Molecular Pathogenesis of Cholangiocarcinoma

BACKGROUND: Cholangiocarcinomas are a heterogeneous group of malignancies arising from a number of cells of origin along the biliary tree. Although most cases in Western countries are sporadic, large population-based studies have identified a number of risk factors. This review summarises the evidence behind reported risk factors and current understanding of the molecular pathogenesis of cholangiocarcinoma, with a focus on inflammation and cholestasis as the driving forces in cholangiocarcinoma development. RISK FACTORS FOR CHOLANGIOCARCINOGENESIS: Cholestatic liver diseases (e.g. primary sclerosing cholangitis and fibropolycystic liver diseases), liver cirrhosis, and biliary stone disease all increase the risk of cholangiocarcinoma. Certain bacterial, viral or parasitic infections such as hepatitis B and C and liver flukes also increase cholangiocarcinoma risk. Other risk factors include inflammatory disorders (such as inflammatory bowel disease and chronic pancreatitis), toxins (e.g. alcohol and tobacco), metabolic conditions (diabetes, obesity and non-alcoholic fatty liver disease) and a number of genetic disorders. MOLECULAR PATHOGENESIS OF CHOLANGIOCARCINOMA: Regardless of aetiology, most risk factors cause chronic inflammation or cholestasis. Chronic inflammation leads to increased exposure of cholangiocytes to the inflammatory mediators interleukin-6, Tumour Necrosis Factor-ɑ, Cyclo-oxygenase-2 and Wnt, resulting in progressive mutations in tumour suppressor genes, proto-oncogenes and DNA mismatch-repair genes. Accumulating bile acids from cholestasis lead to reduced pH, increased apoptosis and activation of ERK1/2, Akt and NF-κB pathways that encourage cell proliferation, migration and survival. Other mediators upregulated in cholangiocarcinoma include Transforming Growth Factor-β, Vascular Endothelial Growth Factor, Hepatocyte Growth Factor and several microRNAs. Increased expression of the cell surface receptor c-Met, the glucose transporter GLUT-1 and the sodium iodide symporter lead to tumour growth, angiogenesis and cell migration. Stromal changes are also observed, resulting in alterations to the extracellular matrix composition and recruitment of fibroblasts and macrophages that create a microenvironment promoting cell survival, invasion and metastasis. CONCLUSION: Regardless of aetiology, most risk factors for cholangiocarcinoma cause chronic inflammation and/or cholestasis, leading to the activation of common intracellular pathways that result in reactive cell proliferation, genetic/epigenetic mutations and cholangiocarcinogenesis. An understanding of the molecular pathogenesis of cholangiocarcinoma is vital when developing new diagnostic biomarkers and targeted therapies for this disease

5-aminolevulinic acid as a potential contrast agent for image-guided surgery in pancreatic cancer

Introduction: Pancreatic cancer often recurs following surgery suggesting new operative approaches are required. Fluorescence-guided surgery aims to assist surgeons in identifying tumour intraoperatively to facilitate complete resection. However, the ideal contrast agent for this purpose is not yet determined. The Rose criterion states that accurate imageguided surgery requires a Tumour-to-Background Ratio of contrast agent greater than 5. We investigated the potential of 5-aminolevulinic acid (ALA) for this purpose. Methods: Pancreatic cancer cell lines CFPAC-1 and PANC-1 were compared with the control pancreatic ductal cell line H6c7. Cells were seeded on day 1 and fluorescence measured on day 4 following 4, 8, 24 or 48 hours incubation with 0.25, 0.50, 0.75 or 1.00mM ALA. Fluorescence was measured using a plate reader and microscopy. Results: The maximum ALA-induced fluorescence for CFPAC-1 and PANC-1 was achieved after 48 hours incubation with 0.50mM ALA. Compared to cells incubated without ALA, a relative fluorescence increase of 39.4-fold in CFPAC-1 and 2.7-fold in PANC-1 was seen. ALA concentrations above 0.50mM did not result in higher fluorescence. In contrast, the control cell line H6c7 showed progressively increasing fluorescence with increasing ALA concentrations. The highest cancer/control cell fluorescence ratios for ALA were after 48 hours incubation with 0.25mM ALA; 122.9 in CFPAC-1 and 9.7 in PANC-1. Conclusion: ALA-induced fluorescence in CFPAC-1 is significantly higher than the control cell line H6c7. PANC-1 achieved only mildly increased fluorescence compared to H6c7. ALA has the potential to provide an adequate level of fluorescence for image-guided pancreatic surgery in ALA-susceptible cancers

Endoscopic ultrasound in the assessment of advanced duodenal adenomatosis in familial adenomatous polyposis

Objective: Current surveillance strategies for duodenal adenomatosis in familial adenomatous polyposis (FAP) miss malignancies and underestimate cancer risk in ampullary disease. This study aimed to evaluate the utility of endoscopic ultrasound (EUS) in the assessment of FAP patients with duodenal and/or ampullary polyposis referred for surgical intervention. Design: A retrospective analysis of FAP patients undergoing index EUS between December 2006 and May 2015 was performed. Follow-up was completed in January 2018, including review of all EUS procedures and surgical interventions (median follow-up 6 years). Results: Fifty-five patients underwent 188 EUS procedures. Six patients (11%) developed malignancy (three duodenal, three ampullary). Ampullary cancer risk was underestimated by Spigelman stage and overestimated by Kashiwagi classification. Ultrasound findings were poor predictors of malignancy, with common bile duct dilatation being the only finding present in one EUS prior to a diagnosis of ampullary cancer. The best predictors of ampullary malignancy were an ampullary polyp size >3 cm and an increase >1 cm in ampullary polyp size. Ampullary polyp size >3 cm provided the best predictive value, correctly identifying two of the three cases of ampullary cancer and both patients with high-grade dysplasia. EUS biopsy failed to detect malignancy later confirmed by surgical histology in two patients. Conclusion: EUS surveillance confers little additional benefit to standard endoscopic surveillance in FAP patients. The best predictor of ampullary malignancy is an ampullary polyp >3 cm; this could be regarded as a relative indication for surgery

Endoscopic ultrasound in the assessment of advanced duodenal adenomatosis in familial adenomatous polyposis

Objective Current surveillance strategies for duodenal adenomatosis in familial adenomatous polyposis (FAP) miss malignancies and underestimate cancer risk in ampullary disease. This study aimed to evaluate the utility of endoscopic ultrasound (EUS) in the assessment of FAP patients with duodenal and/or ampullary polyposis referred for surgical intervention. Design A retrospective analysis of FAP patients undergoing index EUS between December 2006 and May 2015 was performed. Follow-up was completed in January 2018, including review of all EUS procedures and surgical interventions (median follow-up 6 years). Results Fifty-five patients underwent 188 EUS procedures. Six patients (11%) developed malignancy (three duodenal, three ampullary). Ampullary cancer risk was underestimated by Spigelman stage and overestimated by Kashiwagi classification. Ultrasound findings were poor predictors of malignancy, with common bile duct dilatation being the only finding present in one EUS prior to a diagnosis of ampullary cancer. The best predictors of ampullary malignancy were an ampullary polyp size >3 cm and an increase >1 cm in ampullary polyp size. Ampullary polyp size >3 cm provided the best predictive value, correctly identifying two of the three cases of ampullary cancer and both patients with high-grade dysplasia. EUS biopsy failed to detect malignancy later confirmed by surgical histology in two patients. Conclusion EUS surveillance confers little additional benefit to standard endoscopic surveillance in FAP patients. The best predictor of ampullary malignancy is an ampullary polyp >3 cm; this could be regarded as a relative indication for surgery

Effect of 5-aminolevulinic acid on the haem biosynthesis pathway in pancreatic cancer and pancreatic ductal epithelial cell lines

Introduction: 5-aminolevulinic acid (ALA) generates protoporphyrin IX (PpIX)-induced fluorescence by acting as a substrate for the haem biosynthesis pathway. Despite suggestions that ALA could be used for pancreatic cancer photodiagnostics, the pancreatic cancer cell line PANC-1 only shows weak fluorescence following ALA administration. A possible explanation was that the haem biosynthesis pathway varies between cancers. Methods: We compared the mRNA expression of the haem biosynthesis pathway of PANC-1 (weak fluorescence) with the pancreatic cancer cell line CFPAC-1 (strong fluorescence) and the pancreatic ductal cell line H6c7 (control) with or without 24 hours ALA incubation. Cells were seeded on day one, fresh media with or without ALA (0.5mM) added on day two, and RNA extracted on day three. Quantitative real-time polymerase chain reaction was performed to assess the relative mRNA expression of four membrane transporters and eight enzymes responsible for haem biosynthesis. Results: Post-ALA incubation, CFPAC-1 demonstrated significant downregulation of cell membrane ALA influx transporter PEPT1, downregulation of ALA synthase and upregulation of the mitochondrial membrane transporter ABCB6. PANC-1, whilst showing similar changes to ALA synthase and ABCB6, showed significant upregulation of the PpIX efflux transporter ABCG2. PANC-1 also had minimal PEPT1 expression pre- and post-ALA. H6c7 demonstrated significant up- or downregulation of three transporters and five enzymes. Conclusion: Poor PpIX-induced fluorescence in PANC-1 is likely to be secondary to decreased ALA influx from low PEPT1 expression and increased ABCG2 expression. The use of nanocarriers to deliver ALA and/or ABCG2 inhibitors may improve ALA-induced fluorescence in PANC-1 and other ALA-resistant cancers

Targeting of epidermal growth factor receptor (EGFR)-positive pancreatic cancer cell lines with cetuximab-conjugated near-infrared silver sulphide quantum dots

Introduction: Fluorescence-guided surgery could potentially reduce local recurrence after pancreatic cancer resection. However, the ideal contrast agent for this purpose is not yet determined. The monoclonal antibody cetuximab targets the EGFR receptor, which is overexpressed in 64% of pancreatic cancers. We investigated the efficacy of near-infrared emitting silver sulphide Quantum Dot (QD)-cetuximab nanoconjugates for targeting EGFR-positive pancreatic cancer. Methods: 2-Mercaptopropionic acid-coated QDs were prepared from AgNO3 and Na2S. Pancreatic cancer cell lines PANC-1 and CFPAC-1 were confirmed EGFR-positive using a commercial AlexaFluor488-cetuximab probe. Nonconjugated QD and cetuximab-conjugated QD (QD-cetuximab) toxicity was assessed after 24 and 48 hours using MTT assay. Fluorescence microscopy was performed following a) formaldehyde-fixed immunofluorescence and b) live staining with QD-cetuximab for four hours at concentrations corresponding to 0, 10, 50, 100, 200, 400 and 600µg ml-1 of silver. Results: Untargeted QDs were non-toxic in both cell lines after 48 hours at all investigated concentrations, whereas QDcetuximab was toxic at 100µg ml-1 after 24 hours in PANC-1 and at 10µg ml-1 in CFPAC-1. Fixed immunofluorescence demonstrated EGFR targeting by QD-cetuximab at concentrations of 50µg ml-1 upwards in both cell lines. Live staining demonstrated similar efficacy of EGFR targeting up to 50µg ml-1 , although a reduction of fluorescence at higher concentrations was observed when compared to fixed immunofluorescence. Conclusion: Silver sulphide QD-cetuximab nanoconjugates have the potential to target live EGFR-positive pancreatic cancer cells at doses of up to 50 µg ml-1 . The reduction in QD fluorescence observed at higher concentrations is likely to be secondary to cetuximab toxicity

Does an extensive diagnostic workup for upfront resectable pancreatic cancer result in a delay which affects survival? Results from an international multicentre study

Backgrounds/Aims: Pancreatoduodenectomy (PD) is recommended in fit patients with a carcinoma (PDAC) of the pancreatic head, and a delayed resection may affect survival. This study aimed to correlate the time from staging to PD with long-term survival, and study the impact of preoperative investigations (if any) on the timing of surgery. // Methods: Data were extracted from the Recurrence After Whipple’s (RAW) study, a multicentre retrospective study of PD outcomes. Only PDAC patients who underwent an upfront resection were included. Patients who received neoadjuvant chemo-/radiotherapy were excluded. Group A (PD within 28 days of most recent preoperative computed tomography [CT]) was compared to group B (> 28 days). // Results: A total of 595 patents were included. Compared to group A (median CT-PD time: 12.5 days, interquartile range: 6–21), group B (49 days, 39–64.5) had similar one-year survival (73% vs. 75%, p = 0.6), five-year survival (23% vs. 21%, p = 0.6) and median time-todeath (17 vs. 18 months, p = 0.8). Staging laparoscopy (43 vs. 29.5 days, p = 0.009) and preoperative biliary stenting (39 vs. 20 days, p 0.99) and endoscopic ultrasonography (28 vs. 32 days, p > 0.99) were not. // Conclusions: Although a treatment delay may give rise to patient anxiety, our findings would suggest this does not correlate with worse survival. A delay may be necessary to obtain further information and minimize the number of PD patients diagnosed with early disease recurrence

ICAR: endoscopic skull‐base surgery

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