Cholangiocarcinoma combined with biliary obstruction: an exosomal circRNA signature for diagnosis and early recurrence monitoring.

Cholangiocarcinoma (CCA) is a highly malignant biliary tract cancer with currently suboptimal diagnostic and prognostic approaches. We present a novel system to monitor CCA using exosomal circular RNA (circRNA) via serum and biliary liquid biopsies. A pilot cohort consisting of patients with CCA-induced biliary obstruction (CCA-BO, n = 5) and benign biliary obstruction (BBO, n = 5) was used to identify CCA-derived exosomal circRNAs through microarray analysis. This was followed by a discovery cohort (n = 20) to further reveal a CCA-specific circRNA complex (hsa-circ-0000367, hsa-circ-0021647, and hsa-circ-0000288) in both bile and serum exosomes. In vitro and in vivo studies revealed the three circRNAs as promoters of CCA invasiveness. Diagnostic and prognostic models were established and verified by two independent cohorts (training cohort, n = 184; validation cohort, n = 105). An interpreter-free diagnostic model disclosed the diagnostic power of biliary exosomal circRNA signature (Bile-DS, AUROC = 0.947, RR = 6.05) and serum exosomal circRNA signature (Serum-DS, AUROC = 0.861, RR = 4.04) compared with conventional CA19-9 (AUROC = 0.759, RR = 2.08). A prognostic model of CCA undergoing curative-intent surgery was established by calculating early recurrence score, verified with bile samples (Bile-ERS, C-index=0.783) and serum samples (Serum-ERS, C-index = 0.782). These models, combined with other prognostic factors revealed by COX-PH model, enabled the establishment of nomograms for recurrence monitoring of CCA. Our study demonstrates that the exosomal triple-circRNA panel identified in both bile and serum samples serves as a novel diagnostic and prognostic tool for the clinical management of CCA.

Octamer-binding transcription factor 4-positive circulating tumor cell predicts worse treatment response and survival in advanced cholangiocarcinoma patients who receive immune checkpoint inhibitors treatment.

BACKGROUND: Octamer-binding transcription factor 4-positive circulating tumor cell (OCT4+CTC) exhibits high stemness and invasive potential, which may influence the efficacy of immune checkpoint inhibitors (ICI). This study aimed to assess the prognostic role of OCT4+CTC in advanced cholangiocarcinoma (CCA) patients who received ICI treatment. METHODS: In total, 40 advanced CCA patients who received ICI treatment were included, and CTC and OCT4 counts were detected via a Canpatrol system and an RNA in situ hybridization method before ICI treatment. Patients were subsequently divided into none CTC, OCT4-CTC, and OCT4+CTC groups. Patients were followed up for a median of 10.4 months. RESULTS: The percentages of patients in none CTC, OCT4-CTC, and OCT4+CTC groups were 25.0%, 30.0%, and 45.0%, respectively. The proportion of patients with lymph node metastasis was highest in OCT4+CTC group, followed by none CTC group, and lowest in OCT4-CTC group (P = 0.025). The objective response rate (ORR) was lowest in OCT4+CTC group, moderate in OCT4-CTC group, and highest in none CTC group (P = 0.009), while disease control rate was not different among three groups (P = 0.293). In addition, progression-free survival (PFS) (P < 0.001) and overall survival (OS) (P = 0.001) were shorter in the OCT4+CTC group than in none CTC & OCT4-CTC group. Moreover, OCT4+CTC (versus none CTC) was independently linked with poorer PFS [hazard ratio (HR) = 6.752, P = 0.001] and OS (HR = 6.674, P = 0.003) in advanced CCA patients. CONCLUSION: OCT4+CTC relates to lymph node metastasis and shows a good predictive value for poor treatment response and survival in advanced CCA patients who receive ICI treatment.

BCAT1, IKZF1 and SEPT9: methylated DNA biomarkers for detection of pan-gastrointestinal adenocarcinomas.

INTRODUCTION: Methylated circulating tumour DNA (ctDNA) blood tests for BCAT1/IKZF1 (COLVERA) and SEPT9 (Epi proColon) are used to detect colorectal cancer (CRC). However, there are no ctDNA assays approved for other gastrointestinal adenocarcinomas. We aimed to characterize BCAT1, IKZF1 and SEPT9 methylation in different gastrointestinal adenocarcinoma and non-gastrointestinal tumours to determine if these validated CRC biomarkers might be useful for pan-gastrointestinal adenocarcinoma detection. METHODS: Tissue DNA methylation data from colorectal (COAD, READ), gastroesophageal (ESCA, STAD), pancreatic (PAAD) and cholangiocarcinoma (CHOL) adenocarcinoma cohorts within The Cancer Genome Atlas were used for differential methylation analyses. Clinicodemographic predictors of BCAT1, IKZF1 and SEPT9 methylation, and the selectivity of hypermethylated BCAT1, IKZF1 and SEPT9 for colorectal adenocarcinomas in comparison to other cancers were each explored with beta regression. RESULTS: Hypermethylated BCAT1, IKZF1 and SEPT9 were each differentially methylated in colorectal and gastroesophageal adenocarcinomas. IKZF1 was differentially methylated in pancreatic adenocarcinoma. Hypermethylated DNA biomarkers BCAT1, IKZF1 and SEPT9 were largely stable across different stages of disease and were highly selective for gastrointestinal adenocarcinomas relative to other cancer types. DISCUSSION: Existing CRC methylated ctDNA blood tests for BCAT1/IKZF1 and SEPT9 might be usefully repurposed for use in other gastrointestinal adenocarcinomas and warrant further prospective ctDNA studies.

Serum ITIH5 as a novel diagnostic biomarker in cholangiocarcinoma.

Cholangiocarcinoma often remains undetected until advanced stages due to the lack of reliable diagnostic markers. Our goal was to identify a unique secretory protein for cholangiocarcinoma diagnosis and differentiation from other malignancies, benign hepatobiliary diseases, and chronic liver conditions. We conducted bulk RNA-seq analysis to identify genes specifically upregulated in cholangiocarcinoma but not in most other cancers, benign hepatobiliary diseases, and chronic liver diseases focusing on exocrine protein-encoding genes. Single-cell RNA sequencing examined subcellular distribution. Immunohistochemistry and enzyme-linked immunosorbent assays assessed tissue and serum expression. Diagnostic performance was evaluated via receiver-operating characteristic (ROC) analysis. Inter-alpha-trypsin inhibitor heavy chain family member five (ITIH5), a gene encoding an extracellular protein, is notably upregulated in cholangiocarcinoma. This elevation is not observed in most other cancer types, benign hepatobiliary diseases, or chronic liver disorders. It is specifically expressed by malignant cholangiocytes. ITIH5 expression in cholangiocarcinoma tissues exceeded that in nontumorous bile duct, hepatocellular carcinoma, and nontumorous hepatic tissues. Serum ITIH5 levels were elevated in cholangiocarcinoma compared with controls (hepatocellular carcinoma, benign diseases, chronic hepatitis B, and healthy individuals). ITIH5 yielded areas under the ROC curve (AUCs) from 0.839 to 0.851 distinguishing cholangiocarcinoma from controls. Combining ITIH5 with carbohydrate antigen 19-9 (CA19-9) enhanced CA19-9's diagnostic effectiveness. In conclusion, serum ITIH5 may serve as a novel noninvasive cholangiocarcinoma diagnostic marker.

A new biomarker panel for differential diagnosis of cholangiocarcinoma: Results from an exploratory analysis.

INTRODUCTION: Diagnosis of cholangiocarcinoma (CCA) can be challenging due to unclear imaging criteria and difficulty obtaining adequate tissue biopsy. Although serum cancer antigen 19-9 and carcinoembryonic antigen have been proposed as potential diagnostic aids, their use remains limited by insufficient sensitivity and specificity. This exploratory analysis aimed to identify individual- and combinations of serum biomarkers to distinguish CCA from hepatocellular carcinoma (HCC) and chronic liver disease (CLD) controls using samples from a published study. METHODS: This prospective, multicenter, case-control study included patients aged ≥18 years at high-risk of HCC. Serum and ethylene diamine tetraacetic acid-plasma samples were collected prior to any treatment and confirmed diagnosis of HCC or CCA. Fourteen biomarkers (measured by electrochemiluminescence immunoassays or enzyme-linked immunosorbent assays) were subjected to univariate analysis and 13 included in a multivariate analysis (per selected combinations and exhaustive search). RESULTS: Overall, 55 CCA, 306 HCC, and 733 CLD control samples were analyzed. For distinguishing CCA from HCC, alpha-fetoprotein and matrix metalloproteinase-2 (MMP-2) showed the best individual performance (area under the curve (AUC) 86.6% and 84.4%, respectively); tissue inhibitor of metalloproteinase-1 (TIMP-1) was most able to distinguish CCA from CLD (AUC 94.5%) and from HCC + CLD (AUC 88.6%). The combination of MMP-2 and TIMP-1 was the best-performing two-marker panel, with AUC >90% for all comparisons. CONCLUSION: MMP-2 and TIMP-1 are promising biomarkers that could support differential diagnosis of CCA. Incorporating these assays into the diagnostic algorithm could provide additional diagnostic information in a non-invasive, rapid manner, and could supplement existing diagnostic methods.

Monitoring of Dynamic Changes and Clonal Evolution in Circulating Tumor DNA From Patients With IDH-Mutated Cholangiocarcinoma Treated With Isocitrate Dehydrogenase Inhibitors.

PURPOSE: IDH mutations occur in about 30% of patients with cholangiocarcinoma. Analysis of mutations in circulating tumor DNA (ctDNA) can be performed by droplet digital polymerase chain reaction (ddPCR). The analysis of ctDNA is a feasible approach to detect IDH mutations. METHODS: We isolated ctDNA from the blood of patients with IDH-mutated advanced cholangiocarcinoma collected at baseline, on therapy, and at progression to isocitrate dehydrogenase (IDH) inhibitors. RESULTS: Of 31 patients with IDH1R132 (n = 26) or IDH2R172 mutations (n = 5) in the tumor, IDH mutations were detected in 84% of ctDNA samples analyzed by ddPCR and in 83% of ctDNA samples analyzed by next-generation sequencing (NGS). Patients with a low variant allele frequency of ctDNA detected by NGS at baseline had a longer median time to treatment failure compared to patients with high variant allele frequency of ctDNA (3.6 v 1.5 months; P = .008). Patients with a decrease in IDH-mutated ctDNA on therapy by ddPCR compared with no change/increase had a trend to a longer median survival (P = .07). Most frequent emergent alterations in ctDNA by NGS at progression were ARID1A (n = 3) and TP53 mutations (n = 3). CONCLUSION: Detection of IDH mutations in ctDNA in patients with advanced cholangiocarcinoma is feasible, and dynamic changes in ctDNA can correspond with the clinical course and clonal evolution.

Thrombospondin-2 as a diagnostic biomarker for distal cholangiocarcinoma and pancreatic ductal adenocarcinoma.

PURPOSE: Distal cholangiocarcinoma and pancreatic ductal adenocarcinoma are malignancies with poor prognoses that can be difficult to distinguish preoperatively. Thrombospondin-2 has been proposed as a novel diagnostic biomarker for early pancreatic ductal adenocarcinoma. The aim of the present study was to evaluate thrombospondin-2 as a diagnostic and prognostic biomarker in combination with current biomarker CA 19-9 for distal cholangiocarcinoma and pancreatic ductal adenocarcinoma. METHODS: Thrombospondin-2 was measured in prospectively collected serum samples from patients who underwent surgery with a histopathological diagnosis of distal cholangiocarcinoma (N = 51), pancreatic ductal adenocarcinoma (N = 52) and benign pancreatic diseases (N = 27) as well as healthy blood donors (N = 52) using an enzyme-linked immunosorbent assay. RESULTS: Thrombospondin-2 levels (ng/ml) were similar in distal cholangiocarcinoma 55 (41-77) and pancreatic ductal adenocarcinoma 48 (35-80) (P = 0.221). Thrombospondin-2 + CA 19-9 had an area under the curve of 0.92 (95% CI 0.88-0.97) in differentiating distal cholangiocarcinoma and pancreatic ductal adenocarcinoma from healthy donors which was superior to CA 19-9 alone (P < 0.001). The diagnostic value of adding thrombospondin-2 to CA 19-9 was larger in early disease stages. Thrombospondin-2 did not provide additional value to CA 19-9 in differentiating the benign disease group; however, heterogeneity was notable in the benign cohort. Three of five patients with autoimmune pancreatitis patients had greatly elevated thrombospondin-2 levels. Thrombospondin-2 levels had no correlation with prognoses. CONCLUSIONS: Serum thrombospondin-2 in combination with CA 19-9 has potential as a biomarker for distal cholangiocarcinoma and pancreatic cancer.

Prognostic value and predication model of microvascular invasion in patients with intrahepatic cholangiocarcinoma: a multicenter study from China.

BACKGROUND: At present, hepatectomy is still the most common and effective treatment method for intrahepatic cholangiocarcinoma (ICC) patients. However, the postoperative prognosis is poor. Therefore, the prognostic factors for these patients require further exploration. Whether microvascular invasion (MVI) plays a crucial role in the prognosis of ICC patients is still unclear. Moreover, few studies have focused on preoperative predictions of MVI in ICC patients. METHODS: Clinicopathological data of 704 ICC patients after curative resection were retrospectively collected from 13 hospitals. Independent risk factors were identified by the Cox or logistic proportional hazards model. In addition, the survival curves of the MVI-positive and MVI-negative groups before and after matching were analyzed. Subsequently, 341 patients from a single center (Eastern Hepatobiliary Hospital) in the above multicenter retrospective cohort were used to construct a nomogram prediction model. Then, the model was evaluated by the index of concordance (C-Index) and the calibration curve. RESULTS: After propensity score matching (PSM), Child-Pugh grade and MVI were independent risk factors for overall survival (OS) in ICC patients after curative resection. Major hepatectomy and MVI were independent risk factors for recurrence-free survival (RFS). The survival curves of OS and RFS before and after PSM in the MVI-positive groups were significantly different compared with those in the MVI-negative groups. Multivariate logistic regression results demonstrated that age, gamma-glutamyl transpeptidase (GGT), and preoperative image tumor number were independent risk factors for the occurrence of MVI. Furthermore, the prediction model in the form of a nomogram was constructed, which showed good prediction ability for both the training (C-index = 0.7622) and validation (C-index = 0.7591) groups, and the calibration curve showed good consistency with reality. CONCLUSION: MVI is an independent risk factor for the prognosis of ICC patients after curative resection. Age, GGT, and preoperative image tumor number were independent risk factors for the occurrence of MVI in ICC patients. The prediction model constructed further showed good predictive ability in both the training and validation groups with good consistency with reality.

Double-Negative α-Fetoprotein and Carbohydrate Antigen 19-9 Predict a Good Prognosis in Intrahepatic Cholangiocarcinoma: A Propensity Score Matching Analysis.

INTRODUCTION: Carbohydrate antigen 19-9 (CA19-9) and α-fetoprotein (AFP) are routinely tested in patients with liver malignancies before surgery. However, few reports have explored the relevance of the expression pattern of these 2 tumor markers regarding the prognosis of intrahepatic cholangiocarcinoma (ICC). We herein combined these 2 tumor markers to investigate the influence on ICC malignancy and patient prognosis. METHODS: From March 2009 to December 2019, 519 consecutive patients with newly diagnosed ICC who underwent R0 resection were enrolled and followed. The relationships between clinicopathological parameters and these 2 tumor markers were analyzed. Propensity score matching was used to eliminate the baseline differences. RESULTS: A lower proportion of patients with double-negative AFP and CA19-9 had advanced tumor-node-metastasis stage, larger tumor diameter, multiple tumors, lymph node metastasis, microvascular invasion, and perineural invasion. With propensity score matching, patients were divided into double-negative and non-double-negative groups, with 128 patients in each group, and the 5-year recurrence-free survival and overall survival rates were 33.8 vs 15.2 (P < 0.001) and 45.3 vs 19.0, respectively (P < 0.001). In the multivariate Cox analyses, double negativity for the 2 tumor markers was an independent factor for recurrence-free survival (hazard ratios, 0.578; 95% CI, 0.442-0.755, P < 0.001) and overall survival (hazard ratios, 0.567; 95% CI, 0.434-0.741, P < 0.001). DISCUSSION: Double negativity for CA19-9 and AFP indicated less invasive tumor characteristics in patients with ICC. Patients with double-negative tumor markers achieved better outcomes than those with non-double-negative markers, which is meaningful for prognostic counseling and therapeutic triage.

Levels of Circulating PD-L1 Are Decreased in Patients with Resectable Cholangiocarcinoma.

Tumor resection represents the only curative treatment option for patients with biliary tract cancers (BTCs), including intrahepatic cholangiocarcinoma (CCA), perihilar and extrahepatic CCA and gallbladder cancer. However, many patients develop early tumor recurrence and are unlikely to benefit from surgery. Therefore, markers to identify ideal surgical candidates are urgently needed. Circulating programmed cell death 1 ligand 1 (PD-L1) has recently been associated with different malignancies, including pancreatic cancer which closely resembles BTC in terms of patients' prognosis and tumor biology. Here, we aim at evaluating a potential role of circulating PD-L1 as a novel biomarker for resectable BTC. METHODS: Serum levels of PD-L1 were analyzed by ELISA in 73 BTC patients and 42 healthy controls. RESULTS: Circulating levels of preoperative PD-L1 were significantly lower in patients with BTC compared to controls. Patients with low PD-L1 levels displayed a strong trend towards an impaired prognosis, and circulating PD-L1 was negatively correlated with experimental markers of promalignant tumor characteristics such as CCL1, CCL21, CCL25 and CCL26. For 37 out of 73 patients, postoperative PD-L1 levels were available. Interestingly, after tumor resection, circulating PD-L1 raised to almost normal levels. Notably, patients with further decreasing PD-L1 concentrations after surgery showed a trend towards an impaired postoperative outcome. CONCLUSION: Circulating PD-L1 levels were decreased in patients with resectable BTC. Lack of normalization of PD-L1 levels after surgery might identify patients at high risk for tumor recurrence or adverse outcome.

Noninvasive Detection of Polyclonal Acquired Resistance to FGFR Inhibition in Patients With Cholangiocarcinoma Harboring FGFR2 Alterations.

PURPOSE: Fibroblast growth factor receptor (FGFR) 2 alterations, present in 5%-15% of intrahepatic cholangiocarcinomas (IHC), are targets of FGFR-directed therapies. Acquired resistance is common among patients who respond. Biopsies at the time of acquired resistance to targeted agents may not always be feasible and may not capture the genetic heterogeneity that could exist within a patient. We studied circulating tumor DNA (ctDNA) as a less invasive means of potentially identifying genomic mechanisms of resistance to FGFR-targeted therapies. MATERIALS AND METHODS: Serial blood samples were collected from eight patients with FGFR-altered cholangiocarcinoma for ctDNA isolation and next-generation sequencing (NGS) throughout treatment and at resistance to anti-FGFR-targeted therapy. ctDNA was sequenced using a custom ultra-deep coverage NGS panel, incorporating dual index primers and unique molecular barcodes to enable high-sensitivity mutation detection. RESULTS: Thirty-one acquired mutations in FGFR2, 30/31 located in the kinase domain, were identified at resistance in six of eight patients with detectable ctDNA. Up to 13 independent FGFR2 mutations were detected per patient, indicative of striking genomic concordance among resistant subclones. CONCLUSION: ctDNA could be an effective means to longitudinally monitor for acquired resistance in FGFR2-altered IHC. The numerous acquired genetic alterations in FGFR2 suggest frequent polyclonal mechanisms of resistance that cannot be detected from single-site tissue biopsies.

Cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy vs. cytoreductive surgery alone for intrahepatic cholangiocarcinoma with peritoneal metastases: A retrospective cohort study.

BACKGROUND: Cytoreductive surgery (CRS) combined with hyperthermic intraperitoneal chemotherapy (HIPEC) has survival benefits in patients with intraperitoneal malignant lesions, but there is no study specific to intrahepatic cholangiocarcinoma (ICC). PURPOSE: To compare the prognosis of patients with advanced ICC undergoing CRS + HIPEC compared with CRS alone. METHODS: This study was a retrospective cohort study of patients with advanced ICC treated at the Shanghai Eastern Hepatobiliary Surgery Hospital between 01/2014 and 12/2018. The patients were divided into either CRS + HIPEC or CRS group based on the treatment they received. Overall survival (OS), complications, hospital stay, biochemical indicators, tumor markers, and number of HIPEC were examined. RESULTS: There were 51 and 61 patients in the CRS + HIPEC and CRS groups, respectively. There were no differences between the groups regarding preoperative CA19-9 levels (421 ± 381 vs. 523 ± 543 U/mL, P = 0.208). The hospital stay was longer in the CRS + HIPEC group (22.2 ± 10.0 vs. 18.6 ± 7.6 days, P = 0.033). The occurrence of overall complications was similar in the two groups (37.2% vs. 34.4%, P = 0.756). The postoperative CA19-9 levels were lower in the CRS + HIPEC group compared with the CRS group (196 ± 320 vs. 337 ± 396 U/mL, P = 0.044). The median OS was longer in the CRS + HIPEC group than in the CRS group (25.53 vs. 11.17 months, P < 0.001). Compared with the CRS group, the CRS + HIPEC group showed a higher occurrence of leukopenia (7.8% vs. 0, P = 0.040) but a lower occurrence of total bilirubin elevation (15.7% vs. 37.7%, P = 0.032). CONCLUSION: CRS + HIPEC could be a treatment option for patients with advanced ICC, with improved OS and similar complications and adverse events compared with CRS alone.

A Novel Serum Glycobiomarker for Diagnosis and Prognosis of Cholangiocarcinoma Detected by Butea monosperma Agglutinin.

Plant lectins are widely used in medical glycosciences and glycotechnology. Many lectin-based techniques have been applied for the detection of disease-associated glycans and glycoconjugates. In this study, Butea monosperma agglutinin (BMA), a lectin purified from seeds of the medicinal plant Butea monosperma, was used for the detection of cholangiocarcinoma (CCA)-associated glycans. Expression of BMA-binding N-acetyl galactosamine/galactose (GalNAc/Gal)-associated glycan (BMAG) in CCA tissues was determined using BMA lectin histochemistry; the results showed that BMAG was undetectable in normal bile ducts and drastically increased in preneoplastic bile ducts and CCA. The study in hamsters showed that an increase of BMAG was associated with carcinogenesis of CCA. Using an in-house double BMA sandwich enzyme-linked lectin assay, BMAG was highly detected in the sera of CCA patients. The level of serum BMAG in CCA patients (N = 83) was significantly higher than non-CCA controls (N = 287) and it was applicable for diagnosis of CCA with 55.4% sensitivity, 81.9% specificity, and 76.0% accuracy. A high level of serum BMAG (≥82.5 AU/mL) was associated with unfavorable survival of CCA patients; this information suggested the potential of serum BMAG as a poor prognostic indicator of CCA. In summary, BMAG was aberrantly expressed in preneoplastic bile ducts and CCA, it was also highly detected in patient serum which potentially used as a marker for diagnosis and prognostic prediction of CCA.

The prognostic value of sarcopenia combined with preoperative fibrinogen-albumin ratio in patients with intrahepatic cholangiocarcinoma after surgery: A multicenter, prospective study.

BACKGROUND: To explore the prognostic value of the fibrinogen-albumin ratio (FAR) combined with sarcopenia in intrahepatic cholangiocarcinoma (ICC) patients after surgery and to develop a nomogram for predicting the survival of ICC patients. MATERIALS AND METHODS: In this prospective cohort study, 116 ICC patients who underwent radical surgery were enrolled as the discovery cohort and another independent cohort of 68 ICC patients was used as the validation cohort. Kaplan-Meier method was used to analyze prognosis. The independent predictor of overall survival (OS) and recurrence-free survival (RFS) was evaluated by univariable and multivariable Cox regression analyses, then developing nomograms. The performance of nomograms was evaluated by concordance index (C-index), calibration curve, receiver operating characteristic curve analysis (ROC), and decision curve analysis (DCA). RESULTS: Patients with high FAR had lower OS and RFS. FAR and sarcopenia were effective predictors of OS and RFS. Patients with high FAR and sarcopenia had a poorer prognosis than other patients. OS nomogram was constructed based on age, FAR, and sarcopenia. RFS nomogram was constructed based on FAR and sarcopenia. C-index for the nomograms of OS and RFS was 0.713 and 0.686. Calibration curves revealed great consistency between actual survival and nomogram prediction. The area under ROC curve (AUC) for the nomograms of OS and RFS was 0.796 and 0.791 in the discovery cohort, 0.823 and 0.726 in the validation cohort. The clinical value of nomograms was confirmed by the DCA. CONCLUSIONS: ICC patients with high FAR and sarcopenia had a poor prognosis, the nomograms developed based on these two factors were accurate and clinically useful in ICC patients who underwent radical resection.

Prognostic biomarkers for cholangiocarcinoma (CCA): state of the art.

Introduction:Although advances in understanding the molecular basis of cholangiocarcinoma (CCA) have been made, surgery is the only curative therapy option and the overall prognosis of patients suffering from the disease remains poor. Therefore, estimation of prognosis based on known and novel biomarkers is essential for therapy guidance of CCA in both, curative and palliative settings.Areas covered:An extensive literature search on biomarkers for CCA with special emphasis on prognosis was performed. Based on this, prognostic biomarkers from serum, tumor tissue and other compartments that are currently in use or under evaluation for CCA were summarized in this review. Furthermore, an overview of new biomarkers was provided including those determined from extracellular vesicles (EVs), metabolites and nucleic acids. Finally, prognostic markers associated with potential new therapy options for the treatment of CCA were summed up.Expert opinion:So far, an optimal prognostic biomarker for CCA has not been described. However, based on the increasing knowledge about the molecular basis of CCA but also due to novel, innovative technologies, a plethora of novel prognostic biomarkers is currently under evaluation and will be available for CCA in future.

The prognostic impact of lymphocyte-to-C-reactive protein score in patients undergoing surgical resection for intrahepatic cholangiocarcinoma: A comparative study of major representative inflammatory / immunonutritional markers.

BACKGROUND: In many malignancies including intrahepatic cholangiocarcinoma (iCCA), prognostic significance of host-related inflammatory / immunonutritional markers have attracted a lot of attention. However, it is unclear which is the strongest prognostic indicator for iCCA among these markers. The aim of this study was to firstly evaluate the prognostic utility of inflammatory / immunonutritional markers in resected iCCA patients using a multiple comparison in addition to a new marker, lymphocyte-to-C-reactive protein (CRP) score. METHODS: A total of sixty iCCA patients, who underwent surgical resection between October 2004 and April 2019, were enrolled in this study. Their clinical and pathological data were retrospectively assessed using univariate and multivariate analysis to determine prognostic predictors for disease specific survival (DSS). Moreover, these patients, who were divided into high and low groups based on lymphocyte-to-CRP score, were compared these survival outcomes using Kaplan-Meier analysis with a log-rank test. RESULTS: In multivariate analysis, the significant prognostic factors were preoperative lymphocyte-to-CRP score (p = 0.008), preoperative CRP-to-albumin ratio (CAR; p = 0.017), pathological T category (p = 0.003), and pathological vascular invasion (p < 0.001). Resected iCCA patients with a low lymphocyte-to-CRP score (score 0) had significant better prognosis than patients with a high score (score 1 or 2) (p = 0.016). Notably, the mortality of the high lymphocyte-to-CRP score group did not show statistically difference from the poor mortality of unresected iCCA patients (p = 0.204). CONCLUSIONS: Preoperative lymphocyte-to-CRP score was the strongest prognostic indicator in iCCA patients with surgical resection. In these patients, early intervention with nutritional support should be considered prior to operation.

Identification of Circulating Serum miRNAs as Novel Biomarkers in Pancreatic Cancer Using a Penalized Algorithm.

Pancreatic cancer (PC) is difficult to detect in the early stages; thus, identifying specific and sensitive biomarkers for PC diagnosis is crucial, especially in the case of early-stage tumors. Circulating microRNAs are promising non-invasive biomarkers. Therefore, we aimed to identify non-invasive miRNA biomarkers and build a model for PC diagnosis. For the training model, blood serum samples from 63 PC patients and 63 control subjects were used. We selected 39 miRNA markers using a smoothly clipped absolute deviation-based penalized support vector machine and built a PC diagnosis model. From the double cross-validation, the average test AUC was 0.98. We validated the diagnosis model using independent samples from 25 PC patients and 81 patients with intrahepatic cholangiocarcinoma (ICC) and compared the results with those obtained from the diagnosis using carbohydrate antigen 19-9. For the markers miR-155-5p, miR-4284, miR-346, miR-7145-5p, miR-5100, miR-661, miR-22-3p, miR-4486, let-7b-5p, and miR-4703-5p, we conducted quantitative reverse transcription PCR using samples from 17 independent PC patients, 8 ICC patients, and 8 healthy individuals. Differential expression was observed in samples from PC patients. The diagnosis model based on the identified markers showed high sensitivity and specificity for PC detection and is potentially useful for early PC diagnosis.

Carcinoembryonic antigen, α-fetoprotein, and Ki67 as biomarkers and prognostic factors in intrahepatic cholangiocarcinoma: A retrospective cohort study.

INTRODUCTION AND OBJECTIVE: The purpose of this study was to investigate the expression levels and prognostic roles of α-fetoprotein (AFP), carcinoembryonic antigen (CEA), and Ki67 in tumor tissues of intrahepatic cholangiocarcinoma (ICC) patients. PATIENTS OR MATERIALS AND METHODS: The study involved ninety-two ICC patients with complete clinicopathological data and follow-up information, who had previously undergone radical surgery. AFP, CEA, CD10, CD34, and Ki67 were detected in tumor tissues using immunohistochemistry. Statistical tests were used to identify independent risk factors and their associations with overall survival (OS) and disease-free survival (DFS). RESULTS: AFP, CEA and Ki67 were strongly correlated with prognosis. Univariate analysis indicated that higher AFP (P = 0.002), CEA (P < 0.0001), Ki67 (P < 0.0001), CA19-9 (P = 0.039), and CA12-5 (P = 0.002), and larger tumor size (P = 0.001), as well as more advanced tumor node metastasis (TNM) staging (P < 0.0001) were all associated with worse OS. Meanwhile, higher AFP (P = 0.002), CEA (P = 0.001), and Ki67 (P < 0.0001), as well as more advanced TNM staging (P = 0.005) were associated with worse DFS. Multivariate analysis showed that higher AFP (HR = 2.004, 95%CI: 1.146-3.504 P = 0.015), CEA (HR = 2.226, 95%CI: 1.283-3.861 P = 0.004), and Ki67 (HR = 3.785, 95%CI: 2.073-6.909 P < 0.0001), as well as more advanced TNM staging (HR = 2.900, 95%CI: 1.498-5.757 P = 0.002) had associations with worse OS. Furthermore, higher AFP (HR = 2.172, 95%Cl: 1.291-3.654 P = 0.003), CEA (HR = 1.934, 95%Cl: 1.180-3.169 P = 0.009), and Ki67 (HR = 2.203, 95%Cl: 1.291-3.761 P = 0.004) had associations with worse DFS. CONCLUSION: High AFP, CEA, and Ki67 are significant prognostic indicators in ICC patients, and can be used to evaluate ICC biological behavior and prognosis.

A Circular RNA, Cholangiocarcinoma-Associated Circular RNA 1, Contributes to Cholangiocarcinoma Progression, Induces Angiogenesis, and Disrupts Vascular Endothelial Barriers.

BACKGROUND AND AIMS: Circular RNAs (circRNAs) and extracellular vesicles (EVs) are involved in various malignancies. We aimed to clarify the functions and mechanisms of dysregulated circRNAs in the cells and EVs of cholangiocarcinoma (CCA). APPROACH AND RESULTS: CircRNA microarray was used to identify circRNA expression profiles in CCA tissues and bile-derived EVs (BEVs). CCA-associated circRNA 1 (circ-CCAC1) expression was measured by quantitative real-time PCR. The clinical importance of circ-CCAC1 was analyzed by receiver operating characteristic curves, Fisher's exact test, Kaplan-Meier plots, and Cox regression model. The functions of circ-CCAC1 and exosomal circ-CCAC1 were explored in CCA cells and human umbilical vein endothelial cells (HUVECs), respectively. Different animal models were used to verify the in vitro results. RNA sequencing, bioinformatics, RNA immunoprecipitation, RNA pulldown, chromatin immunoprecipitation followed by sequencing, and luciferase reporter assays were used to determine the regulatory networks of circ-CCAC1 in CCA cells and HUVECs. Circ-CCAC1 levels were increased in cancerous bile-resident EVs and tissues. The diagnostic and prognostic values of circ-CCAC1 were identified in patients with CCA. For CCA cells, circ-CCAC1 increased cell progression by sponging miR-514a-5p to up-regulate Yin Yang 1 (YY1). Meanwhile, YY1 directly bound to the promoter of calcium modulating ligand to activate its transcription. Moreover, circ-CCAC1 from CCA-derived EVs was transferred to endothelial monolayer cells, disrupting endothelial barrier integrity and inducing angiogenesis. Mechanistically, circ-CCAC1 increased cell leakiness by sequestering enhancer of zeste homolog 2 in the cytoplasm, thus elevating SH3 domain-containing GRB2-like protein 2 expression to reduce the levels of intercellular junction proteins. In vivo studies further showed that increased circ-CCAC1 levels in circulating EVs and cells accelerated both CCA tumorigenesis and metastasis. CONCLUSIONS: Circ-CCAC1 plays a vital role in CCA tumorigenesis and metastasis and may be an important biomarker/therapeutic target for CCA.

Analysis of bile acid profile in plasma to differentiate cholangiocarcinoma from benign biliary diseases and healthy controls.

Bile acids (BAs) are currently considered as causative agents for Cholangiocarcinoma (CCA). However, the profile of circulating BAs in CCA have not been well characterized. The aim of this study was to describe the alterations of BAs metabolism in patients with CCA compared to benign biliary diseases (BBD) and healthy controls (HC), and to discover the specific BAs as biomarkers for CCA diagnosis. The concentrations of 15 BAs in plasma were measured in a total of 329 subjects, including patients with BBD, CCA, gallbladder cancer (GC), hepatocellular carcinoma (HCC), and healthy subjects, using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Binary logistic regression analysis was used to build a diagnostic model for CCA. An imbalance in the ratio of conjugated to unconjugated BAs was observed in CCA patients compared to BBD and HC groups, with higher conjugated BAs and lower unconjugated BAs. A panel of 2 BA metabolites consisting of CDCA and TCDCA showed high diagnostic performance for CCA versus BBD and CCA versus HC, with higher AUC, sensitivity and specificity than carbohydrate antigen 19-9 (CA 199), clinically employed CCA biomarker. Importantly, HCC and GC samples were also included to confirm specificity of the BA biomarkers for CCA diagnosis. In summary, specific changes in plasma concentrations of BAs may serve as diagnostic biomarkers for distinguishing CCA from BBD and HC, with higher performance than CA199.