Mesenteric Vein in Gastric Carcinoma: a Retrospective Analysis

Xing Xu Cancer Hospital of China Medical University, Liaoning Cancer Hospital & Institute Guoliang Zheng Cancer Hospital of China Medical University, Liaoning Cancer Hospital & Institute Tao Zhang Cancer Hospital of China Medical University, Liaoning Cancer Hospital & Institute Yan Zhao Cancer Hospital of China Medical University, Liaoning Cancer Hospital & Institute Zhichao Zheng (  zhengzhichaoedu@163.com ) Cancer Hospital of China Medical University, Liaoning Cancer Hospital & Institute


Introduction
Gastric cancer (GC) was one the most common cancer-related deaths carcinoma throughout the world [1,2]. Radical gastrectomy is the optimal choice to cure locally advanced resectable gastric cancer. As the 15 years long-term result of Dutch trial showed that D2 lymphadenectomy was associated with lesser rate of recurrence and improved the overall survival than D1 dissection, gastrectomy plus D2 lymph node dissection has been increasingly considered as the standard surgical procedure for advanced resectable gastric cancer [3]. However, over 50% patients suffering from worse survival would subsequently relapse or die after radical surgery, due to node, distant metastasis or locoregional recurrence [4]. Among the metastatic routes in GC (direct in ltration or spread, lymph node metastasis, hematogenous metastasis and implant metastasis), lymph node metastasis remains the most common pathway. However, the extent of lymphadenectomy during the surgical procedure to improve the survival to the utmost extent with less complications remains controversial.
According to 6th edition of International Union Against Cancer (UICC), lymph node metastasis (LNM) is classi ed into isolated tumor cells (ITC), micrometastasis (MI) and macrometastasis (MA) depending on the size of metastatic deposit [5]. Methods to investigate the presence of MI varies from serial slices with Hematoxylin and eosin (HE) staining, immunohistochemical (IHC) staining to real-time reverse transcription-polymerase chain reaction (RT-PCR) [6][7][8][9]. Cytokeratin is one component of the cytoskeleton of epithelial cell, which is not present in normal lymph nodes. And its antibody was widely used to detect the minute deposit of tumor cells in lymph node by IHC staining. There are still controversies about the clinical features of MI and its prognostic signi cance on GC [10][11][12][13].
The JCOG9501 trial didn't reach the conclusion that D2 lymphadenectomy plus para-aortic lymph node dissection obtains the superiority of improving the survival in comparison to D2 lymphadenectomy [14,15]. There were controversies that whether it was necessary for patients with GC to receive 14v dissection and how to identify the subgroup for whom the bene t was maximized from 14v dissection. The study was mainly designed to analyze the characteristics associated with 14v metastasis or micrometastasis and the clinical features and prognostic signi cance of MI for GC. Otherwise, oncological outcomes were also analyzed. Hospital. To analyze the characteristics associated with 14v metastasis or micrometastasis, the inclusion criteria included: Age ranged from 18 to 75 years old; Histopathological examination diagnosis of gastric carcinoma on well-established criteria; Intraoperative exploration and postoperative TNM stage revealed no distant metastases (M0); No unresective factors were founded; Radical gastrectomy plus the No.14v lymph node dissection was performed (D2 or D2+). The study excluded cases for which: R0 surgery after conversion therapy; Gastric remnant carcinoma; 14v lymph node biopsy; Suffering from other malignancies; Without complete follow-up data. A total of 303 patients receiving radical gastrectomy plus the No.14v lymph node dissection were retrospectively collected to make comparisons between groups. Patients with clinical or pathologic TNM stage ( -) or with positive lymph nodes received a perioperative chemotherapy regimen of SOX or XELOX, divided into 2-4 preoperative and several postoperative cycles. 255 cases without No.14v lymph node metastasis underwent CK8/18 immunohistochemistry to detect the minute deposit of tumor cells in lymph nodes. Patients without micrometastatic lymph node were classi ed into negative group, and patients with micrometastatic or metastatic lymph nodes were classi ed into positive group.

Methods And Materials
No.14v lymph node dissection The No.14v lymph nodes were classi ed as lymph nodes along the superior mesenteric vein. All radical gastrectomy procedures plus the No.14v lymph node dissection were performed by 3 experienced surgeons. The dissection criteria of the No.14v lymph node were as follow: Firstly, omentobursectomy and lymphadenectomy of No.6 was performed. And then No.14v lymph nodes were completely removed from the root of nodes. In order to expose the superior mesenteric vein, Henle trunk and middle colic vein, the soft tissue around the superior mesenteric vein was completely removed as well.
Immunohistochemical stain A total of 576 lymph nodes without No.14v metastasis from 255 patients were reexamined by one pathologist to con rm the absence of lymph node metastasis by HE staining section. Lymph nodes were stored in 261 para n-embedded blocks and 3 serial tissue sections were made from each block with 4 mm in thickness and immunohistochemical stain (CK8/18, 1:200 dilution) were performed to evaluate metastasis of lymph nodules. The tumor tissues of GC were used as the positive control to stain in the same manner as experiment group (Supplementary Figure 2).

Assessing staining results
We evaluated the staining results according the location, structure, morphology and staining color. CK8/18 was mainly located in the cytoplasm at the marginal sinus of lymph nodes ( Figure 2). Positive staining cells were lled with brown-yellow color component, while the negative cells are unstained.
Furthermore, positive samples were recon rmed by detecting the structure and morphology of the cells. Only the size of the cell nest was 0.2-2 mm, it was de ned as MI and the sample was classi ed as positive sample. For any one of serial sections was positive staining, we would categorize it as positive group. All of the slices were reviewed by two experienced pathologists by independently observing the CK staining sections under the microscope (×100 and ×200). When disagreements between pathologists happened, a consensus was sought by pathologists rereviewing the slices and discussing it together.

Statistical analysis
Mean ± standard deviation was used to represent continuous values and categorical data were expressed as percentage. Chi-square test or Fisher's test was applied to evaluate the relationship between clinicopathological characteristics and 14v metastasis or micrometastasis variance. Independent factors associated with 14v metastasis or micrometastasis were analyzed in Logistic regression method. Variables with P value of 0.05 in univariate analysis were selected for the multivariate analysis. To predict prognostic risk factors, we use the multivariable Cox proportional hazard model and univariate analysis. The Kaplan-Meier method and log-rank test was applied to distinguish difference of survival data between two groups. Data was proceeded by SPSS 23.0 software. The P value 0.05 was considered to be statistically signi cant.

Clinicopathological features and clinical outcomes
A total of 303 patients underwent radical surgery plus 14v dissection in the study and clinicopathological features were shown in Table 1. Most patients were male (66.0%) and their age ranged from 28 to 75 years old (mean: 55.92 y). A majority of tumors were located in L area (67.7%), presented with poor differentiation (77.2%) and canalicular adenocarcinoma histological type (73.9%). A minority of patients underwent total or proximal gastrectomy surgery (24.1%). And cases were classi ed into stage (17.5%), (20.1%), (62.4%) (postsurgical pathological stage). The metastastic incidence of No.14v lymph node was 15.8% and the micrometastatic incidence was 3.9%. The total metastatic rate of 14v was 19.1%. The median number of overall harvested lymph nodes and harvested 14v lymph nodes was 28.48 and 2.03, respectively. Patients were followed up every 3 months during the rst 3 years, subsequently every 6 months for the following 2 years and once a year after 5 years until the time of death or the deadline, December 31th, 2018. The following-up time ranged from 3 to 178 months (median: 46m). And the 3-, 5- year overall survival rate of patients with radical gastrectomy plus 14v dissection was 71.2% and 50.7%, respectively. We sought to identify subgroups that were likely to be presented with 14v metastasis or micrometastasis. It revealed the metastasis or micrometastasis of 14v was associated with tumor size (P = 0.001), location (P = 0.027), Borrmann type (P = 0.003), pT category (P = 0.001), pN category (P = 0.000), pTNM stage (P = 0.000) and the number of metastatic lymph node (P = 0.000) (    LN14v metastasis nor micrometastasis (P = 0.000, Fig. 3). The bene t of lymphadenectomy of LN14v in gastric cancer Having established that 14v metastatic status was prognosis signi cance for adequately staged patients treated by radical gastrectomy plus the 14v dissection, we sought to identify patient subgroups for whom the bene t was maximized and those for whom TRG was not of prognostic signi cance. In matched analysis, patients with gastric cancer of stage , U/M area, pN2-3 and LN 6(+) underwent lymphadenectomy of 14v suffered better survival than those without lymphadenectomy of 14v (P = 0.006, Fig. 4).

Discussion
Immunohistochemical staining for cytokeratinnodes CK8/18 to evaluate micrometastases has been reported in other elds. But the study rstly introduced CK8/18 to evaluate micrometastases of lymph nodes to formulate clinicopathological characteristics and prognosis of gastric cancer with metastasis or micrometastasis to LN14v, repressing the largest numbers cases associated with LN14v metastasis or micrometastasis. It demonstrated location, pN stage, Borrmann type and the LN6 metastatic status were predictive factor of LN14v metastasis or micrometastasis, which implied that tumor located at M or L area, presented with pN3a or N3b stage,Borrmann or subtype and 6 metastasis were likely to present with 14v metastasis or micrometastasis. It screened out patient subgroup for whom the bene t may be maximized form LN14 dissection and those for whom LN14v dissection seemed to be not prognostic signi cant. Secondly, it revealed that micrometastatic status of LN14v lymph mode was one of important prognostic factors. Lymph node micrometastasis could provide accurate prognostic information for patients with GC. Thus, immunohistochemical detection of micrometastasis of lymph nodes should be recommended.
It was a contentious issue whether LN14v metastasis was categorized to regional lymph node (local disease) or systemic disease [16]. When compared to those with locoregional lymph nodes metastasis, patients with LN14v metastasis had the worse 5-year survival rate (< 10%), and it was similar to that with LN16 metastasis, which was categorized to stage , implying its systemic disease role. However, several studies demonstrated some patients with 14v metastasis would bene t from LN14v dissection and prolonged their survival, indicating at least some patients with 14v metastasis were at the state of local disease rather than systemic disease [16]. According to the second edition of Japanese Classi cation of Gastric Carcinoma, LN14v dissection was included in N2 group for tumors located at the lower third of the stomach [17]. But it was once classi ed as M1 status in the third edition of Japanese Classi cation of Gastric Carcinoma, which recommended it was unnecessary to dissect LN14v for patients with gastric cancer [17]. Furthermore, the 3th edition of Japanese gastric cancer treatment guidelines 2010 proposed patients with tumor located at the lower third of the stomach with No.6 metastasis need dissect LN14v [18]. When we evaluate whether it is essential for patients with GC to dissect LN14v, the 14v metastatic rate, chinicopathological features associated with 14v metastasis, security and feasibility of 14v dissection, the signi cance of dissection should be taken into account.
Lymphatic metastasis is considered to spread from lymphatic ows of the primary tumor site and the lymphatic ows from any special point have some preferred pathway [19][20][21][22][23][24]. There mainly exist three pathways in the region of lower stomach. The lymphatic drainage from LN6 directly ows to LN14v, and then lymphatic ows reach LN16, which nally joins to thoracic duct. In terms of lymphatic ow pathway, LN6 is anatomically upstream of LN14v, whose metastatic status is stringently correlated to that of LN6.
The previous study investigated the impact of regional nodes metastatic status on LN14v metastasis [16]. It revealed that the metastatic status of LN6 and LN9 were predictive factors of LN14v metastasis.
Our study was consistent with the previous research and the stepwise lymphatic metastasis theory. In our study, we found LN6 metastatic status was one signi cant independent variable for the metastatic status of LN14v. Of 146 patients with LN6 metastasis, 34.2% cases were presented with the metastasis or micrometastasis of LN14v. Similar to previous studies, the LN6 status predicted the absence of LN14v metastases with a low false-negative rate (10.7%). Otherwise, the study also demonstrated tumor site, Borrmann classi cation and pN stage were also correlated with LN14v metastasis or micrometastasis.
Tumor located at region of low or middle stomach, presented with Borrmann / subtype and pN2-3 stage were likely to be presented with metastasize or micrometastasize to 14v.
Various interactions factors promoted the occurrence and development of GC, presenting with complicated biological characteristics, high heterogeneity and undesirable prognosis [25]. One study reported that the 5-year survival rate of patients with GC with lymph node metastasis to LN14v was extremely low (11.3%), which was also described in another study with an undesired 5-year survival rate (9.0%) [26,27] At present, detection methods of lymph node micrometastase mainly include serial section, PCR, and immunohistochemistry. Serial sections can signi cantly improve the positive rate of lymph node micrometastasis, but it is di cult to promote it in clinical practice in large scale because of its heavy workload. PCR is characterized by high sensitivity and speci city for lymph node micrometastasis detection, but the requirement of fresh samples, the relatively complicated operation process and high costs hindered the routine application in clinical pathological diagnosis. Compared with previous one, immunohistochemical method seems to be more useful in clinical work. Cytokeratin makes up one component of the cytoskeleton of epithelial cell, which is not present in normal lymph nodes. Ishii et al reported that the usage of CK8/18 monoclonal antibody represented one accurate method to detect lymph node micrometastasis in gastric cancer [7]. For 35 cases underwent lymph node micrometastatic detection, the positive rate was 11.4%. Yun et al also con rmed that CK8/18 monoclonal antibody is one reliable method in detecting lymph node micrometastasis, and its positive rate is as high as 31.3% [13].
Our study suggests that lymph node micrometastatic rate of 14v is only 3.9%. The discrepancy may result from different interval between serial sections, the harvested number of sections from different para n-embedded blocks, the included cases and the focused lymph node. In this study, patients with gastric cancer stage III accounted for 62.4%.
Although a majority of studies demonstrated that patients with lymph node micrometastasis would be presented with worse prognosis, the questions whether lymph node micrometastasis resulted in postoperative recurrence or metastasis and it consequently affected patients' prognosis remained controversial [10,12,29,30]. For the micrometastatic status can be promoted or inhibited by various factors, such as the host's immune status, postoperative radiotherapy or chemotherapy, tumor microenvironment and so on. In accord with previous study, we revealed that the micrometastatic status is a signi cant variable associated with patients' survival. But the difference between survival of patients with 14v micrometastasis and that of 14v metastasis was not obvious, which may result from the limited samples of positive micrometastatic cases. So more multi-center, randomized trials are required to further investigate the extent of the impact of micrometastatic status on survival for GC. There is no doubt that micrometastatic status can be considered as one promising prognostic predictor in GC, which can provide accurate pathological staging and favorable treatment guidelines. We recommend patients with LN14v micrometastasis or on suspect of LN14v micrometastasis receive LN14v dissection.
Our study has some limitations, mainly for its retrospective case-control design and the limited participants. The small sample size may produce selection bias. Secondly, in comparison with the previous study, different interval between serial sections and the harvested number of sections from different para n-embedded blocks would in uence the micrometastatic rate of LN14v. Thirdly, although the study revealed that LN14v metastatic or micmetastatic status was an independent risk factor associated with survival, randomized studies were required to clarify the advantages of 14v dissection on survival for patients with LN14v metastasis or micrometastasis.
In conclusion, locally advanced gastric carcinoma, located at middle or low stomach area with LN6 metastasis were likely to be presented with metastasis or micrometastasis of LN14v and the D2 gastrectomy surgery plus LN14v dissection would be recommended for patient with gastric cancer of stage , U/M area, pN2-3 and LN 6(+), if the serious complication of LN14v dissection can be su ciently controlled. Micrometastatic status of LN14v can be considered as one promising prognostic predictor for GC.

Declarations
This research did not receive any speci c grant from funding agencies in the public, commercial, or notfor-pro t sectors.

Competing interests
There were no relevant a liations or nancial involvement with any organization or entity with a nancial interest in or nancial con ict with the subject matter or materials discussed in the manuscript.
Availability of data and materials The datasets used during the current study are available from the corresponding author on reasonable request.