Clonal and subclonal TP53 molecular impairment is associated with prognosis and progression in multiple myeloma.

Aberrations on TP53, either as deletions of chromosome 17p (del17p) or mutations, are associated with poor outcome in multiple myeloma (MM), but conventional detection methods currently in use underestimate their incidence, hindering an optimal risk assessment and prognostication of MM patients. We have investigated the altered status of TP53 gene by SNPs array and sequencing techniques in a homogenous cohort of 143 newly diagnosed MM patients, evaluated both at diagnosis and at first relapse: single-hit on TP53 gene, either deletion or mutation, detected both at clonal and sub-clonal level, had a minor effect on outcomes. Conversely, the coexistence of both TP53 deletion and mutation, which defined the so-called double-hit patients, was associated with the worst clinical outcome (PFS: HR 3.34 [95% CI: 1.37-8.12] p = 0.008; OS: HR 3.47 [95% CI: 1.18-10.24] p = 0.02). Moreover, the analysis of longitudinal samples pointed out that TP53 allelic status might increase during the disease course. Notably, the acquisition of TP53 alterations at relapse dramatically worsened the clinical course of patients. Overall, our analyses showed these techniques to be highly sensitive to identify TP53 aberrations at sub-clonal level, emphasizing the poor prognosis associated with double-hit MM patients.

Interlaboratory evaluation of Mucorales PCR assays for testing serum specimens: A study by the fungal PCR Initiative and the Modimucor study group.

Interlaboratory evaluations of Mucorales qPCR assays were developed to assess the reproducibility and performance of methods currently used. The participants comprised 12 laboratories from French university hospitals (nine of them participating in the Modimucor study) and 11 laboratories participating in the Fungal PCR Initiative. For panel 1, three sera were each spiked with DNA from three different species (Rhizomucor pusillus, Lichtheimia corymbifera, Rhizopus oryzae). For panel 2, six sera with three concentrations of R. pusillus and L. corymbifera (1, 10, and 100 genomes/ml) were prepared. Each panel included a blind negative-control serum. A form was distributed with each panel to collect results and required technical information, including DNA extraction method, sample volume used, DNA elution volume, qPCR method, qPCR template input volume, qPCR total reaction volume, qPCR platform, and qPCR reagents used. For panel 1, assessing 18 different protocols, qualitative results (positive or negative) were correct in 97% of cases (70/72). A very low interlaboratory variability in Cq values (SD = 1.89 cycles) were observed. For panel 2 assessing 26 different protocols, the detection rates were high (77-100%) for 5/6 of spiked serum. There was a significant association between the qPCR platform and performance. However, certain technical steps and optimal combinations of factors may also impact performance. The good reproducibility and performance demonstrated in this study support the use of Mucorales qPCR as part of the diagnostic strategy for mucormycosis.

Azole-resistant Aspergillus fumigatus: A global phenomenon originating in the environment?

Aspergillus fumigatus is the predominant etiological agent of invasive aspergillosis (IA), a difficult-to-manage fungal disease associated with a high case fatality rate. Azole antifungals, particularly voriconazole, have significantly improved the survival rate of patients with IA. However, the clinical advances made possible through the use of medical azoles could be threatened by the emergence of azole-resistant strains which has been reported in an ever-increasing number of countries over the last 10 years. The major resistance mechanism, that combines point mutation(s) in the coding sequence of cyp51A gene and an insertion of a tandem repeat in the promoter region of this gene which leads to its overexpression (TR34/L98H and TR46/Y121F/T289A), is presumed to be of environmental origin. However, the emergence of clinical and environmental azole-resistant strains without the cyp51A gene mutation suggests that other mechanisms could also be responsible for azole resistance (for example, overexpression of efflux pumps). The development of resistance may be linked to either long-term use of azole antifungals in patients with chronic aspergillosis (patient-acquired route) or selection pressure of the fungicides in the environment (environmental route). The fungicide-driven route could be responsible for resistance in azole-naive patients with IA. This literature review aims to summarize recent findings, focusing on the current situation of azole-resistance in A. fumigatus, and provides better understanding of the importance of the environmental route in resistance acquisition.

Identifying indoor air Penicillium species: a challenge for allergic patients.

PURPOSE: Penicillium is the most common mould isolated in housing. Penicillium chrysogenum is the only species tested by prick test or serology for allergic patients. The American Institute of Medicine has accepted Penicillium as an aetiological agent of rhinitis in children and adults and as an asthma agent in children. However, few studies have identified Penicillium in housing to the species level (354 species). Phenotypic identification is difficult. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) should be an alternative. The aim of this study was (1) to identify the Penicillium species present in dwellings in Eastern France and (2) to evaluate the reliability of MALDI-TOF MS for identification, by comparing it to DNA sequencing and phenotypic identification. METHODOLOGY: Identification to the species level was performed by MALDI-TOF MS on 275 strains isolated from 48 dwellings. These results were compared to beta-tubulin gene sequencing and to the phenotypic aspects. RESULTS: Thanks to MALDI-TOF, 235/275 strains could be identified (85.5 %). Fourteen species were identified among 23 Penicillium species included in the Filamentous Fungi Library 1.0 (Bruker Daltonics). However, 72.2  % of the strains belonged to five main taxa: P. chrysogenum (27.3 %), Penicillium glabrum (22.9 %), Penicilliumcommune (11.3 %), Penicillium brevicompactum (6.5 %) and Penicillium expansum (4.2 %). CONCLUSION: Complete coherence between MALDI-TOF MS and sequence-based identification was found for P. chrysogenum, P. expansum, P. glabrum, Penicillium italicum and Penicillium corylophilum. The main drawback was observed for Penicillium crustosum, which included 21 strains (7.6 %) that could not be identified using MALDI-TOF MS.

Alps to Apennines zircon roller coaster along the Adria microplate margin.

We have traced the particle path of high-pressure metasedimentary rocks on Elba Island, Northern Apennines, with the help of a U-Pb-Hf detrital zircon study. One quarter of the analysed zircons are surprisingly young, 41-30 Ma, with a main age peak at ca. 32 Ma, indicating an unexpected early Oligocene maximum deposition age. These Oligocene ages with negative εHf indicate a volcanic source region in the central-southern Alps. Though young by geological means, these zircons record an extraordinary geodynamic history. They originated in a volcanic arc, during the convergence/collision of the the Adria microplate with Europe from ca. 65 to 30 Ma. Thereafter, the Oligocene zircons travelled ca. 400 km southward along the Adria margin and the accretionary prism to present-day Tuscany, where they were subducted to depths of at least 40 km. Shortly thereafter, they were brought to the surface again in the wake of hinge roll back of the Apennine subduction zone and the resulting rapid extensional exhumation. Such a zircon roller coaster requires a microplate that has back-to-back subduction zones with opposing polarities on two sides.

Real-time polymerase chain reaction detection of Lichtheimia species in bandages associated with cutaneous mucormycosis in burn patients.

BACKGROUND: Cutaneous mucormycoses, mainly due to Lichtheimia (Absidia), have occurred on several occasions in the Burn Unit of the University Hospital of Lille, France. AIM: To investigate the potential vector role of non-sterile bandages used to hold in place sterile gauze used for wound dressing. METHODS: Mycological analysis by conventional culture, Mucorales real-time polymerase chain reaction (qPCR), and Lichtheimia species-specific qPCR were performed on eight crepe and six elasticized bandages that were sampled on two independent occasions in March 2014 and July 2016. Characteristics of the seven Lichtheimia mucormycoses which occurred in burn patients between November 2013 and July 2016 were also collected to assess the epidemiological relationship between potentially contaminated bandages and clinical infections. FINDINGS: One Lichtheimia corymbifera strain was isolated from a crepe bandage by culture, and Lichtheimia spp. qPCR was positive in six out of eight crepe and four out of six elasticized bandages. Using species-specific qPCR, Lichtheimia ramosa, Lichtheimia ornata, and L. corymbifera were identified in six out of ten, five out of ten, and four out of ten bandages, respectively. In patients with mucormycosis, L. ramosa and L. ornata were present in five and two cases, respectively. CONCLUSION: Our data support the utility of Mucorales qPCR for epidemiological investigations, the potential role of these bandages in cutaneous mucormycoses in burn patients in our centre, and, consequently, the need for sterile bandages for the dressing of extensive wounds.

[European birth cohorts: Early life exposure to microorganisms and health impact]. / Cohortes de naissances européennes : exposition aux micro-organismes et impact sanitaire.

In recent years, many birth cohorts have been initiated in Europe, to assess the early life microbiological exposure of children in the indoor environment and better understanding the different effects (adverse/protectors) on health. The results of 12 European cohorts, with different methodologies for exposure and allergic risk assessment are summarized in this review. Four meta-analyzes of cohort are presented too. Microbiological researches in indoor environment seem to turn to a metrology of microbiological exposure, but few studies provide real quantitative data. Thus, the establishment of dose-effect relationship is not possible and can only be done by having a global view of the situation, provided by an identical metrological approach in the different studies, in a large-scale, in the context of large birth cohorts with children followed with strict criteria to establish the clinical diagnosis.

Azole-resistant Aspergillus fumigatus in sawmills of Eastern France.

AIMS: Emergence of azole-resistant Aspergillus fumigatus complicates management of Aspergillus diseases. Currently, selection pressure caused by azole fungicide use in farming is strongly suspected of creating resistance. As sawmills also use azole fungicides, we investigated the presence of azole-resistant strains in this environment and studied the relationship between azole fungicide use and development of resistance. METHODS AND RESULTS: Air (n = 200) and substrate (n = 600) samples were taken in 20 sawmills. Azole-resistant strains (Etest and EUCAST methods) were confirmed by sequencing the cyp51A gene and its promoters. Dosage of propiconazole and tebuconazole was performed by gas chromatography coupled with mass spectrometry. Twenty-four azole-resistant A. fumigatus strains were collected among 20 of the 600 substrate samples (3%). Eighty-three percent of theses strains had TR34 /L98H mutation. A significantly higher number of resistant strains was collected in sawmills using fungicide products made with propiconazole mixed with a high concentration of tebuconazole (P = 0·009). The presence of resistant strains was significantly linked to propiconazole quantities in substrates (P = 0·03). CONCLUSIONS: The outcome of azole-resistant A. fumigatus carrying TR34 /L98H mutation seems to greatly depend on the azole fungicide formulation and quantities of azole. These preliminary results are valuable to propose new approaches limiting the emergence of azole-resistant strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Azole resistance is an emerging problem in A. fumigatus and threatens clinical advances made possible by the use of azole antifungals in the treatment of Aspergillus-related diseases. Azole fungicides are also used in the wood industry, notably in sawmills, to protect wood from wood-destroying fungi. Through our study, we show that sawmills represent another professional environment affected by the presence of azole-resistant A. fumigatus strains carrying the TR34 /L98H mutation. Moreover, this study provides valuable preliminary results to propose some new approaches to limit the emergence of azole-resistant A. fumigatus strains.

Opposite activation of the Hedgehog pathway in CD138+ plasma cells and CD138-CD19+ B cells identifies two subgroups of patients with multiple myeloma and different prognosis.

Hyperactivation of the Hedgehog (Hh) pathway, which controls refueling of multiple myeloma (MM) clones, might be critical to disease recurrence. Although several studies suggest the Hh pathway is activated in CD138- immature cells, differentiated CD138+ plasma cells might also be able to self-renew by producing themselves the Hh ligands. We studied the gene expression profiles of 126 newly diagnosed MM patients analyzed in both the CD138+ plasma cell fraction and CD138-CD19+ B-cell compartment. Results demonstrated that an Hh-gene signature was able to cluster patients in two subgroups characterized by the opposite Hh pathway expression in mature plasma cells and their precursors. Strikingly, patients characterized by Hh hyperactivation in plasma cells, but not in their B cells, displayed high genomic instability and an unfavorable outcome in terms of shorter progression-free survival (hazard ratio: 1.92; 95% confidence interval: 1.19-3.07) and overall survival (hazard ratio: 2.61; 95% confidence interval: 1.26-5.38). These results suggest that the mechanisms triggered by the Hh pathway ultimately led to identify a more indolent vs a more aggressive biological and clinical subtype of MM. Therefore, patient stratification according to their molecular background might help the fine-tuning of future clinical and therapeutic studies.

Selective zircon accumulation in a new benthic foraminifer, Psammophaga zirconia, sp. nov.

Benthic foraminifera are single-celled eukaryotes that make a protective organic, agglutinated or calcareous test. Some agglutinated, single-chambered taxa, including Psammophaga Arnold, 1982, retain mineral particles in their cytoplasm, but the selective mechanism of accumulation is not clear. Here, we report the ability of a foraminiferal species to select and accumulate zircons and other heavy minerals in their cytoplasm. In particular, the use of Scanning Electron Microscope coupled with an Energy Dispersive X-ray microanalysis system (SEM-EDS) enabled a representative overview of the mineral diversity and showed that the analysed Psammophaga zirconia sp. nov. individuals contained dominantly crystals of zircon (51%), titanium oxides (27%), and ilmenite (11%) along with minor magnetite and other minerals. The studied specimens occur in the shallow central Adriatic Sea where the sediment has a content of zircon below 1% and of other heavy minerals below 4%. For that reason we hypothesize that: (i) P. zirconia may be able to chemically select minerals, specifically zircon and rutile; (ii) the chemical mechanism allowing the selection is based on electrostatic interaction, and it could work also for agglutinated foraminifera (whether for ingestion, like Xenophyophores, or incorporation in the test as in many other described taxa). In particular, this aptitude for high preferential uptake and differential ingestion or retention of zircon is reported here for the first time, together with the selection of other heavy minerals already described in members of the genus Psammophaga. They are generally counted among early foraminifera, constructing a morphologically simple test with a single chamber. Our molecular phylogenetic study confirms that P. zirconia is a new species, genetically distinctive from other Psammophaga, and occurs in the Adriatic as well as in the Black Sea.

NF-kB2 induces senescence bypass in melanoma via a direct transcriptional activation of EZH2.

Enhancer of Zeste homologue 2 (EZH2) belongs to the polycomb repressive complex 2 and catalyzes the methylation of histone H3 lysine 27. These pivotal epigenetic marks are altered in many cancers, including melanoma, as a result of EZH2 overexpression. Here, we show that the non-canonical-NF-kB pathway accounts for most of the NF-kB activity in melanoma cells, in contrast to non-cancer cells. We identify the non-canonical-NF-kB pathway as a key regulator of EZH2 expression in melanoma. We show a striking correlation between NF-kB2 and EZH2 expression in human melanoma metastases. We demonstrate that inhibition of the non-canonical NF-kB pathway by targeting NF-kB2/p52 or the upstream kinase NIK restores the senescence program in melanoma cells through the decrease of EZH2. On the contrary, the overexpression of NF-kB2/p52 in normal human melanocytes prevents stress- and oncogene-induced senescence. Finally, we show in mouse models that the inhibition of the non-canonical NF-kB pathway restores senescence and induces a dramatic reduction in tumor growth compared with controls, thus providing potential drug targets for the re-induction of senescence in melanoma and other cancers where EZH2 is overexpressed.

Current and emerging triplet combination therapies for relapsed and refractory multiple myeloma.

Despite significant improvement in outcomes have been observed for multiple myeloma (MM) patients over the past 10-15 years, mainly due to the introduction of novel agents targeting the tumor clone and the bone marrow microenvironment, treatment of refractory and/or relapsed (RR) disease remains a challenge, particularly for patients who have failed prior bortezomib- and lenalidomide-based therapies. More recently, new drugs with different mechanisms of action, including second generation proteasome inhibitors, third generation immunomodulatory drugs, histone deacetylase inhibitors and monoclonal antibodies, have been developed and are under investigation, further increasing treatment options for RRMM patients. Overall, novel agent-based triplet combinations demonstrated superior response rates and prolonged disease control when compared with two-drug regimens in several randomized clinical trials, without adding any relevant additional toxicity. Salvage triplet therapies are likely to play a key role in overcoming drug-resistance and hold promise to further improve long-term outcomes of RRMM patients.

Early diagnosis and monitoring of mucormycosis by detection of circulating DNA in serum: retrospective analysis of 44 cases collected through the French Surveillance Network of Invasive Fungal Infections (RESSIF).

The main objective of this study was to assess the diagnostic performance of a set of three Mucorales quantitative PCR assays in a retrospective multicentre study. Mucormycosis cases were recorded thanks to the French prospective surveillance programme (RESSIF network). The day of sampling of the first histological or mycological positive specimen was defined as day 0 (D0). Detection of circulating DNA was performed on frozen serum samples collected from D-30 to D30, using quantitative PCR assays targeting Rhizomucor, Lichtheimia, Mucor/Rhizopus. Forty-four patients diagnosed with probable (n = 19) or proven (n = 25) mucormycosis were included. Thirty-six of the 44 patients (81%) had at least one PCR-positive serum. The first PCR-positive sample was observed 9 days (range 0-28 days) before diagnosis was made using mycological criteria and at least 2 days (range 0-24 days) before imaging. The identifications provided with the quantitative PCR assays were all concordant with culture and/or PCR-based identification of the causal species. Survival rate at D84 was significantly higher for patients with an initially positive PCR that became negative after treatment initiation than for patients whose PCR remained positive (48% and 4%, respectively; p <10-6). The median time for complete negativity of PCR was 7 days (range 3-19 days) after initiation of l-AmB treatment. Despite some limitations due to the retrospective design of the study, we showed that Mucorales quantitative PCR could not only confirm the mucormycosis diagnosis when other mycological arguments were present but could also anticipate this diagnosis. Quantification of DNA loads may also be a useful adjunct to treatment monitoring.

18F-FDG PET/CT focal, but not osteolytic, lesions predict the progression of smoldering myeloma to active disease.

Identification of patient sub-groups with smoldering multiple myeloma (SMM) at high risk of progression to active disease (MM) is an important goal. 18F-FDG PET/CT (positron emission tomography (PET) integrated with computed tomography (PET/CT) using glucose labelled with the positron-emitting radionuclide (18)F) allows for assessing early skeletal involvement. Identification of osteolytic lesions by this technique has recently been incorporated into the updated International Myeloma Working Group criteria for MM diagnosis. However, no data are available regarding the impact of focal lesions (FLs) without underlying osteolysis on time to progression (TTP) to MM. We hence prospectively studied a cohort of 120 SMM patients with PET/CT. PET/CT was positive in 16% of patients (1 FL: 8, 2 FLs: 3, >3 FLs: 6, diffuse bone marrow involvement: 2). With a median follow-up of 2.2 years, 38% of patients progressed to MM, in a median time of 4 years, including 21% with skeletal involvement. The risk of progression of those with positive PET/CT was 3.00 (95% confidence interval 1.58-5.69, P=0.001), with a median TTP of 1.1 versus 4.5 years for PET/CT-negative patients. The probability of progression within 2 years was 58% for positive versus 33% for negative patients. In conclusion, PET/CT positivity significantly increased the risk of progression of SMM to MM. PET/CT could become a new tool to define high-risk SMM.

[Azole resistance with environmental origin: What alternatives for the future?]. / Résistance aux antifongiques azolés d'origine environnementale : quelles alternatives pour l'avenir ?

Azole resistant Aspergillus fumigatus strains are increasingly reported in many countries. One resistance mechanism is attributed to the use of azole fungicides in environment. Two mutations, TR34/L98H and TR46/Y121F/T289A, on the cyp51A gene, have been described. Results of 40 publications about azole resistant strain detections, with TR34/L98H and TR46/Y121F/T289A mutations, in clinical and/or environmental samples, are presented in this review. These cases, observed in many countries, suggest spreading phenomenon. Measures to moderate fungicides treatments and/or alternative treatments in environment should be established to preserve the effectiveness of azole antifungal therapy for at-risk patients.

Microbiological characterization of 3193 French dwellings of Elfe cohort children.

Although exposure to indoor microorganisms in early life has already been associated with respiratory illness or allergy protection, only a few studies have performed standardized samplings and specific microbial analysis. Moreover, most do not target the different groups of microorganisms involved in respiratory diseases (fungi, bacteria, dust mites). In our study, ten specific qPCR targets (6 fungal species, 1 family and 2 genera of bacteria, 1 house dust mite) were used to analyze the microorganism composition of electrostatic dust fall collector (EDC) from 3193 dwellings of the Elfe French cohort study. Multivariate analyses allowed us to show that the microbial composition of dwellings, assessed with simultaneous analysis of 10 microorganisms, can be characterized by four entities: three bacteria, house dust mite Dermatophagoïdes pteronyssinus, fungi Alternaria alternata, and five other molds. Some dwellings' intrinsic characteristics (occupational ratio, type of dwelling and presence of pets) clearly influence microorganism distribution, and six different profiles of dwellings, characterized by their composition in microorganisms, have been described across France. The use of these clusters seems promising in the evaluation of allergic risk. Allergic respiratory diseases will develop in the near future in some children of the Elfe cohort and will indicate to what extent our approach can be predictive of respiratory disease.

Evaluation of invasive aspergillosis risk of immunocompromised patients alternatively hospitalized in hematology intensive care unit and at home.

UNLABELLED: Contrary to hospital exposure, little is known about the indoor fungal exposure of hematology patients at home. The aim of our study was to investigate the mold exposure of hematology patients both at home and at hospital to assess their invasive aspergillosis (IA) risk. Fungal exposure was assessed by quantifying opportunistic molds at hospital during hospitalization and in homes of 53 hematology patients. IA was diagnosed in 13 of 53 patients and invasive fungal infection (IFI) in one patient. In hospital, no opportunistic species, or low levels of opportunistic species, were found in 98% of weekly controls. Only 2% of hematology intensive care unit (ICU) controls showed a high level of Aspergillus fumigatus spores in corridor air. Five patients IA were hospitalized during these periods. Seven dwellings of 53 (5/14 dwellings of patients with IA/IFI and 2/39 dwellings of non-IA patients) had a percentage of A. fumigatus and Aspergillus flavus to total mold (significant predictor variable of IA/IFI in our study, general linear model, P-value = 0.02) as high as 15%. Maintaining a 'zero Aspergillus' goal at hospital is essential, and establishing specific and individually opportunistic mold monitoring at home could help to further reduce the IA risk through continuous surveillance. PRACTICAL IMPLICATIONS: This study emphasizes the fact that preventive measures should not be aimed only at the hospital setting: among patients diagnosed with invasive aspergillosis/invasive fungal infection (IA/IFI), 5 of 14 (36%) were exposed to opportunistic fungal species at home exclusively. Moreover, four of these five patients were living in homes having the highest percentage of Aspergillus fumigatus and Aspergillus flavus (>15%), one of which had 48% of A. fumigatus. Therefore, our work supports the need for a counselor to carry out an environmental survey in patients' homes.

[Fungal and bacterial contamination of 30 general practitioners' consultation room in Franche-Comté (eastern France)]. / Contamination fongique et bactérienne de 30 salles de consultation de cabinets médicaux en Franche-Comté

OBJECTIVE: To evaluate the level of microbial contamination of general pratice environment. METHODS: Ten points, including air, water and surfaces were sampled in general practitioner's consultation room of 30 general practices in Franche-Comté region (eastern France). For each sample, a global microbial count was assessed as well as the presence of potential pathogen microorganisms: Aspergillus fumigatus, Staphylococcus aureus, Pseudomonas aeruginosa, and Enterobacteriaceae. RESULTS: We note that 66.3% of the samples (n=202) were on the target level (<51 ufc/25 cm (2), <101 ufc/m(3), <100 ufc/mL in total bacteria, without pathogenic species and without A. fumigatus respectively on surfaces, air and water, without isolation of coliforme bacteria or P. aeruginosa for 100mL of water). A. fumigatus, S. aureus, P. aeruginosa and Enterobacteriaceae were recovered in 8.0%, 2.1%, 0.7%, 0% respectively. However, 66.7% of air sampling were positive to A. fumigatus with 2 to 12 cfu/m(3). CONCLUSION: This study shows a low level of bacterial contamination of general practitioner's consultation room. A. fumigatus frequency in air samples seems equivalent to that found in housing without air treatment.