The landscape of etiological patterns of hepatocellular carcinoma and intrahepatic cholangiocarcinoma in Thailand.

Thailand is among countries with the highest global incidence and mortality rates of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA). While viral hepatitis and liver fluke infections have been associated with HCC and iCCA, respectively, other environmental risk factors, overall risk factor commonality and combinatorial roles, and effects on survival have not been systematically examined. We conducted a TIGER-LC consortium-based population study covering all high-incidence areas of both malignancies across Thailand: 837 HCC, 1474 iCCA, and 1112 controls (2011-2019) were comprehensively queried on lifelong environmental exposures, lifestyle, and medical history. Multivariate logistic regression and Cox proportional hazards analyses were used to evaluate risk factors and associated survival patterns. Our models identified shared risk factors between HCC and iCCA, such as viral hepatitis infection, liver fluke infection, and diabetes, including novel and shared associations of agricultural pesticide exposure (OR range of 1.50; 95% CI: 1.06-2.11 to 2.91; 95% CI: 1.82-4.63) along with vulnerable sources of drinking water. Most patients had multiple risk factors, magnifying their risk considerably. Patients with lower risk levels had better survival in both HCC (HR 0.78; 95% CI: 0.64-0.96) and iCCA (HR 0.84; 95% CI: 0.70-0.99). Risk factor co-exposures and their common associations with HCC and iCCA in Thailand emphasize the importance for future prevention and control measures, especially in its large agricultural sector. The observed mortality patterns suggest ways to stratify patients for anticipated survivorship and develop plans to support medical care of longer-term survivors, including behavioral changes to reduce exposures.

Characterization of tumor evolution by functional clonality and phylogenetics in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a molecularly heterogeneous solid malignancy, and its fitness may be shaped by how its tumor cells evolve. However, ability to monitor tumor cell evolution is hampered by the presence of numerous passenger mutations that do not provide any biological consequences. Here we develop a strategy to determine the tumor clonality of three independent HCC cohorts of 524 patients with diverse etiologies and race/ethnicity by utilizing somatic mutations in cancer driver genes. We identify two main types of tumor evolution, i.e., linear, and non-linear models where non-linear type could be further divided into classes, which we call shallow branching and deep branching. We find that linear evolving HCC is less aggressive than other types. GTF2IRD2B mutations are enriched in HCC with linear evolution, while TP53 mutations are the most frequent genetic alterations in HCC with non-linear models. Furthermore, we observe significant B cell enrichment in linear trees compared to non-linear trees suggesting the need for further research to uncover potential variations in immune cell types within genomically determined phylogeny types. These results hint at the possibility that tumor cells and their microenvironment may collectively influence the tumor evolution process.

Pesticide exposure and risk of cholangiocarcinoma: A hospital-based matched case-control study.

BACKGROUND: Cholangiocarcinoma (CCA) caused by Opisthorchis viverrini is a well-known and significant public health issue in northeastern Thailand; however, a link between pesticide exposure (PE) and CCA risk has not yet been established. Therefore, our research objective was to investigate the relationship between PE and CCA risk. METHODS: A hospital-based matched case-control study was carried out. All cases (in-patients) and controls (out-patients) were volunteers at a tertiary hospital in northeast Thailand. Between 2015 and 2019, 178 incident cases of pathologically-confirmed CCA and 356 controls were selected from the check-up clinic from the Srinagarind Hospital outpatient database (two controls per case). The recruited controls were individually-matched to the CCA cases based on sex, age (±5 years) and admission date (±3 months). During face-to-face interviews, a standardised pre-tested questionnaire was used to collect data. Multivariable conditional logistic regression was used to analyse the data. RESULTS: The respective frequency of PE between the 178 CCA cases and 356 controls was 77.0% versus 87.6% for never used, 14.6% versus 5.3% for have used but stopped and 8.4% versus 7.0% for currently using. After adjusting for the highest educational attainment, smoking behaviour, alcohol use and family history of cancer, PE was not significantly associated with CCA (p-value = 0.086). Using volunteers who have never used PE as the reference group, the respective odds of developing CCA for those who have ever used but have since stopped and are currently using was 2.04 (adjusted OR = 2.04; 95% CI: 1.03-4.04) versus 0.83 (adjusted OR = 0.83; 95% CI: 0.39-1.76) times more likely to develop CCA than those who had never used PE. CONCLUSION: There is no association between PE and the risk of CCA. Notwithstanding the finding, future research should focus on enhancing PE assessment methods that consider complex chemical mixtures, chemicals of interest, historical exposure and exposure pathways. Moreover, there is need for more extensive and longer population-based cohort studies that include younger, non-occupationally exposed individuals during periods of developmental susceptibility.

Pan-viral serology uncovers distinct virome patterns as risk predictors of hepatocellular carcinoma and intrahepatic cholangiocarcinoma.

This study evaluates the pan-serological profiles of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA) compared to several diseased and non-diseased control populations to identify risk factors and biomarkers of liver cancer. We used phage immunoprecipitation sequencing, an anti-viral antibody screening method using a synthetic-phage-displayed human virome epitope library, to screen patient serum samples for exposure to over 1,280 strains of pathogenic and non-pathogenic viruses. Using machine learning methods to develop an HCC or iCCA viral score, we discovered that both viral scores were positively associated with several liver function markers in two separate at-risk populations independent of viral hepatitis status. The HCC score predicted all-cause mortality over 8 years in patients with chronic liver disease at risk of HCC, while the viral hepatitis status was not predictive of survival. These results suggest that non-hepatitis viral infections may contribute to HCC and iCCA development and could be biomarkers in at-risk populations.

Compliance with enhanced recovery after surgery predicts long-term outcome after hepatectomy for cholangiocarcinoma.

BACKGROUND: Enhanced recovery after surgery (ERAS) program has been proved to improve postoperative outcome for many surgical procedures, including liver resection. There was limited evidence regarding the feasibility and benefit of ERAS in patients who underwent liver resection for cholangiocarcinoma. AIM: To evaluate the feasibility of ERAS in patients who underwent liver resection for cholangiocarcinoma and its association with patient outcomes. METHODS: We retrospectively analyzed 116 cholangiocarcinoma patients who underwent hepatectomy at Srinagarind Hospital, Khon Kaen University between January 2015 and December 2016. The primary outcome was the compliance with ERAS. To determine the association between ERAS compliance and patient outcomes. the patients were categorized into those adhering more than and equal to 50% (ERAS ≥ 50), and below 50% (ERAS < 50) of all components. Details on type of surgical procedure, preoperative and postoperative care, tumor location, postoperative laboratory results, and survival time were evaluated. The compliance with ERAS was measured by the percentage of ERAS items achieved. The Kaplan-Meier curve was used for survival analysis. RESULTS: The median percentage of ERAS goals achieved was 40% (± 12%). Fourteen patients (12.1%) were categorized into the ERAS ≥ 50 group, and 102 patients were in the ERAS < 50 group. Postoperative hospital stay was significantly shorter in the ERAS ≥ 50 group [8.9 d, 95% confidence interval (CI): 7.3-10.4 d] than in the ERAS < 50 group (13.7 d, 95%CI: 12.2-15.2 d) (P = 0.0217). No hepatobiliary-related complications or in-hospital mortality occurred in the ERAS ≥ 50 group. Overall survival was significantly higher in the ERAS ≥ 50 group. The median survival of the patients in the ERAS < 50 group was 1257 d (95%CI: 853.2-1660.8 d), whereas that of the patients in the ERAS ≥ 50 group was not reached. CONCLUSION: Overall ERAS compliance for patients who underwent liver resection for cholangiocarcinoma is poor. Greater ERAS compliance could predict in-hospital, short-term, and long-term outcomes of the patients.

Infrahepatic Inferior Vena Cava Clamping Reduces Blood Loss during Liver Transection for Cholangiocarcinoma.

BACKGROUND: Major hepatectomy is the mainstay of the treatment for cholangiocarcinoma. Infrahepatic inferior vena cava (IVC) clamping is an effective maneuver for reducing blood loss during liver transection. The impact of this procedure on major hepatectomy for cholangiocarcinoma is unknown. This study evaluated the effect of infrahepatic IVC clamping on blood loss during liver transection. METHODS: Clinical and pathological data were collected retrospectively for 116 cholangiocarcinoma patients who underwent major hepatectomy between January 2015 and December 2016, to investigate the benefit of infrahepatic IVC clamping. Two of five surgeons adapted the policy performing infrahepatic IVC clamping during liver transection in all cases. Patients, therefore, were divided into those (n = 39; 33.6%) who received infrahepatic IVC clamping during liver transection (C1) and those (n = 77; 66.4%) who did not (C0). RESULTS: The patients' backgrounds, operative parameters, and extent of hepatectomy did not differ significantly between the 2 groups, except for gender. A significantly lower blood loss (p = 0.028), blood transfusion (p = 0.011), and rate of vascular inflow occlusion requirement (p < 0.001) were observed in the C1 group. The respective blood losses in the C1 group and the C0 group were 498.9 (95% CI: 375.8-622.1) and 685.6 (95% CI: 571-800.2) millilitres. CONCLUSIONS: The current study found infrahepatic IVC clamping during liver transection for cholangiocarcinoma reduces blood loss, blood transfusion, and rate of vascular inflow occlusion requirement.

Decreasing trends in cholangiocarcinoma incidence and relative survival in Khon Kaen, Thailand: An updated, inclusive, population-based cancer registry analysis for 1989-2018.

BACKGROUND: Cholangiocarcinoma (CCA) is a leading cause of cancer death in northeastern Thailand. We reported on the incidence of CCA using only one method. In the current study, we used three different statistical methods to forecast future trends and estimate relative survival. METHODS: We reviewed the CCA cases diagnosed between 1989 and 2018 recorded in the population-based Khon Kaen Cancer Registry (KKCR). Annual percent change (APC) was calculated to quantify the incidence rate trends using Joinpoint regression. Age-period-cohort models (APC model) were used to examine the temporal trends of CCA by age, calendar year, and birth cohort. We projected the incidence of CCA up to 2028 using three independent approaches: the Joinpoint, Age-period-cohort, and Nordpred models. Survival assessments were based on relative survival (RS). RESULTS: The respective APC in males and females decreased significantly (-3.1%; 95%CI: -4.0 to -2.1 and -2.4%; 95%CI: -3.6 to -1.2). The APC model-AC-P for male CCA-decreased according to a birth-cohort. The CCA incidence for males born in 1998 was 0.09 times higher than for those born in 1966 (Incidence rate ratios, IRR = 0.09; 95%CI: 0.07 to 0.12). The relative incidence for female CCA similarly decreased according to a birth-cohort (IRR = 0.11; 95%CI: 0.07 to 0.17). The respective projection for the age-standardized rate for males and females for 2028 will be 7.6 per 100,000 (102 patients) and 3.6 per 100,000 (140 patients). The five-year RS for CCA was 10.9% (95%CI: 10.3 to 11.6). CONCLUSION: The incidence rate of CCA has decreased. The projection for 2028 is that the incidence will continue to decline. Nevertheless, the survival of patients with CCA remains poor.

Re-Rising of Total Bilirubin Level after Postoperative Day 3 (The V Pattern) Predicting Liver Failure and Survival of Patients who Underwent Hepatectomy for Cholangiocarcinoma.

OBJECTIVE: All types of cholangiocarcinoma (CCA) require a major hepatectomy, which has many post-operative complications. All complications usually present with persistent hyperbilirubinemia; however, studies on the prediction of post-operative hyperbilirubinemia after hepatectomy for patients with CCA are lacking. We evaluated the causes and patterns of persistent hyperbilirubinemia among the patients who underwent hepatectomy for CCA. METHODS: We retrospectively reviewed the records of 216 CCA patients who underwent curative-intent hepatic resection between January 2015 and December 2016. We identified five patterns of hyperbilirubinemia for predicting the cause of persistent hyperbilirubinemia and the respective patient outcome. All clinical parameters and outcomes were analyzed for any significant associations. RESULTS: Twenty-eight patients (24%) had post-operative persistent hyperbilirubinemia. Of these, liver failure was the most common cause (42.9%), followed by bile leakage (14.3%), then cholangitis (3.6%). Re-rising of the bilirubin level after post-operative day 3(the 'V' pattern), very well predicted liver failure. Moreover, this pattern was associated with poor survival of the patient. CONCLUSION: The current study provided a picture of persistent hyperbilirubinemia after hepatectomy for CCA. The proportion of post-operative liver failure was 12 percent. The pattern of serum bilirubin level could be used as a predictor of liver failure and long-term outcomes of CCA patients. The 'V' pattern was significantly associated with a high rate of liver failure and poor survival.

Predictive utility of neutrophil-to-lymphocyte ratio and platelet-to-lymphocyte ratio in intraductal papillary neoplasm of the bile duct.

AIM OF THE STUDY: Intraductal papillary neoplasm of the bile duct (IPNB) can present at various stages of the disease. Each stage needs different treatment. The neutrophil-to-lymphocyte ratio (NLR) and the platelet-to-lymphocyte ratio (PLR) have been described as predictive markers for several tumors. There has been no investigation on the role of NLR and PLR in IPNB. MATERIAL AND METHODS: We retrospectively reviewed the medical records of 112 patients who underwent curative-intent hepatic resection for IPNB between January 2007 and December 2011. All clinical parameters and survival were analyzed for their association with NLR and PLR. RESULTS: For prediction of malignancy, the best respective cut-off for NLR and PLR was 2.74 and 130, with area under the ROC curve being 0.662 and 0.763. For micro-papillary IPNB, both markers well predict malignancy and lymph node involvement. The respective area under the ROC curve of NLR and PLR for prediction of malignancy was 0.78 and 0.88. Both markers had an area under the ROC curve for prediction of lymph node involvement of 1.0. The median overall survival of those with PLR < 130 was 86.4 months compared with 45.0 months for those with PLR > 130 (p = 0.02). CONCLUSIONS: NLR and PLR seem likely candidates for predicting malignancy, lymph node involvement, and survival of the patients. PLR performed better than NLR for all predictions. The markers worked very well for micro-papillary IPNB; however, we recommend using these markers in conjunction with the radiologic appearance of tumors.

Urinary Metabolites Diagnostic and Prognostic of Intrahepatic Cholangiocarcinoma.

BACKGROUND: Liver cancer is the second leading cause of cancer-related deaths worldwide. With a predicted 2.4-fold rise in liver cancer incidence by 2020, there is an urgent need for early, inexpensive diagnostic biomarkers to deploy in the clinic. METHODS: We employed ultraperformance liquid chromatography tandem mass-spectrometry (UPLC/MS-MS) for the quantitation of four metabolites, creatine riboside (CR), N-acetylneuraminic acid (NANA), cortisol sulfate, and a lipid molecule designated as 561+, in urine samples from the NCI-MD cohort comprising 98 hepatocellular carcinoma (HCC) cases, 101 high-risk subjects, and 95 controls. Validation was carried out in the TIGER-LC cohort [n = 370 HCC and intrahepatic cholangiocarcinoma (ICC) cases, 471 high-risk subjects, 251 controls], where ICC, the second most common primary hepatic malignancy, is highly prevalent. Metabolite quantitation was also conducted in TIGER-LC tissue samples (n = 48 ICC; n = 51 HCC). RESULTS: All profiled metabolites were significantly increased in liver cancer when compared with high-risk subjects and controls in the NCI-MD study. In the TIGER-LC cohort, the four-metabolite profile was superior at classifying ICC than a clinically utilized marker, CA19-9, and their combination led to a significantly improved model (AUC = 0.88, P = 4E-8). Metabolites CR and NANA were significantly elevated in ICC when compared with HCC cases in both urine and tissue samples. High levels of CR were associated with poorer prognosis in ICC. CONCLUSIONS: Four metabolites are significantly increased in HCC and ICC and are robust at classifying ICC in combination with the clinically utilized marker CA19-9. IMPACT: Noninvasive urinary metabolite biomarkers hold promise for diagnostic and prognostic evaluation of ICC.

Establishment of Highly Transplantable Cholangiocarcinoma Cell Lines from a Patient-Derived Xenograft Mouse Model.

Cholangiocarcinoma (CCA) is a deadly malignant tumor of the liver. It is a significant health problem in Thailand. The critical obstacles of CCA diagnosis and treatment are the high heterogeneity of disease and considerable resistance to treatment. Recent multi-omics studies revealed the promising targets for CCA treatment; however, limited models for drug discovery are available. This study aimed to develop a patient-derived xenograft (PDX) model as well as PDX-derived cell lines of CCA for future drug screening. From a total of 16 CCA frozen tissues, 75% (eight intrahepatic and four extrahepatic subtypes) were successfully grown and subpassaged in Balb/c Rag-2-/-/Jak3-/- mice. A shorter duration of PDX growth was observed during F0 to F2 transplantation; concomitantly, increased Oct-3/4 and Sox2 were evidenced in 50% and 33%, respectively, of serial PDXs. Only four cell lines were established. The cell lines exhibited either bile duct (KKK-D049 and KKK-D068) or combined hepatobiliary origin (KKK-D131 and KKK-D138). These cell lines acquired high transplantation efficiency in both subcutaneous (100%) and intrasplenic (88%) transplantation models. The subcutaneously transplanted xenograft retained the histological architecture as in the patient tissues. Our models of CCA PDX and PDX-derived cell lines would be a useful platform for CCA precision medicine.

Papillomatosis of the Biliary Tree and Gallbladder: Successful Treatment With Repeated Resection and Liver Transplant.

Intraductal papillary neoplasm of the bile duct is now considered to be a specific type of bile duct tumor. The progenitor cells of intraductal papillary neoplasms of the bile duct are located in the peribiliary gland, which are distributed along the intrahepatic bile duct, extrahepatic bile duct, and gallbladder; therefore, these neoplasms could arise in any area. The mainstay of treatment for patients with intraductal papillary neoplasm of the bile duct is complete surgical resection. Neoplasms involving both lobes of the liver can only be treated with liver transplant. There have been 11 reported cases of patients with biliary papillomatosis treated with liver transplant. In all of these cases, involvement was limited to the bile duct. Herein, we present the first case of papillomatosis with extensive involvement in the intrahepatic bile duct, the extrahepatic bile duct, and the gallbladder, which was successfully treated with repeated resection and liver transplant.

Cholangiocarcinoma Trends, Incidence, and Relative Survival in Khon Kaen, Thailand From 1989 Through 2013: A Population-Based Cancer Registry Study.

BACKGROUND: Cholangiocarcinoma (CCA) is a common malignancy in northeastern Thailand. Over the last 4 decades, several policies have been implemented for its prevention, but there has been no update on the trends and relative survival (RS). Our aim was (a) to perform a statistical assessment of the incidence trends of CCA and project future trends, and (b) to estimate relative survival. METHODS: All cases of CCA diagnosed from 1989 through 2013 were abstracted from the Khon Kaen Cancer Registry (KKCR). A jointpoint regression model was used to estimate the annual percentage change (APC) and to project future trends. We also calculated RS. RESULTS: There were 11,711 cases of CCA. The incidence rate increased with an APC of 1.79% (95% confidence interval [CI], -0.2 to 3.8) from 1989 through 2002, and decreased with an APC of -6.09% (95% CI, -8.2 to -3.9) from 2002 through 2013. The projected incidence of CCA should stable over the next 10 years, albeit higher than the world rate. The respective 5-year RS for both sexes for age groups of 30-40, 41-45, 51-60, and 61-98 years was 22.3% (95% CI, 16.8-29.5), 14.3% (95% CI, 12.0-17.0), 8.6% (95% CI, 7.8-10.0), and 7.2% (95% CI, 6.4-8.0). CONCLUSION: The incidence rate of CCA has decreased since 2002, representing a real decline in the risk of CCA. The incidence of CCA is projected to stabilize by 2025. The survival of patients with CCA remains poor.

The Curious Case of Cholangiocarcinoma: Opportunities for Environmental Health Scientists to Learn about a Complex Disease.

Deaths from complex, noncommunicable diseases such as cancer are predicted to continue to increase worldwide, with low- and middle-income countries bearing the brunt of the burden. This problem requires a concentrated global effort to avoid devastating losses of life as well as economic losses. Cholangiocarcinoma (CCA) is a readily studied model of complex, noncommunicable disease, but it receives little attention outside of the scientific community in Southeast Asia. Here, we bring attention to the opportunity to study CCA as a model to understand the role of multiple, complex factors in cancer. These factors include allostatic load, individual genetic susceptibility, and environmental exposures such as chemicals, diet, socioeconomic factors, and psychosocial stress. The study of CCA offers a unique opportunity to make novel observations that could advance progress in prevention and intervention approaches for prevalent diseases that involve complex, multifactorial interactions.

Association between Repeated Praziquantel treatment and Papillary, and Intrahepatic Cholangiocarcinoma.

INTRODUCTION AND AIM: The carcinogenesis of tubular and papillary cholangiocarcinoma (CCA) differ. The available epidemiologic studies about risk factors for CCA do not differentiate between the tubular and papillary type. The current study investigated the relationship between the number of repeated use of Praziquantel (PZQ) treatments and each type of CCA. MATERIAL AND METHODS: This was a hospital-based, matched, case-control study of patients admitted to Srinagarind Hospital, Khon Kaen University. The patients were 210 pathologically-confirmed cases of CCA, while the controls were 840 subjects diagnosed with other diseases. The 4 controls were individually matched with each case by sex, age, and date of admission. The cases were classified according to location (intrahepatic vs. extrahepatic) and cell type (papillary vs. tubular). Multivariable conditional logistic regression was used for the analysis. RESULTS: After adjusting for confounders, there were statistically significant associations between intrahepatic and papillary CCA and repeated use of PZQ treatment. The respective odds of developing intrahepatic CCA for those who used PZQ once, twice, or more was 1.54 (95%CI:0.92-2.55 ), 2.28 (95%CI:0.91-5.73), and 4.21 (95%CI:1.61-11.05). The respective odds of developing papillary CCA for those who used PZQ once, twice, or more was 1.45 (95%CI:0.80-2.63), 2.96 (95%CI:1.06-8.24), and 3.24 (95%CI:1.09-9.66). There was no association between number of uses of PZQ treatment and developing extrahepatic or tubular CCA. CONCLUSION: The current study found an association between papillary and intrahepatic CCA and repeated use of PZQ treatment. We suggest further study on the risk factors for papillary and tubular CCA should be performed separately.

Decreased argininosuccinate synthetase expression in Thai patients with cholangiocarcinoma and the effects of ADI-PEG20 treatment in CCA cell lines.

Cholangiocarcinoma (CCA) is a severe cancer with poor prognosis. The aim of the present study was to explore the expression of argininosuccinate synthetase (ASS), as well as the possibility of using pegylated arginine deiminase (ADI-PEG20) for the treatment of CCA. ASS expression was determined in CCA specimens from 40 patients in Thailand. Immunohistochemical detection of ASS and determination of the proliferative index, Ki-67, were carried out in paraffin-embedded sections of these specimens, as well as in two CCA cell lines, HuCCA and RmCCA-1, derived from CCA samples from patients in Thailand. In total, ~45% of the CCA specimens had low ASS expression, and the level of expression was significantly negatively associated with cell differentiation (P<0.05) and Ki-67 expression (P<0.05). The level of ASS expression in tumor cells was significantly lower than that in non-tumor cells (1.3-fold, P<0.05). The HuCCA cell line had significantly lower levels (P<0.05) of ASS expression at the mRNA and protein levels relative to those of normal human immortalized fibroblast cells (BJ-1). By contrast, the RmCCA-1 cell line showed no significant difference. In addition, the effects of ADI-PEG20 on growth inhibition, apoptosis and cell cycle arrest were determined in HuCCA and RmCCA-1 cells. ADI-PEG20 treatment reduced cell viability and cell proliferation in the two CCA cell lines, though it had no effect in immortalized BJ-1 cells. Furthermore, ADI-PEG20 treatment significantly increased G0/G1 cell cycle arrest in HuCCA, though not in RmCCA-1 cells. ASS silencing in the RmCCA-1 cell line significantly enhanced its sensitivity to ADI-PEG20 treatment. Results from the in vitro study demonstrated that ADI-PEG20 has antitumor activity against CCA with low ASS expression.

Clustering of patients with intrahepatic cholangiocarcinoma based on serum periostin may be predictive of prognosis.

An effective serum biomarker may improve cholangiocarcinoma (CCA) management. Periostin (PN) has been demonstrated to be associated with aggressive CCA. The current study evaluated PN in blood serum for its diagnostic and prognostic potential in patients with CCA. Sera of 68 patients with CCA were collected prior to treatment, and PN levels were measured using an ELISA. Sera from 50 normal controls, 6 patients with benign liver diseases, 2 with hepatocellular carcinoma and 21 with breast cancer were analyzed. Immunohistochemistry of PN in CCA tissues was also investigated. The data were analyzed using the Mann-Whitney U test, Kaplan-Meier log rank tests, Cox proportional hazard regression models and Fisher's exact tests. The median serum PN level in patients with CCA was significantly increased compared with that in healthy controls, patients with benign liver diseases and patients with breast cancer (all P<0.05). Using an optimal threshold value of 94 ng/ml PN, the diagnostic values for CCA compared with other conditions demonstrated a sensitivity level of 0.38 [95% confidence interval (CI), 0.27-0.51], specificity of 0.90 (95% CI, 0.81-0.96), accuracy of 0.66 (95% CI, 0.58-0.74), positive predictive value of 0.76 (95% CI, 0.59-0.89) and negative predictive value of 0.63 (95% CI, 0.53-0.72) (P<0.001). Furthermore, PN stain in stromal fibroblasts in CCA tissues was associated with serum PN levels (P=0.001), and patients with CCA were classified as low (≤94 ng/ml) or high PN (>94 ng/ml) accordingly. High serum and tissue PN levels were significantly associated with reduced survival rate (P<0.001 and P=0.033, respectively). Serum PN was an independent prognostic factor with a hazard ratio of 3.197 (P=0.001). In conclusion, serum PN may be used to divide patients with intrahepatic CCA into high and low PN groups. Elevated serum PN may be utilized as a marker of poor prognosis in patients with CCA.

Common Molecular Subtypes Among Asian Hepatocellular Carcinoma and Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) and hepatocellular carcinoma (HCC) are clinically disparate primary liver cancers with etiological and biological heterogeneity. We identified common molecular subtypes linked to similar prognosis among 199 Thai ICC and HCC patients through systems integration of genomics, transcriptomics, and metabolomics. While ICC and HCC share recurrently mutated genes, including TP53, ARID1A, and ARID2, mitotic checkpoint anomalies distinguish the C1 subtype with key drivers PLK1 and ECT2, whereas the C2 subtype is linked to obesity, T cell infiltration, and bile acid metabolism. These molecular subtypes are found in 582 Asian, but less so in 265 Caucasian patients. Thus, Asian ICC and HCC, while clinically treated as separate entities, share common molecular subtypes with similar actionable drivers to improve precision therapy.

The Combination of Arginine Deprivation and 5-Fluorouracil Improves Therapeutic Efficacy in Argininosuccinate Synthetase Negative Hepatocellular Carcinoma.

Argininosuccinate synthetase (ASS), a key enzyme to synthesize arginine is down regulated in many tumors including hepatocellular carcinoma (HCC). Similar to previous reports, we have found the decrease in ASS expression in poorly differentiated HCC. These ASS(-) tumors are auxotrophic for arginine. Pegylated arginine deiminase (ADI-PEG20), which degrades arginine, has shown activity in these tumors, but the antitumor effect is not robust and hence combination treatment is needed. Herein, we have elucidated the effectiveness of ADI-PEG20 combined with 5-Fluorouracil (5-FU) in ASS(-)HCC by targeting urea cycle and pyrimidine metabolism using four HCC cell lines as model. SNU398 and SNU387 express very low levels of ASS or ASS(-) while Huh-1, and HepG2 express high ASS similar to normal cells. Our results showed that the augmented cytotoxic effect of combination treatment only occurs in SNU398 and SNU387, and not in HepG2 and Huh-1 (ASS(+)) cells, and is partly due to reduced anti-apoptotic proteins X-linked inhibitor of apoptosis protein (XIAP), myeloid leukemia cell differentiation protein (Mcl-1) and B-cell lymphoma-2 (Bcl-2). Importantly, lack of ASS also influences essential enzymes in pyrimidine synthesis (carbamoyl-phosphate synthetase2, aspartate transcarbamylase and dihydrooratase (CAD) and thymidylate synthase (TS)) and malate dehydrogenase-1 (MDH-1) in TCA cycle. ADI-PEG20 treatment decreased these enzymes and made them more vulnerable to 5-FU. Transfection of ASS restored these enzymes and abolished the sensitivity to ADI-PEG20 and combination treatment. Overall, our data suggest that ASS influences multiple enzymes involved in 5-FU sensitivity. Combining ADI-PEG20 and 5-FU may be effective to treat ASS(-)hepatoma and warrants further clinical investigation.