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Primer sequences and PCR conditions.

Univariate HRs of 24 single genes overall and for the stage 2, MSS, BRAF-wild type subgroup and stage 3, MSS, BRAF-wild type subgroup within the study population.

Purpose: Data on the prognostic significance of promoter CpG island methylation in colorectal cancer (CRC) are conflicting, possibly due to associations between methylation and other factors affecting survival such as genetic alterations and use of adjuvant therapy. Here, we examine the prognostic impact of promoter methylation in patients with CRC treated with surgery alone in the context of microsatellite instability (MSI), BRAF and KRAS mutations.Experimental Methods: One hundred and seventy-three CRCs were analyzed for promoter methylation of 19 tumor suppressor and DNA repair genes, the CpG island methylator phenotype (CIMP), MSI, the exon 15 V600E BRAF mutation and KRAS codon 12 and 13 mutations.Results: Unsupervised hierarchical clustering based on methylation status of 19 genes revealed three subgroups: cluster 1 [CL1, 57% (98/173) of CRCs], cluster 2 [CL2, 25% (43/173) of CRCs], and cluster 3 [CL3, 18% (32/173) of CRCs]. CL3 had the highest methylation index (0.25, 0.49, and 0.69, respectively, P = P < 0.01). Subgroup analysis for tumor stage, MSI, and BRAF status showed no statistically significant differences in survival between CL1, CL2, and CL3 nor between CIMP and non-CIMP CRCs. Analyzing genes separately revealed that CHFR promoter methylation was associated with a poor prognosis in stage II, microsatellite stability (MSS), BRAF wild-type (WT) CRCs: multivariate Cox proportional HR = 3.89 [95% confidence interval (CI), 1.58–9.60, P < 0.01; n = 66] and HR = 2.11 (95% CI, 0.95–4.69, P = 0.068, n = 136) in a second independent population-based study.Conclusions:CHFR promoter CpG island methylation, which is associated with MSI, also occurs frequently in MSS CRCs and is a promising prognostic marker in stage II, MSS, BRAF WT CRCs. Clin Cancer Res; 20(12); 3261–71. ©2014 AACR.

CIMP and CL1, CL2 and CL3 in relation to clinicopathological characteristics of the study population.

Epi)genetic alterations in relation to clinicopathological characteristics of the study population.

Stage II colon cancer (CC), although diagnosed early, accounts for 16% of CC deaths. Predictors of recurrence risk could mitigate this but are currently lacking. By using a DNA methylation-based clinical screening in real-world (n = 383) and in TCGA-derived cohorts of stage II CC (n = 134), we have devised a novel 40 CpG site-based classifier that can segregate stage II CC into four previously undescribed disease sub-classes that are characterised by distinct molecular features, including activation of MYC/E2F-dependant proliferation signatures. By multivariate analyses, hypermethylation of 2 CpG sites at genes CDH17 and LRP2, respectively, was found to independently confer either significantly increased (CDH17; p-value, 0.0203) or reduced (LRP2; p-value, 0.0047) risk of CC recurrence. Functional enrichment and immune cell infiltration analyses, on RNAseq data from the TCGA cohort, revealed cases with hypermethylation at CDH17 to be enriched for KRAS, epithelial-mesenchymal transition and inflammatory functions (via IL2/STAT5), associated with infiltration by ‘exhausted’ T cells. By contrast, LRP2 hypermethylated cases showed enrichment for mTORC1, DNA repair pathways and activated B cell signatures. These findings will be of value for improving personalised care paths and treatment in stage II CC patients.

Resume Introduction Afin de valider notre strategie d’amelioration continue et chercher d’eventuelles nouvelles pistes pour une performance accrue, nous avons audite l’ensemble des processus du service en utilisant les principes du lean management. Materiel et methodes L’application de la methodologie Lean-6-sigma (Gemba Walk, Value Stream Mapping, diagramme spaghetti, atelier Kaizen et matrice de priorisation) a permis d’analyser les processus des secteurs conventionnels et moleculaires, de definir les goulets d’etranglement, d’evaluer les actions sans valeur ajoutee et de proposer des solutions. Resultats L’audit a identifie les principaux goulets d’etranglement qui interessaient les secteurs pre-analytique (enregistrement), analytique (cytologie, immunohistochimie, sequencage, pathologistes) et post-analytique (absence de secretaire et diffusion du courrier). Il a souligne une marche en avant insuffisante, l’impact important de l’augmentation de la charge de travail (8 %/an) sur la reception et la microscopie malgre l’externalisation et une maquette de postes techniques souvent critique qui ne permettait pas de realiser des taches sans valeur ajoutee (qualite, validation de methodes, protocoles) et parfois quotidiennes (coupes, immunohistochimie). Au terme des 72 tâches du plan d’action pour gerer la surproduction, l’amelioration des conditions de travail et le developpement de nouvelles activites, les delais de reponse sont partiellement maitrises et notre demarche d’automatisation bien avancee. Discussion et conclusion L’audit a valide notre strategie d’amelioration et mieux standardise nos conditions de travail. Meme s’il n’a permis qu’une maitrise partielle des delais de reponse, il a initie une dynamique medicale et technique positive vertueuse pour continuer a avancer vers les prochaines etapes de notre reorganisation (automatisation, agrandissement du service).

Idylla epidermal growth factor receptor (577 comparative tests between IdyllaPreanalytical thresholds were established (20% tumour cell content, 50 ng DNA input) and challenged prospectively in routine practice. 16.8% of samples referred for screening were considered non eligible for IdyllaIdylla

AimsIdylla epidermal growth factor receptor (EGFR) is a fast and fully automated mutation assay that is easy to implement. However, under the Biocartis-recommended technical conditions, tissue sections are directly introduced into the cartridge, at the risk of exhausting the tumour sample. In this study, we evaluate the performance of Idylla EGFR on extracted DNA and discuss its place within the global non-small-cell lung cancer (NSCLC) screening strategy.Methods577 comparative tests between Idylla EGFR on extracted DNA and next-generation sequencing (NGS) were performed across two centres.ResultsPreanalytical thresholds were established (20% tumour cell content, 50 ng DNA input) and challenged prospectively in routine practice. 16.8% of samples referred for screening were considered non eligible for Idylla EGFR testing. Due to discordant by design cases, Idylla EGFR sensitivity was 86.9% for currently actionable EGFR mutations. Idylla EGFR specificity was 100% in first-line screening. NGS was always feasible on the same DNA.ConclusionIdylla EGFR on extracted DNA is feasible and enables tumour material to be saved compared with tissue section use. It is not necessary to replace the analytical thresholds of the Biocartis algorithm. Due to both the limits of the mutational repertoire and the high increase of targetable genes in NSCLC, the use of Idylla EGFR should be restricted to clinical emergency situations accompanied by NGS.

Diagnosis of B-cell chronic lymphocytic leukemia (B-CLL) is usually straightforward, involving clinical, immunophenotypic (Matutes score), and (immuno)genetic analyses (to refine patient prognosis for treatment). CLL cases with atypical presentation (e.g., Matutes ≤ 3) are also encountered, and for these diseases, biology and prognostic impact are less clear. Here we report the genomic characterization of a case of atypical B-CLL in a 70-yr-old male patient; B-CLL cells showed a Matutes score of 3, chromosomal translocation t(14;18)(q32;q21) (BCL2/IGH), mutated IGHV, deletion 17p, and mutations in BCL2, NOTCH1 (subclonal), and TP53 (subclonal). Quite strikingly, a novel PAX5 mutation that was predicted to be loss of function was also seen. Exome sequencing identified, in addition, a potentially actionable BRAF mutation, together with novel somatic mutations affecting the homeobox transcription factor NKX2-3, known to control B-lymphocyte development and homing, and the epigenetic regulator LRIF1, which is implicated in chromatin compaction and gene silencing. Neither NKX2-3 nor LRIF1 mutations, predicted to be loss of function, have previously been reported in B-CLL. Sequencing confirmed the presence of these mutations together with BCL2, NOTCH1, and BRAF mutations, with the t(14;18)(q32;q21) translocation, in the initial diagnostic sample obtained 12 yr prior. This is suggestive of a role for these novel mutations in B-CLL initiation and stable clonal evolution, including upon treatment withdrawal. This case extends the spectrum of atypical B-CLL with t(14;18)(q32;q21) and highlights the value of more global precision genomics for patient follow-up and treatment in these patients.

In order to validate our strategy of continuous improvement and to identify new ways to increase performance, an evaluation of all the procedures was conducted in our department using the principles of lean management.Lean-6-sigma methodology (Gemba Walk, Value StreamMapping, spaghetti diagram, Kaizen workshop and priorization matrix) was used to analyze the procedures of the conventional and molecular sectors, and to identify bottlenecks, actions without added value and solutions.The audit identified bottlenecks in pre-analytical (registration), analytical (cytology, immunohistochemistry, sequencing, pathologists) and post-analytical processes (absence of secretaries, delivery of reports by mail). It underlined a suboptimal flow of people and materials, the heavy impact of an increasing work load (8%/year) in reception and microscopy even though we had outsourced, and an often critical work place schedule for technicians which prevent them from achieving tasks without added value (quality control, validation of methods and protocols) or even daily tasks (cutting, immunohistochemistry). After completing the 72 actions aimed at managing overproduction, improving working conditions and developing new activities, turn-around time was partially under control and the automation process was well advanced.The audit validated our strategy of continuous improvement and advanced the standardization of our working conditions. Even if the turn-around time for reports was shortened, the audit initiated a positive medical and technical dynamic that should help us to implement the next steps of our reorganization (automation and extension of the department).

Resume Le demembrement moleculaire des cancers et en particulier des cancers colorectaux conduit a des prises en charge de plus en plus adaptees et de plus en plus complexes. Ce demembrement a abouti a de nombreuses classifications et finalement a une classification moleculaire consensuelle en 2015. Cette classification consensuelle reste pour l’instant non utilisable au quotidien. Plusieurs facteurs moleculaires (statut MSS/MSI, statut BRAF, statut RAS) sont desormais indispensables a la prise en charge du cancer colorectal que ce soit en situation adjuvante ou metastatique. D’autres facteurs tels que le statut HER2 et l’expression de mir-31-3p sont regulierement pris en compte au cas par cas. L’etude des profils de methy-lation pourrait etre une voie d’avenir pour determiner des facteurs pronostiques et predictifs, ameliorer la classification des differents sous-groupes de cancers et ameliorer in fine la prise en charge therapeutique.

INTRODUCTION : Minimal residual disease (MRD) has emerged as an important predictor of clinical outcome in patients with mantle cell lymphoma (MCL). Its use in everyday clinical practice, however, remains uncertain since standardized MRD monitoring strategies and response criteria are not yet formally established. To address this question, we conducted the LyMa-MRD project as an ancillary biology study in a prospective phase III trial in MCL patients < 66 years of age (NCI NCT00921414; LyMa Trial; Le Gouill et al, NEJM 2017). Briefly, in this trial all patients were previously untreated and received 4 courses of R-DHAP followed by ASCT. After ASCT, patients were randomized between observation (obs) versus Rituximab maintenance (RM) which was associated to superior event free, progression free and overall survival, respectively. Herein, we present the final MRD analysis. METHODS : The final MRD analysis was performed in a total of 220 MRD evaluable patients [defined as patients with an available MRD assay and appropriate follow-up sample in peripheral blood (PB) and/or bone marrow, (BM), at the time point of interest for statistical analyses] and aimed to assess MRD response rates and prognostic impact [progression free and overall survival, (PFS, and OS, respectively)] at the pre-ASCT (after induction phase) (n=195 ; 86 RM and 84 obs) or (2 ; 90 RM and 92 obs). Sequential MRD monitoring was a predefined secondary objective. An additional aim was to assess the value of combining molecular MRD and PET (positron emission tomography) for outcome prediction pre- and post-ASCT. MRD was quantitatively assessed in PB and / or BM after induction or after ASCT by using gold standard EURO-MRD RQ-PCR assays targeted to clonal immunoglobulin gene rearrangements, in 3 French national reference laboratories. MRD data for survival analysis was generated from assays with a minimal sensitivity of at least 10-4. MRD status was assigned according to EURO-MRD interpretation guidelines. MRD negativity at a given time point was defined as absence of RQ-PCR amplification product in the follow-up sample (minimal assay sensitivity of 10-4). Non evaluable MRD was mostly due to no follow-up sample, no MRD target or MRD assay failure. RESULTS: In a total of 195 MRD evaluable patients who completed R-DHAP induction therapy, MRD was found negative in 77% (n=144) and 64% (n=115) of PB and BM samples, respectively. MRD status pre-ASCT was predictive of PFS and OS: p< 0.0001, respectively, for PB; p= 0.0295 and 0.0407, respectively, for BM). Post-ASCT, MRD in PB and BM was negative in 94% (n=181) and 81% (n=137) of patients, respectively, and was predictive of PFS (but not OS) (PB, p= 0.0452 ; BM, p=0.0261). In patients that were MRD negative either pre (PB, p= 0.0260; BM, p=0.0405) or post-ASCT (PB, p=0.001; BM, p=0.007), RM was associated to improved PFS and also OS, albeit to a lesser extent [pre-ASCT BM MRD and post-ASCT PB MRD neg groups only ; p= 0.0395 and p =0.02, respectively]. Finally, combining PET and MRD status offered improved prediction of PFS both pre- (p< 0.0001, PB; p=0.0028, BM MRD) and post-ASCT (p CONCLUSION: Pre-ASCT MRD status in both BM and PB is an early predictor of PFS and OS in younger MCL patients receiving ASCT. RM provides longer PFS and OS regardless of MRD status pre- and post-ASCT. Early sequential MRD monitoring at the pre-ASCT treatment phase and directly post-ASCT thus offers strong potential for early clinical outcome prediction, as a surrogate clinical end point, and for MRD-guided, risk-adapted treatment in MCL. Disclosures Delfau-Larue: MUNDIPHARMA: Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); AMGEN: Honoraria; GILEAD: Honoraria; ROCHE: Honoraria. Thieblemont:Roche, Hospita: Research Funding; Roche, Amgen, Kyte Gilead, Celgene, Abbvie, Novartis, Cellectis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Cellectis: Speakers Bureau. Oberic:Roche, Janssen: Consultancy; Roche, Janssen: Other: Travel, Accommodations, Expenses; Roche: Honoraria. Bouabdallah:Gilead Sciences: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Roche: Consultancy, Honoraria. Casasnovas:MSD: Consultancy, Honoraria; AMGEN: Consultancy, Honoraria; BMS: Consultancy, Honoraria; abbvie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding. Ribrag:BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Expenses; argenX: Current equity holder in publicly-traded company, Research Funding; Infinity: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Honoraria; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; arGEN-X-BVBA: Research Funding; BAY1000394 studies on MCL: Patents & Royalties; AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Eisai: Honoraria; MSD: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Immune Design: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Epizyme: Consultancy, Current equity holder in publicly-traded company, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AZD: Honoraria, Other; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Institut Gustave Roussy: Current Employment; Nanostring: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Expenses; Pharmamar: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Hermine:Alexion: Research Funding; AB Science: Consultancy, Current equity holder in publicly-traded company, Honoraria, Patents & Royalties, Research Funding; Novartis: Research Funding; Roche: Consultancy; Celgene BMS: Consultancy, Research Funding. Le Gouill:Roche Genentech, Janssen-Cilag and Abbvie, Celgene, Jazz pharmaceutical, Gilead-kite, Loxo, Daiichi-Sankyo and Servier: Honoraria; Loxo Oncology at Lilly: Consultancy.

Background Testicular germ cell tumor such as seminoma is strongly associated with male reproductive problems commonly associated with the alteration of sperm parameters as described in testicular dysgenesis syndrome. Interestingly, numerous studies have reported that the precursor of germ cell cancer, germ cell neoplasia in situ (GCNIS), present similarities to fetal gonocytes, specifically characterized by global DNA hypomethylation particularly on imprinting sequences. These disorders may have a common origin derived from perturbations of embryonal programming during fetal development. Presently, there is no available information concerning the sperm DNA methylation patterns of testicular cancer patients. For the first time, we evaluated the sperm imprinting of seminoma patients. A total of 92 cryopreserved sperm samples were included, 31 before seminoma treatment (S): 23 normozoospermic (SN) and 8 oligozoospermic (SO) and 61 sperm controls samples: 31 normozoospermic (N) and 30 oligozoospermic (O). DNA methylation levels of seven differentially methylated regions (DMRs) of imprinted genes [H19/IGF2: IG-DMR (CTCF3 and CTCF6 of H19 gene); IGF2-DMRs (DMR0 and DMR2); MEG3/DLK1:IG-DMR; SNURF:TSS-DMR; KCNQ1OT1:TSS-DMR] were assessed by pyrosequencing. All comparative analyses were adjusted for age. Results Comparisons of sperm DNA methylation levels between seminoma (S) and normozoospermic (N) samples showed a significant difference for the SNURF sequence (p = 0.017), but after taking into account the sperm parameters, no difference was observed. However, we confirmed a significant association between oligozoospermia (O) and imprinting defects for H19/IGF2-CTCF6 (p = 0.001), MEG3/DLK1 (p = 0.017), IGF2-DMR2 (p = 0.022), and SNURF (p = 0.032) in comparison with control groups (N). Conclusions This study highlights the high risk of sperm imprinting defects in cases of oligozoospermia and shows for the first time that seminoma patients with normal spermatogenesis present sperm imprinting integrity. These data suggest a low probability of the involvement of a common imprinting defect in fetal cells leading to both TGCT and subfertility. Electronic supplementary material The online version of this article (10.1186/s13148-018-0559-z) contains supplementary material, which is available to authorized users.

Improved prognostic stratification of patients with TNM stage II colorectal cancer (CRC) is desired, since 20–30% of high-risk stage II patients may die within five years of diagnosis. This study was conducted to investigate REarranged during Transfection ( RET ) gene promoter CpG island methylation as a possible prognostic marker for TNM stage II CRC patients. The utility of RET promoter CpG island methylation in tumors of stage II CRC patients as a prognostic biomarker for CRC related death was studied in three independent series (including 233, 231, and 294 TNM stage II patients, respectively) by using MSP and pyrosequencing. The prognostic value of RET promoter CpG island methylation was analyzed by using Cox regression analysis. In the first series, analyzed by MSP, CRC stage II patients ( n = 233) with RET methylated tumors had a significantly worse overall survival as compared to those with unmethylated tumors (HR multivariable = 2.51, 95%-CI: 1.42–4.43). Despite a significant prognostic effect of RET methylation in stage III patients of a second series, analyzed by MSP, the prognostic effect in stage II patients ( n = 231) was not statistically significant (HR multivariable = 1.16, 95%-CI 0.71–1.92). The third series ( n = 294), analyzed by pyrosequencing, confirmed a statistically significant association between RET methylation and poor overall survival in stage II patients (HR multivariable = 1.91, 95%-CI: 1.04–3.53). Our results show that RET promoter CpG island methylation, analyzed by two different techniques, is associated with a poor prognosis in stage II CRC in two independent series and a poor prognosis in stage III CRC in one series. RET methylation may serve as a useful and robust tool for clinical practice to identify high-risk stage II CRC patients with a poor prognosis. This merits further investigation.

Retinoid receptors (RRs) play a key role in cell proliferation and differentiation. We characterized the expression of RA receptors and retinoid X receptors (RARs and RXRs) in a series of 111 thyroid tumors and investigated the mechanisms responsible for their deregulation: hypermethylation of the RARB2 promoter, loss of heterozygosity (LOH) in the regions of RARB and RXRA, and altered expression of CRBP1 and enzymes involved in RA biosynthesis (RDH10 and RALDH2). Expression of RALDH2 and RDH10 was conserved in 100 % of adenomas and in 90 and 98 %, respectively, of carcinomas, whereas staining for CRBP1 was decreased in 9 % of FAs and 28 % of carcinomas, mainly anaplastic carcinomas (55 %). We found an abnormal expression of RARA, RARB, RXRA, and RXRB in 67, 69, 66, and 73 %, respectively, of thyroid carcinomas (n = 78) and in 9, 9, 9, and 33 % of follicular adenomas (n = 33) (p < 0.001). An abnormal staining pattern of at least two of these markers had 90 % sensitivity and 91 % specificity for a diagnosis of malignancy. Promoter hypermethylation of RARB2 was observed in some anaplastic carcinomas (14 %). LOH was found to be common at the RARB locus (3p24–3p25) and the RXRA locus (9q34), respectively, in 44 and 55 % of carcinomas and in 27 and 43 % of adenomas. In conclusion, immunohistochemical staining for RARs and RXRs may help in the differential diagnosis between well-differentiated carcinoma and follicular adenoma. Further investigation should be carried out to determine whether the characterization of RR expression might identify patients who could benefit from therapy with RA derivatives.

Background Already since the 1990s, promoter CpG island methylation markers have been considered promising diagnostic, prognostic, and predictive cancer biomarkers. However, so far, only a limited number of DNA methylation markers have been introduced into clinical practice. One reason why the vast majority of methylation markers do not translate into clinical applications is lack of independent validation of methylation markers, often caused by differences in methylation analysis techniques. We recently described RET promoter CpG island methylation as a potential prognostic marker in stage II colorectal cancer (CRC) patients of two independent series. Methods In the current study, we analyzed the RET promoter CpG island methylation of 241 stage II colon cancer patients by direct methylation-specific PCR (MSP), nested-MSP, pyrosequencing, and methylation-sensitive high-resolution melting (MS-HRM). All primers were designed as close as possible to the same genomic region. In order to investigate the effect of different DNA methylation assays on patient outcome, we assessed the clinical sensitivity and specificity as well as the association of RET methylation with overall survival for three and five years of follow-up. Results Using direct-MSP and nested-MSP, 12.0 % (25/209) and 29.6 % (71/240) of the patients showed RET promoter CpG island methylation. Methylation frequencies detected by pyrosequencing were related to the threshold for positivity that defined RET methylation. Methylation frequencies obtained by pyrosequencing (threshold for positivity at 20 %) and MS-HRM were 13.3 % (32/240) and 13.8 % (33/239), respectively. The pyrosequencing threshold for positivity of 20 % showed the best correlation with MS-HRM and direct-MSP results. Nested-MSP detected RET promoter CpG island methylation in deceased patients with a higher sensitivity (33.1 %) compared to direct-MSP (10.7 %), pyrosequencing (14.4 %), and MS-HRM (15.4 %). While RET methylation frequencies detected by nested-MSP, pyrosequencing, and MS-HRM varied, the prognostic effect seemed similar (HR 1.74, 95 % CI 0.97–3.15; HR 1.85, 95 % CI 0.93–3.86; HR 1.83, 95 % CI 0.92–3.65, respectively). Conclusions Our results show that upon optimizing and aligning four RET methylation assays with regard to primer location and sensitivity, differences in methylation frequencies and clinical sensitivities are observed; however, the effect on the marker’s prognostic outcome is minimal. Electronic supplementary material The online version of this article (doi:10.1186/s13148-016-0211-8) contains supplementary material, which is available to authorized users.

IF 7.760; International audience; CD8(+) T cells participate in the pathogenesis of some vasculitides. However, little is known about their role in Giant Cell Arteritis (GCA). This study was conducted to investigate CD8(+) T cell involvement in the pathogenesis of GCA.Analyses were performed at diagnosis and after 3 months of glucocorticoid treatment in 34 GCA patients and 26 age-matched healthy volunteers . Percentages of CD8(+) T-cell subsets, spectratype analysis of the TCR v beta families of CD8(+) T cells, levels of cytokines and chemokines and immunohistochemistry of temporal artery biopsies (TAB) were assessed.Among total CD8+ T cells, percentages of circulating cytotoxic CD8 T lymphocytes (CTL, CD3(+)CD8(+)perforin(+)granzymeB(+)), Tc17 (CD3(+)CD8(+)IL-17(+)), CD63(+)CD8(+) T cells and levels of soluble granzymes A and B were higher in patients than in controls, whereas the percentage of Tc1 cells (CD3(+)CD8(+)IFN-gamma(+)) was similar. Moreover, CD8+ T cells displayed a restricted TCR repertoire in GCA patients. Percentages of circulating CTL, Tc17 and soluble levels of granzymes A and B decreased after treatment. CXCR3 expression on CD8(+) T cells and its serum ligands (CXCL9, -10, -11) were higher in patients. Analyses of TAB revealed high expression of CXCL9 and -10 associated with infiltration by CXCR3(+)CD8(+) T cells expressing granzyme B and TiA1. The intensity of the CD8 T-cell infiltrate in TAB was predictive of the severity of the disease.This study demonstrates the implication and the prognostic value of CD8(+) T-cells in GCA and suggests that CD8(+) T-cells are recruited within the vascular wall through an interaction between CXCR3 and its ligands. (C) 2016 Elsevier Ltd. All rights reserved.

Purpose: Data on the prognostic significance of promoter CpG island methylation in colorectal cancer (CRC) are conflicting, possibly due to associations between methylation and other factors affecting survival such as genetic alterations and use of adjuvant therapy. Here, we examine the prognostic impact of promoter methylation in patients with CRC treated with surgery alone in the context of microsatellite instability (MSI), BRAF and KRAS mutations. Experimental Methods: One hundred and seventy-three CRCs were analyzed for promoter methylation of 19 tumor suppressor and DNA repair genes, the CpG island methylator phenotype (CIMP), MSI, the exon 15 V600E BRAF mutation and KRAS codon 12 and 13 mutations. Results: Unsupervised hierarchical clustering based on methylation status of 19 genes revealed three subgroups: cluster 1 [CL1, 57% (98/173) of CRCs], cluster 2 [CL2, 25% (43/173) of CRCs], and cluster 3 [CL3, 18% (32/173) of CRCs]. CL3 had the highest methylation index (0.25, 0.49, and 0.69, respectively, P = Conclusions: CHFR promoter CpG island methylation, which is associated with MSI, also occurs frequently in MSS CRCs and is a promising prognostic marker in stage II, MSS, BRAF WT CRCs. Clin Cancer Res; 20(12); 3261–71. ©2014 AACR.

Les ferriers romains du domaine des Forges (Les Martys, Aude) sont aujourd'hui assez bien connus : chronologie (de 60/50 avant J.-C. a 260 environ apres J.-C.), volume de scories, production minimale de fer assuree (37 000 t), bas fourneaux et ateliers du Ier s. avant J.-C., etc. En 1998, une prospection menee au voisinage du domaine, dans le bassin superieur de la Dure, a eu pour but de situer ces vestiges dans leur contexte siderurgique. Elle a permis de reperer 33 ferriers, qui s'ajoutent aux 11 deja connus. La plupart d'entre eux sont surement d'epoque romaine, comme le montre le materiel qu'ils renferment (amphores Dressel 1 principalement et/ou sigillee). On remarque que l'activite du secteur s'est concentree, sans doute a partir de la fin du Ier s. avant J.-C., sur deux sites principaux, le Grand Ferrier des Forges et le ferrier de Laprade-Basse. Enfin, une estimation calculee du volume de tous ces ferriers represente un total de pres de 300 000 m3, ce qui correspond a une production d'environ 80 000 t de fer pendant un peu plus de trois siecles. Il est difficile d'interpreter historiquement ces donnees archeologiques. Peut-etre au Ier s. avant J.-C. le secteur etait-il aux mains d'une societe de publicains, comme invite a le penser une comparaison avec les mines de la haute vallee de l'Orb (Herault). Par la suite, cette concentration s'est maintenue, mais on ignore tout de son organisation.

Background In colorectal carcinoma, extensive gene promoter hypermethylation is called the CpG island methylator phenotype (CIMP). Explaining why studies on CIMP and survival yield conflicting results is essential. Most experiments to measure DNA methylation rely on the sodium bisulfite conversion of unmethylated cytosines into uracils. No study has evaluated the performance of bisulfite conversion and methylation levels from matched cryo-preserved and Formalin-Fixed Paraffin Embedded (FFPE) samples using pyrosequencing. Methods Couples of matched cryo-preserved and FFPE samples from 40 colon adenocarcinomas were analyzed. Rates of bisulfite conversion and levels of methylation of LINE-1, MLH1 and MGMT markers were measured. Results For the reproducibility of bisulfite conversion, the mean of bisulfite-to-bisulfite standard deviation (SD) was 1.3%. The mean of run-to-run SD of PCR/pyrosequencing was 0.9%. Of the 40 DNA couples, only 67.5%, 55.0%, and 57.5% of FFPE DNA were interpretable for LINE-1, MLH1, and MGMT markers, respectively, after the first analysis. On frozen samples the proportion of well converted samples was 95.0%, 97.4% and 87.2% respectively. For DNA showing a total bisulfite conversion, 8 couples (27.6%) for LINE-1, 4 couples (15.4%) for MLH1 and 8 couples (25.8%) for MGMT displayed significant differences in methylation levels. Conclusions Frozen samples gave reproducible results for bisulfite conversion and reliable methylation levels. FFPE samples gave unsatisfactory and non reproducible bisulfite conversions leading to random results for methylation levels. The use of FFPE collections to assess DNA methylation by bisulfite methods must not be recommended. This can partly explain the conflicting results on the prognosis of CIMP colon cancers.

Aim: Genomic quantitative alteration is a backbone event during the carcinogenesis process in microsatellite stable colon cancer. However, the prognostic value of loss or gain of the main genomic regions is still controversial. We conducted a prospective study to assess the prognostic impact of the main genomic quantitative alterations in stage II-III MSS colon cancer (NCT02110329). Methods: Patients treated for stage II-III colon cancer from 01/2002 to 01/2009 with available frozen tissue were included. The Quantitative Multiplex PCR of Short Fluorescent Fragments (QMPSF) method was used for molecular screening (1). The QMPSF was based on the simultaneous amplification, from normal and tumoral tissues, of 8 selected target genomic sequences according to literature data: DCC (18q21); EGFR (7p12); TP53 (17p13.1); BLK (8p23-p22); MYC (8q24.12); APC (5q22.2); ERBB2 (17q12); STK6 (20q13.31); and two control: PCBD2 (5q31.1); HMBS (11q23.3). The primary end-point was to determine the association between molecular alterations and disease-free survival (DSF). Combinations of alterations were defined according to univariate results (p Results: A total of 401 patients were included with a median follow-up of 48 months. There were 236 stage II and 165 stage III and the recurrence rate was 30.2%. Loss of DCC/18q, TP53/17p, BLK/8p, and APC/5q were detected in respectively 33.2%, 26.7%, 22.7%, 9.7% and gain of EGFR/7p, MYC/8q, ERBB2/17q and STK6/20q were observed in 31.7%, 35.7%, 18.7% and 48.9% of cases, respectively. There was no significant interaction between alterations and disease stage. DFS was significantly associated with loss of DCC/18q (57.5 vs 70.6%, p = 0.02) with a trend for BLK/8p loss (59.8 vs 68.6%, p = 0.08) and ERBB2/17q gain (59.2 vs 68.5%, p = 0.14). In multivariate analysis, the combination of loss DCC/18q and/or loss BLK/8p and/or gain ERBB2/17q was significantly associated with DFS. The DFS significantly decreased from 74.2% to 61.7% (HR = 1.78, 95CI: 1.14-2.77) and to 57.2% (HR =2.06, 95CI: 1.26-3.37) in patients with respectively none, 1 and at least 2 of these 3 alterations. Conclusions: Combination of DCC/18q, BLK/8p and ERBB2/17q gene copy number alterations is associated with disease-free survival in stage II-III colon cancer. Disclosure: All authors have declared no conflicts of interest.

Tournier Benjamin. Sur un usage particulier des outils en pierre polie chez les populations pastorales des Hautes-Alpes françaises.. In: Bulletins de la Société d'anthropologie de Paris, II° Série. Tome 9, 1874. pp. 686-689.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy characterized by an accumulation of immature T cells. Although patient outcomes have improved, novel targeted therapies are needed to reduce the intensity of chemotherapy and improve the prognosis of high-risk patients. Interleukin-7 (IL-7) modulates the survival and proliferation of normal and malignant T cells. Targeting the IL-7 signaling pathway is thus a potentially effective therapeutic strategy. To achieve such aim, it is essential to first understand how the IL-7 signaling pathway is activated. Although IL-7 production has been observed from multiple stromal tissues, T-ALL autocrine IL-7 secretion has not yet been described. Interestingly, using T-ALL cell lines, primary and patient-derived xenotransplanted (PDX) T-ALL cells, we demonstrate that T-ALL cells produce IL-7 whereas normal T cells do not. Finally, using knock down of IL7 gene in T-ALL cells, we describe to what extent IL-7 autocrine secretion is involved in the T-ALL cells propagation in bone marrow and how it affects the number of leukemia-initiating cells in PDX mice. Together, these results demonstrate how the autocrine production of the IL-7 cytokine mediated by T-ALL cells can be involved in the oncogenic development of T-ALL and offer novel insights into T-ALL spreading.