The Impact of Anaesthesia and Sedation on Inflammation and NETosis in Hepatocellular Carcinoma Ablation

Overview

According to recent literature, liver disease causes approximately 2 million deaths annually; 1 million are attributed to the complications of cirrhosis, while the remaining deaths are caused by hepatocellular carcinoma (HCC). The latter currently ranks 16th among the leading causes of death worldwide. Current research focuses on investigating novel therapeutic modalities, optimizing surgical and anesthetic practices, and identifying biological biomarkers to predict disease progression and severity. In this study, we address radiofrequency ablation (RFA) with the goal of improving periprocedural prognosis, recurrence rates, and overall survival. Specifically, we will quantify the expression of NETosis, a relatively novel biomarker with potential therapeutic and prognostic value in HCC progression. NETosis is a regulated form of cell death through which granulocytes release decondensed chromatin and various proteases into the extracellular space, forming a web-like meshwork known as neutrophil extracellular traps (NETs). These structures are actively involved in hepatic tumorigenesis by promoting tumor growth and metastasis. Recent clinical data reveal that NETs play a key role in: Local tumor progression The incidence of hepatic metastasis Direct modulation of the immune response Hepatic ischemia-reperfusion injury Several recent studies demonstrate that the in vivo blockade of NETosis reduces tumor recurrence, mitigates the pro-inflammatory state, and serves as a valuable prognostic indicator in advanced liver disease \[2,3,4\]. To suppress NETosis expression and ultimately lower recurrence rates, our protocol evaluates the intraprocedural administration of 1% lidocaine followed by a continuous postoperative intravenous lidocaine infusion.

HCC is the most common type of primary liver cancer. It predominantly develops in individuals with chronic liver diseases, such as cirrhosis resulting from hepatitis B or C. According to the International Agency for Research on Cancer, approximately 826,000 new cases of HCC were recorded globally in 2018, accounting for roughly 4.5% of all malignancies. Projections indicated that by 2025, over one million individuals would be affected by HCC annually. The incidence of HCC varies significantly by region, with the highest rates observed in East and Southeast Asia, and the lowest rates reported in Northern Europe and North America. Therapeutic Landscape \& Thermal Ablation Surgical resection remains the first-line treatment for early-stage HCC. While some studies report that only 20%-25% of HCC patients are candidates for surgical intervention, up to 60% of patients in developed countries undergo surgery, RFA, or microwave ablation (MWA). This variation depends heavily on the patient's performance status, comorbidities, and the clinical stage of the tumor. Furthermore, due to donor shortages and high associated costs, the proportion of HCC patients undergoing liver transplantation remains exceedingly low. For patients with tumors under 3 cm in diameter, RFA demonstrates no significant difference in long-term survival compared to surgical resection and liver transplantation. Moreover, it offers distinct clinical advantages, including a lower risk of complications, reduced costs, superior preservation of functional hepatic volume, and shorter hospital stays. Consequently, RFA has been established as a core local curative technique for HCC, whereas therapies such as tyrosine kinase inhibitors and transarterial chemoembolization (TACE) are typically reserved for palliative management. RFA has become the standard of care for unresectable HCC and has proven highly competitive with surgery for single nodules smaller than 3 cm. However, a high incidence of local and distant recurrence persists, which correlates directly with the ablation technique and tumor volume. Among newer ablative modalities, microwave ablation has shown promising results by increasing the zone of coagulation necrosis and reducing distant metastasis rates. Nonetheless, a comprehensive meta-analysis of these techniques conducted by Facciorusso et al. in 2020 failed to demonstrate a statistically significant difference between them. Optimizing these treatments is critical given the elevated risk of recurrence. Currently, no definitive consensus exists regarding adjuvant regimens that can successfully improve post-procedural outcomes. In light of the high recurrence rates associated with these thermal techniques, we aim to combine the benefits of minimally invasive methods with the potential therapeutic advantages of systemic intravenous lidocaine administration. Scientific Justification of the Research We initiated this study based on its potential to significantly enhance the clinical management of patients with hepatocellular carcinoma. This research aligns directly with the core objectives of the Lancet Oncology Commission on Global Surgery and Anesthesia 2030: "Investment in surgical and anesthesia services is affordable, saves lives, and promotes economic growth." If the findings of this project confirm that lidocaine exerts an inhibitory effect on hepatic neoplasm progression and post-RFA metastasis, it could prompt a paradigm shift in oncological care, demonstrating that periprocedural drug selection can actively improve survival outcomes in HCC patients. Furthermore, we aim to investigate the inflammatory response by analyzing both conventional biomarkers and recently characterized indices, such as NETosis. If it is established that NET formation accelerates recurrence and metastatic rates, and that a specific anesthetic intervention dampens this pathway, it would introduce a vital new variable into peri-RFA management strategies. If validated, our results will provide high-quality evidence that anesthetic techniques can optimize disease-free survival (DFS) in cancer patients undergoing interventions, ultimately improving patient-centered outcomes. Study Aim \& Objectives Study Aim: The aim of this study is to evaluate the involvement of lidocaine and NETosis in the clinical course and progression of hepatocellular carcinoma. Primary Objective: To assess the influence of the anesthetic technique and periprocedural management during radiofrequency ablation of HCC on recurrence rates and overall survival. Secondary Objectives: To evaluate the impact of intravenous lidocaine supplementation on recurrence rates in patients with HCC eligible for thermal ablation. To investigate the effect of periprocedural lidocaine administration on NETosis formation and its subsequent correlation with recurrence incidence. Study Design: Prospective, interventional, longitudinal, randomized controlled trial. Materials and Methods This is a single-center, randomized, interventional pilot study, we include adult patients with HCC undergoing RFA. Patients are randomly assigned into two groups via an online system (random.org). Group 1 receives a procedural sedation and analgesia (PSA) with propofol and fentanyl, combined with a 1.5 mg/kg slow bolus of lidocaine followed by an intravenous lidocaine infusion of 1 mg/kg/h for 24 hours (PF-L group). Group 2 receives PSA with propofol and fentanyl alone (PF group). Primary endpoints includ the assessment of the impact on recurrence rates and survival of 1 year. Secondary outcomes are the assessment of the effect of intravenous lidocaine supplementation on the recurrence rate and on the formation of NETs and their correlation with the incidence of recurrence at 1 year. Ethics and registration All patients are enrolled in the study following the provision of informed consent and are subsequently monitored in accordance with the prevailing regulations set forth in the Research Ethics Guidelines of the "Iuliu Hațieganu" University of Medicine and Pharmacy, as well as the recommendations and directives of the Research Ethics Committee, within the framework of international regulations and with full observance of human rights. The study is conducted with approval of the Ethics Committee of the University of Medicine and Pharmacy Cluj nr.: 212/07.11.2024, the Ethics Committee of the Regional Institute of Gastroenterology and Hepatology (IRGH) nr.:7042/15.05.2024. Study population We includ adult (18-80 years old) patients with a hepatocellular carcinoma suitable for RFA, meaning the possibility of an ultrasound-guided approach and the presence of a tumor measuring up to 3 cm in diameter. Tumor staging follow the AJCC (American Joint Committee on Cancer) and BCLC (Barcelona Clinic Liver Cancer Criteria) guidelines. Patients with ASA I, II, or III anesthetic risk are included in the study. We exclud patients who are candidates for surgery, patients on chronic immunosuppressive medication, patients with contraindications to any of the study medications, patients with psychiatric disorders (depression, bipolar disorder, schizophrenia), autoimmune disorders, or corticosteroid-dependent bronchial asthma; congenital or acquired coagulation disorders; chronic users of anticoagulants and/or antiplatelet agents; HIV-positive patients; and pregnant women. Patients receiving antiarrhythmic therapy (verapamil, propafenone, amiodarone) that could interfere with the antiarrhythmic effect of lidocaine are also excluded. During and after the procedure, all patients receive standard monitoring in accordance with ASA guidelines: ECG, blood pressure, pulse, and SpO2. PSA is administered in accordance with guidelines for procedural sedation in gastroenterology using fentanyl and propofol. Patients are followed for one year to determine the incidence of metastases and postoperative complications. Randomisation The patients are randomized to one of two groups using the online computer system random.org in a 1:1 ratio. The group allocation and patient study number are concealed in a sealed opaque envelope, which is opened after the patient has given their written, informed consent before RFA. While the involved anaesthetists are not masked to study group allocation, all the investigators involved in the postprocedural follow-up, blood sampling, data analysis, and interpretation are unaware of the group allocation. A saline infusion packaged identically to i.v. lidocaine is used as placebo control for the groups without lidocaine infusion. This is prepared and started after induction, by the anaesthetist and continued for 24 h after operation. The infusion rates mirror lidocaine infusion rates. Neutrophil extracellular traps assays. Peripheral venous blood samples (10 mL) are collected from each participant at two distinct time points: prior to sedation and 24 hours following the RFA. This time point is determined based on established data regarding the kinetics of post-stimulation NETosis described in literature, which reaches peak expression in 24 hours. Blood is drawn from venous site, utilizing BD Vacutainer™ serum tubes (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Within 1 hour of collection, the samples underwent centrifugation at 3000 rpm for 15 minutes at room temperature. The isolated serum is subsequently divided into 2 mL aliquots and preserved at -80°C until further assay using the enzyme-linked immunosorbent assay (ELISA) technique. NETosis specific marker, citrullinated histone H3 (H3Cit) is analyzed pre- and postoperatively. These measurements are made using commercially available ELISA kits for: H3Cit (Human H3Cit, MyBi-oSource, San Diego, CA, USA; sensitivity: 1.0 ng ml\_1). 100 μL of serum sample and 50 μL of H3Cit-HRP conjugate are added to the wells and incubated for 1 hour at 37°C. Following incubation, the wells are decanted and rinsed five times with wash buffer. The plate is then incubated with HRP substrate in the dark for 15-20 minutes at 37°C. The enzymatic reaction is halted by adding 50 μL of stop solution. Optical density (OD) was measured at 450 nm using a Dynex DSX Microplate Reader (Dynex Technologies, USA). H3Cit concentrations are determined by plotting sample absorbance values against a standard reference curve. The manufacturer-specified intra-assay coefficient of variation (CV) is \<10%. The manufacturer provides no minimum detectable dose or range, and advises against calculating values falling outside the standard curve limits. Prior literature indicates baseline serum H3Cit levels typically range between 10-15 ng/mL, with a standard deviation of approximately 3 ng/ml. The NLR is determined by dividing the absolute neutrophil count by the absolute lymphocyte count baseline (pre-ablation) and at 24 hours post-ablation. Data management and statistics Based on previous findings from our research group, a 20% reduction (equivalent to 2.0 ng/mL) in H3Cit levels is anticipated. Setting a type I error of 0.05 and a type II error of 0.10 (90% statistical power), a minimum of 25 patients per cohort is required. All data management complies strictly with the EU General Data Protection Regulation (GDPR). Data are compiled in Microsoft Excel and transferred to GraphPad Prism (v8, GraphPad Software, USA) for statistical evaluation.Statistical Tests: Data distributions are verified for normality. Continuous, normally distributed variables across independent cohorts are evaluated using analysis of variance (ANOVA) paired with a post-hoc Bonferroni adjustment. Intra-group comparisons of serum biomarkers before and after anesthesia/surgery are conducted using a paired Student's t-test. Statistical significance is defined as P \< 0.05.