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Introduction

Kurtz: Genentech: Consultancy; Foresight Diagnostics: Other: Ownership; Roche: Consultancy. Khodadoust:Kyowa Kirin: Consultancy; Seattle Genetics: Consultancy. Nadel:Innate Pharma: Research Funding; Institut Roche: Research Funding. Diehn:RefleXion: Consultancy; Varian Medical Systems: Research Funding; Illumina: Research Funding; Roche: Consultancy; BioNTech: Consultancy; AstraZeneca: Consultancy. Roulland:Celgene/BMS: Research Funding; Roche: Honoraria. Alizadeh:Pharmacyclics: Consultancy; Genentech: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Chugai: Consultancy; Gilead: Consultancy; Pfizer: Research Funding; Roche: Consultancy.

Background:Follicular lymphoma (FL) is genetically characterized by translocations involving the BCL2locus on chromosome 18q21. However, up to 70% of healthy individuals also carry detectable t(14;18)-positive cells, suggesting BCL2translocation is critical but not sufficient for FL development.

Background:

Introduction:Plamotamab (XmAb13676) is a humanized bispecific antibody that binds both CD20 and CD3 to recruit cytotoxic T cells to kill CD20 expressing malignant cells. Interim results of an ongoing first-in-human (FIH), dose-escalation study (XmAb13676-01; NCT02924402) in subjects with relapsed/refractory (R/R) non-Hodgkin's lymphoma (NHL) are reported.

Il ressort de l'étude de deux cent quatre-vingt-dix souches de Bacillus provenant de quatre milieux aquatiques différents : lac hypersalé, atoll du Pacifique, eaux de rivières et de mer, que dix espèces prédominent, les plus fréquentes étant B. cereus, B. megaterium, B. licheniformis, B. subtilis ; les espèces B. pumilus, B. firmus, B. lentus, B. laterosporus, B. brevis et B. sphaericus sont présentes, mais fréquemment isolées.En fonction des milieux étudiés certaines espèces sont favorisées ; au sein de chaque espèce des différences portant sur les caractères biochimiques sont notées en fonction de l'environnement.

L'atrazine est un des herbicides les plus couramment utilisés en agriculture. Ses propriétés toxiques au niveau hépatique chez le rat et ses produits de transformation in vivo sont connus.Cette étude a consisté à comparer la métabolisation in vitro de ce pesticide sur différents modèles : d'une part, sur lignées cellulaires d'hépatomes humains (cellules Hep G2 et Hep 3B) et d'autre part, sur des microsomes hépatiques humains et de rat. L'influence de la concentration en atrazine sur le taux de métabolisation a été étudiée. La détermination des metabolites de ce xénobiotique a été effectuée par CLHP, en employant deux méthodes de traitement des échantillons : extraction au dichlorométhane et déprotéinisation-lyophilisation.Les cellules Hep G2 et les microsomes produisent les mêmes composés : métabolites monodéalkylés, mais en proportions différentes selon les modèles.

In a study of a lake having a higher concentration of salts than the Dead Sea, all of the heterotrophic bacteria isolated were aerobes; no strictly anaerobic strains were found. Ninety percent of the strains were euryhalines and ten percent were strict halophiles. The extreme halophiles belonged to the species Halobacterium trapanicumand Halococcus morrhuae.

Molecular magnetic resonance imaging allows noninvasive evaluation of intestinal mucosal inflammation in clinically relevant mouse models.

A 34-year-old man had an unusual mummified cutaneous mixed tumor that developed shortly after trauma. The apocrine type of tumor cells and pilar differentiation could explain the presence of mummification.

Follicular lymphoma (FL) is a prototypical example of germinal center (GC) derived B-cell lymphoma. Using a mouse model recapitulating the sporadic occurrence of the FL hallmark BCL2/IGHtranslocation in healthy individuals, our previous work demonstrated that FL genesis is a dynamic process that requires multiple re-entries of BCL2+memory B-cells into the GC to ultimately accumulate in lymphoid organs. In line with this, using single-cell gene expression analysis of human FL vs normal GC B-cells, we recently discovered that FL cells are not ‘frozen’ at a particular GC maturation stage but instead exhibit a major desynchronization of the GC-specific expression program (Milpied et al. Nat Immunol in press). Since KMT2Dloss-of-function mutations and BCL2translocations are the 2 main alterations in FL, we hypothesized these 2 genetic events might explain the GC program desynchronization we observe in humans.

New insights in the pathogenesis of the splenic marginal zone lymphoma (SMZL) has been recently enlightened by next generation sequencing technology discovering recurrent mutations in several genes implicated in major signalling pathways. In 2 recent reports (JEM 2012), mutations in NOTCH2, a gene encoding a protein required for marginal zone B-cell development, were identified in nearly 25% of the patients, with controversial impact on clinical outcome. The aim of our analysis was to analyse the incidence and the prognostic impact of somatic NOTCH2 mutations in a large cohort of patients with SMZL homogeneously treated.A series of 105 consecutive SMZL patients requiring treatment and referred in 2 expert centers was analysed. Genomic DNA was extracted from frozen tumor samples of involved spleen or blood or bone marrow obtained at diagnosis. Tumor cell clonality was established by amplification of the rearranged IGH genes. Targeted sequencing of the NOTCH2 C-terminal coding exons 26, 27 and 34 was performed using Sanger sequencing and primers described by Kiel MJ et al (J.Exp.Med.209:9,2012). All mutations were verified in at least two independent PCR amplification and sequencing reactions. Log-rank tests were used to compare survival times -overall survival (OS) and progression-free survival (PFS)- between patients with mutated NOTCH2 and patient with wild-type NOTCH2. We controlled the effects of prognostic factors on outcome using multivariate Cox model analysis.The median follow-up was 7 years. At diagnosis, median age was 63 years. Sex ratio was M/F 1:1.35. Nearly all patients presented with a disseminated disease and a good performance status. Forty-four percent of the patients had a high LDH level. A monoclonal component was detected in 31% of the patients. An immunological event such as haemolytic anemia, thrombocytopenia, neutropenia, coagulation disorder, and cryoglobulinemia, was observed in 27% of the patients. None of the patients was infected with hepatitis C virus. Patients were scored according to the age-adjusted International Prognostic Index (n=95), and to the IIL score system (n=76). Splenectomy was realized in 97% of the patients. Three patients were treated with R-Bendamusine without splenectomy. Adjuvant treatment after splenectomy was proposed for 24% of the patients, and included CHOP+/-R (n=16), HLX-VP16 (n=1), chlorambucil (n=4), and rituximab alone (n=1). Five and 10-year OS was estimated at 82% and 69%, respectively. Five and 10-year PFS was estimated at 61% and 46%, respectively. Age>60 was significantly associated to a shorter OS (p=0.0135).We identified NOTCH2 mutations in 10 (10%) of the patients. The detected mutations occurred only within the exon 34 (TAD and PEST domains) and represented mostly truncating or insertional events leading to frameshift mutations (8 of the 10 cases) (also 1 nonsense and 1 missense mutations). Comparison of the clinical characteristics at diagnosis between patients with or without NOTCH2 mutations did not show any significant differences. Patients with NOTCH2 mutations were characterized by a significantly worse OS (at 5 years, 32% vs 87%, p<0.0001). Presence of NOTCH2 mutations was also significantly associated with a shorter PFS (at 5-years, 16% vs 66%; p=.0008), and with a shorter time to histological transformation (at 5 years, 26% vs 6%; p=0.0051). NOTCH2 mutational status did not influence the time to first treatment. Multivariate analysis showed an independent prognostic impact of NOTCH2 mutational status on OS (RR 4.7, p=0.02).NOTCH2 mutation was identified in 10% of the SMZL patients in our series. Our data demonstrate also, as described, than patients with NOTCH2 mutations have a significant poorer prognosis than patients without NOTCH2 mutations, with a shorter PFS and a shorter OS. NOTCH2 mutational status seems to be an independent prognostic factor. This needs to be confirmed in prospective trials in the context of new therapeutics. CA and GB equally contributed Coiffier: Millennium Pharmaceuticals : Consultancy.

New insights in the pathogenesis of the splenic marginal zone lymphoma (SMZL) has been recently enlightened by next generation sequencing technology discovering recurrent mutations in several genes implicated in major signalling pathways. In 2 recent reports (JEM 2012), mutations in NOTCH2, a gene encoding a protein required for marginal zone B-cell development, were identified in nearly 25% of the patients, with controversial impact on clinical outcome. The aim of our analysis was to analyse the incidence and the prognostic impact of somatic NOTCH2mutations in a large cohort of patients with SMZL homogeneously treated.

No relevant conflicts of interest to declare.

The JAK2V617F point mutation has been described in patients with Philadelphia negative myeloproliferative disorders. Improvement in the sensitivity of different methods has lowered the detection threshold of the percentage of mutant allele but currently, there is no consensus on which level of sensitivity is adequate and how to deal with the lowest levels of mutant allele. Moreover, development of JAK2 inhibitors sets the question of monitoring the molecular residual disease. These reinforce the need of the availability of reliable quantitative assays with high sensitivity and of the definition of the limit of significance for a positive result. The aim of this study was thus to establish a threshold for JAK2V617F detection between normal and potential MPD samples. We used the Jak2 MutaQuant™ kit (Ipsogen, Marseille, France), a quantitative highly sensitive allele specific RQ-PCR test on 200 randomly selected healthy individuals (among 366 volunteers). The study was approved by the Institutional Review Board and informed consent was provided according to the Declaration of Helsinki. Exclusion criteria were: age under 18, medical history of deep thrombosis or blood disease, haemoglobin level over 185 g/L for men and over 165 g/L for women, hematocrit over 52% for men and over 48% for women, white blood cell count (WBC) over 12 G/L and platelet count over 400 G/L. Median age was 37 years (19–88), 69% were male, median haemoglobin level was 142 g/L (SD 17.8), median hematocrit was 41.9% (SD 4.8), median WBC was 6.82 (SD 1.8) and median platelet count was 248 (SD 58.2). In parallel, 24 patients presenting with a thrombocytosis or a polycythemia were tested. Genomic DNA was extracted from whole blood. The technology used in this assay allows a sensitive, accurate and highly reproducible detection of single-nucleotide polymorphisms. This technique is based on the use of specific forward primers, for the wild type (WT) and the V617F allele, a common reverse primer, and a double dye TaqMan probe. Only perfect match between primer and target DNA allows extension and amplification in the PCR reaction. This assay benefits from the plasmid technology which allows the precise calibration and normalisation of RQ-PCR results, by generating standard curves based on the known concentration of plasmid dilutions, and precise measurement of the JAK2 WT and V617F alleles copy number in human cell samples without the need of subsequent handling such as capillary electrophoresis or melting curve analysis. Normalized results can be compared between RQ-PCR systems and testing sites. Results are expressed as percentage of JAK2V617F among total JAK2 detected sequences. Limit of blank was determined (95th percentile) on negative samples (n=56) and is approximately equal to 0.04%. Limit of detection was determined (95th percentile) on known low positive samples (n=96) and is approximately equal to 0.2%. As expected, the sensitivity of the test varies according to the total number of JAK2 sequences detectable in 25ng of genomic DNA. In our hands the median sensitivity of the test was 0.1% of mutant alleles (range 0.28–0.038). In all our healthy donor samples the percentage of mutant alleles was lower than 0.1% (% range 0.002–0.085). Among the 24 MPD suspected patients, 11 were positive (range 0.6–62%). Our data contribute to on-going questions regarding the interpretation of JAK2V617F allele burden. Levels of mutation that are significantly above those seen in healthy individuals, even low, should be clearly of clinical interest. They should benefit of a haematological follow up as they might represent a very early stage of a myeloproliferative disorder.

The JAK2V617Fpoint mutation has been described in patients with Philadelphia negative myeloproliferative disorders. Improvement in the sensitivity of different methods has lowered the detection threshold of the percentage of mutant allele but currently, there is no consensus on which level of sensitivity is adequate and how to deal with the lowest levels of mutant allele. Moreover, development of JAK2 inhibitors sets the question of monitoring the molecular residual disease. These reinforce the need of the availability of reliable quantitative assays with high sensitivity and of the definition of the limit of significance for a positive result. The aim of this study was thus to establish a threshold for JAK2V617Fdetection between normal and potential MPD samples. We used the Jak2 MutaQuant™ kit (Ipsogen, Marseille, France), a quantitative highly sensitive allele specific RQ-PCR test on 200 randomly selected healthy individuals (among 366 volunteers). The study was approved by the Institutional Review Board and informed consent was provided according to the Declaration of Helsinki. Exclusion criteria were: age under 18, medical history of deep thrombosis or blood disease, haemoglobin level over 185 g/L for men and over 165 g/L for women, hematocrit over 52% for men and over 48% for women, white blood cell count (WBC) over 12 G/L and platelet count over 400 G/L. Median age was 37 years (19–88), 69% were male, median haemoglobin level was 142 g/L (SD 17.8), median hematocrit was 41.9% (SD 4.8), median WBC was 6.82 (SD 1.8) and median platelet count was 248 (SD 58.2). In parallel, 24 patients presenting with a thrombocytosis or a polycythemia were tested. Genomic DNA was extracted from whole blood. The technology used in this assay allows a sensitive, accurate and highly reproducible detection of single-nucleotide polymorphisms. This technique is based on the use of specific forward primers, for the wild type (WT) and the V617F allele, a common reverse primer, and a double dye TaqMan probe. Only perfect match between primer and target DNA allows extension and amplification in the PCR reaction. This assay benefits from the plasmid technology which allows the precise calibration and normalisation of RQ-PCR results, by generating standard curves based on the known concentration of plasmid dilutions, and precise measurement of the JAK2 WT and V617F alleles copy number in human cell samples without the need of subsequent handling such as capillary electrophoresis or melting curve analysis. Normalized results can be compared between RQ-PCR systems and testing sites. Results are expressed as percentage of JAK2V617Famong total JAK2 detected sequences. Limit of blank was determined (95thpercentile) on negative samples (n=56) and is approximately equal to 0.04%. Limit of detection was determined (95thpercentile) on known low positive samples (n=96) and is approximately equal to 0.2%. As expected, the sensitivity of the test varies according to the total number of JAK2 sequences detectable in 25ng of genomic DNA. In our hands the median sensitivity of the test was 0.1% of mutant alleles (range 0.28–0.038). In all our healthy donor samples the percentage of mutant alleles was lower than 0.1% (% range 0.002–0.085). Among the 24 MPD suspected patients, 11 were positive (range 0.6–62%). Our data contribute to on-going questions regarding the interpretation of JAK2V617Fallele burden. Levels of mutation that are significantly above those seen in healthy individuals, even low, should be clearly of clinical interest. They should benefit of a haematological follow up as they might represent a very early stage of a myeloproliferative disorder.