Evaluating the Degradation of a Novel Form of Ethanol Ablation in a Large Animal Model of the Cervix

Introduction: Over a quarter of a million women die from cervical cancer each year, with 85% of those deaths occurring in low- and middle-income countries (LMICs). The precursor to cervical cancer is cervical neoplasia (CIN), in which abnormal, precancerous cells grow on the surface of a woman’s cervix. If detected, CIN is highly treatable via surgical excision or ablative therapies. Yet, it remains the second leading cause of cancer-related deaths in women in LMICs, due to limitations of current technologies such as high costs, bulkiness, specialized equipment, and the need for constant electricity. Additionally, in some devices, maximum treatment depths don’t adequately treat high-grade CIN. To address these limitations, we developed a low-cost therapy based on ethanol ablation. Specifically, we combined pure ethanol with a polymer, ethyl cellulose (EC), which forms a biocompatible gel when introduced into tissue. This gel increases the efficacy of ethanol by decreasing leakage from the injection site. Notably, our technology does not require specialized equipment, or electricity, and can be delivered at a variety of tissue depths. In a recent study, we investigated the degradation of 48-hour and 7-day EC-ethanol treatments in one in vivo swine model. We found that EC deposits are visible in swine cervical tissue 7 days post-treatment. To better inform patient follow-up after treatment, here we further examined how EC deposits degrade in the cervix over time.

Materials and

Methods: The swine study protocol was approved by the University of Maryland Institutional Animal Care and Use Committee (Protocol number: R-AUG-23-40). To measure differences in degradation, we randomly assigned 10 female Yorkshire cross pigs (Archer Farms, Darlington, MD, USA) into two treatment groups. One group was treated with EC-ethanol (12% w/v, 200-proof ethanol) 7 days before sample collection (n=5). The other group was treated with EC-ethanol 14 days before sample collection (n=5). Throughout the procedure, vitals, side effects, and adverse events were monitored. After each endpoint, the pig cervices were excised, flash-frozen, and cryosectioned. Three sequential 20-micron sections were taken throughout the cervix every 400 microns. Sections were stained using hematoxylin and eosin (H&E), Congo red, and Nicotinamide adenine dinucleotide (NADH). H&E was used for histopathology assessment and to identify cell death mechanisms. Congo Red was used to identify the EC deposit size. NADH staining was used to calculate necrotic volume. All samples were then imaged using an inverted bright-field microscope. All images were collected and analyzed using a custom MATLAB code to calculate necrotic tissue volume and EC depot size. Using the semi-automated code, background, noise, holes, and non-cervical tissue were removed from the images. The stained and unstained regions of cervical tissue were identified and separated from each other. The code then calculated the area and volume of cellulose and necrotic regions.

Results, Conclusions, and Discussions: Vitals collected during all procedures were stable and confirmed by a veterinarian to be within normal ranges. No side effects or adverse events were observed in either treatment group. Staining showed EC deposit size decreased at 2 weeks. This signifies that the gel deposits degrade over time in the body. We also saw no significant difference in necrotic volume between the two treatment groups. H&E staining showed necrotic cell death and wound healing at 14 days.
Data gathered in this study will be used to support a regulatory application and will inform the study design for a clinical trial. This will also inform providers about healing and when to schedule follow-up visits after EC-ethanol treatment.

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