Single-shot multi-channel plasmonic real-time polymerase chain reaction for multi-target point-of-care testing.

Plasmonic nucleic acid amplification tests demand high-throughput and multi-target detection of infectious diseases as well as short turnaround time and small size for point-of-care molecular diagnostics. Here, we report a multi-channel plasmonic real-time reverse-transcription polymerase chain reaction (mpRT-qPCR) assay for ultrafast and on-chip multi-target detection. The mpRT-qPCR system features two pairs of plasmonic thermocyclers for rapid nanostructure-driven amplification and microlens array fluorescence microscopes for in situ multi-color fluorescence quantification. Each channel shows a physical dimension of 32 mm, 75 mm, and 25 mm in width, length, and thickness. The ultrathin microscopes simultaneously capture four different fluorescence images from two PCR chambers of a single cartridge at a single shot exposure per PCR cycle of four different excitation light sources. The experimental results demonstrate a single assay result of high-throughput amplification and multi-target quantification for RNA-dependent RNA polymerase, nucleocapsid, and human ribonuclease P genes in SARS-CoV-2 RNA detection. The mpRT-PCR increases the number of tests four times over the single RT-PCR and exhibits a short detection time of 15 min for the four RT-PCR reactions. This point-of-care molecular diagnostic platform can reduce false negative results in clinical applications of virus detection and decentralize healthcare facilities with limited infrastructure.

Highly Efficient On-Chip Photothermal Cell Lysis for Nucleic Acid Extraction Using Localized Plasmonic Heating of Strongly Absorbing Au Nanoislands.

Cell lysis serves as an essential role in the sample preparation for intracellular material extraction in lab-on-a-chip applications. However, recent microfluidic-based cell lysis chips still face several technical challenges such as reagent removal, complex design, and high fabrication cost. Here, we report highly efficient on-chip photothermal cell lysis for nucleic acid extraction using strongly absorbed plasmonic Au nanoislands (SAP-AuNIs). The highly efficient photothermal cell lysis chip (HEPCL chip) consists of a PDMS microfluidic chamber and densely distributed SAP-AuNIs with large diameters and small nanogaps, allowing for broad-spectrum light absorption. The SAP-AuNIs induce photothermal heat, resulting in a uniform temperature distribution within the chamber and rapidly reaching the target temperature for cell lysis within 30 s. Furthermore, the localized plasmonic heating of SAP-AuNIs expeditiously triggers phase transition and photoporation in the directly contacted lipid bilayer of the cell membrane, resulting in rapid and highly efficient cell lysis. The HEPCL chip successfully lysed 93% of PC9 cells at 90 °C for 90 s without nucleic acid degradation. This on-chip cell lysis offers a new sample preparation platform for integrated point-of-care molecular diagnostics.

Ultrafast Plasmonic Nucleic Acid Amplification and Real-Time Quantification for Decentralized Molecular Diagnostics.

Point-of-care real-time reverse-transcription polymerase chain reaction (RT-PCR) facilitates the widespread use of rapid, accurate, and cost-effective near-patient testing that is available to the public. Here, we report ultrafast plasmonic nucleic acid amplification and real-time quantification for decentralized molecular diagnostics. The plasmonic real-time RT-PCR system features an ultrafast plasmonic thermocycler (PTC), a disposable plastic-on-metal (PoM) cartridge, and an ultrathin microlens array fluorescence (MAF) microscope. The PTC provides ultrafast photothermal cycling under white-light-emitting diode illumination and precise temperature monitoring with an integrated resistance temperature detector. The PoM thin film cartridge allows rapid heat transfer as well as complete light blocking from the photothermal excitation source, resulting in real-time and highly efficient PCR quantification. Besides, the MAF microscope exhibits close-up and high-contrast fluorescence microscopic imaging. All of the systems were fully packaged in a palm size for point-of-care testing. The real-time RT-PCR system demonstrates the rapid diagnosis of coronavirus disease-19 RNA virus within 10 min and yields 95.6% of amplification efficiency, 96.6% of classification accuracy for preoperational test, and 91% of total percent agreement for clinical diagnostic test. The ultrafast and compact PCR system can decentralize point-of-care molecular diagnostic testing in primary care and developing countries.

Ultrafast and Real-Time Nanoplasmonic On-Chip Polymerase Chain Reaction for Rapid and Quantitative Molecular Diagnostics.

Advent and fast spread of pandemic diseases draw worldwide attention to rapid, prompt, and accurate molecular diagnostics with technical development of ultrafast polymerase chain reaction (PCR). Microfluidic on-chip PCR platforms provide highly efficient and small-volume bioassay for point-of-care diagnostic applications. Here we report ultrafast, real-time, and on-chip nanoplasmonic PCR for rapid and quantitative molecular diagnostics at point-of-care level. The plasmofluidic PCR chip comprises glass nanopillar arrays with Au nanoislands and gas-permeable microfluidic channels, which contain reaction microchamber arrays, a precharged vacuum cell, and a vapor barrier. The on-chip configuration allows both spontaneous sample loading and microbubble-free PCR reaction during which the plasmonic nanopillar arrays result in ultrafast photothermal cycling. After rapid sample loading less than 3 min, two-step PCR results for 40 cycles show rapid amplification in 264 s for lambda-DNA, and 306 s for plasmids expressing SARS-CoV-2 envelope protein. In addition, the in situ cyclic real-time quantification of amplicons clearly demonstrates the amplification efficiencies of more than 91%. This PCR platform can provide rapid point-of-care molecular diagnostics in helping slow the fast-spreading pandemic.

Development and Validation of a Simple Index Based on Non-Enhanced CT and Clinical Factors for Prediction of Non-Alcoholic Fatty Liver Disease.

OBJECTIVE: A widely applicable, non-invasive screening method for non-alcoholic fatty liver disease (NAFLD) is needed. We aimed to develop and validate an index combining computed tomography (CT) and routine clinical data for screening for NAFLD in a large cohort of adults with pathologically proven NAFLD. MATERIALS AND METHODS: This retrospective study included 2218 living liver donors who had undergone liver biopsy and CT within a span of 3 days. Donors were randomized 2:1 into development and test cohorts. CTL-S was measured by subtracting splenic attenuation from hepatic attenuation on non-enhanced CT. Multivariable logistic regression analysis of the development cohort was utilized to develop a clinical-CT index predicting pathologically proven NAFLD. The diagnostic performance was evaluated by analyzing the areas under the receiver operating characteristic curve (AUC). The cutoffs for the clinical-CT index were determined for 90% sensitivity and 90% specificity in the development cohort, and their diagnostic performance was evaluated in the test cohort. RESULTS: The clinical-CT index included CTL-S, body mass index, and aspartate transaminase and triglyceride concentrations. In the test cohort, the clinical-CT index (AUC, 0.81) outperformed CTL-S (0.74; p < 0.001) and clinical indices (0.73-0.75; p < 0.001) in diagnosing NAFLD. A cutoff of ≥ 46 had a sensitivity of 89% and a specificity of 41%, whereas a cutoff of ≥ 56.5 had a sensitivity of 57% and a specificity of 89%. CONCLUSION: The clinical-CT index is more accurate than CTL-S and clinical indices alone for the diagnosis of NAFLD and may be clinically useful in screening for NAFLD.

CT of acute rejection after liver transplantation: a matched case-control study.

PURPOSE: This study was conducted in order to investigate computed tomography (CT) findings associated with acute cellular rejection (ACR) following liver transplantation (LT) and their relevance to clinical outcomes. MATERIALS AND METHODS: We analyzed 120 patients with newly diagnosed ACR following LT for various liver diseases and 119 controls matched for age, sex, type of liver graft, and date of CT exam following LT. Two radiologists analyzed the images for morphological characteristics of the graft, morphological change in the major draining vein, graft enhancement in the portal venous phase, graft attenuation on noncontrast CT, and periportal halo. Univariate analysis was used to determine the association between radiological findings and ACR. Clinical outcomes, including treatment response and graft survival, were compared between patients with and without associated radiological findings. RESULTS: Morphological characteristics of the graft (i.e., globular swelling), morphological change in the major draining vein (i.e., nonanastomotic luminal narrowing), and heterogeneous enhancement were significantly associated with ACR (all p < 0.001). On univariate analysis, the severity of morphological characteristics of the grafts (mild/severe: odds ratio [OR], 19.98/32.24) and morphological change in the major draining vein (without/with prestenotic dilatation: OR, 4.17/22.5) were significantly associated with the increased possibility of an ACR diagnosis. Clinical outcomes for treatment response and graft survival were not significantly different between patients with and without associated radiological findings. CONCLUSIONS: Globular swelling, nonanastomotic stenosis with or without prestenotic dilatation of the major draining vein, and heterogeneous enhancement of the graft on portal venous-phase CT were significantly associated with ACR. KEY POINTS: • Globular swelling of the graft, nonanastomotic narrowing in the major vein, and heterogeneous graft enhancement on CT were significantly associated with acute cellular rejection (ACR). • Associated CT findings were highly specific but not sensitive for differentiating ACRs from matched controls.

CT indices for the diagnosis of hepatic steatosis using non-enhanced CT images: development and validation of diagnostic cut-off values in a large cohort with pathological reference standard.

OBJECTIVES: To compare the performances of CT indices for diagnosing hepatic steatosis (HS) and to determine and validate the CT index cut-off values. METHODS: Three indices were measured on non-enhanced CT images of 4413 living liver donor candidates (2939 men, 1474 women; mean age, 31.4 years): hepatic attenuation (CTL), hepatic attenuation minus splenic attenuation (CTL-S), and hepatic attenuation divided by splenic attenuation (CTL/S). The performances of these CT indices in diagnosing HS, relative to pathologic diagnosis, were compared in the development cohort of 3312 subjects by receiver operating characteristic (ROC) analysis. The cut-off values for diagnosing HS > 33% in the development cohort were determined at 95% specificity and 95% sensitivity using bootstrap ROC analysis, and the diagnostic performance of these cut-off values was validated in the test cohort of 1101 subjects. RESULTS: CTL-S showed the highest performance for diagnosing HS ≥ 5% and HS > 33% (areas under the curve (AUCs) = 0.737 and 0.926, respectively), followed by CTL/S (AUCs = 0.732 and 0.925, respectively) and CTL (AUCs = 0.707 and 0.880, respectively). For CT scans using 120 kVp, the CTL-S cut-off values for highly specific (i.e., - 2.1) and highly sensitive (i.e., 7.6) diagnosis of HS > 33% resulted in a specificity of 96.4% with a sensitivity of 64.0% and a sensitivity of 97.3% with a specificity of 54.9%, respectively, in the test cohort. CONCLUSION: CT indices using liver and spleen attenuations have higher performance for diagnosing HS than indices using liver attenuation alone. The CTL-S cut-off values in this study may have utility for diagnosing HS in clinical practice and research. KEY POINTS: • CT indices based on both liver attenuation and spleen attenuation (CTL-Sand CTL/S) have higher diagnostic performance than CTLbased on liver attenuation alone in diagnosing HS using various CT techniques. • The CT index cut-off values determined in this study can be utilized for reliable diagnosis or to rule out subjects with moderate to severe HS in clinical practice and research, including the selection of living liver donors and the development of cohorts with HS or healthy controls.

Development and Validation of a Deep Learning System for Staging Liver Fibrosis by Using Contrast Agent-enhanced CT Images in the Liver.

Purpose To develop and validate a deep learning system (DLS) for staging liver fibrosis by using CT images in the liver. Materials and Methods DLS for CT-based staging of liver fibrosis was created by using a development data set that included portal venous phase CT images in 7461 patients with pathologically confirmed liver fibrosis. The diagnostic performance of the DLS was evaluated in separate test data sets for 891 patients. The influence of patient characteristics and CT techniques on the staging accuracy of the DLS was evaluated by logistic regression analysis. In a subset of 421 patients, the diagnostic performance of the DLS was compared with that of the radiologist's assessment, aminotransferase-to-platelet ratio index (APRI), and fibrosis-4 index by using the area under the receiver operating characteristic curve (AUROC) and Obuchowski index. Results In the test data sets, the DLS had a staging accuracy of 79.4% (707 of 891) and an AUROC of 0.96, 0.97, and 0.95 for diagnosing significant fibrosis (F2-4), advanced fibrosis (F3-4), and cirrhosis (F4), respectively. At multivariable analysis, only pathologic fibrosis stage significantly affected the staging accuracy of the DLS (P = .016 and .013 for F1 and F2, respectively, compared with F4), whereas etiology of liver disease and CT technique did not. The DLS (Obuchowski index, 0.94) outperformed the radiologist's interpretation, APRI, and fibrosis-4 index (Obuchowski index range, 0.71-0.81; P ˂ .001) for staging liver fibrosis. Conclusion The deep learning system allows for accurate staging of liver fibrosis by using CT images. © RSNA, 2018 Online supplemental material is available for this article.

Downregulation of Raf-1 kinase inhibitory protein as a sorafenib resistance mechanism in hepatocellular carcinoma cell lines.

PURPOSE: Although sorafenib enhances overall survival, sorafenib resistance has been reported to be a significant limiting factor for improved prognosis in patients with hepatocellular carcinoma (HCC). Therefore, it is important to identify the mechanism of sorafenib resistance. This study aimed to identify the causative factor of sorafenib resistance and suggest methods for overcoming it. METHODS: The sensitivity to sorafenib was compared in human HCC cell lines and patient-derived HCC primary cells. Based on its cytotoxicity, signaling pathways altered by sorafenib and the causative factors were examined through assays. The mechanism by which sorafenib modified the sorafenib-resistance inducer through gene or protein expression or stability was also investigated. We also designed a treatment option to overcome sorafenib resistance. RESULTS: Sorafenib activated the Raf/MEK/ERK pathway and caused sorafenib resistance in HCC cell lines and patient-derived HCC primary cells. Sorafenib reactivated the MAPK pathway by down-regulating RKIP at the post-translational level. Knockdown of RKIP increased phosphorylated ERK and thus suppressed sorafenib-mediated cell death. We also found that sorafenib-reactivated ERK maybe an attractive target for second-line therapy for patients with sorafenib resistance. Sequential combination treatment with sorafenib and PD98059 significantly reduced the viability and proliferation of sorafenib-resistant cells, while their increasing apoptosis efficacy. CONCLUSION: Reactivation of the Raf/MEK/ERK pathway through aberrant expression of RKIP is one of the mechanisms behind sorafenib resistance in HCC. Sequential combination treatment with sorafenib and PD98059 could provide a new strategy to overcome sorafenib resistance in future clinical studies.

Clinicopathological Features and Prognosis of Intrahepatic Cholangiocarcinoma After Liver Transplantation and Resection.

BACKGROUND Intrahepatic cholangiocarcinoma (ICC) can be incidentally diagnosed after liver transplantation (LT). We investigated the clinicopathological features of LT recipients with ICC and compared prognosis with that of the control group. MATERIAL AND METHODS We identified 16 recipients with ICC in our institutional database. The propensity score-matched control group comprised 100 ICC patients who underwent hepatic resection (HR). RESULTS ICC incidence was 0.5% in all adult LT patients and 1.2% in adult recipients with primary liver cancer. Mean age was 58.0±4.8 years and 15 were male. All ICCs were diagnosed incidentally in the explanted livers. Mean ICC tumor diameter was 2.5±1.1 cm and 14 recipients had a single tumor. Tumor stages were I in 9, II in 5, and IV in 2. Concurrent second primary liver cancer was detected as hepatocellular carcinoma in 7 and combined hepatocellular carcinoma-cholangiocarcinoma in 1. Tumor recurrence and patient survival rates were 56.2% and 81.3% at 1 year and 78.1% and 52.4% at 5 years, respectively. Presence of second cancer did not affect tumor recurrence (p=0.959) or patient survival (p=0.737). All 3 patients with very early ICC did not show ICC recurrence. Compared with the control group, the tumor recurrence rate was higher after LT (p=0.024), but this difference disappeared after analysis was confined to recipients with ICC alone (p=0.121). Post-recurrence survival was not different after HR and LT (p=0.082). CONCLUSIONS ICC is rarely diagnosed after LT and half of such patients have second liver cancer. Post-transplant prognosis of ICC is poor except for very early ICC; thus, strict surveillance is mandatory.

Post-resection Prognosis of Combined Hepatocellular Carcinoma-Cholangiocarcinoma According to the 2010 WHO Classification.

BACKGROUND: Combined hepatocellular carcinoma and cholangiocarcinoma (cHC) has wide histological diversity. We intended to investigate the prognostic influence of tumor types of cHC. METHODS: We analyzed the clinical and pathological features of cHC along 2010 WHO classification. Study group was 100 cHC patients who underwent primary resection. Control group comprised 200 propensity score-matched patients with intrahepatic cholangiocarcinoma (ICC). RESULTS: In cHC group, tumor diameter was 4.4 ± 2.8 cm and 95 patients had single tumor. They were classified as classical type in 46 and subtypes with stem cell (SC) features in 54. Subtypes with SC features included typical in 16, intermediate cell in 22, and cholangiolocellular in 16. Their 1- and 3-year tumor recurrence rates were 31.7 and 59.8%; and 1- and 3-year patient survival rates were 92.5 and 77.3%, respectively. Tumor recurrence (p = 0.008) and patient survival (p = 0.005) rates were different according to tumor types. Further stratification by subtypes with SC features resulted in prognostic stratification in tumor recurrence (p = 0.045) and patient survival (p = 0.042). However, tumor stage was the only independent risk factor for tumor recurrence and patient survival. Comparing with ICC control group, cHC group showed no significant difference in rates of tumor recurrence (p = 0.523), but better survival outcomes (p = 0.008). Median post-recurrence patient survival period was 20 months in cHC patients and 6 months in ICC patients (p = 0.001). CONCLUSIONS: Our results indicated that there would be close relationship between the post-resection prognosis and histological types according to the 2010 WHO classification, but these histological types did not become an independent prognostic factor.

Clinicopathological Features and Post-resection Prognosis of Double Primary Hepatocellular Carcinoma and Intrahepatic Cholangiocarcinoma.

BACKGROUND: Simultaneous double primary hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) (dpHCC-ICC) is very rare. This study investigated the clinicopathological features and post-resection prognosis of dpHCC-ICC. METHODS: We identified 10 patients with dpHCC-ICC through an institutional database search. Three control groups with HCC, ICC and combined HCC-cholangiocarcinoma (cHCC-CC) were selected (each n = 120) using propensity score matching. RESULTS: The incidence of dpHCC-ICC was 0.23%. The mean age was 57.4 ± 11.7 years, and 8 were male. Hepatitis B virus infection was associated with 8 patients. All dpHCC-ICC were diagnosed incidentally from surgical specimens. Only two patients demonstrated simultaneous elevation of alpha-fetoprotein/des-γ-carboxy prothrombin and carbohydrate antigen 19-9. All patients underwent macroscopic curative resection. The HCC component was classified as stage I in 7 and stage II in 3, and ICC component was classified as stage I in 5, stage II in 2 and stage IV in 3. Tumor recurrence and patient survival rates were 30.0 and 90.0% at 1 year and 52.0 and 77.1% at 3 years, respectively. Tumor recurrence rates were not different between the dpHCC-ICC and the three control groups (p = 0.505). The overall and post-recurrence patient survival rates were similar between the dpHCC-ICC and cHCC-CC groups (p > 0.2); however, these were inferior to those in the HCC group but comparable with those in the ICC group. CONCLUSIONS: The post-resection prognosis of dpHCC-ICC was more dependent on the tumor stage of the ICC component than that of the HCC component. Therefore, they can be clinically regarded as ICC with concurrent HCC.

Longterm prognosis of combined hepatocellular carcinoma-cholangiocarcinoma following liver transplantation and resection.

Combined hepatocellular carcinoma-cholangiocarcinoma (cHCC-CC) is a rare disease. We investigated the clinicopathological features of cHCC-CC and compared the longterm outcomes following liver transplantation (LT) and hepatic resection (HR). We identified 32 LT patients with cHCC-CC through an institutional database search. The HR control group (n = 100) was selected through propensity score-matching. The incidence of cHCC-CC among all adult LT patients was 1.0%. Mean patient age was 53.4 ± 6.7 years, and 26 patients were male. Thirty patients had hepatitis B virus infection. All patients of cHCC-CC were diagnosed incidentally in the explanted livers. Mean tumor diameter was 2.5 ± 1.3 cm, and 28 patients had single tumors. Tumor stage was stage I in 23 and II in 9. Concurrent hepatocellular carcinoma (HCC) was detected in 12 patients with stage I in 5 and II in 7. Mean tumor diameter was 1.9 ± 1.2 cm, and 5 had single tumors. Tumor recurrence and survival rates were 15.6% and 84.4% at 1 year and 32.2% and 65.8% at 5 years, respectively. Patients with very early stage cHCC-CC (1 or 2 tumors ≤ 2.0 cm) showed 13.3% tumor recurrence and 93.3% patient survival rates at 5 years, which were significantly improved than those with advanced tumors (P = 0.002). Tumor recurrence and survival rates did not differ significantly between the LT and HR control groups (P = 0.22 and P = 0.91, respectively); however, postrecurrence patient survival did (P = 0.016). In conclusion, cHCC-CC is rarely diagnosed following LT, and one-third of such patients have concurrent HCC. The longterm posttransplant prognosis was similar following LT and HR. Very early cHCC-CC resulted in favorable posttransplant prognosis, thus this selection condition can be prudently considered for LT indication. Liver Transplantation 23 330-341 2017 AASLD.

Hepatic Angiomyolipoma Versus Hepatocellular Carcinoma in the Noncirrhotic Liver on Gadoxetic Acid-Enhanced MRI: A Diagnostic Challenge.

OBJECTIVE: The purpose of this study is to describe the imaging characteristics of hepatic angiomyolipoma (AML) on gadoxetic acid-enhanced MRI and to identify imaging features that are helpful for differentiating it from hepatocellular carcinoma (HCC) in a noncirrhotic liver. MATERIALS AND METHODS: We retrospectively identified 18 patients with pathologically proven hepatic AMLs who had undergone gadoxetic acid-enhanced MRI between 2008 and 2012. We randomly chose 36 patients with noncirrhotic liver who had a single HCC diagnosed radiologically during the same period. None of the HCCs was of the fibrolamellar variant. Two readers reviewed images in consensus to assess the lesion size, the presence of fat, signal intensity characteristics, enhancement profile, early draining veins, intratumoral vessels, and tumor capsules. The tumor-to-liver contrast ratios were measured. These features and the measurements were compared between the two groups. RESULTS: AMLs are more commonly found in women (83.3%), whereas HCCs are more common in men (75%) (p < 0.01). The size of AMLs (3.4 cm) and HCCs (4.3 cm) did not differ significantly. Intratumoral fat was identified in both AMLs (50.0%) and HCCs (30.6%). The dynamic enhancement profile (arterial hypervascularity and hypointensity during the delayed phase) was similar qualitatively and quantitatively except for the portal phase. AMLs and HCCs differed significantly with regard to isointensity on DWI (16.7% vs 0.0%; p = 0.03), washout in the portal phase (61.1% vs 88.9%; p = 0.03), early draining veins (27.8% vs 2.8%; p = 0.01), intratumoral vessels (55.6% vs 22.2%; p = 0.03), and presence of capsule (11.1% vs 50.0%; p = 0.01). CONCLUSION: On gadoxetic acid-enhanced MRI of noncirrhotic liver, AML is often indistinguishable from HCC on the basis of the enhancement profiles. Female sex and some imaging features including DWI could facilitate the differentiation.

AKT-induced PKM2 phosphorylation signals for IGF-1-stimulated cancer cell growth.

Pyruvate kinase muscle type 2 (PKM2) exhibits post-translational modifications in response to various signals from the tumor microenvironment. Insulin-like growth factor 1 (IGF-1) is a crucial signal in the tumor microenvironment that promotes cell growth and survival in many human cancers. Herein, we report that AKT directly interacts with PKM2 and phosphorylates it at Ser-202, which is essential for the nuclear translocation of PKM2 protein under stimulation of IGF-1. In the nucleus, PKM2 binds to STAT5A and induces IGF-1-stimulated cyclin D1 expression, suggesting that PKM2 acts as an important factor inducing STAT5A activation under IGF-1 signaling. Concordantly, overexpression of STAT5A in cells deficient in PKM2 expression failed to restore IGF-induced growth, whereas reconstitution of PKM2 in PKM2 knockdown cells restored the IGF-induced growth capacity. Our findings suggest a novel role of PKM2 in promoting the growth of cancers with dysregulated IGF/phosphoinositide 3-kinase/AKT signaling.

Time-course changes in the expression levels of miR-122, -155, and -21 as markers of liver cell damage, inflammation, and regeneration in acetaminophen-induced liver injury in rats.

Drug-induced liver injury (DILI) is a significant threat to patient health and a major concern during drug development. Recently, multiple circulating microRNAs (miRNAs) have been reported to be potential biomarkers for DILI. To adapt and validate miRNAs for clinical use, we investigated the time-course changes in miR-122 expression levels in an acetaminophen-induced liver injury model in rats. In addition, miR-155 and miR-21 were evaluated as makers of inflammation and regeneration, respectively, to characterize liver status. Our results revealed that miR-122 is an early and sensitive biomarker of hepatocellular injury at a stage when alanine transaminase, aspartate transaminase, and total bilirubin were not detectable. However, no significant differences in the expression levels of other miRNAs (miR-155 and -21) were observed between treatment and vehicle groups. Collectively, these time-course changes in the expression levels of miRNAs may be useful as markers for clinical decision-making, in the diagnosis and treatment of DILI.

Postresection Outcomes of Combined Hepatocellular Carcinoma-Cholangiocarcinoma, Hepatocellular Carcinoma and Intrahepatic Cholangiocarcinoma.

INTRODUCTION: Combined hepatocellular carcinoma and cholangiocarcinoma (cHCC-CC) is rare. This study investigated the clinicopathological features of cHCC-CC and compared the postresection survival outcomes of cHCC-CC, hepatocellular carcinoma (HCC), and intrahepatic cholangiocarcinoma (IHC). METHODS: Between January 2000 and September 2012, 53 patients with cHCC-CC underwent tumor resection, accounting for 1.1 % of surgeries for primary liver malignancies. Control groups included patients with HCC (n = 1452) and IHC (n = 149) who underwent R0 resection of stage I/II tumors ≤5 cm. RESULTS: Mean tumor diameter of cHCC-CC group was 5.5 ± 2.9 cm, and single tumor was identified in 50. Pathological classification included combined (n = 41), mixed (n = 11), and double (n = 1) tumors. The 1-, 3-, 5-, and 10-year tumor recurrence rates were 60.8, 71.8, 80.7, and 80.7 %, respectively. The 1-, 3-, 5-, and 10-year overall survival rates were 73.3, 35.6, 30.5, and 11.1 %, respectively. Tumor recurrence and patient survival did not differ significantly according to AJCC tumor staging and histological type (all p ≥ 0.2). Tumor recurrence rates did not differ significantly between the cHCC-CC, HCC, and IHC groups (p = 0.43), whereas differences in survival rates were significant (p = 0.000), with a median survival after tumor recurrence of 8, 51, and 6 months, respectively (p = 0.000). CONCLUSIONS: Patients with cHCC-CC showed similar recurrence rates to those of control patients with HCC and IHC, whereas their survival outcomes were worse than those of control HCC patients because of poor responses to recurrence treatment. Further evaluation of differences in tumor characteristics and tumor biology is necessary to accurately predict the prognosis of patients with cHCC-CC.

Metformin inhibits early stage diethylnitrosamine­induced hepatocarcinogenesis in rats.

Antitumor effects of metformin have recently emerged despite its original use for type II diabetes. In the present study, the effects of metformin on the development and recurrence of hepatocellular carcinoma (HCC) were investigated using the diethylnitrosamine (DEN)­induced rat model of HCC. Tumor foci were characterized by gross examination and by histopathological characteristics, including proliferation, hepatic progenitor cell content and the expression of hepatocarcinoma­specific molecular markers. Potential target molecules of metformin were investigated to determine the molecular mechanism underlying the inhibitory effects of metformin on chemically induced liver tumorigenesis. The antitumor effects of metformin were increased by the reduction of surface nodules and decreased the incidence of altered hepatocellular foci, hepatocellular adenoma and carcinoma. Also, decreased expression levels of glutathione S­transferase placental form, proliferating cell nuclear antigen and cytokeratin 8 described the inhibitory effects of metformin on HCC. In the present study, Wistar rats receiving treatment with DEN were administered metformin for 16 weeks. In addition, metformin suppressed liver tumorigenesis via an AMPK­dependent pathway. These results suggested that metformin has promising effects on the early stage of HCC in rats. Therefore, metformin may be used for the prevention of HCC recurrence following primary chemotherapy for HCC and/or for high­risk patients, including chronic hepatitis and cirrhosis.

Evaluation of Nonalcoholic Fatty Liver Disease in C57BL/6J Mice by Using MRI and Histopathologic Analyses.

Nonalcoholic fatty liver disease (NAFLD) can lead to cirrhosis, hepatocellular carcinoma, and ultimately death. Magnetic resonance techniques are accurate, noninvasive methods for evaluating hepatic steatosis but, in animals, have not been fully validated against histologic findings. We sought to validate the MRI fat-signal fraction (MRI-FSF) used for diagnosing NAFLD in human nonclinical trials by comparing MRI data with histopathologic findings in C57BL/6J mice (n = 24) fed normal chow (controls) or a methionine- and choline-deficient (MCD) diet to induce NAFLD. Axial T2-weighted fast spin-echo images were used to examine the entire liver. For histopathologic analyses, liver slides were evaluated for hepatic steatosis according to the NAFLD activity score. Pearson correlation coefficient and receiver operating characteristics analyses were performed. According to the fat-fraction signal, the mean percentage of liver fat in mice with induced NAFLD was 57%, which correlated with the histologically determined steatosis grade. The proton-density fat fraction effectively distinguished severe from mild hepatic steatosis, with an AUC of 0.92. Evaluation accuracy decreased when lobular inflammation and hepatocellular ballooning were considered. This study showed strong concurrence between MRI-FSF and histopathologic steatosis in a murine model of NAFLD. MRI-FSF had moderate sensitivity and specificity in this context. These results confirm that the MRI is a useful biomarker of hepatic steatosis in NAFLD in murine model.

Fibroblast growth factor receptor isotype expression and its association with overall survival in patients with hepatocellular carcinoma.

BACKGROUND/AIMS: Fibroblast growth factor signaling is involved in hepatocarcinogenesis. The aim of this study was to determine the fibroblast growth factor receptor (FGFR) isotype expression in hepatocellular carcinoma (HCC) and neighboring nonneoplastic liver tissue, and elucidate its prognostic implications. METHODS: Immunohistochemical staining of FGFR1, -2, -3, and -4 was performed in the HCCs and paired neighboring nonneoplastic liver tissue of 870 HCC patients who underwent hepatic resection. Of these, clinical data for 153 patients who underwent curative resection as a primary therapy were reviewed, and the relationship between FGFR isotype expression and overall survival was evaluated (development set). This association was also validated in 73 independent samples (validation set) by Western blot analysis. RESULTS: FGFR1, -2, -3, and -4 were expressed in 5.3%, 11.1%, 3.8%, and 52.7% of HCCs, respectively. Among the development set of 153 patients, FGFR2 positivity in HCC was associated with a significantly shorter overall survival (5-year survival rate, 35.3% vs. 61.8%; P=0.02). FGFR2 expression in HCC was an independent predictor of a poor postsurgical prognosis (hazard ratio, 2.10; P=0.02) in the development set. However, the corresponding findings were not statistically significant in the validation set. CONCLUSIONS: FGFR2 expression in HCC could be a prognostic indicator of postsurgical survival.