Two updates on adenocarcinoma of gastroesophageal junction from the fth WHO classication of tumors of the digestive system: alteration of gastroesophageal junction denition and the emphasis on HER2 test.

Background: The incidence of gastroesophageal junction (GEJ) adenocarcinoma has increased rapidly but remains controversial over the last decades. There are two crucial updates of the fth WHO classication including the alteration of GEJ location and the emphasize on human epidermal growth factor receptor 2 (HER2) test. Methods: We retrospectively analyzed the clinicopathological features of 566 patients suffered from gastric adenocarcinoma. We comprehensively compared the clinicopathological features among GEJ and non-GEJ tumors, GEJ and proximal and distal gastric tumors with fourth and fth edition, respectively. Besides, we discussed the correlation of the HER2 expression with clinicopathological features according to the fth WHO classication. Results: The results showed that the difference was mainly between GEJ and distal adenocarcinoma in the fourth edition, but some were between proximal and distal adenocarcinoma in the fth edition. Tumors with longer invasion of the oesophagus were still mainly concentrated in GEJ tumors. The expression of HER2 in GEJ and proximal gastric adenocarcinoma was still higher than that in gastric body and distal site, which was basically consistent with the conclusion of the fourth edition classication. Conclusions: The clinicopathological parameters of the GEJ tumors partly changed with the narrowing scope of the GEJ adenocarcinoma. The proximal gastric tumors rather than GEJ tumors tended to be more invasive. But the GEJ tumors with longer oesophageal invasion required additional management. The HER2-expression of GEJ adenocarcinoma is still higher than that of other gastric sites. The classication of the fth edition is reasonable and worthy of recommendation.


Introduction
Gastric cancer is the fth most common cancer worldwide and gastroesophageal junction (GEJ) carcinoma has drew considerable attention because of its remarkable increase in incidence [1].
Compared with oesophageal and gastric carcinoma, GEJ tumor need different surgical procedure and lymph nodes dissection. However, it's often unclear whether the tumor is of gastric or oesophageal origin even though examined with computed tomography or endoscopic. For example, the computed tomography just have a low speci city (44%) when classifying GEJ Siewert type II tumors [2] [3]. The location of pathological anatomy seems the most accurate after operation, but the de nition of GEJ adenocarcinoma (AEGs) remains controversial in last decades. Siewert et al. classi ed GEJ carcinoma into three types including type I with its center 1-5cm above the GEJ, type II with its center between 1 cm above and 2 cm below the junction, type III with its center 2-5 cm distal from the GEJ [4]. On the other hand, Nishi et al. proposed that the tumor is located 2 cm above and 2 cm below the GEJ carcinoma, regardless of its different histological subtype [5]. In the west and east, the Siewert's classi cation has been widely used to distinguish the AEGs and adopted in the fourth WHO classi cation of tumors of the digestive system in past decades except in Japan.
In 2019, the fth WHO classi cation of tumors of the digestive system changes the epicenter distance from the GEJ of the AEGs from 5 cm into 2 cm, showing no difference with Nishi's classi cation [6]. In virtually, the new de nition of GEJ is similar to Siewert II and there are some studies have previously discussed the differences of clinicopathological features among the three Siewert types. A study demonstrates that patients with Siewert III tend to have later stages, more extensive lymph node involvement and worse survival than patients with type I or type II [7]. However, some data show the oncological outcomes of Siewert II tumors are similar to Siewert III in Eastern [8]. Moreover, when compared with distal gastric tumors, GEJ tumors are correlated with poor outcomes [9]. There is no consistent conclusion on the differences of clinicopathological features among GEJ subtypes. Therefore, a detailed and comprehensive analysis among the clinicopathological features is necessary not only between the fourth and the fth edition of GEJ tumors, but also between the GEJ and other gastric tumors.
Besides, there is another signi cant point in fth WHO of GEJ tumors that HER2 should be applied routinely to identify patients who may bene t from the target therapy of trastuzumb [6]. HER2 is a prooncogene encoded by erbB2 on chromosome 17 and its ampli cation might result in angiogenesis, tumorigenesis and excessive cell growth in several tissues such as colon, ovary, bladder, uterine and breast [10]. Although HER2 has been evaluated in many studies with gastric or GEJ adenocarcinomas, the scoring criteria and the de nition of GEJ-location have varied, leading to discrepancies in HER2amplication rates, which vary with an extremely wide range from 4-53% and a median rate of 20.2% [11] [12]. Previous studies showed that HER2-positivity rates were higher in GEJ than gastric cancer [13] [14], whereas some studies suggested that the presence of HER2-expression wasn't in uenced by tumor location [15] [16]. HER2-expression is heterogeneous in gastric or GEJ carcinoma, thus it's important to review the HER2 status by immunohistochemistry (IHC) or in situ hybridization and its correlating clinicopathological features according to the fth WHO classi cation of tumors of the digestive system.
In the fth edition, the two crucial updates are the alteration of GEJ adenocarcinomas' de nition and the emphasis on HER2 test. There is no consistent conclusion about the differences of clinicopathological parameters between each GEJ adenocarcinomas with different location. The clinicopathological features related to HER2 status perhaps have a variation as well. In this study, we compared the clinicopathological features between AEGs and other gastric carcinomas (GCs) with fourth and fth edition respectively. Then, we assessed the relationship between clinicopathological features and HER2expression with fth edition, to make an in-depth and comprehensive understanding about the GEJ adenocarcinomas of the fourth edition WHO of the digestive system.

Case Selection Study Design
We conducted a retrospective analysis including 566 cases that 464 of them were gastric carcinomas and 102 were GEJ adenocarcinoma (fourth edition of WHO). All patients were treated at Sichuan Cancer Hospital & Institute between 2016 and 2019 and this study received the approval from the ethics committee of Sichuan Cancer Hospital. The selected patients underwent radical resection of the tumor except for the ones who received radiotherapy or chemotherapy earlier.
The primary objective of this study was to compare the clinicopathological features of GEJ tumors with other gastric tumors such as proximal (excluding GEJ), body and distal gastric tumors. In addition, we also made a comparison of GEJ tumors between the fth edition/Nishi classi cation and the fourth edition. The secondary objective was to discuss the correlation of HER2-expression with the clinicopathological features according to the fth WHO classi cation of tumors of the digestive system.

Histological evaluation
Hematoxylin and eosin-stained (HE) sections from surgical excisions of specimen in all cases were reviewed by two pathologists using a multi-headed microscope. The histologic features were assessed as following: T classi cation (depth of tumor invasion), N-classi cation (nodal involvement), degree of tumor differentiation, lymphovascular invasion, nerve invasion and histologic type (Lauren's classi cation). The TNM classi cation was followed the AJCC eight edition [17]. Moreover, the sites of lymph nodes metastasis included four groups: lower mediastinal/periesophageal (No110-No.112), perigastric (No.1-No.6), suprapancreatic (No.7-No.11), para-aortic (No.16) [18]. The rate of lymph node metastasis was divided into three grades: score 1 means there was no lymph node metastasis, score 2 stood for 1-2 groups metastasis and score 3 stood for 3-4 groups metastasis.

Immunohistochemistry (IHC)
The rabbit monoclonal antibodies included anti-CDX-2 (RMA-0631, Maxim, Fuzhou, China), anti-CK7 (Kit-0021, Maxim), anti-CK20 (Kit-0025, Maxim) anti-KI67 (MIB-1, Maxim), anti-C-erbB-2 (EP3, Maxim) and antibodies for mismatch repair protein (MMR:MLH1, PMS2, MSH2 and MSH6). All procedures were performed in the EnVision System by a Benchmark-ULTRA automatic immunohistochemical staining instrument (Asia-core, China). HER2 scoring system proposed by Hoffman et al. was used as the criteria [19]. HER2 status was considered negative (HER2-) with scores of 0 and 1 (No membranous reactivity in <10% or faint or barely perceptible reactivity in ≥10% of tumor cells). HER2 with score of 3 (strong and complete basolateral membranous reactivity in ≥10 of tumor cells). HER2 status with score 2 was considered positive unless tested for gene ampli cation. Microsatellite instability (MSI) was evaluated by four markers of MMR, including microsatellite stability with positive for all four markers and microsatellite and high frequency MSI (MSI-H) with de ciency for more than two markers [20].

Fluorescence in situ hybridization (FISH)
The levels of HER2 ampli cation were tested with the cases whose IHC score was two. FISH test of HER2ampli cation was performed with PanthVysion kit (GSP, LBP, Guangzhou, China). The evaluation towards HER2-ampli cation was based on the ratio of HER2 to centromere 17 copy number, according to the guidelines of 2007 ASCO/CAP [21]. Cases were considered as gene ampli ed for the HER2/CEP17 ratio of 2.2, equivocal with ratio less than 2.2 but more than 1.8 and negative with the ratio less than 1.8. The equivocal cases were not selected in our cohort.

Statistical analysis
Findings were analyzed using the SPSS software for Windows, Version 18. The clinicopathological parameters were collected according to a standardized protocol. The difference of clinicopathological features among GEJ and proximal and distal gastric tumors were tested using the Mann-Whitney U test and Kruskal-Wallis test. The difference of clinicopathological features between GEJ and non-GEJ tumors and the correlation between clinicopathological features and HER2-expression were tested using the Mann-Whitney U test and Fisher's exact test. Differences were considered to be signi cant with two-side and P-value of <0.05.

Results
Clinicopathological features of AEGs (5cm from GEJ) and GCs The Clinicopathological features of AEGs(fourth edition/Siewert's classi cation) and GCs (proximal gastric excluding GEJ, gastric body and distal gastric) were summarized in Table1. There was no signi cant difference in patient age, histological type, degree of differentiation, M-classi cation and HER2-status between AEGs and GCs. In distal gastric cancer, female patients accounted for more than AEGs (P=0.005). AEGs had longer tumor diameter (P<0.001), higher T-classi cation (P<0.001), more frequent lymphovascular invasion (P=0.022) and more frequent nodal metastases (P=0.011) than distal gastric tumors. In addition, AEGs had higher rate of lymph node metastasis than gastric body (P=0.015) and distal gastric tumors (P=0.006). There was no difference in other parameters between AEGs and proximal gastric and gastric body. Her2 status was merely different between proximal and distal gastric (P=0.001).

Clinicopathological features of AEGs (2cm from GEJ) and GCs
According to the new fth edition of tumors of the digestive system, the clinicopathological features of AEGs (Nishi's classi cation: 2cm from GEJ) and GCs were compared in Table2. There was no signi cant difference in patient age, histological type, N-classi cation, M-classi cation, rate of lymph node metastasis and HER2 status between AEGs and GCs. Compared with proximal gastric, AEGs had higher degree of differentiation (P=0.029). Besides, AEGs had shorter tumor diameter than proximal gastric (P=0.041) and gastric body (P=0.032), but showed higher T-classi cation than distal gastric (P=0.012).
Her2 status was also merely different between proximal and distal gastric (P=0.021).
Comparison of AEGs' clinicopathological features between fourth edition and fth edition groups.
In order to have a more comprehensive understanding of the impact of the change in the de nition of AEGs, we compared the Clinicopathological features of the new fth edition with the fourth edition of the AEGs. All AEGs were divided into two groups: 2cm-5cm group meant ones of 2cm to 5cm from GEJ and the 2cm group stood for the ones of 2cm from GEJ. There were 103 cases in fourth edition group (Siewert's classi cation) including 57 (56%) cases of 2cm from GEJ (the fth edition) and 45 (44%) cases of 2cm to 5cm from GEJ. The 2cm group showed shorter in tumor diameter (P<0.001), lower Tclassi cation (P=0.021) and lower N-classi cation (P=0.035) than tumors of 2cm-5cm group. There was no signi cant difference in other parameters between the two groups.

Correlation between HER2 status and clinicopathological characteristics
We evaluated the HER2 status and clinicopathological features among all 566 cases and the GEJ tumors were chosen with the new fth edition. The incidence of HER2-expression in GEJ (25%) and proximal gastric (22%) were higher than gastric body (13%) and distal gastric (11%) tumors; this correlation was statistically signi cant (P=0.001). HER2-positivity was more common in low-moderate differentiation cases than high degree ones (P<0.001). Moreover, tumors of HER2-expression were distinctly associated with larger tumor diameter (0.001) and higher M-classi cation (0.039). Interestingly, there was no statistically difference in T-classi cation and N-classi cation. Although no difference in MMR status, there was merely one case of MSI-H in HER2-expression tumors.

Discussion
The Siewert's classi cation is widely used for surgical procedure of AEGs, while the Nishi's classi cation from Japan is almost the same as the Siewert II. Previous studies have been more focused on comparing the differences among Siewert I to Siewert III [22], or comparing the clinicopathological features between GEJ tumors with GCs. In the fth WHO classi cation of tumors of the digestive system, Nishi's classi cation is used in the de nition of AEGs. The change in the de nition of GEJ tumors is signi cantly important for clinicopathological assessment and clinical management, such as the surgical choice and lymph node dissection.
In this study, we compared the clinicopathological features between AEGs and GCs with fourth and fth edition respectively according to the fth WHO classi cation. The difference between the AEGs and the GCs was mainly focused on the AEGs and distal gastric adenocarcinoma with the criteria of the fourth edition. The AEGs had higher T-classi cation than distal gastric with the criteria of both the fourth and fth edition. This was in accord with previous studies, that proximal (GEJ and cardia) tumors were associated with poor outcomes [23] [24]. However, when analyzed the N-classi cation and the rate of lymph node metastasis, the difference between AEGs and distal gastric with the fourth edition criteria turned into the difference between the proximal and the distal gastric adenocarcinoma with the fth edition criteria. This was perhaps due to the tumors of 2cm-5cm from the GEJ classi ed to the proximal gastric tumors in the fth edition. Moreover, the tumors of 2cm-5cm was same as the Siewert III.A multivariate analysis showed only lymph node metastases predicted the gastric carcinoma survival [25].
The majority of patients were found to have more lymph node involvement in the Siewert III [26], which was consistent with our results. From the results of this part, the Siewert III tumors were no longer included in GEJ adenocarcinoma, the treatment for GEJ tumors need update as well. Total gastrectomy or more extensive distal gastric lymph node dissection may not be required. On the other hand, the scope of proximal gastric tumors (excluding GEJ) was also changed with the update of the de nition of GEJ. The proximal gastric adenocarcinoma statistically exhibited longer tumor diameter, higher T-classi cation and higher N-classi cation than distal gastric adenocarcinoma, that didn't appear in the fourth edition. What should be noted is that proximal gastric tumors rather than GEJ may have the worse survival and need to be treated especially and comprehensively. Besides, there was no signi cant difference in histologic types between AEGs and GCs, indicating that the tumor morphology could not help to distinguish AEGs from GCs. Furthermore, we also compared the clinicopathological features between the AEGs (2cm from GEJ) and non-AEGs (2cm-5cm from GEJ) according to the fth edition. The data showed AEGs of 2cm from GEJ had shorter tumor diameter, lower T-classi cation and lower N-classi cation than non-AEGs of 2cm to 5cm from GEJ. It seemed the AEGs were less invasive than non-AEGs. Notably, the extent of lymph node dissection in the mediastinum and the choice of distal esophagectomy made great important in GEJ tumors treatment. A multicenter retrospective study considered only the distance from the GEJ was signi cantly related to metastasis, and the longer the distance, the higher rate of lymph node metastasis [27]. In this study, AEGs were shorter in diameter than non-AEGs, which stood that AEGs were less likely to be lymph node involvement. Besides, some studies suggested lower mediastinal lymphadenectomy was recommended for oesophageal invasion of 3 cm or less; the extent of upper or middle mediastinal lymphadenectomy for oesophageal invasion of ≥ 3 cm [28] [29]. Although shorter in diameter, the AEGs in our cohort had a higher rate than non-AEGs in terms of the length of oesophageal invasion more than 3cm, which showed AEGs need upper or middle mediastinal lymphadenectomy. Therefore, mediastinal lymph node dissection and surgical resection were not completely uni ed in AEGs, even though the scope of AEGs became narrowed under the fth edition. The tumor's diameter and the length of oesophageal invasion both required special attention. What's more, we also compared the difference of mediastinal lymph node involvement between the two groups. The proportion of AEGs (57%) were slightly higher than non-AEGs, but with no statistical difference. This may be due to the lack of an accurate assessment of the length of oesophageal invasion before operation and the incomplete extent of lymph node dissection.
HER2 test is another signi cant point which was formally required in the fth edition. This marker's expression and its relevant clinicopathological features were studied in previous researches with Siewert's classi cation [30] [31]. On the basis of the fth edition, we re-evaluated the clinicopathological features of total 566 cases and their HER2 status. The HER2-expression in GEJ and proximal tumors were statistically higher than that in body and distal tumors, which was consistent with other studies [30] [32]. On the contrary, a study of 612 cases in Japan reported that the HER2-overexpression was not in uenced by tumor location with Siewert's classi cation [16]. We assessed the HER2 status with both IHC and FISH and classi ed the tumor locations with Nishi's classi cation ( fth edition). Our results demonstrated GEJ tumors had a higher expression of HER2 than body and distal tumors even if the number of GEJ tumors was decreased at the new criteria. This should be a crucial factor to emphasize the need for HER2 test in GEJ tumors. In addition, our analyses showed a statistically signi cant association between HER2expression and pathological grade, tumor diameter and M-classi cation of gastric tumors. HER2expression tumors had poor differentiation, longer diameter and more metastasis than HER2-negative ones, that meant HER2-expression tumors presented more invasive than HER2-negative ones. These results were agreed with other studies [33]. Therefore, the relevant clinicopathological features of HER2expressing GEJ tumors were almost not changed in the fth edition. Microsatellite instability was another signi cant molecular detection in gastric carcinoma, that was related to the contraction or expansion or of microsatellite sequences owing to the replication errors caused by mutations in the mismatch repair (MMR) in most cases [34]. Patients with MSI-H were more than 30 percent likely to develop Lynch syndrome. Even though no statistically signi cant difference between MSI and HER2-expression, there was merely one of the 19 MSI-H cases showing positive for HER2, while other 18 cases were all negative for HER2. This demonstrated that the HER2-expression cases probably did not suffer from MSI-H, but more data were needed for further veri cation.
In conclusion, our study comprehensively compared the clinicopathological features among AEGs and non-AEGs tumors, GEJ and proximal and distal gastric tumors. The analyses showed that the difference was mainly between GEJ and distal adenocarcinoma in the fourth edition, but some of these differences were between proximal and distal adenocarcinoma in the fth edition. And the invasiveness of proximal gastric tumors appeared to be relatively more invasive than GEJ and distal gastric tumors. The clinical management of proximal gastric tumors may need more attention and further research. In addition, the criteria of GEJ adenocarcinoma ( fth edition) are equivalent to the tumors of Siewert II (fourth edition). The Siewert III, with the worst prognosis, is no longer included in the category of the GEJ tumors. The treatment of the GEJ tumors does not have to be too aggressive. However, our data showed that tumors with longer invasion of the oesophagus were still mainly concentrated in GEJ tumors of fth edition. it should be noted that these tumors require additional middle even upper mediastinal lymph node dissection and longer scope of esophagectomy. Besides, although the scope of GEJ carcinoma became narrowed after the revision, the expression of HER2 in GEJ and proximal gastric adenocarcinoma was still higher than that in gastric body and distal site, which was basically consistent with the conclusion of the fourth edition classi cation.

Conclusions
The clinicopathological parameters of the GEJ tumors change partly with the narrower scope of the GEJ adenocarcinoma. Although the treatment tends to be more uni ed and standardized. The GEJ tumors with longer oesophageal invasion required additional management. The HER2-expression in the fth edition of GEJ adenocarcinoma is still higher than that of other sites of gastric. Therefore, the emphasis on the detection of HER2 in GEJ tumors is of great signi cance in clinical work. The overall analyses show the rationality with GEJ tumors criteria of the fth WHO classi cation of tumors of the digestive system, which is worth recommending in pathological diagnosis and clinical work.

Declarations
Ethics approval and consent to participate This article was approved by the Ethnic Committee of Sichuan Cancer Hospital & Institute.

Consent for publication
The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

Availability of data and materials
The data and materials are available to be shared.

Declaration of Interest
The author(s) declared no potential con icts of interest with respect to the research, authorship, and/or publication of this article.