circPVT1 and PVT1/AKT3 show a role in cell proliferation, apoptosis, and tumor subtype-definition in small cell lung cancer.

Small cell lung cancer (SCLC) is treated as a homogeneous disease, although the expression of NEUROD1, ASCL1, POU2F3, and YAP1 identifies distinct molecular subtypes. The MYC oncogene, amplified in SCLC, was recently shown to act as a lineage-specific factor to associate subtypes with histological classes. Indeed, MYC-driven SCLCs show a distinct metabolic profile and drug sensitivity. To disentangle their molecular features, we focused on the co-amplified PVT1, frequently overexpressed and originating circular (circRNA) and chimeric RNAs. We analyzed hsa_circ_0001821 (circPVT1) and PVT1/AKT3 (chimPVT1) as examples of such transcripts, respectively, to unveil their tumorigenic contribution to SCLC. In detail, circPVT1 activated a pro-proliferative and anti-apoptotic program when over-expressed in lung cells, and knockdown of chimPVT1 induced a decrease in cell growth and an increase of apoptosis in SCLC in vitro. Moreover, the investigated PVT1 transcripts underlined a functional connection between MYC and YAP1/POU2F3, suggesting that they contribute to the transcriptional landscape associated with MYC amplification. In conclusion, we have uncovered a functional role of circular and chimeric PVT1 transcripts in SCLC; these entities may prove useful as novel biomarkers in MYC-amplified tumors.

Unravelling similarities and differences in the role of circular and linear PVT1 in cancer and human disease.

The plasmacytoma variant translocation 1 (PVT1) is a long non-coding RNA gene involved in human disease, mainly in cancer onset/progression. Although widely analysed, its biological roles need to be further clarified. Notably, functional studies on PVT1 are complicated by the occurrence of multiple transcript variants, linear and circular, which generate technical issues in the experimental procedures used to evaluate its impact on human disease. Among the many PVT1 transcripts, the linear PVT1 (lncPVT1) and the circular hsa_circ_0001821 (circPVT1) are frequently reported to perform similar pathologic and pro-tumorigenic functions when overexpressed. The stimulation of cell proliferation, invasion and drug resistance, cell metabolism regulation, and apoptosis inhibition is controlled through multiple targets, including MYC, p21, STAT3, vimentin, cadherins, the PI3K/AKT, HK2, BCL2, and CASP3. However, some of this evidence may originate from an incorrect evaluation of these transcripts as two separate molecules, as they share the lncPVT1 exon-2 sequence. We here summarise lncPVT1/circPVT1 functions by mainly focusing on shared pathways, pointing out the potential bias that may exist when the biological role of each transcript is analysed. These considerations may improve the knowledge about lncPVT1/circPVT1 and their specific targets, which deserve further studies due to their diagnostic, prognostic, and therapeutic potential.

A single-center study on 140 patients with cerebral cavernous malformations: 28 new pathogenic variants and functional characterization of a PDCD10 large deletion.

Cerebral cavernous malformation (CCM) is a capillary malformation arising in the central nervous system. CCM may occur sporadically or cluster in families with autosomal dominant transmission, incomplete penetrance, and variable expressivity. Three genes are associated with CCM KRIT1, CCM2, and PDCD10. This work is a retrospective single-center molecular study on samples from multiple Italian clinical providers. From a pool of 317 CCM index patients, we found germline variants in either of the three genes in 80 (25.2%) probands, for a total of 55 different variants. In available families, extended molecular analysis found segregation in 60 additional subjects, for a total of 140 mutated individuals. From the 55 variants, 39 occurred in KRIT1 (20 novel), 8 in CCM2 (4 novel), and 8 in PDCD10 (4 novel). Effects of the three novel KRIT1 missense variants were characterized in silico. We also investigated a novel PDCD10 deletion spanning exon 4-10, on patient's fibroblasts, which showed significant reduction of interactions between KRIT1 and CCM2 encoded proteins and impaired autophagy process. This is the largest study in Italian CCM patients and expands the known mutational spectrum of KRIT1, CCM2, and PDCD10. Our approach highlights the relevance of seeking supporting information to pathogenicity of new variants for the improvement of management of CCM.

A novel method for the isolation of single cells mimicking circulating tumour cells adhered on Smart Bio Surface slides by Laser Capture Microdissection.

In recent years, the importance of isolating single cells from blood circulation for several applications, such as non-invasive tumour diagnosis, the monitoring of minimal residual disease, and the analysis of circulating fetal cells for prenatal diagnosis, urged the need to set up innovative methods. For such applications, different methods were developed. All show some weaknesses, especially a limited sensitivity, and specificity. Here we present a new method for isolating a single or a limited number of cells adhered to SBS slides (Tethis S.p.a.) (a glass slide coated with Nanostructured Titanium Dioxide) by Laser Capture Microdissection (LCM) and subsequent Whole Genome Amplification. SBS slides have been shown to have an optimal performance in immobilizing circulating tumour cells (CTCs) from early breast cancer patients. In this work, we spiked cancer cells in blood samples to mimic CTCs. By defining laser parameters to cut intact samples, we were able to isolate genetically intact single cells. We demonstrate that SBS slides are optimally suited for isolating cells using LCM and that this method provides high-quality DNA, ideal for gene-specific assays such as PCR and Sanger sequencing for mutation analysis.

Endometrial Cancer: A Pilot Study of the Tissue Microbiota.

BACKGROUND: The endometrium remains a difficult tissue for the analysis of microbiota, mainly due to the low bacterial presence and the sampling procedures. Among its pathologies, endometrial cancer has not yet been completely investigated for its relationship with microbiota composition. In this work, we report on possible correlations between endometrial microbiota dysbiosis and endometrial cancer. METHODS: Women with endometrial cancer at various stages of tumor progression were enrolled together with women with a benign polymyomatous uterus as the control. Analyses were performed using biopsies collected at two specific endometrial sites during the surgery. This study adopted two approaches: the absolute quantification of the bacterial load, using droplet digital PCR (ddPCR), and the analysis of the bacterial composition, using a deep metabarcoding NGS procedure. RESULTS: ddPCR provided the first-ever assessment of the absolute quantification of bacterial DNA in the endometrium, confirming a generally low microbial abundance. Metabarcoding analysis revealed a different microbiota distribution in the two endometrial sites, regardless of pathology, accompanied by an overall higher prevalence of pathogenic bacterial genera in cancerous tissues. CONCLUSIONS: These results pave the way for future studies aimed at identifying potential biomarkers and gaining a deeper understanding of the role of bacteria associated with tumors.

Amplicon-Based Microbiome Profiling: From Second- to Third-Generation Sequencing for Higher Taxonomic Resolution.

The 16S rRNA amplicon-based sequencing approach represents the most common and cost-effective strategy with great potential for microbiome profiling. The use of second-generation sequencing (NGS) technologies has led to protocols based on the amplification of one or a few hypervariable regions, impacting the outcome of the analysis. Nowadays, comparative studies are necessary to assess different amplicon-based approaches, including the full-locus sequencing currently feasible thanks to third-generation sequencing (TGS) technologies. This study compared three different methods to achieve the deepest microbiome taxonomic characterization: (a) the single-region approach, (b) the multiplex approach, covering several regions of the target gene/region, both based on NGS short reads, and (c) the full-length approach, which analyzes the whole length of the target gene thanks to TGS long reads. Analyses carried out on benchmark microbiome samples, with a known taxonomic composition, highlighted a different classification performance, strongly associated with the type of hypervariable regions and the coverage of the target gene. Indeed, the full-length approach showed the greatest discriminating power, up to species level, also on complex real samples. This study supports the transition from NGS to TGS for the study of the microbiome, even if experimental and bioinformatic improvements are still necessary.

Natural and after colon washing fecal samples: the two sides of the coin for investigating the human gut microbiome.

To date several studies address the important role of gut microbiome and its interplay with the human host in the health and disease status. However, the selection of a universal sampling matrix representative of the microbial biodiversity associated with the gastrointestinal (GI) tract, is still challenging. Here we present a study in which, through a deep metabarcoding analysis of the 16S rRNA gene, we compared two sampling matrices, feces (F) and colon washing feces (CWF), in order to evaluate their relative effectiveness and accuracy in representing the complexity of the human gut microbiome. A cohort of 30 volunteers was recruited and paired F and CWF samples were collected from each subject. Alpha diversity analysis confirmed a slightly higher biodiversity of CWF compared to F matched samples. Likewise, beta diversity analysis proved that paired F and CWF microbiomes were quite similar in the same individual, but remarkable inter-individual variability occurred among the microbiomes of all participants. Taxonomic analysis in matched samples was carried out to investigate the intra and inter individual/s variability. Firmicutes, Bacteroidota, Proteobacteria and Actinobacteriota were the main phyla in both F and CWF samples. At genus level, Bacteirodetes was the most abundant in F and CWF samples, followed by Faecalibacterium, Blautia and Escherichia-Shigella. Our study highlights an inter-individual variability greater than intra-individual variability for paired F and CWF samples. Indeed, an overall higher similarity was observed across matched F and CWF samples, suggesting, as expected, a remarkable overlap between the microbiomes inferred using the matched F and CWF samples. Notably, absolute quantification of total 16S rDNA by droplet digital PCR (ddPCR) revealed comparable overall microbial load between paired F and CWF samples. We report here the first comparative study on fecal and colon washing fecal samples for investigating the human gut microbiome and show that both types of samples may be used equally for the study of the gut microbiome. The presented results suggest that the combined use of both types of sampling matrices could represent a suitable choice to obtain a more complete overview of the human gut microbiota for addressing different biological and clinical questions.

PVT1: A long non-coding RNA recurrently involved in neoplasia-associated fusion transcripts.

NGS technologies and bioinformatics tools allow the rapid identification of chimeric transcripts in cancer. More than 40,000 fusions are so far reported in the literature; however, for most of them, the role in oncogenesis is still not fully understood. This is the case for fusions involving the long non-coding RNA (lncRNA) Plasmacytoma variant translocation 1 (PVT1) (8q24.21). This lncRNA displays oncogenic functions in several cancer types interacting with microRNAs and proteins, but the role of PVT1 fusion transcripts is more obscure. These chimeras have been identified in both hematological malignancies and solid tumors, mainly arising from rearrangements and/or amplification of the 8q24 chromosomal region. In this review, we detail the full spectrum of PVT1 fusions in cancer, summarizing current knowledge about their genesis, function, and role as biomarkers.

CircRNAs and Fusion-circRNAs in cancer: New players in an old game.

Circular RNAs (circRNAs) are generated from 'back-splicing' events. Their circular structure makes them stable in cells and body fluids. These entities are involved in several human diseases including cancer, as they affect the expression of genes promoting proliferation, invasion, apoptosis, and angiogenesis. Moreover, they are secreted in extracellular vesicles, such as exosomes, having a potential role as messengers in cell-to-cell communications. CircRNAs are also generated by the back-splicing of linear fusion transcripts derived from genomic rearrangements, giving rise to fusion circRNAs (f-circRNAs). Here we discuss the most relevant results achieved by studying the role of circRNAs in cancer onset and progression, particularly focusing on f-circRNAs in hematological and solid tumors. Moreover, we report recent advances in the application of circRNAs as novel "liquid biopsy" biomarkers for early and non-invasive diagnosis of tumors, and as therapeutic targets in human cancer. Their use as engineered molecules sponging oncogenic miRNAs or stably expressing proteins/drugs is also discussed. All these achievements suggest the crucial importance of circRNAs and f-circRNAs in the future setup of personalized therapies in molecular medicine.