BACKGROUND/AIM:Advanced melanoma treatment remains debated, particularly in patients with B-Raf proto-oncogene serine/threonine kinase (BRAF) V600 mutations eligible for both immune checkpoint inhibitors (ICI) and BRAF/MEK inhibitors. Additional biomarkers are needed to improve treatment selection. Mutations of the telomerase reverse transcriptase (TERT) promoter are frequent in melanoma, although their clinical role remains unclear. PATIENTS AND METHODS:We conducted a retrospective single-center study including patients with metastatic melanoma treated with first-line ICI or BRAF/MEK inhibitors. TERT mutational status and co-mutations were assessed using a customized next-generation sequencing panel. Overall (OS) and progression-free (PFS) survival were estimated using the Kaplan-Meier method and compared with the log-rank test. RESULTS:Among 159 patients, TERT mutations were detected in 73% of cases and frequently co-occurred with BRAFV600 mutations (84%). In the overall population, TERT status was not associated with OS or PFS. Brain and liver metastases, lactate dehydrogenase levels, and metastatic burden were the main determinants of outcome. In the BRAFV600-mutant subgroup, exploratory analyses suggested a potential interaction between TERT status and the agents used in first-line treatment. Patients with BRAF/TERT co-mutations showed longer OS when treated with ICI than with BRAF/MEK inhibitors (50.3 vs. 14.6 months), whereas the opposite trend was observed in patients with wild-type TERT (7.8 vs. 20.9 months). Similar patterns were observed for PFS, although findings were based on small subgroups and not consistently supported by multivariable analyses. CONCLUSION:TERT promoter mutations were not independently associated with survival outcomes in metastatic melanoma. Although exploratory findings suggest a possible interaction between TERT status and treatment type, further investigation of its biological relevance in well-designed prospective studies are needed.

MicroRNAs (miRNAs) are gene expression regulators that play a fundamental role in developmental and biological processes. Dysregulated expression of miRNAs has been associated with most human conditions, and their accurate quantification is an essential activity in molecular biology laboratories. Indeed, the levels of specific tissue or circulating miRNAs have been tested as disease biomarkers in many clinical and experimental settings. Droplet digital PCR (ddPCR) technology is a sensitive and accurate method to obtain the absolute or relative quantification of specific miRNAs, bypassing several issues related to low abundance targets and PCR efficiency. This chapter addresses the workflow and methods for miRNA quantification in formalin-fixed paraffin-embedded (FFPE) samples, cells, and plasma/serum/extracellular vesicles using EvaGreen-based ddPCR or probe-based ddPCR, as well as how to analyze and interpret results. In addition, we provide a ddPCR method to quantify miRNA isoforms (isomiRs).

Transarterial chemoembolization (TACE) is the standard treatment for patients with intermediate-stage hepatocellular carcinoma (HCC), yet nearly half of treated patients fail to achieve durable benefit, and reliable biomarkers enabling early therapeutic stratification are still lacking. Treatment response is typically assessed by imaging one month after TACE and at three-month intervals, potentially delaying timely access to alternative therapies in non-responding patients. Circulating microRNAs (miRNAs) represent promising biomarkers due to their stability in body fluids and ease of detection. Here, we evaluated circulating miR-22 as an early predictor of TACE non-responder status and as a mechanistically relevant therapeutic target. Circulating miR-22 levels were measured by microarray and quantitative RT–PCR in three independent cohorts of early-to-intermediate-stage HCC patients undergoing TACE. Circulating miR-22 increased significantly in non-responders as early as 48 h after treatment, and fold changes consistently predicted treatment failure across two independent validation cohorts. Mechanistically, we identified the G2/M checkpoint kinase WEE1 as a direct functional target of miR-22. Modulation of the miR-22/WEE1 axis affected cell-cycle progression, proliferation, apoptosis, and DNA damage response in HCC cell lines and xenograft models. Under hypoxia-mimicking conditions combined with doxorubicin exposure, pharmacological inhibition of WEE1 induced mitotic catastrophe in highly proliferative miR-22-silenced cells. Collectively, these findings identify early post-TACE elevation of circulating miR-22 as a biomarker of non-response and highlight the miR-22/WEE1 axis as a potential target for precision treatment strategies in HCC.

Cancer cells actively release extracellular vesicles (EVs) into the tumor microenvironment, where they interact with both malignant and non-malignant cells, activating signaling pathways and reshaping the microenvironment. In this study, we investigated EVs secreted by FGFR2 -amplified cancers of unknown primary (CUPs), which generate extrachromosomal circular DNA (ecDNA) as a mechanism of oncogene amplification. We found that FGFR2 -containing ecDNA is packaged into both small and large EVs, horizontally transferred to recipient cells, and remains functionally active. Upon exposure to CUP-derived EVs—either by direct administration or co-culture—cancer (NCI-N87, THP1) and non-cancer (HUVEC, fibroblasts) cells internalized FGFR2 ecDNA, which was subsequently transcribed and translated to some extent. Functionally, CUP-derived EVs polarized THP1 cells toward an M2-like phenotype and promoted HUVEC proliferation. In vivo , xenografts generated from CUP cell lines released circulating FGFR2 + EVs, which mediated the systemic transfer of FGFR2 ecDNA to distant organs. Collectively, these findings demonstrate that tumor-derived EVs can propagate and horizontally transfer oncogenic ecDNA both in vitro and in vivo , providing a possible mechanistic basis for the high metastatic potential of this tumor type.

IntroductionCancer of unknown primary (CUP) is a metastatic malignancy with no identifiable site of origin, accounting for 2–5% of cancer diagnoses. Its marked heterogeneity and the limited availability of tumor tissue pose major challenges to genomic profiling.MethodsIn this retrospective observational study, we applied a 92-gene CUP-specific targeted sequencing panel to liquid biopsy samples from 39 CUP patients, analyzing circulating cell-free DNA (ccfDNA) together with paired germline DNA (gDNA), when available. Somatic, germline, and CHIP-related variants were classified using predefined variant allele frequency (VAF) thresholds and paired ccfDNA/gDNA comparison.ResultsWe identified somatic mutations in 78 of 92 genes and 44 clinically relevant variants (Tier I–III), most frequently affecting NF1, KRAS, ARID1A, and PIK3CA. Mutated genes were primarily involved in cell-cycle regulation, receptor tyrosine kinase signaling, and NOTCH pathways. Recurrent genetic alterations were detected in 15 genes, including canonical hotspot mutations in TP53, KRAS, and PIK3CA, 10 of which were shared by three or more patients. Actionable mutations, as defined by current clinical annotation guidelines, were identified in 13 genes, supporting the potential role of molecularly guided therapies in CUP. Likely CHIP-associated variants, defined as mutations in CHIP-associated genes with VAF ≥ 2% in PBMC-derived genomic DNA, were detected in 17 patients. In addition, pathogenic germline variants in MITF, NTRK1, and BAP1 were identified in three patients; notably, the NTRK1 germline variant was accompanied by an independent somatic mutation in the same gene.DiscussionOverall, these findings support liquid biopsy as a valuable approach for molecular profiling of CUP and highlight the critical importance of paired ccfDNA/gDNA analysis, including CHIP assessment, to accurately distinguish somatic, germline, and hematopoiesis-related variants.

Cutaneous melanoma is a malignant tumor and remains a clinical challenge due to unmet needs in risk stratification, real-time treatment monitoring, and early detection of progression or resistance. Early detection is crucial, as prognosis significantly worsens once metastasis occurs. Liquid biopsy has emerged as a powerful, minimally invasive tool for early diagnosis, real-time disease monitoring, and therapeutic guidance. Among its components, circulating tumor cells (CTCs) are rare yet clinically informative, providing insights into tumor heterogeneity, metastatic potential, and prognosis. Given its high heterogeneity, melanoma could particularly benefit from CTC analysis to inform personalized treatment strategies and improve clinical outcomes. Quantification of melanoma CTCs remains limited by low abundance and heterogeneity, as well as the lack of standardized isolation methods across laboratories. This review summarizes current methods for melanoma CTC detection and isolation. It also explores the clinical significance of melanoma CTCs, addressing the challenges and future opportunities for their implementation in clinical practice.

Background High-grade serous tubo-ovarian cancer (HGSOC) frequently presents with malignant ascites, a biologically relevant compartment rich in tumor cells. While circulating tumor cells (CTCs) in peripheral blood (PB) are often scarce, ascitic fluid (AF) may represent a more accessible and abundant source of tumor-derived material. This study evaluated the feasibility of isolating AF-derived CTCs using the CellSearch platform for subsequent molecular characterization. Methods Paired PB and AF samples from 12 HGSOC patients were collected at surgery and analyzed using the CellSearch system. After CTC enumeration, DNA was extracted from CellSearch cartridges containing AF-derived CTC-enriched fractions and subjected to next-generation sequencing (NGS) using a panel clinically validated for solid tumors. Associations between AF-derived CTC burden and clinical variables were explored. Results CTCs were detected in 100% of AF samples and in 50% of PB samples. AF contained markedly higher numbers of CTCs than PB, whereas the latter showed low or undetectable levels. AF-derived CTCs frequently formed multicellular aggregates, potentially leading to underestimation by automated counting. An optimized protocol for DNA extraction from post-processing CellSearch cartridges enabled successful downstream NGS analysis from CTC-enriched AF fractions. Tumor-specific TP53 mutations identified in AF-derived CTC-enriched fractions showed complete concordance with matched tumor tissue in all analyzed cases, supporting the feasibility of using these fractions for molecular characterization of the tumor. No significant associations were observed between AF-derived CTC burden and CA125, PCI, progression-free survival (PFS), or overall survival (OS), although higher CTC levels were associated with a trend toward shorter PFS. Conclusions Compared to PB, AF is a richer source of CTCs in HGSOC. Importantly, AF-derived CellSearch fractions enabled reliable molecular profiling through downstream NGS analysis, suggesting that this approach may be exploited to enrich for tumor-poor peritoneal washings usually considered for staging IC HGSOC. The complete mutational concordance between AF-derived CTCs and matched tumor tissue supports the biological validity of this approach and highlights its potential as a minimally invasive liquid biopsy for tumor characterization and longitudinal disease monitoring in advanced HGSOC.

Abstract Cancer cells actively release extracellular vesicles (EVs) into the tumor microenvironment, where they interact with both malignant and non-malignant cells, activating signaling pathways and reshaping the microenvironment. In this study, we investigated EVs secreted by FGFR2-amplified cancers of unknown primary (CUPs), which generate extrachromosomal circular DNA (ecDNA) as a mechanism of oncogene amplification. We found that FGFR2-containing ecDNA is packaged into both small and large EVs, horizontally transferred to recipient cells, and remains functionally active. Upon exposure to CUP-derived EVs—either by direct administration or co-culture—cancer (NCI-N87, THP1) and non-cancer (HUVEC, fibroblasts) cells internalized FGFR2 ecDNA, which was subsequently transcribed and translated to some extent. Functionally, CUP-derived EVs polarized THP1 cells toward an M2-like phenotype and promoted HUVEC proliferation. In vivo, xenografts generated from CUP cell lines released circulating FGFR2+ EVs, which mediated the systemic transfer of FGFR2 ecDNA to distant organs. Collectively, these findings demonstrate that tumor-derived EVs can propagate and horizontally transfer oncogenic ecDNA both in vitro and in vivo, providing a possible mechanistic basis for the high metastatic potential of this tumor type. Citation Format: Irene Salamon, Giulia Gallerani, Jens Luebeck, Gianluca Storci, Simone Spandau, Beatrice Fontana, Alessia Soru, Mattia Riefolo, Marco Pagano Mariano, Ilaria Pace, Andrea Cavazzoni, Vineet Bafna, Massimiliano Bonafe', Manuela Ferracin. Horizontal transfer of functional extrachromosomal DNA via extracellular vesicles in FGFR2-amplified cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3345.

The identification of predictive factors for sentinel lymph node (SLN) positivity in melanoma patients is crucial for accurate staging, prognosis, and personalized therapeutic decisions. This review synthesizes recent advancements in molecular and clinicopathological predictors, with a particular focus on liquid biopsy and gene expression profiling (GEP) tools. Emerging evidence highlights the significant role of miRNAs in melanoma progression, metastatic potential, and lymphatic spread. Clinicopathological factors such as Breslow thickness, ulceration, and mitotic rate remain critical, while GEP provides additional precision by uncovering tumor-specific molecular pathways. By integrating these tools, clinicians can improve risk stratification, reduce unnecessary procedures, and personalize management strategies.

Psoriasis is characterized by aberrant keratinocyte activity and immune cell infiltration, driven by immune-mediated pathways. MicroRNAs (miRNAs) play crucial roles in regulating these processes, offering insights into disease mechanisms and therapeutic targets. This study aimed to investigate changes in circulating miRNAs in psoriasis patients undergoing risankizumab therapy, an anti-IL-23 monoclonal antibody, to understand its impact on disease pathogenesis and treatment response. Plasma samples from 12 psoriasis patients were collected before (T0) and after 1 year (T1) of risankizumab treatment and analyzed using small RNA sequencing. Findings were validated in a separate cohort of 23 patients using quantitative real-time PCR (qRT-PCR). T-regulatory cell (Treg) numbers and pro-inflammatory cytokine levels were also assessed. Significant clinical improvement was observed in all patients after 1 year of treatment, accompanied by increased Treg counts and reduced levels of pro-inflammatory cytokines. Twenty-four miRNAs exhibited differential expression post-treatment; 9 were downregulated and 15 upregulated. Notably, miR-200a-3p showed a significant correlation with baseline Psoriasis Area Severity Index (PASI), indicating its potential as a severity marker. Risankizumab therapy also decreased peripheral blood levels of IL-23, IL-1β, and IL-8. This study identifies specific circulating miRNAs, including miR-200a-3p, as potential biomarkers for monitoring treatment responses in psoriasis patients. The findings underscore the therapeutic efficacy of risankizumab in modulating miRNA profiles and immune pathways associated with psoriasis pathogenesis. Overall, these results provide new insights into the mechanisms of risankizumab action and highlight miRNAs as promising candidates for personalized medicine approaches in psoriasis management.

BACKGROUND:The neuromuscular junction (NMJ) establishment occurs through complex communication events between motor neurons and muscle fibers; however, the molecular mechanisms leading to NMJ formation have yet to be fully elucidated. Little is known about the significance of extracellular vesicles (EVs) in mediating the interaction between motor neurons and muscle fiber in the NMJ establishment; this study investigates the role of motor neuron-derived EVs during the earliest stages of NMJ formation. METHODS:NSC-34 cells have been used as a model of motor neurons; EVs have been isolated during neurite development using a serial ultracentrifugation protocol specifically adjusted to isolate large and small EVs. Isolated EVs were quantified through Nanoparticles Tracking Assay and characterized by Western Blot and TEM analyses. The microRNA (miRNA) cargo of EV subpopulations was identified by small-RNA sequencing and the predicted miRNA downstream targets were investigated. RESULTS:NGS analysis of small RNAs carried by NSC-34-derived EVs identified a total of 245 EV specific miRNAs, most of which are up-regulated in NSC-34 cells and EVs during neurite stretching. Target prediction analysis evidenced how these miRNAs synergically target the Wnt signaling pathway. Moreover, we found that NSC-34-derived EVs carry Wnt proteins, including Wnt11, Wnt4 and Wnt3a. Since several studies suggested a role for the Wnt-associated signaling network in NMJ formation, we investigated the potential role of NSC-34 EVs in NMJ development and demonstrated that EV administration to myotubes increases acetylcholine receptor (AChR) cluster formation, as revealed by immunofluorescence staining with α-bungarotoxin. Moreover, myotube treatment with NSC-34-derived EVs led to GSK3β and JNK phosphorylation, followed by β-catenin nuclear translocation, suggesting that neuron-derived EVs can induce AChR clustering through Wnt pathway activation. CONCLUSION:These data demonstrate that EVs released from differentiated motor neurons carry multimodal signals, miRNAs, and Wnts, which can stimulate AChR clustering in myotubes, a fundamental preparatory stage for NMJ formation. These new data highlight that EVs may play a role in the NMJ establishment and function under physiological and pathological conditions, particularly neurodegenerative diseases.

Viral infections such as human papillomavirus (HPV) and Epstein–Barr virus (EBV) play a critical role in the onset of oropharyngeal (OPC) and nasopharyngeal cancer (NPC), respectively. Despite advancements in targeted therapies and immunotherapies, in the recurrent/metastatic setting, these tumors remain incurable diseases with poor prognosis. The development of therapeutic tumor vaccines, utilizing either neoantigens or oncoviral antigens, represents a promising addition to the cancer immunotherapy arsenal. Research on vaccine-based immunotherapy for OPC and NPC focuses on targeting viral antigens, particularly HPV E6/E7 and EBV EBNA1/LMP2. The potential for vaccine platforms, including peptide-based, DNA, RNA, and viral vector-based vaccines, to induce durable immune responses against viral antigens is reported. The early-phase clinical trials evaluating vaccine-based therapies for HPV-related OPC and EBV-related NPC revealed safety and preliminary signs of efficacy; however, further clinical trials are crucial for validation. This review provides an overview of the current landscape of vaccine-based strategies for HPV-related OPC and EBV-related NPC, discussing their biological mechanisms and immune processes involved in anti-HPV and anti-EBV vaccine treatments, with a particular focus on the immune factors that influence these therapies.

Background/objectives: Despite advancements in early diagnosis and clinical practices guided by standardized care protocols, Merkel cell carcinoma (MCC) is marked by an unfavorable prognosis with a 5-year relative survival rate of 65%, based primarily on data collected prior to the introduction of immunotherapy. Regional nodal metastases affect 40–50% of MCC patients, while approximately 33% experience distant dissemination. Among these, bone and bone marrow metastases are particularly notable, although the characteristics and clinical implications of this metastatic disease in MCC remain poorly understood. Methods: A comprehensive review was conducted using the Medline database (via PubMed) up to January 2025. The search strategy included the string “(Merkel cell carcinoma AND (bone OR marrow))”. Results: A total of 1133 (69.3% male and 30.7% female) patients diagnosed with advanced MCC were collected. The median (IQR) age at diagnosis was 67.5 (12.65) years old. Overall, 201 (20.8%) cases of bone and/or bone marrow metastases were identified and linked to a primary known MCC in 75.7% of cases. Bone metastases (BMs) appear as the third most common metastatic site, following the liver (second) and lymph nodes (first). They show mixed biological and radiological behavior, with a marked preference for the axial skeleton over the appendicular one. Addressing the characteristics of metastatic bone disease, neurological symptoms were the most documented, whereas bone marrow involvement and leukemic spread seemed to be primarily related to immunosuppression. Multimodal treatment strategies, including platinum-based chemotherapy and radiotherapy, were the primary approaches adopted, reflecting therapeutic practices from the pre-immunotherapy era. Conclusions: The pattern of metastatic spread in MCC differs among studies, with the bones resulting as the third most common site of distant spread. Excluding head and neck MCC, which seems to be more regularly associated with liver metastases, the relationship between the primary tumor site and the development of bone or bone marrow metastases appears inconsistent. Overall, BMs mostly correlated with advanced MCC stages and poorer survival outcomes, with a median overall survival (OS) of 8 months (range 12.75–4). The integration of international guidelines, evolving evidence from clinical trials, and the expanding role of immune checkpoint inhibitors (ICIs) will contribute to improving systemic disease control and enhance patient care.

The extracellular vesicle (EV) route is essential for cell-to-cell communication. Cancer cells release EVs in the extracellular space, where they can interact with cancer and non-cancer cells, activating specific signaling pathways, modulating tumor microenvironment remodeling and inducing gene expression alterations. We investigated the functional role of EVs released by cancer of unknown primary (CUP), a rare disease (1-3% of novel cancer diagnoses) that presents with metastasis of unknown or uncertain origin and no apparent primary tumor. We derived three CUP cell lines from patient’s tumor cells, CUP#55A and S and CUP#96, both characterized by FGFR2 gene amplification, either in the form of trisomy (CUP#55A), chromosomal homogeneously staining region (CUP#55S) or double minute chromosomes (CUP#96). It has been recently recognized that tumors use extrachromosomal circular DNA (eccDNA) as a way to increase oncogenic amplification, thus conferring resistance to therapy and contributing to a worse survival. We demonstrated that FGFR2 amplification in CUP cell lines is associated with ecDNA generation and that this ecDNA is loaded as cargo inside EVs and exert a functional activity in nearby cells. The full-length FGFR2 DNA was detectable inside both small and large vesicles isolated from cell culture medium and we confirmed the circular nature of a fraction of this FGFR2 ecDNA using DNAse based strategies and visualizing DNA circles in cells and in EVs with atomic force microscopy. The protein surface profile of small and large EV populations revealed the presence of cancer related molecules that mirrored the cells of origin, including EpCAM and CD44, and variable degrees of FGFR2pos EVs. Cancer (NCI-N87) and non-cancer cell exposure (THP1 and HUVEC) to CUP EVs (by direct administration and co-culture) revealed an increase of FGFR2 DNA copies at 24 hours and its functional transcription in FGFR2 mRNA after 48 hours in all exposed cells. In addition to oncogenic delivery, CUP#96 EVs induced a polarization of THP1 cells towards M2 subtype, while CUP#55S EVs exerted a proliferative effect on HUVEC. In conclusion, we identified a model of oncogene amplification and EV-mediated delivery recurrent in cancer of unknown primary, whose DNA content was demonstrated to be functionally active in recipient cells and proportional with extrachromosomal DNA generation. This mechanism could contribute to the high metastatic potential of this cancer type. (The research leading to these results has received funding from AIRC under IG 2021 - ID. 25789 project - P.I. Ferracin Manuela) Irene Salamon, Giulia Gallerani, Gianluca Storci, Beatrice Fontana, Salvatore Serravalle, Francesco Valle, Marco Brucale, Marco Pagano Mariano, Andrea Cavazzoni, Roberta Roncarati, Spartaco Santi, Massimiliano Bonafè, Manuela Ferracin. Extracellular vesicle delivery of functional extrachromosomal DNA in FGFR2-amplified cancer of unknown primary [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6569.

Tumor dissemination is a key event in tumor progression. During this event, a main role is played by circulating tumor cells (CTCs), immune cells, and their interaction. How the immune system supports the survival and proliferation of CTCs is not fully elucidated. In this study we established an in-vitro co-culture system consisting of immune cells and CTCs from the same patient, which increased the success rate in the establishment of CTC-derived long-term cell cultures. In this system, we characterized the immune cells of successful co-cultures and the signals they exchange with cancer cells, including cytokines and extracellular vesicle (EV) content. Using this protocol, we stabilized four CTC-derived cell lines from patients with metastatic gastroesophageal cancer, which were cultured for over a year and characterized from a genetic and molecular point of view. The four cell lines harbor shared chromosomal aberrations including the amplification at 8q24.21 containing MYC and deletion 9p21.3 containing CDKN2A/B and the IFN type I cluster. The transcriptomic profile of CTC cell lines is distinct from primary tumors, and we detected the activation of E2F, G2M and MYC pathways and the downregulation of interferon response pathway. Each cell line shows a degree of invasiveness in zebrafish in-vivo, and the most invasive ones share the same mutation in RAB14 gene. In addition, the four cell lines secrete cell-line specific EVs containing microRNAs that target YAP, BRG1-AKT1, TCF8-HDAC pathways. Overall, we highlight how the immune system plays a key role in the proliferation of CTCs through EV signaling, and how CTC cell line genomic and transcriptomic alterations make these cells less visible from the immune system and likely responsible for the survival advantage in sites distant from the microenvironment of origin.

The persistence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) on substrates, and the impact of fomites on Coronavirus Disease 19 (COVID-19) transmission, is until now, widely discussed. Consequently, further investigations are required for a correct risk assessment in high-risk facilities such as hospitals, healthcare facilities (HCFs), and long-term care facilities (LTCFs). Therefore, appropriate surveillance and disinfection programs represent the best approach to guarantee the safety of these communities. This study proposes an environmental SARS-CoV-2 surfaces routine monitoring approach in HCF and communities' settings, to provide rapid and effective evaluation of surface hygienic conditions and the effectiveness of applied sanitization measures. Surfaces samples (n = 118) were collected using the SRK® kit (Copan Italia) from 2020 to 2023. Three molecular techniques were compared: Reverse Transcription Loop mediated isothermal AMPlification (RT-LAMP, Enbiotech), Reverse-Transcription quantitative polymerase chain reaction (RT-qPCR) (RT-qPCR, Seegene) and droplet digital PCR (ddPCR, Bio-Rad). For ddPCR, two RNA extraction methods were compared: TRIzol LS (Invitrogen) versus QIAmp Viral Mini kit (QIAGEN), showing how the latter is more suitable for surfaces. Regarding the quantitative ddPCR results, the ROC analysis allowed to reduce the manufacturer cut-off for droplets number (from 3 to 1) for the positive samples. Moreover, a new cut-off for the viral RNA copies' number/μL for each target (N1 and N2) on environmental monitoring was fixed at 2,82. The results obtained using the QIAmp kit, suggested that the N2 target is more stable in the environment and could be most suitable for the virus environmental detection. The percentage of positive samples was similar among the techniques (26% for RT-LAMP, 36% for ddPCR and 23% for RT-qPCR). Using RT-qPCR as reference method, a sensitivity (SE) of 30% for RT-LAMP and 41% for ddPCR was observed. By contrast, specificity (SP) was higher for RT-LAMP (75%) respect to ddPCR (66%). Comparing the faster RT-LAMP with the sensitive ddPCR the 26% and 74% of SE and SP for RT-LAMP, were reported. The low sensitivity for RT-LAMP and ddPCR could be explained with the use of clinical rather than environmental kits, other than the changing in the virus prevalence during the sampling campaign. Although the RT-LAMP requires improvements in term of SE and SP, this research presents an innovative environmental monitoring and prevention method for SARS-CoV-2, that could be extended to other pathogens that are under environmental surveillance.

Emery–Dreifuss muscular dystrophy (EDMD) is caused by mutations in EMD, LMNA, SYNE1, SYNE2, and other related genes. The disease is characterized by joint contractures, muscle weakening and wasting, and heart conduction defects associated with dilated cardiomyopathy. Previous studies demonstrated the activation of fibrogenic molecules such as TGFbeta 2 and CTGF in preclinical models of EDMD2 and increased secretion of TGFbeta 2 in patient serum. A wide screening of patient cells suggested fibrosis, metabolism, and myogenic signaling as the most affected pathways in various EDMD forms. In this study, we show that alpha-smooth muscle actin-positive myofibroblasts are overrepresented in patient fibroblast cultures carrying EMD, LMNA, or SYNE2 mutations, and profibrotic miRNA-21 is upregulated. Upon CRISPR/Cas correction of the mutated EMD or LMNA sequence in EDMD1 or EDMD2 fibroblasts, respectively, we observe a reduced expression of fibrogenic molecules. However, in patient myoblasts, neither fibrogenic proteins nor miRNA-21 were upregulated; instead, miRNA-21-5p was downregulated along with muscle-specific miRNA-133b and miRNA-206, which have a crucial role in muscle cell homeostasis. These observations suggest that the conversion of laminopathic fibroblasts into a profibrotic phenotype is a determinant of EDMD-associated muscle fibrosis, while miRNA-206-dependent defects of laminopathic myoblasts, including altered regulation of VEGF levels, contribute to muscle cell deterioration. Notably, our study provides a proof-of-principle for the application of gene correction to EDMD1 and EDMD2 and presents EDMD1 isogenic cells that exhibit an almost complete rescue of a disease-specific miRNA signature. These cells can be used as experimental models for studying muscular laminopathies.

Lung cancer remains one of the leading causes of cancer-related deaths worldwide. It is classified into two main histological groups: non-small cell lung cancer (NSCLC) and small cell lung cancer. Improving the outcome of cancer patients could be possible by enhancing the early diagnosis. In the current study, we evaluated the levels of three microRNAs - miR-21-5p, miR-155-5p, and miR-181a-5p in tumor (TT) vs adjacent normal tissue (NT), as well as their expression levels in plasma and extracellular vesicles (EVs) from plasma in lung squamous cell carcinoma (LUSC) male patients vs healthy individuals as means to identify a panel of miRNAs that could serve as novel biomarkers for the diagnosis of LUSC in male patients. Matched paired tissue samples from male LUSC (n=40) patients were used for miRNA expression analysis. MiR-21-5p and miR-155-5p in tumor tissue were overexpressed, while underexpression of miR-181a-5p was observed in LUSC TT vs NT. These results were further validated in the TCGA LUSC dataset, considering 279 male samples. These alterations of miR-21-5p, miR-181a-5p, and miR-155-5p in tumor tissue are also present in plasma and plasma extracellular vesicles in LUSC male patients. In addition, ROC curves were performed to assess the sensitivity and specificity of different combinations of these miRNAs, confirming a high diagnostic accuracy for LUSC of up to 88 % in male subjects. The expression levels in tissue samples and the abundance in plasma and plasma EVs of the three miRNAs combined - miR-21-5p, miR-155-5p and miR-181a-5p – could be considered for further studies on biomarkers for the early detection of LUSC in male subjects.