Laparoscopic cholecystectomy based on Laennec approach via the cystic plate with lymphadenectomy in Calot's triangle for gallbladder neoplasms:Initial experience and technical details

Background:It is still challenging to define the exact stage of early gallbladder carcinoma with preoperative imaging.Generally,subserous gallbladder is dissected for the potential early gallbladder carcinoma,which may cause incomplete tumor resection or tumor spread especially for the patients with T2 stage.Here,we reported our experience and safety of Laennec approach via the cystic plate to dissect the whole gallbladder with lymphadenectomy in Calot's triangle for accurate diagnosis and stage in gallbladder neoplasms.Methods:The anatomical gap between Laennec capsule and the cystic plate serves as the landmark to dissect the whole gallbladder through Laennec approach.Laparoscopic cholecystectomy based on Laennec approach via the cystic plate,together with lymphadenectomy in Calot's triangle,was performed in 17 patients with gallbladder neoplasms.Results:All patients had less intraoperative bleeding,no gallbladder breakage,no bile leakage,and accurate intraoperative rapid pathological staging under the corresponding strategies.The duration of surgery was comparable to that of traditional laparoscopic cholecystectomy.Conclusion:Laparoscopic cholecystectomy based on Laennec approach via the cystic plate,together with lymphadenectomy in Calot's triangular is safe for gallbladder neoplasms.In the future,the prospective clinical trial is going on to confirm the feasibility and effectiveness of this approach.

Cholecystoenteric fistula in a patient with advanced gallbladder cancer: A case report and review of literature

BACKGROUND Cholecystoenteric fistula(CEF)involves the formation of a spontaneous ano-malous tract between the gallbladder and the adjacent gastrointestinal tract.Chronic gallbladder inflammation can lead to tissue necrosis,perforation,and fistulogenesis.The most prevalent cause of CEF is chronic cholelithiasis,which rarely results from malignancy.Because the symptoms and laboratory findings associated with CEF are nonspecific,the condition is often misdiagnosed,pre-senting a challenge to the surgeon when detected intraoperatively.Therefore,a preoperative diagnosis of CEF is crucial.We present the case of a 57-year-old male with advanced gallbladder cancer(GBC)who arrived at the emergency room with persistent vomiting,abdominal pain,and diarrhea.An abdominopelvic computed tomography scan revealed a contracted gallbladder with bubbles in the fundus connected to the second por-tion of the duodenum and transverse colon.We suspected that GBC had invaded the adjacent gastrointestinal tract through a cholecystoduodenal fistula(CDF)or a cholecystocolonic fistula(CCF).He underwent multiple examinations,including esophagogastroduodenoscopy,an upper gastrointestinal series,colo-noscopy,and magnetic resonance cholangiopancreatography;the results of these tests con-firmed a diagnosis of synchronous CDF and CCF.The patient underwent a Roux-en-Y gastrojejunostomy and loop ileostomy to address the severe adhesions that were previously observed to cover the second portion of the duodenum and hepatic flexure of the colon.His symptoms improved with supportive treatment while hospitalized.He initiated oral targeted therapy with lenvatinib for further anticancer treatment.CONCLUSION The combination of imaging and surgery can enhance preoperative diagnosis and alleviate symptoms in patients with GBC complicated by CEF.

Effects of cytoreductive surgery combined with hyperthermic perfusion chemotherapy on prognosis of patients with advanced gallbladder cancer

BACKGROUND Gallbladder cancer(GC)is a common malignant tumor and one of the leading causes of cancer-related death worldwide.It is typically highly invasive,difficult to detect in the early stages,and has poor treatment outcomes,resulting in high mortality rates.The available treatment options for GC are relatively limited.One emerging treatment modality is hyperthermic intraperitoneal chemotherapy(HIPEC).HIPEC involves delivering heated chemotherapy directly into the abdominal cavity.It combines the strategies of surgical tumor resection and localized chemotherapy administration under hyperthermic conditions,aiming to enhance the concentration and effectiveness of drugs within the local tumor site while minimizing systemic toxicity.AIM To determine the effects of cytoreductive surgery(CRS)combined with HIPEC on the short-term prognosis of patients with advanced GC.METHODS Data from 80 patients treated at the Punan Branch of Renji Hospital,Shanghai Jiao Tong University School of Medicine between January 2018 and January 2020 were retrospectively analyzed.The control group comprised 44 patients treated with CRS,and the research group comprised 36 patients treated with CRS combined RESULTS The baseline data of the research and control groups were similar(P>0.05).Six days after surgery,the alanine aminotransferase,aspartate aminotransferase,total bilirubin,and direct bilirubin levels significantly decreased compared to the preoperative levels in both groups(P<0.05).However,the values did not differ between the two groups six days postoperatively(P>0.05).Similarly,the postoperative creatinine and blood urea nitrogen levels were significantly lower than the preoperative levels in both groups(P<0.05),but they did not differ between the groups six days postoperatively(P>0.05).Furthermore,the research group had fewer postoperative adverse reactions than the control group(P=0.027).Finally,a multivariate Cox analysis identified the tumor stage,distant metastasis,and the treatment plan as independent factors affecting prognosis(P<0.05).The three-year survival rate in the study group was higher than that in the control group(P=0.002).CONCLUSION CRS combined with HIPEC lowers the incidence of adverse reactions and improves survival in patients with advanced GC.

Regional lymphadenectomy for gallbladder cancer: Rational extent, technical details, and patient outcomes

AIM: To define the rational extent of regional lymphadenectomy for gallbladder cancer and to clarify its effect on long-term survival. METHODS: A total of 152 patients with gallbladder cancer who underwent a minimum of "extended" portal lymph node dissection (defined as en bloc removal of the first-and second-echelon nodes) from 1982 to 2010 were retrospectively analyzed. Based on previous studies, regional lymph nodes of the gallbladder were divided into first-echelon nodes (cystic duct or pericholedochal nodes), second-echelon nodes (node groups posterosuperior to the head of the pancreas or around the hepatic vessels), and more distant nodes. RESULTS: Among the 152 patients (total of 3352 lymph nodes retrieved, median of 19 per patient), 79 patients (52%) had 356 positive nodes. Among nodepositive patients, the prevalence of nodal metastasis was highest in the pericholedochal (54%) and cystic duct (38%) nodes, followed by the second-echelon node groups (29% to 19%), while more distant node groups were only rarely (5% or less) involved. Disease-specific survival after R0 resection differed according to the nodal status (P < 0.001): most node-negative patients achieved long-term survival (median, not reached; 5-year survival, 80%), whereas among nodepositive patients, 22 survived for more than 5 years (median, 37 mo; 5-year survival, 43%). CONCLUSION: The rational extent of lymphadenectomy for gallbladder cancer should include the first-and second-echelon nodes. A considerable proportion of node-positive patients benefit from such aggressive lymphadenectomy.

"Extended" radical cholecystectomy for gallbladder cancer:Long-term outcomes, indications and limitations

AIM:To delineate indications and limitations for "ex tended" radical cholecystectomy for gallbladder cancer:a procedure which was instituted in our department in 1982. METHODS:Of 145 patients who underwent a radi cal resection for gallbladder cancer from 1982 through 2006, 52 (36%) had an extended radical cholecystec tomy, which involved en bloc resection of the gallblad der, gallbladder fossa, extrahepatic bile duct, and the regional lymph nodes (first-and second-echelon node groups). A retrospective analysis of the 52 patients was conducted including at least 5 years of follow up. Residual tumor status was judged as no residual tumor (R0) or microscopic/macroscopic residual tumor (R1 2). athological findings were documented according tothe American Joint Committee on Cancer Cancer Stag ing anual (7th edition). RESULTS:he primary t mor as classified as patho-logical T1 (pT1) in 3 patients, pT2 in 36, pT3 in 12, and pT4 in 1. Twenty three patients had lymph node metastases; 11 had a single positive node, 4 had two positive nodes, and 8 had three or more positive nodes. None of the three patients with pT1 tumors had nodal disease, whereas 23 of 49 (47%) with pT2 or more advanced tumors had nodal disease. One patient died during the hospital stay for definitive resection, giv ing an in hospital mortality rate of 2%. Overall survival (OS) after extended radical cholecystectomy was 65% at 5 years and 53% at 10 years in all 52 patients. OS differed according to the p classification ( < 0.001) and the nodal status ( = 0.010). All of 3 patients with pT1 tumors and most (29 of 36) patients with pT2 tu mors survived for more than 5 years. Of 12 patients with pT3 tumors, 8 who had an R1 2 resection, distant metastasis, or extensive extrahepatic organ involve ment died soon after resection. Of the remaining four pT3 patients who had localized hepatic spread through the gallbladder fossa and underwent an R0 resection, 2 survived for more than 5 years and another survived for 4 years and 2 mo. The only patient with pT4 tumor died of disease soon after resection. Among 23 node positive patients, 11 survived for more than 5 years, and of these, 10 had a modest degree of nodal disease (one or two positive nodes). CONCLUSION:Extended radical cholecystectomy is indicated for pT2 tumors and some pT3 tumors with localized hepatic invasion, provided that the regional nodal disease is limited to a modest degree (up to two positive nodes). Extensive pT3 disease, pT4 disease, or marked nodal disease appears to be beyond the scope of this radical procedure.

Cyclooxygenase-2 promotes angiogenesis by increasing vascular endothelial growth factor and predicts prognosis in gallbladder carcinoma

AIM: To investigate the relationships between the expression of cyclooxygenase-2 (COX-2), vascular endothelial growth factor (VEGF) and the degree of vascularization, clinicopathologic feature, survival time of patients with gallbladder carcinomas. METHODS: Sixty-four gallbladder carcinoma specimens were evaluated for COX-2, VEGF expression by immunohi stochemical methods. Microvessel counts (MVC) were determined using CD34. The relationships between COX-2, VEGF expression, CD34-stained MVC, clinicopathologic features and survival time were analyzed. The correlations between COX-2 and VEGF expression, CD34-stained MVC were also investigated. RESULTS: COX-2, VEGF immunoreactivity were observed in 71.9% (46/64) and 54.7% (35/64) specimens, respectively. The average MVC in 64 cases of gallbladder carcinoma was 57±14 per high power vision field. The status of MVC was closely correlated with Nevin staging, tumor differentiation and lymph node metastasis (P<0.01, 0.002, and 0.003, 0.000, respectively). Increased VEGF expression was significantly correlated with tumor differentiation (poorly and moderately>well differentiated, P<0.05, P = 0.016). Clinical stages had no relation with the expression of VEGF (P>0.05, P = 0.612). There was a positive correlation between COX-2 expression and clinical stages. The positive rate of COX-2 was higher in cases of Nevin stages S4-S5 (81.8%) than in those of Nevin stages S1-S3 (50.0%) with a statistical significance (P0.01, P = 0.009). The expression of COX-2 did not vary with differentiation (P>0.05, P= 0.067). Statistically significant differences were also observed according to lymph node metastasis, COX-2 expression and VEGF expression (P<0.01,0.000, and 0.001, respectively). There was no relation between VEGF, COX-2 expression, MVC and the age and sex of patients. MVC and VEGF positive rate in the COX-2 positive gallbladder carcinoma tissue was higher than that in the COX-2 negative tissue (P<0.05, 0.000, and 0.032, respectively). Patients with VEGF, COX-2 positive tumors had a significantly shorter survival time than those with negative tumors (P<0.05,0.004, 0.01, respectively). CONCLUSION: Augmented tumor neovascularization induced by VEGF may be one of the several effects of COX-2 responsible for poor prognosis of human gallbladder carcinoma. COX-2 inhibitor, either in combination therapy with other agents, or for chemoprevention, may be effective via suppression of angiogenesis in this fatal disease.

Unsuspected Gallbladder Cancer During or After Laparoscopic Cholecystectomy

Objective To summarize the clinical features and outcomes of unsuspected gallbladder carcinoma （HGC） detected during or after laparoscopic cholecystectomy. Methods Medical records of 8005 patients, who underwent laparoscopic cholecystectomy in Peking Hnion Medical College Hospital between June 1993 and June 2011, were reviewed. Patients that pathologically diagnosed as HGC were retrospectively studied in terms of clinical features, preoperative and postoperative diagnosis, surviving period, and complications. Results In the 8005 patients who received laparoscopic cholecystectomy, 36 （0.45%） were diagnosed as LIGC during （25 patients） or after （11 patients） laparoscopic cholecystectomy. The gallbladder cancer was staged as T1 in 16 patients, T2 in 11 patients, and T3 in 9 patients. The 1-, 3-, and 5-year survival rates of all the patients were 88.9% （32/36）, 63.9% （23/36）, and 58.3% （21/36）. The 5-year survival rates in T1 stage, T2 stage, and T3 stage patients were 100%, 75.0%, and 0.0%, respectively. Conclusions The survival rate of HGC is associated with tumor stage, not with operation approaches. Laparoscopic cholecystectomy is appropriate for T 1 patients.

Genetic changes of p53,K-ras,and microsatellite instability in gallbladder carcinoma in high-incidence areas of Japan and Hungary

AIM： To disclose geographic differences in genetic changes involved in gallbladder carcinogenesis between two distinct high-incidence areas of Japan and Hungary. METHODS： We examined 42 cases of gallbladder carcinoma： 22 Japanese and 20 Hungarian cases, p53 mutations at exons 5 to 8 and K-ras mutations at codon 12 were tested by direct sequencing. Microsatellite instability was determined from fluorescent dye-labeled PCR amplifications of flve-microsatellite markers （BAT-25, BAT-26, D2S123, DSS346, and D17S250）. RESULTS： Mutations of p53 were detected in 11 of 22 Japanese cases and 6 of 18 Hungarian cases （11/22 vs 6/18, P = 0.348）. Transition at CpG sites was found in none of 11 Japanese cases and 2 of 6 Hungarian cases; the difference was marginally significant （0/11 vs 2/6,P = 0.110）. K-ras mutations were detected in only one of the Hungarian cases. Eight of 19 （42.1%） ]apanese cases were MSI-high （presence of novel peaks in more than one of the five loci analyzed）, whereas only 1 of 15 （6.7%） Hungarian cases was MSI-high （P = 0.047）. CONCLUSION： It appears that the p53 mutations and MSI differ in patients with gallbladder carcinoma between two distinct high-incidence areas. Geographic variation might exist in the process of gallbladder carcinogenesis.

Analysis of p53 and vascular endothelial growth factor expression in human gallbladder carcinoma for the determination of tumor vascularity

AIM： To examine the expression of p53 and vascular endothelial growth factor （VEGF） as well as microvessel count （MVC） and to investigate the role of VEGF as an angiogenic marker and the possible role of p53 in the regulation of angiogenesis in human gallbladder carcinoma. METHODS： Surgically resected specimens of 49 gallbladder carcinomas were studied by immunohistochemical staining for p53 protein, VEGF, and factor VIII-related antigen. VEGF expression and mutant p53 expression were then correlated with Nevin stage, differentiation grade, MVC, and lymph node metastasis. RESULTS： Positive p53 protein and VEGF expressions were found in 61.2% and 63.3% of tumors, respectively. p53 and VEGF staining status was identical in 55.1% of tumors. The Nevin staging of p53- or VEGF-positive tumors was significantly later than that of negative tumors. The MVC in p53- or VEGF-positive tumors was significantly higher than that in negative tumors, and MVC in both p53- and VEGF-negative tumors was significantly lower than that in the other subgroups. CONCLUSION： Our findings suggest that pS3-VEGF pathway can regulate tumor angiogenesis in human gallbladder carcinoma. Combined analysis of p53 and VEGF expression might be useful for predicting the tumor vascularity of gallbladder cancer.

A clinicopathological analysis in unsuspected gallbladder carcinoma: A report of 23 cases

AIM： To study the clinicopathological characteristics of unsuspected gallbladder carcinoma （UGC）. METHODS： We retrospectively studied 23 cases of UGC in Tongji Hospital, and compared their clinicopathological characteristics with 33 cases of preoperatively diagnosed gallbladder carcinoma （PDGC）. RESULTS： The proportion of UGC coexisting with cholecystolithiasis was significantly higher than that of PDGC （x^2 = 13.53, P 〈 0.01）. The infection rate of hepatitis B virus was 21.74% （5/23） in UGC and 30.30% （10/33） in PDGC. Nine （39.13%） of 23 patients with UGC and 8/33 （24.24） PDGC had contact with schistosome pestilent water. The rate of multiple pregnancies was 56.52% （13/23） in the patients with UGC and 42.42% （14/33） in PDGC. The primary location of the UGC was mostly in the neck and body of the gallbladder, and that of the PDGC was often in the body and bottom. The incidence of Nevin stage I and 11 UGC was significantly higher than that of PDGC （x^2 = 4.44, P 〈 0.05 and 2 = 4.96, P 〈 0.05） while that of Nevin stage V UGC was significantly lower than that of PDGC （x^2 = 7.59, P 〈 0.01）. According to the grading of carcinoma, the incidence of well-differentiated UGC was significantly higher than that of PDGC （2 = 4.16, P 〈 0.05）, and that of poorlydifferentiated UGC was significantly lower than that of PDGC （x^2 = 4.48, P 〈 0.05）.CONCLUSION： There are different characteristics between UGC and PDGC, such as in primary location, malignant degree and incidence of coexistence with cholecystolithiasis. Cholecystolithiasis, hepatitis B, schistosome and multiple pregnancies were high risk factors for gallbladder carcinoma.

Intraluminal versus infiltrating gallbladder carcinoma:Clinical presentation,ultrasound and computed tomography

AIM： To compare clinical presentation and ultrasound （US） and computed tomography （CT） sensitivity between intraluminal and infiltrating gallbladder carcinoma （GBCA）. METHODS： This retrospective study evaluated 65 cases of GBCA that were categorized morphologically into the intraluminaI-GBCA （n = 37） and infiltrating-GBCA （n = 28） groups. The clinical and laboratory findings, presence of gallstones, gallbladder size, T-staging, nodal status, sensitivity of preoperative US and CT studies, and outcome were compared between the two groups. RESULTS： There were no significant differences between the two groups with respect to female predominance, presence of abdominal pain, serum aminotransferases level, T2-T4 staging, and regional metastatic nodes. Compared with the patients with intraluminaI-GBCA, those with infiltrating-GBCA were significantly older （65.49 ± 1.51 years vs 73.07 ± 1.90 years）, had a higher frequency of jaundice （3/37 patients vs 13/28 patients） and fever （3/37 patients vs 10/28 patients）, higher alkaline phosphatase （119.36 ± 87.80 IU/L vs 220.68 ± 164.84 IU/L） and total bilirubin （1.74 ± 2.87 mg/L vs 3.50 ± 3.51 mg/L） levels, higher frequency of gallstones （12/37 patients vs 22/28 patients）, smaller gallbladder size （length, 7.47± 1.70 cm vs 6.47 ± 1.83 cm; width, 4.21 ± 1.43 cm vs 2.67 ± 0.93 cm）, and greater proportion of patients with 〈 12 mo survival （16/37 patients vs 18/28 patients）. The sensitivity for diagnosing intraluminal- GBCA with and without gallstones was 63.6% and 91.3% by US, and 80% and 100% by CT, respectively. The sensitivity for diagnosing infiltrating-GBCA with and without gallstones was 12.5% and 25% by US, and 71.4% and 75% by c-r, respectively. CONCLUSION： In elderly women exhibiting small gallbladder and gallstones on US, especially those with jaundice, fever, high alkaline phosphatase and bilirubin levels, CT may reveal concurrent infiltrating-GBCA.

Clinical relationship between MDR1 gene and gallbladder cancer

BACKGROUND: The most common mechanisms of mul- tidrug resistance (MDR) in cancer cells is the expression of an energy-dependent exfflux pump. P-glycoprotein (P-gp) encoded by MDR1 gene and multidrug associated protein (MRP) are well known proteins associated with MDR. In human cancers, the MDR1 gene expression is common in patients with intrinsic and acquired MDR. It is a major therapeutic problem in cancer chemotherapy. Previously we found that the MDR of HCC is related to MRP gene ex- pression and initiates the intrinsic MDR. The aim of this study is to study the expression of MDR1 gene encoding P-gp and MDR1 mRNA in primary gallbladder carcinoma, and analyze its clinical significance. METHODS: Immunohistochemistry (IHC) S-P method and in situ polymerase chain reaction (ISPCR) were used to detect the expression of P-gp and MDR1 mRNA in 53 cases of untreated primary gallbladder carcinoma and 12 ca- ses of cholecystitis (archival paraffin-embedded tissues). RESULTS: The positive expression rates of P-gp and MDR1 mRNA in the 53 cases and 12 cases were 60.38%, 71.69% and 25.00%, 33.33%, respectively. There was a significant difference between the two groups (P<0.05). The positive expression rate of P-gp and MDRlmRNA were 69.44%, 83.33% and 41.18%, 47.06% respectively in tissues in stage of Nevin against Nevin , (P<0.05). In well, moderately differentiated gallbladder carcinoma tissues, their expressions were 79.49%, 69.23% against 50.00%, 35.71% in low, undifferentiated tissues (P<0.05). CONCLUSIONS: MDR to gallbladder carcinoma is closely related to the intrinsic MDR and it provides an important evidence to reverse the MDR by detection of the MDR1gene. Meanwhile, MDR1 gene expression in gallbladder carcinoma is correlated with some biological characteris- tics , takes part in the carcinogenesis of gallbladder tissues, and acts as a valuable biomarker of prognosis.

Preoperative lymphocyte to C-reactive protein ratio as a new prognostic indicator in patients with resectable gallbladder cancer

Background:Inflammation is often related to cancer,and several inflammatory scores have been established to predict the prognosis of various types of cancer.Our study aimed to determine the prognostic value of the preoperative lymphocyte to C-reactive protein ratio(LCR)for predicting postoperative outcomes in patients with resectable gallbladder cancer(GBC).Methods:A retrospective analysis of 104 GBC patients who received curative surgery at Xinhua Hospital,Affiliated to Shanghai Jiao Tong University School of Medicine from January 2000 to December 2016 was performed.A time-dependent receiver operating characteristic curve was constructed to evaluate the accuracy of different markers.Univariate and multivariate Cox proportional hazard models were used to define factors associated with overall survival.Results:Among the assessed variables,the preoperative LCR showed the highest accuracy in predicting the overall survival of GBC patients(AUC:0.736).Decreased preoperative LCR was significantly associated with advanced tumor stage,including tumor invasion(P=0.018),lymph node metastasis(P=0.011)and TNM stage(P=0.022).A low preoperative LCR(cutoff threshold=145.5)was an independent risk factor for overall survival in patients with resectable GBC(P<0.001).Conclusions:The preoperative LCR is a novel and valuable prognostic indicator of postoperative survival in patients with resectable GBC.

Perimuscular connective tissue contains more and larger lymphatic vessels than the shallower layers in human gallbladders

AIM： To clarify whether perimuscular connective tissue contains more lymphatic vessels than the shallower layers in human gallbladders. METHODS： Lymphatic vessels were stained immunohistochemically with monoclonal antibody D2-40, which is a specific marker of lymphatic endothelium, in representative sections of 12 normal human gallbladders obtained at the time of resection for colorectal carcinoma liver metastases. In individual gallbladder specimens, nine high-power （×200） fields with the highest lymphatic vessel density （LVD）, termed ＂hot spots＂; were identified for each layer （mucosa, muscle layer, and perimuscular connective tissue）. In individual hot spots, the LVD and relative lymphatic vessel area （LVA） were measured microscopically using a computer-aided image analysis system. The mean LVD and LVA values for the nine hot spots in each layer were used for statistical analyses. RESULTS： In the mucosa, muscle layer, and perimuscular connective tissue, the LVD was 16.1 ± 9.2, 35.4 ± 15.7, and 65.5 ± 12.2, respectively, and the LVA was 0.4 ± 0.4, 2.1 ± 1.1, and 9.4 ± 2.6, respectively. Thus, both the LVD and LVA differed significantly （P 〈 0.001 and P 〈 0.001, respectively; KruskaI-Wallis test） among the individual layers of the wall of the gallbladder, with the highest LVD and LVA values in the perimuscular connective tissue. Most （98 of 108） of the hot spots within the perimuscular connective tissue were located within 500 μm of the lower border of the muscle layer. CONCLUSION： The perimuscular connective tissue contains more and larger lymphatic vessels than the shallower layers in the human gallbladder. This observation partly explains why the incidence of lymph node metastasis is high in T2 （tumor invading the perimuscular connective tissue） or more advanced gallbladder carcinoma.

Fork head box M1 regulates vascular endothelial growth factor-A expression to promote the angiogenesis and tumor cell growth of gallbladder cancer

BACKGROUND Gallbladder cancer(GBC)is an aggressive type of biliary tract cancer that lacks effective therapeutic targets.Fork head box M1(FoxM1)is an emerging molecular target associated with tumor progression in GBC,and accumulating evidence suggests that vascular endothelial growth factor(VEGF)promotes various tumors by inducing neoangiogenesis.AIM To investigate the role of FoxM1 and the angiogenesis effects of VEGF-A in primary GBC.METHODS Using immunohistochemistry,we investigated FoxM1 and VEGF-A expression in GBC tissues,paracarcinoma tissues and cholecystitis tissues.Soft agar,cell invasion,migration and apoptosis assays were used to analyze the malignant phenotype influenced by FoxM1 in GBC.Kaplan-Meier survival analysis was performed to evaluate the impact of FoxM1 and VEGF-A expression in GBC patients.We investigated the relationship between FoxM1 and VEGF-A by regulating the level of FoxM1.Next,we performed MTT assays and Transwell invasion assays by knocking out or overexpressing VEGF-A to evaluate its function in GBC cells.The luciferase assay was used to reveal the relationship between FoxM1 and VEGF-A.BALB/c nude mice were used to establish the xenograft tumor model.RESULTS FoxM1 expression was higher in GBC tissues than in paracarcinoma tissues.Furthermore,the high expression of Foxm1 in GBC was significantly correlated with a malignant phenotype and worse overall survival.Meanwhile,high expression of FoxM1 influenced angiogenesis;high expression of FoxM1 combined with high expression of VEGF-A was related to poor prognosis.Attenuated FoxM1 significantly suppressed cell proliferation,transfer and invasion in vitro.Knockdown of FoxM1 in GBC cells reduced the expression of VEGF-A.Luciferase assay showed that FoxM1 was the transcription factor of VEGF-A,and knockdown VEGF-A in FoxM1 overexpressed cells could partly reverse the malignancy phenotype of GBC cells.In this study,we found that FoxM1 was involved in regulation of VEGF-A expression.CONCLUSION FoxM1 and VEGF-A overexpression were associated with the prognosis of GBC patients.FoxM1 regulated VEGF-A expression,which played an important role in the progression of GBC.

Effect of norcantharidin on proliferation and invasion of human gallbladder carcinoma GBC-SD cells

AIM: To investigate the effect of norcantharidin on proliferation and invasion of human gallbladder carcinoma GBC-SD cells in vitro and its anticancer mechanism. METHODS: Human gallbladder carcinoma GBC-SD cells were cultured by cell culture technique. The growth and the invasiveness of GBC-SD cells in vitro were evaluated by the tetrazolium-based colorimetric assay and by the Matrigel experiment and the crossing-river test. Expression of PCNA, Ki-67, MMP2 and TIMP2 proteins of GBC-SD cells was determined by streptavidin-biotin complex method. RESULTS: In vitro norcantharidin inhibited the growth and proliferation of GBC-SD cells in a dose- and time-dependent manner, with the IC50 value of 56.18 μ/mL at 48 h. Norcantharidin began to inhibit the invasion of GBC-SD cells at the concentration of 5 μg/mL, and the invasive action of GBC-SD cells was inhibited completely and their crossing-river time was prolonged significantly at 40 μg/mL. After treatment with norcantharidin, the expression of PCNA, Ki-67, and MMP2 was significantly decreased. With the increase in TIMP2 expression, the MMP2 to TIMP2 ratio was decreased significantly (P<0.05). CONCLUSION: Norcantharidin inhibits the proliferation and growth of human gallbladder carcinoma cells in vitro at relatively low concentrations by inhibiting PCNA and Ki-67 expression. Its anti-invasive activity may be the result of decrease in MMP2 to TIMP2 ratio and reduced cell motility.

Norcantharidin inhibits growth of human gallbladder carcinoma xenografted tumors in nude mice by inducing apoptosis and blocking the cell cycle in vivo

BACKGROUND: Gallbladder carcinoma, a lethal malignant neoplasm with poor prognosis, has dismal results of surgical resection and chemoradiotherapy. We previously reported that norcantharidin (NCTD) is useful against growth, proliferation, and invasion of human gallbladder carcinoma GBC-SD cells in vitro. In this study, we further studied the inhibitory effect of NCTD on the growth of xenografted tumors of human gallbladder carcinoma in nude mice in vivo and the underlying mechanisms. METHODS: The tumor xenograft model of human gallbladder carcinoma in nude mice in vivo was established with subcutaneous GBC-SD cells. The experimental mice were randomly divided into control, 5-FU, NCTD, and NCTD+5-FU groups which were given different treatments. Tumor growth in terms of size, growth curve, and inhibitory rate was evaluated. Cell cycle, apoptosis, and morphological changes of the xenografted tumors were assessed by flow cytometry and light/electron microscopy. The expression of the cell cycle-related proteins cyclin-D1 and p27 as well as the apoptosis-related proteins Bcl-2, Box, and survivin were determined by the streptavidin-biotin complex (SABC) method and RT-PCR. RESULTS: NCTD inhibited the growth of the xenografted tumors in a dose- and time-dependent manner. Tumor volume decreased (5.61+/-0.39 vs. 9.78+/-0.61 cm(3), P=0.000) with an increased tumor inhibitory rate (42.63% vs. 0%, P=0.012) in the NTCD group compared with the control group. The apoptosis rate increased (15.08+/-1.49% vs. 5.49+/-0.59%, P=0.0001) along with a decreased percentage of cells in S phase (43.47+/-2.83% vs. 69.85+/-1.96%, P=0.0001) in the NTCD group compared with the control group. The morphological changes of apoptosis such as nuclear shrinkage, chromatin aggregation, chromosome condensation, and typical apoptosis bodies in the xenografted tumor cells induced by NCTD were observed by light and electron microscopy. The expression of cyclin-D1, Bcl-2 and survivin proteins/mRNAs decreased significantly, with increased expression of p27 and Bax proteins/mRNAs in the NCTD group compared with the control group. CONCLUSION: NCTD inhibits the growth of xenografted tumors of human gallbladder carcinoma in nude mice by inducing apoptosis and blocking the cell cycle in vivo.

Surgical therapy and prognosis of sarcomatoid carcinoma of the gallbladder

BACKGROUND: Sarcomatoid carcinoma of the gallbladder is rare and its characteristics are poorly understood. This study aimed to understand the behavior and prognosis of sarcomatoid carcinoma of the gallbladder as well as its clinical manifestations and survival rate of patients after radical or palliative surgery, and to review the reported data worldwide and our 10 patients. METHODS: From 2004 to 2009, ten patients were pathologically diagnosed with sarcomatoid carcinoma of the gallbladder and underwent operation at our center. These characteristics, clinical presentations, tumor-node-metastasis (TNM) staging, surgical modes, and prognosis were reviewed, retrospectively. We collected the data of 46 patients reported in the English-language literature worldwide and analyzed the survival with ours. The survival rate was estimated using the Kaplan-Meier method, and was compared using the log-rank test. RESULTS: The median age of the 10 patients was 67 years (inter-quartile range 59-74 years), and the size of tumor inter-quartile ranged from 3.1 to 7.9 cm. In this series, 9 patients received radical surgery, and one undewent palliative surgery. There was no surgical mortality, and one patient underwent a second operation because of liver metastasis. The median survival time of the patients was 9 months (inter-quartile range 6-12 months), with 3 patients still being alive until follow-up; however, two patients had tumor recurrence. The data from the 56 patients (10 patients in our series and 46 reported elsewhere) statistically indicated that the median age was 66 years (inter-quartile range 61-74.5 years) and the overall median survival was 5.5 months (inter-quartile range 2.5-10 months). The survival time in the patients undergoing radical surgery (n=42) was significantly longer than that in the patients undergoing palliative surgery (n=14) (P=0.031). CONCLUSIONS: The survival of the patients with sarcomatoid carcinoma of the gallbladder is poor. Some patients may die shortly after the surgery because of recurrence or metastasis. However, radical surgery is still necessary if possible. (Hepatobiliary Pancreat Dis Int 2010; 9: 175-179)

Influence of norcantharidin on proliferation,proliferation-related gene proteins prolifera-ting cell nuclear antigen and Ki-67 of human gallbladder carcinoma GBC-SD cells

BACKGROUND: Gallbladder carcinoma is a highly lethal and aggressive disease with early metastasis, strong invasion and poor prognosis. Most patients with this disease are at the advanced and un-resectable stage and should be consi- dered for palliative treatment such as chemotherapy and ra- diotherapy. Unfortunately, reports of chemotherapy and radiotherapy for gallbladder carcinoma are disappointing. We investigated the influence of norcantharidin (NCTD) on proliferation, proliferation-related gene proteins PCNA and Ki-67 of human gallbladder carcinoma GBC-SD cells in vitro. METHODS: GBC-SD cell lines of human gallbladder carci- noma were cultured by the cell culture technique. The ex- periment was divided into NCTD group and control group. The tetrazolium-based colorimetric assay was used to evaluate cell growth. The streptavidin-biotin complex method was used to determine the expressions of prolifera- tion-related gene proteins PCNA and Ki-67 of human gall- bladder carcinoma GBC-SD cells. RESULTS: NCTD inhibited the growth and proliferation of GBC-SD cells from 10 mg/L or after 6 hours in a dose- and time-dependent manner, with the IC50 value of 56.18 μg/ ml at 48 hours. After treatment with NCTD, the expression of PCNA (0.932 ±0.031 vs. 0.318 ±0.023, P<0.001) and Ki-67 (0.964 ±0.092 vs. 0.297 ±0.018, P<0.001) proteins were decreased significantly. CONCLUSION: NCTD inhibits the proliferation of human gallbladder carcinoma GBC-SD cells in vitro and the expres- sion of their proliferation-related gene proteins PCNA and Ki-67.

Gallbladder sarcomatoid carcinoma:Seven case reports

BACKGROUND Gallbladder sarcomatoid carcinoma is a rare and aggressive tumor,and little is known about its clinical behavior,prognosis,and optimal treatment.CASE SUMMARY From 1997 to 2017,we collected seven cases of gallbladder sarcomatoid carcinoma at our institution.The median patient age was 68.5 years.Six(85.7%)patients were female.Overall,85.7%(6/7)of the tumors had a maximal diameter greater than 7 cm.Late TNM stage was associated with a significantly poor prognosis.All patients with advanced-stage(III/IV)disease died from metastases or disease progression shortly after surgery.One patient with stage IIIB disease who received adjuvant chemoradiotherapy(gemcitabine and capecitabine)achieved a progression-free survival(PFS)of 12 mo and overall survival of 15 mo,which might be the longest PFS reported among patients who ultimately experienced recurrence or metastasis.CONCLUSION Sarcomatoid carcinoma is a unique and aggressive gallbladder malignancy.Surgery is suggested as the first and only recognized treatment.There is a significant difference in prognosis between patients with early-stage and advanced-stage disease.Postoperative adjuvant therapy may bring survival benefits for locally advanced patients.Gemcitabine combined with fluorouracil and radiotherapy could be a potential strategy.