The Risk of Lymph Node Metastasis in Early Gastric Cancer Conforming to Indications of Endoscopic Resection and Pylorus-Preserving Gastrectomy: A Single-Center Retrospective Study

Background: Lymph node metastasis (LNM) status is an important prognostic factor that strongly inuences the treatment decision of early gastric cancer (EGC). This study aimed to evaluate the pattern and clinical signicance of LNM in EGC. Methods: Patients with EGC who underwent radical gastrectomy were enrolled. Their clinicopathological features, pathological reports, and prognostic data were collected and analyzed. Results: Three hundred fty-four patients with EGC were enrolled. The incidence of LNM in patients with EGC was 18.36% (65/354). The rates of D1 and D2 station metastases were 12.1% (43/354) and 6.214% (22/354), respectively. The rates of LNM in absolute indication of endoscopic resection and expanded indication were 3.27% (2/61) and 28.55% (4/14), respectively. Skip LNM was observed in 3.67% (13/354) of patients. For those with middle-third EGC, the metastasis rate of the No. 5 lymph node was 3.05% (5/164). The independent risk factors for LNM were tumors measuring >30 mm, poorly differentiated tumors, and lymphovascular invasion (all P < 0.05 (cid:0) area under the curve = 0.783). Five-year disease-free survival rates of patients with EGC with and without LNM were 96.26% and 79.17%, respectively (P = 0.011). Tumors measuring >20 mm and LNM were independent predictive factors for poor survival outcome in patients with EGC. Conclusions: Patients with EGC conforming to expanded indications have a relatively high risk of LNM and may not be suitable for endoscopic submucosal dissection. Pylorus-preserving gastrectomy for patients with middle-third EGC remains controversial due to the high metastasis rate of the No. 5 lymph node. cancer, LNM:lymph node metastasis, LVI:lymphovascular invasion, EMR:endoscopic mucosal resection, ESD:endoscopic submucosal dissection, PPG:pylorus-preserving gastrectomy, CA:cancer antigen, CEA:carcinoembryonic antigen, HR:hazard ratio, CI:condence interval, OR:odds ratio, ER:endoscopic resection, RR:risk ratio, ROC:receiver operating characteristic, AUC:area under the curve, CT:computed tomography

treatments for patients with EGC with indications. In addition to ER treatment, some modi ed surgical procedures such as pylorus-preserving gastrectomy (PPG), segmental gastrectomy, and local resection can be considered for EGC with a low risk of LNM and are not suitable for EMR/ESD to improve quality of life.
LNM status is an important prognostic factor of EGC [4][5][6]. According to the 2018 Japanese GC treatment guidelines [3], the choice of ER for EGC treatment is mainly dependent on the risk of LNM. LNM in EGC within absolute indications for EMR or ESD has been hypothesized to be negligible (less than 1%).
Currently, ESD is widely used as a standard method for EGC in Japan, and its indications are expanded.
However, several problems remain. First, although most evidence suggests that the risk of LNM in patients with absolute indications is negligible, the results between these studies have been inconsistent [3]. Second, the expanded indications for ESD are still controversial. Third, the management of cases with noncurative resection after ER has been controversial. Fourth, whether ER treatment is superior or not inferior to traditional D2 lymphadenectomy with gastrectomy in terms of long-term outcomes is still unclear.
Furthermore, skip LNM in GC refers to the presence of extraperi-gastric lymph node involvement without peri-gastric LNM [7]. Some studies have revealed that skip metastasis occurs frequently in cases of GC [7][8][9][10]. The existence of skip LNM may seriously in uence the treatment decision of EGC. Therefore, a better understanding of the precise pattern of LNM in patients with EGC is important.
There have been relatively few studies on the positive rate of each lymph node station and skip metastasis in EGC [7][8][9][10][11]. In the present study, we aimed to elucidate the precise distribution of LNM in EGC by analyzing the metastasis status of each lymph node station in patients with EGC who underwent D2 lymphadenectomy with gastrectomy and to explore the clinical signi cance of LNM pattern and skip metastasis in making treatment decisions for EGC.

Patient cohort and data collection
The clinicopathological data of patients (n = 2,245) who underwent radical gastrectomy at the First A liated Hospital of Sun Yat-Sen University (January 2010 to December 2018) were retrospectively analyzed. All clinicopathologic data, including age, sex, tumor location, tumor size, histology classi cation, lymphovascular invasion (LVI), depth of tumor invasion, and LNM were drawn from hospital and pathological records. This study was approved by the Ethics Committee of the First A liated Hospital of Sun Yat-sen University and conducted in accordance with the principles of the Declaration of Helsinki. The need for informed consent for participation and for approval of all patients was waived.
Three hundred fty-four cases histologically proven to be EGC following the inclusion and exclusion criteria were enrolled (Fig. 1). All patients with EGC were divided into the LNM+ group (n = 65, LNM+: the presence of LNM) or LNM-group (n = 289, LNM-: the absence of LNM). To analyze the LNM rate for the patients with EGC selected by the indications of ESD/EMR, all patients were also divided into four different groups according the absolute and expanded indications of ESD/EMR (Table 1). For submucosal invasive (T1b) EGC, the LNM status was analyzed according to two conditions (≤ 2 cm, differentiated type; ≤ 2 cm, undifferentiated type) ( Table 1).

Follow-up
All patients included in this study were regularly followed up using a standardized protocol. Follow-up assessment included abdominal ultrasonography, computed tomography (CT) imaging (of the chest, abdomen and pelvis), and tumor marker tests (including cancer antigen [CA]-19-9, carcinoembryonic antigen [CEA], CA125, squamous cell carcinoma) at each visit. Deaths due to cancer were recorded as events, and deaths secondary to other causes were censored.

Statistical analysis
All statistical analyses were conducted using the SPSS 24.0 statistical package (IBM Inc., New York) and R (https://www.r-project.org/). Continuous variables are described as the mean and standard deviation, and an analysis of variance test was used to compare continuous variables. For categorical variables, Pearson's chi-square test or Fisher's exact test was used to compare the differences between patient groups. Univariate and multivariate logistic regression models, in which all covariates were adjusted simultaneously, was used to determine independent risk factors for LNM in EGC. Kaplan-Meier curves were plotted to evaluate survival outcomes in patients with EGC, and comparisons of prognostic differences between patient groups were performed using the log-rank test. Independent prognostic factors were identi ed by multivariate analysis using the Cox proportional-hazard model with a stepwise selection procedure. Hazard ratios (HRs) and 95% con dence intervals (CIs) were calculated to quantify the relationship between survival outcome and each clinicopathologic factor. Statistical signi cance was accepted at a P value < 0.05.

Results
The metastasis status of different lymph node groups in patients with EGC A total of 2,245 patients with GC who underwent radical gastrectomy with lymphadenectomy at the First A liated Hospital of Sun Yat-Sen University between January 1, 2010 and December 31, 2018 were reviewed retrospectively. Three hundred fty-four cases histologically proven to be EGC following the inclusion and exclusion criteria were enrolled for the next analysis. Among these 354 cases, there were 27 cases of upper-third EGC, 136 of middle-third EGC, and 142 of low-third EGC.
In this study, the incidence of LNM in EGC was 18.36% (65/354). In order to elucidate the role of LNM in EGC further, we analyzed the positive rate (Table 2) and location distribution (Table 3) of LNM for each lymph node station. As shown in Table 2 Clinicopathological characteristics of patients with EGC according to LNM As shown in Table 4, the current study consisted of 224 male patients (63.27%) and 130 female patients (36.72%), with a median age of 57.50 ± 11.399 years (range, 24-85 years). Among these 354 patients, 35  Table 4). There was no signi cant difference in the mean age of patients between the two groups, but there was a signi cant difference between patients aged <40 years and those aged ≥40 years (P=0.006), suggesting that young patients have a higher risk of LNM (risk ratio [RR] = 2.297; 95% CI, 1.333-3.947). Tumor sizes were signi cantly larger for LNM+ than LNM− (P = 0.009). Compared with LNM−, tumor invasion was deeper (P < 0.001; RR = 2.256; 95% CI, 1.447-3.518) and showed poor differentiation (P < 0.001; RR = 3.328; 95% CI, 1.914-5.787) in LNM+. However, the distribution of other variables including sex, body mass index, tumor maker, and tumor location were similar between the LNM− and LNM+ groups.

Correlation factors analysis of the extent of LNM in EGC
The rates of D1 station metastasis and D2 station metastasis in patients with EGC were 12.1% (43/354) and 6.214% (22/354), respectively (Table 6). An analysis of the clinical pathological characteristics was performed on patients with D1 station or D2 station LNM. There was no signi cant difference between the occurrence of D2 station LNM and the age, sex, tumor size, differentiation, location, depth of tumor invasion, and LVI. The levels of CA 19-9 and CEA were signi cantly different between the two groups (10.113 vs. 30.125 U/mL, P = 0.001 and 3.189 vs. 6.861 U/mL, respectively; P = 0.003). However, the difference in CA 125 was not signi cant ( Table 6).

Analysis of the clinicopathological characteristics of patients with EGC with skip metastasis
According to the Japanese classi cation of gastric carcinoma (3rd edition) [12] and the de nition of skip metastasis, patients with LNM (n = 65) were classi ed into a no skip metastasis group (n = 52) or a skip metastasis group (n = 13). The possibility of skip metastasis was 3.67% (13/354) in all patients with EGC. There was no signi cant difference between the two groups with respect to clinicopathological characteristics (Table 7).

Univariate and multivariate analyses of prognostic factors in patients with EGC
The 5-year survival rates of EGC between the LNM− and LNM+ groups were 96.26% and 79.17%, respectively (P = 0.011) ( Table 4). The prognostic outcome of patients who were LNM+ was worse than that of LNM-patients (P = 0.008) (Fig. 4). The results of the univariate and multivariate analyses for prognostic factors are listed in Table 8. Tumor size (HR, 3.473; 95% CI, 1.372-8.791; P = 0.009) and LNM (HR, 4.895; 95% CI, 1.588-15.095; P = 0.006) were independent predictive factors for poor survival outcome in patients with EGC.

LNM rate in patients with EGC selected by the indications of ESD/EMR
The 2018 Japanese GC treatment guidelines [3] revealed that the indication for ER depends on the depth of invasion, differentiation type, diameter, and ulcerative ndings. The LNM rates of these factors are demonstrated in Table 4. All patients with EGC (n = 354) were analyzed according to the absolute and expanded indications of ESD/EMR (Table 1), and only 75 (21.18%) patients conformed to the absolute and expanded indications of ESD/EMR. The rates of LNM in absolute and expanded indications were 2/61 (3.27%) and 4/14 (28.55%), respectively. Subgroup analysis showed that the rates of LNM with respect to the absolute indication of EMR/ESD and absolute indication of ESD 2 group were 0%. The rate of LNM with respect to the absolute indication of the ESD 1 group was 20%. For the submucosal invasive (T1b) EGC, the LNM status was analyzed with two conditions (≤2 cm, differentiated type: 7.40%; ≤2 cm, undifferentiated type: 35.375%), which was consistent with the outcome of the multivariable logistic analysis (Table 5).

Discussion
EGC was rst de ned in 1962 by the Japanese Research Society for Gastric Cancer as tumors with invasion limited to the mucosa or submucosa of the stomach, irrespective of lymph node involvement [2]. In the 8th American Joint Committee on Cancer TNM staging system, EGC corresponds to GC with Tis, T1a (mucosa), and T1b (submucosa) stages [13]. Recently, the treatment techniques and strategies for EGC have been updated rapidly. According to the latest Japanese GC treatment guidelines (5th version) [3], EMR or ESD is considered as a standard treatment for patients with EGC with absolute indications and an alternative treatment for EGC with expanded indications. With the development and prevalence of ER (ESD and EMR), the criteria for the indications of ER for EGC have continually expanded. However, there is debate as to whether ER can be used in patients with expanded indications.
Although the 2018 Japanese GC treatment guidelines declared that the possibility of harboring LNM in the tumor with absolute indication is less than 1%. However, most of the data referred to in the guidelines were from Japan [3]. It is still unclear whether the data can be extrapolated to other countries. Here, we revealed that the incidence of LNM in patients with EGC, which con rmed the absolute or expanded indications of ESD/EMR, was obviously higher than that in the Japanese cohort [2,[14][15][16]. The rates of LNM in the absolute indication of the ESD 1 group (20%) and expanded indication group (28.55%) were obviously higher than 1%. In additional, a meta review [2] in 2018 indicated that the incidence rate of LNM was 2.6% (25/972) in patients who met the expanded criteria. Moreover, a Korean study [17] in 2020 reported that LNMs were found in 6.7% (18/270) of patients with undifferentiated-type EGC who underwent additional surgery after non-curative endoscopic resection. Therefore, caution should be exercised before applying ESD to patients with undifferentiated-type adenocarcinoma and those with tumors bigger than 2 cm despite T1a and differentiated-type adenocarcinoma without ulcerative ndings. Further studies are urgently needed to nd new methods to distinguish populations with high risk of LNM from EGC conforming to the indications of ER.
It is worth mentioning that the sample size of this study was small. We screened for desirable cases from 2,245 cases of gastric cancer. However, only 15.77% of patients with GC were diagnosed with EGC in our center. The data were consistent with the results of other centers in China. The proportion of cases of EGC in China varies from 10-20%, compared to about 50% in Japan [18,19]. Moreover, only 21.18% of patients with EGC conformed to the absolute and expanded indications of ESD/EMR in this study. Therefore, more data are needed to draw a rm conclusion.
The Japanese Gastric Cancer Association guidelines suggested a gastrectomy procedure with D1/D1 + lymph node dissection as the standard surgical procedure for cases in which the depth of invasion is clinically diagnosed as T1b without LNM and T1a without LNM, which are not suitable for EMR and ESD. However, our data showed that the rate of D2 LNM in EGC was 6.214%. For these cases, the D1 or D1 + dissection is not su cient. Furthermore, skip LNM is another factor in uencing the determination of the extent of lymph node dissection. Skip metastasis in GC refers to the presence of extra-perigastric LNM without peri-gastric lymph node involvement [10]. There have been few studies on the phenomenon of jump metastasis and its related mechanism in patients with GC, especially those with EGC [7][8][9][10][11]20]. The incidence of skip metastasis in patients with LNM in EGC has been reported to range from 2.7-21.6% [9,20,21]. In this study, the incidence of skip metastasis in patients with LNM was 3.67% (13/354), which is consistent with prior research results. Liu et al. [8] revealed that tumor size was the only clinicopathologic factor that could predict lymph node skip metastasis in patients with N1 stage cancer (the number of metastatic lymph nodes among the regional lymph nodes is 1-2) undergoing radical surgery. However, no signi cant related clinical characteristics were found for skip metastasis in our study. Considering the relatively high incidences of D2 LNM and skip LNM in EGC, it is not suitable for these patients to receive D1 or D1 + dissection. Therefore, the identi cation of these high-risk portions from EGC is urgently needed so that the patients can undergo radical lymphadenectomy.
Regarding the extent of gastric resection, the Japanese GC treatment guidelines have revealed that the standard surgical procedure for cN + or T2-T4a tumor is total or distal gastrectomy. For cT1N0 tumors, PPG and proximal gastrectomy can be considered depending on the tumor position. PPG is a lessinvasive function-preserving procedure that has been applied for the cT1N0M0 middle-third EGC with a distal tumor border at least 4 cm proximal to the pylorus according to the Japanese GC treatment guidelines [3]. The survival and recovery bene ts of PPG have already been reported in several retrospective studies [22][23][24]. However, PPG remains controversial. One of the reasons is that the dissection of the No. 5 lymph node may be incomplete in PPG because the pyloric branches of the vagus nerve are kept to reduce postoperative gastric stasis complications. In previous studies, it was reported that the metastasis rate of No. 5 lymph node in middle-third EGC was only 0.5% [25]. Kong et al. [26] reported that the metastasis rate of the No. 5 lymph node in middle-third EGC with a distal tumor border at least 6 cm proximal to the pylorus was 0% in T1a stage and 0.9% in T1b stage EGC. However, the metastasis rate of the No. 5 lymph node was 3.03% (5/165) for the middle-third EGC in this study (Table 2 and Table 3), which was similar to the result of Seung et al. [27], who reported that the metastasis rate to the No. 5 lymph nodes was 4.2% (52/1245). Seung et al. [27] also pointed out that the presence or absence of metastasis in the No. 5 and No. 6 lymph nodes should be carefully evaluated preoperatively using endoscopic ultrasonography and CT. Therefore, caution should be exercised before performing PPG for EGC given that the risk of No. 5 LNM is high according to our data. Further prospective studies using large case series are necessary to con rm this conclusion. It is worth mentioning that PPG should be performed for GC located in the middle third of the stomach and at least 4.0 cm away from the pylorus according to the guidelines. However, information about the distance from the tumor to the pylorus was unavailable in our database. This limitation weakens our conclusion.
LNM is one of the most important factors in uencing the prognosis of EGC. The risk of LNM is a major concern in choosing the optimal treatment for EGC. According to previous studies, the incidence of LNM in patients with EGC regardless of T1a or T1b was 15-24% [28,29]. Recently, Chen et al. reported that the LNM rate was 16.7% in EGC (8.7% in T1a, 24.6% in T1b) in their retrospective study that enrolled 1,033 patients with EGC [30]. In agreement with results of previously reported studies, our data showed that the incidence rates of LNM were 12.38% in T1a stage, 27.94% in T1b stage, and 18.36% in whole EGC. Classifying the low and high risks of LNM in patients with EGC is important in EGC studies.
A number of studies have identi ed the risk factors associated with LNM in EGC [20,[31][32][33][34][35]. They also revealed that LNM in EGC is related to differentiation, tumor size, depth of invasion, and LVI, which is consistent with the results of this study ( Table 5). The AUC of the ROC curve (Fig. 2), which validated this multivariable regression model, was 0.783 in this study. In other studies on the prediction of LNM with clinicopathological characteristics, the AUCs of the ROC curves were about 0.69-0.86 [32,36,37].
Similarly, the 2018 Japanese GC treatment guidelines [3] predicted the risk of LNM in EGC according to the clinicopathological characteristics including histological types (ulcerative ndings), and tumor sizes < 2 cm (non-ulcerative) and < 3 cm (ulcerative). However, the prediction of LNM in EGC by these factors is still not ideal given that the incidence risk of LNM was high in the population of patients with EGC with absolute or expanded indication, as shown in this study. The prediction of LNM in EGC based only on the current routine detection items and pathological examination may not be reliable. Hence, the discovery of more factors that could more accurately predict LNM is still a research interest in the eld of EGC. Finally, this study also analyzed the prognosis of patients with EGC and revealed that patients with LNM had a worse prognosis (Fig. 4).

Conclusions
In summary, the risks of LNM were high in patients with EGC with undifferentiated-type adenocarcinoma and with > 2-cm tumor and expanded indications of ER. In addition, PPG remains controversial due to the high metastasis rate of the No. 5 LN in the patients with middle-third EGC. Hence, physicians should be cautious when choosing a minimally invasive treatment (e.g., EMR, ESD, or PPG) that could carry a risk if the dissection of metastatic lymph nodes is neglected. Abbreviations EGC early gastric cancer, GC:gastric cancer, LNM:lymph node metastasis, LVI:lymphovascular invasion, EMR:endoscopic mucosal resection, ESD:endoscopic submucosal dissection, PPG:pylorus-preserving gastrectomy, CA:cancer antigen, CEA:carcinoembryonic antigen, HR:hazard ratio, CI:con dence interval, OR:odds ratio, ER:endoscopic resection, RR:risk ratio, ROC:receiver operating characteristic, AUC:area under the curve, CT:computed tomography Declarations Ethics approval and consent to participate This study was approved by the Ethics Committee of the First A liated Hospital of Sun Yat-sen University and was conducted in accordance with the principles of the Declaration of Helsinki. The need for informed consent to participate and for approval of all patients was waived.

Consent to publish
Not applicable.

Availability of data and materials
The datasets used and/or analyzed in the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.  Absolute indication of ESD 1: A differentiated-type adenocarcinoma without ulcerative ndings, in which the depth of invasion is clinically diagnosed as T1a and the diameter is >2 cm.
Absolute indication of ESD 2: A differentiated-type adenocarcinoma with ulcerative ndings, in which the depth of invasion is clinically diagnosed as T1a and the diameter is ≤3 cm.
Expanded indication: An undifferentiated-type adenocarcinoma without ulcerative ndings in which the depth of invasion is clinically diagnosed as T1a and the diameter is ≤2 cm.       Nomogram for the prediction of lymph node metastasis in patients with EGC.