Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive neoplasia, characterized by early metastasis, low diagnostic rates at early stages, resistance to drugs, and poor prognosis. There is an urgent need to better characterize this disease in order to identify efficient diagnostic/prognostic biomarkers. Since microRNAs (miRNAs) contribute to oncogenesis and metastasis formation in PDAC, they are considered potential candidates for fulfilling this task. In this work, the levels of two miRNA subsets (involved in chemoresistance or with oncogenic/tumor suppressing functions) were investigated in a panel of PDAC cell lines and liquid biopsies of a small cohort of patients. We used RT-qPCR and droplet digital PCR (ddPCR) to measure the amounts of cellular- and vesicle-associated, and circulating miRNAs. We found that both PDAC cell lines, also after gemcitabine treatment, and patients showed low amounts of cellular-and vesicle-associated miR-155-5p, compared to controls. Interestingly, we did not find any differences when we analyzed circulating miR-155-5p. Furthermore, vesicle-related miR-27a-3p increased in cancer patients compared to the controls, while circulating let-7a-5p, miR-221-3p, miR-23b-3p and miR-193a-3p presented as dysregulated in patients compared to healthy individuals. Our results highlight the potential clinical significance of these analyzed miRNAs as non-invasive diagnostic molecular tools to characterize PDAC.

Background/Objectives: Hepatocellular carcinoma (HCC) is the most common primary liver malignancy, often arising in cirrhosis. Its early non-invasive diagnosis and prognostic assessment remain challenging due to the limited availability of reliable circulating biomarkers, creating the need to establish non-invasive biomarkers that distinguish malignant transformation. This preliminary study aimed to: discover EV-associated miRNAs that differ among healthy subjects, patients with liver cirrhosis, and patients with HCC; validate selected candidates by ddPCR in an independent cohort; compare their abundance in plasma-derived EVs, whole plasma, and paired tumoral/peritumoral tissues; and explore their diagnostic performance for HCC versus cirrhosis and their associations with clinical outcomes. Methods: miRNA sequencing was performed on plasma-derived EVs from an exploratory cohort of 10 healthy subjects, 10 cirrhotic patients, and 10 HCC patients. Three candidate miRNAs, miR-98-5p, miR-130a-3p, and miR-1246, were subsequently quantified by droplet digital PCR (ddPCR) in an independent cohort of 29 HCC patients, 30 cirrhotic patients, and 20 healthy subjects using plasma-derived EVs, plasma, and paired tumoral/peritumoral liver tissues. Results: In plasma EVs, miR-98-5p and miR-130a-3p were reduced in HCC and cirrhotic patients compared with healthy subjects, whereas miR-1246 was increased in HCC compared with cirrhosis. In plasma, all three miRNAs were elevated in HCC compared with cirrhosis and healthy controls. Plasma miR-1246 showed the highest diagnostic performance to discriminate HCC from cirrhosis (AUC = 0.87). In tissue, miR-98-5p was upregulated and miR-130a-3p downregulated in tumoral versus peritumoral samples, whereas miR-1246 showed no significant difference. Higher plasma miR-130a-3p and miR-1246 levels were associated with improved overall survival, while increased miR-98-5p levels were associated with lower relapse rates. Conclusions: Among the three miRNAs, plasma miR-1246 showed the strongest diagnostic performance for distinguishing HCC from liver cirrhosis. Combination with miR-98-5p and/or miR-130a-3p did not improve discrimination. Thus, plasma miR-1246 emerged as the leading candidate diagnostic indicator in the HCC cohort, whereas the three miRNAs showed distinct potential associations with prognosis.

Head and neck squamous cell carcinomas (HNSCCs) develop from the mucosa of the oral cavity, pharynx, and larynx. The methylation levels of Septin 9 (SEPT9) and short stature homeobox 2 (SHOX2) genes in ccfDNA are considered pan-cancer biomarkers and have shown prognostic value in preliminary reports in HNSCC. Liquid biopsy is a non-invasive procedure that collects tumor-derived molecules including circulating cell-free DNA (ccfDNA). Here, we developed a ddPCR-based assay to detect the DNA methylation levels of plasma circulating SEPT9 and SHOX2 in patients with HNSCC. We first set up the assay on commercial methylated and un-methylated DNA. Then, the dynamic changes of the methylation levels of SEPT9 and SHOX2 were quantified in 20 patients during the follow-up. The results highlighted: i) the capability of the ddPCR-based assay in detecting very low copies of methylated molecules; ii) the significant decrease in methylation levels of SEPT9 and SHOX2 in plasma of HNSCC patients at the first time points of follow-up respect to T0; iii) a different trend of longitudinally DNA methylation variations associable to the clinicopathological features of the patients. The absolute quantification of the methylation levels of SEPT9 and SHOX2 in HNSCC may be used for risk stratification and disease monitoring.