Ake Pugkhem

Orotate phosphoribosyl transferase mRNA expression and the response of cholangiocarcinoma to 5-fluorouracil

AIM To determine whether expression of certain enzymes related to 5-fluorouracil (5-FU) metabolism predicts 5-FU chemosensitivity in cholangiocarcinoma (CCA). METHODS The histoculture drug response assay (HDRA) was performed using surgically resected CCA tissues. Tumor cell viability was determined morphologically with hematoxylin and eosin- and terminal deoxynucleotide transferase-mediated dUTP nick-end labeling-stained tissues. The mRNA expression of thymidine phosphorylase (TP), orotate phosphoribosyl transferase (OPRT), thymidylate synthase (TS), and dihydropyrimidine dehydrogenase (DPD) was determined with real-time reverse transcriptase-polymerase chain reaction. The levels of gene expression and the sensitivity to 5-FU were evaluated. RESULTS Twenty-three CCA tissues were obtained from patients who had been diagnosed with intrahepatic CCA and who underwent surgical resection at Srinagarind Hospital, Khon Kaen University from 2007 to 2009. HDRA was used to determine the response of these CCA tissues to 5-FU. Based on the dose-response curve, 200 μg/mL 5-FU was selected as the test concentration. The percentage of inhibition index at the median point was selected as the cut-off point to differentiate the responding and non-responding tumors to 5-FU. When the relationship between TP, OPRT, TS and DPD mRNA expression levels and the sensitivity of CCA tissues to 5-FU was examined, only OPRT mRNA expression was significantly correlated with the response to 5-FU. The mean expression level of OPRT was significantly higher in the responder group compared to the non-responder group (0.41 ± 0.25 vs 0.22 ± 0.12, P < 0.05). CONCLUSION OPRT mRNA expression may be a useful predictor of 5-FU chemosensitivity of CCA. Whether OPRT mRNA could be used to predict the success of 5-FU chemotherapy in CCA patients requires confirmation in patients.

Molecular analysis of Helicobacter pylori virulent‐associated genes in hepatobiliary patients

OBJECTIVES The Helicobacter pylori virulence-associated genes in hepatobiliary patients, including vacA, iceA, babA2, cagA and cagE, have not been reported. The aim of this study was to investigate these genes and the association of those and the clinical outcomes in hepatobiliary diseases. METHODS Eighty H. pylori-PCR-positive cases were obtained from hepatobiliary patients, representing both cholangiocarcinoma (CCA) (n= 58) and cholelithiasis (n= 22). The diversity of virulence genes was examined by polymerase chain reaction and DNA sequencing. Phylogenetic analysis of cagA was determined using the maximum parsimony method. RESULTS The vacAs1a + c/m1, iceA1 and babA2 genes were the most predominant genotypes in both CCA and cholelithiasis patients. The cagA and cagE genes were found significantly more frequently in patients with CCA than those with cholelithiasis (P < 0.05). The cagA positive samples were the Western-type cagA and showed that almost all of the detected sequences in Thai hepatobiliary and Thai gastric cancer patients were classified in the same cluster but separated from the cluster of Japan and other countries. CONCLUSIONS The cagA and cagE genes may be associated in the pathogenesis of hepatobiliary diseases, especially of CCA. Besides the bacterial variation, other host factors may be involved in the pathogenesis of hepatobiliary cancer.

Aberrant expression of NF-κB in liver fluke associated cholangiocarcinoma: implications for targeted therapy.

Background Up-regulation and association of nuclear factor kappa B (NF-κB) with carcinogenesis and tumor progression has been reported in several malignancies. In the current study, expression of NF-κB in cholangiocarcinoma (CCA) patient tissues and its clinical significance were determined. The possibility of using NF-κB as the therapeutic target of CCA was demonstrated. Methodology Expression of NF-κB in CCA patient tissues was determined using immunohistochemistry. Dehydroxymethylepoxyquinomicin (DHMEQ), a specific NF-κB inhibitor, was used to inhibit NF-κB action. Cell growth was determined using an MTT assay, and cell apoptosis was shown by DNA fragmentation, flow cytometry and immunocytofluorescent staining. Effects of DHMEQ on growth and apoptosis were demonstrated in CCA cell lines and CCA-inoculated mice. DHMEQ-induced apoptosis in patient tissues using a histoculture drug response assay was quantified by TUNEL assay. Principal Findings Normal bile duct epithelia rarely expressed NF-κB (subunits p50, p52 and p65), whereas all CCA patient tissues (n  =  48) over-expressed all NF-κB subunits. Inhibiting NF-κB action by DHMEQ significantly inhibited growth of human CCA cell lines in a dose- and time-dependent manner. DHMEQ increased cell apoptosis by decreasing the anti-apoptotic protein expressions–Bcl-2, XIAP–and activating caspase pathway. DHMEQ effectively reduced tumor size in CCA-inoculated mice and induced cell apoptosis in primary histocultures of CCA patient tissues. Conclusions NF-κB was over-expressed in CCA tissues. Inhibition of NF-κB action significantly reduced cell growth and enhanced cell apoptosis. This study highlights NF-κB as a molecular target for CCA therapy.

Surgical management of perihilar cholangiocarcinoma: a Khon Kaen experience

Cholangiocarcinoma is the most common cancer in the northeast of Thailand. Most of the patients present when the disease is in an advanced stage. Improvement of preoperative diagnoses and surgical techniques provide more satisfactory results. Herein we reviewed our 30‐year experience in management of perihilar cholangiocarcinoma in Khon Kaen northeast Thailand. Between 1982 and 2012 we reviewed four specific studies of perihilar cholangiocarcinoma in Srinagarind Hospital, Khon Kaen, Thailand. The first study focused on advanced surgical pathology and palliative surgery, which were used to treat obstructive jaundice cholangiocarcinoma patients. Long‐term survival in this study was rare with a one‐year survival of just 15%. The second study was conducted on 30 consecutive cases of perihilar cholangiocarcinoma who presented with obstructive jaundice without preoperative biliary drainage. All the patients underwent major liver resection with bilio‐enteric reconstruction. Perioperative mortality was 6.7% without a 5‐year survival. The third study aimed to analyze the survival rates and factors affecting survival in extrahepatic CCA patients following surgical treatment at Srinagarind Hospital and concluded that resection margins are an important prognostic factor. The last study objective was the analysis of curative surgical attempt in 99 consecutive perihilar cholangiocarcinoma and results showed that R0 resection could improve long‐term survival. We evaluated four studies of perihilar cholangiocarcinoma in Srinagarind Hospital, Khon Kaen, Thailand from 1982–2012. Viewed chronologically there has been a progressive improvement of diagnosis and surgical treatment during the past 30 years. Despite these advances the 5‐year survival rate remains unsatisfactorily low. Future improvement of patient selection and surgical techniques can lead to a greater survival rate for patients.

Cholangiocarcinoma Patient Outcome in Northeastern Thailand: Single-Center Prospective Study

BACKGROUND Cholangiocarcinoma is relatively rare worldwide. Most previous reports collected only patients with pathological diagnosis. In fact, however, many patients coming to hospital are diagnosed by clinical suspicion with radiologic imaging and receive treatment without histological confirmation. Real survival data and outcome of each treatment, especially for patients that do not have histologic confirmation, are lacking. In this study, therefore, we aimed to analyze the survival rates of CCA patients and the proportions of patients receiving different treatments. MATERIALS AND METHODS A total of 270 patients clinically suspected of CCA and visiting Srinagarind Hospital in May-July 2010, were prospectively followed until December 2014. After checking their clinical records, 163 of 270 patients were finally diagnosed as having CCA, and the data of this group were analyzed for survival rate and received treatments. RESULTS Of the 163 patients, 96 (58.9%) had intrahepatic, 56 (34.4%) had perihilar and 11 (6.7%) had distal CCA. The majority [107 (65.6%, 95%CI, 57.8-73.0)] received only supportive care. Overall median survival was 4 months (95%CI, 3.3-4.7), and 2-years survival was only 8.1% (95%CI,4.5-12.9). However, the 4 year survival of the R0 resection group was 100%. CONCLUSIONS The present results show that the prognosis of CCA is very poor in North-east Thailand. Most CCA patients receive only treatment to alleviate symptoms due to their advanced stage of disease. Complete surgical resection at the early stage is the only treatment that significantly improves patient survival.

Long-term outcome of surgical resection for intraductal papillary neoplasm of the bile duct

Intraductal papillary neoplasm of the bile duct (IPNB) is a specific type of bile duct tumor. Studies about the surgical outcomes for IPNB are few; therefore, we investigated the survival of patients who underwent curative surgical resection of IPNB.

Outcome of curative resection for perihilar cholangiocarcinoma in Northeast Thailand

AIM To examine survival outcomes of perihilar cholangiocarcinoma (PCCA) resection including mortality, morbidity and prognostic factors. METHODS Multivariate analyses were carried out based on the survival data of all patients with histologically confirmed PCCA who underwent curative resection at Srinagarind Hospital from January 2006 to December 2011. RESULTS There were 29 (19%) cases of intrahepatic CCA that involved hilar and 124 (81%) with hilar bile-duct cancer. R0 resection was carried out on 66 (43.1%) patients of whom 50 (32.7%) also had lymph node metastasis. The other patients underwent R1 resection. The overall 5-year survival rate was 20.6% (95%CI: 13.8-28.4) and median survival time was 19.9 mo. Postoperative mortality was 2%, and 30% of patients had complications. Patients without lymph node metastasis were 60% less likely to die than those with metastasis. Achieving R0 led to a 58% reduction in the chance of mortality as compared to R1. CONCLUSION To achieve a better survival outcome, focus should center on performing radical surgery and detection of patients with early stage cancer.

Differential Protein Expression Marks the Transition From Infection With Opisthorchis viverrini to Cholangiocarcinoma.

Parts of Southeast Asia have the highest incidence of intrahepatic cholangiocarcinoma (CCA) in the world because of infection by the liver fluke Opisthorchis viverrini (Ov). Ov-associated CCA is the culmination of chronic Ov-infection, with the persistent production of the growth factors and cytokines associated with persistent inflammation, which can endure for years in Ov-infected individuals prior to transitioning to CCA. Isobaric labeling and tandem mass spectrometry of liver tissue from a hamster model of CCA was used to compare protein expression profiles from inflammed tissue (Ovinfected but not cancerous) versus cancerous tissue (Ov-induced CCA). Immunohistochemistry and immunoblotting were used to verify dysregulated proteins in the animal model and in human tissue. We identified 154 dysregulated proteins that marked the transition from Ov-infection to Ov-induced CCA, i.e. proteins dysregulated during carcinogenesis but not Ov-infection. The verification of dysregulated proteins in resected liver tissue from humans with Ov-associated CCA showed the numerous parallels in protein dysregulation between human and animal models of Ov-induced CCA. To identify potential circulating markers for CCA, dysregulated proteins were compared with proteins isolated from exosomes secreted by a human CCA cell line (KKU055) and 27 proteins were identified as dysregulated in CCA and present in exosomes. These data form the basis of potential diagnostic biomarkers for human Ov-associated CCA. The profile of protein dysregulation observed during chronic Ovinfection and then in Ov-induced CCA provides insight into the etiology of an infection-induced inflammation-related cancer.

Morphological Classification of Intraductal Papillary Neoplasm of the Bile Duct with Survival Correlation

Background: Intraductal papillary neoplasm of the bile duct (IPNB) is a specific entity for which there has been no classification that correlates clinical presentation with patient survival. We, therefore, propose a new classification based on radio-pathological appearance correlated with clinical findings including outcome. Methods: We retrospectively reviewed the medical and pathological records of 103 IPNB patients who underwent curative-intent hepatic resection between January 2008 and December 2011. A morphological classification was then created based on the presence of (a) bile duct dilatation, (b) intraductal mass(es), (c) cystic lesion(s), and (d) macro-invasion of the liver. All clinical parameters and survival were analyzed. Results: The median survival of IPNB patients was 1,728 days (95% CI: 1,485 to 1,971 days). The proposed classification predicted survival very well (log-rank test; p < 0.01). For patients with the cystic variant and micro-papillary IPNB, there were no tumor-related deaths within 3 years of surgery and median survival was not reached during the follow-up. The respective median survival times for IPNBs with unilateral intrahepatic duct dilatation, bilateral intrahepatic duct dilatation, and macro-invasion were 1,888 days (95%CI 1,118- 2,657), 673 days (95% CI: 392- 953), and 578 days (95% CI: 285- 870). Conclusion: We propose a new classification for IPNBs which not only provides a view of patients in terms of their radio-pathologic status but also should help in guiding planning of surgical procedures.

Simultaneous Extensive Intraductal Papillary Neoplasm of the Bile Duct and Pancreas: A Very Rare Entity

Intraductal papillary neoplasm of the bile duct (IPNB) is a specific type of bile duct tumor. It has been proposed that it could be the biliary counterpart of the intraductal papillary neoplasm of the pancreas (IPMN-P). This hypothesis is supported by the presence of simultaneous intraductal tumors of both the bile duct and pancreas. There have been five reports of patients with simultaneous IPNB and IPMN-P. In all of these cases, biliary involvement was limited to the intrahepatic and perihilar bile duct, which had characteristics similar to IPMN-P and usually had slow progression in nature. Herein, we present the first case of extensive intraductal neoplasm involving the extrahepatic bile duct, intrahepatic bile duct, and entire length of the pancreas with a poor outcome, even after being treated aggressively with radical surgery and adjuvant chemotherapy. Additionally, we summarize previous case reports of simultaneous intraductal lesions of the bile duct and pancreas.