Effect of Thoracic Duct Ligation During VATS Esophagectomy On T Cell Subset and DFS in Squamous Cell Carcinoma Patients With T1b-3N0M0 Stage

Objective: To study whether the ligation of thoracic duct during video-assisted thoracic surgery esophagectomy will cause damage to the immune system, thus affecting the disease-free survival(DFS) of patients with cT1b-3N0M0 stage. Methods: We studied the esophageal squamous cell carcinoma conrmed by endoscopic ultrasound biopsy and PET-CT. They were randomly divided into thoracic duct ligation group and non ligation group. In addition to thoracoscopic resection of esophageal cancer, thoracic duct ligation was also performed in the experimental group. The peripheral blood T lymphocyte subsets were detected by ow cytometry during perioperation. All patients were reexamined regularly after operation in order to nd recurrence or metastasis early. The Chi-square test and t-test were employed for statistical analysis with statistical signicance at p<0.05.The effect of thoracic duct ligation on DFS curves were calculated by the Kaplan– Meier method and compared by the log-rank test. A Cox regression model with stepwise selection was used for the multivariate analyses. Result: After early screening and late exclusion, a total of 67 patients entered the study and completed the follow-up. There was no signicant difference in gender, age, tumor location, depth of invasion, degree of differentiation and presence of tumor thrombus between the ligation group (32 cases) and the non ligation group (35 cases). There was no signicant difference in T lymphocyte subsets before and 3 weeks after operation, but there was signicant difference on the 1st days after operation. Cox regression analysis showed that depth of invasion (P= 0.0020), degree of differentiation (P= 0.0262), presence of tumor thrombus (P = 0.0158) and thoracic duct ligation (P= 0.0036) were independent factors affecting DFS. Conclusion: Thoracic duct ligation can affect the short-term immune function after thoracoscopic esophagectomy in patients with pT1b-3N0M0 stage, duct ligation, of invasion, of differentiation and of tumor thrombus


Introduction
The immunologists, honored with the 2018 award in Physiology or Medicine, pioneered immunotherapy, which harnesses the body's immune system to ght cancer and achieved great success [1]. The rapid progress has once again proved the in nite power of immune system, and has become the most promising development direction of tumor therapy. Therefore, we have to look back at all aspects of our treatment for tumor from the perspective of immunology. As we all know, lymphocyte recycling is a process of repeated circulation in which lymphocytes, which are designated to reside in the peripheral immune organs, enter the blood circulation through the lymphatic trunk, thoracic duct or right lymphatic duct, pass through the blood circulation to the peripheral immune organs, pass through the high endothelial venules and redistribute in the systemic lymphatic organs and tissues [2]. It is because of this kind of lymphocyte recycling that lymphocytes can be reasonably distributed in various lymphoid tissues and organs in the body. T cells and B cells with speci c antigen receptors constantly travel around the body, increasing the chance of contact with antigen and antigen presenting cells [2]. As the last terminal pathway of lymphatic system into blood circulation, whether the ligation of thoracic duct will cause serious damage to the immune system, thus affecting the recognition and killing of tumor by the immune system of patients, is a subject worthy of study.
There have been different opinions on whether to preventively ligate the thoracic duct during esophagectomy [3][4][5]. With the development of medicine, video-assisted thoracic surgery (VATS) has gradually become the mainstream surgical method for the treatment of early and middle stage of esophageal cancer due to its advantages of small trauma and rapid recovery [6]. Under the new operation method, whether the thoracic duct should be ligated is still in front of us.

Patients
Patients with esophageal cancer admitted to our hospital from June 1, 2017 to June 1, 2021, without serious cardiopulmonary and immune diseases. Squamous cell carcinoma with cT1b-3N0M0 stage invasion con rmed by endoscopic ultrasonography and biopsy were enrolled. Patients with distant metastasis and lymph node metastasis were excluded by positron emission tomography-Computed Tomography(PET-CT). The general conditions of heart Color Doppler ultrasound, lung function, liver and kidney function, blood routine and coagulation were evaluated. Patients with anastomotic leakage, severe pulmonary infection, heart failure and other serious complications were excluded. Postoperative pathology con rmed that patients beyond T1b-3N0M0 stage should also be excluded. The study has been approved by the Committee on Medical Ethics of Taian City Central Hospital, and written informed consent was obtained from all patients.

Surgical procedure
Video-assisted esophagectomy and cervical gastroesophagostomy were performed by the same group of surgeons accordance with the Chinese standard for diagnosis and treatment of esophageal cancer. To identify thoracic duct easily, 100-150 ml olive oil was drunk by the patient 8-12 h before operation.
During operation all arch of azygos vein need to be ligated and transected. Behind lower segment esophagus, the thoracic duct could be easily recognized and separated between the descending aorta and the azygos vein. According to random grouping, the thoracic duct was separated and ligated between the level of diaphragm and inferior pulmonary vein.

Detection of T lymphocyte subsets
Venous blood was drawn before operation and on the 1st day after operation and three weeks after operation. First, the samples were stained with Tritest CD4-FITC/CD8-PE/CD3-PerCP three-color reagents(BD company, USA) and then analyzed by ow cytometer with CellQuest software(BD Biosciences, Franklin Lakes, NJ, USA). By forward scatter and side scatter monitoring, white blood cell was displayed, and lymphocyte populations were gated according to their size and granularity. The nonspeci c binding was determined with isotype control tube, and marker was set for distinguishing uorescence negative and positive cell populations. A minimum of 2,000 lymphocytes were initially acquired. The number of CD3, CD3/CD4, and CD3/CD8 positive lymphocytes could be analyzed based on their Percp, FITC and PE uorescence respectively. By this ow cytometric analysis,the percentages of T lymphocyte, CD4 + T lymphocyte, and CD8 + T lymphocyte in total lymphocytes could be reported [7] .

Follow-up
All patients were reexamined every 3 months in 2 years, every 6 months in 5 years and every year after 5 years. Reexamination contents: blood routine, gastroscope, chest CT (plain scan or enhanced scan), Color Doppler ultrasound for neck and abdominal, etc. when necessary other examinations such as PET-CT, bone scan, MRI, etc. should be supplemented according to the patient's symptoms, signs and routine examination.

Statistical analysis
In the follow-up study we collect the age, gender, T stage (T1b, T2, T3), degree of differentiation (G1, G2, G3), tumor location (upper, middle, lower), whether the thoracic duct was ligated, whether the postoperative pathology indicated vascular cancer thrombus, etc.The data of the two groups were compared by Chi-square test. The t-test were employed for T lymphocyte subsets statistical analysis with statistical signi cance at p < 0.05. DFS (status = 1 for tumor recurrence or metastasis, 0 for deletion) was calculated from the date of operation to metastasis or last follow-up. The results were analysised according to Kaplan-Meier and Cox (proportional hazard) regression with SAS9.2 software. Results 1. After early screening and pathological stage exclusion, a total of 69 patients were enrolled in the study.
In the ligation group, 1 case of anastomotic leakage and 1 case of severe pulmonary infection were excluded. No chylothorax occurred in both groups. Finally, 67 patients entered the study and completed the follow-up. There was no signi cant difference in gender, age, tumor location, depth of invasion, degree of differentiation and presence of tumor thrombus between the thoracic duct ligation group (32 cases) and the non ligation group (35 cases) (P > 0.05). The results are shown in Table 1. weeks after operation (P > 0.05), but there was signi cant difference in T lymphocyte subsets on 1st days after operation (P < 0.05). The results are shown in Table 2.  Table 3 and Fig. 1.

Discussion
With the development of medicine, people pay more and more attention to the unlimited potential of immune system in tumor treatment. Ligation of thoracic duct is an effective measure for the treatment of chylothorax [8]. There have been academic disagreements on whether to preventive ligation during operation [3][4][5]. Our previous study was to explore the effect of thoracic duct ligation on immune function and absorptive function [7,9]. This time, we not only increased the monitoring time to dynamically record the changes of T cell subsets after ligation but also chose T1b-3N0M0 patients to expand the study population and increase the number of participants. In addition, we conducted a longterm follow-up of the patients to detect early metastasis and explore the impact of thoracic duct ligation on DFS.
According to the guidelines, for intramucosal esophageal cancer, we generally choose endoscopic resection rather than thoracoscopic resection [10]. At the same time, PET-CT combined with postoperative pathology was used to exclude the patients beyond T1b-3N0M0 who often need chemoradiotherapy and then have interference factors on immune function and DFS [10] .
Our previous studies for T1N0M0 stage showed that there was no difference between the two groups before operation, the T cells and CD4 + T cell subsets of ligation group were signi cantly lower than those of non ligation group on 1st days after operation, and the CD8 + T cell subsets of ligation group were signi cantly higher than those of non ligation group [7]. In this study, the subjects were extended to T1b-3N0M0 stage, and the same results were obtained. This study was carried out in patients undergoing VATS esophagectomy during which the azygos vein arch often needs to be transected for easier to free esophagus and clean lymphonode. We know there are a lot of communicating branches between azygos vein and thoracic duct [11]. When the thoracic duct and azygous arch are transected at the same time, lymph uid and a large number of lymphocytes in it are di cult return to the blood in a short time. As time goes on, the accumulated lymph always ows back into the blood through the reestablishment of collateral circulation [12]. It's not hard to explain that there was no difference between the two groups again 3 weeks after operation. It can be imagined that the decrease of immune function in a short time after operation might give the residual tumor cells an opportunity to escape and metastasize.
Postoperative follow-up also con rmed this result. Univariate analysis showed that gender, age and tumor location had no signi cant effect on DFS (P > 0.05), but depth of invasion, degree of differentiation, presence of tumor thrombus and thoracic duct ligation had signi cant effect on DFS (P < 0.05). Cox regression analysis showed the same results, especially the preventive thoracic duct ligation was a high risk factor for postoperative metastasis(P = 0.0036, β = 1.48379). Combined with the results of this study, we suggest that prophylactic ligation should be taken when there are signs of thoracic duct injury, otherwise it is not recommended.
The de ciency of this study is that the number of samples is small, and T cell subsets can not be monitored at more time points. Because of the di culty of follow-up,OS evaluation index was not used. We are also trying to add additional follow-up data to improve and perfect our study.

Conclusion
Our study suggests that the prophylactic ligation of thoracic duct during thoracoscopic esophagectomy can lead to the change of T cell subsets, especially the decrease of CD4 + T cells in the early stage after surgery. At the same time, the late follow-up con rmed that the ligation of thoracic duct, the poor differentiation of tumor, the depth of invasion and the vascular tumor thrombus are the independent risk factors for postoperative recurrence and metastasis. The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.