Common variants at 21q22.3 locus influence MX1 and TMPRSS2 gene expression and susceptibility to severe COVID-19

The established risk factors of coronavirus disease 2019 (COVID-19) are advanced age, male sex and comorbidities, but they do not fully explain the wide spectrum of disease manifestations. Genetic factors implicated in the host antiviral response provide for novel insights into its pathogenesis. We performed an in-depth genetic analysis of chromosome 21 exploiting the genome-wide association study data, including 6,406 individuals hospitalized for COVID-19 and 902,088 controls with European genetic ancestry from the COVID-19 Host Genetics Initiative. We found that five single nucleotide polymorphisms within TMPRSS2 and near MX1 gene show associations with severe COVID-19. The minor alleles of the five SNPs correlated with a reduced risk of developing severe COVID-19 and high level of MX1 expression in blood. Our findings demonstrate that host genetic factors can influence the different clinical presentations of COVID-19 and that MX1 could be a potential therapeutic target

Genetic Predisposition to Solid Pediatric Cancers

Progresses over the past years have extensively improved our capacity to use genome-scale analyses-including high-density genotyping and exome and genome sequencing-to identify the genetic basis of pediatric tumors. In particular, exome sequencing has contributed to the evidence that about 10% of children and adolescents with tumors have germline genetic variants associated with cancer predisposition. In this review, we provide an overview of genetic variations predisposing to solid pediatric tumors (medulloblastoma, ependymoma, astrocytoma, neuroblastoma, retinoblastoma, Wilms tumor, osteosarcoma, rhabdomyosarcoma, and Ewing sarcoma) and outline the biological processes affected by the involved mutated genes. A careful description of the genetic basis underlying a large number of syndromes associated with an increased risk of pediatric cancer is also reported. We place particular emphasis on the emerging view that interactions between germline and somatic alterations are a key determinant of cancer development. We propose future research directions, which focus on the biological function of pediatric risk alleles and on the potential links between the germline genome and somatic changes. Finally, the importance of developing new molecular diagnostic tests including all the identified risk germline mutations and of considering the genetic predisposition in screening tests and novel therapies is emphasized

Single-cell transcriptomics of neuroblastoma identifies chemoresistance-associated genes and pathways

High-Risk neuroblastoma (NB) survival rate is still <50%, despite treatments being more and more aggressive. The biggest hurdle liable to cancer therapy failure is the drug resistance by tumor cells that is likely due to the intra-tumor heterogeneity (ITH). To investigate the link between ITH and therapy resistance in NB, we performed a single cell RNA sequencing (scRNAseq) of etoposide and cisplatin resistant NB and their parental cells. Our analysis showed a clear separation of resistant and parental cells for both conditions by identifying 8 distinct tumor clusters in etoposide-resistant/parental and 7 in cisplatin-resistant/parental cells. We discovered that drug resistance can affect NB cell identities; highlighting the bi-directional ability of adrenergic-to-mesenchymal transition of NB cells. The biological processes driving the identified resistant cell subpopulations revealed genes such as (BARD1, BRCA1, PARP1, HISTH1 axis, members of RPL family), suggesting a potential drug resistance due to the acquisition of DNA repair mechanisms and to the modification of the drug targets. Deconvolution analysis of bulk RNAseq data from 498 tumors with cell subpopulation signatures showed that the transcriptional heterogeneity of our cellular models reflected the ITH of NB tumors and allowed the identification of clusters associated with worse/better survival. Our study demonstrates the distinct cell populations characterized by genes involved in different biological processes can have a role in NB drug treatment failure. These findings evidence the importance of ITH in NB drug resistance studies and the chance that scRNA-seq analysis offers in the identification of genes and pathways liable for drug resistance

Somatic mutations enriched in cis-regulatory elements affect genes involved in embryonic development and immune system response in neuroblastoma

: Noncoding cis-regulatory variants have gained interest as cancer drivers, yet progress in understanding their significance is hindered by the numerous challenges and limitations of variant prioritization. To overcome these limitations, we focused on active cis-regulatory elements (aCRE) in order to design a customized panel for the deep sequencing of 56 neuroblastoma tumor and normal DNA sample pairs. In order to search for driver mutations, aCREs were defined by reanalysis of H3K27ac ChiP-seq peaks in 25 neuroblastoma cell lines. These regulatory genomic regions were tested for an excess of somatic mutations and assessed for statistical significance using a global approach that accounted for chromatin accessibility and replication timing. Additional validation was provided by whole genome sequence analysis of 151 neuroblastomas. Analysis of Hi-C data determined the presence of candidate target genes interacting with mutated regions. An excess of somatic mutations in aCREs of diverse genes were identified, including IPO7, HAND2, and ARID3A. CRISPR-Cas9 editing was utilized to assess the functional consequences of mutations in the IPO7 aCRE. Patients with noncoding mutations in aCREs showed inferior overall and event-free survival independent of age at diagnosis, stage, risk stratification, and MYCN status. Expression of aCRE-interacting genes correlated strongly with negative prognostic markers and low survival rates. Moreover, a convergence between the biological functions of aCRE target genes and transcription factors with mutated binding motifs was associated with embryonic development and immune system response. Overall, this strategy enabled the identification of somatic mutations in regulatory elements that collectively promote neuroblastoma tumorigenesis

HIF-1 transcription activity: HIF1A driven response in normoxia and in hypoxia

Abstract Background HIF1A (Hypoxia-Inducible-Factor 1A) expression in solid tumors is relevant to establish resistance to therapeutic approaches. The use of compounds direct against hypoxia signaling and HIF1A does not show clinical efficiency because of changeable oxygen concentrations in solid tumor areas. The identification of HIF1A targets expressed in both normoxia and hypoxia and of HIF1A/hypoxia signatures might meliorate the prognostic stratification and therapeutic successes in patients with high-risk solid tumors. Methods In this study, we conducted a combined analysis of RNA expression and DNA methylation of neuroblastoma cells silenced or unsilenced for HIF1A expression, grown in normoxia and hypoxia conditions. Results The analysis of pathways highlights HIF-1 (heterodimeric transcription factor 1) activity in normoxia in metabolic process and HIF-1 activity in hypoxia in neuronal differentiation process. HIF1A driven transcriptional response in hypoxia depends on epigenetic control at DNA methylation status of gene regulatory regions. Furthermore, low oxygen levels generate HIF1A-dependent or HIF1A-independent signatures, able to stratify patients according to risk categories. Conclusions These findings may help to understand the molecular mechanisms by which low oxygen levels reshape gene signatures and provide new direction for hypoxia targeting in solid tumor

Exploring Shared Susceptibility between Two Neural Crest Cells Originating Conditions:Neuroblastoma and Congenital Heart Disease

In the past years, genome wide association studies (GWAS) have provided evidence that inter-individual susceptibility to diverse pathological conditions can reveal a common genetic architecture. Through the analysis of congenital heart disease (CHD) and neuroblastoma (NB) GWAS data, we aimed to dissect the genetic susceptibility shared between these conditions, which are known to arise from neural crest cell (NCC) migration or development abnormalities, via identification and functional characterization of common regions of association. Two loci (2q35 and 3q25.32) harbor single nucleotide polymorphisms (SNPs) that are associated at a p-value < 10−3 with conotruncal malformations and ventricular septal defect respectively, as well as with NB. In addition, the lead SNP in 4p16.2 for atrial septal defect and the lead SNP in 3q25.32 for tetralogy of Fallot are less than 250 Kb distant from the lead SNPs for NB at the same genomic regions. Some of these shared susceptibility loci regulate the expression of relevant genes involved in NCC formation and developmental processes (such as BARD1, MSX1, and SHOX2) and are enriched in several epigenetic markers from NB and fetal heart cell lines. Although the clinical correlation between NB and CHD is unclear, our exploration of a possible common genetic basis between NB and a subset of cardiac malformations can help shed light on their shared embryological origin and pathogenetic mechanisms

Inherited rare variants in homologous recombination and neurodevelopmental genes are associated with increased risk of neuroblastoma

Background: Neuroblastoma (NB) is the most common solid extracranial paediatric tumour. Genome-wide association studies have driven the discovery of common risk variants, but no large study has investigated the contribution of rare variants to NB susceptibility. Here, we conducted a whole-exome sequencing (WES) of 664 NB cases and 822 controls and used independent validation datasets to identify genes with rare risk variants and involved pathways. Methods: WES was performed at 50× depth and variants were jointly called in cases and controls. We developed two models to identify mutations with high clinical impact (P/LP model) and to discover less penetrant risk mutations affecting non-canonical cancer pathways (RPV model). We performed a gene-level collapsing test using Firth's logistic regression in 242 selected cancer predisposition genes (CPGs) and a gene-sets burden analysis of biologically-informed pathways. Findings: Twelve percent of patients carried P/LP variants in CPGs and showed a significant enrichment (P = 2.3 × 10-4) compared to controls (6%). We identified P/LP variants in 45 CPGs enriched in homologous recombination (HR) pathway. The most P/LP enriched genes in NB were BRCA1, ALK and RAD51C. Additionally, we found higher RPV burden in gene-sets of neuron differentiation, neural tube development and synapse assembly, and in gene-sets associated with neurodevelopmental disorders (NDD). Interpretation: The high fraction of NB patients with P/LP variants indicates the need of genetic counselling. Furthermore, inherited rare variants predispose to NB development by affecting mechanisms related to HR and neurodevelopmental processes, and demonstrate that NDD genes are altered in NB at the germline level. Funding: Associazione Italiana per la Ricerca sul Cancro, Fondazione Italiana per la Lotta al Neuroblastoma, Associazione Oncologia Pediatrica e Neuroblastoma, Regione Campania, Associazione Giulio Adelfio onlus, and Italian Health Ministry

Therapeutic Targeting of ALK in Neuroblastoma: Experience of Italian Precision Medicine in Pediatric Oncology

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood. Patients with relapsed/refractory disease have a poor prognosis, and additional therapeutic options are needed. Mutations and amplifications in the ALK (Anaplastic Lymphoma Kinase) gene constitute a key target for treatment. Our goal, within the Italian project of PeRsonalizEdMEdicine (PREME), was to evaluate the genomic status of patients with relapsed/refractory NB and to implement targeted therapies in those with targetable mutations. From November 2018 to November 2021, we performed Whole Exome Sequencing or Targeted Gene Panel Sequencing in relapsed/refractory NB patients in order to identify druggable variants. Activating mutations of ALK were identified in 8(28.57%) of 28 relapsed/refractory NB patients. The mutation p.F1174L was found in six patients, whereas p.R1275Q was found in one and the unknown mutation p.S104R in another. Three patients died before treatment could be started, while five patients received crizotinib: two in monotherapy (one with p.F1174L and the other with p.S104R) and three (with p.F1174L variant) in combination with chemotherapy. All treated patients showed a clinical improvement, and one had complete remission after two cycles of combined treatment. The most common treatment-related toxicities were hematological. ALK inhibitors may play an important role in the treatment of ALK-mutated NB patients

Therapeutic Targeting of ALK in Neuroblastoma: Experience of Italian Precision Medicine in Pediatric Oncology

: Neuroblastoma (NB) is the most common extracranial solid tumor in childhood. Patients with relapsed/refractory disease have a poor prognosis, and additional therapeutic options are needed. Mutations and amplifications in the ALK (Anaplastic Lymphoma Kinase) gene constitute a key target for treatment. Our goal, within the Italian project of PeRsonalizEdMEdicine (PREME), was to evaluate the genomic status of patients with relapsed/refractory NB and to implement targeted therapies in those with targetable mutations. From November 2018 to November 2021, we performed Whole Exome Sequencing or Targeted Gene Panel Sequencing in relapsed/refractory NB patients in order to identify druggable variants. Activating mutations of ALK were identified in 8(28.57%) of 28 relapsed/refractory NB patients. The mutation p.F1174L was found in six patients, whereas p.R1275Q was found in one and the unknown mutation p.S104R in another. Three patients died before treatment could be started, while five patients received crizotinib: two in monotherapy (one with p.F1174L and the other with p.S104R) and three (with p.F1174L variant) in combination with chemotherapy. All treated patients showed a clinical improvement, and one had complete remission after two cycles of combined treatment. The most common treatment-related toxicities were hematological. ALK inhibitors may play an important role in the treatment of ALK-mutated NB patients