BL-MOL-AR Project, Preliminary Results about Liquid Biopsy: Molecular Approach Experience and Research Activity in Oncological Settings.

Background Liquid biopsy is mainly used to identify tumor cells in pulmonary neoplasms. It is more often used in research than in clinical practice. The BL-MOL-AR study aims to investigate the efficacy of next-generation sequencing (NGS) and clinical interpretation of the circulating free DNA (cfDNA) levels. This study reports the preliminary results from the first samples analyzed from patients affected by various neoplasms: lung, intestinal, mammary, gastric, biliary, and cutaneous. Methods The Biopsia Liquida-Molecolare-Arezzo study aims to enroll cancer patients affected by various malignancies, including pulmonary, intestinal, advanced urothelial, biliary, breast, cutaneous, and gastric malignancies. Thirty-nine patients were included in this preliminary report. At time zero, a liquid biopsy is executed, and two types of NGS panels are performed, comprising 17 genes in panel 1, which is already used in the routine tissue setting, and 52 genes in panel 2. From the 7th month after enrollment, 10 sequential liquid biopsies are performed up to the 17th month. The variant allele frequency (%) and cfDNA levels (ng/mL) are measured in every plasmatic sample. Results The NGS results obtained by different panels are similar even though the number of mutations is more concordant for lung pathologies. There are no significant differences in the actionability levels of the identified variants. Most of the molecular profiles of liquid biopsies reflect tissue data. Conclusions Preliminary data from this study confirm the need to clarify the limitations and potential of liquid biopsy beyond the lung setting. Overall, parameters related to cfDNA levels and variant allele frequency could provide important indications for prognosis and disease monitoring.

In silico assessment of Y1795C and Y1795H SCN5A mutations: implication for inherited arrhythmogenic syndromes.

The effects of two SCN5A mutations (Y1795C, Y1795H), previously identified in one Long QT syndrome type 3 (LQT3) and one Brugada syndrome (BrS) families, were investigated by means of numerical modeling of ventricular action potential (AP). A Markov model capable of reproducing a wild-type as well as a mutant sodium current (I(Na)) was identified and was included into the Luo-Rudy ventricular cell model for action potential (AP) simulation. The characteristics of endocardial, midmyocardial, and epicardial cells were reproduced by differentiating the transient outward current (I(TO)) and the ratio of slow delayed rectifier potassium (I(Ks)) to rapid delayed rectifier current (I(Kr)). Administration of flecainide and mexiletine was simulated by appropriately modifying I(Na), calcium current (I(Ca)), I(TO), and I(Kr). Y1795C prolonged AP in a rate-dependent manner, and early afterdepolarizations (EADs) appeared during bradycardia in epicardial and midmyocardial cells; flecainide and mexiletine shortened AP and abolished EADs. Y1795H resulted in minimal changes in the APs; flecainide but not mexiletine induced APs heterogeneity across the ventricular wall that accounts for the ST segment elevation induced by flecainide in Y1795H carriers. The AP abnormalities induced by Y1795H and Y1795C can explain the clinically observed surface ECG phenotype. For the first time by modeling the effects of flecainide and mexiletine, we are able to gather mechanistic insights on the response to drugs administration observed in affected patients.

Computer simulation of wild-type and mutant human cardiac Na+ current.

Long QT syndrome (LQTS) and Brugada syndrome (BrS) are inherited diseases predisposing to ventricular arrhythmias and sudden death. Genetic studies linked LQTS and BrS to mutations in genes encoding for cardiac ion channels. Recently, two novel missense mutations at the same codon in the gene encoding the cardiac Na+ channel (SCN5A) have been identified: Y1795C (causing the LQTS phenotype) and Y1795H (causing the BrS phenotype). Functional studies in HEK293 cells showed that both mutations alter the inactivation of Na+ current and cause a sustained Na+ current upon depolarisation. In this paper, a nine state Markov model was used to simulate the Na+ current in wild-type Na+ cardiac channel and the current alterations observed in Y1795C and Y1795H mutant channels. The model includes three distinct closed states, a conducting open state and five inactivation states (one fast-, two intermediate- and two closed-inactivation). Transition rates between these states were identified on the basis of previously published voltage-clamp experiments. The model was able to reproduce the experimental Na+ current in mutant channels just by altering the assignment of model parameters with respect to wild-type case. Parameter assignment was validated by performing action potential clamp experiments and comparing experimental and simulated I(Na) current. The Markov model was subsequently introduced in the Luo-Rudy model of ventricular myocyte to investigate "in silico" the consequences on the ventricular cell action potential of the two mutations. Coherently with their phenotypes, the Y1795C mutation prolongs the action potential, while the Y1795H mutation causes only negligible changes in action potential morphology.

Effect of premature ventricular beats on manual and automatic repolarization measurements.

OBJECTIVE: To compare QT interval and QT dispersion in ventricular ectopic beats with measurements from the preceding and the immediately following sinus beats, and investigate differences between manual and automatic measurements. PATIENTS: Eleven chronic uremic patients. MAIN OUTCOME MEASURES: ECGs were recorded during hemodialysis treatment and 12-lead sections containing five consecutive beats were extracted, each containing four sinus beats and one centrally-positioned premature ventricular beat. QT measurements were performed both manually and with a computer-automated technique. RESULTS: T wave amplitude was greater in the ectopic beats compared to the sinus beats (0.61 +/- 0.18 vs. 0.23 +/- 0.06 mV, P <.001). The ectopic beats had a greater QT than the sinus beats when measured manually (415 +/- 35 ms vs. 386 +/- 28 ms, P <.001), or automatically (375 +/- 30 vs. 366 +/- 27 ms, P<.01). The sinus beats following the ectopics had a greater QT than the preceding sinus beats (400 +/- 27 vs. 386 +/- 28 ms, P<.001, manual; 382 +/- 24 vs. 366 +/- 27 ms, P<.001, automatic). Differences in QT dispersion were seen only between the ectopic and sinus beats (91 +/- 31 vs. 58 +/- 27 ms, P <.001, manual; 68 +/- 33 vs. 49 +/- 35 ms, P <.001, automatic). CONCLUSIONS: Manual measurement resulted in greater QT values than automatic measurement. Both techniques identified differences between sinus and ectopic beats. The ventricular ectopic beats resulted in an increase in the QT of the immediately following sinus beats. These results confirm the need to interpret QT measurements with care in the presence of ectopic beats.

Electrocardiographic changes during hemodiafiltration with different potassium removal rates.

BACKGROUND/AIMS: Sudden K removal is thought to be implicated in ECG alterations observed during hemodialysis (HD). The effects of the K removal rate on ECG-derived parameters have been investigated. METHODS: Two different hemodiafiltration (HDF) schedules were used for 10 HD patients: the dialysate K concentration was kept constant in HDFst, while in HDFK it was decreased during the session in order to maintain a uniform plasma-dialysate K gradient. A 12-lead Holter monitor was used to acquire the ECG in the course of the treatments. Classical ECG parameters and overall indices for quantifying ventricular repolarization abnormalities were evaluated. RESULTS: Several ECG parameters were affected by both HD therapies (ST depression, QRS amplitude and QT dispersion), but only indices of the homogeneity of repolarization (PCA-T, E1-T) were significantly affected by the K removal rate. CONCLUSION: The present study confirms the large impact of HD therapy on ECG. The analysis of the spatial T wave complexity points out the intrinsic arrhythmogenic implications of the K removal rate.