Clinical And Basic Research On The Prevention Of Postoperative Recurrence Of Well-differentiated Pancreatic Neuroendocrine Tumors By Traditional Chinese Medicin

Objective:Pancreatic neuroendocrine tumors(pNENs)are a group of rare tumors with strong heterogeneity.Recently,its incidence has gradually increased,which has attracted the attention of clinicians.According to the differentiation,pNENs were divided into well-differentiated pancreatic neuroendocrine tumors(pNETs)and poorly differentiated pancreatic neuroendocrine carcinomas(pNECs).Surgery is the only way to cure early-stage pNETs patients,but some patients still have recurrence after operation,but the guidelines do not recommend adjuvant treatment for patients after complete excision,even though the patients have high-risk factors of postoperative recurrence.Our center used traditional Chinese medicine to intervene patients after excision,and achieved good results,but the internal mechanism is still unclear.This work aims to explore the high-risk factors of postoperative recurrence of pNETs and whether traditional Chinese medicine can prevent postoperative recurrence of pNETs,and to study its molecular mechanism.Methods:(1)The literatures on postoperative recurrence factors of pNETs in PubMed,EMBASE,Web of science and Cochrane library databases were searched,and the related factors affecting postoperative recurrence were meta-analyzed.(2)The clinical and pathological data of patients with pNETs after radical resection in China Japan Friendship Hospital from August 2012 to March 2021 were retrospectively analyzed.They were divided into traditional Chinese medicine group(35 cases)and simple follow-up group(42 cases).The difference of disease-free survival(DFS)and the factors affecting postoperative recurrence were analyzed.(3)Human pNETs cell lines(QGP-1,BON-1)was used to observe the effect of curcuma zedoary active ingredient curcumin on pNETs cells,such as cell proliferation toxicity test,plate cloning test,scratch test,cell cycle and apoptosis test.Transcriptome sequencing of QGP-1 cells after curcumin intervention were conducted to explore possible tumor related molecular pathways,and the results were verified by western blotting.Results:(1)Ten literatures were finally included in the meta-analysis.Between 1993 and 2018,2863 cases underwent radical resection and 358 cases recurred.The combined recurrence rate was 13%.In addition,G2,lymph node metastasis,positive surgical margin.vascular invasion,and nerve invasion were found to be high-risk factors for postoperative recurrence and the combined HR were 5.40,3.99,2.62,4.13 and 3.39 respectively.There was no significant relationship between gender and functional pNETs postoperative recurrence.(2)In the study of traditional Chinese medicine in preventing postoperative recurrence of pNETs,77 patients,35 patients in traditional Chinese medicine group and 42 patients in simple follow-up group were included.The median follow-up time was 59.7 months(2-134.9 months).The 1-year,3-year and 5-year cumulative disease-free survival rates of patients in the traditional Chinese medicine group were 97.0%,80.8%and 71.8%respectively.The median DFS was not reached.The 1-year,3-year and 5-year cumulative disease-free survival rates of patients in the follow-up group were 78.6%,40.1%and 27.4%respectively,and the median DFS was 26.5 months(95%CI 13.8-39.3).There was significant difference(P=0.001).The results of DFS univariate analysis showed that pathological grade G3,lymph node metastasis,vascular tumor thrombus and stage Ⅲ were the risk factors associated with DFS(P<0.05).Multivariate results showed that traditional Chinese medicine treatment was an independent protective factor for tumor recurrence and metastasis(HR=0.244),and DFS was significantly longer than that in the follow-up group(P=0.005).Multivariate results showed that traditional Chinese medicine treatment was an independent protective factor for tumor recurrence and metastasis(HR=0.244),and DFS was significantly longer than that in the follow-up group(P=0.005).Through the analysis of the traditional Chinese medicine prescription of the traditional Chinese medicine group,it was found that the core pathogenesis of pNETs after operation was the lack of healthy qi and the loss of movement of the spleen.The treatment was to replenish qi and transport the spleen,strengthen the health and suppress tumor,and summed up the experience prescription Qizhen Yiliu decoction.(3)We further explored the internal molecular mechanism of Qizhen Yiliu Decoction in preventing postoperative recurrence of pNETs.With reference to the literature,we selected curcumin,the effective anti-tumor component of zedoary turmeric in the prescription for basic experiments.The results showed that curcumin could significantly inhibit the proliferation of pNETs cell line,weaken the ability of cell cloning,inhibit cell migration,block cell cycle and induce apoptosis.The transcriptome sequencing of QGP-1 cells after curcumin intervention showed that compared to control group,groups with different concentrations(20μM、40 μM、60 μM)curcumin has some changes in tumor related signal pathways,which are commonly seen in PI3K-AKT,ferroptosis,breast cancer,MAPK,basal cell carcinoma,hepatocellular carcinoma signaling pathway.The common signal pathway of the three concentrations is the PI3K-AKT signal pathway,which can be verified by the hint mechanism exploration experiment.The results of western blotting revealed that after 48h curcumin intervention(QGP-1:60 μM,BON-1:30 μM),the total protein contents of PI3K-85 α,AKT and mTOR did not change significantly,but the phosphorylated PI3K-85 α、AKT and mTOR decreased significantly,and the activity of PI3K/AKT/mTOR pathway decreased.Conclusions:(1)Meta analysis found that postoperative recurrence of pNETs is not rare.In addition,G2,positive lymph nodes,positive cutting edges,vascular invasion and peripheral nerve invasion may be high-risk factors for postoperative recurrence of well differentiated pNETs.Patients with these high-risk factors need closer postoperative follow-up,and these patients may benefit from postoperative adjuvant therapy,which still needs to be further verified by a carefully designed large sample randomized controlled trial.(2)The retrospective study of our center found that Zhenqi Yiliu decoction can prevent postoperative recurrence of pNETs.The possible risk factors of recurrence include pathological grade G3,lymph node metastasis,vascular tumor thrombus and stage Ⅲ.Traditional Chinese medicine may be an independent protective factor.(3)Curcumin down regulated the activity of PI3K/AKT/mTOR pathway,which may be the mechanism of its inhibition of the proliferation of pNETs cell lines.