Current surgical treatment for bile duct cancer

Since extrahepatic bile duct cancer is difficult to diagnose and to cure, a safe and radical surgical strategy is needed. In this review, the modes of infiltration and spread of extrahepatic bile duct cancer and surgical strategy are discussed. Extended hemihepatectomy, with or without pancreatoduodenectomy （PD）, plus extrahepatic bile duct resection and regional lyrnphadenectomy has recently been recognized as the standard curative treatment for hilar bile duct cancer. On the other hand, PD is the choice of treatment for middle and distal bile duct cancer. Major hepatectomy concomitant with PD （hepatopancreatoduodenectomy） has been applied to selected patients with widespread tumors. Preoperative biliary drainage （BD） followed by portal vein embolization （PVE） enables major hepatectomy in patients with hilar bile duct cancer without mortality. BD should be performed considering the surgical procedure, especially, in patients with separated intrahepatic bile ducts caused by hilar bile duct cancer. Right or left trisectoriectomy are indicated according to the tumor spread and biliary anatomy. As a result, extended radical resection offers a chance for cure of hilar bile duct cancer with improved resectability, curability, and a 5-year survival rate of 40%. A 5-year survival rate has ranged from 24% to 39% after PD for middle and distal bile duct cancer.

Effect of preoperative biliary drainage on surgical results after pancreaticoduodenectomy in patients with distal common bile duct cancer:Focused on the rate of decrease in serum bilirubin

AIM: To examine if the rate of decrease in serum bilirubin after preoperative biliary drainagecan be used as a predicting factor for surgical complications and postoperative recovery after pancreaticoduodenectomy in patients with distal common bile duct cancer.METHODS: A retrospective study was performed in 49 consecutive patients who underwent pancrea-ticoduodenectomy for distal common bile duct cancer. Potential risk factors were compared between the complicated and uncomplicated groups. Also, the rates of decrease in serum bilirubin were compared pre- and postoperatively. RESULTS: Preoperative biliary drainage (PBD) was performed in 40 patients (81.6%). Postoperative morbidity and mortality rates were 46.9% (23/49) and 6.1% (3/49), respectively. The presence or absence of PBD was not different between the complicated and uncomplicated groups. In patients with PBD, neither the absolute level nor the rate of decrease in serum bilirubin was significantly different. Patients with rapid decrease preoperatively showed faster decrease during the first postoperative week (5.5 ± 4.4 μmol/L vs -1.7 ± 9.9 μmol/L, P = 0.004).CONCLUSION: PBD does not affect the surgical outcome of pancreaticoduodenectomy in patients with distal common bile duct cancer. There is a certain group of patients with a compromised hepatic excretory function, which is represented by the slow rate of decrease in serum bilirubin after PBD.

Prognostic factors in patients with middle and distal bile duct cancers

AIM: To identify the influence of the surgery type and prognostic factors in middle and distal bile duct cancers.

Early bile duct cancer

Bile duct cancers are frequently diagnosed as advanced diseases. Over half of patients with advanced bile duct cancer present with unresectable malignancies and their prognosis has been very poor even after curative resections. Although there has been a need to diagnose bile duct cancer at its eady stage, it has been a difficult goal to achieve due to our lack of knowledge regarding this disease entity. Early bile duct cancer may be defined as a carcinoma whose invasion is confined within the fibromuscular layer of the extrahepatic bile duct or intrahepatic large bile duct without distant metastasis irrespective of lymph node involvement. Approximately 3%-10% of resected bile duct cancers have been reported to be early cancers in the literature. The clinicopathological features of patients with early bile duct cancer differ from those of patients with advanced bile duct cancer, with more frequent asymptomatic presentation, characteristic histopathological findings, and excellent prognosis. This manuscript is organized to emphasize the need for convening an international consensus to develop the concept of early bile duct cancer.

Relationship between the GH-IGFs axis and the proliferation of bile duct cancer cell line QBC939 in vitro

BACKGROUND: In recent years, recombined human growth hormone (rhGH) has been increasingly used in patients to help them recover from operation. But GH, as a mitogen, can promote cell renewal and increase malignant transformation. In the current study, we assessed the proliferation of a bile duct cancer cell line (QBC939) in vitro with GH and explored the possible relationship with the axis of GH-IGFs (insulin-like growth factors). METHODS: QBC939 cells in the exponential growth stage were harvested and divided into an experimental group (GH group) and a control group (NS group). The GH group was divided into four sub-groups according to the dose of GH and culture time (50 mu g/L for 2 hours, 50 mu g/L for 24 hours, 100 mu g/L for 2 hours, 100 mu g/L for 24 hours). The NS group was divided into two sub-groups (NS for 2 hours and NS for 24 hours). After 2 or 24 hours, IGF-1 and IGF-2 were detected using the enzyme-linked immunosorbent assay. The QBC939 cells cultured for 24 hours with two GH concentrations were made into single cell suspensions and samples underwent subsequent cell cycle evaluation. Messenger RNA of IGF-1 and IGF-2 receptor (IGF-1RmRNA and IGF-2RmRNA) were tested with the method of in situ hybridization. RESULTS: There was no statistically significant difference between the GH and NS groups after 2 hours of culture (P>0.05). But after 24 hours of culture, GH stimulated cell growth in vitro and also elevated the percentage in S phase and the proliferation index (P<0.05). IGF-1RmRNA and IGF-2RmRNA were expressed in QBC939 in contrast to the blank group. The expression of IGF-1RmRNA increased with the dose of GH, but IGF-2RmRNA did not. CONCLUSION: GH can stimulate QBC939 cell growth and proliferation in vitro and the mechanism is most likely by the GH-IGF-1-IGF-1R axis.

Effect of external beam radiotherapy on patency of uncovered metallic stents in patients with inoperable bile duct cancer

BACKGROUND： Although biliary decompression with metallic stenting is the preferred treatment for inoperable bile duct cancer（BDC）, maintenance of patency is still unsatisfactory.We tried to assess the effectiveness and safety of external beam radiotherapy（EBRT） for prolonging stent patency in patients having uncovered metallic stents.METHOD： We retrospectively reviewed 50 patients who received endoscopic stenting, of whom 18 received EBRT（RT group） and 32 did not（non-RT group）.RESULTS： No difference was found in baseline characteristics between the two groups. Although stent patency was longer in the RT group than that in the non-RT group（140.7±51.3 vs136.4±34.9 days, P=0.94）, the difference was not statistically significant. There were a lower rate of stent occlusion（27.8% vs50.0% of patients, P=0.12） and a longer overall survival（420.1 ±73.2 vs 269.1±41.7 days, P=0.11） in the RT group than in the non-RT group, and the difference again was not statistically significant. The development of adverse reactions did not differ（55.6% vs 53.1% of patients, P=0.91）. There was no serious adverse reaction in both groups（P=0.99）.CONCLUSIONS： EBRT did not significantly improve stent patency in patients with inoperable BDC having uncovered metallic stents. However, EBRT was safe. Future trials withrefined protocols for better efficacy are expected.

Metachronous bile duct cancer nine years after resection of gallbladder cancer

We report a rare case of a 74-year-old man with metachronous gallbladder cancer and bile duct cancer who underwent curative resection twice, with the operations nine years apart. At the age of 65 years, the patient underwent a cholecystectomy and resection of the liver bed for gallbladder cancer. This was a welldifferentiated adenocarcinoma, with negative resection margins （T2NOM0, stage Ⅰ B）. Nine years later, during a follow-up examination, abdominal computed tomography and MRCP showed an enhanced 1.7 cm mass in the hilum that extended to the second branch of the right intrahepatic bile duct. We diagnosed this lesion as a perihilar bile duct cancer, Bismuth type Ⅲ a, and performed bile duct excision, right hepatic Iobectomy and Roux-en-Y hepaticojejunostomy. The histological diagnosis was a well-differentiated adenocarcinoma with one regional lymph node metastasis （TINIM0, stage Ⅱ B）. Twelve months after the second operation, the patient is well, with no signs of recurrence. This case is compared with 11 other cases of metachronous biliary tract cancer published in the world medical literature.

Diagnostic accuracy of administrative database for bile duct cancer by ICD-10 code in a tertiary institute in Korea

Background:Administrative database provides valuable information for large cohort studies,especially when tissue diagnosis is rather difficult such as the diagnosis for bile duct cancer(BDC).The aim of this study was to evaluate the diagnostic accuracy of administrative database for BDC by International Classification of Diseases(ICD)-10 codes in a tertiary institute.Methods:BDC and control groups were collected from 2003 to 2016 at Seoul National University Bundang Hospital.Cases of BDC were identified in the National Health Insurance Service(NHIS)database by ICD 10-code supported by V code.The control group was selected from cases without ICD-10 codes for BDC.A definite or possible diagnosis was defined according to pathologic reports.Medical records,images,and pathology reports were analyzed to evaluate ICD-10 codes for BDC.Sensitivity,specificity,positive predictive value,and negative predictive value for BDC were analyzed according to diagnostic criteria and cancer locations.Results:A total of 1707 patients with BDC and 1707 controls were collected.Among those with BDC,1320(77.3%)were diagnosed by definite criteria.Most(99.4%)of them had adenocarcinoma.Rate of definite diagnosis was the highest for ampulla of Vater(88.9%),followed by that for extrahepatic(84.9%)and intrahepatic(68.3%)BDCs.False positive cases commonly had hepatocellular carcinomas.For overall diagnosis of BDC,sensitivity,specificity,positive predictive value,and negative predictive value were 99.94%,98.33%,98.30%,and 99.94%,respectively.Diagnostic accuracies were similar regardless of diagnostic criteria or tumor locations.Conclusions:Administrative database for BDC collected according to ICD-10 code with V code shows good accuracy.

Renal Collecting Duct Cancer： a Report of 2 Cases

Renal collecting duct cancer is a rare malignant tumor, which accounts for 1% to 2% of epithelial kidney tumors. Its pathologial appearance has been easily misdiagnosed as a mammilliform renal cell carcinoma or as other tumors. The malignancy of renal collecting duct cancer is high, with early metastasis and poor prognosis. The clinical data for 2 cases of the tumor are discussed in this report, including reports on the histopathology and the changes in immunohistochemistry.

Cystic duct cancer: Should it be deemed as a type of gallbladder cancer?

BACKGROUND According to the latest American Joint Committee on Cancer and Union for International Cancer Control manuals,cystic duct cancer(CC)is categorized as a type of gallbladder cancer(GC),which has the worst prognosis among all types of biliary cancers.We hypothesized that this categorization could be verified by using taxonomic methods.AIM To investigate the categorization of CC based on population-level data.METHODS Cases of biliary cancers were identified from the Surveillance,Epidemiology,and End Results 18 registries database.Together with routinely used statistical methods,three taxonomic methods,including Fisher’s discriminant,binary logistics and artificial neuron network(ANN)models,were used to clarify the categorizing problem of CC.RESULTS The T staging system of perihilar cholangiocarcinoma[a type of extrahepatic cholangiocarcinoma(EC)]better discriminated CC prognosis than that of GC.After adjusting other covariates,the hazard ratio of CC tended to be closer to that of EC,although not reaching statistical significance.To differentiate EC from GC,three taxonomic models were built and all showed good accuracies.The ANN model had an area under the receiver operating characteristic curve of 0.902.Using the three models,the majority(75.0%-77.8%)of CC cases were categorized as EC.CONCLUSION Our study suggested that CC should be categorized as a type of EC,not GC.Aggressive surgical attitude might be considered in CC cases,to see whether long-term prognosis could be immensely improved like the situation in EC.

CD24 expression predicts distant metastasis in extrahepatic bile duct cancer

AIM: To evaluate the prognostic significance of CD24 expression in patients undergoing adjuvant chemoradiotherapy for extrahepatic bile duct (EHBD) cancer. METHODS: Eighty-four patients with EHBD cancer who underwent curative resection followed by adjuvant chemoradiotherapy were enrolled in this study. Postoperative radiotherapy was delivered to the tumor bed and regional lymph nodes up to a median of 40 Gy (range: 40-56 Gy). All patients also received fluoropyrimidine chemotherapy for radiosensitization during radiotherapy. CD24 expression was assessed with immunohistochemical staining on tissue microarray. Clinicopathologic factors as well as CD24 expression were evaluated in multivariate analysis for clinical outcomes including loco-regional recurrence, distant metastasisfree and overall survival. RESULTS: CD24 was expressed in 36 patients (42.9%). CD24 expression was associated with distant metastasis, but not with loco-regional recurrence nor with overall survival. The 5-year distant metastasis-free survival rates were 55.1% and 29.0% in patients with negative and positive expression, respectively (P=0.0100). On multivariate analysis incorporating N stage, histologic differentiation and CD24 expression, N stage was the only significant factor predicting distant metastasis-free survival (P=0.0089), while CD24 expression had borderline significance (P=0.0733). In subgroup analysis, CD24 expression was significantly associated with 5-year distant metastasis-free survival in node-positive patients (38.4% with negative expression vs 0% with positive expression, P=0.0110), but not in nodenegative patients (62.0% with negative expression vs 64.0% with positive expression,P=0.8599). CONCLUSION: CD24 expression was a significant predictor of distant metastasis for patients undergoing curative resection followed by adjuvant chemoradiotherapy especially for node-positive EHBD cancer.

Preoperative Diagnosis of Thyroglossal Duct Cancer: A Case Report and Literature Review

Objective: To clarify the preoperative diagnostic rate and elucidate the morphological features of thyroglossal duct cancer through a literature search on cases reported in Japan. Methods: A search of a medical database (Japan Medical Abstracts Society) identified 40 studies on thyroglossal duct cancer in Japanese patients between 1976 and 2014. A total of 47 cases, including the present case, are summarized herein. Patient characteristics, preoperative diagnosis, and morphological features were reviewed and analyzed. Morphological features of the internal portions in the cystic lesions were classified using the previously reported Yokosuka Kyosai Hospital criteria for ultrasonography findings of thyroid cystic tumors. Results: Preoperative diagnosis was described for 43 of the 47 cases. Malignancy was suspected in 18 (41.9%) of the 43 cases on the basis of fine needle aspiration (FNA) cytology (presence of suspected papillary carcinoma cells) and imaging studies (presence of calcifications), 12 and 6 cases, respectively. Preoperative FNA was performed in 24 cases with a correct diagnosis obtained in only 12 (50%) cases. Morphological features were evaluated by preoperative imaging studies and/or postoperative histopathology. We found 6 cases (15%) with solid lesions, 32 cases (80%) with cystic lesions containing a solid part, and 2 cases (5%) with solo cystic lesions, respectively. Calcification was observed in 28 (72.5%) cases. We further examined the internal morphology of 32 cases with cystic lesions according to the criteria for ultrasonography findings of thyroid cystic tumors described in Methods. Of the 32 cases, 25 (62.5%) and 7 (17.5%) were classified as “eccentric acute angle type (Ea)” and “multiseptate type (M)”, respectively. The boundary between the solid part and the cystic part was irregular in all 7 “M” cases. No “eccentric and blunt angle type (Eb)” or “concentric type (C)” lesions were observed. Conclusions: The preoperative diagnostic rate for thyroglossal duct cancer using FNA is low, and it is important that diagnosis be performed in conjunction with imaging findings. The presence of solid parts or calcified lesions classified as “Ea” or “M with irregular boundaries” on the basis of imaging findings is suggestive of malignancy.

Diagnostic-therapeutic management of bile duct cancer

Biliary tract cancer, or cholangiocarcinoma, comprises a heterogeneous group of malignant tumors that can emerge at any part of the biliary tree. This group is the second most common type of primary liver cancer. Diagnosis is usually based on symptoms, which may be heterogeneous, and nonspecific biomarkers in serum and biopsy specimens, as well as on imaging techniques. Endoscopy-based diagnosis is essential, since it enables biopsy specimens to be taken. In addition, it can help with locoregional staging of distal tumors. Endoscopic retrograde cholangiopancreatography is a key technique for the evaluation and treatment of malignant biliary tumors. Correct staging of cholangiocarcinoma is essential in order to be able to determine the degree of resectability and assess the results of treatment. The tumor is staged based on the TNM classification of the American Joint Committee on Cancer. The approach will depend on the classification of the tumor. Thus, some patients with early-stage disease could benefit from surgery;complete surgical resection is the cornerstone of cure. However, only a minority of patients are diagnosed in the early stages and are suitable candidates for resection. In the subset of patients diagnosed with locally advanced or metastatic disease, chemotherapy has been used to improve outcome and to delay tumor progression. The approach to biliary tract tumors should be multidisciplinary, involving experienced endoscopists, oncologists, radiologists, and surgeons.

Radiation therapy for extrahepatic bile duct cancer: Current evidences and future perspectives

Extrahepatic bile duct cancer (EBDC) is a rare malignancy that involves neoplastic changes extending from both hepatic ducts to the common bile duct. The treatment of choice is surgical resection, but the predominant pattern of initial treatment failure is locoregional recurrence. Accordingly, adjuvant radiotherapy has been administered after surgical resection based on these rationales. At this time, there is minimal evidence supporting adjuvant radiotherapy, because there have been no phase III trials evaluating its benefit. Relatively small retrospective studies have tried to compare outcomes associated with EBDC treated with or without radiotherapy. We aimed to review studies investigating adjuvant radiotherapy for resected EBDC. Because less than onethird of EBDC cases are amenable to curative resection at diagnosis, other locoregional treatment modalities need to be considered, including radiotherapy. The next aim of this review was to summarize reports of definitive radiotherapy for unresectable EBDC. Patients with advanced EBDC often experience biliary obstruction, which can lead to jaundice and progress to death. Biliary stent insertion is an important palliative procedure, but stents are prone to occlusion after subsequent ingrowth of the EBDC. Radiotherapy can be effective for maintaining the patency of inserted stents. We also reviewed the benefit of palliative radiotherapy combined with the biliary stent insertion. Lastly, we discuss the existing gaps in the evidence supporting radiotherapy in the management of EBDC.

Synchronous double cancers of the common bile duct

We report an extremely rare case of synchronous double cancers of the common bile duct without pancreaticobiliary maljunction.Only two similar cases have been reported in the English literature.Endoscopic retrograde cholangiopancreatography showed a tuberous filling defect in the middle and superior parts of the common bile duct,and mild stenosis in the inferior duct.Computed tomography(CT) showed a well enhanced mass in the middle and superior parts of the common bile duct.A single cancer of the middle and superior bile duct was suspected and extra-hepatic bile duct resection was performed.CT eleven months after surgery revealed enhanced inferior bile duct wall and a slightly enhanced tumor within it.Retrospective review of the CT images taken before first surgery showed enhanced inferior bile duct wall without intrabiliary tumor only on the delayed phase.The inferior bile duct tumor was suspected to have originally co-existed with the middle and superior bile duct tumor.Pancreaticoduodenectomy was performed subsequently.Histopathological examination revealed that the middle and superior bile duct tumor was a moderately differentiated tubular adenocarcinoma while the inferior bile duct tumor was a papillary adenocarcinoma.The two tumors were separated and had different histological findings and growth patterns,further suggesting that they were two primary cancers.

Detection of K-ras gene mutation at codon 12 by pancreatic duct brushing for pancreatic cancer

OBJECTIVE: To assess the diagnostic value of endoscopic pancreatic duct brushing in detecting mutation of the K-ras gene at codon 12 in cytologic specimens from patients with pancreatic cancer. METHODS: Thirty-five patients treated at Changhai Hospital, Shanghai between 1999 and 2001 were enrolled. Their cells obtained by pancreatic duct brushing during endoscopic retrograde tholangiopancreatography (ERCP) were suspended with phosphate buffer solution (PBS). DNA of the cells was extracted and mutation of the K-ras gene at codon 12 detected by means of PCR-SSCP. RESULTS: The K-ras gene mutation rate of pancreatic cancer was 70%, which was higher than that of chronic pancreatitis (14%, P<0.05). K-ras gene mutation was not found in patients with pancreatic cystorcarcinoma and duodenum carcinoma. As to the location of pancreatic cancer, no significant difference was observed between the head, the body and tail. The sensitivity, specificity, accuracy of pancreatic duct brushing in detecting pancreatic cancer was 70%, 94%, and 83%, respectively. CONCLUSION: K-ras analysis of pancreatic brushing samples is helpful in the diagnosis of patients with early pancreatic cancer.

Müllerian duct anomalies and their effect on the radiotherapeutic management of cervical cancer

Radiotherapy plays a major role in the treatment of cervical cancer. A successful radiotherapy program integrates both external beam and brachytherapy components. The principles of radiotherapy are strongly based on the anatomy of the organ and patterns of local and nodal spread. However, in patients with distorted anatomy, several practical issues arise in the delivery of optimal radiotherapy, especially with brachytherapy. Müllerian duct anomalies result in congenital malformations of the female genital tract. Though being very commonly studied for their deleterious effects on fertility and pregnancy, they have not been recognized for their potential to interfere with the delivery of radiotherapy among patients with cervical cancer. Here, we discuss the management of cervical cancer among patients with Müllerian duct anomalies and review the very sparse amount of published literature on this topic.

Presence of CCK-A, B receptors and effect of gastrin and cholecystokinin on growth of pancreatobiliary cancer cell lines

AIM: To investigate the effects of gastrin and cholecystokinin (CCK) and their specific antagonists on the growth of pancreatic and biliary tract cancer cell lines. METHODS: Five pancreatic and 6 biliary cancer cell lines with 2 conrtol cells were used in this study. Cell proliferation study was done using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) test and direct cell count method. Reverse transcription-polymerase chain reaction (RT-PCR) and slot blot hybridization were performed to examine and quantify the expression of hormonal receptors in these cell lines. RESULTS: SNU-308 showed a growth stimulating effect by gastrin-17, as did SNU-478 by both gastrin-17 and CCK-8. The trophic effect of these two hormones was completely blocked by specific antagonists (L-365, 260 for gastrin and L-364, 718 for CCK). Other cell lines did not respond to gastrin or CCK. In RT-PCR, the presence of CCK-A receptor and CCK-B/gastrin receptor mRNA was detected in all biliary and pancreatic cancer cell lines. In slot blot hybridization, compared to the cell lines which did not respond to hormones, those that responded to hormones showed high expression of receptor mRNA. CONCLUSION: Gastrin and CCK exert a trophic action on some of the biliary tract cancers.

Carcinoma of the middle bile duct:Is bile duct segmental resection appropriate?

AIM:To compare survival between bile duct segmental resection(BDSR)and pancreaticoduodenectomy(PD) for treating distal bile duct cancers. METHODS:Retrospective analysis was conducted for 45 patients in a BDSR group and for 149 patients in a PD group. RESULTS:The T-stage(P<0.001),lymph node invasion (P=0.010)and tumor differentiation(P=0.005)were significant prognostic factors in the BDSR group.The 3-and 5-year overall survival rates for the BDSR group and PD group were 51.7%and 36.6%,respectively and 46.0%and 38.1%,respectively(P=0.099).The BDSR group and PD group did not show any significant difference in survival when this was adjusted for the TNM stage.The 3-and 5-year survival rates were: stageⅠa[BDSR(100.0%and 100.0%)vs PD(76.9% and 68.4%)(P=0.226)];stageⅠb[BDSR(55.8% and 32.6%)vs PD(59.3%and 59.3%)(P=0.942)]; stageⅡb[BDSR(19.2%and 19.2%)vs PD(31.9%and 14.2%)(P=0.669)]. CONCLUSION:BDSR can be justified as an alternative radical operation for patients with middle bile duct inselected patients with no adjacent organ invasion and resection margin is negative.

Cytotoxic effects of Thai noni juice product ethanolic extracts against cholangiocarcinoma cell lines

Objective:To investigate the cytotoxic activity and molecular mechanism(s)of two Thai noni juice(TNJ)products ethanolic extracts against cholangiocarcinoma(CCA)cell lines and non-cancerous cells,and to explore phenolic acid compositions of TNJ products.Methods:Phenolic acid profiles of TNJ Chiangrai(TNJ-Cr)and TNJ Buasri(TNJ-Bs)ethanolic extracts were determined by HPLC.The cytotoxicity of TNJ ethanolic extracts on cancer and non-cancerous cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and trypan blue assays.Mechanism(s)underlying the anti-CCA activity of TNJ ethanolic extracts were determined by cell cycle,apoptosis,and reactive oxygen species(ROS)generation assays.The expression levels of proteins involved in apoptosis and ERK signaling were evaluated by Western blot analysis.Results:Phenolic acid profiles of both TNJ ethanolic extracts showed that the p-hydroxybenzoic,vanillic,and protocatechuic acids were the major phenolic acids in TNJ products.Cytotoxicity assays revealed that the TNJ-Cr and TNJ-Bs ethanolic extracts reduced viability of CCA cell lines through induction of apoptosis by up-regulation of p53 and Bax proapoptotic proteins.Both TNJ ethanolic extracts promoted ROS generation by activating the ERK1/2 signaling in well-differentiated CCA cells KKU-213B.Meanwhile,TNJ ethanolic extracts did not induce ROS production in poorly differentiated CCA cells KKU-100.Both TNJ ethanolic extracts showed no toxicity to human peripheral blood mononuclear cells.Conclusions:TNJ ethanolic extracts could inhibit CCA cell proliferation by inducing ROS generation and apoptosis and may be applicable for combination therapies in CCA treatment.