Key Points• The MYD88 locus is altered in 91% of patients with WM. • MYD88 might be new target for therapeutic in WM.Mutation of the MYD88 gene has recently been identified in activated B-cell-like diffuse cell lymphoma and enhanced Janus kinase/signal transducer and activator of transcription (JAK-STAT) and nuclear factor kB (NF-kB) signaling pathways. A whole exome-sequencing study of Waldenstrom macroglobulinemia (WM) suggested a high frequency of MYD88 L265P mutation in WM. The genetic background is not fully deciphered in WM, although the role of NF-kB and JAK-STAT has been demonstrated. We analyzed MYD88 mutation in exon 5 and characterized the clinical significance of this genetic alteration in 67 WM patients. Clinical features; immunophenotypic markers; and conventional cytogenetic, fluorescence in situ hybridization, and single nucleotide polymorphism array data were analyzed. MYD88 L265P mutation was acquired in 79% of patients. Overall, we have identified alteration of the MYD88 locus in 91% of WM patients, including 12% with gain on chromosome 3 at the 3p22 locus that included the MYD88 gene. Patients with absence of MYD88 mutation were WM characterized with a female predominance, a splenomegaly, gain of chromosome 3, and CD27 expression. Importantly, inhibition of MYD88 signaling induced cytotoxicity and inhibited cell growth of cell lines issued from patients with WM. In conclusion, these results confirm a high frequency of MYD88 L265P mutation in WM. The discovery of MYD88 L265P mutation may contribute to a better understanding of the physiopathogeny of WM. (Blood. 2013;121(22):4504-4511)

Automated selection was used to evolve an Escherichia coli strain unable to synthesize thymine nucleotides into a chemically modified organism whose DNA genome is composed of adenine, cytosine, guanine, and an artificial base, the thymine analogue 5‐chlorouracil. Evolving cells were initially observed as irregular filaments and progressively recovered the appearance of short rods typical of wild‐type E. coli (see picture).

BackgroundClostridium sticklandii belongs to a cluster of non-pathogenic proteolytic clostridia which utilize amino acids as carbon and energy sources. Isolated by T.C. Stadtman in 1954, it has been generally regarded as a "gold mine" for novel biochemical reactions and is used as a model organism for studying metabolic aspects such as the Stickland reaction, coenzyme-B12- and selenium-dependent reactions of amino acids. With the goal of revisiting its carbon, nitrogen, and energy metabolism, and comparing studies with other clostridia, its genome has been sequenced and analyzed.ResultsC. sticklandii is one of the best biochemically studied proteolytic clostridial species. Useful additional information has been obtained from the sequencing and annotation of its genome, which is presented in this paper. Besides, experimental procedures reveal that C. sticklandii degrades amino acids in a preferential and sequential way. The organism prefers threonine, arginine, serine, cysteine, proline, and glycine, whereas glutamate, aspartate and alanine are excreted. Energy conservation is primarily obtained by substrate-level phosphorylation in fermentative pathways. The reactions catalyzed by different ferredoxin oxidoreductases and the exergonic NADH-dependent reduction of crotonyl-CoA point to a possible chemiosmotic energy conservation via the Rnf complex. C. sticklandii possesses both the F-type and V-type ATPases. The discovery of an as yet unrecognized selenoprotein in the D-proline reductase operon suggests a more detailed mechanism for NADH-dependent D-proline reduction. A rather unusual metabolic feature is the presence of genes for all the enzymes involved in two different CO2-fixation pathways: C. sticklandii harbours both the glycine synthase/glycine reductase and the Wood-Ljungdahl pathways. This unusual pathway combination has retrospectively been observed in only four other sequenced microorganisms.ConclusionsAnalysis of the C. sticklandii genome and additional experimental procedures have improved our understanding of anaerobic amino acid degradation. Several specific metabolic features have been detected, some of which are very unusual for anaerobic fermenting bacteria. Comparative genomics has provided the opportunity to study the lifestyle of pathogenic and non-pathogenic clostridial species as well as to elucidate the difference in metabolic features between clostridia and other anaerobes.

Purpose: Whole-genome sequencing has revealed MYD88 L265P and CXCR4 mutations (CXCR4 mut ) as the most prevalent somatic mutations in Waldenstr€ om macroglobulinemia. CXCR4 mutation has proved to be of critical importance in Waldenstr€ om macroglobulinemia, in part due to its role as a mechanism of resistance to several agents. We have therefore sought to unravel the different aspects of CXCR4 mutations in Waldenstr€ om macroglobulinemia.Experimental Design: We have scanned the two coding exons of CXCR4 in Waldenstr€ om macroglobulinemia using deep nextgeneration sequencing and Sanger sequencing in 98 patients with Waldenstr€ om macroglobulinemia and correlated with SNP array landscape and mutational spectrum of eight candidate genes involved in TLR, RAS, and BCR pathway in an integrative study.Results: We found all mutations to be heterozygous, somatic, and located in the C-terminal domain of CXCR4 in 25% of the Waldenstr€ om macroglobulinemia. CXCR4 mutations led to a truncated receptor protein associated with a higher expression of CXCR4. CXCR4 mutations pertain to the same clone as to MYD88 L265P mutations but were mutually exclusive to CD79A/CD79B mutations (BCR pathway). We identified a genomic signature in CXCR4 mut Waldenstr€ om macroglobulinemia traducing a more complex genome. CXCR4 mutations were also associated with gain of chromosome 4, gain of Xq, and deletion 6q.Conclusions: Our study panned out new CXCR4 mutations in Waldenstr€ om macroglobulinemia and identified a specific signature associated to CXCR4 mut , characterized with complex genomic aberrations among MYD88L265P Waldenstr€ om macroglobulinemia. Our results suggest the existence of various genomic subgroups in Waldenstr€ om macroglobulinemia.

Key Points• Superiority of R-ACVBP over R-CHOP14 was not established, as IHP criteria driving consolidation did not properly reflect disease control.• The 26% PET2 2 /PET4 2 patients using IHP criteria increased to 79% using DSUVmax, which may help better select those needing an alternative to SIC.Dose-dense induction and up-front consolidation with autologous stem cell transplantation (ASCT) remain controversial issues when treating patients with high-risk diffuse large B-cell lymphoma. GELA designed a randomized phase 2 trial evaluating the efficacy of either rituximab, doxorubicin, cyclophosphamide, vindesine, bleomycin, prednisone (R-ACVBP) or rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone (R-CHOP14) induction and a positron emission tomography (PET)-driven ASCT or standard immunochemotherapy (SIC) consolidation in age-adjusted international prognosis index 2 (aaIPI2)-aaIPI3 patients. PET was performed at baseline, after 2 (PET2) and 4 (PET4) induction cycles, and centrally assessed using international harmonization project (IHP) criteria. PET2 2 / PET4 2 patients were assigned SIC, PET2 1 /PET4 2 patients were assigned ASCT, and PET4 1 patients were treated with the investigator's choice. The primary end-point was the 2007 international working group complete response (CR) rate after induction. Change in maximum standard uptake value (DSUVmax) after PET assessment was explored. Two hundred eleven patients were randomly assigned to R-ACVBP (n 5 109) or R-CHOP14 (n 5 102). PET4 2 /CR rates were 53%/47% with R-ACVBP and 41%/39% with R-CHOP14 (CR 95% confidence interval [CI], 38%-67% and 28%-54%, respectively; P 5 .076). Consolidation in the R-ACVBP and R-CHOP14 groups was SIC in 26% and 23% of patients and ASCT in 28% and 18% of patients, respectively. PET4 positivity was higher with R-CHOP14 vs R-ACVBP (54% vs 41%; P 5 .08), leading to more salvage therapy (37% vs 26%; P 5 .07) and lower event-free survival (EFS; 4-year EFS, 31% vs 43%; P < .01), but progression-free survival (PFS) and overall survival (OS) were similar in both groups. PET2 2 /PET42 and PET2 1 /PET4 2 patients had similar outcomes. Using DSUVmax, 79% of the patients were PET2 2 /PET4 2 . DSUVmaxPET0-4 >70% was associated with better outcome (4-year PFS, 84% vs 35%; 4-year OS, 91% vs 57%; P < .0001), whatever the consolidation. Superiority of R-ACVBP over R-CHOP14 was not established, as IHP criteria did not properly reflect disease control. DSUVmax may help better select patients needing an alternative to SIC, including ASCT. (Blood. 2017;130(11):1315-1326

The recent introduction of mass cytometry, a technique coupling a cell introduction system generating a stream of single cells with mass spectrometry, has greatly increased the number of parameters that can be measured per single cell. As with all new technology there is a need for dissemination of standardization and quality control procedures. Here, we characterize variations in sensitivity observed across the mass range of a mass cytometer, using different lanthanide tags. We observed a five-fold difference in lanthanide detection over the mass range and demonstrated that each instrument has its own sensitivity pattern. Therefore, the selection of lanthanide combinations is a key step in the establishment of a staining panel for mass cytometry-based experiments, particularly for multicenter studies. We propose the sensitivity pattern as the basis for panel design, instrument standardization and future implementation of normalization algorithms. V C 2015 International Society for Advancement of Cytometry Key terms Key terms: mass cytometry; CyTOF; standardization; flow cytometry CYTOMETRY by time-of-flight (CyTOF) is a recently developed technique allowing the simultaneous detection of more than 40 parameters at the single cell level. CyTOF technology is novel in that it makes use of antibodies or other specific probes conjugated with pure metal isotopes to label cells that are finally detected by atomic mass spectrometry (1). CyTOF technology has recently been used to characterize signaling pathways in hematopoietic cells (2,3), to analyze cytokine expression and phenotype in detail in antigen-specific CD8 T cells (4) and to map cell-cycle phases in PBMCs and bone marrow aspirates (5). The advantages of this new technology over conventional fluorescence-based flow cytometry were described in detail in a recent review (6). In short, whereas conventional flow cytometry can be used to measure fluorescence in a maximum of 18 channels, with considerable spectral overlap requiring ad hoc mathematical processing, CyTOF technology makes it possible to measure more than 40 different parameters, with minimal spectral overlap. The fluorochromes used in conventional flow cytometry include small organic molecules, such as FITC, and larger proteins, such as allophycocyanin, which may differ considerably in molecular weight and chemical properties. In addition, each fluorochrome has its own unique excitation and emission spectra and its spillover characteristics are therefore different from those of other fluorochromes. These properties strongly affect the sensitivity of the assay, the performance of which is dependent on the combination

is a tumor-suppressor gene that functions as a regulator influencing cellular responses to DNA damage, and alterations are associated with pejorative outcome in most B-lymphoid disorders. Little is known regarding alteration in Waldenstrom's macroglobulinemia (WM). Here, we have explored the incidence of alteration using Sanger sequencing and ultradeep-targeted sequencing in 125 WM and 10 immunoglobulin M (IgM) monoclonal gammopathy of undetermined significance (MGUS), along with the clinical features and the associated genomic landscape using single-nucleotide polymorphism array and mutational landscape in an integrative study. Overall, we have identified alteration of locus including mutation, deletion, and copy-neutral LOH in 11.2% of WM. mutation was acquired in 7.3% of patients with WM at diagnosis, being absent in IgM MGUS, and was highly correlated to deletion 17p. No correlation with mutations was observed. Patients with alteration had a greater number of genomic abnormalities. Importantly, WM with alteration had a significantly shorter overall survival, particularly in symptomatic WM, and independently of the international prognostic scoring system for Waldenstrom macroglobulinemia (IPSSWM) score. Specific treatment for WM with may have to be studied. Nutlin-3a-targeted p53 signaling induced cytotoxicity preclinically, along with new compounds such as ibrutinib, Prima, or CP31398 that bypass p53 pathway in WM, paving the path for future treatment-tailored options. Our results highlight the clinical significance of detection of 3 alteration in WM to determine the prognosis of WM and guide the treatment choice..

For the ornithine fermentation pathway, described more than 70 years ago, genetic and biochemical information are still incomplete. We present here the experimental identification of the last four missing genes of this metabolic pathway. They encode L-ornithine racemase, (2R,4S)-2,4-diaminopentanoate dehydrogenase, and the two subunits of 2-amino-4-ketopentanoate thiolase. While described only for the Clostridiaceae to date, this pathway is shown to be more widespread.