Machine learning-based immune phenotypes correlate with STK11/KEAP1 co-mutations and prognosis in resectable NSCLC: a sub-study of the TNM-I trial.

BACKGROUND: We aim to implement an immune cell score model in routine clinical practice for resected non-small-cell lung cancer (NSCLC) patients (NCT03299478). Molecular and genomic features associated with immune phenotypes in NSCLC have not been explored in detail. PATIENTS AND METHODS: We developed a machine learning (ML)-based model to classify tumors into one of three categories: inflamed, altered, and desert, based on the spatial distribution of CD8+ T cells in two prospective (n = 453; TNM-I trial) and retrospective (n = 481) stage I-IIIA NSCLC surgical cohorts. NanoString assays and targeted gene panel sequencing were used to evaluate the association of gene expression and mutations with immune phenotypes. RESULTS: Among the total of 934 patients, 24.4% of tumors were classified as inflamed, 51.3% as altered, and 24.3% as desert. There were significant associations between ML-derived immune phenotypes and adaptive immunity gene expression signatures. We identified a strong association of the nuclear factor-κB pathway and CD8+ T-cell exclusion through a positive enrichment in the desert phenotype. KEAP1 [odds ratio (OR) 0.27, Q = 0.02] and STK11 (OR 0.39, Q = 0.04) were significantly co-mutated in non-inflamed lung adenocarcinoma (LUAD) compared to the inflamed phenotype. In the retrospective cohort, the inflamed phenotype was an independent prognostic factor for prolonged disease-specific survival and time to recurrence (hazard ratio 0.61, P = 0.01 and 0.65, P = 0.02, respectively). CONCLUSIONS: ML-based immune phenotyping by spatial distribution of T cells in resected NSCLC is able to identify patients at greater risk of disease recurrence after surgical resection. LUADs with concurrent KEAP1 and STK11 mutations are enriched for altered and desert immune phenotypes.

Molecular screening program to select molecular-based recommended therapies for metastatic cancer patients: analysis from the ProfiLER trial.

BACKGROUND: Antitumor activity of molecular-targeted agents is guided by the presence of documented genomic alteration in specific histological subtypes. We aim to explore the feasibility, efficacy and therapeutic impact of molecular profiling in routine setting. PATIENTS AND METHODS: This multicentric prospective study enrolled adult or pediatric patients with solid or hematological advanced cancer previously treated in advanced/metastatic setting and noneligible to curative treatment. Each molecular profile was established on tumor, relapse or biopsies, and reviewed by a molecular tumor board (MTB) to identify molecular-based recommended therapies (MBRT). The main outcome was to assess the incidence rate of genomic mutations in routine setting, across specific histological types. Secondary objectives included a description of patients with actionable alterations and for whom MBRT was initiated, and overall response rate. RESULTS: Four centers included 2579 patients from February 2013 to February 2017, and the MTB reviewed the molecular profiles achieved for 1980 (76.8%) patients. The most frequently altered genes were CDKN2A (N = 181, 7%), KRAS (N = 177, 7%), PIK3CA (N = 185, 7%), and CCND1 (N = 104, 4%). An MBRT was recommended for 699/2579 patients (27%), and only 163/2579 patients (6%) received at least one MBRT. Out of the 182 lines of MBRT initiated, 23 (13%) partial responses were observed. However, only 0.9% of the whole cohort experienced an objective response. CONCLUSION: An MBRT was provided for 27% of patients in our study, but only 6% of patients actually received matched therapy with an overall response rate of 0.9%. Molecular screening should not be used at present to guide decision-making in routine clinical practice outside of clinical trials.This trial is registered with ClinicalTrials.gov, number NCT01774409.

Cophylogeny reconstruction via an approximate Bayesian computation.

Despite an increasingly vast literature on cophylogenetic reconstructions for studying host-parasite associations, understanding the common evolutionary history of such systems remains a problem that is far from being solved. Most algorithms for host-parasite reconciliation use an event-based model, where the events include in general (a subset of) cospeciation, duplication, loss, and host switch. All known parsimonious event-based methods then assign a cost to each type of event in order to find a reconstruction of minimum cost. The main problem with this approach is that the cost of the events strongly influences the reconciliation obtained. Some earlier approaches attempt to avoid this problem by finding a Pareto set of solutions and hence by considering event costs under some minimization constraints. To deal with this problem, we developed an algorithm, called Coala, for estimating the frequency of the events based on an approximate Bayesian computation approach. The benefits of this method are 2-fold: (i) it provides more confidence in the set of costs to be used in a reconciliation, and (ii) it allows estimation of the frequency of the events in cases where the data set consists of trees with a large number of taxa. We evaluate our method on simulated and on biological data sets. We show that in both cases, for the same pair of host and parasite trees, different sets of frequencies for the events lead to equally probable solutions. Moreover, often these solutions differ greatly in terms of the number of inferred events. It appears crucial to take this into account before attempting any further biological interpretation of such reconciliations. More generally, we also show that the set of frequencies can vary widely depending on the input host and parasite trees. Indiscriminately applying a standard vector of costs may thus not be a good strategy.

Superfluid high REynolds von Kármán experiment.

The Superfluid High REynolds von Kármán experiment facility exploits the capacities of a high cooling power refrigerator (400 W at 1.8 K) for a large dimension von Kármán flow (inner diameter 0.78 m), which can work with gaseous or subcooled liquid (He-I or He-II) from room temperature down to 1.6 K. The flow is produced between two counter-rotating or co-rotating disks. The large size of the experiment allows exploration of ultra high Reynolds numbers based on Taylor microscale and rms velocity [S. B. Pope, Turbulent Flows (Cambridge University Press, 2000)] (Rλ > 10000) or resolution of the dissipative scale for lower Re. This article presents the design and first performance of this apparatus. Measurements carried out in the first runs of the facility address the global flow behavior: calorimetric measurement of the dissipation, torque and velocity measurements on the two turbines. Moreover first local measurements (micro-Pitot, hot wire,…) have been installed and are presented.

Liquid helium inertial jet for comparative study of classical and quantum turbulence.

We present a new cryogenic wind tunnel facility developed to study the high Reynolds number developed classical or quantum turbulence in liquid (4)He. A stable inertial round jet flow with a Reynolds number of 4 × 10(6) can be sustained in both He I and He II down to a minimum temperature of 1.7 K. The circuit can be pressurized up to 3.5 × 10(5) Pa. The system has been designed to exploit the self-similar properties of the jet far field in order to adapt to the spatial resolution of the existing probes. Multiple and complementary sensors can be simultaneously installed to obtain spatial and time resolved measurements. The technical difficulties and design details are described and the system performance is presented.

Synthesis of alcoholic ZnO nanocolloids in the presence of piperidine organic base: nucleation-growth evidence of Zn5(OH)8Ac2.2H2O fine particles and ZnO nanocrystals.

Piperidine as a new free OH* organic base has been successfully used to prepare Zn5(OH)8(Ac).22H2O particles (named Zn-HDS) or concentrated alcoholic ZnO sols. Considering the applications of Zn-HDS and ZnO compounds, as well as interests of these synthesis mechanisms for fundamental chemistry, such investigations are of importance. This strategy not only allows preparing Zn-HDS compounds at room temperature but also brings evidence of some new nucleation-growth, and permits the preparation of well crystalline ZnO nanocrystals at low temperature (maximum 60 degrees C). It was possible to convincingly prove that the formation of Zn-HDS phase is concomitant to the ZnO nanocrystals formation and that Zn-HDS could be considered as an intermediate initiator of ZnO nanocrystals. A parallel approach was used for the fast screening of the synthesis progress.

Experimental evidence of accelerated energy transfer in turbulence.

We investigate the vorticity dynamics in a turbulent vortex using scattering of acoustic waves. Two ultrasonic beams are adjusted to probe simultaneously two spatial scales in a given volume of the flow, thus allowing a dual channel recording of the dynamics of coherent vorticity structures. Our results show that this allows one to measure the average energy transfer time between different spatial length scales, and that such transfer goes faster at smaller scales.

Analysis of slipped sequences in EST projects

Slippage is an important sequencing problem that can occur in EST projects. However, very few studies have addressed this. We propose three new methods to detect slippage artifacts: arithmetic mean method, geometric mean method, and echo coverage method. Each method is simple and has two different strategies for processing sequences: suffix and subsequence. Using the 291,689 EST sequences produced in the SUCEST project, we performed comparative tests between our proposed methods and the SUCEST method. The subsequence strategy is better than the suffix strategy, because it is not anchored at the end of the sequence, so it is more flexible to find slippage at the beginning of the EST. In a comparison with the SUCEST method, the advantage of our methods is that they do not discard the majority of the sequences marked as slippage, but instead only remove the slipped artifact from the sequence. Based on our tests the echo coverage method with subsequence strategy shows the best compromise between slippage detection and ease of calibration.

Statistics of Fourier modes of velocity and vorticity in turbulent flows: intermittency and long-range correlations.

We perform a statistical analysis of experimental fully developed turbulence longitudinal velocity data in the Fourier space. We address the controversial issue of statistical intermittency of spatial Fourier modes by acting on the finite spectral resolution. We derive a link between velocity structure functions and the flatness of Fourier modes thanks to cascade models. Similar statistical behaviors are recovered in the analysis of spatial Fourier modes of vorticity obtained in an acoustic scattering experiment. We conclude that vorticity is long-range correlated and found more intermittent than longitudinal velocity.

Activation by GDNF of a transcriptional program repressing neurite growth in dorsal root ganglia.

Glial cell line-derived neurotrophic factor (GDNF) promotes the survival of postnatal-but not embryonic-mouse dorsal root ganglion cells in vitro, despite the fact that its receptors are expressed at both ages. To understand this difference, we have performed an oligonucleotide microarray experiment. We found that several hundred genes were regulated between embryonic and postnatal stages, and that several important classes of genes were differentially regulated by GDNF treatment, including genes related to translation and to phenotypic specification and maturation. Interestingly, a set of genes related to cell adhesion, cytoskeleton and cellular morphology were consistently down-regulated by GDNF, suggesting a previously uncharacterized role for GDNF in repressing neurite growth and/or branching. This nuclear program initiated by GDNF was functionally confirmed in cultures of embryonic wild-type neurons sustained with nerve growth factor and in bax(-/-) neurons that survive in the absence of trophic support.

Attenuation of a caspase-3 dependent cell death in NT4- and p75-deficient embryonic sensory neurons.

Neuronal survival during the developmental period of naturally occurring cell death is mediated through a successful competition for limiting concentrations of neurotrophic factors, and the deprived neurons will die. New results show that induced death through the p75 neurotrophin receptor (p75(NTR)), a member of the p55TNF/Fas family of cell death receptors, may also influence survival during development. We find that eliminating p75(NTR) or neurotrophin 4 (NT4) in mice leads to a marked attenuation of apoptosis during the programmed cell death period of the trigeminal ganglion neurons, suggesting that NT4 can induce the death of these neurons through the p75(NTR). These in vivo findings were reproduced in primary cell cultures, where NT4 was found to induce death in a p75(NTR)-dependent pathway. Analysis of p75 deficient and wild-type cells revealed two separate cell death pathways, a p75(NTR)- and caspase-3-independent pathway activated by trophic factor deprivation, and a p75(NTR)- and caspase-3-dependent pathway initiated by NT4. Crossing in the NT4 null alleles in brain-derived neurotrophic factor (BDNF) null mutant mice led to a rescue of a large proportion of BDNF-dependent neurons from excessive cell death, indicating that trophic factor deprivation is not sufficient for the death of many neurons and that additional death inducing signals might be required. Our results suggest that NT4 competitively signals survival and death of sensory neurons through trkB and p75(NTR), respectively.

Positive and negative interactions of GDNF, NTN and ART in developing sensory neuron subpopulations, and their collaboration with neurotrophins.

Glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN) and neublastin/artemin (ART) are distant members of the transforming growth factor beta family, and have been shown to elicit neurotrophic effects upon several classes of peripheral and central neurons. Limited information from in vitro and expression studies has also substantiated a role for GDNF family ligands in mammalian somatosensory neuron development. Here, we show that although dorsal root ganglion (DRG) sensory neurons express GDNF family receptors embryonically, they do not survive in response to their ligands. The regulation of survival emerges postnatally for all GDNF family ligands. GDNF and NTN support distinct subpopulations that can be separated with respect to their expression of GDNF family receptors, whereas ART supports neurons in populations that are also responsive to GDNF or NTN. Sensory neurons that coexpress GDNF family receptors are medium sized, whereas small-caliber nociceptive cells preferentially express a single receptor. In contrast to brain-derived neurotrophic factor (BDNF)-dependent neurons, embryonic nerve growth factor (NGF)-dependent nociceptive neurons switch dependency to GDNF, NTN and ART postnatally. Neurons that survive in the presence of neurotrophin 3 (NT3) or neurotrophin 4 (NT4), including proprioceptive afferents, Merkel end organs and D-hair afferents, are also supported by GDNF family ligands neonatally, although at postnatal stages they lose their dependency on GDNF and NTN. At late postnatal stages, ART prevents survival elicited by GDNF and NTN. These data provide new insights on the roles of GDNF family ligands in sensory neuron development.

1,25-Dihydroxyvitamin D3 regulates the expression of VDR and NGF gene in Schwann cells in vitro.

The vitamin D receptor (VDR) is a nuclear receptor that mediates the effect of the active metabolite of vitamin D3, the 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). To investigate the potential role of this hormone in the peripheral nervous system, we have studied the VDR expression in Schwann cells. The VDR mRNA was detected by Northern blot analysis in rat primary cultures of Schwann cells, and its levels were strongly increased in the presence of 1,25-(OH)2D3. Using the mouse Schwann cell line, MSC80, we showed that concentrations as low as 10(-10) M of hormone stimulated the expression of the VDR gene and strongly increased the amounts of activated VDR, capable of binding to the specific vitamin D responsive element (VDRE). We also found that 1,25-(OH)2D3 stimulated the expression of the nerve growth factor gene in MSC80. These data suggest a role for the hormone in the peripheral nervous system, possibly as a mediator active in trauma.

Expression and regulation of GFRalpha3, a glial cell line-derived neurotrophic factor family receptor.

We report the identification of an additional member of the glial cell line-derived neurotrophic factor (GDNF) family receptor, termed GFRalpha3, that is homologous to the previously identified GDNF and neurturin ligand binding receptors GFRalpha1 and GFRalpha2. GFRalpha3 is 32% and 37% identical to GFRalpha1 and GFRalpha2, respectively. RNase protection assays show that whereas gfralpha1 and gfralpha2 are abundant in both developing and adult brain, gfralpha3 is exclusively expressed during development. All receptors are widely present in both the developing and adult peripheral nervous system and in peripheral organs. For instance, in situ hybridization shows that the developing liver, stomach, intestine, kidney, and sympathetic chain, which all contain ret-expressing cells, transcribe unique complementary and overlapping patterns of most or all of the GDNF family receptors and ligands. In sensory neurons of the trigeminal ganglion gfralpha2 and gfralpha3 are expressed in different subpopulations of neurons, whereas gfralpha1 is coexpressed in some gfralpha2 and gfralpha3-positive neurons. We find that the gfralpha1 population of trigeminal neurons is absent in GDNF null mutant mice, suggesting that GDNF signals in vivo by interacting with GFRalpha1. Thus, our results show that there are at least three members in the GDNF family of ligand binding receptors and that these receptors may be crucial in conferring ligand specificity in vivo. The unique complementary and overlapping expression of gfralpha3 implies distinct functions in the developing and adult mouse from that of GFRalpha1 and GFRalpha2.

Differentially expressed genes in C6.9 glioma cells during vitamin D-induced cell death program.

C6.9 rat glioma cells undergo a cell death program when exposed to 1, 25-dihydroxyvitamin D3 (1,25-D3). As a global analytical approach, we have investigated gene expression in C6.9 engaged in this cell death program using differential screening of a rat brain cDNA library with probes derived from control and 1,25-D3-treated cells. Using this methodology we report the isolation of 61 differentially expressed cDNAs. Forty-seven cDNAs correspond to genes already characterized in rat cells or tissues. Seven cDNAs are homologous to yeast, mouse or human genes and seven are not related to known genes. Some of the characterized genes have been reported to be differentially expressed following induction of programmed cell death. These include PMP22/gas3, MGP and beta-tubulin. For the first time, we also show a cell death program induced up-regulation of the c-myc associated primary response gene CRP, and of the proteasome RN3 subunit and TCTP/mortalin genes. Another interesting feature of this 1,25-D3 induced-cell death program is the down-regulated expression of transcripts for the microtubule motor dynein heavy chain/MAP 1C and of the calcium-binding S100beta protein. Finally 15 upregulated cDNAs encode ribosomal proteins suggesting a possible involvement of the translational apparatus in this cell program. Alternatively, these ribosomal protein genes could be up-regulated in response to altered rates of cellular metabolism, as has been demonstrated for most of the other isolated genes which encode proteins involved in metabolic pathways. Thus, this study presents to our knowledge the first characterization of genes which are differentially expressed during a cell death program induced by 1, 25-D3. Therefore, this data provides new information on the fundamental mechanisms which participate in the antineoplastic effects of 1,25-D3 and on the machinery of a cell death program in a glioma cell line.

Was the formation of 1,25-dihydroxyvitamin D3 initially a catabolic pathway?

Following solar ultraviolet radiation, epidermal 7-dehydrocholesterol is converted to previtamin D3, which then undergoes a thermal isomerization into vitamin D3. The metabolism of vitamin D3, which is usually considered as an inactive compound, gives rise to the active hormone 1,25-dihydroxyvitamin D3, following two hydroxylation steps occurring in liver and kidney. Here, we propose that this anabolic pathway can also be interpreted as a catabolic one leading to the degradation of the photoproducts of 7-dehydrocholesterol, for which a specific biological role in the skin is proposed.

Noradrenaline inhibits the programmed cell death induced by 1,25-dihydroxyvitamin D3 in glioma.

The rat glioma cell line C6.9 has been recently reported to respond to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) by the induction of a programmed cell death. Since, in vivo, glial cells are thought to be exposed to several neurotransmitters, we investigated the possibility of a neurotransmitter-mediated inhibition of this active cell death process. Noradrenaline and the beta-adrenoceptor agonist isoproterenol showed significant inhibition of the 1,25(OH)2D3-induced programmed cell death. The beta-adrenoceptor antagonist propanolol reversed this inhibition, while the alpha-adrenoceptor antagonist yohimbin was devoid of any effect. This suggests that the efficiency of antiproliferative vitamin D-related therapies could be influenced by endogenous levels of noradrenaline.

1,25-Dihydroxyvitamin D3 induces programmed cell death in a rat glioma cell line.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3), a seco-steroid hormone with potential antitumoral activities, has been recently reported to exert cytotoxic effects on C6 glioma cells. However, the molecular mechanisms which trigger this cell death remain unknown. We show here that this 1,25(OH)2D3-induced cell death is dependent upon protein synthesis and is accompanied by the expression of c-myc, p53, and gadd45 genes. Two other genes, coding for interleukin-6 and vaso-endothelial growth factor, are also upregulated after addition of 1,25(OH)2D3. This programmed cell death can be suppressed when cells are treated with forskolin, a drug which increases intracellular cAMP concentration, or with genistein, an inhibitor of tyrosine protein kinases. However, in spite of the demonstration of fragmented DNA in 1,25(OH)2D3-treated cells, the C6.9 cells used in this study do not show the classical morphological features of apoptosis. These results provide the first evidence for the existence of a programmed cell death triggered by 1,25(OH)2D3 in glioma cells and may provide a basis for the development of new therapeutic strategies. In addition, these data also suggest that the treatment of C6.9 cells with 1,25(OH)2D3 may be a useful model to study the molecular mechanisms involved in the programmed cell death of a cell of glial origin.

Differential expression of M-CSF, LIF, and TNF-alpha genes in normal and malignant rat glial cells: regulation by lipopolysaccharide and vitamin D.

The effect of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on the expression of macrophage colony-stimulating factor (M-CSF), leukemia inhibitory factor (LIF), and tumor necrosis factor-alpha (TNF-alpha) genes in primary rat astrocytes and C6 glioma cells was examined. The results show that the hormone differentially regulates the cytokine mRNA in the two cell types. 1,25-(OH)2D3 augments M-CSF and LIF mRNA in C6 glioma cells, while lipopolysaccharide (LPS) has minimal effects. When LPS and 1,25-(OH)2D3 are used in combination, a strong synergistic effect upon the induction of M-CSF and LIF genes is observed. No TNF-alpha transcript has been detected in C6 glioma cells under any stimulus conditions used. In contrast, 1,25-(OH)2D3 has no pronounced effect on M-CSF, LIF, and TNF-alpha transcripts in primary astrocytes when used as a sole stimulus, while treatment with LPS strongly enhances the levels of the three cytokines. However, when 1,25-(OH)2D3 is used in combination with LPS, a partial reduction in LPS-induced levels of M-CSF and TNF-alpha mRNA is observed. The overall results indicate that genes coding for some inflammatory cytokines obey distinct regulatory mechanisms in C6 cells and in primary astrocytes. They also suggest that 1,25-(OH)2D3, by altering the response of astrocytes to an inflammatory stimulus, could participate in the regulation of the CNS immune response.