Evaluation of a novel cryoballoon swipe ablation system in bench, porcine, and human esophagus models

Evaluation of a novel cryoballoon swipe ablation system in bench, porcine, and human esophagus... SUMMARY Current ablation devices for dysplastic Barrett's esophagus are effective but have significant limitations. This pilot study aims to evaluate the safety, feasibility, and dose response of a novel cryoballoon swipe ablation system (CbSAS) in three experimental in vitro and in vivo models. CbSAS is a through-the-scope compliant balloon that is simultaneously inflated and cooled by liquid nitrous oxide delivered from a disposable handheld unit. When the cryogen is applied through a special diffuser it covers a 90° section of the circumference of the esophagus for 3 cm length. Doses range from 0.9 to 0.5 mm/second. (1) Bench model: The fixture consisted of an ‘esophagus-like’ tube lined with agar at 37°C to create an inner diameter of 20 or 30 mm, within which thermocouples were embedded. (2) In vivo porcine esophagus: CbSAS ablations were performed in animals that were euthanized and histological assessments of depth and percentage of esophageal mucosa successfully ablated were done. (3) In vivo, pre-esophagectomy human esophagus. After CbSAS ablations, histological assessments were performed (at 0, 4, and 28 days) to assess the depth and percentage of ablated mucosa. As outcomes, we assessed the safety and tolerability (pain and serious, device-related adverse events); efficacy (depth and uniformity) of ablation; and device performance (ease of deployment and device malfunction). In the bench model, during CbSAS, thermocouples measured minimal temperatures of −40 to −48 °C at all doses. In the porcine model, maximal effect on the mucosa was reached with a dose of 0.8 mm/second that extended to superficial submucosa, while 0.5 mm/second extended through the submucosa. All animals tolerated the treatments and, regardless of ablation dose, continued oral intake and gained weight. In the human model, six patients (5 male, 1 female, mean age 68) tolerated the procedure without adverse events. CbSAS was simple to operate, and balloon contact with tissue was easily and uniformly maintained. The maximal effect on the mucosa is achieved with a 0.8 mm/second dose. We concluded that the CbSAS device enables uniform 3 cm long, quarter-circumferential mucosal ablation in a one-step process by using a novel, through-the-scope balloon. The CbSAS delivers predictable ablation with mucosal and limited submucosal necrosis in bench, animal, and human esophagus. Because of its ease of use, this new device merits further clinical study in the treatment of patients with dysplastic Barrett's esophagus. Abbreviations BE Barrett's esophagus CbSAS cryoballoon swipe ablation system; cryotherapy; mucosal ablation INTRODUCTION Barrett's esophagus (BE) is a precursor lesion to esophageal adenocarcinoma, through a stepwise progression from metaplasia to low- and high-grade dysplasia.1 Current guidelines advise endoscopic surveillance of patients with BE to identify early neoplastic lesions that can be cured with endoscopic therapies, such as endoscopic resection and mucosal ablation.2 Whereas nodular areas of dysplastic BE are removed by endoscopic resection, flat dysplastic BE without visible lesions is typically ablated. In addition, residual BE after endoscopic resection of a dysplastic lesion is ablated regardless of the presence of dysplasia to prevent metachronous lesions.3 Current ablation devices for dysplastic BE, such as radiofrequency, argon plasma coagulation and spray cryotherapy are effective but none provide the necessary attributes for widespread adoption, because of side effects, lack of predictability as to depth of ablation, length of procedure time or cost (e.g. inventory requirements, need for capital equipment).4 A novel technology for delivering esophageal cryotherapy is the CryoBalloon focal ablation system (C2 Therapeutics, Redwood City, CA, USA), a through-the-scope (TTS) device that allows focal ablation (approximately 2 cm2) by releasing nitrous oxide into a highly compliant balloon that adapts to the diameter of the esophagus. Preliminary clinical studies have shown that this approach is simple, safe, and effective and it eliminates extensive resource requirements such as the use of large controller units that are required for radiofrequency and cryogen spray systems.5,6 Cryoablation causes cell death by dehydrating the cell through inducing ice crystal formation in the intracellular space. Cryoablation does not cause permanent changes in protein structure after thawing, thereby preserving the extracellular collagen matrix architecture and enabling deeper mucosal ablation and possibly resulting in less pain due to an anesthetic effect of tissue and nerve cooling.7 This study was designed to evaluate the safety, feasibility, and dose response of a novel cryoballoon swipe ablation system (CbSAS) in three models: bench, animal (swine) esophagus and pre-esophagectomy human esophagus. CbSAS is a through-the-scope compliant balloon that is simultaneously inflated and cooled by liquid nitrous oxide delivered from a disposable handheld unit. When the cryogen is applied through a special diffuser it covers a 90° section of the circumference of the esophagus for 3 cm length. METHODS This is a prospective 3-prong, in vitro and in vivo (animal and human) study, designed to assess the safety, performance, and dosing of a novel through-the-scope ablation balloon system that enables uniform 3 cm long, quarter-circumferential mucosal ablation in a one-step process. The human portions of the study were approved by the local institutional review boards of the participating hospitals. Similarly, the animal protocol was also IRB-approved, separately, by the Institutional Animal Care & Use Committee. Technology The CbSAS is a through-the-scope cryosurgical device that is compatible with commercially available endoscopes with a minimum working channel of 3.7 mm and maximum length of 100 cm. It is comprised of a catheter with a balloon at its tip, a controller, a foot pedal, and cartridges containing nitrous oxide (Fig. 1). The balloon is highly compliant and automatically sizes to the esophageal lumen; it is simultaneously inflated and cooled by liquid nitrous oxide delivered from a small, disposable, handheld unit through a diffuser that traverses a length of 3 cm while emitting cryogen. The treatment site is selected by advancing or retracting the balloon to position the diffuser contained within the balloon. When the cryogen is applied, it covers a 90° section of the circumference of the esophagus in the treatment area for over a 3 cm length without the need for a venting tube. The controller software can allow adjustment of the ablation speed prior to treatment. Doses range from 0.9 mm/second (lowest dose) to 0.5 mm/second (highest dose). Dose is the rate at which the diffuser traverses the axis of the balloon catheter while emitting cryogen (Fig. 2). Fig. 1 View largeDownload slide Illustration of the CbSAS (CryoBalloon 90), a cryosurgical device with a nitrous oxide cooled balloon catheter probe that is compatible with upper gastrointestinal therapeutic endoscopes. CbSAS is comprised of the controller with cryogen cartridge (A) and a foot pedal (B) that are intended to work in conjunction with the balloon catheter (c), controlling the catheter-specific fluid mechanics and diffuser movement to facilitate tissue ablation. The catheter (C) consists of a proximal connector, catheter shaft, balloon and distal atraumatic tip, and is provided with the balloon constrained within a protective sheath. The balloon is a low-pressure design constructed of highly compliant material such that its contour accommodates the esophageal lumen and achieves apposition with the esophageal wall upon inflation. The nitrous oxide from the cartridge exits the refrigerant delivery lumen via the diffuser inside the balloon (the cryogen does not directly contact patient's tissue). The controller contains the cartridge holder and refrigerant delivery valve which is controlled by the trigger. Fig. 1 View largeDownload slide Illustration of the CbSAS (CryoBalloon 90), a cryosurgical device with a nitrous oxide cooled balloon catheter probe that is compatible with upper gastrointestinal therapeutic endoscopes. CbSAS is comprised of the controller with cryogen cartridge (A) and a foot pedal (B) that are intended to work in conjunction with the balloon catheter (c), controlling the catheter-specific fluid mechanics and diffuser movement to facilitate tissue ablation. The catheter (C) consists of a proximal connector, catheter shaft, balloon and distal atraumatic tip, and is provided with the balloon constrained within a protective sheath. The balloon is a low-pressure design constructed of highly compliant material such that its contour accommodates the esophageal lumen and achieves apposition with the esophageal wall upon inflation. The nitrous oxide from the cartridge exits the refrigerant delivery lumen via the diffuser inside the balloon (the cryogen does not directly contact patient's tissue). The controller contains the cartridge holder and refrigerant delivery valve which is controlled by the trigger. Fig. 2 View largeDownload slide Illustration of CbSAS set-up. Once positioned within the esophagus at the target location, the balloon is simultaneously inflated and cooled with nitrous oxide. The system emits nitrous oxide through a diffuser in the inner lumen inside the balloon. The balloon remains stationary during the delivery of the nitrous oxide while the diffuser traverses the length of the balloon starting at the distal end and moving in a proximal direction to ablate a quarter of the circumference (90°) of an approximate 3 cm length segment of tissue. The dose varies based on the rate (in millimeters [mm] per second) at which the diffuser traverses the length of the balloon while emitting cryogen. The lower the rate, the higher the dose of cryogen released. Fig. 2 View largeDownload slide Illustration of CbSAS set-up. Once positioned within the esophagus at the target location, the balloon is simultaneously inflated and cooled with nitrous oxide. The system emits nitrous oxide through a diffuser in the inner lumen inside the balloon. The balloon remains stationary during the delivery of the nitrous oxide while the diffuser traverses the length of the balloon starting at the distal end and moving in a proximal direction to ablate a quarter of the circumference (90°) of an approximate 3 cm length segment of tissue. The dose varies based on the rate (in millimeters [mm] per second) at which the diffuser traverses the length of the balloon while emitting cryogen. The lower the rate, the higher the dose of cryogen released. Experimental testing Bench model The bench fixture consisted of a plastic tube simulating the tubular esophagus. Its interior surface was lined with agar at 37°C to create an inner diameter of 20 or 30 mm, with thermocouples embedded at depths of 0.5, 1, and 1.5 mm in the subluminal surface. The thermocouples in the test structure covered a 90o portion (+10o, to simulate overlap of side-by-side ablations) of the agar tube circumference for a length of 3 cm. The model allows for evaluation of temperature uniformity as the cryogen sprayer traverses the length of the balloon (Fig. 3). Data were collected at various speeds and depths. Fig. 3 View largeDownload slide Illustration of the bench fixture consisting of a plastic tube simulating the tubular esophagus. Its interior surface was lined with agar at 37°C to create an inner diameter of 20 or 30 mm, with thermocouples embedded at depths of 0.5, 1, and 1.5 mm in the subluminal surface. The thermocouples in the test structure covered a 90° portion (+10°, to simulate overlap of side-by-side ablations) of the agar tube circumference for a length of 3 cm. This model allows for evaluation of temperature uniformity as the cryogen sprayer traverses the length of the balloon. Data were collected at various speeds and depths. The 90°CbSAS at sprayer speed of 0.8 mm/second (shown) achieved at least a temperature of −40°C at a depth of 0.5 mm. Fig. 3 View largeDownload slide Illustration of the bench fixture consisting of a plastic tube simulating the tubular esophagus. Its interior surface was lined with agar at 37°C to create an inner diameter of 20 or 30 mm, with thermocouples embedded at depths of 0.5, 1, and 1.5 mm in the subluminal surface. The thermocouples in the test structure covered a 90° portion (+10°, to simulate overlap of side-by-side ablations) of the agar tube circumference for a length of 3 cm. This model allows for evaluation of temperature uniformity as the cryogen sprayer traverses the length of the balloon. Data were collected at various speeds and depths. The 90°CbSAS at sprayer speed of 0.8 mm/second (shown) achieved at least a temperature of −40°C at a depth of 0.5 mm. In vivo porcine esophagus CbSAS ablations (0.9, 0.8, 0.6 and 0.5 m/second, each 3 cm long) were performed in 3 anesthetized animals that were then euthanized at 0 (3 hours), 4 and 28 days post-treatment. Histological assessments of the depth and percentage of esophageal mucosa ablated were made following a standard protocol (see below). In vivo, pre-esophagectomy human esophagus After CbSAS ablations, histological assessments were performed (at 0 and 7 days) to assess the depth and percentage of esophageal mucosa successfully ablated. The following outcomes were examined: Device performance, patient tolerability, safety, and efficacy (depth and percentage of esophageal mucosa ablated, using standard protocol). Histopathological assessment Appendix  1 depicts the details of the standardized histopathological assessment. Such assessment was intended to quantify the degree of the initial ablative injury of the epithelial surface and the submucosal structures (depth of ablation), the preservation of the muscularis propria and adventitia (risk of perforation) as well as the subsequent reparation to integrity (risk of stricture formation). RESULTS In vitro (bench) studies Several factors affected accurate temperature acquisition, such as the controller's motor speed, thermocouple accuracy, thickness of agar (0.5–1.5 mm) covering the thermocouples, consistency of the agar, the embedding of the thermocouples into the agar and the presence of air bubbles between thick and thin agar layers. As shown in Figure 3, the 90°CbSAS at sprayer speeds of 0.8 mm/second achieved at least a temperature of −40 °C at a depth of 0.5 mm. Sprayer speeds of 0.7 mm/second and below achieved at least a temperature of −40 °C at a depth of 1.0 mm. A temperature of −40 °C was not reached at a depth of 1.5 mm (data not shown). Porcine model Figure 4 displays representative images of the histologic effects of CbSAS at doses of 0.5 and 0.8 mm/second applied over a 3 cm length. There were no significant differences between the 2 doses. On day 0 (3 hours), there was full thickness necrosis of the epithelium with separation at epithelial–submucosal margin, indicative of tissue weakening at this junction. The epithelial cells revealed cytoplasmic change and condensed nuclei, and there was scant fibrin and neutrophilic infiltration, as well as submucosal edema, neutrophilic infiltration, and congestion of the smooth muscle layers, and mild to moderate edema of the adventitia. On day 4, there were multifocal areas of mucosal ulceration lined by fibrin, degenerated neutrophils and mucus. The submucosa was edematous with infiltration by acute and chronic inflammatory cells. The submucosal vessels exhibited congestion and vascular necrosis while the muscle layers exhibited neutrophilic infiltration and necrosis. The adventitia was expanded by edema, acute, and chronic inflammation and fibrin exudation. On day 28, the treated areas were characterized by intact and well-differentiated epithelium, minimal lymphocytic and plasma cell infiltration, and fibrosis of the submucosa and the inner and outer muscle layers with lesser involvement of the adventitia. Overall, the gross and histological findings showed progressive changes of injury and subsequent healing, from initial necrosis and epithelial–submucosal separation and acute inflammation, to mucosal edema and ulceration, to re-epithelialization and regional fibrosis involving the submucosa, muscularis propria, and adventitia. Table 1 presents the data from the animal study in which the 90°CbSAS was used at doses of 0.5 to 0.8 mm/second in 3 animals euthanized at 0, 4, and 28 days, respectively. Fig. 4 View largeDownload slide Representative images of the histologic effects of CbSAS at a dose of 0.5 mm/second in the swine model. (A; Day 0). Full thickness epithelial necrosis with separation at epithelial–submucosal margin, indicative of tissue weakening at this junction. (B; Day 4). Maximal effect of cryoablation exhibiting injured sloughed epithelium resulting in areas of ulceration associated with moderate edema and moderate to large numbers of infiltrating neutrophils. (C; Day 28). Re-epithelialization of the treatment site and the predominant histologic finding was a regional area of fibrosis involving the submucosa, muscularis propria and adventitia. The overlying epithelial integrity was restored. Fig. 4 View largeDownload slide Representative images of the histologic effects of CbSAS at a dose of 0.5 mm/second in the swine model. (A; Day 0). Full thickness epithelial necrosis with separation at epithelial–submucosal margin, indicative of tissue weakening at this junction. (B; Day 4). Maximal effect of cryoablation exhibiting injured sloughed epithelium resulting in areas of ulceration associated with moderate edema and moderate to large numbers of infiltrating neutrophils. (C; Day 28). Re-epithelialization of the treatment site and the predominant histologic finding was a regional area of fibrosis involving the submucosa, muscularis propria and adventitia. The overlying epithelial integrity was restored. Table 1 Quantitative assessment of histopathology of swine esophagus at 0, 4, and 28 days after CbSAS at doses of 0.5 mm/second and 0.8 mm/second, showing mucosal and submucosal injury with preservation of muscularis propria and adventitia at all time points. At the doses tested there was ablation of the mucosa and partly the submucosa with preservation of the muscularis propria and adventitia with restoration to integrity at 28 days. There were no significant differences between the 2 doses used   Day 0  Day 4  Day 28  Dose  0.5 mm/second  0.8 mm/second  0.5 mm/second  0.8 mm/second  0.5 mm/second  0.8 mm/second  Squamous epithelium  0.5  0.5  3  3  0  0  Submucosa  1  2  3  2  1  1  Muscularis propria  2  2  3  2  4  4  Adventitia  1  1  3  2  0.5  1    Day 0  Day 4  Day 28  Dose  0.5 mm/second  0.8 mm/second  0.5 mm/second  0.8 mm/second  0.5 mm/second  0.8 mm/second  Squamous epithelium  0.5  0.5  3  3  0  0  Submucosa  1  2  3  2  1  1  Muscularis propria  2  2  3  2  4  4  Adventitia  1  1  3  2  0.5  1  View Large Table 1 Quantitative assessment of histopathology of swine esophagus at 0, 4, and 28 days after CbSAS at doses of 0.5 mm/second and 0.8 mm/second, showing mucosal and submucosal injury with preservation of muscularis propria and adventitia at all time points. At the doses tested there was ablation of the mucosa and partly the submucosa with preservation of the muscularis propria and adventitia with restoration to integrity at 28 days. There were no significant differences between the 2 doses used   Day 0  Day 4  Day 28  Dose  0.5 mm/second  0.8 mm/second  0.5 mm/second  0.8 mm/second  0.5 mm/second  0.8 mm/second  Squamous epithelium  0.5  0.5  3  3  0  0  Submucosa  1  2  3  2  1  1  Muscularis propria  2  2  3  2  4  4  Adventitia  1  1  3  2  0.5  1    Day 0  Day 4  Day 28  Dose  0.5 mm/second  0.8 mm/second  0.5 mm/second  0.8 mm/second  0.5 mm/second  0.8 mm/second  Squamous epithelium  0.5  0.5  3  3  0  0  Submucosa  1  2  3  2  1  1  Muscularis propria  2  2  3  2  4  4  Adventitia  1  1  3  2  0.5  1  View Large Pre-esophagectomy studies We studied 6 patients (5M:1F), all Caucasian, median age 69 (IQR 58–77), median BMI 26.3 (IQR 22.0–26.9). Four (67%) had history of GERD and BE. In the 2 patients with delayed esophagectomy (six and ten days post-ablation), pain scores were zero after ablation and remained at zero through follow-up in both patients. One patient had a dysphagia score of 1 at 24 hours and reported a score of zero at 7-day follow-up (of note is that this patient had pre-existing dysphagia prior to ablation). The second patient had no dysphagia postablation. Table 2 summarizes the histopathology of human esophagus prior to esophagectomy using a range of CbSAS doses (0.5–0.9 mm/second) and various time intervals postablation (0–10 days) revealing surface epithelial injury and preservation of the muscularis propria and adventitia. Based on collective experience with bench, animal and human data the 0.8 mm/second dose was ultimately deemed ideal for planned human Barrett's ablation trials. Table 2 Descriptive histopathology of human esophagus ablated prior to esophagectomy using a range of CbSAS doses (0.5 mm/second to 0.9 mm/second) and various time intervals post ablation (0–10 days) revealing surface epithelial injury and preservation of the muscularis propria and adventitia. Based on collective experience with bench, animal and human data the 0.8 mm/second dose was ultimately deemed ideal for planned human Barrett's ablation trials Study ID  Dose (mm/second)  Time (days)  Pathology findings (ISQ, inner squamous mucosa; SM, submucosa)  Grade  1  0.5  0  ISQ: Expansion of mature cells, pyknosis of epithelial nuclei  0.5        SM: Normal  0        Adventitia: Normal  0  2  0.6  0  ISQ: Expansion of mature cells, pyknosis of epithelial nuclei  0.5        SM: Mild separation of muscle fibers with inflammatory cell infiltration  2        Adventitia: Mild edematous thickening  3  3  0.8  0  ISQ: Necrosis of basal epithelial layer  3        SM: Moderate separation of muscle fibers with inflammation  2        Adventitia: Moderate thickening of adventitia with inflammatory cell infiltration  3  4  0.9  0  ISQ: Erosion of cells to level of basal layer  1        SM: Dilation with edema, necrosis of muscle fibers, inflammatory cell infiltration  1        Adventitia: Mild edematous thickening  1  5  0.5  5  ISQ: Necrosis of basal epithelial layer  3        SM: Dilation with edema, necrosis of muscle fibers, inflammatory cell infiltration  1        Adventitia: Moderate thickening with inflammatory cell infiltration  1  6  0.8  10  ISQ: Necrosis of basal epithelial layer  3        SM: Dilation with edema, necrosis of muscle fibers, inflammatory cell infiltration  1        Adventitia: Moderate thickening with inflammatory cell infiltration  1  Study ID  Dose (mm/second)  Time (days)  Pathology findings (ISQ, inner squamous mucosa; SM, submucosa)  Grade  1  0.5  0  ISQ: Expansion of mature cells, pyknosis of epithelial nuclei  0.5        SM: Normal  0        Adventitia: Normal  0  2  0.6  0  ISQ: Expansion of mature cells, pyknosis of epithelial nuclei  0.5        SM: Mild separation of muscle fibers with inflammatory cell infiltration  2        Adventitia: Mild edematous thickening  3  3  0.8  0  ISQ: Necrosis of basal epithelial layer  3        SM: Moderate separation of muscle fibers with inflammation  2        Adventitia: Moderate thickening of adventitia with inflammatory cell infiltration  3  4  0.9  0  ISQ: Erosion of cells to level of basal layer  1        SM: Dilation with edema, necrosis of muscle fibers, inflammatory cell infiltration  1        Adventitia: Mild edematous thickening  1  5  0.5  5  ISQ: Necrosis of basal epithelial layer  3        SM: Dilation with edema, necrosis of muscle fibers, inflammatory cell infiltration  1        Adventitia: Moderate thickening with inflammatory cell infiltration  1  6  0.8  10  ISQ: Necrosis of basal epithelial layer  3        SM: Dilation with edema, necrosis of muscle fibers, inflammatory cell infiltration  1        Adventitia: Moderate thickening with inflammatory cell infiltration  1  View Large Table 2 Descriptive histopathology of human esophagus ablated prior to esophagectomy using a range of CbSAS doses (0.5 mm/second to 0.9 mm/second) and various time intervals post ablation (0–10 days) revealing surface epithelial injury and preservation of the muscularis propria and adventitia. Based on collective experience with bench, animal and human data the 0.8 mm/second dose was ultimately deemed ideal for planned human Barrett's ablation trials Study ID  Dose (mm/second)  Time (days)  Pathology findings (ISQ, inner squamous mucosa; SM, submucosa)  Grade  1  0.5  0  ISQ: Expansion of mature cells, pyknosis of epithelial nuclei  0.5        SM: Normal  0        Adventitia: Normal  0  2  0.6  0  ISQ: Expansion of mature cells, pyknosis of epithelial nuclei  0.5        SM: Mild separation of muscle fibers with inflammatory cell infiltration  2        Adventitia: Mild edematous thickening  3  3  0.8  0  ISQ: Necrosis of basal epithelial layer  3        SM: Moderate separation of muscle fibers with inflammation  2        Adventitia: Moderate thickening of adventitia with inflammatory cell infiltration  3  4  0.9  0  ISQ: Erosion of cells to level of basal layer  1        SM: Dilation with edema, necrosis of muscle fibers, inflammatory cell infiltration  1        Adventitia: Mild edematous thickening  1  5  0.5  5  ISQ: Necrosis of basal epithelial layer  3        SM: Dilation with edema, necrosis of muscle fibers, inflammatory cell infiltration  1        Adventitia: Moderate thickening with inflammatory cell infiltration  1  6  0.8  10  ISQ: Necrosis of basal epithelial layer  3        SM: Dilation with edema, necrosis of muscle fibers, inflammatory cell infiltration  1        Adventitia: Moderate thickening with inflammatory cell infiltration  1  Study ID  Dose (mm/second)  Time (days)  Pathology findings (ISQ, inner squamous mucosa; SM, submucosa)  Grade  1  0.5  0  ISQ: Expansion of mature cells, pyknosis of epithelial nuclei  0.5        SM: Normal  0        Adventitia: Normal  0  2  0.6  0  ISQ: Expansion of mature cells, pyknosis of epithelial nuclei  0.5        SM: Mild separation of muscle fibers with inflammatory cell infiltration  2        Adventitia: Mild edematous thickening  3  3  0.8  0  ISQ: Necrosis of basal epithelial layer  3        SM: Moderate separation of muscle fibers with inflammation  2        Adventitia: Moderate thickening of adventitia with inflammatory cell infiltration  3  4  0.9  0  ISQ: Erosion of cells to level of basal layer  1        SM: Dilation with edema, necrosis of muscle fibers, inflammatory cell infiltration  1        Adventitia: Mild edematous thickening  1  5  0.5  5  ISQ: Necrosis of basal epithelial layer  3        SM: Dilation with edema, necrosis of muscle fibers, inflammatory cell infiltration  1        Adventitia: Moderate thickening with inflammatory cell infiltration  1  6  0.8  10  ISQ: Necrosis of basal epithelial layer  3        SM: Dilation with edema, necrosis of muscle fibers, inflammatory cell infiltration  1        Adventitia: Moderate thickening with inflammatory cell infiltration  1  View Large DISCUSSION The novel CbSAS device described herein allows controlled cooling and quarter-circumferential (90o) ablation of esophageal mucosa under direct endoscopic vision over a length of 3 cm. As such, it offers the advantage of larger surface ablation when compared to the focal device previously tested in vitro as well as in vivo in animals and patients with BE. Pressing a button on the handheld instrument results in simultaneous inflation and cooling of the balloon using nitrous oxide delivered from a small canister located inside the handle to the diffuser that moves vertically within the balloon at a rate corresponding to the dose. The relatively low pressure of nitrous oxide gas in the CbSAS is advantageous because it permits the use of a highly compliant balloon that can conform to the shape of the esophagus and can achieve mucosal contact over a range of diameters from 20 to 35 mm, even when the surface geometry is challenging. The CbSAS is designed to expand until contact with the esophagus is achieved, up to a maximal diameter of 35 mm. Once in contact with the esophagus, the balloon stops expanding and applies a constant low pressure of up to 0.4 atm until the ablation is complete; the balloon then rapidly deflates. By advancing or retracting the CbSAS the diffuser contained within the balloon is positioned over the targeted area for treatment. When the cryogen is applied, it covers a 90° section of the circumference of the esophagus in the treatment area for 3 cm length without the need for a venting tube. In this study, we found that the optimal dose, that is, the rate at which the diffuser traverses the axis of the balloon catheter while emitting cryogen, was 0.8 mm/second (Fig. 2). Cryoablation, or the destruction of tissue by freezing, is a multifactorial process. Primary cell necrosis caused by intracellular ice formation occurs between −15 and −40°C, with some necrosis occurring at −15°C, and complete necrosis achieved by −40°C. Further, secondary effects caused by vascular injury and induced cellular apoptosis occur to some degree in all tissue subjected to freezing. In the CbSAS device, the balloon temperature is −80°C, and a mucosal temperature exceeding −40°C is achieved with good contact between the inflated balloon and the esophageal surface, which is sufficient to cause immediate primary cell necrosis of the superficial mucosa. As the application time is lengthened, progressively deeper sections of the esophageal wall are exposed to lower temperatures, resulting in deeper ablation. Histologic studies that have measured the thickness of BE epithelium (0.4–0.6 mm), squamous epithelium (0.4–0.6 mm), and esophageal wall thickness (2.4 mm for normal esophagus, 3.1 mm nondysplastic BE, 3.4 mm BE with high-grade dysplasia) serve as a guide to the optimal depth of ablation. The application time must be long enough to cause necrosis of up to 0.6 mm of BE epithelial thickness (or perhaps slightly more in cases of high-grade dysplasia) while minimizing damage to the deeper portions of the esophageal wall.8 In a similar fashion to previous experiments, our animal testing of the CbSAS was conducted in Yorkshire swine, and the results are summarized in Figure 3. The device was positioned and ablation was carried out without difficulty in all animals and humans. There was no difficulty in maintaining the position of the balloon during ablation, and we did not observe any tendency of esophageal peristalsis to pull the balloon down during the procedure. As the ablation time increased the percentage of mucosa ablated increased, reaching a desired effect with an application time of 0.8 cm/second. Further design improvements could lead to a half- or full-circumferential surface freeze and possibly increase the percentage of mucosa that could be ablated or slightly reduce the required ablation time. In the porcine model 2–4 days after cryoablation, necrosis was observed in the deeper esophageal wall layers, however, only very little damage and no strictures were seen after 28 days. This was pleasantly surprising, since pigs are more prone to stricture formation than humans after endoscopic resection or ablation.9,10 Although not directly comparable, in the six human cases, the depth and severity of injury appeared to be significantly less than in the porcine model. Our panel of experimental studies has some inherent limitations. First, although the swine model is frequently used given its size and anatomical similarities to the human, the esophageal wall layers are generally thinner and the cryoablation effects may be overestimated compared to the human esophagus. Yet four days after ablation, at a timepoint at which the tissue response is at its most extensive, we encountered no serious perforative injury and there was full recovery at 28 days. Second, we have limited dosimetry data in the human, pre-esophagectomy model, reflecting the inherent recruitment difficulties of such patients. Third, CbSAS was performed mostly on squamous esophageal epithelium, since there are no preclinical models of BE available. Since BE is known to have thicker wall layers compared to the normal squamous esophagus and even has an overlying protective mucus gel layer, safety is not likely to be an issue when the technology is tested clinically in patients with dysplastic Barrett's esophagus when the dose (0.8 mm/second) was proven safe in squamous epithelium in vivo.10,11 Nevertheless, there are several significant remaining issues in further development of the CbSAS. With the current balloon design, ablation over the length of the balloon must be optimized and the overall circumferential effect increased to reduce the number of necessary applications for a long and circumferential segment of BE since it is desirable to achieve 100% ablation. This may require a second application or overlapping segments during stepwise ablation of extended areas of BE. Further studies will be needed to investigate these issues. Historically, focal cryoballoon eradication rates have been comparable with rates reported for RFA for the treatment of dysplastic BE (88–93%).12,13 However, cryoablation might be favorable over RFA because of the lack of capital equipment, and it therefore has the potential for widespread implementation into the community, especially in rural areas. Moreover, unlike heat-based technologies, cryoablation does not disrupt the extracellular collagen matrix, which theoretically might allow for deeper ablation without causing perforation or strictures.14 Endoscopic resection, by EMR or ESD, will remain the modality of choice for nodular dysplasia eradication and for staging. Cryoballoon and RFA play a role in the management of flat dysplasia and residual metaplasia after resection because of their simplicity and low likelihood for stricture formation when applied circumferentially. In conclusion, CbSAS enables uniform 3 cm long, quarter-circumferential mucosal ablation in a one-step process by using a novel, through-the-scope balloon. At a dose of 0.8 mm/second, the system delivers uniform and predictable ablation with mucosal and submucosal necrosis in bench, animal and human esophagus. Because of its ease of use, this new device merits further clinical study in the treatment of dysplastic Barrett's esophagus. Notes Grant Support: This study was funded by C2 Therapeutics, Redwood City, CA. Disclosures: GT is a consultant to C2 Therapeutics. BEL, WH and BLW have no conflicts to declare. Specific author contributions: Planning and/or conducting the study: Brian E. Louie, Wayne Hofstetter, George Triadafilopoulos, Bas L. Weusten; Collecting and/or interpreting data: Brian E. Louie, Wayne Hofstetter, George Triadafilopoulos, Bas L. Weusten; Drafting the manuscript and revision: Brian E. Louie, Wayne Hofstetter, George Triadafilopoulos, Bas L. Weusten. References 1. Spechler S J, Souza R F. Barrett's esophagus. N Engl J Med  2014; 371: 836– 45. Google Scholar CrossRef Search ADS PubMed  2. Shaheen N J, Falk G W, Iyer P G et al.   ACG Clinical Guideline: Diagnosis and Management of Barrett's Esophagus. Am J Gastroenterol  2016; 111: 30– 50. Google Scholar CrossRef Search ADS PubMed  3. Overwater A, Weusten BLAM. Cryoablation in the management of Barrett's esophagus. Curr Opin Gastroenterol  2017; 33: 261. Google Scholar CrossRef Search ADS PubMed  4. Gosain S, Mercer K, Twaddell W S et al.   Liquid nitrogen spray cryotherapy in Barrett's esophagus with high-grade dysplasia: long-term results. Gastrointest Endosc  2013; 78: 260– 5. Google Scholar CrossRef Search ADS PubMed  5. Schölvinck D W, Künzli H T, Kestens C et al.   Treatment of Barrett's esophagus with a novel focal cryoablation device: a safety and feasibility study. Endoscopy  2015; 47: 1106– 12. Google Scholar CrossRef Search ADS PubMed  6. Künzli H T, Schölvinck D W, Meijer S L, Seldenrijk K A, Bergman J G, Weusten B L. Efficacy of the CryoBalloon Focal Ablation System for the eradication of dysplastic Barrett's esophagus islands. Endoscopy  2017; 49: 169– 75. Google Scholar PubMed  7. Baust J G, Gage A A, Bjerklund Johansen T E, Baust J M. Mechanisms of cryoablation: clinical consequences on malignant tumors. Cryobiology  2014; 68: 1– 11. Google Scholar CrossRef Search ADS PubMed  8. Gill K R, Ghabril M S, Jamil L H et al.   Variation in Barrett's esophageal wall thickness: is it associated with histology or segment length? J Clin Gastroenterol  2010; 44: 411– 5. Google Scholar CrossRef Search ADS PubMed  9. Kamler J P, Borsatto R, Binmoeller K F. Circumferential endoscopic mucosal resection in the swine esophagus assisted by a cap attachment. Gastrointest Endosc  2002; 55: 923– 8. Google Scholar CrossRef Search ADS PubMed  10. Scholvinck D W, Alvarez Herrero L, Visser M, Bergman J J, Weusten B L. Effects of Lugol staining on stenosis formation induced by radiofrequency ablation of esophageal squamous epithelium: a study in a porcine model. Dis Esophagus  2015; 28: 603– 11. Google Scholar CrossRef Search ADS PubMed  11. Dixon J, Strugala V, Griffin S M et al.   Esophageal mucin: an adherent mucus gel barrier is absent in the normal esophagus but present in columnar-lined Barrett's esophagus. Am J Gastroenterol  2001; 96: 2575– 83. Google Scholar CrossRef Search ADS PubMed  12. Phoa K N, Pouw R E, van Vilsteren F G et al.   Remission of Barrett's esophagus with early neoplasia 5 years after radiofrequency ablation with endoscopic resection: a Netherlands cohort study. Gastroenterology  2013; 145: 96– 104. Google Scholar CrossRef Search ADS PubMed  13. Shaheen N J, Overholt B F, Sampliner R Eet al. Durability of radiofrequency ablation in Barrett's esophagus with dysplasia. Gastroenterology  2011; 141: 460– 8. Google Scholar CrossRef Search ADS PubMed  14. Weusten B L, Bergman J J. Cryoablation for managing Barrett's esophagus refractory to radiofrequency ablation? Don’t embrace the cold too soon!. Gastrointest Endosc  2015; 82: 449– 51. Google Scholar CrossRef Search ADS PubMed  APPENDIX 1 Grading scale (0–4) Area  Grading  Squamous mucosa  Normal  0    Cell expansion with nuclear pyknosis  0.5    Cell erosion  1    Cell erosion to basal layer  2    Necrosis of all basal layer  3  Submucosa  Normal  0    Inflammatory cell infiltration  1    Separation with inflammation  2    Edema and necrosis  3  Muscularis propria  Normal  0    Edema and inflammatory infiltration  1    Focal necrosis and inflammation  2    Fiber separation with inflammation  3    Fibrosis  4  Adventitia  Normal  0    Edema  0.5    Inflammatory cell infiltration  1    Necrosis and inflammation  2    Thickening with necrosis and inflammation  3  Area  Grading  Squamous mucosa  Normal  0    Cell expansion with nuclear pyknosis  0.5    Cell erosion  1    Cell erosion to basal layer  2    Necrosis of all basal layer  3  Submucosa  Normal  0    Inflammatory cell infiltration  1    Separation with inflammation  2    Edema and necrosis  3  Muscularis propria  Normal  0    Edema and inflammatory infiltration  1    Focal necrosis and inflammation  2    Fiber separation with inflammation  3    Fibrosis  4  Adventitia  Normal  0    Edema  0.5    Inflammatory cell infiltration  1    Necrosis and inflammation  2    Thickening with necrosis and inflammation  3  Area  Grading  Squamous mucosa  Normal  0    Cell expansion with nuclear pyknosis  0.5    Cell erosion  1    Cell erosion to basal layer  2    Necrosis of all basal layer  3  Submucosa  Normal  0    Inflammatory cell infiltration  1    Separation with inflammation  2    Edema and necrosis  3  Muscularis propria  Normal  0    Edema and inflammatory infiltration  1    Focal necrosis and inflammation  2    Fiber separation with inflammation  3    Fibrosis  4  Adventitia  Normal  0    Edema  0.5    Inflammatory cell infiltration  1    Necrosis and inflammation  2    Thickening with necrosis and inflammation  3  Area  Grading  Squamous mucosa  Normal  0    Cell expansion with nuclear pyknosis  0.5    Cell erosion  1    Cell erosion to basal layer  2    Necrosis of all basal layer  3  Submucosa  Normal  0    Inflammatory cell infiltration  1    Separation with inflammation  2    Edema and necrosis  3  Muscularis propria  Normal  0    Edema and inflammatory infiltration  1    Focal necrosis and inflammation  2    Fiber separation with inflammation  3    Fibrosis  4  Adventitia  Normal  0    Edema  0.5    Inflammatory cell infiltration  1    Necrosis and inflammation  2    Thickening with necrosis and inflammation  3  © The Author(s) 2018. Published by Oxford University Press on behalf of International Society for Diseases of the Esophagus. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Diseases of the Esophagus Oxford University Press

Evaluation of a novel cryoballoon swipe ablation system in bench, porcine, and human esophagus models

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© The Author(s) 2018. Published by Oxford University Press on behalf of International Society for Diseases of the Esophagus. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
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10.1093/dote/dox155
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Abstract

SUMMARY Current ablation devices for dysplastic Barrett's esophagus are effective but have significant limitations. This pilot study aims to evaluate the safety, feasibility, and dose response of a novel cryoballoon swipe ablation system (CbSAS) in three experimental in vitro and in vivo models. CbSAS is a through-the-scope compliant balloon that is simultaneously inflated and cooled by liquid nitrous oxide delivered from a disposable handheld unit. When the cryogen is applied through a special diffuser it covers a 90° section of the circumference of the esophagus for 3 cm length. Doses range from 0.9 to 0.5 mm/second. (1) Bench model: The fixture consisted of an ‘esophagus-like’ tube lined with agar at 37°C to create an inner diameter of 20 or 30 mm, within which thermocouples were embedded. (2) In vivo porcine esophagus: CbSAS ablations were performed in animals that were euthanized and histological assessments of depth and percentage of esophageal mucosa successfully ablated were done. (3) In vivo, pre-esophagectomy human esophagus. After CbSAS ablations, histological assessments were performed (at 0, 4, and 28 days) to assess the depth and percentage of ablated mucosa. As outcomes, we assessed the safety and tolerability (pain and serious, device-related adverse events); efficacy (depth and uniformity) of ablation; and device performance (ease of deployment and device malfunction). In the bench model, during CbSAS, thermocouples measured minimal temperatures of −40 to −48 °C at all doses. In the porcine model, maximal effect on the mucosa was reached with a dose of 0.8 mm/second that extended to superficial submucosa, while 0.5 mm/second extended through the submucosa. All animals tolerated the treatments and, regardless of ablation dose, continued oral intake and gained weight. In the human model, six patients (5 male, 1 female, mean age 68) tolerated the procedure without adverse events. CbSAS was simple to operate, and balloon contact with tissue was easily and uniformly maintained. The maximal effect on the mucosa is achieved with a 0.8 mm/second dose. We concluded that the CbSAS device enables uniform 3 cm long, quarter-circumferential mucosal ablation in a one-step process by using a novel, through-the-scope balloon. The CbSAS delivers predictable ablation with mucosal and limited submucosal necrosis in bench, animal, and human esophagus. Because of its ease of use, this new device merits further clinical study in the treatment of patients with dysplastic Barrett's esophagus. Abbreviations BE Barrett's esophagus CbSAS cryoballoon swipe ablation system; cryotherapy; mucosal ablation INTRODUCTION Barrett's esophagus (BE) is a precursor lesion to esophageal adenocarcinoma, through a stepwise progression from metaplasia to low- and high-grade dysplasia.1 Current guidelines advise endoscopic surveillance of patients with BE to identify early neoplastic lesions that can be cured with endoscopic therapies, such as endoscopic resection and mucosal ablation.2 Whereas nodular areas of dysplastic BE are removed by endoscopic resection, flat dysplastic BE without visible lesions is typically ablated. In addition, residual BE after endoscopic resection of a dysplastic lesion is ablated regardless of the presence of dysplasia to prevent metachronous lesions.3 Current ablation devices for dysplastic BE, such as radiofrequency, argon plasma coagulation and spray cryotherapy are effective but none provide the necessary attributes for widespread adoption, because of side effects, lack of predictability as to depth of ablation, length of procedure time or cost (e.g. inventory requirements, need for capital equipment).4 A novel technology for delivering esophageal cryotherapy is the CryoBalloon focal ablation system (C2 Therapeutics, Redwood City, CA, USA), a through-the-scope (TTS) device that allows focal ablation (approximately 2 cm2) by releasing nitrous oxide into a highly compliant balloon that adapts to the diameter of the esophagus. Preliminary clinical studies have shown that this approach is simple, safe, and effective and it eliminates extensive resource requirements such as the use of large controller units that are required for radiofrequency and cryogen spray systems.5,6 Cryoablation causes cell death by dehydrating the cell through inducing ice crystal formation in the intracellular space. Cryoablation does not cause permanent changes in protein structure after thawing, thereby preserving the extracellular collagen matrix architecture and enabling deeper mucosal ablation and possibly resulting in less pain due to an anesthetic effect of tissue and nerve cooling.7 This study was designed to evaluate the safety, feasibility, and dose response of a novel cryoballoon swipe ablation system (CbSAS) in three models: bench, animal (swine) esophagus and pre-esophagectomy human esophagus. CbSAS is a through-the-scope compliant balloon that is simultaneously inflated and cooled by liquid nitrous oxide delivered from a disposable handheld unit. When the cryogen is applied through a special diffuser it covers a 90° section of the circumference of the esophagus for 3 cm length. METHODS This is a prospective 3-prong, in vitro and in vivo (animal and human) study, designed to assess the safety, performance, and dosing of a novel through-the-scope ablation balloon system that enables uniform 3 cm long, quarter-circumferential mucosal ablation in a one-step process. The human portions of the study were approved by the local institutional review boards of the participating hospitals. Similarly, the animal protocol was also IRB-approved, separately, by the Institutional Animal Care & Use Committee. Technology The CbSAS is a through-the-scope cryosurgical device that is compatible with commercially available endoscopes with a minimum working channel of 3.7 mm and maximum length of 100 cm. It is comprised of a catheter with a balloon at its tip, a controller, a foot pedal, and cartridges containing nitrous oxide (Fig. 1). The balloon is highly compliant and automatically sizes to the esophageal lumen; it is simultaneously inflated and cooled by liquid nitrous oxide delivered from a small, disposable, handheld unit through a diffuser that traverses a length of 3 cm while emitting cryogen. The treatment site is selected by advancing or retracting the balloon to position the diffuser contained within the balloon. When the cryogen is applied, it covers a 90° section of the circumference of the esophagus in the treatment area for over a 3 cm length without the need for a venting tube. The controller software can allow adjustment of the ablation speed prior to treatment. Doses range from 0.9 mm/second (lowest dose) to 0.5 mm/second (highest dose). Dose is the rate at which the diffuser traverses the axis of the balloon catheter while emitting cryogen (Fig. 2). Fig. 1 View largeDownload slide Illustration of the CbSAS (CryoBalloon 90), a cryosurgical device with a nitrous oxide cooled balloon catheter probe that is compatible with upper gastrointestinal therapeutic endoscopes. CbSAS is comprised of the controller with cryogen cartridge (A) and a foot pedal (B) that are intended to work in conjunction with the balloon catheter (c), controlling the catheter-specific fluid mechanics and diffuser movement to facilitate tissue ablation. The catheter (C) consists of a proximal connector, catheter shaft, balloon and distal atraumatic tip, and is provided with the balloon constrained within a protective sheath. The balloon is a low-pressure design constructed of highly compliant material such that its contour accommodates the esophageal lumen and achieves apposition with the esophageal wall upon inflation. The nitrous oxide from the cartridge exits the refrigerant delivery lumen via the diffuser inside the balloon (the cryogen does not directly contact patient's tissue). The controller contains the cartridge holder and refrigerant delivery valve which is controlled by the trigger. Fig. 1 View largeDownload slide Illustration of the CbSAS (CryoBalloon 90), a cryosurgical device with a nitrous oxide cooled balloon catheter probe that is compatible with upper gastrointestinal therapeutic endoscopes. CbSAS is comprised of the controller with cryogen cartridge (A) and a foot pedal (B) that are intended to work in conjunction with the balloon catheter (c), controlling the catheter-specific fluid mechanics and diffuser movement to facilitate tissue ablation. The catheter (C) consists of a proximal connector, catheter shaft, balloon and distal atraumatic tip, and is provided with the balloon constrained within a protective sheath. The balloon is a low-pressure design constructed of highly compliant material such that its contour accommodates the esophageal lumen and achieves apposition with the esophageal wall upon inflation. The nitrous oxide from the cartridge exits the refrigerant delivery lumen via the diffuser inside the balloon (the cryogen does not directly contact patient's tissue). The controller contains the cartridge holder and refrigerant delivery valve which is controlled by the trigger. Fig. 2 View largeDownload slide Illustration of CbSAS set-up. Once positioned within the esophagus at the target location, the balloon is simultaneously inflated and cooled with nitrous oxide. The system emits nitrous oxide through a diffuser in the inner lumen inside the balloon. The balloon remains stationary during the delivery of the nitrous oxide while the diffuser traverses the length of the balloon starting at the distal end and moving in a proximal direction to ablate a quarter of the circumference (90°) of an approximate 3 cm length segment of tissue. The dose varies based on the rate (in millimeters [mm] per second) at which the diffuser traverses the length of the balloon while emitting cryogen. The lower the rate, the higher the dose of cryogen released. Fig. 2 View largeDownload slide Illustration of CbSAS set-up. Once positioned within the esophagus at the target location, the balloon is simultaneously inflated and cooled with nitrous oxide. The system emits nitrous oxide through a diffuser in the inner lumen inside the balloon. The balloon remains stationary during the delivery of the nitrous oxide while the diffuser traverses the length of the balloon starting at the distal end and moving in a proximal direction to ablate a quarter of the circumference (90°) of an approximate 3 cm length segment of tissue. The dose varies based on the rate (in millimeters [mm] per second) at which the diffuser traverses the length of the balloon while emitting cryogen. The lower the rate, the higher the dose of cryogen released. Experimental testing Bench model The bench fixture consisted of a plastic tube simulating the tubular esophagus. Its interior surface was lined with agar at 37°C to create an inner diameter of 20 or 30 mm, with thermocouples embedded at depths of 0.5, 1, and 1.5 mm in the subluminal surface. The thermocouples in the test structure covered a 90o portion (+10o, to simulate overlap of side-by-side ablations) of the agar tube circumference for a length of 3 cm. The model allows for evaluation of temperature uniformity as the cryogen sprayer traverses the length of the balloon (Fig. 3). Data were collected at various speeds and depths. Fig. 3 View largeDownload slide Illustration of the bench fixture consisting of a plastic tube simulating the tubular esophagus. Its interior surface was lined with agar at 37°C to create an inner diameter of 20 or 30 mm, with thermocouples embedded at depths of 0.5, 1, and 1.5 mm in the subluminal surface. The thermocouples in the test structure covered a 90° portion (+10°, to simulate overlap of side-by-side ablations) of the agar tube circumference for a length of 3 cm. This model allows for evaluation of temperature uniformity as the cryogen sprayer traverses the length of the balloon. Data were collected at various speeds and depths. The 90°CbSAS at sprayer speed of 0.8 mm/second (shown) achieved at least a temperature of −40°C at a depth of 0.5 mm. Fig. 3 View largeDownload slide Illustration of the bench fixture consisting of a plastic tube simulating the tubular esophagus. Its interior surface was lined with agar at 37°C to create an inner diameter of 20 or 30 mm, with thermocouples embedded at depths of 0.5, 1, and 1.5 mm in the subluminal surface. The thermocouples in the test structure covered a 90° portion (+10°, to simulate overlap of side-by-side ablations) of the agar tube circumference for a length of 3 cm. This model allows for evaluation of temperature uniformity as the cryogen sprayer traverses the length of the balloon. Data were collected at various speeds and depths. The 90°CbSAS at sprayer speed of 0.8 mm/second (shown) achieved at least a temperature of −40°C at a depth of 0.5 mm. In vivo porcine esophagus CbSAS ablations (0.9, 0.8, 0.6 and 0.5 m/second, each 3 cm long) were performed in 3 anesthetized animals that were then euthanized at 0 (3 hours), 4 and 28 days post-treatment. Histological assessments of the depth and percentage of esophageal mucosa ablated were made following a standard protocol (see below). In vivo, pre-esophagectomy human esophagus After CbSAS ablations, histological assessments were performed (at 0 and 7 days) to assess the depth and percentage of esophageal mucosa successfully ablated. The following outcomes were examined: Device performance, patient tolerability, safety, and efficacy (depth and percentage of esophageal mucosa ablated, using standard protocol). Histopathological assessment Appendix  1 depicts the details of the standardized histopathological assessment. Such assessment was intended to quantify the degree of the initial ablative injury of the epithelial surface and the submucosal structures (depth of ablation), the preservation of the muscularis propria and adventitia (risk of perforation) as well as the subsequent reparation to integrity (risk of stricture formation). RESULTS In vitro (bench) studies Several factors affected accurate temperature acquisition, such as the controller's motor speed, thermocouple accuracy, thickness of agar (0.5–1.5 mm) covering the thermocouples, consistency of the agar, the embedding of the thermocouples into the agar and the presence of air bubbles between thick and thin agar layers. As shown in Figure 3, the 90°CbSAS at sprayer speeds of 0.8 mm/second achieved at least a temperature of −40 °C at a depth of 0.5 mm. Sprayer speeds of 0.7 mm/second and below achieved at least a temperature of −40 °C at a depth of 1.0 mm. A temperature of −40 °C was not reached at a depth of 1.5 mm (data not shown). Porcine model Figure 4 displays representative images of the histologic effects of CbSAS at doses of 0.5 and 0.8 mm/second applied over a 3 cm length. There were no significant differences between the 2 doses. On day 0 (3 hours), there was full thickness necrosis of the epithelium with separation at epithelial–submucosal margin, indicative of tissue weakening at this junction. The epithelial cells revealed cytoplasmic change and condensed nuclei, and there was scant fibrin and neutrophilic infiltration, as well as submucosal edema, neutrophilic infiltration, and congestion of the smooth muscle layers, and mild to moderate edema of the adventitia. On day 4, there were multifocal areas of mucosal ulceration lined by fibrin, degenerated neutrophils and mucus. The submucosa was edematous with infiltration by acute and chronic inflammatory cells. The submucosal vessels exhibited congestion and vascular necrosis while the muscle layers exhibited neutrophilic infiltration and necrosis. The adventitia was expanded by edema, acute, and chronic inflammation and fibrin exudation. On day 28, the treated areas were characterized by intact and well-differentiated epithelium, minimal lymphocytic and plasma cell infiltration, and fibrosis of the submucosa and the inner and outer muscle layers with lesser involvement of the adventitia. Overall, the gross and histological findings showed progressive changes of injury and subsequent healing, from initial necrosis and epithelial–submucosal separation and acute inflammation, to mucosal edema and ulceration, to re-epithelialization and regional fibrosis involving the submucosa, muscularis propria, and adventitia. Table 1 presents the data from the animal study in which the 90°CbSAS was used at doses of 0.5 to 0.8 mm/second in 3 animals euthanized at 0, 4, and 28 days, respectively. Fig. 4 View largeDownload slide Representative images of the histologic effects of CbSAS at a dose of 0.5 mm/second in the swine model. (A; Day 0). Full thickness epithelial necrosis with separation at epithelial–submucosal margin, indicative of tissue weakening at this junction. (B; Day 4). Maximal effect of cryoablation exhibiting injured sloughed epithelium resulting in areas of ulceration associated with moderate edema and moderate to large numbers of infiltrating neutrophils. (C; Day 28). Re-epithelialization of the treatment site and the predominant histologic finding was a regional area of fibrosis involving the submucosa, muscularis propria and adventitia. The overlying epithelial integrity was restored. Fig. 4 View largeDownload slide Representative images of the histologic effects of CbSAS at a dose of 0.5 mm/second in the swine model. (A; Day 0). Full thickness epithelial necrosis with separation at epithelial–submucosal margin, indicative of tissue weakening at this junction. (B; Day 4). Maximal effect of cryoablation exhibiting injured sloughed epithelium resulting in areas of ulceration associated with moderate edema and moderate to large numbers of infiltrating neutrophils. (C; Day 28). Re-epithelialization of the treatment site and the predominant histologic finding was a regional area of fibrosis involving the submucosa, muscularis propria and adventitia. The overlying epithelial integrity was restored. Table 1 Quantitative assessment of histopathology of swine esophagus at 0, 4, and 28 days after CbSAS at doses of 0.5 mm/second and 0.8 mm/second, showing mucosal and submucosal injury with preservation of muscularis propria and adventitia at all time points. At the doses tested there was ablation of the mucosa and partly the submucosa with preservation of the muscularis propria and adventitia with restoration to integrity at 28 days. There were no significant differences between the 2 doses used   Day 0  Day 4  Day 28  Dose  0.5 mm/second  0.8 mm/second  0.5 mm/second  0.8 mm/second  0.5 mm/second  0.8 mm/second  Squamous epithelium  0.5  0.5  3  3  0  0  Submucosa  1  2  3  2  1  1  Muscularis propria  2  2  3  2  4  4  Adventitia  1  1  3  2  0.5  1    Day 0  Day 4  Day 28  Dose  0.5 mm/second  0.8 mm/second  0.5 mm/second  0.8 mm/second  0.5 mm/second  0.8 mm/second  Squamous epithelium  0.5  0.5  3  3  0  0  Submucosa  1  2  3  2  1  1  Muscularis propria  2  2  3  2  4  4  Adventitia  1  1  3  2  0.5  1  View Large Table 1 Quantitative assessment of histopathology of swine esophagus at 0, 4, and 28 days after CbSAS at doses of 0.5 mm/second and 0.8 mm/second, showing mucosal and submucosal injury with preservation of muscularis propria and adventitia at all time points. At the doses tested there was ablation of the mucosa and partly the submucosa with preservation of the muscularis propria and adventitia with restoration to integrity at 28 days. There were no significant differences between the 2 doses used   Day 0  Day 4  Day 28  Dose  0.5 mm/second  0.8 mm/second  0.5 mm/second  0.8 mm/second  0.5 mm/second  0.8 mm/second  Squamous epithelium  0.5  0.5  3  3  0  0  Submucosa  1  2  3  2  1  1  Muscularis propria  2  2  3  2  4  4  Adventitia  1  1  3  2  0.5  1    Day 0  Day 4  Day 28  Dose  0.5 mm/second  0.8 mm/second  0.5 mm/second  0.8 mm/second  0.5 mm/second  0.8 mm/second  Squamous epithelium  0.5  0.5  3  3  0  0  Submucosa  1  2  3  2  1  1  Muscularis propria  2  2  3  2  4  4  Adventitia  1  1  3  2  0.5  1  View Large Pre-esophagectomy studies We studied 6 patients (5M:1F), all Caucasian, median age 69 (IQR 58–77), median BMI 26.3 (IQR 22.0–26.9). Four (67%) had history of GERD and BE. In the 2 patients with delayed esophagectomy (six and ten days post-ablation), pain scores were zero after ablation and remained at zero through follow-up in both patients. One patient had a dysphagia score of 1 at 24 hours and reported a score of zero at 7-day follow-up (of note is that this patient had pre-existing dysphagia prior to ablation). The second patient had no dysphagia postablation. Table 2 summarizes the histopathology of human esophagus prior to esophagectomy using a range of CbSAS doses (0.5–0.9 mm/second) and various time intervals postablation (0–10 days) revealing surface epithelial injury and preservation of the muscularis propria and adventitia. Based on collective experience with bench, animal and human data the 0.8 mm/second dose was ultimately deemed ideal for planned human Barrett's ablation trials. Table 2 Descriptive histopathology of human esophagus ablated prior to esophagectomy using a range of CbSAS doses (0.5 mm/second to 0.9 mm/second) and various time intervals post ablation (0–10 days) revealing surface epithelial injury and preservation of the muscularis propria and adventitia. Based on collective experience with bench, animal and human data the 0.8 mm/second dose was ultimately deemed ideal for planned human Barrett's ablation trials Study ID  Dose (mm/second)  Time (days)  Pathology findings (ISQ, inner squamous mucosa; SM, submucosa)  Grade  1  0.5  0  ISQ: Expansion of mature cells, pyknosis of epithelial nuclei  0.5        SM: Normal  0        Adventitia: Normal  0  2  0.6  0  ISQ: Expansion of mature cells, pyknosis of epithelial nuclei  0.5        SM: Mild separation of muscle fibers with inflammatory cell infiltration  2        Adventitia: Mild edematous thickening  3  3  0.8  0  ISQ: Necrosis of basal epithelial layer  3        SM: Moderate separation of muscle fibers with inflammation  2        Adventitia: Moderate thickening of adventitia with inflammatory cell infiltration  3  4  0.9  0  ISQ: Erosion of cells to level of basal layer  1        SM: Dilation with edema, necrosis of muscle fibers, inflammatory cell infiltration  1        Adventitia: Mild edematous thickening  1  5  0.5  5  ISQ: Necrosis of basal epithelial layer  3        SM: Dilation with edema, necrosis of muscle fibers, inflammatory cell infiltration  1        Adventitia: Moderate thickening with inflammatory cell infiltration  1  6  0.8  10  ISQ: Necrosis of basal epithelial layer  3        SM: Dilation with edema, necrosis of muscle fibers, inflammatory cell infiltration  1        Adventitia: Moderate thickening with inflammatory cell infiltration  1  Study ID  Dose (mm/second)  Time (days)  Pathology findings (ISQ, inner squamous mucosa; SM, submucosa)  Grade  1  0.5  0  ISQ: Expansion of mature cells, pyknosis of epithelial nuclei  0.5        SM: Normal  0        Adventitia: Normal  0  2  0.6  0  ISQ: Expansion of mature cells, pyknosis of epithelial nuclei  0.5        SM: Mild separation of muscle fibers with inflammatory cell infiltration  2        Adventitia: Mild edematous thickening  3  3  0.8  0  ISQ: Necrosis of basal epithelial layer  3        SM: Moderate separation of muscle fibers with inflammation  2        Adventitia: Moderate thickening of adventitia with inflammatory cell infiltration  3  4  0.9  0  ISQ: Erosion of cells to level of basal layer  1        SM: Dilation with edema, necrosis of muscle fibers, inflammatory cell infiltration  1        Adventitia: Mild edematous thickening  1  5  0.5  5  ISQ: Necrosis of basal epithelial layer  3        SM: Dilation with edema, necrosis of muscle fibers, inflammatory cell infiltration  1        Adventitia: Moderate thickening with inflammatory cell infiltration  1  6  0.8  10  ISQ: Necrosis of basal epithelial layer  3        SM: Dilation with edema, necrosis of muscle fibers, inflammatory cell infiltration  1        Adventitia: Moderate thickening with inflammatory cell infiltration  1  View Large Table 2 Descriptive histopathology of human esophagus ablated prior to esophagectomy using a range of CbSAS doses (0.5 mm/second to 0.9 mm/second) and various time intervals post ablation (0–10 days) revealing surface epithelial injury and preservation of the muscularis propria and adventitia. Based on collective experience with bench, animal and human data the 0.8 mm/second dose was ultimately deemed ideal for planned human Barrett's ablation trials Study ID  Dose (mm/second)  Time (days)  Pathology findings (ISQ, inner squamous mucosa; SM, submucosa)  Grade  1  0.5  0  ISQ: Expansion of mature cells, pyknosis of epithelial nuclei  0.5        SM: Normal  0        Adventitia: Normal  0  2  0.6  0  ISQ: Expansion of mature cells, pyknosis of epithelial nuclei  0.5        SM: Mild separation of muscle fibers with inflammatory cell infiltration  2        Adventitia: Mild edematous thickening  3  3  0.8  0  ISQ: Necrosis of basal epithelial layer  3        SM: Moderate separation of muscle fibers with inflammation  2        Adventitia: Moderate thickening of adventitia with inflammatory cell infiltration  3  4  0.9  0  ISQ: Erosion of cells to level of basal layer  1        SM: Dilation with edema, necrosis of muscle fibers, inflammatory cell infiltration  1        Adventitia: Mild edematous thickening  1  5  0.5  5  ISQ: Necrosis of basal epithelial layer  3        SM: Dilation with edema, necrosis of muscle fibers, inflammatory cell infiltration  1        Adventitia: Moderate thickening with inflammatory cell infiltration  1  6  0.8  10  ISQ: Necrosis of basal epithelial layer  3        SM: Dilation with edema, necrosis of muscle fibers, inflammatory cell infiltration  1        Adventitia: Moderate thickening with inflammatory cell infiltration  1  Study ID  Dose (mm/second)  Time (days)  Pathology findings (ISQ, inner squamous mucosa; SM, submucosa)  Grade  1  0.5  0  ISQ: Expansion of mature cells, pyknosis of epithelial nuclei  0.5        SM: Normal  0        Adventitia: Normal  0  2  0.6  0  ISQ: Expansion of mature cells, pyknosis of epithelial nuclei  0.5        SM: Mild separation of muscle fibers with inflammatory cell infiltration  2        Adventitia: Mild edematous thickening  3  3  0.8  0  ISQ: Necrosis of basal epithelial layer  3        SM: Moderate separation of muscle fibers with inflammation  2        Adventitia: Moderate thickening of adventitia with inflammatory cell infiltration  3  4  0.9  0  ISQ: Erosion of cells to level of basal layer  1        SM: Dilation with edema, necrosis of muscle fibers, inflammatory cell infiltration  1        Adventitia: Mild edematous thickening  1  5  0.5  5  ISQ: Necrosis of basal epithelial layer  3        SM: Dilation with edema, necrosis of muscle fibers, inflammatory cell infiltration  1        Adventitia: Moderate thickening with inflammatory cell infiltration  1  6  0.8  10  ISQ: Necrosis of basal epithelial layer  3        SM: Dilation with edema, necrosis of muscle fibers, inflammatory cell infiltration  1        Adventitia: Moderate thickening with inflammatory cell infiltration  1  View Large DISCUSSION The novel CbSAS device described herein allows controlled cooling and quarter-circumferential (90o) ablation of esophageal mucosa under direct endoscopic vision over a length of 3 cm. As such, it offers the advantage of larger surface ablation when compared to the focal device previously tested in vitro as well as in vivo in animals and patients with BE. Pressing a button on the handheld instrument results in simultaneous inflation and cooling of the balloon using nitrous oxide delivered from a small canister located inside the handle to the diffuser that moves vertically within the balloon at a rate corresponding to the dose. The relatively low pressure of nitrous oxide gas in the CbSAS is advantageous because it permits the use of a highly compliant balloon that can conform to the shape of the esophagus and can achieve mucosal contact over a range of diameters from 20 to 35 mm, even when the surface geometry is challenging. The CbSAS is designed to expand until contact with the esophagus is achieved, up to a maximal diameter of 35 mm. Once in contact with the esophagus, the balloon stops expanding and applies a constant low pressure of up to 0.4 atm until the ablation is complete; the balloon then rapidly deflates. By advancing or retracting the CbSAS the diffuser contained within the balloon is positioned over the targeted area for treatment. When the cryogen is applied, it covers a 90° section of the circumference of the esophagus in the treatment area for 3 cm length without the need for a venting tube. In this study, we found that the optimal dose, that is, the rate at which the diffuser traverses the axis of the balloon catheter while emitting cryogen, was 0.8 mm/second (Fig. 2). Cryoablation, or the destruction of tissue by freezing, is a multifactorial process. Primary cell necrosis caused by intracellular ice formation occurs between −15 and −40°C, with some necrosis occurring at −15°C, and complete necrosis achieved by −40°C. Further, secondary effects caused by vascular injury and induced cellular apoptosis occur to some degree in all tissue subjected to freezing. In the CbSAS device, the balloon temperature is −80°C, and a mucosal temperature exceeding −40°C is achieved with good contact between the inflated balloon and the esophageal surface, which is sufficient to cause immediate primary cell necrosis of the superficial mucosa. As the application time is lengthened, progressively deeper sections of the esophageal wall are exposed to lower temperatures, resulting in deeper ablation. Histologic studies that have measured the thickness of BE epithelium (0.4–0.6 mm), squamous epithelium (0.4–0.6 mm), and esophageal wall thickness (2.4 mm for normal esophagus, 3.1 mm nondysplastic BE, 3.4 mm BE with high-grade dysplasia) serve as a guide to the optimal depth of ablation. The application time must be long enough to cause necrosis of up to 0.6 mm of BE epithelial thickness (or perhaps slightly more in cases of high-grade dysplasia) while minimizing damage to the deeper portions of the esophageal wall.8 In a similar fashion to previous experiments, our animal testing of the CbSAS was conducted in Yorkshire swine, and the results are summarized in Figure 3. The device was positioned and ablation was carried out without difficulty in all animals and humans. There was no difficulty in maintaining the position of the balloon during ablation, and we did not observe any tendency of esophageal peristalsis to pull the balloon down during the procedure. As the ablation time increased the percentage of mucosa ablated increased, reaching a desired effect with an application time of 0.8 cm/second. Further design improvements could lead to a half- or full-circumferential surface freeze and possibly increase the percentage of mucosa that could be ablated or slightly reduce the required ablation time. In the porcine model 2–4 days after cryoablation, necrosis was observed in the deeper esophageal wall layers, however, only very little damage and no strictures were seen after 28 days. This was pleasantly surprising, since pigs are more prone to stricture formation than humans after endoscopic resection or ablation.9,10 Although not directly comparable, in the six human cases, the depth and severity of injury appeared to be significantly less than in the porcine model. Our panel of experimental studies has some inherent limitations. First, although the swine model is frequently used given its size and anatomical similarities to the human, the esophageal wall layers are generally thinner and the cryoablation effects may be overestimated compared to the human esophagus. Yet four days after ablation, at a timepoint at which the tissue response is at its most extensive, we encountered no serious perforative injury and there was full recovery at 28 days. Second, we have limited dosimetry data in the human, pre-esophagectomy model, reflecting the inherent recruitment difficulties of such patients. Third, CbSAS was performed mostly on squamous esophageal epithelium, since there are no preclinical models of BE available. Since BE is known to have thicker wall layers compared to the normal squamous esophagus and even has an overlying protective mucus gel layer, safety is not likely to be an issue when the technology is tested clinically in patients with dysplastic Barrett's esophagus when the dose (0.8 mm/second) was proven safe in squamous epithelium in vivo.10,11 Nevertheless, there are several significant remaining issues in further development of the CbSAS. With the current balloon design, ablation over the length of the balloon must be optimized and the overall circumferential effect increased to reduce the number of necessary applications for a long and circumferential segment of BE since it is desirable to achieve 100% ablation. This may require a second application or overlapping segments during stepwise ablation of extended areas of BE. Further studies will be needed to investigate these issues. Historically, focal cryoballoon eradication rates have been comparable with rates reported for RFA for the treatment of dysplastic BE (88–93%).12,13 However, cryoablation might be favorable over RFA because of the lack of capital equipment, and it therefore has the potential for widespread implementation into the community, especially in rural areas. Moreover, unlike heat-based technologies, cryoablation does not disrupt the extracellular collagen matrix, which theoretically might allow for deeper ablation without causing perforation or strictures.14 Endoscopic resection, by EMR or ESD, will remain the modality of choice for nodular dysplasia eradication and for staging. Cryoballoon and RFA play a role in the management of flat dysplasia and residual metaplasia after resection because of their simplicity and low likelihood for stricture formation when applied circumferentially. In conclusion, CbSAS enables uniform 3 cm long, quarter-circumferential mucosal ablation in a one-step process by using a novel, through-the-scope balloon. At a dose of 0.8 mm/second, the system delivers uniform and predictable ablation with mucosal and submucosal necrosis in bench, animal and human esophagus. Because of its ease of use, this new device merits further clinical study in the treatment of dysplastic Barrett's esophagus. Notes Grant Support: This study was funded by C2 Therapeutics, Redwood City, CA. Disclosures: GT is a consultant to C2 Therapeutics. BEL, WH and BLW have no conflicts to declare. Specific author contributions: Planning and/or conducting the study: Brian E. Louie, Wayne Hofstetter, George Triadafilopoulos, Bas L. Weusten; Collecting and/or interpreting data: Brian E. Louie, Wayne Hofstetter, George Triadafilopoulos, Bas L. Weusten; Drafting the manuscript and revision: Brian E. Louie, Wayne Hofstetter, George Triadafilopoulos, Bas L. Weusten. References 1. Spechler S J, Souza R F. Barrett's esophagus. N Engl J Med  2014; 371: 836– 45. Google Scholar CrossRef Search ADS PubMed  2. Shaheen N J, Falk G W, Iyer P G et al.   ACG Clinical Guideline: Diagnosis and Management of Barrett's Esophagus. Am J Gastroenterol  2016; 111: 30– 50. Google Scholar CrossRef Search ADS PubMed  3. Overwater A, Weusten BLAM. Cryoablation in the management of Barrett's esophagus. Curr Opin Gastroenterol  2017; 33: 261. Google Scholar CrossRef Search ADS PubMed  4. Gosain S, Mercer K, Twaddell W S et al.   Liquid nitrogen spray cryotherapy in Barrett's esophagus with high-grade dysplasia: long-term results. Gastrointest Endosc  2013; 78: 260– 5. Google Scholar CrossRef Search ADS PubMed  5. Schölvinck D W, Künzli H T, Kestens C et al.   Treatment of Barrett's esophagus with a novel focal cryoablation device: a safety and feasibility study. Endoscopy  2015; 47: 1106– 12. Google Scholar CrossRef Search ADS PubMed  6. Künzli H T, Schölvinck D W, Meijer S L, Seldenrijk K A, Bergman J G, Weusten B L. Efficacy of the CryoBalloon Focal Ablation System for the eradication of dysplastic Barrett's esophagus islands. Endoscopy  2017; 49: 169– 75. Google Scholar PubMed  7. Baust J G, Gage A A, Bjerklund Johansen T E, Baust J M. Mechanisms of cryoablation: clinical consequences on malignant tumors. Cryobiology  2014; 68: 1– 11. Google Scholar CrossRef Search ADS PubMed  8. Gill K R, Ghabril M S, Jamil L H et al.   Variation in Barrett's esophageal wall thickness: is it associated with histology or segment length? J Clin Gastroenterol  2010; 44: 411– 5. Google Scholar CrossRef Search ADS PubMed  9. Kamler J P, Borsatto R, Binmoeller K F. Circumferential endoscopic mucosal resection in the swine esophagus assisted by a cap attachment. Gastrointest Endosc  2002; 55: 923– 8. Google Scholar CrossRef Search ADS PubMed  10. Scholvinck D W, Alvarez Herrero L, Visser M, Bergman J J, Weusten B L. Effects of Lugol staining on stenosis formation induced by radiofrequency ablation of esophageal squamous epithelium: a study in a porcine model. Dis Esophagus  2015; 28: 603– 11. Google Scholar CrossRef Search ADS PubMed  11. Dixon J, Strugala V, Griffin S M et al.   Esophageal mucin: an adherent mucus gel barrier is absent in the normal esophagus but present in columnar-lined Barrett's esophagus. Am J Gastroenterol  2001; 96: 2575– 83. Google Scholar CrossRef Search ADS PubMed  12. Phoa K N, Pouw R E, van Vilsteren F G et al.   Remission of Barrett's esophagus with early neoplasia 5 years after radiofrequency ablation with endoscopic resection: a Netherlands cohort study. Gastroenterology  2013; 145: 96– 104. Google Scholar CrossRef Search ADS PubMed  13. Shaheen N J, Overholt B F, Sampliner R Eet al. Durability of radiofrequency ablation in Barrett's esophagus with dysplasia. Gastroenterology  2011; 141: 460– 8. Google Scholar CrossRef Search ADS PubMed  14. Weusten B L, Bergman J J. Cryoablation for managing Barrett's esophagus refractory to radiofrequency ablation? Don’t embrace the cold too soon!. Gastrointest Endosc  2015; 82: 449– 51. Google Scholar CrossRef Search ADS PubMed  APPENDIX 1 Grading scale (0–4) Area  Grading  Squamous mucosa  Normal  0    Cell expansion with nuclear pyknosis  0.5    Cell erosion  1    Cell erosion to basal layer  2    Necrosis of all basal layer  3  Submucosa  Normal  0    Inflammatory cell infiltration  1    Separation with inflammation  2    Edema and necrosis  3  Muscularis propria  Normal  0    Edema and inflammatory infiltration  1    Focal necrosis and inflammation  2    Fiber separation with inflammation  3    Fibrosis  4  Adventitia  Normal  0    Edema  0.5    Inflammatory cell infiltration  1    Necrosis and inflammation  2    Thickening with necrosis and inflammation  3  Area  Grading  Squamous mucosa  Normal  0    Cell expansion with nuclear pyknosis  0.5    Cell erosion  1    Cell erosion to basal layer  2    Necrosis of all basal layer  3  Submucosa  Normal  0    Inflammatory cell infiltration  1    Separation with inflammation  2    Edema and necrosis  3  Muscularis propria  Normal  0    Edema and inflammatory infiltration  1    Focal necrosis and inflammation  2    Fiber separation with inflammation  3    Fibrosis  4  Adventitia  Normal  0    Edema  0.5    Inflammatory cell infiltration  1    Necrosis and inflammation  2    Thickening with necrosis and inflammation  3  Area  Grading  Squamous mucosa  Normal  0    Cell expansion with nuclear pyknosis  0.5    Cell erosion  1    Cell erosion to basal layer  2    Necrosis of all basal layer  3  Submucosa  Normal  0    Inflammatory cell infiltration  1    Separation with inflammation  2    Edema and necrosis  3  Muscularis propria  Normal  0    Edema and inflammatory infiltration  1    Focal necrosis and inflammation  2    Fiber separation with inflammation  3    Fibrosis  4  Adventitia  Normal  0    Edema  0.5    Inflammatory cell infiltration  1    Necrosis and inflammation  2    Thickening with necrosis and inflammation  3  Area  Grading  Squamous mucosa  Normal  0    Cell expansion with nuclear pyknosis  0.5    Cell erosion  1    Cell erosion to basal layer  2    Necrosis of all basal layer  3  Submucosa  Normal  0    Inflammatory cell infiltration  1    Separation with inflammation  2    Edema and necrosis  3  Muscularis propria  Normal  0    Edema and inflammatory infiltration  1    Focal necrosis and inflammation  2    Fiber separation with inflammation  3    Fibrosis  4  Adventitia  Normal  0    Edema  0.5    Inflammatory cell infiltration  1    Necrosis and inflammation  2    Thickening with necrosis and inflammation  3  © The Author(s) 2018. Published by Oxford University Press on behalf of International Society for Diseases of the Esophagus. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

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Diseases of the EsophagusOxford University Press

Published: Feb 21, 2018

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