Evolution of the experimental models of cholangiocarcinoma

Cholangiocarcinoma (CCA) is a rare, aggressive disease with poor overall survival. In advanced cases, surgery is often not possible or fails; in addition, there is a lack of effective and specific therapies. Multidisciplinary approaches and advanced technologies have improved the knowledge of CCA molecular pathogenesis, highlighting its extreme heterogeneity and high frequency of genetic and molecular aberrations. Effective preclinical models, therefore, should be based on a comparable level of complexity. In the past years, there has been a consistent increase in the number of available CCA models. The exploitation of even more complex CCA models is rising. Examples are the use of CRISPR/Cas9 or stabilized organoids for in vitro studies, as well as patient-derived xenografts or transgenic mouse models for in vivo applications. Here, we examine the available preclinical CCA models exploited to investigate: (i) carcinogenesis processes from initiation to progression; and (ii) tools for personalized therapy and innovative therapeutic approaches, including chemotherapy and immune/targeted therapies. For each model, we describe the potential applications, highlighting both its advantages and limits

Could specific EKG markers identify a pharmacologically induced type 1 Brugada pattern? Insights from a large, single-centre cohort

Abstract Funding Acknowledgements Type of funding sources: None. Background. Pharmacological (Ajmaline) induction of a type 1 Brugada pattern is currently considered mandatory for the diagnosis of Brugada syndrome. However, performing the test requires time and healthcare resources. Some EKG markers have been proposed as predictors of positive result at Ajmaline test. Aim. To evaluate in a large population the predictive value of multiple EKG markers for Ajmaline test results. Methods. We retrospectively analysed consecutive patients (pts) referred to our Centre to perform Ajmaline test. All pts had type 2 Brugada pattern detected at a conventional EKG or were relatives of pts with positive Ajmaline test, with or without type 2 Brugada pattern at EKG. All pts performed the Ajmaline pharmacological test (1 mg/Kg iv) with EKG "superior" right precordial unipolar derivations monitoring. To determine whether clinical parameters (age, gender, cardiomyopathy, history of arrhythmias, symptoms, familiarity) and EKG markers (heart rate (HR), PR duration, R1V1 and SV6 duration and amplitude, QRSV1/QRSV6 duration, V1 and V2 ST amplitude (coved or saddle back pattern) were independently associated to positivity at Ajmaline test, a logistic regression model was applied. Results. From January 2010 to December 2019 we evaluated 442 consecutive pts: mean age 40.1 ± 14.5 years; 273 (65%) male; 352 (80%) pts were included because of type 2 Brugada pattern at EKG and 90 (20%) for familial screening. The Ajmaline test was positive in 150 (34%) pts. At multivariate logistic regression analysis adjusted for baseline confounders, age > 45 years (OR= 1.64, 95%CI: 1.03 to 2.54; p = 0.0385), female gender (OR = 1.79, 95%CI: 1.12 to 2.85; p = 0.0141), HR > 60 bpm (OR = 2.44, 95%CI: 1.48 to 4.03; p = 0.0005), QRSV1/QRSV6 duration (msec) >1 (OR = 5.34, 95%CI: 3.28 to 8.69; p < 0.0001) and non isoelectric pattern (coved/saddle back) in V2 (OR = 1.93, 95%CI: 1.03 to 3.63, p = 0.0416) remained associated with a positive Ajmaline test. The percentage of pts with positive Ajmaline test increased according to the presence of significant EKG markers in their risk profile: 11.3% (8 out 71, absence of both QRSV1/QRSV6 duration (msec) >1 and V2 non isoelectric pattern), 24.3% (50 out 206, presence of only V2 non isoelectric pattern), 48.5% (16 out 33, presence of only QRSV1/QRSV6 duration (msec) >1), 57.6% (76 out 132, presence of both factors). Conclusions. In our large population: 1) we confirmed the positive predictive power of QRSV1/QRSV6 duration (msec) >1 and of a non isoelectric pattern (coved/saddle back) in V2 for a pharmacologically induced type 1 Brugada pattern; 2) we observed a non-negligible percentage of pts who would not be correctly diagnosed for type 1 Brugada pattern, if selected according to an EKG parameters-based prescreening

A novel multidrug‐resistant cell line from an italian intrahepatic cholangiocarcinoma patient

Chemotherapy resistance is a relevant clinical issue in tumor treatment, in particular in biliary tract carcinoma (BTC), for which there are no effective therapies, neither in the first nor in the second line. The development of chemoresistant cell lines as experimental models to investigate the mechanisms of resistance and identify alternative druggable pathways is mandatory. In BTC, in which genetics and biological behavior depend on the etiology, ethnicity, and anatomical site of origin, the creation of models that better recapitulate these characteristics is even more crucial. Here we have established and characterized an intrahepatic cholangiocarcinoma (iCCA) cell line derived from an Italian patient, called 82.3. Cells were isolated from a patient-derived xenograft (PDX) and, after establishment, immunophenotypic, biological, genetic, molecular characteristics, and tumorigenicity in vivo in NOD/SCID mice were investigated. 82.3 cells exhibited epithelial morphology and cell markers (EPCAM, CK7, and CK19); they also expressed different cancer stem markers (CD44, CD133, CD49b, CD24, Stro1, PAX6, FOXA2, OCT3/4), α–fetoprotein and under anchorage-independent and serum-free conditions were capable of originating cholangiospheres. The population doubling time was approximately 53 h. In vitro, they demonstrated a poor ability to migrate; in vivo, 82.3 cells retained their tumorigenicity, with a long latency period (16 weeks). Genetic identity using DNA fingerprinting analysis revealed 16 different loci, and the cell line was characterized by a complex hyperdiploid karyotype. Furthermore, 82.3 cells showed cross-resistance to gemcitabine, 5-fluorouracil, carboplatin, and oxaliplatin; in fact, their genetic profile showed that 60% of genes (n = 168), specific for drug resistance and related to the epithelial-mesenchymal transition, were deregulated in 82.3 cells compared to a control iCCA cell line sensitive to chemotherapeutics. RNA sequencing analysis revealed the enrichment for genes associated with epithelial to mesenchymal transition (EMT), vasculature development, and extracellular matrix (ECM) remodeling, underlining an aggressive phenotype. In conclusion, we have created a new iCCA cell line of Caucasian origin: this could be exploited as a preclinical model to study drug resistance mechanisms and to identify alternative therapies to improve the prognosis of this tumor type

Identification of novel circulating microRNAs in advanced heart failure by next-generation sequencing

Abstract Aims Risk stratification in patients with advanced chronic heart failure (HF) is an unmet need. Circulating microRNA (miRNA) levels have been proposed as diagnostic and prognostic biomarkers in several diseases including HF. The aims of the present study were to characterize HF‐specific miRNA expression profiles and to identify miRNAs with prognostic value in HF patients. Methods and results We performed a global miRNome analysis using next‐generation sequencing in the plasma of 30 advanced chronic HF patients and of matched healthy controls. A small subset of miRNAs was validated by real‐time PCR (P < 0.0008). Pearson's correlation analysis was computed between miRNA expression levels and common HF markers. Multivariate prediction models were exploited to evaluate miRNA profiles' prognostic role. Thirty‐two miRNAs were found to be dysregulated between the two groups. Six miRNAs (miR‐210‐3p, miR‐22‐5p, miR‐22‐3p, miR‐21‐3p, miR‐339‐3p, and miR‐125a‐5p) significantly correlated with HF biomarkers, among which N‐terminal prohormone of brain natriuretic peptide. Inside the cohort of advanced HF population, we identified three miRNAs (miR‐125a‐5p, miR‐10b‐5p, and miR‐9‐5p) altered in HF patients experiencing the primary endpoint of cardiac death, heart transplantation, or mechanical circulatory support implantation when compared with those without clinical events. The three miRNAs added substantial prognostic power to Barcelona Bio‐HF score, a multiparametric and validated risk stratification tool for HF (from area under the curve = 0.72 to area under the curve = 0.82). Conclusions This discovery study has characterized, for the first time, the advanced chronic HF‐specific miRNA expression pattern. We identified a few miRNAs able to improve the prognostic stratification of HF patients based on common clinical and laboratory values. Further studies are needed to validate our results in larger populations

Impact of Glycemic and Blood Pressure Variability on Surrogate Measures of Cardiovascular Outcomes in Type 2 Diabetic Patients

OBJECTIVE—The effect of glycemic variability (GV) on cardiovascular risk has not been fully clarified in type 2 diabetes. We evaluated the effect of GV, blood pressure (BP), and oxidative stress on intima-media thickness (IMT), left ventricular mass index (LVMI), flow-mediated dilation (FMD), and sympathovagal balance (low frequency [LF]/high frequency [HF] ratio) in 26 type 2 diabetic patients (diabetes duration 4.41 6 4.81 years; HbA1c 6.70 6 1.25%) receiving diet and/or metformin treatment, with no hypotensive treatment or complications. RESEARCH DESIGN AND METHODS—Continuous glucose monitoring (CGM) data were used to calculate mean amplitude of glycemic excursion (MAGE), continuous overall net glycemic action (CONGA)-2, mean blood glucose (MBG), mean postprandial glucose excursion (MPPGE), and incremental area under the curve (IAUC). Blood pressure (BP), circadian rhythm, and urinary 15-F2t-isoprostane (8-iso-prostaglandin F2a [PGF2a]) were also evaluated. Subjects were divided into dipper (D) and nondipper (ND) groups according to DBP. RESULTS—IMT and LVMIwere increased inNDversusD(0.7760.08 vs. 0.6860.13 [P=0.04] and 67 6 14 vs. 55 6 11 [P = 0.03], respectively). MBG, MAGE, and IAUC were significantly associated with LF/HF ratio at night (r = 0.50, P = 0.01; r = 0.40, P = 0.04; r = 0.41, P = 0.04, respectively), MPPGE was negatively associated with FMD (r =20.45, P = 0.02), andCONGA-2was positively associatedwith LVMI (r=0.55, P=0.006).TheDsystolic BP was negatively associated with IMT (r =20.43, P = 0.03) andwith LVMI (r =20.52, P = 0.01). Urinary 8-iso-PGF2a was positively associated with LVMI (r = 0.68 P , 0.001). CONCLUSIONS—An impaired GV and BP variability is associated with endothelial and cardiovascular damage in short-term diabetic patients with optimal metabolic control. Oxidative stress is the only independent predictor of increased LV mass and correlates with glucose and BP variability