Prognostic and predictive value of human equilibrative nucleoside transporter 1 (hENT1) in extrahepatic cholangiocarcinoma: a translational study.

Introduction: Effective (neo) adjuvant chemotherapy for cholangiocarcinoma is lacking due to chemoresistance and the absence of predictive biomarkers. Human equilibrative nucleoside transporter 1 (hENT1) has been described as a potential prognostic and predictive biomarker. In this study, the potential of rabbit-derived (SP120) and murine-derived (10D7G2) antibodies to detect hENT1 expression was compared in tissue samples of patients with extrahepatic cholangiocarcinoma (ECC), and the predictive value of hENT1 was investigated in three ECC cell lines. Methods: Tissues of 71 chemonaïve patients with histological confirmation of ECC were selected and stained with SP120 or 10D7G2 to assess the inter-observer variability for both antibodies and the correlation with overall survival. Concomitantly, gemcitabine sensitivity after hENT1 knockdown was assessed in the ECC cell lines EGI-1, TFK-1, and SK-ChA-1 using sulforhodamine B assays. Results: Scoring immunohistochemistry for hENT1 expression with the use of SP120 antibody resulted in the highest interobserver agreement but did not show a prognostic role of hENT1. However, 10D7G2 showed a prognostic role for hENT1, and a potential predictive role for gemcitabine sensitivity in hENT1 in SK-ChA-1 and TFK-1 cells was found. Discussion: These findings prompt further studies for both preclinical validation of the role of hENT1 and histochemical standardization in cholangiocarcinoma patients treated with gemcitabine-based chemotherapy.

Treatment patterns and survival in older adults with unresected nonmetastatic biliary tract cancers.

INTRODUCTION: The optimal treatment for unresected nonmetastatic biliary tract cancer (uBTC) is not well-established. The objective of this study was to analyze the treatment patterns and compare the differences in overall survival (OS) between different treatment strategies amongst older adults with uBTC. MATERIALS AND METHODS: We identified patients aged ≥65 years with uBTC using the Surveillance, Epidemiology, and End Results (SEER)-Medicare database (2004-2015). Treatments were classified into chemotherapy, chemoradiotherapy, and radiotherapy. The primary outcome was OS. The differences in OS were analyzed using Kaplan-Meier curves and multivariable Cox proportional hazard regression. RESULTS: A total of 4352 patients with uBTC were included. The median age was 80 years and median OS was 4.1 months. Most patients (67.3%, n = 2931) received no treatment, 19.1% chemotherapy (n = 833), 8.1% chemoradiotherapy (n = 354), and 5.4% radiotherapy alone (n = 234). Patients receiving no treatment were older and had more comorbidities. Chemotherapy was associated with significantly longer OS than no treatment in uBTC (hazard ratio [HR] 0.87, 95% confidence interval [CI] 0.79-0.95), but no difference was found in the subgroups of intrahepatic cholangiocarcinoma (iCCA; HR 0.87, 95% CI 0.75-1.00) and gallbladder carcinoma (GBC; HR 1.09, 95% CI 0.86-1.39). In the sensitivity analyses, capecitabine-based chemoradiotherapy showed significantly longer OS in uBTC compared to chemotherapy (adjusted HR 0.71, 95% CI 0.53-0.95). DISCUSSION: A minority of older patients with uBTC receive systemic treatments. Chemotherapy was associated with longer OS compared to no treatment in uBTC, but not in the subgroups of iCCA and GBC. The efficacy of chemoradiotherapy, especially in perihilar cholangiocarcinoma using capecitabine-based chemoradiotherapy, may be further evaluated in prospective clinical trials.

Experimental Conditions That Influence the Utility of 2'7'-Dichlorodihydrofluorescein Diacetate (DCFH2-DA) as a Fluorogenic Biosensor for Mitochondrial Redox Status.

Oxidative stress has been causally linked to various diseases. Electron transport chain (ETC) inhibitors such as rotenone and antimycin A are frequently used in model systems to study oxidative stress. Oxidative stress that is provoked by ETC inhibitors can be visualized using the fluorogenic probe 2',7'-dichlorodihydrofluorescein-diacetate (DCFH2-DA). Non-fluorescent DCFH2-DA crosses the plasma membrane, is deacetylated to 2',7'-dichlorodihydrofluorescein (DCFH2) by esterases, and is oxidized to its fluorescent form 2',7'-dichlorofluorescein (DCF) by intracellular ROS. DCF fluorescence can, therefore, be used as a semi-quantitative measure of general oxidative stress. However, the use of DCFH2-DA is complicated by various protocol-related factors that mediate DCFH2-to-DCF conversion independently of the degree of oxidative stress. This study therefore analyzed the influence of ancillary factors on DCF formation in the context of ETC inhibitors. It was found that ETC inhibitors trigger DCF formation in cell-free experiments when they are co-dissolved with DCFH2-DA. Moreover, the extent of DCF formation depended on the type of culture medium that was used, the pH of the assay system, the presence of fetal calf serum, and the final DCFH2-DA solvent concentration. Conclusively, experiments with DCFH2-DA should not discount the influence of protocol-related factors such as medium and mitochondrial inhibitors (and possibly other compounds) on the DCFH2-DA-DCF reaction and proper controls should always be built into the assay protocol.

Development and external validation of a prediction model for overall survival after resection of distal cholangiocarcinoma.

BACKGROUND: Various prognostic factors are associated with overall survival (OS) after resection of distal cholangiocarcinoma (dCCA). The objective of this study was to develop and validate a prediction model for 3-year OS after pancreatoduodenectomy for dCCA. METHODS: The derivation cohort consisted of all patients who underwent pancreatoduodenectomy for dCCA in the Netherlands (2009-2016). Clinically relevant variables were selected based on the Akaike information criterion using a multivariate Cox proportional hazards regression model, with model performance being assessed by concordance index (C-index) and calibration plots. External validation was performed using patients from the Belgium Cancer Registry (2008-2016), and patients from two university hospitals of Southampton (U.K.) and Verona (Italy). RESULTS: Independent prognostic factors for OS in the derivation cohort of 454 patients after pancreatoduodenectomy for dCCA were age (HR 1.02, 95% CI 1.01-1.03), pT (HR 1.43, 95% CI 1.07-1.90) and pN category (pN1: HR 1.78, 95% CI 1.37-2.32; pN2: HR 2.21, 95% CI 1.63-3.01), resection margin status (HR 1.79, 95% CI 1.39-2.29) and tumour differentiation (HR 2.02, 95% CI 1.62-2.53). The prediction model was based on these prognostic factors. The optimism-adjusted C-indices were similar in the derivation cohort (0.69), and in the Belgian (0.66) and Southampton-Verona (0.68) validation cohorts. Calibration was accurate in the Belgian validation cohort (slope = 0.93, intercept = 0.12), but slightly less optimal in the Southampton-Verona validation cohort (slope = 0.88, intercept = 0.32). Based on this model, three risk groups with different prognoses were identified (3-year OS of 65.4%, 33.2% and 11.8%). CONCLUSIONS: The prediction model for 3-year OS after resection of dCCA had reasonable performance in both the derivation and geographically external validation cohort. Calibration slightly differed between validation cohorts. The model is readily available via www. pancreascalculator.com to inform patients from Western European countries on their prognosis, and may be used to stratify patients for clinical trials.

Adjuvant treatment for the elderly patient with resected gallbladder cancer: a SEER-Medicare analysis.

Background: In patients with resected gallbladder cancer (GBC), the role of adjuvant chemotherapy (aCT) remains ill-defined, especially in elderly patients. This study evaluates the value of aCT in elderly patients with GBC and assesses response according to tumor stage. Methods: Patients of ≥65 years of age with resected GBC diagnosed from 2004-2015 were identified using a Surveillance, Epidemiology and End Results (SEER)/Medicare linked database. After propensity score matching, survival of patients treated with aCT was compared to survival of patients who did not receive aCT using Kaplan-Meier and Cox proportional hazards analysis. Results: Of 2,179 patients with resected GBC, 876 (25%) received aCT. In the full cohort of 810 propensity-score matched patients, survival did not differ between patients treated with aCT (17.6 months ) and without aCT (19.5 months, P=0.7720). Subgroup analysis showed that survival was significantly better after aCT in T3/T4 disease (12.3 vs. 7.2 months, P=0.013). Interaction analysis showed that benefit of aCT was primarily seen in combined T3/T4, node-positive disease (HR 0.612 , P=0.006). Conclusions: In this large cohort of elderly patients with resected GBC, aCT was not associated with increased survival. However, aCT may provide a survival benefit in T3/4, node-positive disease.

Efficacy and Safety of Gemcitabine Plus Cisplatin as Potential Preoperative Chemotherapy in Locally Advanced Intrahepatic, Perihilar, and Mid-Cholangiocarcinoma: A Retrospective Cohort Study.

BACKGROUND: In this retrospective cohort study, the potential of gemcitabine (gem)/cisplatin (cis) chemotherapy as future preoperative therapy for patients with unresectable locally advanced or borderline resectable intrahepatic, perihilar, and mid-cholangiocarcinoma was investigated. METHODS: All patients with intrahepatic, perihilar, and mid-cholangiocarcinoma presented at Amsterdam UMC between January 2016 and October 2019 were included. The radiologic response after 3 and/or 6 cycles of gem/cis chemotherapy in patients with unresectable locally advanced or borderline resectable disease was derived from the original radiologic reports and subsequently re-evaluated for surgical exploration by consensus reading of 2 HPB surgeons and 1 radiologist. RESULTS: Overall, 65 of 364 patients had a locally advanced or borderline resectable disease. Twenty-eight patients were treated with palliative chemotherapy, including 25 (89.3%) patients who received more than 3 cycles. Twenty-two patients (88.0%) and 13 patients (46.4%) showed RECIST stable disease or partial response after 3 and 6 cycles of chemotherapy, respectively. Three patients experienced grade 3 adverse events. Consensus reading concluded that exploration could have been reconsidered in 7 of 28 patients (25.0%). CONCLUSION: Gem/cis may be a safe and feasible preoperative treatment in initially unresectable locally advanced or borderline resectable cholangiocarcinoma. In addition, the findings of this study support to always rediscuss patients with stable or responsive disease in multidisciplinary team meetings to reconsider resection. Besides, prospective studies are needed to investigate this effect further and, based on these preliminary data, seem feasible in this setting.

Efficacy and safety of systemic induction therapy in initially unresectable locally advanced intrahepatic and perihilar cholangiocarcinoma: A systematic review.

BACKGROUND: According to international guidelines, induction therapy may be considered in selected patients with initially unresectable locally advanced cholangiocarcinoma. The criteria for (un)resectability in cholangiocarcinoma varies between studies and no consensus-based agreement is available about these criteria. By performing a systematic literature review, we aimed to investigate the efficacy and safety of systemic induction therapy in initially unresectable locally advanced perihilar (pCCA) and intrahepatic cholangiocarcinoma (iCCA) and summarize resectability criteria used across studies. METHODS: A literature search was performed in PubMed, EMBASE, Web of Science and Cochrane library to identify studies on systemic induction therapy in locally advanced pCCA and/or iCCA. The primary outcome was resection rate (RR) after induction therapy and secondary outcomes were overall survival (OS) and objective response rate (ORR). RESULTS: Ten studies with a total of 1167 patients met the inclusion criteria and were included in this review. Among these patients, 334 (28.6%) were treated with systemic induction therapy. Across the studies, different types of chemotherapy regimens were administered (e.g., gemcitabine (based) chemotherapy and 5-FU (based) chemotherapy). Only six studies provided sufficient data and were used to analyze pooled (radical) resection rates. After induction therapy, 94 patients (39.2%) underwent a resection, of which R0 resections (22.9%). Pooled data on OS showed, better OS for chemotherapy plus resection versus chemotherapy only (pooled HR = 0.31, 95% CI = 0.19-0.50; P value < 0.0001). CONCLUSION: Adequately selected patients with locally advanced pCCA or iCCA may benefit from induction therapy followed by surgical resection. Prospective randomized controlled trials are warranted.

Efficacy and safety of FOLFIRINOX as salvage treatment in advanced biliary tract cancer: an open-label, single arm, phase 2 trial.

BACKGROUND: No standard treatment is available for advanced biliary tract cancer (BTC) after first-line therapy with gemcitabine plus cisplatin (GEMCIS). The objective of this study was to evaluate safety and anti-tumour activity of fluorouracil, leucovorin, irinotecan plus oxaliplatin (FOLFIRINOX) as salvage treatment in patients with previously treated advanced BTC. METHODS: In this two-stage phase 2 study, patients with advanced BTC who had disease progression or unacceptable toxicity after ≥3 cycles of GEMCIS were eligible. Primary endpoints were safety and efficacy (defined as objective response rate, ORR). In stage one, ten patients were treated with FOLFIRINOX every 2 weeks. In stage two, an additional 20 patients were enrolled at a starting dose as defined in stage one, provided that in stage ≥1 objective response or ≥2 stable diseases were observed and ≤3 patients had serious adverse events (SAEs) within the first 6 weeks of treatment. Secondary endpoints were progression-free survival (PFS) and overall survival (OS). RESULTS: Forty patients were screened for eligibility and 30 patients were enrolled. In stage one, one patient had a partial response and five patients had stable disease. One patient had a SAE during the first 6 weeks of treatment, and five patients required a dose reduction due to adverse events. The most common grade 3-4 adverse events in stage one were neutropaenia, mucositis and diarrhoea. Stage two was initiated with FOLFIRINOX in an adapted dose. In stage two, grade 3-4 neutropaenia, diarrhoea, nausea and vomiting were the most common adverse events. The ORR, median PFS and OS in all patients were 10%, 6.2 and 10.7 months, respectively. CONCLUSIONS: In patients with advanced BTC who progressed after or were intolerant to GEMCIS, FOLFIRINOX can be administered safely and could be considered as an option for salvage treatment in these patients. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT02456714.

Prognostic immunohistochemical biomarkers of chemotherapy efficacy in biliary tract cancer: A systematic review and meta-analysis.

INTRODUCTION: Chemotherapy is the mainstay of systemic treatment of biliary tract cancer (BTC). However, the treatment response to chemotherapy varies between patients. Currently, no prognostic biomarkers for chemotherapy efficacy have been considered for use in clinical practice. A systematic review was conducted to evaluate the prognostic value of immunohistochemical biomarkers for chemotherapy in patients with resected as well as with advanced BTC. METHOD: Medline and EMBASE databases were searched up to March 2017 for studies that evaluated biomarker expression by immunohistochemistry in resected or advanced BTC patients treated with chemotherapy. The primary endpoints were overall survival (OS) and disease or progression free survival (DFS or PFS). RESULT: Twenty-six studies, including a total of 1348 patients and 26 different biomarkers, met the inclusion criteria and were included in this review. The most frequently studied prognostic biomarkers in BTC were the human Equilibrative Nucleoside Transporter 1 (hENT1), Ribonucleotide Reductase M1 (RRM1), and excision repair cross-complementation 1 (ERCC1). In the meta-analysis of patients treated with gemcitabine-based chemotherapy, high hENT1 expression was associated with longer OS (HR 0.43, 95% CI: 0.28 to 0.64) and DFS/PFS (HR 0.45, 95% CI: 0.33 to 0.61). CONCLUSION: hENT1 is a promising prognostic biomarker for gemcitabine-based chemotherapy in resected as well as in advanced BTC and should be further validated for the selection of patients for chemotherapy.

Treatment and survival of resected and unresected distal cholangiocarcinoma: a nationwide study.

Background: Population-based data on distal cholangiocarcinoma (DCC) from the Western world are not available, albeit essential to identify areas for improvement. This study investigated the incidence, treatment and outcomes, including time trends and predictors for survival, in a nationwide cohort of DCC. Methods: This is a retrospective cohort study of patients diagnosed with DCC (2009-2016) derived from the Netherlands Cancer Registry. Overall survival (OS) and its predictors were analyzed using Kaplan-Meier and Cox regression analysis. Time trends (2009-2012 versus 2013-2016) were assessed. Results: Overall, 1338 patients with DCC were included, with 1-, 3- and 5-year OS of 46%, 18%, and 11%. Incidence of DCC was 0.55-0.90 per 100.000 per year. Median OS was 10.4 months across all stages; 21.9 months for resected (n = 620, 46.3%), 6.7 months for unresected nonmetastatic (n = 445, 33.3%), and 3.6 months for metastatic DCC (n = 273, 20.4%) (p < .001). After resection, 30-day mortality was 4.8% and 90-day mortality 7.7%. Patients with metastatic DCC who received chemotherapy (n = 78, 28.6%) had a median OS of 8.2 versus 2.8 months for those not treated (p < .001). Over time, resection rates (53.6% to 61.7%, p = .008) and use of palliative chemotherapy in metastatic DCC (22.3% to 32.9%, p = .05) increased, without improvement in OS (10.3 vs 10.6 months, p = .55). Independent poor prognostic factors for OS in resected disease were increasing age, pT3/T4 stage, higher lymph node ratio, poor differentiation, and R1 resection. Conclusions: In a nationwide cohort of DCC, resection rates and the use of chemotherapy increased whereas OS remained stable at 10.4 months.