**Does Circular Stapler Size in Surgical Management of Esophageal Cancer A**ff**ect Anastomotic Leak Rate? 4-Year Experience of a European High-Volume Center**

### **Dolores T. Müller, Benjamin Babic, Veronika Herbst, Florian Gebauer, Hans Schlößer, Lars Schi**ff**mann, Seung-Hun Chon, Wolfgang Schröder, Christiane J. Bruns and Hans F Fuchs \***

Department of General, Visceral, Cancer and Transplant Surgery, University of Cologne, Kerpener Str. 62, D-50937 Cologne, Germany; dolores.mueller@uk-koeln.de (D.T.M.); benjamin.babic@uk-koeln.de (B.B.); veronika.herbst3@gmail.com (V.H.); florian.gebauer@uk-koeln.de (F.G.); hans.schloesser@uk-koeln.de (H.S.); lars.schiffmann@uk-koeln.de (L.S.); seung-hun.chon@uk-koeln.de (S.-H.C.); wolfgang.schroeder@uk-koeln.de (W.S.); christiane.bruns@uk-koeln.de (C.J.B.)

**\*** Correspondence: hans.fuchs@uk-koeln.de

Received: 28 October 2020; Accepted: 19 November 2020; Published: 22 November 2020

**Simple Summary:** One of the most severe postoperative complications after a transthoracic esophagectomy for esophageal cancer is a leakage of the anastomosis created between the remnant esophagus and the stomach. There is substantial debate on which surgical technique and which stapler are the best. The aim of this study was to retrospectively analyze whether the stapler diameter had an impact on postoperative anastomotic leak rates during a 4-year time frame from 2016 to 2020. A total of 632 patients (open, hybrid, and totally minimally invasive esophagectomy) met the inclusion criteria. A total of 214 patients underwent an anastomosis with a 25 mm stapler vs. 418 patients with a 28 mm stapler. Anastomotic leak rates were 15.4% vs. 10.8%, respectively. Stapler size should be chosen according to the individual anatomical situation of the patient and may be of higher relevance in patients undergoing totally minimally invasive reconstruction.

**Abstract:** Anastomotic leak is one of the most severe postoperative complications and is therefore considered a benchmark for the quality of surgery for esophageal cancer. There is substantial debate on which anastomotic technique is the best for patients undergoing Ivor Lewis esophagectomy. Our standardized technique is a circular stapled anastomosis with either a 25 or 28 mm anvil. The aim of this study was to retrospectively analyze whether the stapler diameter had an impact on postoperative anastomotic leak rates during a 4-year time frame from 2016 to 2020. A total of 632 patients (open, hybrid, and totally minimally invasive esophagectomy) met the inclusion criteria. A total of 214 patients underwent an anastomosis with a 25 mm stapler vs. 418 patients with a 28 mm stapler. Anastomotic leak rates were 15.4% vs. 10.8%, respectively (*p* = 0.0925). Stapler size should be chosen according to the individual anatomical situation of the patient. Stapler size may be of higher relevance in patients undergoing totally minimally invasive reconstruction.

**Keywords:** esophagectomy; esophageal anastomosis; minimally invasive surgery

#### **1. Introduction**

Due to its increasing incidence, a curative treatment of esophageal carcinoma has gained more importance than ever in recent years. For locally advanced but resectable carcinomas, a transthoracic esophagectomy with reconstruction using a gastric conduit and a high intrathoracic anastomosis (Ivor Lewis esophagectomy) depicts the current curative treatment of choice, mostly in a multimodal setting [1]. Despite improvements of perioperative care and surgical technique, this surgical procedure is

still related to specific risks, such as anastomotic leak, conduit necrosis, chylothorax, and recurrent nerve injury. In particular, anastomotic leak, as one of the most severe and early postoperative complications, is considered a benchmark for the quality of the esophagectomy and is known to increase postoperative mortality and morbidity, leading to a decreased long-term survival [2–4]. Many surgical factors, including procedure type, localization of the anastomosis, and operative technique, are known to affect the integrity and quality of the anastomosis, and there is substantial debate on which anastomotic technique is the best for patients undergoing Ivor Lewis esophagectomy [5,6]. Our standardized technique and the most common technique in minimally invasive surgery is a circular stapled end-to-side anastomosis with purse string using either a 25 or 28 mm anvil. The current literature has shown this technique to be safe and efficient, leading to a comparatively low anastomotic leak rate of 14% in the EsoBench database [4]. The aim of this study was to retrospectively analyze whether the stapler diameter had an impact on postoperative anastomotic leak rates according to Esophagectomy Complications Consensus Group (ECCG) criteria during a 4-year time frame from 2016 to 2020 at our certified center of excellence for surgery of upper gastrointestinal cancer.

#### **2. Results**

A total of 632 patients met the inclusion criteria. In 214 patients (34%), a 25 mm circular stapler was used for the construction of a transthoracic esophagogastric anastomosis, and in 418 patients (66%), a size of 28 mm. For further analysis of results, the patients were grouped according to stapler size (25 mm—small; 28 mm—large). Demographic and oncological data of both patient cohorts are shown in Table 1. In addition, a statistical comparison of the baseline characteristics of both groups was performed, and the *p*-values are shown.


**Table 1.** Demographic characteristics and oncological data of the patients undergoing an Ivor Lewis esophagectomy for esophageal cancer with either a 25 or 28 mm circular stapler. The *p*-values for statistical comparison of the baseline characteristics of both groups were calculated.

BMI: body mass index; CROSS and FLOT are well defined neoadjuvant treatments.

Furthermore, the operative approach was analyzed for both groups and compared with each other. Figure 1 shows the distribution of open, hybrid, and totally minimally invasive procedures among patient cohorts.

**Figure 1.** (**a**) Distribution of surgical approach in the 25 mm stapler patient group. Percentages of patients undergoing an Ivor Lewis esophagectomy using an open, hybrid, or totally minimally invasive approach are shown. (**b**) Distribution of surgical approach in the 28 mm stapler patient group. Percentages of patients undergoing an Ivor Lewis esophagectomy using an open, hybrid, or totally minimally invasive approach are shown.

While an open approach was used equally often in both cohorts (*p* = 0.6809), a hybrid approach was more often performed in the 28 mm group (*p* < 0.0001), compared with a totally minimally invasive approach, which was more frequently performed in the 25 mm group (*p* < 0.0001). In line with these findings, a two-stage procedure was more often used in the 28 mm stapler size group (6.5% vs. 11.5%; *p* = 0.0490).

A total of 72 patients in the given time frame were operated on using an either completely robotic or hybrid robotic approach. Thirty-one (14.5%) of the patients in the 25 mm stapler group were operated on using a robotic technique compared with 41 (9.8%) in the 28 mm stapler group. No statistically significant difference was shown for the utilization of a robotic technique between both groups (*p* = 0.0863).

A total of 114 patients in the given time frame were operated on using a totally minimally invasive approach (laparoscopic or robotic gastrolysis/thoracoscopic or robotic esophagectomy). Sixty-three (29.4%) of the patients in the 25 mm stapler group were operated on using a totally minimally invasive technique, compared with 51 (12.2%) in the 28 mm stapler group. A totally minimally invasive technique was significantly more often used in the smaller stapler group (*p* < 0.0001).

#### *Postoperative Complications*

Table 2 shows the severity of postoperative complications classified according to Clavien–Dindo (CD) of our patient groups. In addition, *p*-values were obtained to analyze whether statistically significant differences between postoperative outcomes of both cohorts were present.

The median length of stay (LOS) was 15 days in both groups with a range of 9–112 days (standard deviation (SD) 12) in the small stapler group and a range of 7–99 days (SD 11) in the large stapler group with no statistically significant difference between the groups (*p* = 0.3993).

An anastomotic leak was detected in a total of 78 patients (12.3%), 33 in the small stapler group and 45 in the large stapler group. Figure 2 depicts the anastomotic leak rates among patient cohorts. No statistical significance was noted between the groups (*p* = 0.09878); however, a trend approaching statistical significance shows that anastomotic leaks were more frequent in the small stapler size group.

**Table 2.** Severity of postoperative complications among patient cohorts. The Clavien–Dindo classification was used to objectify the severity of postoperative complications among both patient cohorts. *p*-Values were calculated to analyze whether statistically significant difference between both stapler sizes was present. In addition, further analysis of patients with severe postoperative complications, here classified as CD ≥ IIIa, was performed.


**Figure 2.** Anastomotic leak rates for the small stapler size (25 mm), the large stapler size (28 mm), and the overall cohort shown as percentages.

Further details on demographic information, preoperative comorbidities, and risk factors of patients who developed an anastomotic leak are shown in Table 3.


**Table 3.** Demographic information, comorbidities, and risk factors of patients who developed an anastomotic leak. Data are shown for both subgroups (25 mm and 28 mm circular staplers), and percentages of patients from the respective subgroups were calculated.

\* Information was given voluntarily; therefore, not all patients answered this question. COPD: chronic obstructive pulmonary disease, FEV1: forced expiratory pressure.

In addition, independent predictors of anastomotic leak were identified by a multivariate logistic regression analysis adjusting for stapler size, operative technique, gender, type of cancer, neoadjuvant therapy, tobacco, alcohol consumption, BMI, and cardiac comorbidities. Besides operative approach (open, hybrid, totally minimally invasive) and gender, no other independent predictors for anastomotic leak were found (*p* > 0.05). With totally minimally invasive approach set as a reference, both other approaches had a significantly higher leak rate when adjusted for all factors mentioned above (*p* < 0.05).

Table 4 shows further details about the distribution of the type of anastomotic leaks as well as the severity of complications using the Clavien–Dindo classification among patients that developed an anastomotic leak. The anastomotic leak rate for a Type II leak was 11.2% in the 25 mm group vs. 8.4% in the 28 mm group and 4.2% vs. 2.2% for Type III leaks, respectively. In addition, the percentage of patients with a certain CD class from the total of anastomotic leaks among the respective stapler sizes is shown. No patient with an anastomotic leak was classified as CD I or II. To investigate how many patients with an anastomotic leak developed organ failure, we included a subgroup analysis of patients classified as CD ≥ IV.


**Table 4.** Anastomotic leak types and severity of postoperative complications among patients that developed an anastomotic leak. Stapler sizes of 25 and 28 mm were analyzed separately, and *p*-values for statistical comparison of both groups were calculated. Percentages were calculated as percentage from the cohort that developed an anastomotic leak.

#### **3. Discussion**

Anastomotic leakage is among the most feared complications in surgery due to its consequences, especially in esophageal cancer surgery. Many technical variations in performing the esophagogastric anastomosis are still used without expert consensus. Our institution has contributed significantly in the past to find innovative and new ways using minimally invasive technology to treat these complications with an interventional approach and mostly without redo surgery, leading to more ECCG Type II anastomotic leaks in recent years [7,8]. Schröder et al. have published in their recent multicenter analysis of high-volume centers from 2011 to 2016 a leakage rate of 13.9% for an intrathoracic circular stapled anastomosis. Our own data from 2016 to 2020 in this present study show an overall leakage rate of 12.3%, meeting the benchmarks in Schröder's and Schmidt's studies [2,4]. When looking into more detail as presented above, we were able to show that in addition to the technology and technique used, even the stapler size may play an essential role in developing an anastomotic leak. In this context, it is important to note that anatomical reasons may play an essential role when choosing the stapler size, and sometimes both options are technically not possible.

Interestingly, we were able to show that a 28 mm stapler was overall significantly more often used at our institution. This result may be biased by the fact that more hybrid than totally minimally invasive procedures were performed in this collective, as the 25 mm stapler was the more commonly chosen technology in the totally minimally invasive subgroup. Also, patients with squamous cell carcinoma more often underwent a 25 mm anastomosis, a fact that can be attributed to the usually higher mediastinal location of the tumors and anatomical reasons not to perform a 28 mm stapled anastomosis.

Few previous studies have focused on the technical factor of the stapler diameter itself, but more often evaluated general technical options, such as circular, linear, and handsewn (technical factors), and anatomical options, such as intrathoracic vs. cervical (anatomical factors) [4,5]. Whereas Markar et al. published a meta-analysis in 2013 showing no significant differences among the technical factors, they were able to show significant differences and an almost fivefold increased leakage rate for cervical vs. intrathoracic anastomosis. In contrast, Schröder's analysis of the EsoBenchmark database showed no difference among the anatomical factors. Even if the difference in leakage rate in our patient collective (10.8% vs. 15.4%) is in favor of the 28 mm stapler group, this technical factor was only nearing statistical significance (*p* = 0.0925).

In our analysis of complications, focusing on the patients with an anastomotic leak (Table 4), we looked at the severity of the leaks according to the ECCG group [9]. No significant difference was found between the two analyzed stapler groups. Nevertheless, there was again a trend of less severe leaks in favor of the 28 mm staplers. No clear differences could be shown for the Clavien–Dindo score between the groups.

In our study, we found a relatively high number of CD ≥ IIIa complications, namely, 51.4% (28 mm) and 55.6% (25 mm). This exceeds the benchmarks set by Schmidt et al. with 30.8%, defined as the "best possible outcome" [2]. Truly, our collective does not comprise a selection of patients with low comorbidities, and esophagectomy was performed both by experts and by trainees under expert supervision at our institution, meaning that our results represent an unbiased, unselected analysis of a prospective cohort. In addition, complications at our institution are thoroughly recorded according to ECCG guidelines, meaning that postoperative interventions such as chest tube placement or postoperative EGD are automatically classified as a IIIa complication. As postoperative endoscopic interventions are considered a "standard of care" in some other centers, these might not be classified in the same way everywhere.

#### **4. Materials and Methods**

#### *4.1. Patients*

Our academic center is a certified center of excellence for surgery of the upper gastrointestinal tract with more than 250 upper gastrointestinal cancer surgeries being performed annually. All patients undergoing esophagectomy for esophageal cancer in our high-volume center are entered into an IRB-approved prospective database. A retrospective chart review was performed for all patients undergoing an Ivor Lewis esophagectomy for esophageal cancer from May 2016 to May 2020. Patients were included in the analysis if a 25 or 28 mm circular stapler was used for the esophagogastric transthoracic anastomosis. An intraoperative subjective assessment of the patient's anatomy was used to choose the appropriate stapler size. Patients with handsewn anastomoses or other stapler diameters were excluded from the analysis. Retrospective analysis of our prospectively collected data was conducted with approval from the ethical committee at the University of Cologne (IRB reference 13-091). Demographics, endoscopic findings, and biopsies at different follow-up time points, as well as tumor histology and stage, were recorded in our prospective database.

#### *4.2. Assessment of Postoperative Complications*

The Clavien–Dindo classification was used to classify the severity of postoperative complications [10]. In addition, our institution contributes to the well-established database of the Esophagectomy Complications Consensus Group (ECCG), which provides a standardized and international assessment of complications following esophagectomy [9]. Therefore, definitions established by the ECCG are used at our clinic to ensure precise documentation. An anastomotic leak was defined as a "full thickness GI defect involving esophagus, anastomosis, staple line, or conduit irrespective of presentation or method of identification." Further subgrouping into three types was applied with Type I being a local defect requiring no change in therapy or being treated medically or with dietary modification, Type II being a localized defect requiring interventional but not surgical therapy, and Type III being a localized defect requiring surgical therapy. Length of hospital stay was calculated in days from the day of the surgical procedure to discharge of the patient.

#### *4.3. Treatment Pathway of Patients with Resectable Esophageal Cancer*

Treatment of patients with esophageal cancer at our National Center of Excellence follows a standardized protocol in line with national and international guidelines [1,11–13]. Following restaging, usually 4–6 weeks after neoadjuvant therapy, either a standardized Ivor Lewis esophagectomy with reconstruction using a gastric conduit and a high thoracic esophagogastric anastomosis is performed at

our institution, or if suitable, patients with an adenocarcinoma of the gastroesophageal junction Siewert Type II are enrolled into the CARDIA trial, which aims to compare the oncological and surgical outcome after transthoracic esophagectomy and transhiatal extended gastrectomy [14,15]. For a transthoracic esophagectomy, a hybrid procedure (abdominal part—laparoscopically/thoracic part—open) depicts the current standard at our institution. Whenever possible, however, dependent on the patient's anatomy and whether the patient is classified as low risk, a totally minimally invasive approach is chosen (abdominal part—laparoscopically/thoracic part—thoracoscopically). Our thorough risk assessment preoperatively includes a standardized and validated risk scoring system [16]. In addition, the DaVinci Xi robotic surgical system (Intuitive Surgical, Inc., Sunnyvale, CA, USA) is available at our clinic since February 2017. A robotic approach is often especially used for the thoracic part, as the great advantage of the system becomes evident during the thoracic dissection [17]. Furthermore, complete robotic and minimally invasive Ivor Lewis esophagectomies are increasingly performed at our institution.

#### *4.4. Surgical Technique—Abdominal Part*

The following steps for preparation of the gastric conduit are performed in a standardized fashion using either a robotic or a laparoscopic approach. Our standardized steps of the operation are the same for the robotic and the laparoscopic procedure: The patient is placed in a French and anti-Trendelenburg position. For the robotic approach, an 8 mm DaVinci trocar is inserted through a supraumbilical median incision using the open technique, and a pneumoperitoneum is established. Four additional trocars are then placed, one 5 mm trocar on the right and one 12 mm trocar on the left edge of the costal arch, a 12 mm trocar in the right upper abdomen and an 8 mm trocar in the left upper abdomen depicting the standard for a minimally invasive robotic DaVinci gastrolysis. If performed laparoscopically, one 5 mm and four 11 mm abdominal ports are used. A 45-degree angled scope (5 mm Stryker indocyanine green (ICG) or robotic 8 mm Intuitive ICG) is inserted through the subxiphoidal trocar. The hiatus is then exposed by elevating the liver with a Cuschieri retractor through the right 5 mm trocar. From here, the peritoneum on the right diaphragmatic crus is incised, and the lower mediastinum outside the hernia sac is dissected and circumferentially mobilized up to the left diaphragmatic crus to dissect the lower esophagus. Opening the right and the left pleura is avoided at any time during the hiatal dissection. Dissection of the lymph nodes along the lesser curvature of the stomach onto the stomach wall follows. The upper margin of the retroperitoneal pancreas is now exposed and can be inspected. A D2 lymphadenectomy following the hepatic ligament, the common hepatic artery, along the celiac trunk continuing along the splenic artery and of the retroperitoneum is performed. The left gastric artery and the left gastric vein are ligated, clipped, and divided. The right gastric artery is preserved. Subsequently, lymph nodes along the retroperitoneum via the crus of the diaphragm up to the lower mediastinum are mobilized, and the lymphadenectomy is completed above the splenic artery all the way up to the hilum of the spleen. Opening the gastrocolic omentum access to the omental bursa is gained, and the greater curvature of the stomach is mobilized starting from the corpus region beyond the epiploic vessels toward the left crus of the diaphragm, while the gastroepiploic arcade is preserved, and the short gastric vessels are divided until visualization of the left diaphragmatic crus is achieved. To later create an omentum wrap covering the anastomosis, a part of the greater omentum just below the spleen is preserved. Mobilization is completed by separating the colon all the way until the splenic flexure, confirming sufficient blood supply for the greater curvature. Dissection at the gastric crow's foot region is performed followed by the construction of the gastric conduit. A tristapler (Endo Gia (Covidien), violet, 45 mm) is applied for the first bite of the construction of the gastric sleeve. Using at least two additional Endo Gia 60 mm violet stapling magazines, construction of the gastric conduit is completed. Intraoperative angiography using indocyanine green (ICG) can, in combination with the robotic DaVinci Xi system of the laparoscopic Stryker system, demonstrate sufficient blood supply of the fundus by showing the gastroepiploic vessels via fluorescence.

#### *4.5. Surgical Technique—Thoracic Part*

The following steps for completion of the esophagectomy and reconstruction of the gastrointestinal passage using a gastric conduit are performed in a standardized fashion using either a minimally invasive thoracoscopic, robotic, or open approach: The patient is placed in a left lateral semiprone position for a robotic procedure or in a left lateral decubitus position for an open procedure. Using a double-lumen intubation, artificial atelectasis of the right lung is achieved. Our standardized steps of the operation are the same for the robotic or the open procedure. For a robotic approach, three DaVinci ports and two assistance ports are placed on the right according to the standard, and the robot is docked from the patient's right side, creating a view from the left for the operating surgeon. A right-sided transthoracic approach is used for an open procedure. Using the robotic monopolar cautery hook, the pulmonary ligament is dissected with the lymph nodes adhering to the esophagus upward toward the pericardial layer and the azygos vein. Using a tristapler (Endo Gia (Covidien), gold, 45 mm), the azygos arch is divided. The thoracic duct is identified and clipped with two polymer clips (Grena Click'aV®). Dissection of the periesophageal fat tissue along the aorta dividing small aortic branches and along the pericardium is performed. Especially when using the robotic technique, a radical but controlled dissection of the carinal, retrotracheal, and paratracheal tissue can be performed. Vagal and recurrent nerves are preserved during this step. Opening the hiatus, a connection to the abdominal surgical field is made. A monofilament purse string suture is performed, and the gastric conduit is pulled into the right thoracic cavity. If a minimally invasive approach is chosen, a minithoracotomy of 7 cm length is then created from the incision of the 12 mm upper assistance trocar, and an Alexis S wound protector/retractor (Alexis Laparoscopic System, Applied Medical) is inserted.

#### *4.6. Surgical Technique—The Esophagogastric Anastomosis*

Either a 25 or 28 mm stapler head depending on the patient's anatomy is inserted and guided into the esophagus. The prepared purse string suture is used to suture the stapler head into the esophageal remnant. If necessary, a second purse string suture may be placed. Figure 3 shows the setup for the creation of the esophagogastric anastomosis. The gastric conduit is then gently pulled upward into the chest. We always ensure that the fundus lies alongside with the esophageal stump without tension, which further proofs a sufficient length of the conduit. Intraoperative angiography using ICG can be again used with the robotic system to demonstrate sufficient blood supply of the graft. If a robotic approach is chosen, the DaVinci is then disconnected, and the assistant surgeon holds the camera similar to a thoracoscopic approach. Using a variable number of loads of the Endo GIA stapler (Covidien), preparation of the gastric conduit is completed. The specimen is removed and preserved for histopathologic evaluation. A 25 or 28 mm stapler is inserted through the minor curvature of the stomach, and an esophagogastric anastomosis is made, retrieving two complete donuts. Another Endo GIA stapler load is used to staple off the open end of the stomach. The previously prepared omentum wrap is then used to cover the anastomosis. In addition, final control of blood perfusion using ICG fluorescence can be used.

#### *4.7. Data Analysis and Statistical Evaluation*

For analysis of data, patients were divided into two groups based on CS (circular stapler) size ("small" = 25 mm circular stapler and "large" = 28 mm circular stapler). In addition, a subgroup analysis of patients who underwent a totally minimally invasive and a robotic esophagectomy was performed. Continuous variables are presented as means and range. Categorical data are presented as numbers and percentages. Student's *t*-test (for continuous variables) and Fisher's exact test (for nominal or categorical variables) were used for all bivariate analyses. Independent predictors of anastomotic leak were identified by a multivariate logistic regression analysis. All tests were two-sided, with statistical significance set at *p* ≤ 0.05. Data were analyzed by GraphPad Software (San Diego, CA, USA) and SPSS Statistics for Mac (version 21, SPSS).

(**a**) (**b**)

**Figure 3.** Robotic-assisted minimally invasive esophagectomy. The top left picture (**a**) shows a minithoracotomy of 7 cm length created from the incision of the 12 mm upper assistance trocar and secured with an Alexis S wound protector/retractor. The top right picture (**b**) shows intraoperative angiography using indocyanine green (ICG). The bottom pictures display how the prepared purse string suture is used to suture the stapler head into the esophageal remnant (**c**,**d**).

#### **5. Conclusions**

This large single-center analysis clearly defines anastomotic leak rates of a standardized, unselected consecutive patient cohort in a high-volume center. We highly recommend that stapler size be always chosen according to the individual anatomical situation of the patient, but when in doubt, we suggest choosing the larger diameter. This suggestion may be of even higher relevance to patients undergoing minimally invasive thoracic reconstruction.

**Author Contributions:** Conceptualization, D.T.M., B.B., W.S., C.J.B., H.F.F.; methodology, D.T.M., H.F.F.; formal analysis, D.T.M., H.F.F., V.H.; data curation, V.H., D.T.M.; writing—original draft preparation, D.T.M., H.F.F.; writing—review and editing, D.T.M., B.B., F.G., H.S., L.S., S.-H.C., W.S., C.J.B., H.F.F.; supervision, H.F.F., C.J.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


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### *Article* **Outcomes of Radiotherapy for Mesenchymal and Non-Mesenchymal Subtypes of Gastric Cancer**

**Jeong Il Yu 1, Hee Chul Park 1,2,\*, Jeeyun Lee 3,\*, Changhoon Choi 1, Won Ki Kang 3, Se Hoon Park 3, Seung Tae Kim 3, Tae Sung Sohn 4, Jun Ho Lee 4, Ji Yeong An 4, Min Gew Choi 4, Jae Moon Bae 4, Kyoung-Mee Kim 5, Heewon Han 6, Kyunga Kim 6, Sung Kim <sup>7</sup> and Do Hoon Lim <sup>1</sup>**


Received: 25 March 2020; Accepted: 9 April 2020; Published: 10 April 2020

**Abstract:** *Background:* The purpose of this study was to evaluate the clinical outcomes following postoperative chemotherapy (XP) versus chemoradiotherapy (XP-RT) according to mesenchymal subtype based on RNA sequencing in gastric cancer (GC) in a cohort of the Adjuvant chemoRadioTherapy In Stomach Tumor (ARTIST) trial. Methods: Of the 458 patients enrolled in the ARTIST trial, formalin-fixed, paraffin-embedded (FFPE) specimens were available from 106 (23.1%) patients for RNA analysis. The mesenchymal subtype was classified according to a previously reported 71-gene MSS/EMT signature using the NanoString assay. Results: Of the 106 patients analyzed (50 in XP arm, 56 in XP-RT arm), 36 (34.0%) patients were categorized as mesenchymal subtype by NanoString assay. Recurrence-free survival (RFS, p = 0.009, hazard ratio (HR) = 2.11, 95% confidence interval (CI): 1.21–3.70) and overall survival (OS, p = 0.003, HR = 2.28, 95% CI: 1.31–3.96) were significantly lower in the mesenchymal subtype than in the non-mesenchymal subtype. In terms of post-operative radiotherapy (RT), mesenchymal subtype was not an independent variable to predict RFS or OS regardless to the assigned arm (XP with or without RT) in this patient cohort. However, there was a trend in the adjuvant XP arm, which showed higher OS than the XP-RT arm for the mesenchymal subtype and lower OS than the XP-RT arm for the non-mesenchymal subtype. *Conclusions:* We could not determine any significant differences between the mesenchymal and non-mesenchymal subtypes with respect to the effects of adjuvant XP with or without RT in gastric cancer following curative surgery.

**Keywords:** adjuvant therapy; gastrointestinal tract; genetic diagnosis; radiosensitivity

#### **1. Introduction**

Gastric cancer (GC) remains an unresolved major health problem in the world, ranking fifth among the most common cancers, with an estimated incidence of 951,000 cases in 2012 [1]. Furthermore, it is the third leading cause of cancer-related deaths, with 723,100 patients dying because of it in 2012. Nevertheless, a gradual improvement in GC management has been achieved, based on pathophysiological studies, novel surgical techniques, and/or emerging systemic therapies.

Despite significant improvements in clinical outcomes related to the implementation of screenings, advances in surgical techniques and/or usage of (neo-)adjuvant chemotherapy and/or concurrent chemoradiotherapy, a considerable proportion of patients still experience recurrences and die of GC [2–4].

With the recent development of genetic analysis techniques, the molecular classification of GC and the prognostic value of the different subtypes have been actively studied. The Cancer Genome Atlas Research Network suggested four types of GC molecular classes [5], whereas the Asian Cancer Research Group (ACRG) proposes another classification [6]. In particular, the ACRG suggested prognostic differences according to each molecular subtype and validated the survival differences in an independent cohort. According to this study, the mesenchymal (microsatellite-stable with epithelial-to-mesenchymal transition phenotype, MSS/EMT) tumors showed the worst prognosis with younger age at diagnosis, and higher recurrence rate with first presentation of peritoneal seeding. In addition to these clinical features of the mesenchymal subtype, the association of hypoxia and the increase in poly [adenosine diphosphate-ribose] polymerase-1 (PARP-1), which repairs deoxyribonucleic acid (DNA) damage, has been reported [7–9].

Recently, our group performed a study to predict outcomes in patients with the mesenchymal subtype using a NanoString assay in 70 ACRG specimens [10]. The mesenchymal subtype showed significantly worse survival compared to the non-mesenchymal subtype following curative surgery in GC [11–13]. The impact of mesenchymal subtype which could be clearly related with radioresistance, like hypoxia and increment of PARP-1, in terms of radiotherapy (RT) efficacy has not been defined yet although several studies have demonstrated that mesenchymal subtype predicts poor outcome following chemotherapy in GC [11–13]. It would be appropriate to evaluate the efficacy of RT on mesenchymal subtype of GC through the ARTIST trial which had been conducted to compare postoperative chemotherapy (XP) versus chemoradiotherapy (XP-RT) following complete curative resection with D2 lymph node dissection in GC (clinical trials.gov identifier NCT00323830).

In this study, we investigated the clinical outcomes according to the application of adjuvant XP-RT or XP for mesenchymal and non-mesenchymal subtype cancers in a cohort of the ARTIST trial.

#### **2. Methods**

#### *2.1. Patients and Samples*

This study was performed on patients who participated in the ARTIST trial who agreed to have their tissue studied, and whose tissue surgical specimens were available and sufficient for ribonucleic acid (RNA) extraction. A total of 458 patients (228 assigned to XP and 230 to XP-RT arm), who had received curative D2 resection without preoperative treatment, were enrolled in the randomized phase III study, ARTIST trial. The XP arm was expected to receive six cycles of XP regimen (capecitabine 1000 mg/m2 twice daily on days 1 to 14; cisplatin 60 mg/m2 on day 1 every 3 weeks) and the XP-RT arm would receive 25 fractions of 45 gray (Gy) RT with capecitabine (825 mg/m<sup>2</sup> twice daily) after two cycles of XP (as in the XP arm) followed by two additional cycles of planned XP. The planned treatment was completed in 75.4% of patients in the XP arm and 81.7% in the XP-RT arm [11]. Other details of the ARTIST trial, including chemotherapy and RT protocols, were described in previous reports [8–10]. From the 458 patients enrolled, 106 tissue samples of 359 patients except 99 patients with stage I were available for evaluation in the present study. Of these, 56 patients were in the XP-RT arm and the remaining 50 patients were in the XP arm (Figure 1).

**Figure 1.** Flow diagram of patient inclusion.

#### *2.2. Mesenchymal Gene Signature*

Details about the development of the 71-gene MSS/EMT signature (consisting of 60 upregulated and 11 downregulated genes) using the NanoString assay, and the validation procedure using the conventional Affymetrix method, are described in a previous report [10]. In that study, 73 samples from the ARTIST cohort were tested using the 71-gene MSS/EMT signature to validate their mesenchymal subtype. Twenty out of 73 samples were classified as mesenchymal subtype tumors, which is equivalent to the MSS/EMT subtype in terms of their dismal outcome, typical characteristics of whole stomach involvement, poorly-differentiated or signet ring cell carcinoma, and low microsatellite instability.

In this study, we used the outcomes of previous study evaluating mesenchymal and non-mesenchymal subtype using MSS/EMT gene signature analysis using the NanoString assay in a total of 106 samples, which consisted of 73 samples used as a validation set and 33 additional specimens from patients of the ARTIST trial evaluated through further work after publication of the previous study.

The first site of recurrence was used for the classification and/or analysis of recurrence. Simultaneous recurrence was defined as any recurrence detected within 2 weeks after the first detection of recurrence. Loco-regional recurrence (LRR) was defined as recurrence at one of following sites: anastomosis area, remnant stomach, tumor bed, duodenal stump, or regional lymph nodes (LN) within the RT field in the XP-RT group or the hypothetical RT field in the XP group. All cases of suspected LRR were reviewed and evaluated by dedicated radiation oncologists (JIY and DHL; specialists in gastrointestinal tumors, including stomach cancer) as described in a previous study [13].

### *2.3. Ethical Approval and Informed Consent Statement*

The authors stated that all methods of this study were carried out in accordance with the Declaration of Helsinki, and the protocol for the present study was reviewed and approved by the Samsung Medical Center Institutional Review Board (IRB No. 2010-12-088) and all participants in the ARTIST trial consented to this study after being informed about the purpose and investigational nature.

#### *2.4. Statistical Analysis*

Baseline characteristics were compared between the mesenchymal and non-mesenchymal subtypes, using chi-square or Fisher's exact test for categorical variables and Student's t-test or Mann–Whitney U-test for continuous variables, as appropriate. For each survival-related event, such as LRR, recurrence, or death, survival times were calculated from the date of surgery to the date of event detection, or the date of the last follow-up visit. LRR-free survival (LRRFS), recurrence-free survival (RFS) and

overall survival (OS) curves were estimated and compared between the XP and XP-RT groups, both for mesenchymal and non-mesenchymal subtypes, with the adjustment for stage, operation type and classification of Lauren based on the multivariate Cox proportional hazards model. Statistical analysis was performed using SAS software version 9.4 (SAS Institute Inc., Cary, NC, USA) and *p* < 0.05 was considered statistically significant.

#### **3. Results**

#### *3.1. Patients*

Among the 458 patients who participated in the ARTIST trial, a total of 106 (23.1%) formalin-fixed, paraffin-embedded (FFPE) samples that were available for targeted profiling by the NanoString nCounter assay were evaluated and analyzed in the present study. The baseline characteristics of patients enrolled in the present study and all patients in the ARTIST trial are shown in Table S1. Patients enrolled in this study had significantly more metastatic lymph nodes, and lymphovascular invasion (LVI) and perineural invasion (PNI) were more common than those of all the patients in the ARTIST trial. Clinical-Trials.gov identifier: NCT0176146. Trial Registration: clinical trials.gov identifier NCT00323830 (date of registration: May 10, 2006).

Among the 106 patients enrolled in this study, 56 were assigned to the XP-RT group and the remaining 50 patients were assigned to the XP group. When testing molecular subtypes using the developed MSS/EMT signature, 36 out of 106 patients were classified as having the mesenchymal subtype.

Table 1 displays the detailed characteristics of the enrolled patients having mesenchymal or non-mesenchymal subtypes of GC. In the mesenchymal subtype group, the ratio of diffuse-type GC, classified according to Lauren, was significantly higher than that of the non-mesenchymal subtype group (88.9% versus 60.0%, *p* = 0.004). Additionally, the proportion of patients who received total gastrectomy was higher in the mesenchymal subtype group (55.6% versus 40.0%, *p* = 0.128), although no statistical significance was found. No differences in the percentage of patients receiving XP or XP-RT as adjuvant treatment, nor significant differences in the pathologic staging, were found between the two subtypes.


**Table 1.** Baseline characteristics of patients.


**Table 1.** *Cont.*

Abbreviations: LN: lymph node, AJCC: American Joint Committee on Cancer, LVI: lymphovascular invasion, PNI: perineural invasion, XP: capecitabine and cisplatin, XP-RT: XP-radiation therapy.

Baseline characteristics of patients randomly assigned to the XP-RT and XP arms of the ARTIST trial are displayed in Table S2. Age was slightly but significantly lower in the XP-RT arm than in the XP arm.

#### *3.2. Patterns of Recurrence*

During follow up (median: 43.6 months, range: 1.8–72.0 months), recurrence was identified in 50 patients (47.2%). Among them, 20 (57.1%) recurrences had developed in the mesenchymal subtype group and the remaining 30 (42.3%) recurrences occurred in the non-mesenchymal subtype group. Median time to any recurrence was 12.9 months (range: 3.8–75.1 months) in the mesenchymal subtype group and 18.0 months (range: 4.5–81.3 months) in the non-mesenchymal subtype group. LRR developed in 20 out of 50 patients, and seven of them were classified as mesenchymal subtype. Median time to LRR was 26.9 months (range: 3.8–81.3 months) in the mesenchymal subtype group and 23.7 months (range: 1.77–154.9 months) in the non-mesenchymal subtype group. Detailed patterns of recurrence according to subtype and adjuvant treatment are shown in Table S3. Peritoneal seeding was the major recurrence pattern in the XP-RT arm, especially in the mesenchymal subtype, and LRR was more frequent in the XP arm. Fifty-one deaths were registered during this period (30 in the nonmesenchymal and 21 in the mesenchymal subtype group).

#### *3.3. Prognostic Factors and Survival Outcomes*

Table 2 shows the univariate analysis outcomes of LRRFS, RFS and OS according to the variables, including mesenchymal versus non-mesenchymal subtypes. LRRFS was not different for the mesenchymal and non-mesenchymal subtypes (*p* = 0.275); PNI was the only significant prognostic factor for LRRFS (*p* = 0.044, hazard ratio (HR) = 3.57, 95% confidence interval (CI): 1.03–12.20). RFS was significantly lower in the mesenchymal subtype group (*p* = 0.009, HR = 2.11, 95% CI: 1.21–3.70). Other significant prognostic factors for RFS were type IV of the macroscopic type (*p* < 0.001), total gastrectomy (*p* = 0.007), stage IV (*p* < 0.001), PNI (*p* = 0.014), and Lauren classification other than diffuse-type (*p* = 0.028). The RFS was not different according to the adjuvant treatment of XP or XP-RT (*p* = 0.500). OS was also significantly lower in the mesenchymal subtype group (*p* = 0.003, HR = 2.28, 95% CI: 1.31–3.96). Other significant prognostic factors for OS were: type IV of the macroscopic type (*p* < 0.001), PNI (*p* = 0.034), total gastrectomy (*p* = 0.002), and stage IV (*p* < 0.001). Adjuvant treatment of XP or XP-RT did not affect OS.


**Table 2.** Univariate analysis of loco-regional recurrence-free survival (LRRFS), recurrence-free survival (RFS), and overall survival

PNI: perineural invasion, TG: total gastrectomy, STG: subtotal gastrectomy, XP: capecitabine and cisplatin, XP-RT: XP-radiation

 therapy.

#### *3.4. Survival Outcomes for Mesenchymal and Non-Mesenchymal Subtypes*

To evaluate the different roles of adjuvant XP-RT or XP between mesenchymal and non-mesenchymal subtypes, multivariate analysis in both mesenchymal and non-mesenchymal subtype was performed including the following factors, which were identified as significant factors not only in the present study but also in many other studies: stage, type of operation, and Lauren classification. Table 3 shows the outcomes of multivariate analyses of RFS and OS for mesenchymal and non-mesenchymal subtypes. Additional results of multivariate analyses of RFS and OS for mesenchymal and non-mesenchymal subtypes including another well-known prognostic factor of PNI are presented in Table S4.

**Table 3.** Multivariate analysis of recurrence-free survival (RFS), and overall survival (OS) in mesenchymal and non-mesenchymal subtypes.


Abbreviations: RFS: recurrence-free survival, OS: overall survival, HR: hazard ratio, CI: confidence interval, TG: total gastrectomy, STG: subtotal gastrectomy, XP: capecitabine and cisplatin, XP-RT: XP-radiation therapy.

In the multivariate analysis of RFS for the mesenchymal subtype, stage and total gastrectomy were significant prognostic factors, compared to the non-mesenchymal subtype, which was significantly affected by stage, and type of Lauren classification. As displayed in Figure 2A,B, adjusted RFS was not significantly different according to the adjuvant treatment of XP or XP-RT, either for the mesenchymal or non-mesenchymal subtype. Forest plots were used to represent adjusted RFS with HR and 95% CI for the mesenchymal and non-mesenchymal subtypes (Figure 3).

No significant prognostic factors were found in the multivariate analysis of OS for the mesenchymal subtype. On the contrary, stage, total gastrectomy, and type of Lauren classification were significant prognostic factors of OS for the non-mesenchymal subtype. OS was not significantly different according to the adjuvant treatment of XP or XP-RT, either for the mesenchymal or non-mesenchymal subtype. As displayed in Figure 2C,D, however, there was a minor difference in OS between mesenchymal and non-mesenchymal subtypes according to the adjuvant treatment group. The adjuvant XP group showed slightly higher adjusted OS than the XP-RT group for the mesenchymal subtype, and lower adjusted OS than the XP-RT group for the non-mesenchymal subtype. Forest plots were used to represent adjusted OS with HR and 95% CI for the mesenchymal and non-mesenchymal subtypes (Figure 3).

**Figure 2.** Adjusted curve of recurrence-free survival (RFS, **A**,**B**) and overall survival (OS, **C**,**D**) according to the use of adjuvant XP-RT or XP for the mesenchymal (**B**,**D**) and non-mesenchymal subtypes (**A**,**C**). In these curves which were displayed for the average value of the covariates in the study population, no significant differences related to the use of adjuvant XP-RT or XP were detected, although OS curves were inverted depending on adjuvant modality for the mesenchymal and non-mesenchymal subtypes (The presented p-values were based on HR test in the Cox proportional hazards model.).

**Figure 3.** Forest plot for adjusted RFS and OS. No significant differences in RFS related to the use of adjuvant XP-RT or XP for the mesenchymal and non-mesenchymal subtypes were detected (OP, operation; TG, total gastrectomy; STG, subtotal gastrectomy; O, others, M, mesenchymal subtype; N, non-mesenchymal subtype)**.**

#### **4. Discussion**

In the present study we evaluated the clinical outcomes according to the adjuvant treatment (XP-RT or XP) for mesenchymal and non-mesenchymal subtypes of GC in a cohort of the ARTIST trial. The mesenchymal subtype showed worse prognostic factors, such as frequent recurrence and lower OS, than the non-mesenchymal subtype, as shown in other reports. However, we could not find any differences between the mesenchymal and non-mesenchymal subtypes in terms of OS as well as RFS.

Beyond the previous histologic and/or anatomic classification of cancer [14–16], there is a growing body of research and evidence supporting the use of molecular analysis for precise classification and tailored management [5,6,17,18]. Although many advances have been made in the management of GC, it still has a poor prognosis in advanced tumors, becoming one of the areas where molecular classification is being actively attempted. Our group, which is the third referral institution of Korea, proposed four subtypes of molecular classification linked to recurrence patterns and prognosis, as well as to distinct patterns of genomic alterations based on ACRG data which have been validated in institutional cohorts [6].

Among the four subtypes of our molecular classification, the mesenchymal-like type, which showed loss of expression of the *CDH1* which encodes the protein E-cadherin. It is known that loss of E-cadherin function decreases the power of cell-to-cell adhesion and increases the cellular motility [19]. It is suggested that the efficacy of local treatment including RT could be limited, because of high metastatic potential originated from these characteristics of the mesenchymal-like type. Actually, it is associated with a younger age of occurrence and is diagnosed as diffuse-type at stage III/IV, showed a significantly higher recurrence rate, higher probability of developing peritoneal seeding at the first site of recurrence, and extremely poor survival compared to other subtypes. Furthermore, it has been reported that PARP-1, which is involved in the mechanism of radiation induced DNA damage repair, is increased in the mesenchymal phenotype, and it could reduce the cell killing effects of RT in prostate cancer [7,8]. In addition, hypoxia, which is a hallmark of tumor and the most important cause of radioresistance, was reported to be associated with the loss of E-cadherin [9].

Our group also proposed the classification of certain GC as mesenchymal type by means of a targeted NanoString gene expression profile [10]. This unique subtype classification may positively impact the standard management of GC, promoting modifications on current treatment protocols which would improve their clinical outcomes.

RT is one of the main therapeutic modalities in the oncology fields. Though adjuvant RT combined with chemotherapy showed survival advantages over surgery alone in the Intergroup trial 0116 [20,21], there is still controversy as to the real efficacy of RT as an adjuvant modality in complete D2-resection GC [22]. The ARTIST trial was a randomized phase III trial designed to evaluate the advantages of adjuvant XP-RT over adjuvant XP after complete D2-resection in GC [11–13]. Superiority of adjuvant XP-RT over XP was not detected in these patients, except for those with LN metastasis.

Although the optimal indications of adjuvant XP-RT remain controversial, RT is still one of the most valuable and important treatment modalities in the management of GC, especially in the neoadjuvant or palliative setting. Furthermore, there is a rapid development of RT technology [23], also accompanied by an increased understanding of radiation biology [24,25]. In this respect, evaluating and comparing the effects of RT on GC of the mesenchymal subtype is of paramount importance, considering its heterogeneity and difficult therapeutic management.

The mesenchymal subtype is known to be closely related to younger age of occurrence and diffuse type of the Lauren classification, showing a worse prognosis and higher recurrence rate compared with other subtypes [6,17,18]. Those characteristics of the mesenchymal subtype were observed in the present study as well. Our group has continued to conduct studies to screen out which GC patients might benefit from adjuvant XP-RT over XP [4,11–13,22]. We found and reported that the benefit of adjuvant XP-RT over XP in GC is reduced to patients with diffuse-type GC and younger age, which correspond to characteristics of the mesenchymal type [12].

The effect of adjuvant XP-RT in mesenchymal subtype of GC in the present study was not significantly different than that of non-mesenchymal subtype in terms of RFS, in contrast to the possibility that the effect of RT might be reduced when considering the characteristics of this subtype. On the other hand, the adjuvant XP-RT group showed a lower OS curve than the XP group for the mesenchymal subtype, although without statistical significance. Although we failed to detect significant difference of RFS between the subtypes according to the adjuvant XP-RT and XP, this marginal difference of OS might be related to the different patterns of recurrence between the subtypes, especially LRR and/or peritoneal seeding. Therefore, the possibility of poorer outcomes after adjuvant XP-RT than after XP for the mesenchymal subtype with complete D2-resection cannot be ruled out. Further research on this issue is needed.

The present study has some important limitations. First, this study was evaluated in a fraction of the participants in the ARTIST trial, mainly because of availability of tissue specimens. Therefore, it is not possible to avoid selection bias in the XP-RT and XP groups, which is minimized by the random allocation nature of phase III. Second, the patients enrolled in this study have characteristics that differ from those of all patients in the ARTIST trial. Third, there could be problems originating from variation in outcomes due to ethnic differences, since the ARTIST trial was conducted at a single Korean institution. Further similar studies are essential for validation in other ethnicities in order to generalize present outcomes, because it is well-known that the characteristics and/or clinical outcomes of gastric cancer are quite distinct according to ethnicity [26–28].

#### **5. Conclusions**

We could not determine any significant differences on the effect of adjuvant XP-RT on RFS between mesenchymal and non-mesenchymal subtypes in this cohort of the ARTIST trial. There was a minor difference, however, in the adjuvant XP-RT group showing lower OS than the XP group for the mesenchymal subtype and higher OS for the non-mesenchymal subtype.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6694/12/4/943/s1, Table S1. Baseline characteristics of enrolled patients of present study and all patients of ARTIST trial; Table S2. Baseline characteristics of patients according to adjuvant treatment; Table S3. Detailed patterns of recurrence according to subtype and adjuvant treatment; Table S4. Multivariate analysis of recurrence-free survival (RFS), and overall survival (OS) in mesenchymal and non-mesenchymal subtypes including perineural invasion.

**Author Contributions:** Conceptualization and design: J.I.Y., H.C.P., and J.L.; Development of methodology: J.I.Y., H.C.P., J.L., C.C., K.-M.K., D.H.L., H.H., and K.K.; Acquisition of data: J.I.Y., H.C.P., J.L., C.C., W.K.K., S.H.P., S.T.K., S.K., T.S.S., J.H.L., J.Y.A., M.G.C., J.M.B., K.-M.K., and D.H.L.; Analysis and interpretation of data: J.I.Y., H.C.P., J.L., K.-M.K., H.H., and K.K.; Writing, review, and/or revision of the manuscript: all authors. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was partly supported by a Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2017R1D1A1B03031275).

**Conflicts of Interest:** The authors declare no conflict of interest.

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