) and a clinically validated-chemotherapeutic regimen (C+F) were examined in an orthotopic (floor of the mouth) model of human HNSCC (CAL33 cell line, over-expressing EGFR, secreting VEGF-A, H1047R PIK3CA mutated). In parallel, in-vitro experiments were conducted in order to strengthen the data arising from the in-vivo study. On day 0, cells were injected in nude NMRI mice. Treatment was started on day 3 and consisted of 8 groups (10 mice/group) treated as follows: Control, Tem (5 mg/kg i.p. five times a week), Cetux (2.5 mg/kg i.p. once a week), cisplatin (6 mg/kg) + 5-fluorouracil (17 mg/kg) (i.p. once a week), concomitant treatment combining Tem with either Cetux, C+F, or Cetux+C+F. The tumor growth was recorded by IVIS imaging and on day 13 after animal euthanasia with a caliper ruler. Results: All tested treatments produced significant growth inhibition as compared to controls (p Conclusion: These data on the association of temsilorimus with cetuximab could serve as a strong basis for innovative treatments aiming at an optimal management of patients with recurrent/metastatic HNSCC. Citation Format: Alexandre Bozec, Nathalie Ebran, Martino Monteverde, Nicolas Toussan, Anne Cayre, Marie-Cristine Etienne-Grimaldi, Cristiana Lo Nigro, Anne Sudaka, Marco Merlano, Frederique Penault-Llorca, Gerard Milano. Combination of mTOR targeting with cetuximab and conventional chemotherapy: a preclinical study on an orthotopic xenograft model of head and neck cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2069. doi:10.1158/1538-7445.AM2013-2069

Abstract 4428 The Fas death receptor (CD95/TNFSFR6) conveys death and non-death signals through binding to its cognate ligand, FasL (CD95L)1,2. Fas is expressed constitutively in CD34+ cells of patients with chronic myeloid leukemia3. In order to explore the implication of Fas in CML patient's response, we aimed at analyzing expression and function of Fas in the imatinib-sensitive human Bcr-abl+ Fas+ AR 230S cell line, and in its imatinib-resistant counterpart AR 230R. We analysed the Fas-mediated apoptosis of AR230S and showed that not only imatinib (Figure 1) but also nilotinib and dasatinib (data not shown) potentiate Fas-L mediated apoptosis. This potentiation is dose dependent (Figure 2). Interestingly, there is no potentiation of TKI-mediated apoptosis by FasL in the imatinib-resistant counterpart AR 230R which is Fas negative. The Fas knockdown with a lentivirus-expressing a FAS-shRNA in AR 230S was associated with a disappearance of potentiation effect, whereas the up regulation of Fas expression in AR230 S cell line using lentivirus-expressing human Fas (hFas) induced an emphasized potentiation effect confirming the pivotal role of Fas in the potentiation of TKI-mediated apoptosis. Notably, IFN-α is able to enhance expression of Fas on CD34+ cells from CML patients3. We thus explored the action of IFN-α on the Fas potentiation of TKI-mediated apoptosis. We observed that preincubation with IFN-α increased potentiation in a dose-dependant manner (Data not shown). In conclusion, our preliminary results demonstrate the pivotal role of Fas in the potentiation of TKI-mediated apoptosis and highlight the role of IFN-α in the design of CML treatments. Figure 1: Percentage of FasL-mediated apoptosis in AR230 cell line. Apoptosis induced by FasL in presence or in absence of imatinib was analyzed by propidium iodide staining of cellular DNA content and shown as percentage of sub-G1 to the whole population. Figure 1:. Percentage of FasL-mediated apoptosis in AR230 cell line. Apoptosis induced by FasL in presence or in absence of imatinib was analyzed by propidium iodide staining of cellular DNA content and shown as percentage of sub-G1 to the whole population. Figure 2: Dose-dependentof FasL mediated apoptosis in absence or in combination with imatinib. Apoptosis induced by FasL in presence or in absence of imatinib analyzed by propidium iodide staining of cellular DNA content and shown as percentage of sub-G1 to the whole population. Each point represents the mean ± S.E.M. of 3 experiments. Figure 2:. Dose-dependentof FasL mediated apoptosis in absence or in combination with imatinib. Apoptosis induced by FasL in presence or in absence of imatinib analyzed by propidium iodide staining of cellular DNA content and shown as percentage of sub-G1 to the whole population. Each point represents the mean ± S.E.M. of 3 experiments. Disclosures: Legros: Novartis: Research Funding, Speakers Bureau; BMS: Speakers Bureau; Celgene: Speakers Bureau.