Clinical Study And Relating Mechanism For The Effect Of Pringle Manoeuvre On Postoperative HCC Recurrence

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, especially in China, where it has been ranked as the second leading cancer killer since the 1990s. Chronic hepatitis B virus (HBV) infection is the most common etiology here. Until now hepatic resection remains the best choice of liver cancer therapeutic strategies, but the long-term survival is still unsatisfactory. Although great advances have achieved in the diagnosis and treatment of HCC, the long-term prognosis is unsatisfactory due to a high incidence of tumor recurrence, ranging from 50% to 60%. The reason for high recurrence rate is not totally clear.From previous experience, we know that Pringle Manoeuvre(PM)was a surgical art adopted widely in hepatectomy during the past whole century and it could limit blood loss greatly. However, the surgical stress such as ischemia-reperfusion (I/R) injury elicited by PM seems to be inevitable and results in complex metabolic, immunological, and microvascular changes, which altogether might contribute to hepatocellular damage and dysfunction. In the past, different effects of PM have been widely discussed. To our interests, its effect on long-term prognosis of oncologic patients and behavior of the tumor cell was not deliberately mentioned. Recent studies indicated that I/R injury of the liver might be a significant factor, which can promote the tumor recurrence and metastasis in animal models. If it is also a truth in human, there must be a big challenge to the PM. To date, there are no such reported data or RCT studies conducted for review. Based on above-mentioned information, we hypothesized that the long-term prognosis of HCC patients could be worsened by the PH due to its side effect on tumor recurrence of the liver remnant.In clinical situation, there are more impact factors such as blood loss, blood transfusion, liver function, HBV loads, etc, that affect the prognosis of cancer patients in different ways. Results obtained in laboratory may be conflicted or even reversed by clinical factors. Whatever, it's an important and urgent issue in liver surgery, which may lead to a big change to nowadays'knowledge. Therefore we conducted an ambispective cohort study, case-control study and a prospective multicenter RCT to answer this question,also with its relating mechanism.Materials and methods1. Ambispective Cohort studyFrom December 2000 to July 2008, 730 consecutive HCC patients received hepatectomy in the Institute of Hepatobiliary Surgery of Southwest Hospital, and 469 eligible patients were selected for ambispective cohort study. The exclusion criteria included tumor positive lymph nodes in the abdomen and other exclusion items. Then they were divided into two subgroups as Pringle group and non-Pringle group according to the exposure of PM during hepatectomy. Tumor recurrence-free survival and overall survival were adopted to evaluate the primary outcome. Intraoperative blood loss and transfusion, time consumption of the hepatectomy, hospital stay and severe postoperative complications were taken as the secondary outcome measures. This study was started on the August 1, 2008 and ended on the July 31, 2009.2. Case control studyBased on cohort population, we conducted a 1-year nested case control study. The observation period was set to 1 year following the primary hepatectomy. During this period, patients with the diagnosis of tumor recurrence and cancer related death were determined and taken as"Cases"respectively in case group. Those who had a disease free survival (DFS) and overall survival more than one year were enrolled as the candidates of the control group. Then we matched each case to a control candidate by sex, age and HBsAg. Finally, we got 199 pairs for 1-year DFS analysis and 124 pairs for 1-year overall survival analysis, respectively. For the sake of high prevalence incidence of tumor recurrence at the end 3-year after the primary hepatectomy, there will be much more patients in case group than control group. Then the design of nested case control study is not feasible. Hence we conducted a case cohort analysis for the 5-y survivors.400 eligible patients were randomly selected from the previous cohort just beginning of the follow-up as the control cohort. Then we enrolled all the patients with tumor recurrence during the five years into the case group. We aimed to find out the risk factors correlated with tumor recurrence through the comparison and statistical analysis of case group and control cohort.Patients with the diagnosis of cancer-related death during five years were enrolled into case group. Compared with the same control cohort, we conducted a case cohort study to find out the risk factors correlated with 5-year overall survival.3. Based on 142 HCC samples, we construct a tissue microarrays and aim to detect the expression of E-Cadherin, N-Cadherin, Vimentin, HIF1α, TWIST and SNAIL1, then to analyze their internal correlation and correlation with DFS and OS of HCC patients.4. By using 50μM H2O2, we built up a short-term cell hypoxic model to examine the changes of E-Cadherin, Vimentin, HIF1α, TWIST and SNAIL1. After then, a model of nude mice was used to investigate the biological changes of HepG2 cell line undergone short-term hypoxic and reoxygenation.5. Ad-HIF1αadenovirus vector and Ad-HIF1αsiRNA- pSES-HUS were transferred separately into HepG2 cells in order to overexpression and interfere expression of HIF1αto study the key role of HIF1αon the regulation of down stream nuclear transcription factors and E-Cadherion, Vimentin.6. We conducted a multi-center prospective randomized surgical trial to assess the long-term effect of PM on the tumor recurrence of HCC after curative resection. The trial will test whether a hepatic resection without Pringle maneuver for the HCC patients can reduce the recurrence rate and improve the recurrence-free period of patients. At least 760 eligible patients from five participating centers will be included and randomized into the Pringle group and non-Pringle group at the ratio of 1:1 according to a permuted-randomization protocol. Each patient will received a 5-year follow-up schedule. The primary objective is to compare the disease free survival of patients in the Pringle group with non-Pringle group.Results1. We have built up a cohort of 469 patients who had undergone curative resection and also met with the inclusion of this study. 261 patients who had undergone hepatectomy with PM during the operation were enrolled into"Pringle"group and the other 208 patients without application of PM were enrolled into"Non-Pringle"group. For the 469 patients, median DFS period was 447.00 days. The 1-, 3- and 5-year accumulative DFS rate was 54.0%, 29.0% and 19.1%. Overall survival period of the 469 patients was 1275.48±63.46 days with a median of 849.00±92.75 days in this study. Accordingly, 1-, 3-, 5-year overall survival rate was 74.5%, 44.9% and 31.2%, respectively.2. Median DFS period was 294.00 days in Pringle group,and 624.00 days in Non-Pringle group, respectively. The 1-,3-,5-year accumulative DFS rate was 47.6%,26.0% and 15.1% in Pringle group, and 61.7%,32.7% and 26.8% for 208 patients in Non-Pringle group. Obviously, there was a superior DFS rate in Non-Pringle(P=0.001) with a indication that PM may facilitate postoperative tumor recurrence. Overall survival period (median) in Pringle and non-Pringle group was 701.00 days and 1168.00 days following the primary hepatectomy. The 1-,3-,5-year overall survival rate (69.9%,39.2%,26.1%) in Pringle group was lower than Non-pringle group(80.2%,51.3%,38.0%),which indicated that hepatectomy without Pringle may benefit the HCC patients in overall survival(P=0.002).3. For stratified analysis,it was showed that PM has a significant effect on the prognosis at the level of tumors≤5cm, HBV-DNA <103, presence of liver cirrhosis, without portal hypertension, segments involvement less than 3, Edmondson grading ofⅠ/Ⅱ, T1 stage and no need of blood transfusion.4. According to the result of RR and AR value, we found out that curative resection of HCC without PM might reduce the risk of tumor recurrence, especially in the first year.5. A reciprocal effect was observed between PM and other risk factors including numbers of segment involvement, boundary of tumor foci, Edmondson grade, numbers of tumor nodules and blood loss.6. Whether the PM was used or not during hepatectomy was not correlated with secondary outcome measures including intraoperative bleeding/transfusion requirement, overall/ postoperative hospital stay and severe operative complications, but the different types (portal hepatis, hemihepatic, total vascular occlusion) and duration of blood occlusion may affect time consumption of hepatectomy and postoperative recovery of serum AST.7. PM was the independent risk factor for 1-year DFS and 1-, 5-year overall survival.8. The result from TMA analysis showed that EMT was popular in HCC and a close relationship with worse prognosis was found. HIF1αpositive was closely correlated with the overexpression of TWIST and SNAIL1 in sample of HCC and they all had influence on the expression of E-Cadherin and Vimentin.9. By treating L02 and HepG2 cell with 50μM H2O2, it was showed that transient hypoxic of 30 min had no effect on cell morphologic changes, however it increased the expression of HIF1α, SNAIL1, TWIST, Vimentin and decrease the expression of E-Cadherin. Meanwhile, an increased production of ROS was found in HepG2 cell.10. Interfere the expression of HIF1αcould diminish the effect of hypoxic on TWIST, SNAIL1, E-Cadherion and Vimentin in HepG2 cell line. But overexpression of HIF1αwill improve such a effect. HIF1αmight play a key role in such a cell model. 11.This multicenter prospective RCT have been registered and approved by the local ethics committee, primary result will be anticipated in 2015.Main conclusion1. Whether the PM is performed or not is not correlated with perioperative recovery, but there lies a disadvantage of PM on long-term outcome of HCC patients, especially for 1-year DFS and OS.2. EMT was popular in the HCC and also correlated with a worse prognosis. E-Cadherin may play important roles in such a pathological course.3. A transient hypoxic state of HepG2 cell result a significant increase in HIF1α, TWIST, SNAIL1 and Vimentin, also with an enhancement of capability of invasion and tumorgenesis which indicated that short-term hypoxic might be a risk factor for HCC recurrence.4. HIF1αacts as a key upstream regulator of TWIST, SNAIL1 in the molecular changes of such a short-term hypoxic state.5. We conducted a prospective RCT to evaluate the long-term effect of PM on tumor recurrence of HCC after curative resection, which will afford more powerful evidence of PM'S adverse effect.