CAR+ extracellular vesicles predict ICANS in patients with B cell lymphomas treated with CD19-directed CAR T cells.

BACKGROUND: Predicting Immune-effector Cell Associated Neurotoxicity Syndrome (ICANS) in patients infused with Chimeric Antigen Receptor T cells (CAR-T) is still a conundrum. This complication, thought to be consequent to CAR-T cell activation, arises a few days after infusion, when circulating CAR-T cells are scarce and specific CAR-T cell-derived biomarkers are lacking. METHODS: Human CD19.CAR-T cells were generated to gain insight into CAR+ extracellular vesicle (CAR+EV) release upon target engagement. A prospective cohort of 100 B-cell lymphoma patients infused with approved CD19.CAR-T cell products (axi-cel, brexu-cel and tisa-cel) was assessed for plasma CAR+EVs as potential biomarkers of in vivo CD19.CAR-T cell activation and predictors of ICANS. Human induced pluripotent stem cells (iPSCs)-derived neural cells were used as a model for CAR+EV-induced neurotoxicity. RESULTS: In vitro, exosome-like CAR+EVs were released by CD19.CAR-T cells upon target engagement. In vivo, CAR+EVs were detectable as early as 1 hour in the plasma of patients. A concentration > 132.8 CAR+EVs/µl at hour +1 or > 224.5 CAR+EVs/µl at day +1 predicted ICANS in advance of 4 days, with a sensitivity up to 96.55% and a specificity up to 80.36%, outperforming other potential ICANS predictors. Enolase 2 (ENO2+) nanoparticles were released by iPSCs-derived neural cells upon CAR+EVs exposure and were increased in the plasma of ICANS patients. CONCLUSIONS: These results convey that plasma CAR+EVs are an immediate signal of CD19.CAR-T cell activation, are suitable predictors of neurotoxicity, and may be involved in ICANS pathogenesis. TRIAL REGISTRATION: NCT04892433, NCT05807789.

Neuronopathic Gaucher disease models reveal defects in cell growth promoted by Hippo pathway activation.

Gaucher Disease (GD), the most common lysosomal disorder, arises from mutations in the GBA1 gene and is characterized by a wide spectrum of phenotypes, ranging from mild hematological and visceral involvement to severe neurological disease. Neuronopathic patients display dramatic neuronal loss and increased neuroinflammation, whose molecular basis are still unclear. Using a combination of Drosophila dGBA1b loss-of-function models and GD patient-derived iPSCs differentiated towards neuronal precursors and mature neurons we showed that different GD- tissues and neuronal cells display an impairment of growth mechanisms with an increased cell death and reduced proliferation. These phenotypes are coupled with the downregulation of several Hippo transcriptional targets, mainly involved in cells and tissue growth, and YAP exclusion from nuclei. Interestingly, Hippo knock-down in the GBA-KO flies rescues the proliferative defect, suggesting that targeting the Hippo pathway can be a promising therapeutic approach to neuronopathic GD.

Molecular Signature of Biological Aggressiveness in Clear Cell Sarcoma of the Kidney (CCSK).

Clear cell sarcoma of the kidney (CCSK) is a rare pediatric renal tumor with a worse prognosis than Wilms' tumor. Although recently, BCOR internal tandem duplication (ITD) has been found as a driver mutation in more than 80% of cases, a deep molecular characterization of this tumor is still lacking, as well as its correlation with the clinical course. The aim of this study was to investigate the differential molecular signature between metastatic and localized BCOR-ITD-positive CCSK at diagnosis. Whole-exome sequencing (WES) and whole-transcriptome sequencing (WTS) were performed on six localized and three metastatic BCOR-ITD-positive CCSKs, confirming that this tumor carries a low mutational burden. No significant recurrences of somatic or germline mutations other than BCOR-ITD were identified among the evaluated samples. Supervised analysis of gene expression data showed enrichment of hundreds of genes, with a significant overrepresentation of the MAPK signaling pathway in metastatic cases (p < 0.0001). Within the molecular signature of metastatic CCSK, five genes were highly and significantly over-expressed: FGF3, VEGFA, SPP1, ADM, and JUND. The role of FGF3 in the acquisition of a more aggressive phenotype was investigated in a cell model system obtained by introducing the ITD into the last exon of BCOR by Crispr/Cas9 gene editing of the HEK-293 cell line. Treatment with FGF3 of BCOR-ITD HEK-293 cell line induced a significant increase in cell migration versus both untreated and scramble cell clone. The identification of over-expressed genes in metastatic CCSKs, with a particular focus on FGF3, could offer new prognostic and therapeutic targets in more aggressive cases.

ERCC6L2-related disease: a novel entity of bone marrow failure disorder with high risk of clonal evolution.

ERCC excision repair 6 like 2 (ERCC6L2) gene encodes for different helicase-like protein members of the Snf2 family involved in transcription-coupled nucleotide excision repair and in cell proliferation. Germline homozygous mutations in children and adults predispose to a peculiar bone marrow failure phenotype characterized by mild hematological alterations with a high risk of developing acute myeloid leukemia. The outcome for patients with leukemia progression is dismal while patients undergoing hematopoietic stem cell transplantation in the early stage have better outcomes. The ERCC6L2-related hematological disease presents a high penetrance, posing important questions regarding the treatment strategies and possible preemptive approaches. This review describes the biological function of ERCC6L2 and the clinical manifestations of the associated disease, trying to focus on the unsolved clinical questions.

Immune dysregulation associated with co-occurring germline CBL and SH2B3 variants.

BACKGROUND: CBL syndrome is a RASopathy caused by heterozygous germline mutations of the Casitas B-lineage lymphoma (CBL) gene. It is characterized by heterogeneous clinical phenotype, including developmental delay, facial dysmorphisms, cardiovascular malformations and an increased risk of cancer development, particularly juvenile myelomonocytic leukemia (JMML). Although the clinical phenotype has been progressively defined in recent years, immunological manifestations have not been well elucidated to date. METHODS: We studied the genetic, immunological, coagulative, and clinical profile of a family with CBL syndrome that came to our observation after the diagnosis of JMML, with homozygous CBL mutation, in one of the members. RESULTS: Variant analysis revealed the co-occurrence of CBL heterozygous mutation (c.1141 T > C) and SH2B3 mutation (c.1697G > A) in two other members. Patients carrying both mutations showed an ALPS-like phenotype characterized by lymphoproliferation, cytopenia, increased double-negative T-cells, impaired Fas-mediated lymphocyte apoptosis, altered cell death in PBMC and low TRECs expression. A coagulative work-up was also performed and showed the presence of subclinical coagulative alterations in patients carrying both mutations. CONCLUSION: In the reported family, we described immune dysregulation, as part of the clinical spectrum of CBL mutation with the co-occurrence of SH2B3.

C5 and SRGAP3 Polymorphisms Are Linked to Paediatric Allergic Asthma in the Italian Population.

Asthma is a complex and heterogeneous disease, caused by the interaction between genetic and environmental factors with a predominant allergic background in children. The role of specific genes in asthmatic bronchial reactivity is still not clear, probably because of the many common pathways shared with other allergic disorders. This study is focused on 11 SNPs possibly related to asthma that were previously identified in a GWAS study. The genetic variability of these SNPs has been analysed in a population of 773 Italian healthy controls, and the presence of an association between the polymorphisms and the asthma onset was evaluated performing genotyping analysis on 108 children affected with asthma compared with the controls. Moreover, a pool of 171 patients with only allergic rhinoconjunctivitis has been included in the case-control analysis. The comparison of allele frequencies in asthmatic patients versus healthy controls identified two SNPs-rs1162394 (p = 0.019) and rs25681 (p = 0.044)-associated with the asthmatic condition, which were not differentially distributed in the rhinoconjunctivitis group. The rs25681 SNP, together with three other SNPs, also resulted in not being homogenously distributed in the Italian population. The significantly higher frequency of the rs25681 and rs1162394 SNPs (located, respectively, in the C5 and SRGAP3 genes) in the asthmatic population suggests an involvement of these genes in the asthmatic context, playing a role in increasing the inflammatory condition that may influence asthma onset and clinical course.

Role of CBL Mutations in Cancer and Non-Malignant Phenotype.

CBL plays a key role in different cell pathways, mainly related to cancer onset and progression, hematopoietic development and T cell receptor regulation. Somatic CBL mutations have been reported in a variety of malignancies, ranging from acute myeloid leukemia to lung cancer. Growing evidence have defined the clinical spectrum of germline CBL mutations configuring the so-called CBL syndrome; a cancer-predisposing condition that also includes multisystemic involvement characterized by variable phenotypic expression and expressivity. This review provides a comprehensive overview of the molecular mechanisms in which CBL exerts its function and describes the clinical manifestation of CBL mutations in humans.

Are Induced Pluripotent Stem Cells a Step towards Modeling Pediatric Leukemias?

Despite enormous improvements in pre-clinical and clinical research, acute leukemia still represents an open challenge for pediatric hematologists; both for a significant relapse rate and for long term therapy-related sequelae. In this context, the use of an innovative technology, such as induced pluripotent stem cells (iPSCs), allows to finely reproduce the primary features of the malignancy and can be exploited as a model to study the onset and development of leukemia in vitro. The aim of this review is to explore the recent literature describing iPSCs as a key tool to study different types of hematological malignancies, comprising acute myeloid leukemia, non-down syndrome acute megakaryoblastic leukemia, B cell acute lymphoblastic leukemia, and juvenile myelomonocytic leukemia. This model demonstrates a positive impact on pediatric hematological diseases, especially in those affecting infants whose onsets is found in fetal hematopoiesis. This evidence highlights the importance of achieving an in vitro representation of the human embryonic hematopoietic development and timing-specific modifications, either genetic or epigenetic. Moreover, further insights into clonal evolution studies shed light in the way of a new precision medicine era, where patient-oriented decisions and therapies could further improve the outcome of pediatric cases. Nonetheless, we will also discuss here the difficulties and limitations of this model.

Peripheral blood cellular profile at pre-lymphodepletion is associated with CD19-targeted CAR-T cell-associated neurotoxicity.

Background: Infusion of second generation autologous CD19-targeted chimeric antigen receptor (CAR) T cells in patients with R/R relapsed/refractory B-cell lymphoma (BCL) is affected by inflammatory complications, such as Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS). Current literature suggests that the immune profile prior to CAR-T infusion modifies the chance to develop ICANS. Methods: This is a monocenter prospective study on 53 patients receiving approved CAR T-cell products (29 axi-cel, 24 tisa-cel) for R/R-BCL. Clinical, biochemical, and hematological variables were analyzed at the time of pre-lymphodepletion (pre-LD). In a subset of 21 patients whose fresh peripheral blood sample was available, we performed cytofluorimetric analysis of leukocytes and extracellular vesicles (EVs). Moreover, we assessed a panel of soluble plasma biomarkers (IL-6/IL-10/GDF-15/IL-15/CXCL9/NfL) and microRNAs (miR-146a-5p, miR-21-5p, miR-126-3p, miR-150-5p) which are associated with senescence and inflammation. Results: Multivariate analysis at the pre-LD time-point in the entire cohort (n=53) showed that a lower percentage of CD3+CD8+ lymphocytes (38.6% vs 46.8%, OR=0.937 [95% CI: 0.882-0.996], p=0.035) and higher levels of serum C-reactive protein (CRP, 4.52 mg/dl vs 1.00 mg/dl, OR=7.133 [95% CI: 1.796-28], p=0.005) are associated with ICANS. In the pre-LD samples of 21 patients, a significant increase in the percentage of CD8+CD45RA+CD57+ senescent cells (median % value: 16.50% vs 9.10%, p=0.009) and monocytic-myeloid derived suppressor cells (M-MDSC, median % value: 4.4 vs 1.8, p=0.020) was found in ICANS patients. These latter also showed increased levels of EVs carrying CD14+ and CD45+ myeloid markers, of the myeloid chemokine CXCL-9, as well of the MDSC-secreted cytokine IL-10. Notably, the serum levels of circulating neurofilament light chain, a marker of neuroaxonal injury, were positively correlated with the levels of senescent CD8+ T cells, M-MDSC, IL-10 and CXCL-9. No variation in the levels of the selected miRNAs was observed between ICANS and no-ICANS patients. Discussion: Our data support the notion that pre-CAR-T systemic inflammation is associated with ICANS. Higher proportion of senescence CD8+ T cells and M-MDSC correlate with early signs of neuroaxonal injury at pre-LD time-point, suggesting that ICANS may be the final event of a process that begins before CAR-T infusion, consequence to patient clinical history.

iPSC-Derived Gaucher Macrophages Display Growth Impairment and Activation of Inflammation-Related Cell Death.

Gaucher disease is a lysosomal storage disorder characterized by ß-glucosidase enzyme deficiency and substrate accumulation, especially in cells of the reticuloendothelial system. Typical features of the disease are the unrestrained activation of inflammatory mechanisms, whose molecular pathways are still unclear. To investigate biological mechanisms underlying the macrophage activation in GD, we derived iPSCs from a healthy donor and a GD patient line and differentiated them into hematopoietic progenitors. While GD iPSCs are able to efficiently give rise to CD33+/CD45+ myeloid progenitors, the maturation towards the CD14+/CD163+ monocyte/macrophages fate resulted enhanced in the GD lines, that in addition displayed a decreased growth potential compared to control cells either in semisolid or in liquid culture. The GD lines growth impairment was associated with a significant upregulation of RIPK3 and MLKL, two key effectors of necroptosis, the inflammation related cell death pathway. The activation of necroptosis, which has already been linked to neuronopathic GD, may play a role in the disease proinflammatory condition and in the identified cell growth defects. Understanding the GD macrophage role in the alteration of mechanisms linked to cellular metabolism imbalance, cell death and inflammation are crucial in identifying new ways to approach the disease.

Gene Expression Landscape of SDH-Deficient Gastrointestinal Stromal Tumors.

BACKGROUND: About 20-40% of gastrointestinal stromal tumors (GISTs) lacking KIT/PDGFRA mutations show defects in succinate dehydrogenase (SDH) complex. This study uncovers the gene expression profile (GEP) of SDH-deficient GIST in order to identify new signaling pathways or molecular events actionable for a tailored therapy. METHODS: We analyzed 36 GIST tumor samples, either from formalin-fixed, paraffin-embedded by microarray or from fresh frozen tissue by RNA-seq, retrospectively collected among KIT-mutant and SDH-deficient GISTs. Pathway analysis was performed to highlight enriched and depleted transcriptional signatures. Tumor microenvironment and immune profile were also evaluated. RESULTS: SDH-deficient GISTs showed a distinct GEP with respect to KIT-mutant GISTs. In particular, SDH-deficient GISTs were characterized by an increased expression of neural markers and by the activation of fibroblast growth factor receptor signaling and several biological pathways related to invasion and tumor progression. Among them, hypoxia and epithelial-to-mesenchymal transition emerged as features shared with SDH-deficient pheochromocytoma/paraganglioma. In addition, the study of immune landscape revealed the depletion of tumor microenvironment and inflammation gene signatures. CONCLUSIONS: This study provides an update of GEP in SDH-deficient GISTs, highlighting differences and similarities compared to KIT-mutant GISTs and to other neoplasm carrying the SDH loss of function. Our findings add a piece of knowledge in SDH-deficient GISTs, shedding light on their putative histology and on the dysregulated biological processes as targets of new therapeutic strategies.

Filaggrin Loss-of-Function Mutations Are Risk Factors for Severe Food Allergy in Children with Atopic Dermatitis.

Atopic dermatitis is frequently associated with the onset of other allergic conditions, such as asthma, rhino-conjunctivitis and food allergy. The etiology of atopic dermatitis is marginally understood in spite of the number of predisposing factors, above all, mutations in the Filaggrin gene (FLG). In this study, the association between loss-of-function variants in the FLG gene and other allergic manifestations, in particular food allergy, was evaluated in an Italian pediatric population affected by atopic dermatitis. The 10 more frequently mutated loci in the FLG gene were genotyped in 238 children affected by atopic dermatitis and tested for association with clinical features of allergic disorders by a multivariate logistic regression model. R501X and 2282del4 were the only two mutations identified; 12.2% of children carry one of these variants, corresponding to an allelic frequency of 6.5%. According to multivariate statistical analysis, loss-of-function variants in the FLG gene represent a risk factor for the onset of severe manifestations of food allergy (OR = 8.9; CI: 3.1-28.3). Peanut and hazelnut were identified as high-risk foods in patients with FLG mutations. This study demonstrates that atopic children carrying FLG mutations represent a high-risk population due to their predisposition to develop severe food allergy reactions, such as anaphylaxis.

Interferon-γ signaling synergizes with LRRK2 in neurons and microglia derived from human induced pluripotent stem cells.

Parkinson's disease-associated kinase LRRK2 has been linked to IFN type II (IFN-γ) response in infections and to dopaminergic neuronal loss. However, whether and how LRRK2 synergizes with IFN-γ remains unclear. In this study, we employed dopaminergic neurons and microglia differentiated from patient-derived induced pluripotent stem cells carrying LRRK2 G2019S, the most common Parkinson's disease-associated mutation. We show that IFN-γ enhances the LRRK2 G2019S-dependent negative regulation of AKT phosphorylation and NFAT activation, thereby increasing neuronal vulnerability to immune challenge. Mechanistically, LRRK2 G2019S suppresses NFAT translocation via calcium signaling and possibly through microtubule reorganization. In microglia, LRRK2 modulates cytokine production and the glycolytic switch in response to IFN-γ in an NFAT-independent manner. Activated LRRK2 G2019S microglia cause neurite shortening, indicating that LRRK2-driven immunological changes can be neurotoxic. We propose that synergistic LRRK2/IFN-γ activation serves as a potential link between inflammation and neurodegeneration in Parkinson's disease.