Role of carbon nanotracers in lymph node dissection of advanced gastric cancer and the selection of preoperative labeling time

INTRODUCTION

Stomach cancer is the fifth most common cancer worldwide and the third leading cause of cancer mortality. There are significant regional differences in the incidence of gastric cancer. The incidence in Asia is significantly higher than that in Europe and the United States. Gastric cancer deaths in China account for > 40% of the global total in the same period[1]. Reducing cancer-related mortality and improving quality of life is one of the current research focuses.

第三阶段：形成融合标准最终版，由两部分构成，第一部分为军民共同表达的要素及代码，第二部分为军用特有的要素及代码。

Grade E (Poor): 0

In this study, preoperative endoscopic injection of carbon nanoparticles was selected due to the obvious contrast after the labeling of carbon nanoparticles, longer tissue fixation time, high lymph node affinity, and lack of need for special instruments and other characteristics. In gastric cancer and other gastrointestinal malignancies, there are still no specific guidelines for when preoperative labeling should be performed. On the basis of previous research, we extended the time of preoperative submucosal tracer injection, and the experimental group was divided into three: operation day (2-6 h)labeling group, preoperative 1-d (18-24 h) labeling group and preoperative 2-d (42-48 h) labeling group. The differences in the number of lymph nodes detected and the number of black-staining lymph nodes between the three groups and the control group were compared. The results provide direction for further research.

由此可见，在社会经济发展相对落后的西部地区，社区居民行为生活方式有待进一步改进，基本公共卫生服务中生活行为方式干预的相关项目有待进一步加强。

MATERIALS AND METHODS

Clinical data

We prospectively analyzed 307 patients with advanced gastric cancer who were hospitalized for surgery in the Department of General Surgery of Weifang People’s Hospital between June 2018 and February 2021. According to the different primary location of the tumor in the stomach, laparoscopic subtotal gastrectomy (LSG) or laparoscopic total gastrectomy (LTG) was selected, and D2 Lymph node dissection was performed for all patients. The lymph nodes that needed to be dissected for different surgical procedures are shown in Table 1. There were 180 patients in the ex

perimental group and 127 in the control group. To determine the preoperative labeling time, patients were randomly allocated to receive endoscopic labeling with nanocarbon suspension in the experimental group on the day of surgery, 1 d before surgery, and 2 d before surgery. The control group was not labeled with nanocarbon and the other treatment measures were the same.

Inclusion criteria

The inclusion criteria were: (1) Signed informed consent was given for surgery; (2)Gastroscopic examination and pathological biopsy confirmed malignant tumor of the stomach; (3) Abdominal enhanced computed tomography or magnetic resonance imaging and other ancillary examinations showed no distant metastasis; (4) No important organ dysfunction and could tolerate surgery; and (5) Clinical stage advanced gastric cancer.

Exclusion criteria

The exclusion criteria were: (1) Secondary examination suggested multiple metastases without surgical indications; (2) Patients with important organ dysfunction or other major diseases and could not tolerate surgery; and (3) Other contraindications and could not undergo D2 radical surgery.

Nanocarbon labeling method

When performing the preoperative nanocarbon labeling, if the submucosal injection level is too shallow, the nanocarbon could not enter the lymphatic flow to mark cancer tissue, and if the injection level is too deep, the nanocarbon would penetrate the serosal membrane and pollute the surgical field of view. Therefore, the sandwich labeling method was adopted: and 2-4 points were selected at 0.5-1.0 cm from the tumor edge. Normal saline was first injected to raise the submucosa, nanocarbon was injected into the submucosal surface, and subsequently, normal saline was injected again to increase the pressure of submucosal carbon nanoparticles suspension, so that the carbon nanoparticles could easily penetrate into the lymphatic tissues. A total of 2.0 mL nanocarbon suspension was injected. Radical proximal gastric cancer resection was not considered, and all patients were labeled at the oral but not at the anal end.Figure 1 shows preoperative carbon nanoparticles injection, and Figure 2 shows intraoperative black staining of lymph nodes and surrounding tissues.

Surgical method

对照组：患者采用口外弓支抗正畸治疗，佩戴Nance弓(杭州慈北医疗器械)矫正。告知患者Nance弓佩戴方法，每天需佩戴8 h以上；定期复诊(1次/月)，对Nance弓适当加力。同时对患者进行口腔卫生宣教。

“市场社会”道德建设的“情”“理”之辩——桑德尔与李泽厚的建构路径比较研究 ……………………………… 王婧菲，袁久红 2·032

LSG lymph node[M1] dissection and anastomosis procedures were: The loose connective tissue between the anterior and posterior lobes of the right transverse mesocolon was extended gradually to the left until the first short gastric vessel behind the root of the left gastroomentum artery. The No. 6 Lymph nodes were dissected from the right omentum vessel. Hepatoduodenal ligament was opened, and No. 5, and 12a lymph nodes were dissected along the proper hepatic artery. The root of the right gastric artery was exposed and along the main trunk of the right gastric artery, the surrounding soft tissue was dissected, the left gastric artery and the beginning part of the coronary vein were exposed, the root of the vessel was ligated, and the surrounding No. 7, 8a, 9, and 11p lymph nodes were dissected. Mesangial tissue was isolated along the lesser curvature of the stomach till the right diaphragm, and No. 1,3a, 3b and 5 Lymph nodes were dissected. Billroth II and Braun anastomosis was performed. All procedures were performed by the same group of surgeons.

Postoperative lymph node sorting

After the surgical specimen was isolated, the senior attending physician placed it according to its anatomical position and took photographs. The lymph nodes in each group around the stomach were cut and marked according to the blood vessels. The tissues in each group were finely separated and the surface tissues of lymph nodes were removed, and bagged separately. All lymph nodes were sent to the pathology department for postoperative analysis according to their corresponding perigastric lymph node groups. For the patients with total gastrectomy, the gastric peripheral lymph nodes on the lower cardia side were classified as No. 1, the gastric peripheral lymph nodes on the greater cardia side were classified as No. 2, and the peripheral tissues of short gastric vessels above the left arteriovenous Hemlok clip were classified as No. 4sa. The left arteriovenous clipped tissue along the gastric omentum was classified as No. 4sb, the right arteriovenous clipped tissue along the gastric omentum was classified as No. 4d, and the subpyloric region was classified as No. 6. Ligation of the right gastric arteriovenous Hemlok clipped to the upper part of the pylorus was classified as No. 5, from the ligation of the left gastric arteriovenous Hemlok clipped to its first branch was classified as No. 7, and the remaining perigastric tissue near the lesser curvature was classified as No. 3. Figures 3 and 4 shows lymph node sorting after gastric cancer.

Observation target

The number of dissected lymph nodes, black-stained lymph nodes were counted, and basic information, including gender, age, pathological types, postoperative complications such as intraoperative blood loss, and anastomotic fistula, were observed.

Statistical analysis

SPSS 25.0 was used for statistical analysis. Measurement data were expressed as mean± SD, and atest was used for comparison between the two groups. One-way analysis of variance and multiple comparisons were used for intragroup comparison, and<0.05 was considered statistically significant.

RESULTS

Basic information

A total of 307 patients were enrolled. Gender, age, pathological type, pathological stage, tumor markers, intraoperative blood loss (significant), postoperative complications and other indicators are shown in Table 2, and the differences were not significant.

Comparison between groups

There were 180 patients in the experimental group (99 treated with LTG and 81 with LSG), and a total of 6105 Lymph nodes (3460 LTG and 2645 LSG) were detected, with an average of 34.95 ± 4.81/case for LTG and 32.65 ± 3.82/case for LSG. There were 127 patients in the control group (77 treated with LTG and 50 with LSG), and a total of 4000 Lymph nodes (2456 LTG and 1544 LSG) were detected, with an average of 31.90 ±4.47/case for LTG and 30.88 ± 2.69/case for LSG (Tables 3 and 4). The differences in the number of dissected lymph nodes, and number of black-stained lymph nodes at D1 and D2 stations under different surgical methods and different preoperative labeling times are shown in Tables 5 and 6.

DISCUSSION

Over the past 20 years, with the progression of medical science, the comprehensive treatment of gastric cancer has made great strides. Surgical treatment is still the most used method, and lymph node dissection is one of the most important techniques in radical gastrectomy. How to remove a sufficient number of lymph nodes in gastric cancer surgery more safely and effectively to achieve the goal of radical resection has been one of the topics studied by gastrointestinal surgeons. For advanced gastric cancer, the surgical criteria were D2 or D2+ radical surgery: tumor resection and regional lymph node dissection. The metastasis of gastric cancer is mainlythelymphatic pathway, so survival depends not only on the primary lesion but also on the presence of regional lymph node metastasis[9]. Baxter[10] found that a certain number of lymph nodes should be dissected during radical gastrectomy for gastric cancer, and there was a correlation between the number of lymph nodes dissected and prognosis. According to the clinical data of gastric cancer patients in the American Surveillance, Epidemiology, and End Results database[11,12], prognosis can be improved by the addition of 10 Lymph nodes in postoperative specimens. Even for patients with negative lymph nodes after surgery, the number of detected lymph nodes is still an independent factor affecting prognosis[12]. Some researchers have reported that, in order to improve the accuracy of pathological lymph node staging of gastric cancer specimens, at least 10-15 Lymph nodes should be detected in the N0 stage, and ≥ 20 should be detected in the N1-3 stage. If ≥ 30 Lymph nodes are collected for examination, postoperative lymph node staging could be more accurate[13,14]. The number of dissected lymph nodes recommended by the 8th Edition of the International Union against Cancer/American Joint Committee on Cancer (UICC/AJCC) in TNM staging of gastric cancer should not be less than 16[15]. According to the Japanese Regulations on the Management of Gastric Cancer[16], the number of lymph nodes dissected during radical gastrectomy for gastric cancer should be ≥ 15, and an insufficient number of dissected lymph nodes will significantly affect the 5-year postoperative survival rate[17-19]. The more lymph nodes sent for examination, the greater the possibility of detection of metastatic lymph nodes[20]. The more reliable the accuracy of lymph node staging is, the more the occurrence of lymph node staging migration can be reduced or avoided[21]. Therefore, effective lymph node dissection is indispensable for thorough radical treatment of gastric cancer. According to the multicenter analysis of postoperative gastric cancer survival data[22], there was an obvious postoperative lymph node stage migration phenomenon in Chinese patients with gastric cancer, especially in early-stage patients with < 15 Lymph nodes dissected andin advanced patients with < 35 Lymph nodes dissected. According to a study by Sano[17] in 2017, the average number of lymph nodes detected in each gastric cancer specimen in Japan reached 39.4/case, followed by 33.0/case in South Korea, while the figure for several major centers included in the survey in China was only 24.8/case–even lower than the 29.5/case in Europe and America[23].

In order to correctly distinguish between lymph node and normal tissue and to dissect the lymph nodes more thoroughly, we could selectively label the pericancerous lymph nodes. The existing lymphatic tracers can be divided into three generations: the first is represented by methylene blue and India ink, the second by iodine oil and activated carbon, and the third by nanocarbon. Nanocarbon lymph node tracers are essentially lymphatic tracers, and their physical and chemical properties have been described in the previous section. At the same time, due to the high contrast of the color, nanocarbon tracers can help surgeons to correctly distinguish the lymph nodes and normal tissues, reduce the damage to normal tissues and the time of surgical dissection, and increase the number of lymph node dissections. In recent years,nanocarbon tracers have gradually matured for malignant melanoma, breast cancer,thyroid cancer, and some digestive malignant tumors[21]. Nanocarbon lymph node tracers can help surgeons to determine lymph node metastasis to a certain extent, and they can improve the effective removal of lymph nodes during surgery[8,24].

In this study, the injection dose of nanocarbon suspension was 2.0 mL at a total of four sites, with an average of 0.5 mL at each site. According to existing literature, the injection dose of nanocarbon was 0.4-0.6 mL in breast cancer patients[25] and 1.0 mL in thyroid cancer patients[26]. In patients with colorectal cancer, the injection dose of carbon nanoparticles was 1.0 Ml[27]. Considering the deep infiltration of advanced gastric cancer and the thickness of gastric wall tissue compared with thyroid, breast and colorectal tissue, an injection dose < 2.0 mL may lead to unclear lymph node display in some patients. If the dose is > 2.0 mL, some patients may have excessively deep staining due to excessive dosing, which will affect the operation, and the sandwich injection method can be selected for preoperative labeling of carbon nanoparticles[28].

在实际对在公路沥青路面开展施工的过程当中所需要用到的原材料，一般要选择干净、干燥且不包含其他的杂质的材料，因为这样的材料才能真正意义上提升施工质量。说明我们在对原材料进行选择时，必须要做到细致认真，最终选择的每一类原材料，都必须要与公路建设施工相关的规章及标准相符合。但是许多企业相对来说更加重视材料成本的控制，所以选择材料时很容易出现偷工减料的问题。此外在对材料进行选择的时候，还要考虑到季节性的要素，不同的原材料有不同的环境要求，受到外界的降水、温度等条件影响，其质量指标也必然会有一定的变动，即便是符合所有施工要求的原材料，如果不能做好合理贮存、合理利用，都无法确保施工成果[2]。

In the present study, the average number of dissected lymph nodes in the experimental group (LTG 34.95 ± 4.81/case; LSG 32.65 ± 3.82/case) was significantly higher than that in the control group (LTG 31.90 ± 4.47/case, LSG 30.88 ± 2.69 /case). Under LTG operation, compared with the control group, the number of lymph nodes dissected at the D1 and D2 stations in the experimental group was significantly better than that in the control group. However, under LSG operation, the number of lymph nodes dissected at the D1 station showed no significant difference between the two groups, and the number of lymph nodes dissected at the D2 station was better than that in the control group. In a study by Cheng[29], the number of lymph nodes detected in the nanocarbon group and the non-nanocarbon group was 32.28 ±4.10/case and 21.28 ± 2.74/case, respectively. In the study of Jia[30], 15484 Lymph nodes were detected in the nanocarbon group and 7963 in the non-nanocarbon group.The average number of lymph nodes detected in each patient in the nanocarbon group and non-nanocarbon group was 31.99 ± 8.99 and 19.81 ± 4.74, respectively. The results in our experimental group are consistent with the previous studies. In our study, there were 180 patients in the experimental group, and no complications such as marker point bleeding or perforation occurred after endoscopic carbon nanolabeling. In addition, there was no significant difference in the operating time, postoperative hospital stay, and incidence of postoperative complications such as postoperative anastomotic fistula, anastomotic bleeding, and obstruction, which proved that the effectiveness and safety of nanocarbon tracers were similar to those in previous studies.

It is worth noting that our study showed that the intraoperative blood loss of the experimental group was less than that of the control group (under the same operation), which may have been due to clearly visualized lymph nodes after preoperative nanocarbon labeling, thus avoiding unnecessary tissue and vascular damage and reducing intraoperative blood loss.

最近几年来，离开家乡外出觅食中，目前暂居于浙江温州。我觉得最理想的生活方式莫过于本人的签名：劈柴喂马，撩拨山水。一个是生活，一个是梦想，我将为此而努力。

Although preoperative injection of nanocarbon tracer is helpful for lymph node dissection, there is still no consensus or guidelines on the optimal time point for preoperative tracer labeling. By comparison among experimental groups in this study,we found that under the premise of the same operation and the same lymph node station the results (number of lymph nodes detected and number stained black) of the nanocarbon labeling group 2 and 1 d before surgery were significantly better than those of the labeling group on the day before surgery. At the same time, there was no significant difference in the number of lymph nodes detected between the 2-d and 1-d preoperative labeling groups. This may be because the optimal time for imaging in tissues after injection of carbon nanoparticles is 2-12 h after injection[31], and there was no significant difference compared with previous results.

西安人大都以古城墙为傲，谈起古城墙就如数家珍。游人眼中的惊奇，对于他们则是生活。这道静默地屹立的古城墙，有如这座城市的巨大脉管，将历史和现实紧密相连，从历史中迤逦而出，延伸到西安人的现代生活，延伸到西安人的灵魂之中，成为了他们的文化之根。每天上班下班、买菜、接送小孩都穿梭在城墙之间;在城墙根下晒太阳、吼秦腔，爬上城墙吹吹埙、望望月……日子就这么过着，古城墙也就默默地守望着。

Our study had some limitations. We only studied the influence of three different labeling time points on the results of lymph node dissection after radical resection of gastric cancer. Further determination of a more accurate labeling time of the tracer needs to be confirmed by more clinical trials. For example, whether better results can be achieved by changing the marker time to 3 or even 4 d before surgery or whether a better result can be achieved between 1 d before and on the day of surgery remains to be answered by more in-depth studies with larger sample sizes.

CONCLUSION

In conclusion, carbon nanoparticle labeling has a good guiding effect for laparoscopic lymph node dissection of gastric cancer, and is safe and effective. Compared with the control group, preoperative submucosal injection of carbon nanoparticles could significantly improve the detection rate of lymph nodes, which is conducive to pathological staging and subsequent comprehensive antitumor therapy.

一是加强思想引领。要引导青年画家用习近平总书记关于文艺工作的讲话精神武装头脑，加强政治理论学习和道德品质修养，继承发扬老一辈艺术家的优良作风和光荣传统，自觉成为党的文艺方针政策的拥护者、践行者和时代风气的先觉者、先行者，积极践行“爱国、为民、崇德、尚艺”的文艺界核心价值观，坚守艺术理想和艺术追求，坚持以人民为中心和德艺双馨的价值取向，抵制虚华浮躁、乖戾自我之气，排斥低俗媚俗、急功近利之风，以高度的政治责任感和历史使命感投入到艺术创作中去，用自己的精品力作弘扬社会主义核心价值观，传播社会正能量。

Retrospective analysis study was performed, all patients were randomly divided into experimental group (preoperative injection of carbon-nano group) and control group(preoperative injection of carbon-nano group) according to the principle of randomization; In the experimental group, according to the different groups of preoperative labeling time, the differences between the groups were studied.

The average number of dissected lymph nodes in the experimental group [34.95 ±4.81/case in the laparoscopic total gastrectomy (LTG) group; 32.65 ± 3.82/case in the laparoscopic subtotal gastrectomy (LSG) group] was higher than that in the control group (31.90 ± 4.47/case in the LTG group; 30.88 ± 2.69/case in the LSG group,<0.05). In comparisons within the experimental group, the experimental results (number of lymph node dissections, number of black-staining lymph nodes) of the nano-carbon labeling group 2 and 1 d before surgery were better than those of the labeling group on the day before surgery (< 0.05).

(1) Nano-carbon labeling has a good guiding effect on lymph node dissection during laparoscopic gastric cancer, and it is safe and effective; and (2) Compared with the control group, submucosal injection of a carbon tracer in the experimental group at a certain time before surgery can significantly improve the lymph node detection rate (< 0.05), which is conducive to pathological staging and follow-up anti-tumor comprehensive treatment.

Gastric cancer is the fifth most common cancer in the world, redical operation is still the preferred treatment method for advanced gastric cancer, postoperative lymph node detection rate is one of the major factors affecting PN staging of lymph node metastasis after radical gastrectomy, In order to help the surgeon correctly distinguish the normal tissue from the lymph nodes and dissect lymph nodes as much as possible,endoscopic injection of carbon nanoparticles was selected.