An Update on Circular RNA in Pediatric Cancers

Circular RNAs (circRNAs) are a class of single-stranded closed noncoding RNA molecules which are formed as a result of reverse splicing of mRNAs. Despite their relative abundance, only recently there appeared an increased interest in the understanding of their regulatory importance. Among their most relevant characteristics are high stability, abundance and evolutionary conservation among species. CircRNAs are implicated in several cellular functions, ranging from miRNA and protein sponges to transcriptional modulation and splicing. Additionally, circRNAs’ aberrant expression in pathological conditions is bringing to light their possible use as diagnostic and prognostic biomarkers. Their use as indicator molecules of pathological changes is also supported by their peculiar covalent closed cyclic structure which bestows resistance to RNases. Their regulatory role in cancer pathogenesis and metastasis is supported by studies involving human tumors that have investigated different expression profiles of these molecules. As endogenous competitive RNA, circRNAs can regulate tumor proliferation and invasion and they arouse great consideration as potential therapeutic biomarkers and targets for cancer. In this review, we describe the most recent findings on circRNAs in the most common pediatric solid cancers (such as brain tumors, neuroblastomas, and sarcomas) and in more rare ones (such as Wilms tumors, hepatoblastomas, and retinoblastomas)

Genotyping and antibiotic resistance traits in Campylobacter jejuni and coli from pigs and wild boars in Italy

The present study investigated the genomic constitution and antimicrobial resistance (AMR) of 238 Campylobacter from pigs and wild boars in Italy between 2012 and 2019. Campylobacter strains were genotyped using multilocus sequence typing (MLST) and whole genome MLST (wgMLST), screened for antimicrobial resistance genes, and tested for phenotypic susceptibility to six different antibiotics. C. coli was detected in 98.31% and 91.66% of pigs and wild boars, while C. jejuni was isolated in the remaining cases. MLST assigned 73 STs and 13 STs in pigs and wild boars, respectively, including 44 novel STs. The predominant ST in pigs was ST-854 (12.36%), followed by ST-9264 (6.18%). ST-1055 and ST-1417 were predominant in wild boars (30% and 13.33%, respectively). The minimum spanning tree using 1,121 global MLST profiles showed specific Italian clusters and a clear separation between pig and wild boar profiles. The wgMLST confirmed the MLST clustering and revealed a high genetic diversity within C. coli population in Italy. Minimum inhibitory concentrations (MIC) of six antibiotics revealed higher resistance in pigs to ciprofloxacin, nalidixic acid, streptomycin and tetracycline, compared to wild boar. In contrast, most strains were susceptible to gentamicin. Worrying levels of multidrug resistance (MDR) were observed mostly in pig isolates. Molecular screening of AMR mechanisms revealed the predominance of gyrA T86I substitution among fluoroquinolone- and quinolone-resistant isolates, and the 23S rRNA A2075G mutation among macrolide-resistant isolates. Other resistance determinants were observed: (i) tet(O) gene was present among tetracycline-resistant isolates; (ii) rpsL and aph(3’)-III genes conferring resistance to aminoglycosides, were identified only in streptomycin or gentamicin-resistant pig isolates; (iii) cmeA, cmeB, cmeC, cmeR genes responsible of pump efflux mechanisms, were observed in almost all the strains; (iv) OXA-61, encoding β-lactamase, was found in the half of the strains. Genotypic and phenotypic AMR profiling was fairly correlated for quinolones/fluoroquinolones. Campylobacter infection is common also in wild boar populations in Italy, suggesting that wild boars could be a reservoir of resistant and multi-resistant Campylobacter species, which may be of public health concern. The present study adds to our knowledge on the epidemiological and ecological traits of this pathogen in domesticated and wild swine

A chart review on the feasibility and safety of the Vincristine Irinotecan Pazopanib (VIPaz) association in children and adolescents with resistant or relapsed sarcomas

Background: Pediatric patients with relapsed or refractory sarcomas have poor outcome and need novel therapies that provide disease control while maintaining an acceptable quality of life. The safety of vincristine, irinotecan, and pazopanib (VIPaz) association has not yet been published in this population. Methods: A chart review was conducted in children and adolescents with relapsed or refractory bone and soft tissue sarcomas who received VIPaz in our institution. Results: One hundred sixty-six patients with a diagnosis of soft or bone sarcoma were admitted to our hospital in the period between March 2015 and August 2018, 30 were relapsed or resistant. Seventeen out of 30 resistant or relapsed patients (median age, 14 years) received 114 VIPaz cycles (median six cycles per patient, range 1–17). Sixteen courses (15%) resulted in gastrointestinal toxicity with Grade two diarrhea; 35 courses (30%) resulted in Grade ≥3 neutropenia. One patient presented Grade two hypothyroidism after nine courses, and another one had Grade two hyperbilirubinemia after 12 courses. Two and five patients required a 25% dose reduction of irinotecan (because of diarrhea) and pazopanib (because of neutropenia four and hyperbilirubinemia 1), respectively. No patient experienced heart failure, hypertension, nor posterior reversible encephalopathy syndrome. Pneumothorax was not reported in any case even in lung metastatic patients. After two and four VIPaz cycles, we observed one complete response (CR), five partial responses (PRs), seven stable diseases (SDs), and four progressive diseases (PDs). With a median follow-up of 15 months (range 3–32), five out of 17 (29%) patients were alive, and four patients were in continuous CR after 12 VIPaz cycles. Conclusions: The VIPaz regimen might be a safe option in children and adolescents with relapsed or refractory sarcomas otherwise unable to be enrolled in other clinical trials; on the other hand, the efficacy of pazopanib observed cannot be sustained from the current study

Antimicrobial resistance genotypes and phenotypes of Campylobacter jejuni isolated in Italy from humans, birds from wild and urban habitats, and poultry

Campylobacter jejuni, a common foodborne zoonotic pathogen, causes gastroenteritis worldwide and is increasingly resistant to antibiotics. We aimed to investigate the antimicrobial resistance (AMR) genotypes of C. jejuni isolated from humans, poultry and birds from wild and urban Italian habitats to identify correlations between phenotypic and genotypic AMR in the isolates. Altogether, 644 C. jejuni isolates from humans (51), poultry (526) and wild- and urban-habitat birds (67) were analysed. The resistance phenotypes of the isolates were determined using the microdilution method with EUCAST breakpoints, and AMR-associated genes and single nucleotide polymorphisms were obtained from a publicly available database. Antimicrobial susceptibility testing showed that C. jejuni isolates from poultry and humans were highly resistant to ciprofloxacin (85.55% and 76.47%, respectively), nalidixic acid (75.48% and 74.51%, respectively) and tetracycline (67.87% and 49.02%, respectively). Fewer isolates from the wild- and urban-habitat birds were resistant to tetracycline (19.40%), fluoroquinolones (13.43%), and quinolone and streptomycin (10.45%). We retrieved seven AMR genes (tet (O), cmeA, cmeB, cmeC, cmeR, blaOXA-61 and blaOXA- 184) and gyrA-associated point mutations. Two major B-lactam genes called blaOXA-61 and blaOXA-184 were prevalent at 62.93% and 82.08% in the poultry and the other bird groups, respectively. Strong correlations between genotypic and phenotypic resistance were found for fluoroquinolones and tetracycline. Compared with the farmed chickens, the incidence of AMR in the C. jejuni isolates from the other bird groups was low, confirming that the food-production birds are much more exposed to antimicrobials. The improper and overuse of antibiotics in the human population and in animal husbandry has resulted in an increase in antibiotic-resistant infections, particularly fluoroquinolone resistant ones. Better understanding of the AMR mechanisms in C. jejuni is necessary to develop new strategies for improving AMR programs and provide the most appropriate therapies to human and veterinary populations

Proteomic profiling of retinoblastoma-derived exosomes reveals potential biomarkers of vitreous seeding

Retinoblastoma (RB) is the most common tumor of the eye in early childhood. Although recent advances in conservative treatment have greatly improved the visual outcome, local tumor control remains difficult in the presence of massive vitreous seeding. Traditional biopsy has long been considered unsafe in RB, due to the risk of extraocular spread. Thus, the identification of new biomarkers is crucial to design safer diagnostic and more effective therapeutic approaches. Exosomes, membrane-derived nanovesicles that are secreted abundantly by aggressive tumor cells and that can be isolated from several biological fluids, represent an interesting alternative for the detection of tumor-associated biomarkers. In this study, we defined the protein signature of exosomes released by RB tumors (RBT) and vitreous seeding (RBVS) primary cell lines by high resolution mass spectrometry. A total of 5666 proteins were identified. Among these, 5223 and 3637 were expressed in exosomes RBT and one RBVS group, respectively. Gene enrichment analysis of exclusively and differentially expressed proteins and network analysis identified in RBVS exosomes upregulated proteins specifically related to invasion and metastasis, such as proteins involved in extracellular matrix (ECM) remodeling and interaction, resistance to anoikis and the metabolism/catabolism of glucose and amino acids

Neuroblastoma-secreted exosomes carrying miR‐375 promote osteogenic differentiation of bone-marrow mesenchymal stromal cells

Bone marrow (BM) is the major target organ for neuroblastoma (NB) metastasis and its involvement is associated with poor outcome. Yet, the mechanism by which NB cells invade BM is largely unknown. Tumour microenvironment represents a key element in tumour progression and mesenchymal stromal cells (MSCs) have been recognized as a fundamental part of the associated tumour stroma. Here, we show that BM-MSCs isolated from NB patients with BM involvement exhibit a greater osteogenic potential than MSCs from non-infiltrated BM. We show that BM metastasis-derived NB-cell lines secrete higher levels of exosomal miR-375, which promotes osteogenic differentiation in MSCs. Of note, clinical data demonstrate that high level of miR-375 correlates with BM metastasis in NB patients. Our findings suggest, indeed, a potential role for exosomal miR-375 in determining a favourable microenvironment in BM to promote metastatic progression. MiR-375 may, thus, represent a novel biomarker and a potential target for NB patients with BM involvement

Integration of multiple platforms for the analysis of multifluorescent marking technology applied to pediatric GBM and dipg

The intratumor heterogeneity represents one of the most difficult challenges for the development of effective therapies to treat pediatric glioblastoma (pGBM) and diffuse intrinsic pontine glioma (DIPG). These brain tumors are composed of heterogeneous cell subpopulations that coexist and cooperate to build a functional network responsible for their aggressive phenotype. Understanding the cellular and molecular mechanisms sustaining such network will be crucial for the identification of new therapeutic strategies. To study more in-depth these mechanisms, we sought to apply the Multifluorescent Marking Technology. We generated multifluorescent pGBM and DIPG bulk cell lines randomly expressing six different fluorescent proteins and from which we derived stable optical barcoded single cell-derived clones. In this study, we focused on the application of the Multifluorescent Marking Technology in 2D and 3D in vitro/ex vivo culture systems. We discuss how we integrated different multimodal fluorescence analysis platforms, identifying their strengths and limitations, to establish the tools that will enable further studies on the intratumor heterogeneity and interclonal interactions in pGBM and DIPG

MYOD-SKP2 axis boosts tumorigenesis in fusion negative rhabdomyosarcoma by preventing differentiation through p57Kip2^{Kip2} targeting

Rhabdomyosarcomas (RMS) are pediatric mesenchymal-derived malignancies encompassing PAX3/7-FOXO1 Fusion Positive (FP)-RMS, and Fusion Negative (FN)-RMS with frequent RAS pathway mutations. RMS express the master myogenic transcription factor MYOD that, whilst essential for survival, cannot support differentiation. Here we discover SKP2, an oncogenic E3-ubiquitin ligase, as a critical pro-tumorigenic driver in FN-RMS. We show that SKP2 is overexpressed in RMS through the binding of MYOD to an intronic enhancer. SKP2 in FN-RMS promotes cell cycle progression and prevents differentiation by directly targeting p27$^{Kip1}$ and p57$^{Kip2}$, respectively. SKP2 depletion unlocks a partly MYOD-dependent myogenic transcriptional program and strongly affects stemness and tumorigenic features and prevents in vivo tumor growth. These effects are mirrored by the investigational NEDDylation inhibitor MLN4924. Results demonstrate a crucial crosstalk between transcriptional and post-translational mechanisms through the MYOD-SKP2 axis that contributes to tumorigenesis in FN-RMS. Finally, NEDDylation inhibition is identified as a potential therapeutic vulnerability in FN-RMS