Overexpression of the GR Riborepressor LncRNA GAS5 Results in Poor Treatment Response and Early Relapse in Childhood B-ALL.

Glucocorticoids (GCs) remain the cornerstone of childhood acute lymphoblastic leukemia (chALL) therapy, exerting their cytotoxic effects through binding and activating of the glucocorticoid receptor (GR). GAS5 lncRNA acts as a potent riborepressor of GR transcriptional activity, and thus targeting GAS5 in GC-treated chALL could provide further insights into GC resistance and support personalized treatment decisions. Herein, to study the clinical utility of GAS5 in chALL prognosis and chemotherapy response, GAS5 expression was quantified by RT-qPCR in bone marrow samples of chB-ALL patients at diagnosis (n = 164) and at end-of-induction (n = 109), treated with ALL-BFM protocol. Patients' relapse and death were used as clinical end-points for survival analysis. Bootstrap analysis was performed for internal validation, and decision curve analysis assessed the clinical net benefit for chALL prognosis. Our findings demonstrated the elevated GAS5 levels in blasts of chALL patients compared to controls and the significantly higher risk for short-term relapse and poor treatment outcome of patients overexpressing GAS5, independently of their clinicopathological data. The unfavorable prognostic value of GAS5 overexpression was strongly validated in the high-risk/stem-cell transplantation subgroup. Finally, multivariate models incorporating GAS5 levels resulted in superior risk stratification and clinical benefit for chALL prognostication, supporting personalized prognosis and precision medicine decisions in chALL.

Novel Nested-Seq Approach for SARS-CoV-2 Real-Time Epidemiology and In-Depth Mutational Profiling in Wastewater.

Considering the lack of effective treatments against COVID-19, wastewater-based epidemiology (WBE) is emerging as a cost-effective approach for real-time population-wide SARS-CoV-2 monitoring. Here, we report novel molecular assays for sensitive detection and mutational/variant analysis of SARS-CoV-2 in wastewater. Highly stable regions of SARS-CoV-2 RNA were identified by RNA stability analysis and targeted for the development of novel nested PCR assays. Targeted DNA sequencing (DNA-seq) was applied for the analysis and quantification of SARS-CoV-2 mutations/variants, following hexamers-based reverse transcription and nested PCR-based amplification of targeted regions. Three-dimensional (3D) structure models were generated to examine the predicted structural modification caused by genomic variants. WBE of SARS-CoV-2 revealed to be assay dependent, and significantly improved sensitivity achieved by assay combination (94%) vs. single-assay screening (30%-60%). Targeted DNA-seq allowed the quantification of SARS-CoV-2 mutations/variants in wastewater, which agreed with COVID-19 patients' sequencing data. A mutational analysis indicated the prevalence of D614G (S) and P323L (RdRP) variants, as well as of the Β.1.1.7/alpha variant of concern, in agreement with the frequency of Β.1.1.7/alpha variant in clinical samples of the same period of the third pandemic wave at the national level. Our assays provide an innovative cost-effective platform for real-time monitoring and early-identification of SARS-CoV-2 variants at community/population levels.

BCL2L12 improves risk stratification and prediction of BFM-chemotherapy response in childhood acute lymphoblastic leukemia.

Background Risk-adjusted treatment has led to outstanding improvements of the remission and survival rates of childhood acute lymphoblastic leukemia (ALL). Nevertheless, overtreatment-related toxicity and resistance to therapy have not been fully prevented. In the present study, we evaluated for the first time the clinical impact of the apoptosis-related BCL2L12 gene in prognosis and risk stratification of BFM-treated childhood ALL. Methods Bone marrow specimens were obtained from childhood ALL patients upon disease diagnosis and the end-of-induction (EoI; day 33) of the BFM protocol, as well as from control children. Following total RNA extraction and reverse transcription, BCL2L12 expression levels were determined by qPCR. Patients' cytogenetics, immunophenotyping and minimal residual disease (MRD) evaluation were performed according to the international guidelines. Results BCL2L12 expression was significantly increased in childhood ALL and correlated with higher BCL2/BAX expression ratio and favorable disease markers. More importantly, BCL2L12 expression was associated with disease remission, while the reduced BCL2L12 expression was able to predict patients' poor response to BFM therapy, in terms of M2-M3 response and MRD≥0.1% on day 15. The survival analysis confirmed the significantly higher risk of the BFM-treated patients underexpressing BCL2L12 at disease diagnosis for early relapse and worse survival. Lastly, evaluation of BCL2L12 expression clearly strengthened the prognostic value of the established disease prognostic markers, leading to superior prediction of patients' outcome and improved specificity of BFM risk stratification. Conclusions The expression levels of the apoptosis-related BCL2L12 predict response to treatment and survival outcome of childhood ALL patients receiving BFM chemotherapy.

Clinical utility of miR-143/miR-182 levels in prognosis and risk stratification specificity of BFM-treated childhood acute lymphoblastic leukemia.

Although childhood acute lymphoblastic leukemia (ALL) is characterized by high remission rates, there are still patients who experience poor response to therapy or toxic effects due to intensive treatment. In the present study, we examined the expression profile of miR-143 and miR-182 in childhood ALL and evaluated their clinical significance for patients receiving Berlin-Frankfurt-Münster (BFM) protocol. Bone marrow specimens from 125 childhood ALL patients upon diagnosis and the end-of-induction (EoI; day 33), as well as from 64 healthy control children undergone RNA extraction, polyadenylation, and reverse transcription. Expression levels of miRNAs were quantified by qPCR analysis. Patients' cytogenetic, immunohistotype and MRD evaluation was performed according to international guidelines. Median follow-up time was 86.0 months (95% CI 74.0-98.0), while patients' mean DFS and OS intervals were 112.0 months (95% CI 104.2-119.8) and 109.2 months (95% CI 101.2-117.3), respectively. Bone marrow levels of miR-143/miR-182 were significantly decreased in childhood ALL patients at diagnosis and increased in more than 90% of patients at the EoI. Patients' survival analysis highlighted that children overexpressing miR-143/miR-182 at the EoI presented significantly higher risk for short-term relapse (log-rank test: p = 0.021; Cox regression: HR = 4.911, p = 0.038) and death (log-rank test: p = 0.028; Cox regression: HR = 4.590, p = 0.046). Finally, the evaluation of the miR-143/miR-182 EoI levels along with the established disease prognostic markers resulted to improved prediction of BFM-treated patients' survival outcome and response to therapy and additionally to superior BFM risk stratification specificity. Concluding, miR-143 and miR-182 could serve as novel prognostic molecular markers for pediatric ALL treated with BFM chemotherapy.

miR-125b predicts childhood acute lymphoblastic leukaemia poor response to BFM chemotherapy treatment.

BACKGROUND: Despite the favourable survival rates of childhood acute lymphoblastic leukaemia (ALL), a significant number of patients present resistance to antileukaemic agents and dismal prognosis. In this study, we analysed miR-125b expression in childhood ALL and evaluated its clinical utility for patients treated with Berlin-Frankfurt-Münster (BFM) protocol. METHODS: The study included 272 bone marrow specimens obtained on diagnosis and on BFM day 33 from 125 patients and 64 healthy children. Following extraction, RNA was polyadenylated and reverse transcribed. miR-125b levels were quantified by quantitative PCR. Cytogenetics, immunohistotype and MRD were analysed according to international guidelines. RESULTS: Downregulated miR-125b levels were detected in childhood ALL patients and correlated with adverse prognosis. Following BFM induction, miR-125b levels were significantly increased, however, elevated day 33/diagnosis miR-125b ratio was associated with unfavourable disease features. Loss of miR-125b during diagnosis and higher day 33/diagnosis ratio were correlated with stronger risk for disease short-term relapse and patients' worse survival. Moreover, multivariate regression models highlighted the independent prognostic value of miR-125b for childhood ALL. Finally, the combination of miR-125b with clinically used disease markers clearly enhanced the prediction of patients' resistance to BFM chemotherapy. CONCLUSIONS: miR-125b significantly improves the prognosis of childhood ALL patients' outcome under BFM treatment.

Cord blood copeptin concentrations in fetal macrosomia.

BACKGROUND/AIM: Excessive fetal growth is associated with increased adiposity and reduced insulin sensitivity at birth. Copeptin, a surrogate marker of arginine vasopressin (AVP) secretion, is upregulated in states of hyperinsulinemia and is considered one of the mediators of insulin resistance. We aimed to investigate cord blood concentrations of copeptin (C-terminal fragment of AVP pro-hormone) in healthy large-for-gestational-age (LGA) infants at term. METHODS: This prospective study was conducted on 30 LGA (n=30) and 20 appropriate-for-gestational-age (AGA, n=20) singleton full-term healthy infants. Cord blood copeptin and insulin concentrations were determined by ELISA and IRMA, respectively. Infants were classified as LGA or AGA, based on customized birth-weight standards adjusted for significant determinants of fetal growth. RESULTS: Cord blood copeptin concentrations were similar in LGA cases, compared to AGA controls, after adjusting for delivery mode. However, in the LGA group, cord blood copeptin concentrations positively correlated with birth-weight (r=0.422, p=0.020). In the AGA group, cord blood copeptin concentrations were elevated in cases of vaginal delivery vs elective cesarean section (p=0.003). Cord blood insulin concentrations were higher in LGA cases, compared to AGA controls (p=0.036). No association was recorded between cord blood copeptin concentrations and maternal age, parity, gestational age or fetal gender in both groups. CONCLUSIONS: Cord blood copeptin concentrations may not be up-regulated in non-distressed LGA infants. However, the positive correlation between cord blood copeptin concentrations and birth-weight in the LGA group may point to the documented association between AVP release and increased fat deposition. Vaginal delivery vs elective cesarean section is accompanied by a marked stress-related increase of cord blood copeptin concentrations.