Identification and bioinformatic characterization of a serum miRNA signature for early detection of laryngeal squamous cell carcinoma.

BACKGROUND: The growing understanding of cancer biology and the establishment of new treatment modalities has not yielded the expected results in terms of survival for Laryngeal Squamous Cell Cancer (LSCC). Early diagnosis, as well as prompt identification of patients with high risk of relapse would ensure greater chance of therapeutic success. However, this goal remains a challenge due to the absence of specific biomarkers for this neoplasm. METHODS: Serum samples from 45 LSCC patients and 23 healthy donors were collected for miRNA expression profiling by TaqMan Array analysis. Additional 20 patients and 42 healthy volunteers were included for the validation set, reaching an equal number of clinical samples for each group. The potential diagnostic ability of the such identified three-miRNA signature was confirmed by ROC analysis. Moreover, each miRNA was analyzed for the possible correlation with HNSCC patients' survival and TNM status by online databases Kaplan-Meier (KM) plotter and OncomiR. In silico analysis of common candidate targets and their network relevance to predict shared biological functions was finally performed by PANTHER and GeneMANIA software. RESULTS: We characterized serum miRNA profile of LSCC patients identifying a novel molecular signature, including miR-223, miR-93 and miR-532, as circulating marker endowed with high selectivity and specificity. The oncogenic effect and the prognostic significance of each miRNA was investigated by bioinformatic analysis, denoting significant correlation with OS. To analyse the molecular basis underlying the pro-tumorigenic role of the signature, we focused on the simultaneously regulated gene targets-IL6ST, GTDC1, MAP1B, CPEB3, PRKACB, NFIB, PURB, ATP2B1, ZNF148, PSD3, TBC1D15, PURA, KLF12-found by prediction tools and deepened for their functional role by pathway enrichment analysis. The results showed the involvement of 7 different biological processes, among which inflammation, proliferation, migration, apoptosis and angiogenesis. CONCLUSIONS: In conclusion, we have identified a possible miRNA signature for early LSCC diagnosis and we assumed that miR-93, miR-223 and miR-532 could orchestrate the regulation of multiple cancer-related processes. These findings encourage the possibility to deepen the molecular mechanisms underlying their oncogenic role, for the desirable development of novel therapeutic opportunities based on the use of short single-stranded oligonucleotides acting as non-coding RNA antagonists in cancer.

Exosomes multiplex profiling, a promising strategy for early diagnosis of laryngeal cancer.

BACKGROUND: Exosomes are nanosized vesicles released from all cells into surrounding biofluids, including cancer cells, and represent a very promising direction in terms of minimally invasive approaches to early disease detection. They carry tumor-specific biological contents such as DNA, RNA, proteins, lipids, and sugars, as well as surface molecules that are able to pinpoint the cellular source. By the above criteria, exosomes may be stratified according to the presence of tissue and disease-specific signatures and, due to their stability in such biofluids as plasma and serum, they represent an indispensable source of vital clinical insights from liquid biopsies, even at the earliest stages of cancer. Therefore, our work aimed to isolate and characterize LCa patients' derived exosomes from serum by Flow Cytometry in order to define a specific epitope signature exploitable for early diagnosis. METHODS: Circulating exosomes were collected from serum collected from 30 LCa patients and 20 healthy volunteers by the use of antibody affinity method exploiting CD63 specific surface marker. Membrane epitopes were then characterized by Flow cytometry multiplex analysis and compared between LCa Patients and Healthy donors. Clinical data were also matched to obtain statistical correlation. RESULTS: A distinct overexpression of CD1c, CD2, CD3, CD4, CD11c, CD14, CD20, CD44, CD56, CD105, CD146, and CD209 was identified in LCa patients compared to healthy controls, correlating positively with tumor presence. Conversely, CD24, CD31, and CD40, though not overexpressed in tumor samples, showed a significant correlation with nodal involvement in LCa patients (p < 0.01). CONCLUSION: This approach could allow us to set up a cost-effective and less invasive liquid biopsy protocol from a simple blood collection in order to early diagnose LCa and improve patients' outcomes and quality of life.

Immune cell infiltration and inflammatory landscape in primary brain tumours.

BACKGROUND: Primary malignant brain tumours are more than one-third of all brain tumours and despite the molecular investigation to identify cancer driver mutations, the current therapeutic options available are challenging due to high intratumour heterogeneity. In addition, an immunosuppressive and inflammatory tumour microenvironment strengthens cancer progression. Therefore, we defined an immune and inflammatory profiling of meningioma and glial tumours to elucidate the role of the immune infiltration in these cancer types. METHODS: Using tissue microarrays of 158 brain tumour samples, we assessed CD3, CD4, CD8, CD20, CD138, Granzyme B (GzmB), 5-Lipoxygenase (5-LOX), Programmed Death-Ligand 1 (PD-L1), O-6-Methylguanine-DNA Methyltransferase (MGMT) and Transglutaminase 2 (TG2) expression by immunohistochemistry (IHC). IHC results were correlated using a Spearman correlation matrix. Transcript expression, correlation, and overall survival (OS) analyses were evaluated using public datasets available on GEPIA2 in Glioblastoma (GBM) and Lower Grade Glioma (LGG) cohorts. RESULTS: Seven out of ten markers showed a significantly different IHC expression in at least one of the evaluated cohorts whereas CD3, CD4 and 5-LOX were differentially expressed between GBMs and astrocytomas. Correlation matrix analysis revealed that 5-LOX and GzmB expression were associated in both meningiomas and GBMs, whereas 5-LOX expression was significantly and positively correlated to TG2 in both meningioma and astrocytoma cohorts. These findings were confirmed with the correlation analysis of TCGA-GBM and LGG datasets. Profiling of mRNA levels indicated a significant increase in CD3 (CD3D, CD3E), and CD138 (SDC1) expression in GBM compared to control tissues. CD4 and 5-LOX (ALOX5) mRNA levels were significantly more expressed in tumour samples than in normal tissues in both GBM and LGG. In GBM cohort, GzmB (GZMB), SDC1 and MGMT gene expression predicted a poor overall survival (OS). Moreover, in LGG cohort, an increased expression of CD3 (CD3D, CD3E, CD3G), CD8 (CD8A), GZMB, CD20 (MS4A1), SDC1, PD-L1, ALOX5, and TG2 (TGM2) genes was associated with worse OS. CONCLUSIONS: Our data have revealed that there is a positive and significant correlation between the expression of 5-LOX and GzmB, both at RNA and protein level. Further evaluation is needed to understand the interplay of 5-LOX and immune infiltration in glioma progression.

Predictive significance of FGFR4 p.G388R polymorphism in metastatic colorectal cancer patients receiving trifluridine/tipiracil (TAS-102) treatment.

BACKGROUND: TAS-102 (Lonsurf®) is an oral fluoropyrimidine consisting of a combination of trifluridine (a thymidine analog) and tipiracil (a thymidine phosphorylation inhibitor). The drug is effective in metastatic colorectal cancer (mCRC) patients refractory to fluorouracil, irinotecan and oxaliplatin. This study is a real-world analysis, investigating the interplay of genotype/phenotype in relation to TAS-102 sensitivity. METHODS: Forty-seven consecutive mCRC patients were treated with TAS-102 at the National Cancer Institute of Naples from March 2019 to March 2021, at a dosage of 35 mg/m2, twice a day, in cycles of 28 days (from day 1 to 5 and from day 8 to 12). Clinical-pathological parameters were described. Activity was evaluated with RECIST criteria (v1.1) and toxicity with NCI-CTC (v5.0). Survival was depicted through the Kaplan-Meyer curves. Genetic features of patients were evaluated with Next Generation Sequencing (NGS) through the Illumina NovaSeq 6000 platform and TruSigt™Oncology 500 kit. RESULTS: Median age of patients was 65 years (range: 46-77). Forty-one patients had 2 or more metastatic sites and 38 patients underwent to more than 2 previous lines of therapies. ECOG (Eastern Cooperative Oncology Group) Performance Status (PS) was 2 in 19 patients. The median number of TAS-102 cycles was 4 (range: 2-12). The most frequent toxic event was neutropenia (G3/G4 in 16 patients). There were no severe (> 3) non-haematological toxicities or treatment-related deaths. Twenty-six patients experienced progressive disease (PD), 21 stable disease (SD). Three patients with long-lasting disease control (DC: complete, partial responses or stable disease) shared an FGFR4 (p.Gly388Arg) mutation. Patients experiencing DC had more frequently a low tumour growth rate (P = 0.0306) and an FGFR4 p.G388R variant (P < 0.0001). The FGFR4 Arg388 genotype was associated with better survival (median: 6.4 months) compared to the Gly388 genotype (median: 4 months); the HR was 0.25 (95% CI 0.12- 0.51; P = 0.0001 at Log-Rank test). CONCLUSIONS: This phenotype/genotype investigation suggests that the FGFR4 p.G388R variant may serve as a new marker for identifying patients who are responsive to TAS-102. A mechanistic hypothesis is proposed to interpret these findings.

Whole-genome analysis suggesting probiotic potential and safety properties of Pediococcus pentosaceus DSPZPP1, a promising LAB strain isolated from traditional fermented sausages of the Basilicata region (Southern Italy).

Introduction: Many lactic acid bacteria (LAB) strains are currently gaining attention in the food industry and various biological applications because of their harmless and functional properties. Given the growing consumer demand for safe food, further research into potential probiotic bacteria is beneficial. Therefore, we aimed to characterize Pediococcus pentosaceus DSPZPP1, a LAB strain isolated from traditional fermented sausages from the Basilicata region of Southern Italy. Methods: In this study, we analyzed the whole genome of the P. pentosaceus DSPZPP1 strain and performed in silico characterization to evaluate its applicability for probiotics and use in the food industry. Results and Discussion: The whole-genome assembly and functional annotations revealed many interesting characteristics of the DSPZPP1 strain. Sequencing raw reads were assembled into a draft genome of size 1,891,398 bp, with a G + C content of 37.3%. Functional annotation identified 1930 protein-encoding genes and 58 RNAs including tRNA, tmRNA, and 16S, 23S, and 5S rRNAs. The analysis shows the presence of genes that encode water-soluble B-group vitamins such as biotin, folate, coenzyme A, and riboflavin. Furthermore, the analysis revealed that the DSPZPP1 strain can synthesize class II bacteriocin, penocin A, adding importance to the food industry for bio-enriched food. The DSPZPP1 genome does not show the presence of plasmids, and no genes associated with antimicrobial resistance and virulence were found. In addition, two intact bacteriophages were identified. Importantly, the lowest probability value in pathogenicity analysis indicates that this strain is non-pathogenic to humans. 16 s rRNA-based phylogenetic analysis and comparative analysis based on ANI and Tetra reveal that the DSPZPP1 strain shares the closest evolutionary relationship with P. pentosaceus DSM 20336 and other Pediococcus strains. Analysis of carbohydrate active enzymes (CAZymes) identified glycosyl transferases (GT) as a main class of enzymes followed by glycoside hydrolases (GH). Our study shows several interesting characteristics of the isolated DSPZPP1 strain from fermented Italian sausages, suggesting its potential use as a promising probiotic candidate and making it more appropriate for selection as a future additive in biopreservation.