Clinical impact of bile-derived exosomal microRNAs as novel diagnostic and prognostic biomarkers for biliary tract cancers

Sampling of bile juice during endoscopic retrograde cholangiopancreatography (ERCP) has potential benefit of being amenable to the identification of novel biomarkers in liquid biopsy. This study reports the results of a global investigation of exosomal microRNAs (miRNAs) in bile to identify potential biomarkers for biliary tract cancers (BTCs). Eighty-eight bile samples collected during ERCP (45 BTC and 43 noncancer control samples) were enrolled in this study. Eleven BTC samples and nine control samples were assigned as the discovery set. Exosomes in bile and serum samples were collected using a glass membrane column with size-controlled macroporous glass (MPG), and exosomal miRNA expression profiles were evaluated using comprehensive miRNA microarray analysis (3D-Gene). For validation, exosomal miRNA in the bile samples of 34 BTCs and 34 controls were comprehensively evaluated using 3D-Gene. In the discovery set, eight exosomal miRNAs in bile were identified as significant aberrant expression markers, while no miRNA with aberrant expression in serum was identified. In a comparison of the discovery and validation sets, miR-451a and miR-3619-3p were identified as reproducible upregulated markers, and the combination of the two bile miRNAs showed an excellent area under the curve (0.819) value for diagnosing BTCs. In addition, high miR-3619-3p expression in bile reflects poorer prognosis of BTCs (hazard ratio = 2.89). The MPG-extracted exosomal miRNAs in bile aspirated during ERCP provide a convenient new approach for diagnosing biliary diseases. Bile-derived miRNA analysis with miR-451a and miR-3619-3p represents a potentially valuable diagnostic strategy for identifying BTCs as well as a predictive indicator of BTC prognosis.     

Multivariate prognostic factors analysis for second-line chemotherapy in advanced biliary tract cancer

Background:

The role of second-line chemotherapy (CT) is not established in advanced biliary tract cancer (aBTC). We investigated the outcome of aBTC patients treated with second-line CT and devised a prognostic model.

Methods:

Baseline clinical and laboratory data of 300 consecutive aBTC patients were collected and association with overall survival (OS) was investigated by multivariable Cox models.

Results:

The following parameters resulted independently associated with longer OS: Eastern Cooperative Oncology Group performance status of 0 (P<0.001; hazard ratio (HR), 0.348; 95% confidence interval (CI) 0.215–0.562), CA19.9 lower than median (P=0.013; HR, 0.574; 95% CI 0.370–0.891), progression-free survival after first-line CT ⩾6 months (P=0.027; HR, 0.633; 95% CI 0.422–0.949) and previous surgery on primary tumour (P=0.027; HR, 0.609; 95% CI 0.392–0.945). We grouped the 249 patients with complete data available into three categories according to the number of fulfilled risk factors: median OS times for good-risk (zero to one factors), intermediate-risk (two factors) and poor-risk (three to four factors) groups were 13.1, 6.6 and 3.7 months, respectively (P<0.001).

Conclusions:

Easily available clinical and laboratory factors predict prognosis of aBTC patients undergoing second-line CT. This model allows individual patient-risk stratification and may help in treatment decision and trial design.     

Prognostic factors in gallbladder and biliary tract cancer

Background. Cancers of the gallbladder and bile ducts are uncommon neoplasms with poor survival. Prognostic factors are not well defined because of the scant number of patients reported through series of cases. Methods. We reviewed the medical records of patients with cancer of the bile ducts and gallbladder between the years 1979 and 1998, and analyzed their characteristics according to location (gallbladder, extrahepatic biliary tract, intrahepatic biliary tract, and Klatskin tumors). Results. One hundred and sixty-eight patients were included; the mean follow-up time was 238 ± 54 d. The tumor found at more advanced stages was the biliary tract tumor. Overall survival time was 254 ± 40 d. Location did not influence survival. The factors significantly associated to increased survival were age at diagnosis less than 50 yr (p=0.0065), surgical treatment (p<0.001), adjuvant chemotherapy and radiotherapy (p<0.001 and p=0.0072, respectively), surgical treatment with curative purpose (p<0.001), stage of the disease (p<0.0001), absence of jaundice (p=0.0425), and absence of weight loss (p=0.0446). In the multivariate analysis the significant variables were age, surgical treatment, adjuvant chemotherapy, surgery with curative purpose, stage of the disease, and absence of jaundice. Conclusions. Cancers of the bile ducts are neoplasms known to have a poor prognosis. Chemotherapy was an independent survival factor despite the context, there is need of future studies to define its role on this disease.     

Biweekly cisplatin and gemcitabine in patients with advanced biliary tract cancer

Treatment with cisplatin and gemcitabine demonstrates a survival benefit in patients with advanced biliary tract cancer (ABTC). However, the weekly administration can add significant toxicities that may prohibit prolonged treatment. Based on previous studies, we implemented a modified biweekly regimen of GC in an attempt to optimize the prescribed regimen with an improved toxicity profile, added convenience to patients while maintaining efficacy. Patients with ABTC were treated with fixed dose rate (FDR) gemcitabine (1,000 mg/m²/min) and cisplatin 20 mg/m² on days 1 and 15 of every 28‐day cycle. Patients received treatment until time of progression, death, or discontinuation due to intolerance. Collected data included demographics, clinico‐pathologic features, toxicities, and survival. Kaplan‐Meier curves were used to calculate the median overall survival (OS) and progression free survival (PFS). The study included 107 evaluable pts with unresectable ABTC who received the biweekly regimen. Sites of tumor included gallbladder (21.5%), ampullary (3.7%), and bile duct (74.8%). Median number of cycles was 6 (1–27). Median PFS was 8.34 (6.74, 9.23) months and median OS was 10.32 (9.10, 11.43) months. Most common grade ≥3 adverse events included neutropenia (11%), fatigue (10%), and thrombocytopenia (6.4%). Biweekly FDR GC in ABTC is associated with a more favorable toxicity profile while maintaining efficacy similar to that observed in prior clinical trials. Minimal toxicities were observed despite a prolonged course for many patients. Further prospective trials should consider evaluating the role of biweekly GC regimen in ABTC, including a potentially more favorable platform in novel experimental strategies.     

Histological features of lymph node metastasis in patients with biliary tract cancer

BACKGROUND AND OBJECTIVES: The presence of lymph node (LN) metastases is an important prognostic factor in patients with biliary cancers. The aim of this study was to characterize systematically the morphological features of metastatic LNs in biliary cancers. METHODS: Four hundred ninety-six LNs (including 112 para-aortic LNs) dissected from 47 patients with biliary cancer were examined. The diameter of the long axis (size) and the percent metastatic area relative to whole-node area were measured from histologic specimens. RESULTS: The average size of metastatic LNs (9.5 mm) was significantly larger than those without metastasis (6.5 mm; P < 0.01). The optimum cut-off size for positive LNs was >7.5 mm, but the sensitivity of this predictor of metastasis was low (60.8%). In general, metastatic area correlated significantly with the size of metastatic LNs (P = 0.023). Para-aortic LNs contained metastasis in 7.1% of cases, and only 25% of para-aortic LNs with a high ratio of metastatic area could be evaluated from preoperative CT scans. CONCLUSIONS: Although large LNs are highly suggestive of metastasis, poor detection of many small LNs with a low percentage of metastatic area can increase risk in patients with biliary tract cancer.     

Prognostic factors and predictive model in patients with advanced biliary tract adenocarcinoma receiving first‐line palliative chemotherapy

BACKGROUND:

Advanced biliary tract adenocarcinoma (BTA) has been a rare but fatal cancer. If unresectable, palliative chemotherapy improved the quality and length of life, but to the authors' knowledge, prognostic factors in such patients have not been well established to date. In the current study, prognostic factors were investigated in patients with advanced BTA receiving first‐line palliative chemotherapy.

METHODS:

Data from 213 patients with advanced BTA who were in prospective phase 2 or retrospective studies from September 2000 through October 2007 were used.

RESULTS:

With a median follow‐up duration of 29.7 months, the median overall survival (OS) was 7.3 months (95% confidence interval [95% CI], 6.3 months‐8.3 months). A Cox proportional hazards model indicated that metastatic disease (hazards ratio [HR], 1.521; P = .011), intrahepatic cholangiocellular carcinoma (HR, 1.368; P = .045), liver metastasis (HR, 1.845; P < .001), Eastern Cooperative Oncology Group performance status (HR, 1.707; P < .001), and alkaline phosphatase level (IU/L) (HR, 1.001; P < .001) were statistically significant independent predictors of poor prognosis. Patients were classified into 3 risk groups based on the prognostic index (PI), which was constructed using the regression coefficients of each variable. The median OS was 11.5 months (95% CI, 9.6 months‐13.5 months) for the low‐risk group (PI ≤ 1.5; n = 67), 7.3 months (95% CI, 5.7 months‐8.9 months) for the intermediate‐risk group (PI > 1.5 but ≤ 2.2; n = 75), and 3.6 months (95% CI, 2.9 months‐4.1 months) for the high‐risk group (PI > 2.2; n = 70 [P < .001]).

CONCLUSIONS:

Five prognostic factors in patients with advanced BTA were identified. The predictive model based on PI appears to be promising and may be used for the management of individual patients and to guide the design of future clinical trials, although external validation is needed. Cancer 2009. © 2009 American Cancer Society.     

Outpatient chemotherapy with gemcitabine and oxaliplatin in patients with biliary tract cancer

This phase II study was conducted to determine the efficacy and toxicity of a gemcitabine (GEM) and oxaliplatin (OX) chemotherapy protocol in patients with unresectable biliary tract cancer (BTC). Patients were treated with GEM 1000 mg m-2 (30 min infusion) on days 1, 8, 15, and OX 100 mg m-2 (2 h infusion) on days 1 and 15 (gemcitabine and oxaliplatin (GEMOX-3 protocol), repeated every 28 days. The data were collected according to the Simon 2-stage design for a single centre phase II study (alpha=0.05; beta=0.2). Primary end point was response rate; secondary end points were time-to-progression (TTP), median survival, and safety profile. Thirty-one patients were enrolled in the study between July 2002 and April 2005. Therapeutic responses were as follows: partial response in eight patients (26%, 95% confidence interval (CI) 14-44), stable disease in 14 patients (45%, 95%CI 29-62), resulting in a disease control rate of 71%. Nine patients (29%, 95%CI 16-47) had progressive disease. Median TTP was 6.5 months. Median overall survival was 11 months. Common Toxicity Criteria (CTC) Grade 3-4 toxicities were transient thrombocytopenia (23%), peripheral sensory neuropathy (19%), leucopenia (16%), and anaemia (10%). In conclusion the GEMOX-3 protocol is active and well tolerated in patients with advanced BTC. It can be applied in an outpatient setting with three visits per month only.     

Survival of patients with primary liver cancer, pancreatic cancer and biliary tract cancer in Europe

The EUROCARE Study is a European Union project to assemble survival data from population-based cancer registries and analyse them according to standard procedures. We investigated and compared liver, pancreatic and biliary tract cancer survival in 17 countries from 1985 to 1989. Time trends in survival over the 1978–1989 period were also investigated in 12 countries. The overall European mean 1 year relative survival was 16% for primary liver cancer, 26% for biliary tract cancer and 15% for pancreatic cancer. The corresponding 5-year relative survival was 5, 12 and 4%, respectively. Taking the European average as the reference, the relative risk (RR) of death was at least 20% higher for the three cancers in Denmark and Estonia. Survival tended to be higher in Spain for primary liver cancer and biliary tract cancer. Gender had little influence on survival whilst age at diagnosis was inversely related to prognosis. There was an improvement in 1-year relative survival rate for primary liver cancer: relative risk (RR) of 0.68 (95% confidence interval (CI) of 0.60–0.77) for 1987–1989 versus 1978–1980 and biliary tract cancer (RR 0.77, 95% CI 0.68–0.87). There was less variation in 5-year relative survival rate over time. Some intercountry survival differences for primary liver, biliary tract and pancreatic cancers exist over Europe. Differences in quality of care, in particular treatment aggressiveness, may explain some of these differences in survival. New approaches to the management of these cancers need to be found.     

Phase II study of S-1 in patients with advanced biliary tract cancer

The aim of this study was to investigate the efficacy and safety of an oral fluoropyrimidine derivative, S-1, in patients with advanced biliary tract cancer. Patients with pathologically confirmed advanced biliary tract cancer, a measurable lesion, and no history of radiotherapy or chemotherapy were enrolled. S-1 was administered orally (40 mg m(-2) b.i.d.) for 28 days, followed by a 14-day rest period. A pharmacokinetic study was performed on day 1 in the initial eight patients. In all, 19 consecutive eligible patients were enrolled in the study between July 2000 and January 2002. The site of the primary tumour was the gallbladder (n=16), the extrahepatic bile ducts (n=2), and the ampulla of Vater (n=1). A median of two courses of treatment (range, 1-12) was administered. Four patients achieved a partial response, giving an overall response rate of 21.1%. The median time-to-progression and median overall survival period were 3.7 and 8.3 months, respectively. Although grade 3 anorexia and fatigue occurred in two patients each (10.5%), no grade 4 toxicities were observed. The pharmacokinetic parameters after a single oral administration of S-1 were similar to those of patients with other cancers. S-1 exhibits definite antitumour activity and is well tolerated in patients with advanced biliary tract cancer.     

Neutrophil/lymphocyte ratio as a prognostic factor in biliary tract cancer

BackgroundBiliary tract cancers (BTCs) include intrahepatic (IHC), hilar, distal bile duct (DBD) and gallbladder carcinoma (GBC). Neutrophil/lymphocyte ratio (NLR), a marker of host inflammation, is prognostic in several cancers but has not been reviewed in large BTC series, or advanced BTC (ABTC) at diagnosis.Patients and methodsBaseline demographics and NLR at diagnosis were retrospectively evaluated in 864 consecutive patients with BTC treated from January 1987 to December 2012. The association between NLR and overall survival (OS) was determined using a multivariable Cox proportional hazards model.ResultsEight hundred and sixty-four patients were included in the analysis, of which 62% had ABTC and 38% had surgery with curative intent. Median age was 65 years, 444 (51%) were male and 727 (84%) had performance status (PS) ⩽2. A NLR ⩾3.0, PS >2, IHC primary, stage, lack of surgery, haemoglobin <110 g/L and albumin <40 g/L were associated with significantly worse OS on multivariable analysis. A NLR ⩾3.0 was an independent prognostic factor for OS for the entire cohort; median OS was 21.6 months versus 12.0 months for patients with NLR <3.0 versus NLR ⩾3.0 respectively (adjusted hazard ratio (HR)-1.26, 95% confidence interval (CI); 1.06–1.50, P = 0.01). NLR was also prognostic in patients with ABTC (HR-1.26, 95% CI; 1.02–1.56, P = 0.035) and hilar cancer: overall group (N = 149) (HR-1.70, 95% CI; 1.10–2.50, P = 0.01) and advanced group (N = 111) (HR-1.57, 95% CI; 1.04–2.44, P = 0.048).ConclusionBaseline NLR is a readily available and inexpensive prognostic biomarker in patients with BTC and likely warrants validation in large prospective clinical trials.     

Current progress in the clinical use of circulating tumor cells as prognostic biomarkers

The process of metastasis is characterized by the shedding of tumor cells into the bloodstream, where they are transported to other parts of the body to seed new tumors. These cells, known as circulating tumor cells (CTCs), have the potential to reveal much about an individual cancer case, and theoretically can aid in the prediction of outcomes and design of precision treatments. Recent advances in technology now allow for the robust and reproducible characterization of CTCs from a simple blood draw. Both the number of circulating cells and important molecular characteristics correlated with clinical phenotypes such as drug resistance can be obtained and used for real-time prognostic analysis. Molecular characterization can provide a snapshot of the activity of the main tumor (serving as a “liquid biopsy”) and early warnings concerning changes such as the development of resistance, and aid in predicting the efficacy of different therapeutic approaches for treatment optimization. Herein, the authors review the current clinical use of CTCs as prognostic biomarkers for several different cancers. The quantification of CTCs can lead to more accurate staging and decision making regarding options such as adjuvant chemotherapy.     

Gemcitabine alone or in combination with cisplatin in patients with biliary tract cancer: a comparative multicentre study in Japan

Background:

A British randomised study of gemcitabine plus cisplatin (GC) combination showed promising results in biliary tract cancer (BTC) patients. In our study, we evaluated the efficacy and safety of this combination compared with gemcitabine alone (G) in Japanese BTC patients.

Methods:

Overall, 84 advanced BTC patients were randomised to either cisplatin 25 mg m⁻² plus gemcitabine 1000 mg m⁻² on days 1, 8 of a 21-day cycle (GC-arm), or single-agent gemcitabine 1000 mg m⁻² on days 1, 8 and 15 of a 28-day cycle (G-arm). Treatments were repeated for at least 12 weeks until disease progression or unacceptable toxicity occurred, up to a maximum of 48 weeks.

Results:

A total of 83 patients were included in the analysis. For the GC and G-arms, respectively, the 1-year survival rate was 39.0 vs 31.0%, median survival time 11.2 vs 7.7 months, median progression-free survival time 5.8 vs 3.7 months and overall response rate 19.5 vs 11.9%. The most common grade 3 or 4 toxicities (GC-arm/G-arm) were neutropenia (56.1%/38.1%), thrombocytopenia (39.0%/7.1%), leukopenia (29.3%/19.0%), haemoglobin decrease (36.6%/16.7%) and γ-GTP increase (29.3%/35.7%).

Conclusions:

Gemcitabine plus cisplatin combination therapy was found to be effective and well tolerated, suggesting that it could also be a standard regimen for Japanese patients.     

肝外胆道癌分子流行病学研究的新进展

胆道癌是一种生存率极低的高致死性疾病,在世界范围内,胆道癌的发病率呈上升趋势。胆道癌不良的预后源于缺乏早期诊断和有效治疗的手段,因此,明确胆道癌的发病机制显得至关重要。基因多态性的关联分析将有助于阐明胆道癌的发生机制,有望发现有价值的肿瘤标记物以确定胆道癌高危人群并进行早期诊断和预后评估,进而成为基因治疗的新靶点。本文从分子流行病学角度对胆道癌基因多态性方面的最新研究进展进行综述。     

Sorafenib in patients with advanced biliary tract carcinoma: a phase II trial

Background:

Advanced biliary tract carcinoma has a very poor prognosis, with chemotherapy being the mainstay of treatment. Sorafenib, a multikinase inhibitor of VEGFR-2/-3, PDGFR-β, B-Raf, and C-Raf, has shown to be active in preclinical models of cholangiocarcinoma.

Methods:

We conducted a phase II trial of single-agent sorafenib in patients with advanced biliary tract carcinoma. Sorafenib was administered at a dose of 400 mg twice a day. The primary end point was the disease control rate at 12 weeks.

Results:

A total of 46 patients were treated. In all, 26 (56%) had received chemotherapy earlier, and 36 patients completed at least 45 days of treatment. In intention-to-treat analysis, the objective response was 2% and the disease control rate at 12 weeks was 32.6%. Progression-free survival (PFS) was 2.3 months (range: 0–12 months), and the median overall survival was 4.4 months (range: 0–22 months). Performance status was significantly related to PFS: median PFS values for ECOG 0 and 1 were 5.7 and 2.1 months, respectively (P=0.0002). The most common toxicities were skin rash (35%) and fatigue (33%), requiring a dose reduction in 22% of patients.

Conclusions:

Sorafenib as a single agent has a low activity in cholangiocarcinoma. Patients having a good performance status have a better PFS. The toxicity profile is manageable.     

Tumor‐infiltrating neutrophils predict prognosis and adjuvant chemotherapeutic benefit in patients with biliary cancer

Tumor‐infiltrating neutrophils (TIN) carry out quite significant but opposite functions in different cancers, and their function in biliary cancer has not been fully characterized. To investigate the prognostic significance of TIN in biliary cancer, a training set (n = 118) and a validation set (n = 127) were involved in this study. TIN were evaluated by immunohistochemical staining of CD66b, and then defined as low (neutrophils <18/high‐power field [HPF]) vs high (neutrophils ≥18/HPF). Kaplan‐Meier curve, Cox proportional hazards models and receiver operating characteristic curve were used to assess the prognostic significance. TIN was identified as an independent prognostic factor for overall survival in the training set (HR: 4.720; 95% CI: 2.623‐8.493; P < .001) which was confirmed in the validation set (HR: 4.993; 95% CI: 2.626‐9.492; P < .001). Notably, among patients with stage III and IV disease, those with low TIN could benefit from adjuvant chemotherapy, with a reduced risk of compromised survival compared with those with high TIN (HR: 0.294; 95% CI: 0.099‐0.873; P = .047 in the training set; and HR: 0.100; 95% CI: 0.022‐0.462; P = .006 in the validation set). In addition, TIN were negatively related to biological pathways as regulation of activated T‐cell proliferation and lymphocyte‐mediated immunity, and showed a negative correlation with CD8 +  T cells (r = −.324, P < .001). Taken together, our results implicate TIN as an independent marker of prognosis and indicator of patients who would benefit from adjuvant chemotherapy in biliary cancer.     

肝胆疾病生物标本库的建立

目的:建立肝胆系统疾病血液及组织标本库,为肝胆各种疾病在分子水平的研究提供基础。方法:选取2013年7月至2014年4月我院肝胆外科以原发性肝癌、胆囊癌及胆囊结石为主要诊断而行手术切除的患者标本,留取血浆、血清及组织标本,制作石蜡包块并行常规病理组织学鉴定,并定期使用Trizol试剂提取总RNA及基因组DNA以进行标本质量控制。结果:以原发性肝癌为主要诊断者血液标本388例,组织标本67例,以胆囊癌为主要诊断者血液及组织标本78例,术后均进行病理诊断,其中胆囊癌47例,胆管癌31例。此外,本研究也收集胆结石患者的血液标本1154例。结论:本实验建立的肝胆疾病标本采集流程有效可行,临床资料收集完备,能够为今后肝胆肿瘤的发生发展机制研究提供较好的标本资源。