Targeting ATP2B1 impairs PI3K/Akt/FOXO signaling and reduces SARS-COV-2 infection and replication.

ATP2B1 is a known regulator of calcium (Ca2+) cellular export and homeostasis. Diminished levels of intracellular Ca2+ content have been suggested to impair SARS-CoV-2 replication. Here, we demonstrate that a nontoxic caloxin-derivative compound (PI-7) reduces intracellular Ca2+ levels and impairs SARS-CoV-2 infection. Furthermore, a rare homozygous intronic variant of ATP2B1 is shown to be associated with the severity of COVID-19. The mechanism of action during SARS-CoV-2 infection involves the PI3K/Akt signaling pathway activation, inactivation of FOXO3 transcription factor function, and subsequent transcriptional inhibition of the membrane and reticulum Ca2+ pumps ATP2B1 and ATP2A1, respectively. The pharmacological action of compound PI-7 on sustaining both ATP2B1 and ATP2A1 expression reduces the intracellular cytoplasmic Ca2+ pool and thus negatively influences SARS-CoV-2 replication and propagation. As compound PI-7 lacks toxicity in vitro, its prophylactic use as a therapeutic agent against COVID-19 is envisioned here.

Improving single nucleotide polymorphisms genotyping accuracy for dihydropyrimidine dehydrogenase testing in pharmacogenetics.

Fluoropyrimidines, crucial in cancer treatment, often cause toxicity concerns even at standard doses. Toxic accumulation of fluoropyrimidine metabolites, culminating in adverse effects, can stem from impaired dihydropyrimidine dehydrogenase (DPYD) enzymatic function. Emerging evidence underscores the role of single nucleotide polymorphisms (SNPs) in DPYD gene, capable of inducing DPYD activity deficiency. Consequently, DPYD genotyping's importance is on the rise in clinical practice before initiating fluoropyrimidine treatment. Although polymerase chain reaction (PCR) followed by Sanger sequencing (SS; PCR-SS) is a prevalent method for DPYD genotyping, it may encounter limitations. In this context, there is reported a case in which a routine PCR-SS approach for genotyping DPYD SNP rs55886062 failed in a proband of African descent. The Clinical Pharmacogenetics Implementation Consortium (CPIC) categorizes the guanine (G) allele of this SNP as non-functional. The enforcement of whole genome sequencing (WGS) approach led to the identification of two adenine (A) insertions near the PCR primers annealing regions in the proband, responsible for a sequence frameshift and a genotyping error for rs55886062. These SNPs (rs145228578, 1-97981199-T-TA and rs141050810, 1-97981622-G-GA) were extremely rare in non-Finnish Europeans (0.05%) but prevalent in African populations (16%). Although limited evidence was available for these SNPs, they were catalogued as benign variants in public databases. Notably, these two SNPs exhibited a high linkage disequilibrium [LD; squared correlation coefficient (R2) = 0.98]. These findings highlighted the importance to consider the prevalence of genetic variants within diverse ethnic populations when designing primers and probes for SNP genotyping in pharmacogenetic testing. This preventive measure is essential to avoid sequence frameshifts or primer misalignments arising from SNP occurrences in the genome, which can compromise PCR-SS and lead to genotyping failures. Furthermore, this case highlights the significance of exploring alternative genotyping approaches, like WGS, when confronted with challenges associated with conventional techniques.

Exploring the role of HLA variants in neuroblastoma susceptibility through whole exome sequencing.

Although a number of susceptibility loci for neuroblastoma (NB) have been identified by genome-wide association studies, it is still unclear whether variants in the HLA region contribute to NB susceptibility. In this study, we conducted a comprehensive genetic analysis of variants in the HLA region among 724 NB patients and 2863 matched controls from different cohorts. We exploited whole-exome sequencing data to accurately type HLA alleles with an ensemble approach on the results from three different typing tools, and carried out rigorous sample quality control to ensure a fine-scale ancestry matching. The frequencies of common HLA alleles were compared between cases and controls by logistic regression under additive and non-additive models. Population stratification was taken into account adjusting for ancestry-informative principal components. We detected significant HLA associations with NB. In particular, HLA-DQB1*05:02 (OR = 1.61; padj = 5.4 × 10-3) and HLA-DRB1*16:01 (OR = 1.60; padj = 2.3 × 10-2) alleles were associated to higher risk of developing NB. Conditional analysis highlighted the HLA-DQB1*05:02 allele and its residue Ser57 as key to this association. DQB1*05:02 allele was not associated to clinical features worse outcomes in the NB cohort. Nevertheless, a risk score derived from the allelic combinations of five HLA variants showed a substantial predictive value for patient survival (HR = 1.53; p = 0.032) that was independent from established NB prognostic factors. Our study leveraged powerful computational methods to explore WES data and HLA variants and to reveal complex genetic associations. Further studies are needed to validate the mechanisms of these interactions that contribute to the multifaceted pattern of factors underlying the disease initiation and progression.

From the identification of actionable molecular targets to the generation of faithful neuroblastoma patient-derived preclinical models.

BACKGROUND: Neuroblastoma (NB) represents the most frequent and aggressive form of extracranial solid tumor of infants. Although the overall survival of patients with NB has improved in the last years, more than 50% of high-risk patients still undergo a relapse. Thus, in the era of precision/personalized medicine, the need for high-risk NB patient-specific therapies is urgent. METHODS: Within the PeRsonalizEd Medicine (PREME) program, patient-derived NB tumors and bone marrow (BM)-infiltrating NB cells, derived from either iliac crests or tumor bone lesions, underwent to histological and to flow cytometry immunophenotyping, respectively. BM samples containing a NB cells infiltration from 1 to 50 percent, underwent to a subsequent NB cells enrichment using immune-magnetic manipulation. Then, NB samples were used for the identification of actionable targets and for the generation of 3D/tumor-spheres and Patient-Derived Xenografts (PDX) and Cell PDX (CPDX) preclinical models. RESULTS: Eighty-four percent of NB-patients showed potentially therapeutically targetable somatic alterations (including point mutations, copy number variations and mRNA over-expression). Sixty-six percent of samples showed alterations, graded as "very high priority", that are validated to be directly targetable by an approved drug or an investigational agent. A molecular targeted therapy was applied for four patients, while a genetic counseling was suggested to two patients having one pathogenic germline variant in known cancer predisposition genes. Out of eleven samples implanted in mice, five gave rise to (C)PDX, all preserved in a local PDX Bio-bank. Interestingly, comparing all molecular alterations and histological and immunophenotypic features among the original patient's tumors and PDX/CPDX up to second generation, a high grade of similarity was observed. Notably, also 3D models conserved immunophenotypic features and molecular alterations of the original tumors. CONCLUSIONS: PREME confirms the possibility of identifying targetable genomic alterations in NB, indeed, a molecular targeted therapy was applied to four NB patients. PREME paves the way to the creation of clinically relevant repositories of faithful patient-derived (C)PDX and 3D models, on which testing precision, NB standard-of-care and experimental medicines.

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.

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.

CFDP1 is a neuroblastoma susceptibility gene that regulates transcription factors of the noradrenergic cell identity.

Pleiotropic genetic factors (e.g., DNA polymorphisms) may be involved in the initiation of neuroblastoma (NB) and coronary artery disease (CAD) given their common origin from defects in neural crest development. To discover novel NB susceptibility genes, we conducted a three-stage survey including a meta-analysis of NB and CAD genome-wide association data, prioritization of NB causal variants, and validation in an independent cohort of affected individuals-control subjects. The lead SNP, rs13337397 at the 16q23.1 locus, associated with both diseases in the meta-analysis and with NB in the validation study. All the SNPs in linkage disequilibrium with rs13337397 were annotated using the H3K27ac epigenetic marker of neural crest cells (NCC) and NB cell lines. Indeed, we identified the functional SNP rs13337017, mapping within an enhancer of NCCs and NB cell lines and showing long-range interactions with CFDP1 by Hi-C analysis. Luciferase assays indicated that the risk allele of rs13337017 increased CFDP1 expression in NB cell lines. Of note, CFDP1 high expression associated with unfavorable prognostic markers in an analysis including 498 NB transcriptomes. Moreover, depletion of CFDP1 markedly decreased viability and migration and increased apoptotic rates in NB cell lines. Finally, transcriptome and qPCR analyses revealed that the depletion of CFDP1 may affect noradrenergic neuron differentiation by downregulating master regulators of sympathetic noradrenergic identity, including PHOX2B, HAND2, and GATA3. Our data strongly suggest that CFDP1 acts as oncogene in NB. In addition, we provide evidence that genetic predisposition to NB can be mediated by the alteration of noradrenergic lineage-specific gene expression.

Italian Precision Medicine in Pediatric Oncology: Moving beyond Actionable Alterations.

Neuroblastoma (NB) is the most common extracranial solid tumor encountered in childhood. Although there has been significant improvement in the outcomes of patients with high-risk disease, the prognosis for patients with metastatic relapse or refractory disease is poor. Hence, the clinical integration of genome sequencing into standard clinical practice is necessary in order to develop personalized therapy for children with relapsed or refractory disease. The PeRsonalizEdMEdicine (PREME) project focuses on the design of innovative therapeutic strategies for patients suffering from relapsed NB. We performed whole exome sequencing (WES) of patient-matched tumor-normal samples to identify genetic variants amenable to precision medicine. Specifically, two patients were studied (First case: a three-year-old male with early relapsed NB; Second case: a 20-year-old male who relapsed 10 years after the first diagnosis of NB). Results were reviewed by a multi-disciplinary molecular tumor board (MTB) and clinical reports were issued to the ordering physician. WES revealed the mutation c.G320C in the CUL4A gene in case 1 and the mutation c.A484G in the PSMC2 gene in case 2. Both patients were treated according to these actionable alterations, with promising results. The effective treatment of NB is one of the main challenges in pediatric oncology. In the era of precision medicine, the need to design new therapeutic strategies for NB is fundamental. Our results demonstrate the feasibility of incorporating clinical WES into pediatric oncology practice.

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.

Germline rare variants of lectin pathway genes predispose to asymptomatic SARS-CoV-2 infection in elderly individuals.

PURPOSE: Emerging evidence suggest that infection-dependent hyperactivation of complement system (CS) may worsen COVID-19 outcome. We investigated the role of predicted high impact rare variants - referred as qualifying variants (QVs) - of CS genes in predisposing asymptomatic COVID-19 in elderly individuals, known to be more susceptible to severe disease. METHODS: Exploiting exome sequencing data and 56 CS genes, we performed a gene-based collapsing test between 164 asymptomatic subjects (aged ≥60 years) and 56,885 European individuals from the Genome Aggregation Database. We replicated this test comparing the same asymptomatic individuals with 147 hospitalized patients with COVID-19. RESULTS: We found an enrichment of QVs in 3 genes (MASP1, COLEC11, and COLEC10), which belong to the lectin pathway, in the asymptomatic cohort. Analyses of complement activity in serum showed decreased activity of lectin pathway in asymptomatic individuals with QVs. Finally, we found allelic variants associated with asymptomatic COVID-19 phenotype and with a decreased expression of MASP1, COLEC11, and COLEC10 in lung tissue. CONCLUSION: This study suggests that genetic rare variants can protect from severe COVID-19 by mitigating the activity of lectin pathway and prothrombin. The genetic data obtained through ES of 786 asymptomatic and 147 hospitalized individuals are publicly available at http://espocovid.ceinge.unina.it/.

FGFR1 is a potential therapeutic target in neuroblastoma.

BACKGROUND: FGFR1 regulates cell-cell adhesion and extracellular matrix architecture and acts as oncogene in several cancers. Potential cancer driver mutations of FGFR1 occur in neuroblastoma (NB), a neural crest-derived pediatric tumor arising in sympathetic nervous system, but so far they have not been studied experimentally. We investigated the driver-oncogene role of FGFR1 and the implication of N546K mutation in therapy-resistance in NB cells. METHODS: Public datasets were used to predict the correlation of FGFR1 expression with NB clinical outcomes. Whole genome sequencing data of 19 paired diagnostic and relapse NB samples were used to find somatic mutations. In NB cell lines, silencing by short hairpin RNA and transient overexpression of FGFR1 were performed to evaluate the effect of the identified mutation by cell growth, invasion and cologenicity assays. HEK293, SHSY5Y and SKNBE2 were selected to investigate subcellular wild-type and mutated protein localization. FGFR1 inhibitor (AZD4547), alone or in combination with PI3K inhibitor (GDC0941), was used to rescue malignant phenotypes induced by overexpression of FGFR1 wild-type and mutated protein. RESULTS: High FGFR1 expression correlated with low relapse-free survival in two independent NB gene expression datasets. In addition, we found the somatic mutation N546K, the most recurrent point mutation of FGFR1 in all cancers and already reported in NB, in one out of 19 matched primary and recurrent tumors. Loss of FGFR1 function attenuated invasion and cologenicity in NB cells, whereas FGFR1 overexpression enhanced oncogenicity. The overexpression of FGFR1N546K protein showed a higher nuclear localization compared to wild-type protein and increased cellular invasion and cologenicity. Moreover, N546K mutation caused the failure in response to treatment with FGFR1 inhibitor by activation of ERK, STAT3 and AKT pathways. The combination of FGFR1 and PI3K pathway inhibitors was effective in reducing the invasive and colonigenic ability of cells overexpressing FGFR1 mutated protein. CONCLUSIONS: FGFR1 is an actionable driver oncogene in NB and a promising therapy may consist in targeting FGFR1 mutations in patients with therapy-resistant NB.

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) to design a customized panel for the deep sequencing of 56 neuroblastoma tumor and normal DNA sample pairs. To search for driver mutations, aCREs were defined by reanalysis of H3K27ac chromatin immunoprecipitation sequencing 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 HiC 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. SIGNIFICANCE: Assessment of noncoding cis-regulatory variants and long-range interaction data highlight the combined effect of somatic mutations in regulatory elements in driving neuroblastoma.

Functional annotation and investigation of the 10q24.33 melanoma risk locus identifies a common variant that influences transcriptional regulation of OBFC1.

The 10q24.33 locus is known to be associated with susceptibility to cutaneous malignant melanoma (CMM), but the mechanisms underlying this association have been not extensively investigated. We carried out an integrative genomic analysis of 10q24.33 using epigenomic annotations and in vitro reporter gene assays to identify regulatory variants. We found two putative functional single nucleotide polymorphisms (SNPs) in an enhancer and in the promoter of OBFC1, respectively, in neural crest and CMM cells, one, rs2995264, altering enhancer activity. The minor allele G of rs2995264 correlated with lower OBFC1 expression in 470 CMM tumors and was confirmed to increase the CMM risk in a cohort of 484 CMM cases and 1801 controls of Italian origin. Hi-C and chromosome conformation capture (3C) experiments showed the interaction between the enhancer-SNP region and the promoter of OBFC1 and an isogenic model characterized by CRISPR-Cas9 deletion of the enhancer-SNP region confirmed the potential regulatory effect of rs2995264 on OBFC1 transcription. Moreover, the presence of G-rs2995264 risk allele reduced the binding affinity of the transcription factor MEOX2. Biologic investigations showed significant cell viability upon depletion of OBFC1, specifically in CMM cells that were homozygous for the protective allele. Clinically, high levels of OBFC1 expression associated with histologically favorable CMM tumors. Finally, preliminary results suggested the potential effect of decreased OBFC1 expression on telomerase activity in tumorigenic conditions. Our results support the hypothesis that reduced expression of OBFC1 gene through functional heritable DNA variation can contribute to malignant transformation of normal melanocytes.

The TNFRSF13C H159Y Variant Is Associated with Severe COVID-19: A Retrospective Study of 500 Patients from Southern Italy.

To identify host genetic determinants involved in humoral immunity and associated with the risk of developing severe COVID-19, we analyzed 500 SARS-CoV-2 positive subjects from Southern Italy. We examined the coding sequences of 10 common variable immunodeficiency-associated genes obtained by the whole-exome sequencing of 121 hospitalized patients. These 10 genes showed significant enrichment in predicted pathogenic point mutations in severe patients compared with the non-severe ones. Moreover, in the TNFRSF13C gene, the minor allele of the p.His159Tyr variant, which is known to increase NF-kB activation and B-cell production, was significantly more frequent in the 38 severe cases compared to both the 83 non-severe patients and the 375 asymptomatic subjects further genotyped. This finding identified a potential genetic risk factor of severe COVID-19 that not only may serve to unravel the mechanisms underlying the disease severity but, also, may contribute to build the rationale for individualized management based on B-cell therapy.

Regulatory Noncoding and Predicted Pathogenic Coding Variants of CCR5 Predispose to Severe COVID-19.

Genome-wide association studies (GWAS) found locus 3p21.31 associated with severe COVID-19. CCR5 resides at the same locus and, given its known biological role in other infection diseases, we investigated if common noncoding and rare coding variants, affecting CCR5, can predispose to severe COVID-19. We combined single nucleotide polymorphisms (SNPs) that met the suggestive significance level (P ≤ 1 × 10-5) at the 3p21.31 locus in public GWAS datasets (6406 COVID-19 hospitalized patients and 902,088 controls) with gene expression data from 208 lung tissues, Hi-C, and Chip-seq data. Through whole exome sequencing (WES), we explored rare coding variants in 147 severe COVID-19 patients. We identified three SNPs (rs9845542, rs12639314, and rs35951367) associated with severe COVID-19 whose risk alleles correlated with low CCR5 expression in lung tissues. The rs35951367 resided in a CTFC binding site that interacts with CCR5 gene in lung tissues and was confirmed to be associated with severe COVID-19 in two independent datasets. We also identified a rare coding variant (rs34418657) associated with the risk of developing severe COVID-19. Our results suggest a biological role of CCR5 in the progression of COVID-19 as common and rare genetic variants can increase the risk of developing severe COVID-19 by affecting the functions of CCR5.

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 single nucleotide polymorphisms (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.

Association of PARP1 polymorphisms with response to chemotherapy in patients with high-risk neuroblastoma.

The genetic aetiology and the molecular mechanisms that characterize high-risk neuroblastoma are still little understood. The majority of high-risk neuroblastoma patients do not take advantage of current induction therapy. So far, one of the main reasons liable for cancer therapeutic failure is the acquisition of resistance to cytotoxic anticancer drugs, because of the DNA repair system of tumour cells. PARP1 is one of the main DNA damage sensors involved in the DNA repair system and genomic stability. We observed that high PARP1 mRNA level is associated with unfavourable prognosis in 3 public gene expression NB patients' datasets and in 20 neuroblastomas analysed by qRT-PCR. Among 4983 SNPs in PARP1, we selected two potential functional SNPs. We investigated the association of rs907187, in PARP1 promoter, and rs2048426 in non-coding region with response chemotherapy in 121 Italian patients with high-risk NB. Results showed that minor G allele of rs907187 associated with induction response of patients (P = .02) and with decrease PARP1 mRNA levels in NB cell line (P = .003). Furthermore, rs907187 was predicted to alter the binding site of E2F1 transcription factor. Specifically, allele G had low binding affinity with E2F1 whose expression positively correlates with PARP1 expression and associated with poor prognosis of patients with NB. By contrast, we did not find genetic association for the SNP rs2048426. These data reveal rs907187 as a novel potential risk variant associated with the failure of induction therapy for high-risk NB.

Functional characterization of full-length BARD1 strengthens its role as a tumor suppressor in neuroblastoma.

BARD1 is associated with the development of high-risk neuroblastoma patients. Particularly, the expression of full length (FL) isoform, FL BARD1, correlates to high-risk neuroblastoma development and its inhibition is sufficient to induce neuroblastoma cells towards a worst phenotype. Here we have investigated the mechanisms of FL BARD1 in neuroblastoma cell lines depleted for FL BARD1 expression. We have shown that FL BARD1 expression protects the cells from spontaneous DNA damage and from damage accumulated after irradiation. We demonstrated a role for FL BARD1 as tumor suppressor to prevent unscheduled mitotic entry of DNA damaged cells and to lead to death cells that have bypassed cell cycle checkpoints. FL BARD1-depleted cells that have survived to checkpoints acquire features of aggressiveness. Overall, our results show that FL BARD1 may defend cells against cancer and prevent malignant transformation of cells.

Neural crest-derived tumor neuroblastoma and melanoma share 1p13.2 as susceptibility locus that shows a long-range interaction with the SLC16A1 gene.

Neuroblastoma (NB) and malignant cutaneous melanoma (CMM) are neural crest cells (NCC)-derived tumors and may have a shared genetic basis, but this has not been investigated systematically by genome-wide association studies (GWAS). We took a three-staged approach to conduct cross-disease meta-analysis of GWAS for NB and CMM (2101 NB cases and 4202 controls; 12 874 CMM cases and 23 203 controls) to identify shared loci. Findings were replicated in 1403 NB cases and 1403 controls of European ancestry and in 636 NB, 508 CMM cases and 2066 controls of Italian origin. We found a cross-association at locus 1p13.2 (rs2153977, odds ratio = 0.91, P = 5.36 × 10-8). We also detected a suggestive (P < 10-7) NB-CMM cross-association at 2q37.1 with opposite effect on cancer risk. Pathway analysis of 110 NB-CMM risk loci with P < 10-4 demonstrated enrichment of biological processes such as cell migration, cell cycle, metabolism and immune response, which are essential of human NCC development, underlying both tumors. In vitro and in silico analyses indicated that the rs2153977-T protective allele, located in an NB and CMM enhancer, decreased expression of SLC16A1 via long-range loop formation and altered a T-box protein binding site. Upon depletion of SLC16A1, we observed a decrease of cellular proliferation and invasion in both NB and CMM cell lines, suggesting its role as oncogene. This is the largest study to date examining pleiotropy across two NC cell-derived tumors identifying 1p13.2 as common susceptibility locus for NB and CMM risk. We demonstrate that combining genome-wide association studies results across cancers with same origins can identify new loci common to neuroblastoma and melanoma arising from tissues which originate from neural crest cells. Our results also show 1p13.2 confer risk to neuroblastoma and melanoma by regulating SLC16A1.