Collagens and Lysyl Oxidases shape the extracellular matrix, with their dysregulation promoting matrix stiffness and pathological conditions. Plant-derived microRNAs (xenomiRs) can act across species, potentially modulating human physiology. Here, we present a robust protocol for isolating and sequencing xenomiRs from plants of the Evia region of Greece and predicting their human targets, highlighting their potential to regulate collagens and lysyl oxidases and providing a framework for cross-kingdom therapeutic approaches.

Alterations in DNA methylation represent one of the earliest molecular events in bladder tumorigenesis and are strongly associated with treatment efficacy and prognosis. Herein, we investigated DNA methylation landscape of MIR141 promoter and elucidated its clinical relevance in bladder cancer (BlCa) patients. miRNA-seq was performed to analyze miRNA profile in bladder tumors. Expression analysis of miR-141-3p and methylation profiling of MIR141 promoter were performed in 15 patient-derived xenografts (PDXs) mouse models, and in our screening patients' cohort (n = 133). TCGA-BLCA (n = 412) was used as independent validation cohort. Survival analysis was performed employing tumor relapse/progression as clinical endpoints in non-muscle-invasive BlCa (NMIBC; TaT1), and mortality in muscle-invasive BlCa (MIBC; T2-T4) patients. Internal validation was conducted through bootstrap analysis, and the clinical benefit of the findings was evaluated using decision curve analysis. miRNA-seq highlighted miR-141-3p as one of the most significantly downregulated miRNAs in muscle-invasive and high-grade tumors, while PDXs, TCGA-BLCA and screening cohort revealed its robust epigenetic regulation through MIR141 core promoter methylation. Consistent with miR-141-3p loss, hypermethylation of MIR141 core promoter was associated with advanced stage and high-grade tumors, reduced survival in PDXs, and significantly higher risk of TaT1 patients for short-term progression to muscle-invasive disease. Multivariate models highlighted MIR141 promoter hypermethylation as independent predictor for NMIBC short-term progression, displaying superior clinical benefit in patients' prognostication. Overall, MIR141 core promoter methylation results in miR-141-3p loss in bladder tumors and correlates with patients' poor treatment outcome, offering improved risk-stratification and addressing prognostic heterogeneity beyond traditional markers.

BACKGROUND:Multiple myeloma (MM) is an incurable malignancy, marked by treatment resistance and frequent relapses, posing ongoing challenges to patients' long-term management. Herein, we have examined tRNA-derived small RNA fragments (3'U-tRFs), generated from precursor tRNAs, to identify MM-related 3'U-tRFs in ameliorating MM precision prognostics. METHODS:3'U-tRF profiles were generated by small RNA-seq data. Target prediction and gene ontology analysis were assessed by tRFtarget and DAVID databases, respectively. CD138 + 3'U-tRFSerTGA levels were quantified in our MM screening cohort (n = 136 patients) by RT-qPCR. Kaplan-Meier and Cox proportional regression analyses were performed, using disease progression and patients' mortality as clinical endpoints. Internal validation was conducted by bootstrap Cox regression while clinical benefit on patients' prognosis was assessed by decision curve analysis (DCA). RESULTS:Small RNA-seq data analysis highlighted the significantly increased 3'U-tRFSerTGA levels in MM cell lines compared to normal cells (FC: 14.03). Our screening cohort confirmed the significantly higher risk for short-term progression and worse survival of the patients presenting elevated 3'U-tRFSerTGA. 3'U-tRFSerTGA-fitted multivariate models demonstrated superior risk-stratification of the patients for treatment response and prognosis. CONCLUSIONS:Our study indicate the elevated 3'U-tRFSerTGA as a strong independent predictor of poor first-line chemotherapy outcomes and MM progression, providing refined stratification of patient risk.

BACKGROUND:Bladder cancer (BlCa) surveillance requires lifelong invasive monitoring, imposing substantial patient burden and healthcare costs. This underscores the need for minimally-invasive precision medicine approaches. Herein, we investigated the circulating miRNome of BlCa patients to assess its clinical and translational utility. METHODS:miRNA-seq was performed on tumors and matched plasma samples from 15 patient-derived xenograft (PDX) mouse models. The screening cohort of the study consisted of 216 patients, and circulating miRNAs were quantified by RT-qPCR following 3'-end poly(A)-tailing. Cox regression models were used to develop liquid biopsy-based miRNA risk-scores (miRisk-scores), for non-muscle invasive (NMIBC) and muscle invasive BlCa (MIBC). RESULTS:miRNA-seq identified six miRNAs (miR-100, miR-1246, miR-1290, miR-193b, miR-375, miR-4488) that were concurrently upregulated in tumors and plasma, and correlated with advanced stage/grade and early progression in PDX mouse models. In our screening cohort, elevated miR-375 and miR-4488 were associated with poorer NMIBC prognosis, whereas miR-1290 and miR-193b with worse survival in MIBC. Higher miRisk-scores were associated with short-term relapse and progression in NMIBC, while miRisk score-fitted multivariate models improved TaT1 risk-stratification. Moreover, higher miRisk-scores correlated with worse clinical outcomes in MIBC, and enhanced the prognostic performance of clinical markers. CONCLUSION:Overall, liquid biopsy miRNA signatures offer a minimally-invasive tool to complement biopsy-based assessments and refine BlCa risk-stratification and prognosis.

BACKGROUND:While long coronavirus disease 2019 (COVID-19) is linked to prolonged vascular dysfunction in adults, research in children remains poor. In this study, we assessed vascular health in children infected with severe acute respiratory syndrome coronavirus 2 about 6.8 months postinfection, comparing them with healthy controls. METHODS:Two hundred twenty-three children were assessed and divided into group 1, which included children with a positive disease history and group 2, which consisted of healthy controls. Anthropometric measurements, lipid profile, biomarkers (interleukin-6, C-reactive protein, tumor necrosis factor-alpha and soluble intracellular adhesion molecule) and long COVID symptoms were assessed, along with pulse wave velocity (PWV) measurements and carotid intima-media thickness (cIMT) to evaluate aortic stiffness. RESULTS:Children in group 1 were older (mean age: 10.8 ± 3.2 years vs. 8.5 ± 2.8 years, P < 0.001) and had higher body mass index (20.3 ± 5.6 kg/m 2 vs. 18.4 ± 3.5 kg/m 2 , P < 0.001). PWV was increased in group 1 (5.02 ± 0.7 m/s vs. 4.7 ± 0.6, P < 0.001). However, vascular differences between the groups disappeared after adjusting for age, body mass index, and blood pressure. Soluble intracellular adhesion molecule-1 levels were elevated in children with a history of moderate/severe COVID-19 infection compared with controls (555.8 ± 113.2 ng/mL vs. 428 ± 42.6 ng/mL, P < 0.001). Cholesterol levels, inflammatory markers and cIMT were comparable between groups. Long COVID symptoms were reported mainly by participants of group 1 [34 (23.6%) vs. 3 (3.8%), P < 0.001]. CONCLUSIONS:This study demonstrates insights into the long-term effects of COVID-19 infection in children. Evidence of endothelial activation without structural arterial changes was found. Persistent inflammation postinfection was absent, yet approximately one-quarter of the participants experienced long COVID symptoms, indicating potential differences in the pathophysiology of postacute COVID-19 infection in childhood.

Uncontrolled proliferation, resistance to apoptosis, inability to maintain genome integrity, and, recently, epigenetic reprogramming are all hallmarks of cancer. A number of gene expression and cell signaling networks control these-often-interconnected processes, while the study of their deregulation is in the forefront of cancer research for decades. Here we present data from cells and patients indicating that KMT2C, one of the most frequently mutated proteins in solid malignancies, is involved in all these processes. Its loss, a bad prognosis marker in bladder cancer, is associated with activation of the PI3K/PDK/AKT oncogenic/antiapoptotic axis, and tolerance to DNA damage during cell cycle progression. On the other hand, these cells suffer from mitotic stress that can be therapeutically exploited. Treatment with a PLK1 inhibitor showed high efficacy in vivo, and was associated with mitotic catastrophe and cellular senescence, providing evidence that targeting genes that promote mitotic progression could be a promising therapeutic approach in the subset of tumors with KMT2C loss.

Due to its highly heterogenous molecular landscape, bladder cancer (BlCa) is still characterized by non-personalized prognosis and treatment decisions. N6-methyladenosine (m6A) has emerged as the most common and conserved internal mRNA modification, regulating RNA metabolism and translation. Herein, we have profiled mutations and copy number variations (CNVs) within m6A RNA machinery genes and assessed their clinical relevance in BlCa patients’ prognosis and treatment outcome. DNA-seq libraries were prepared from 96 bladder specimens (tumors: n=87; normal urothelium: n=9) using a custom-designed panel of enrichment probes for m6A writers (METTL3, METTL14, METTL16, VIRMA, WTAP, RBM15, RBM15B, ZC3H13), erasers (FTO, ALKBH5) and readers (YTHDF1, YTHDF2, YTHDF3, YTHDC1, YTHDC2, IGF2BP1, IGF2BP2, IGF2BP3). METTL3 methyltransferase expression was quantified by RT-qPCR. Mutational and CNV analyses of DNA-seq data were performed by SNPNexus and CNVkit, respectively. Kaplan-Meier curves and Cox regression analysis were implemented for patient’s survival analysis. Internal validation was performed by bootstrap analysis. DNA-seq on DNBSEQ-G400 revealed that ∼30% of bladder tumors (25/87) harbored deleterious mutations in m6A writers, while m6A erasers (3,2%) and readers (5,3%) were less mutated. CNVs were found in ∼60% of tumors (52/87), with VIRMA frequently amplified (22%) in superficial (TaT1) tumors. Reduced METLL3 expression was detected in muscle-invasive tumors (p=0.009) and associated with increased risk for NMIBC relapse (p=0.002) and progression (p=0.028) to MIBC. Moreover, MIBC patients with deleterious mutations in m6A writer’s complex presented significantly higher progression risk (p=0.036) and worse survival (p=0.010), while VIRMA copy-number gain/amplification was associated with significantly increased risk of short-term relapse (p=0.019) in NMIBC. Finally, multivariate Cox regression confirmed m6A writer complex mutations as independent marker for patients’ progression (HR=3.242, p=0.021) and death (HR=5.388, p=0.006) in MIBC, and of VIRMA gain/amplification as an independent indicator of NMIBC relapse risk (HR=3.564, p=0.010).Genomic variations in m6A machinery emerge as modern molecular markers to address BlCa clinical heterogeneity and guide personalized prognosis and treatment/monitoring decisions. Acknowledgements: The research project was supported by the Hellenic Foundation for Research and Innovation (H.F.R.I.) under the “2nd Call for H.F.R.I. Research Projects to support Faculty Members & Researchers” (Project Number: HFRI-FM20-3765) Andreas Scorilas, Katerina-Marina Pilala, Panagiotis Tsiakanikas, Konstantina Panoutsopoulou, Maria-Alexandra Papadimitriou, Konstantinos Soureas, Georgios-Christos Giagkos, Panagiotis Levis, Georgios Kotronopoulos, Zoi Kanaki, Ioannis Prassas, Lampros Dimitrakopoulos, George Yousef, Konstantinos Stravodimos, Stiliani Koroneou, Margaritis Avgeris. Unveiling m6A epitranscriptome-related genomic variations in bladder cancer: predictive biomarkers for disease progression and treatment outcome towards tailored therapeutics [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 3337.

The persistence of high relapse rates and therapy resistance continues to challenge the effective management of multiple myeloma (MM). The identification of novel MM-specific molecular markers could ameliorate risk-stratification tools and accurately identify high-risk patients towards personalized prognosis and therapy. miRNA-seq analysis of CD138+ plasma cells (n = 24) unveiled miR-221-3p and miR-222-3p (miR-221/222 cluster) as the most downregulated miRNAs in R-ISS III compared to R-ISS I/II patients. Subsequently, miR-221/222 levels were quantified by RT-qPCR in CD138+ plasma cells of our screening cohort (n = 141), assessing patients’ mortality and disease progression as clinical endpoints. Internal validation was performed by bootstrap analysis, while clinical benefit was estimated by decision curve analysis. Kryukov et al. (n = 149) and Aass et al. (n = 86) served as institutional-independent validation cohorts. Loss of miR-221/222 cluster was strongly associated with patients’ short-term progression and poor overall survival, which was confirmed by Kryukov et al. and Aass et al. validation cohorts. Intriguingly, miR-221/222-fitted multivariate models offered superior risk-stratification within R-ISS staging and risk-based cytogenetics. Moreover, miR-221/222 loss could effectively discriminate optimal 1st-line treatment responders with inferior treatment outcome. Our study identified the loss of miR-221/222 cluster as a powerful independent predictor of patients’ post-treatment progression, ameliorating prognosis and supporting precision medicine in MM.

Background: The aim of this study was to evaluate whether increased body mass index (BMI) and biochemical and lifestyle parameters linked to obesity and smoke exposure disrupt immune responses of children and adolescents following vaccination with the mRNA BNT162b2 vaccine. Methods: A prospective, single-center, cohort study was conducted. Participants were assigned to receive two doses of the mRNA vaccine. Anti-SARS-CoV-2 IgG and neutralizing antibodies (AB) were measured before vaccination (T0) and 14 days after the second dose (T1). BMI and biochemical parameters were evaluated at T0. A questionnaire on lifestyle characteristics was filled in. Results: IgG optical density (OD) ratio at T1 was lower in the overweight–obese group regardless of COVID-19 disease positive history [p = 0.028 for the seronegative group, p = 0.032 for the seropositive group]. Neutralizing AB were lower in overweight–obese participants in the seronegative group at T1 [p = 0.008]. HDL, fasting glucose/insulin ratio (FGIR), C-reactive protein (CRP), HBA1c, uric acid, and smoke exposure were significantly correlated with BMI [p = 0.006, p < 0.001, p < 0.001, p = 0.006, p = 0.009, p < 0.001, respectively]. The main biochemical parameters that were inversely correlated with IgG and neutralizing AB titers at T1 were uric acid [p = 0.018, p = 0.002], FGIR [p = 0.001, p = 0.008] and HBA1C [p = 0.027, p = 0.038], while smoke exposure negatively affected the humoral immune responses at T0 in the convalescent group [p = 0.004, p = 0.005]. Conclusions: Current data suggests that uric acid, insulin resistance (IR), and smoke exposure could adversely affect the immune responses in overweight–obese vaccinated children, highlighting the need for actions to enhance the protection of this particular subgroup.

Cellular senescence, a state of permanent cell cycle arrest, recapitulates the aging process at the cellular level. It can be triggered by intrinsic or extrinsic factors including telomere shortening (replicative senescence) and in response to various types of stresses such as oncogenic stress (oncogene-induced senescence, OIS). Senescence has been detected in vitro and in premalignant lesions in mice and humans expressing mutant oncogenes. MicroRNAs (miRNAs) are short noncoding RNAs that regulate gene expression at the posttranscriptional level, and have been involved in both replicative senescence and OIS. Several methods have been used to identify miRNAs and compare their expression in normal versus oncogene-induced senescent cells, as well as to analyze their role and their targets in senescence. Here, we describe several methods that can be employed to identify miRNAs in cells undergoing OIS, including miRNA-sequencing, RT-qPCR-based detection and quantification of miRNAs and Nanostring miRNA analysis (nCounter miRNA Expression Assay). Moreover, we perform a meta-analysis of studies employing the above methodologies, pinpoint miRNAs with consistent expression changes across senescence models, and predict their target genes and the pathways in which they partake.

Bladder cancer (BlCa) exhibits a highly heterogeneous molecular landscape and treatment response, underlining the pressing need for personalized prognosis. N6-methyladenosine (m6A) constitutes the most abundant RNA modification, modulates RNA biology/metabolism, and maintains cellular homeostasis, with its dysregulation involved in cancer initiation and progression. Herein, we evaluated the clinical value of METTL3 m6A methyltransferase, the main catalytic component of m6A methylation machinery, in improving BlCa patients' risk stratification and prognosis. The screening cohort of the study included 213 patients. The UROMOL (n = 535) was analyzed as a validation cohort for non-muscle-invasive BlCa (NMIBC), while the TCGA-BLCA (n = 412) and Mariathasan et al. (n = 348) cohorts were analyzed for muscle-invasive BlCa (MIBC). Disease recurrence/progression and patients' mortality were assessed as clinical endpoints for NMIBC and MIBC, respectively. Internal validation of Cox regression models was conducted using bootstrap analysis, while the clinical utility for patient prognosis was evaluated through decision curve analysis. Reduced METTL3 expression was correlated with muscle-invasive disease and tumors of advanced stage. Loss of METTL3 expression at diagnosis was strongly associated with higher risk of short-term progression (HR = 2.903, 95% CI: 1.303-6.464, p = 0.006) to invasive stages in NMIBC and with worse survival of MIBC patients (HR = 1.908, 95% CI: 1.020-3.567, p = 0.042). Consistently, validation cohorts confirmed the poor treatment outcomes in patients exhibiting loss of METTL3. Finally, METTL3-fitted multivariate models improved risk stratification and offered superior clinical benefit for NMIBC and MIBC prognostication compared to clinically established disease markers. Overall, loss of METTL3 expression correlates with inferior treatment outcomes in BlCa, driving more accurate risk stratification and ameliorating patients' prognosis in BlCa.

Childhood acute lymphoblastic leukaemia (chALL) remains the most prevalent malignancy in children and adolescents. Improving risk stratification and providing personalized prognosis and treatment remain major clinical challenges. Herein, we analysed the clinical utility of NEAT1 lncRNA for the prognosis and prediction of treatment outcome of childhood B-cell precursor ALL (chB-ALL). NEAT1_1 isoform was quantified in bone marrow samples of chB-ALL patients at diagnosis (n = 160) and at the end of induction (n = 108) of ALL-BFM protocol, and in age-matched healthy children (n = 68). Relapse and death served as clinical end-points for survival analysis. Bootstrap analysis was performed for internal validation and decision curve analysis assessed the clinical net benefit for chB-ALL prognosis. Our analysis showed that chB-ALL patients with NEAT1 overexpression at diagnosis are at significantly higher risk for progression (HR = 2.957, 95% CI: 1.122-7.790, p = 0.011) and worse survival (HR = 5.832, 95% CI: 1.259-27.01, p = 0.012), independently of clinicopathological and treatment data. Moreover, NEAT1-fitted multivariate models resulted in improved risk stratification compared to the conventional disease markers of white blood cells, bone marrow response and minimal residual disease, while decision curve analysis highlighted the superior clinical net benefit for chB-ALL prognosis. In conclusion, NEAT1 overexpression constitutes a powerful, independent predictor of poor treatment outcomes and disease progression of chB-ALL, providing refined stratification of patient's risk.

The orchestration of dynamic epigenetic and epitranscriptomic modifications is pivotal for the fine-tuning of gene expression. However, these modifications are traditionally examined independently. Recent compelling studies have disclosed an interesting communication and interplay between m6A RNA methylation (m6A epitranscriptome) and epigenetic modifications, enabling the formation of feedback circuits and cooperative networks. Intriguingly, the interaction between m6A and DNA methylation machinery, coupled with the crosstalk between m6A RNA and histone modifications shape the transcriptional profile and translational efficiency. Moreover, m6A modifications interact also with non-coding RNAs, modulating their stability, abundance, and regulatory functions. In the light of these findings, m6A imprinting acts as a versatile checkpoint, linking epigenetic and epitranscriptomic layers toward a multilayer and time-dependent control of gene expression and cellular homeostasis. The scope of the present review is to decipher the m6A-coordinated circuits with DNA imprinting, chromatin architecture, and non-coding RNAs networks in normal physiology and carcinogenesis. Ultimately, we summarize the development of innovative CRISPR-dCas engineering platforms fused with m6A catalytic components (m6A writers or erasers) to achieve transcript-specific editing of m6A epitranscriptomes that can create new insights in modern RNA therapeutics.

Objectives We performed a cross- sectional study in two maternity hospitals in Athens, Greece between 2017 and 2019 assessing seroprevalence and Geometric Mean Titres (GMTs) of diphtheria, pertussis and tetanus antibodies in pregnant women and recorded adherence to Greek National Immunization Program (NIP) regarding Tdap vaccination in pregnancy. Methods Blood samples were collected from women in labour and anti- diphtheria, tetanus and pertussis toxin IgG antibodies were measured by Elisa kits. Seropositivity was defined as anti-diphtheria and anti- tetanus toxin IgG levels ≥0.1, and anti- pertussis >50 IU/mL. Seroprevalence and GMTs were calculated according to demographic factors. Tdap vaccination before and during pregnancy was self-reported by study participants. Results We analysed 253 blood samples and paired questionnaires. Seropositivity was 57.7 % for diphtheria. The lowest rate (38.2 %) was observed in the youngest age group (≤25 years). Increasing age was associated with higher seroprevalence (p = 0.036). 12.5 % of women were seropositive against pertussis. Most were seropositive for tetanus (92.7 %). Anti-pertussis GMTs were 16.98, anti-diphtheria 0.13 and anti- tetanus GMTs 0.63 IU/mL. Women born in Greece and with higher educational level had higher antibodies against tetanus (p = 0.004 & 0.004 respectively). 3/253 (1.2 %) of women assessed reported Tdap vaccination during pregnancy. Conclusion Seropositivity rates were low for diphtheria and pertussis among pregnant women. In addition, less than 2 % were vaccinated with Tdap despite recommendation by Greek NIP. Therefore, many infants end up unprotected during the first months of life. Our study highlights the urgent need for national campaigns targeting to completion of childhood immunization and information of the public about safety and importance of Tdap vaccination in pregnancy.

Background: Antibody levels decline a few months post-acute COVID-19, but humoral memory persists in adults. Age and disease severity may affect antibody responses. This study aims to evaluate the presence and durability of antibody responses in children with COVID-19. Methods: A prospective, single-center study, involving unvaccinated children 0–16 years of age who were hospitalized with COVID-19 between October 2020 and December 2021, was conducted. Serological testing for anti-Spike severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgG and neutralizing antibodies was performed at diagnosis and at 1-, 3-, 6- and 12-months post-infection. Results: A total of 65 immunocompetent children were enrolled [mean age (±SD): 6.7 (±6.4) years; males: 56.9%]. At 3 months, 40/44 (91%) children were seropositive; seropositivity persisted in 22/26 (85%) children at 6 months and in 10/12 (83%) children at 12 months. There was no evidence that age was modifying the prediction of variance of SARS-CoV-2 IgG levels. In contrast, SARS-CoV-2 IgG levels varied with time and disease severity. The association with time was non-linear, so that with increasing time there was a significant reduction in SARS-CoV-2 IgG levels [coef, 0.044 (95% confidence interval {CI}: 0.061–0.028), P < 0.001]. For each increment of time, the higher disease severity group was associated with 0.9 lower SARS-CoV-2 IgG levels. Everyone varied from the average effect of time with an SD of 0.01, suggesting that individuals may have different trajectories across time. Conclusion: Disease severity, but not age, influences antibody titers among children hospitalized with COVID-19. SARS-CoV-2 infection induces durable seroconversion in these children with detectable IgG levels at 1 year after infection.

Introduction: Long-read sequencing with nanopore technology is a groundbreaking approach, particularly useful for identifying circular RNAs (circRNAs), which are regulatory molecules regarded as promising non-invasive biomarkers for various malignancies, including multiple myeloma (MM). Despite the breakthroughs of nanopore sequencing in circRNA profiling, there have been no studies so far thoroughly exploring the wealth of the circular transcriptome in MM. Thus, we aimed to identify novel MM-related circRNAs and elucidate their expression patterns, variety in structure and length, splicing patterns, and biomarker potential. Methods: 8 MM cell lines (L-363, H929, U266, RPMI 8226, KMS-12-BM, LP-1, OPM-2, and SK-MM-2) were propagated. Total RNA was extracted and 1 μg from each cell line was subjected to rRNA depletion; the rRNA-depleted RNAs were treated with RNase R at 37◦C for 15 min to effectively digest linear RNAs, before their rolling-circle reverse transcription using random hexamers. Finally, sequencing libraries were constructed and nanopore sequencing was conducted using PromethION 2, with high-accuracy basecalling enabled. All generated reads were mapped against the GRCh38 reference genome and data were analyzed using CIRI-long 1.1.0 and in-house-developed algorithms. Moreover, bone marrow aspirate (BMA) samples were collected from 145 adult patients with plasma cell disorders. Next, CD138+ selection of plasma cells among mononuclear cells of BMA samples was performed, followed by RNA extraction and cDNA synthesis. A first-round PCR was used to pre-amplify selected circRNAs and GAPDH (reference gene), before their quantification via real-time qPCR. circRNA expression was measured in relative quantification units (RQU). Results: Through this optimized workflow, the circRNA-enriched RNA fractions from the 8 cell lines were effectively used to characterize full-length circRNA sequences. In particular, the total number of identified circRNAs ranged from 3,795 to 14,612 among all cell lines. Remarkably, the percentage of these circRNAs that were not deposited in databases such as circAtlas, MiOncoCirc, and CSCD2, ranged from 42.2% to 68.8% among all cell lines. Most splice sites at the back-splice junction were canonical; however, non-canonical splice sites were also detected, albeit at smaller frequencies, in all datasets. Moreover, although most circRNAs were exonic, many intronic circRNAs were also identified, in agreement with recent findings that support the substantial inclusion of intronic regions in the sequence of circRNAs. Several circRNAs from MM-associated genes, including TP53, BRAF, EGR1, CYL, and PRDM1, were also identified. Interestingly, 2 novel circRNAs, namely circZFAND6 and circLRCH3, were highly expressed in all cell lines. We successfully developed and applied a quantification assay to determine the expression levels of both these circRNAs in patients' CD138+ plasma cells; the cohort of patients consisted of 110 newly diagnosed symptomatic MM cases and 35 smoldering MM (SMM) cases. circZFAND6 was expressed in 64 (58.2%) MM samples and 15 (42.9%) SMM samples, while circLRCH3 was expressed in 50 (45.5%) MM samples and 16 (45.7%) SMM samples. Interestingly, both circZFAND6 and circLRCH3 were significantly downregulated in the MM samples, compared to the SMM samples (median circZFAND6 expression = 61.99 RQU in MM vs. 407.16 RQU in SMM samples; median circLRCH3 expression = 3.76 RQU in MM vs. 128.26 RQU in SMM samples). Receiver operating characteristic (ROC) curve analysis showed that both circRNAs could efficiently distinguish MM from SMM cases (circZFAND6: AUC=0.72, 95% CI=0.57-0.88, P=0.004; circ LRCH3: AUC=0.71, 95% CI=0.55-0.87, P=0.0012). Conclusions: Following our circRNA profiling pipeline, we were able to effectively characterize the landscape of circRNAs in 8 MM cell lines, thus providing a crucial foundation for investigating the role of particular circRNAs in MM pathogenesis. Our findings demonstrate that a wide range of circRNAs are expressed in MM, characterized by diverse splicing events, including the presence of novel exons and/or intron retention. Moreover, the expression levels of 2 newly identified circRNAs, namely circZFAND6 and circLRCH3, differ significantly between SMM and MM patients' CD138+ plasma cells. Further research is expected to investigate their promising prognostic potential for this disease.

Bladder cancer (BlCa) is an extensively heterogeneous disease that leads to great variability in tumor evolution scenarios and lifelong patient surveillance, emphasizing the need for modern, minimally invasive precision medicine. Here, we explored the clinical significance fi cance of copy number alterations (CNAs) in BlCa. CNA profiling fi ling was performed in 15 patient- derived xenografts (PDXs) and validated in The Cancer Genome Atlas BlCa (TCGA-BLCA; n = 408) and Lindgren et al. (n n = 143) cohorts. CDKN2A copy number loss was identifi ed as the most frequent CNA in bladder tumors, associated with reduced CDKN2A expression, tumors of a papillary phenotype, and prolonged PDX survival. The study's ' s screening cohort consisted of 243 BlCa patients, and CDKN2A copy number was assessed in genomic DNA and cell-free DNA (cfDNA) from 217 tumors and 189 pre-treatment serum samples, respectively. CDKN2A copy number loss was correlated with superior disease-free and progression-free survival of non- muscle-invasive BlCa (NMIBC) patients. Moreover, a higher CDKN2A index ( CDKN2A/LEP ratio) in pre-treatment cfDNA was associated with advanced tumor stage and grade and shortterm NMIBC progression to invasive disease, while multivariate models fi tted for CDKN2A index in pre-treatment cfDNA offered superior risk stratification fi cation of T1/high-grade and EORTC high-risk patients, enhancing prediction of treatment outcome. CDKN2A copy number status could serve as a minimally invasive tool to improve risk stratification fi cation and support personalized prognosis in BlCa.

PURPOSE:The lack of personalized management of bladder cancer (BlCa) results in patients' lifelong post-treatment monitoring with invasive interventions, underlying the urgent need for tailored and minimally invasive health care services. On the basis of our previous findings on miR-143/145 cluster methylation in bladder tumors, we evaluated its clinical significance in pretreatment cell-free DNA (cfDNA) of patients with BlCa. MATERIALS AND METHODS:Methylation analysis was performed in our screening cohort (120 patients with BlCa; 20 age-matched healthy donors) by bisulfite-based pyrosequencing. Tumor recurrence/progression for patients with non-muscle-invasive bladder cancer, and progression and mortality for patients with muscle-invasive bladder cancer (MIBC) were used as clinical end point events in survival analysis. Bootstrap analysis was applied for internal validation of Cox regression models and decision curve analysis for assessment of clinical benefit on disease prognosis. RESULTS:Decreased methylation of MIR145 core promoter in pretreatment cfDNA was associated with short-term disease progression (multivariate Cox: hazard ratio [HR], 2.027 [95% CI, 1.157 to 3.551]; P = .010) and poor overall survival (multivariate Cox: HR, 2.098 [95% CI, 1.154 to 3.817]; P = .009) of patients with MIBC after radical cystectomy (RC). Multivariate models incorporating MIR145 promoter methylation in cfDNA with tumor stage clearly ameliorated patients' risk stratification, highlighting superior clinical benefit in MIBC prognostication. CONCLUSION:Reduced pretreatment cfDNA methylation of MIR145 core promoter was markedly correlated with increased risk for short-term progression and worse survival of patients with MIBC after RC and adjuvant therapy, supporting modern personalized and minimally invasive prognosis. Methylation profiling of MIR145 core promoter in pretreatment cfDNA could serve as a minimally invasive and independent predictor of MIBC treatment outcome and emerge as a promising marker for blood-based test in BlCa.

Despite the substantial progress in multiple myeloma (MM) therapy nowadays, treatment resistance and disease relapse remain major clinical hindrances. Herein, we have investigated tRNA-derived fragment (tRF) profiles in MM and precursor stages (smoldering MM/sMM; monoclonal gammopathy of undetermined significance/MGUS), aiming to unveil potential MM-related tRFs in ameliorating MM prognosis and risk stratification. Small RNA-seq was performed to profile tRFs in bone marrow CD138+ plasma cells, revealing the significant deregulation of the mitochondrial internal tRFHisGTG (mt-i-tRFHisGTG) in MM versus sMM/MGUS. The screening cohort of the study consisted of 147 MM patients, and mt-i-tRFHisGTG levels were quantified by RT-qPCR. Disease progression was assessed as clinical end-point for survival analysis, while internal validation was performed by bootstrap and decision curve analyses. Screening cohort analysis highlighted the potent association of reduced mt-i-tRFHisGTG levels with patients' bone disease (p = 0.010), osteolysis (p = 0.023) and with significantly higher risk for short-term disease progression following first-line chemotherapy, independently of patients' clinical data (HR = 1.954; p = 0.036). Additionally, mt-i-tRFHisGTG-fitted multivariate models led to superior risk stratification of MM patients' treatment outcome and prognosis compared to disease-established markers. Notably, our study highlighted mt-i-tRFHisGTG loss as a powerful independent indicator of post-treatment progression of MM patients, leading to superior risk stratification of patients' treatment outcome.