NGS-based stratification refines the risk stratification in T-ALL and identifies a Very High-Risk subgroup of patients.

We previously reported a better outcome in adult and pediatric T-cell acute lymphoblastic leukemia (T-ALL) harboring NOTCH1 and/or FBXW7 mutations without alterations of K-N-RAS and PTEN genes. Availability of high-throughput next-generation sequencing strategies (NGS) led us to refine the outcome prediction in T-ALL. Targeted whole-exome sequencing of 72 T-ALL related oncogenes was performed in 198 adult T-ALLs in first remission (CR1) from the GRAALL-2003/2005 protocols (ClinicalTrial.gov, NCT00222027, NCT00327678) and 242 pediatric T-ALLs from the FRALLE2000T. This approach enabled the identification of the first NGS-based classifier in T-ALL categorizing low-risk patients as those with N/F, PHF6, or EP300 mutations, excluding N-K-RAS, PI3K pathway (PTEN, PIK3CA, and PIK3R1), TP53, DNMT3A, IDH1/2, and IKZF1 alterations, with a 5-year cumulative incidence of relapse (CIR) estimated at 21%. Conversely, the remaining patients were classified as high-risk, exhibiting a 5-year CIR estimated at 47%. We externally validated this stratification in the pediatric cohort. NGS-based classifier was highly prognostic, independently of minimal residual disease (MRD) and white blood cells counts (WBC), in both adult and pediatric cohorts. Integration of the NGS-based classifier into a comprehensive risk stratification model, including WBC count at diagnosis and MRD at the end of induction, enabled the identification of an adverse risk subgroup (25%) with a 5-year CIR estimated at 51%, and a favorable risk group (32%) with a 5-year CIR estimated at 12%. NGS-based stratification combined with WBC and MRD sharpens the prognostic classification in T-ALL and identifies a new subgroup of patients who may benefit from innovative therapeutic approaches.

Genomic imbalances analysis provides new insight into prognostic factors in adult and pediatric T-ALL.

Given the poor outcome of refractory and relapsing T-ALL, identifying prognostic markers is still challenging. Using SNP-array analysis, we provide a comprehensive analysis of genomic imbalances in a cohort of 317 newly-diagnosed T-ALL patients including 135 children and 182 adults with respect to clinical and biological features and outcomes. SNP-array results identified at least one somatic genomic imbalance in virtually all T-ALL patients (~96%). Del(9)(p21) (~70%) and UPD(9)p21)/CDKN2A/B (~28%) were the most frequent genomic imbalances. Unexpectedly del(13q14)/RB1/DLEU1 (~14%) was the second more frequent CNV followed by del(6)(q15)/CASP8AP2 (~11%), del(1)(p33)/SIL-TAL1 (~11%), del(12)(p13)ETV6/CDKN1B (~9%), del(18)(p11)/PTPN2 (~9%), del(1)(p36)/RPL22 (~9%), and del(17)(q11)/NF1/SUZ12 (~8%). SNP-array also revealed distinct profiles of genomic imbalances according to age, immunophenotype, and oncogenetic subgroups. In particular, adult T-ALL patients demonstrated a significantly higher incidence of del(1)(p36)/RPL22, and del(13)(q14)/RB1/DLEU1, and lower incidence of del(9)(p21) and UPD(9p21)/CDKN2A/B. We determined a threshold of 15 genomic imbalances to stratify patients into high- and low-risk groups of relapse. Survival analysis also revealed the poor outcome, despite the low number of affected cases, conferred by the presence of chromothripsis (n=6, ~2%), del(16)(p13)/CREBBP (n=15, ~5%) as well as the newly identified recurrent gain at 6q27 involving MLLT4 (n=10, ~3%). Genomic complexity, del(16)(p13)/CREBBP and gain at 6q27 involving MLLT4 maintained their significance in multivariate analysis for survival outcome. Our study thus demonstrated that whole genome analysis of imbalances provides new insights to refine risk stratification in T-ALL.

Optogenetics 2.0: challenges and solutions towards a quantitative probing of neural circuits.

To exploit the full potential of optogenetics, we need to titrate and tailor optogenetic methods to emulate naturalistic circuit function. For that, the following prerequisites need to be met: first, we need to target opsin expression not only to genetically defined neurons per se, but to specifically target a functional node. Second, we need to assess the scope of optogenetic modulation, i.e. the fraction of optogenetically modulated neurons. Third, we need to integrate optogenetic control in a closed loop setting. Fourth, we need to further safe and stable gene expression and light delivery to bring optogenetics to the clinics. Here, we review these concepts for the human and rodent brain.

CD169 and CD64 could help differentiate bacterial from CoVID-19 or other viral infections in the Emergency Department.

The identification of a bacterial, viral, or even noninfectious cause is essential in the management of febrile syndrome in the emergency department (ED), especially in epidemic contexts such as flu or CoVID-19. The aim was to assess discriminative performances of two biomarkers, CD64 on neutrophils (nCD64) and CD169 on monocytes (mCD169), using a new flow cytometry procedure, in patients presenting with fever to the ED during epidemics. Eighty five adult patients presenting with potential infection were included during the 2019 flu season in the ED of La Timone Hospital. They were divided into four diagnostic outcomes according to their clinical records: no-infection, bacterial infection, viral infection and co-infection. Seventy six patients with confirmed SARS-CoV-2 infection were also compared to 48 healthy volunteers. For the first cohort, 38 (45%) patients were diagnosed with bacterial infections, 11 (13%) with viral infections and 29 (34%) with co-infections. mCD169 was elevated in patients with viral infections, with a majority of Flu A virus or Respiratory Syncytial Virus, while nCD64 was elevated in subjects with bacterial infections, with a majority of Streptococcus pneumoniae and Escherichia coli. nCD64 and mCD169 showed 90% and 80% sensitivity, and 78% and 91% specificity, respectively, for identifying patients with bacterial or viral infections. When studied in a second cohort, mCD169 was elevated in 95% of patients with SARS-CoV-2 infections and remained at normal level in 100% of healthy volunteers. nCD64 and mCD169 have potential for accurately distinguishing bacterial and acute viral infections. Combined in an easy and rapid flow cytometry procedure, they constitute a potential improvement for infection management in the ED, and could even help for triage of patients during emerging epidemics.

Major impact of an early bone marrow checkpoint (day 21) for minimal residual disease in flow cytometry in childhood acute lymphoblastic leukemia.

The early persistence of minimal residual disease (MRD) is considered a poor prognostic factor indicative of chemoresistance in acute lymphoblastic leukemia. In French children, chemosensitivity is assessed at day 21 post-induction by cytomorphology. Here, it was investigated whether a more precise evaluation could be obtained at this time point with multiparameter flow cytometry (MFC). This study enrolled 123 children with de novo acute lymphoblastic leukemia. MRD0 was investigated at day 21 in MFC with a combination of antibodies based on the immunophenotype of diagnosis. It was also evaluated at day 35 by immunoglobulin/T-cell receptor quantitative real-time polymerase chain reaction (MRD1). Three risk groups could be delineated based on MRD0. Patients with MFC/MRD0 levels >10-2 (n = 25) were considered high risk, those with levels between 10-2 and 10-4 (n = 46) intermediate risk, and those <10-4 (n = 50) low risk. Overall survival (p = 0.048) and event-free survival (EFS, p = 0.00017) were significantly different between these three groups. EFS of the 14 corticoresistant patients strongly depended on their MRD0 level (p = 0.004). Similarly, both EFS (p = 0.0004) and overall survival (p = 0.02) were significantly different in the 109 chemosensitive patients, according to MRD0 levels. MRD0 and MRD1 levels, compared with 112 patients, were consistent (-/- or +/+) in 57.2% of the cases. Both MRD0+/MRD1+ and MRD0+/MRD1- patients had a significantly worse EFS (p = 0.0001) than those with undetectable MRD at both MRD0 and MRD1. This study confirms the usefulness and superiority of an early point of MRD detection by MFC. In addition, MRD0 in MFC identifies a subgroup of patients with poorer prognosis (MRD0+/MRD1-). Copyright © 2015 John Wiley & Sons, Ltd.

Fractalkine Signaling and Microglia Functions in the Developing Brain.

Microglial cells are the resident macrophages of the central nervous system (CNS). Besides their classical roles in pathological conditions, these immune cells also dynamically interact with neurons and influence their structure and function in physiological conditions. The neuronal chemokine fractalkine and its microglial receptor CX3CR1 are one important signaling pathway involved in these reciprocal interactions. In the present review, we will discuss recent evidence indicating that fractalkine signaling also determines several functions of microglial cells during normal CNS development. It has been known for a decade that microglial cells influence the neuronal death that normally occurs during CNS development. Surprisingly, recent evidence indicates that they can also support survival of developing neurons, control axon outgrowth, and laminar positioning of subsets of interneurons in the forebrain. Moreover, microglial cells influence the maturation of synaptic circuits at early postnatal stages: their phagocytic activity allows them to eliminate inappropriate synapses and they can also influence the functional expression of synaptic proteins by releasing mediators. Fractalkine signaling controls these functions of microglial cells in part by regulating their timely recruitment at sites of developing synapses. Finally, on-going research suggests that this signaling pathway is also a key player in neurodevelopmental disorders.

Paradoxical effects of minocycline in the developing mouse somatosensory cortex.

Minocycline, a tetracycline derivative, is known to exert neuroprotective effects unrelated to its antimicrobial action. In particular, minocycline prevents microglial activation in pathological conditions and consequently reduces the production of proinflammatory factors contributing to the propagation of diseases. Accumulative evidence indicates that microglial cells contribute to the maturation of neuronal and synaptic networks during the normal development of the central nervous system (CNS) and that perinatal inflammation is a known risk factor for brain lesions. Although minocycline has been used to infer microglia functions during development, mechanisms by which this tetracycline derivative affect the immature CNS have not been analyzed in detail. In this study, we demonstrate that minocycline administration during the first postnatal week of development has paradoxical effects on microglia phenotype and on neuronal survival in the mouse somatosensory cortex. Using a combination of immunohistochemistry and electrophysiology, we show that intraperitoneal injections of minocycline between postnatal days 6 and 8 affect distribution, morphology, and functional properties of microglia cells of the whisker-related barrel cortex, leading to the development of a phenotype resembling that of microglia activated in pathological conditions. Minocyline also induced a massive cell death that developed faster than changes in microglia phenotype, suggesting that the latter is a consequence of the former. Finally, cell death and microglial activation were not observed when minocycline treatment was postponed by only 2 days (i.e., between postnatal days 8 and 10). These observations call into question the use of tetracycline derivatives during CNS development to study microglia or to reduce perinatal inflammation.

[Microglia: immune cells sculpting and controlling neuronal synapses]. / La microglie : des cellules immunitaires qui sculptent et contrôlent les synapses neuronales.

The development of new animal models and functional analysis methods has dramatically changed our understanding of the physiology of microglial cells. These cells which are the resident macrophages of central nervous system have the ability to adapt rapidly to subtle changes of their environment. Recent findings indicate in particular that they can establish contacts with neuronal synapses that they can eliminate and modulate by releasing specific mediators. Here we review the experimental observations that have revealed the occurrence of these interactions not only in pathological conditions but also in the healthy brain and in particular during normal brain development. The discovery of bi-directional communications between synapses and microglia sheds a new light on our understanding of brain functioning and should allow a better understanding of brain functioning and of interactions between immune and nervous systems.

[Suspicion of constitutional abnormality at diagnosis of childhood leukemia: Update of the leukemia committee of the French Society of Childhood Cancers]. / Suspicion d'anomalie constitutionnelle au diagnostic de leucémie chez l'enfant : mise au point du comité leucémies de la Société française des cancers de l'enfant.

The spectrum of childhood leukemia predisposition syndromes has grown significantly over last decades. These predisposition syndromes mainly involve CEBPA, ETV6, GATA2, IKZF1, PAX5, RUNX1, SAMD9/SAMD9L, TP53, RAS-MAPK pathway, DNA mismatch repair system genes, genes associated with Fanconi anemia, and trisomy 21. The clinico-biological features leading to the suspicion of a leukemia predisposition are highly heterogeneous and require varied exploration strategies. The study of the initial characteristics of childhood leukemias includes high-throughput sequencing techniques, which have increased the frequency of situations where a leukemia predisposing syndrome is suspected. Identification of a leukemia predisposition syndrome can have a major impact on the choice of chemotherapy, the indication for hematopoietic stem cell transplantation, and screening for associated malformations and pathologies. The diagnosis of a predisposition syndrome can also lead to the exploration of family members and genetic counseling. Diagnosis and management should be based on dedicated and multidisciplinary care networks.

Deficiency of the microglial receptor CX3CR1 impairs postnatal functional development of thalamocortical synapses in the barrel cortex.

Accumulative evidence indicates that microglial cells influence the normal development of brain synapses. Yet, the mechanisms by which these immune cells target maturating synapses and influence their functional development at early postnatal stages remain poorly understood. Here, we analyzed the role of CX3CR1, a microglial receptor activated by the neuronal chemokine CX3CL1 (or fractalkine) which controls key functions of microglial cells. In the whisker-related barrel field of the mouse somatosensory cortex, we show that the recruitment of microglia to the sites where developing thalamocortical synapses are concentrated (i.e., the barrel centers) occurs only after postnatal day 5 and is controlled by the fractalkine/CX3CR1 signaling pathway. Indeed, at this developmental stage fractalkine is overexpressed within the barrels and CX3CR1 deficiency delays microglial cell recruitment into the barrel centers. Functional analysis of thalamocortical synapses shows that CX3CR1 deficiency also delays the functional maturation of postsynaptic glutamate receptors which normally occurs at these synapses between the first and second postnatal week. These results show that reciprocal interactions between neurons and microglial cells control the functional maturation of cortical synapses.

Adaptive phenotype of microglial cells during the normal postnatal development of the somatosensory "Barrel" cortex.

Accumulative evidence indicates that microglial cells influence the normal development of central nervous system (CNS) synapses. Yet, the functional properties of microglia in relation with synapse development remain unclear. We recently showed that in layer 4 of the whisker-related barrel field of the mouse somatosensory cortex, microglial cells are recruited only after postnatal day (P)5 in the center of the barrels where thalamo-cortical synapses are concentrated and begin their maturation. In the present study, we analyzed the phenotype of microglia during this developmental process. We show that between P5 and P7 microglial cells acquire a more ramified morphology with a smaller soma, they express classical markers of microglia (Iba1, CD11b, and CD68) but never markers of activation (Mac-2 and MHCII) and rarely the proliferation marker Ki67. Electrophysiological recordings in acute cortical slices showed that at P5 a proportion of layer 4 microglia transiently express voltage-dependant potassium currents of the delayed rectifier family, mostly mediated by Kv1.3 subunits, which are usually expressed by activated microglia under pathological conditions. This proportion of cells with rectifying properties doubles between P5 and P6, in concomitance with the beginning of microglia invasion of the barrel centers. Finally, analysis of the responses mediated by purinergic receptors indicated that a higher percentage of rectifying microglia expressed functional P2Y6 and P2Y12 receptors, as compared with nonrectifying cells, whereas all cells expressed functional P2X7 receptors. Our results indicate that during normal cortical development distinct microglia properties mature differentially, some of them being exquisitely influenced by the local environment of the maturating neuronal network.

Flow cytometry thresholds of myeloperoxidase detection to discriminate between acute lymphoblastic or myeloblastic leukaemia.

The World Health Organization 2008 Classification emphasizes myeloperoxidase (MPO) detection as sufficient for assigning a blast population to the myeloid lineage. Published MPO positivity thresholds are 10% for flow cytometry (FCM) but 3% for cytochemistry. Here we re-evaluated the FCM-MPO threshold by comparing retrospectively 128 acute lymphoblastic leukaemias and 75 acute myeloid leukaemias without maturation, all assessed by benzidine-based cytochemistry. A 13% threshold was found to be relevant using an isotype control as background-reference (sensitivity 95·1%, specificity 91·7%). Residual normal lymphocytes proved to be an advantageous alternative reference, a threshold of 28% yielding improved 97·4% sensitivity and 96·1% specificity.

SARS-CoV-2 spike protein induces a differential monocyte activation that may contribute to age bias in COVID-19 severity.

A strong bias related to age is observed in COVID-19 patients with pediatric subjects developing a milder disease than adults. We hypothesized that a specific SARS-CoV-2 effect conjugated with preexisting differences in the immune systems may explain this. Using flow cytometry, we investigated basal immune differences in a cohort consisting of 16 non-infected young and 16 aged individuals and further leveraged an in vitro whole blood model of SARS-CoV-2 infection so that functional differences could be mined as well. In short, blood diluted in culture media was incubated 5 or 24 h with the trimeric spike protein or controls. Following unsupervised analysis, we first confirmed that the immune lymphoid and myeloid systems in adults are less efficient and prone to develop higher inflammation than those in children. We notably identified in adults a higher CD43 lymphocyte expression, known for its potentially inhibitory role. The spike protein induced different responses between adults and children, notably a higher increase of inflammatory markers together with lower monocyte and B cell activation in adults. Interestingly, CD169, a CD43 ligand overexpressed in COVID-19 patients, was confirmed to be strongly modulated by the spike protein. In conclusion, the spike protein exacerbated the preexisting lower immune responsiveness and higher inflammatory potential in adults. Altogether, some of the markers identified may explain the marked age bias and be predictive of severity.