Noninvasive Visualization of Tumor Blood Vessels within Hepatocellular Carcinoma by Application of Superb Microvascular Imaging to Contrast-Enhanced Ultrasonography.

The combination or sequential use of systemic therapies, such as lenvatinib and locoregional therapies, can improve the curability rate of hepatocellular carcinoma. This is based on the notion that lenvatinib remodels abnormal tumor vessels into normal vessels, potentially enhancing the efficacy of locoregional therapies. In this case report, we achieved noninvasive visualization of tumor blood vessels by applying superb microvascular imaging (SMI) to contrast-enhanced ultrasonography (CEUS). A man in his 80s with a borderline resectable hepatocellular carcinoma received preoperative therapy using lenvatinib. The patient achieved a complete response after lenvatinib therapy, underwent hepatectomy, and maintained a cancer-free status. CEUS and SMI revealed a decrease in tumor blood vessels at 1 week after lenvatinib administration and a decrease in tumor perfusion at 2 weeks. Although CEUS alone is adequate for noninvasive real-time evaluation of tumor perfusion, it is not sufficient to achieve accurate assessments of tumor blood vessels. We performed a noninvasive time-course evaluation of vascular normalization after lenvatinib administration by applying SMI. The evaluation of vascular normalization with lenvatinib therapy using CEUS and SMI can support the decision to proceed to conversion therapies.

Contrast-enhanced Ultrasonography Features for Diagnosing Pseudoprogression of Hepatocellular Carcinoma with Immunotherapy: A Case Report of the Response after Pseudoprogression.

A male patient in his 70s with recurrent hepatocellular carcinoma (HCC) after surgery received atezolizumab plus bevacizumab (Atezo+Bev) therapy. Initial computed tomography (CT) revealed tumor growth along with an increase in tumor markers, and contrast-enhanced ultrasonography (CEUS) showed multiple round avascular areas within the nodules with an appearance similar to a slice of Swiss cheese. Continuation of immunotherapy with consideration of the potential for pseudoprogression produced a dramatic response. Although it is difficult to distinguish between true progression and pseudoprogression, the Swiss cheese-like appearance on CEUS may be important for the early diagnosis of pseudoprogression.

Contrast-enhanced ultrasonography for the diagnosis of spontaneous necrosis of hepatocellular carcinoma: A report of 2 cases.

Spontaneous necrosis of hepatocellular carcinoma (HCC) is rare and difficult to diagnose preoperatively if it occurs before the definitive diagnosis of HCC; this is because spontaneous necrosis of HCC exhibits various patterns in imaging studies. We compared imaging and pathological findings, and examined the possibility of diagnosing spontaneous necrosis of HCC using contrast-enhanced ultrasonography (CEUS). We experienced 2 cases of spontaneous necrosis of HCC. In case 1, spontaneous necrosis occurred after HCC diagnosis, while in case 2 it occurred before the first admission. The tumor in case 2 contained internal nodules and outer fibrous tissue. CEUS revealed a vascular spot in the hypovascular area during the vascular phase and a complete defect during the Kupffer phase. These findings accorded with the pathological findings and may be important for diagnosing spontaneous necrosis of HCC.

Extracellular RNA transfer from non-malignant human cholangiocytes can promote cholangiocarcinoma growth.

Extracellular vesicles (EV) within the cellular secretome are emerging as modulators of pathological processes involved in tumor growth through their ability to transfer donor-derived RNA into recipient cells. While the effects of tumor and stromal cell EVs within the tumor microenvironment have been studied, less is known about the contributions of normal, nontransformed cells. We examined the impact of EVs within the cellular secretome from nonmalignant cells on transformed cell growth and behavior in cholangiocarcinoma cells. These effects were enhanced in the presence of the pro-fibrogenic mediator TGF-ß. We identified miR-195 as a TGF-ß responsive miRNA in normal cells that can be transferred via EV to tumor cells and regulate cell growth, invasion, and migration. The effects of miR-195 involve modulation of the epithelial-mesenchymal transition through direct effects on the transcription factor Snail. These studies provide in vitro and in vivo evidence for the impact of normal cellular secretome on transformed cell growth, show the importance of EV RNA transfer, and identify mechanisms of EV-mediated transfer of miRNA as a contributor to tumor development, which may provide new therapeutic opportunities for targeting human cholangiocarcinoma.

B-Mode Ultrasonography versus Contrast-Enhanced Ultrasonography for Surveillance of Hepatocellular Carcinoma: A Prospective Multicenter Randomized Controlled Trial.

BACKGROUND: Current practice guidelines recommend the use of ultrasound (US) as an initial surveillance tool for hepatocellular carcinoma (HCC) in patients with liver cirrhosis. Patients with liver cirrhosis, however, frequently have coarse liver parenchyma, masking the presence of tiny nodules during B-mode US. Contrast-enhanced US (CEUS) with Sonazoid has a long-lasting, stable Kupffer phase, which makes it possible to scan the entire liver to depict small lesions. In addition, defect reperfusion imaging (reinjection imaging) enables to determine whether the detected nodule is HCC or not. This prospective, multicenter, randomized, controlled trial was conducted to demonstrate the usefulness of Kupffer phase surveillance in the detection of small HCC compared to B-mode US. METHODS: A total of 23 institutions joined this study. In total, 656 patients with hepatitis B- or C-related liver cirrhosis were randomized either to the B-mode US surveillance group (n = 313) or the Kupffer phase CEUS with Sonazoid surveillance group (n = 309). The primary endpoint was the maximum size of HCC at the time of the first detection. Secondary endpoints included time to HCC detection, number of tumors, and Barcelona Clinic Liver Cancer stage at the first detection, and sensitivity, specificity, and accuracy of each method in the diagnosis, and the cumulative detection rate of HCC. RESULTS: The mean HCC size at the first detection was significantly smaller in the CEUS (13.0 ± 4.1 mm; n = 28) than in the B-mode US group (16.7 ± 4.1 mm; n = 26) (p = 0.011). Of the 38 patients with HCV cirrhosis diagnosed with HCC by US alone, mean tumor size at the first detection was significantly smaller in the 20 patients diagnosed by CEUS alone than in the 18 diagnosed by B-mode US alone (12.7 ± 3.1 vs. 17.6 ± 7.0 mm, p = 0.012). In contrast, among the 16 patients with HBV cirrhosis diagnosed by US alone, mean tumor size at the first detection was similar in the 8 patients diagnosed by CEUS alone and the 8 diagnosed by B-mode US (13.6 ± 6.0 vs. 14.5 ± 2.7 mm, p = 0.715). CONCLUSION: Kupffer phase CEUS surveillance with Sonazoid is extremely useful for the early detection and confirmation of HCC using a reinjection technique. Kupffer phase CEUS with Sonazoid contrast combined with the reinjection technique is, therefore, recommended as first-line screening tool for HCC in patients with liver cirrhosis, especially those with very coarse liver parenchyma.

Extracellular vesicle-encapsulated miR-30e suppresses cholangiocarcinoma cell invasion and migration via inhibiting epithelial-mesenchymal transition.

Early-staged cholangiocarcinoma (CCA) is difficult to diagnose due to its high potential for invasion and metastasis. Epithelial-mesenchymal transition (EMT) is induced by transforming growth factor-ß (TGF-ß) in a process thought to be important for invasion and metastasis in several cancers, including CCA. Although microRNAs (miRNAs) have been implicated in the pathogenesis of several malignancies, their roles to CCA are not clearly understood. Some miRNAs were reported to be included in extracellular vesicles (EVs) and transferred from their donor cells to other cells, modulating recipient cell behaviors. In this study, the involvement and functional roles of EV-contained miRNAs during EMT in human CCA were determined. Expression profiling identified a subset of miRNAs that were reduced by TGF-ß in CCA cells. Among these, miR-30e was highly downregulated by TGF-ß and predicted to target Snail, which is an EMT-inducible transcription factor. MiR-30e overexpression suppressed cell invasion and migration via inhibiting EMT, whereas miR-30e inhibition promoted EMT, cell invasion and migration. Moreover, miR-30e was enriched in EVs derived from CCA cells after miR-30e overexpression, and miR-30e intercellular transfer through EVs suppressed EMT, cell invasion and migration in recipient CCA cells. Together, our results suggest that EV-mediated miR-30e transfer could inhibit EMT via directly targeting Snail, which subsequently suppresses CCA cell invasion and migration. These findings provide several new insights into regulatory mechanisms of tumor invasion and metastasis in human CCA.

Computer-aided diagnosis for estimating the malignancy grade of hepatocellular carcinoma using contrast-enhanced ultrasound: an ROC observer study.

BACKGROUND & AIMS: We are developing a computer-aided diagnosis (CAD) system for estimating the malignancy grade of hepatocellular carcinoma (HCC) using contrast-enhanced ultrasound (CEUS). In this study, observers estimated the malignancy grade of HCC with and without the cues provided by CAD. MATERIALS AND METHODS: Institutional review board approval was obtained and informed consent was waived. A total of 232 histologically confirmed HCCs were studied: 76 well-differentiated HCC (w-HCC), 133 moderately differentiated HCC (m-HCC), and 23 poorly differentiated HCC (p-HCC). In this observer study, CEUS vascular images acquired using the maximum intensity projection technique were displayed together with static B-mode and Kupffer-phase (defined as 10 min after injection) images. Five hepatologists independently assigned confidence ratings for the malignancy grade of each HCC. Each hepatologist first read each case without CAD and then immediately afterwards with CAD. The observers' rating data were evaluated by multireader multicase receiver operating characteristic (ROC) analysis. RESULTS: The overall sensitivity of our CAD system for discrimination between three histological differentiation grades of HCC was 87.5% (203/232). For discrimination between w-HCC and m/p-HCC, the mean area under the ROC curve (AUC) for the five observers was significantly improved from 0.779 ± 0.074 to 0.872 ± 0.090 with CAD (P = 0.0069). For discrimination between m-HCC and p-HCC, the mean AUC was also significantly improved from 0.713 ± 0.107 to 0.863 ± 0.101 with CAD (P = 0.0321). CONCLUSION: The use of our CAD system can significantly improve the diagnostic performance of hepatologists in discriminating between three histological differentiation grades of HCC using CEUS.

Angiotensin II type 1 receptor antagonist prevents hepatic carcinoma in rats with nonalcoholic steatohepatitis.

BACKGROUND: Angiotensin II type 1 receptor blockers (ARBs) have been reported to attenuate hepatic fibrosis in non-alcoholic steatohepatitis (NASH). However, it is uncertain whether ARBs prevent hepatocarcinogenesis in NASH even after hepatic fibrosis has developed. METHODS: Male Wistar rats were fed with a choline-deficient, L-amino acid-defined (CDAA) diet for 24 weeks, and then fed with the CDAA diet with telmisartan (2 mg/kg/day), a novel ARB, or vehicle for another 24 weeks. The liver histology and the expression of genes and proteins related to angiogenesis were investigated. RESULTS: The 24-week CDAA diet induced liver cirrhosis. The 48-week CDAA diet exacerbated liver cirrhosis, and developed hepatocellular carcinoma (HCC) in 54.6 % of the rats concurrently with increases of hypoxia-inducible factor-1α (HIF-1α) protein and vascular endothelial growth factor (VEGF) mRNA, which are potent angiogenic factors in the liver. Telmisartan inhibited hepatic fibrosis and preneoplastic lesions and prevented the development of HCC along with inducing decreases in HIF-1α protein and VEGF mRNA. CONCLUSIONS: These data indicated that telmisartan may prevent hepatocarcinogenesis through the inhibition of hepatic angiogenesis even after liver cirrhosis has been established.

Hepatic schwannoma: imaging findings on CT, MRI and contrast-enhanced ultrasonography.

A primary benign schwannoma of the liver is extremely rare and is difficult to preoperatively discriminate from a malignant tumor. We compared the imaging and pathological findings, and examined the possibility of preoperatively diagnosing a benign liver schwannoma. A 72-year-old woman was admitted to our hospital because of a 4.6-cm mass in the liver. A malignant tumor was suspected, and a right hepatectomy was performed. After this, the diagnosis of a primary benign schwannoma of the liver was made through pathological examination. Contrast-enhanced ultrasonography (CEUS) with Sonazoid showed minute blood flows into the septum and solid areas of the tumor in the vascular phase; most likely due to increased arterial flow associated with infiltration of chronic inflammatory cells. In the postvascular phase, CEUS showed contrast defect of cystic areas and delayed enhancement of solid areas; most likely due to aggregation of siderophores. Because discriminating between a benign and malignant schwannoma of the liver is difficult, surgery is generally recommended. However, the two key findings from CEUS may be useful in discriminating ancient schwannoma by recognizing the hemorrhage involved in the secondary degeneration and aggregation of siderophores.

Homeobox gene CDX2 inhibits human pancreatic cancer cell proliferation by down-regulating cyclin D1 transcriptional activity.

OBJECTIVES: Homeobox gene caudal related homeobox gene 2 (CDX2) is an intestine-specific tumor suppressor gene. This study is intended to investigate the effect of CDX2 expression on cell proliferation and cyclin D1 expression in pancreatic cancer cells. METHODS: Four pancreatic ductal adenocarcinoma cell lines (PancQGO-1, BxPC-3, MIAPaCa-2, CFPAC-1), 1 islet carcinoma cell line (QGP-1), and 1 adenosquamous carcinoma cell line (KP-3) were analyzed for CDX1 and CDX2 expression using real-time reverse transcription-polymerase chain reaction and Western blot analysis. Proliferation of pancreatic cancer cells was analyzed using WST-1 assay after CDX2 transfection. Luciferase assay was performed to examine the effects of CDX2 on cyclin D1 transcriptional activity. RESULTS: CDX2 was expressed at a significantly higher level in QGP-1 cells than in KP-3 cells. Moreover, CDX2 was expressed at a middle level in 4 pancreatic ductal adenocarcinoma cells. Cell proliferation and cyclin D1 mRNA level were inhibited significantly after CDX2 transfection in pancreatic cancer cells. Furthermore, CDX2 inhibited exogenous nuclear factor kappaB-p65-induced luciferase gene expression in a dose-dependent manner. In addition, CDX2 inhibited pGL2HIVD1kappaB2-luciferase activity. CONCLUSIONS: CDX2 might play a role in inhibiting cell proliferation and repressing cyclin D1 transcriptional activity through the proximal nuclear factor kappaB binding site in pancreatic cancer cells.

An autopsy case of autoimmune pancreatitis.

CONTEXT: Autoimmune pancreatitis is an increasingly recognized type of chronic pancreatitis, but little is known about the long-term outcome of the disease. CASE REPORT: We report an autopsy case of autoimmune pancreatitis. The patient was an 81-year-old Japanese male. He was referred to our department with jaundice in February 1996. ERCP images revealed a severe stricture of the lower part of the common bile duct and irregular narrowing of the main pancreatic duct. A diagnosis of extrahepatic cholangiocarcinoma was made and endoscopic biliary drainage was performed. A stricture of the common bile duct and narrowing of the pancreatic duct had improved on ERCP images when a follow-up examination was performed in November 1998. He was followed up for chronic pancreatitis. The serum IgG and IgG4 levels were increased on serological examination. He died of interstitial pneumonia and congestive heart failure in May 2003. At the autopsy examination, fibrosis was found in the periductal, interlobular and intralobular parts of the pancreas. Focal atrophy of the acinar cells was also identified. There was little infiltration of inflammatory cells into the parenchyma or the stroma of the pancreas. These pathological findings were similar to those of 'conventional' chronic pancreatitis. CONCLUSION: We present an autopsy case of autoimmune pancreatitis which is a rare finding.

Inhibitory effect of angiotensin II receptor antagonist on hepatic stellate cell activation in non-alcoholic steatohepatitis.

AIM: To investigate the efficacy of angiotensin II receptor antagonist on hepatic stellate cells (HSCs) activation in the patients with non-alcoholic steatohepatitis (NASH). METHODS: Seven patients with NASH were prescribed losartan, a selective angiotensin II type 1 receptor antagonist (50 mg/d) for 48 wk. Liver biopsies were performed both at the entry and end of the study in all patients. Quiescent and activated HSCs were identified by double immunostaining using anti-p75 and -smooth muscle actin antibodies, and the number of each phenotype was counted. Similarly, the liver specimens obtained from the eight patients with non-alcoholic fatty liver (NAFL) were also examined as controls. RESULTS: In NASH hepatic tissues, activated HSCs were dominantly distributed as compared with those in NAFL. The 48-wk losartan treatment induced a remarkable decrease in activated HSCs and a mild increase in quiescent phenotypes. CONCLUSION: Our data suggest the crucial involvement of HSCs in anti-fibrotic effect of angiotensin II receptor antagonist on patients with NASH.

Therapeutic efficacy of an angiotensin II receptor antagonist in patients with nonalcoholic steatohepatitis.

The therapeutic efficacy of angiotensin II receptor antagonist, losartan, was studied in patients with nonalcoholic steatohepatitis (NASH). Seven patients with both NASH and hypertension were treated with losartan (50 mg/d) for 48 weeks. Treatment with losartan resulted in a significant decrease in blood markers of hepatic fibrosis, plasma TGF-beta1 and serum ferritin concentration concurrently with an improvement in serum aminotransferase levels. Histological assessment showed improvement of hepatic necroinflammation in five patients, reduction of hepatic fibrosis in four patients, and disappearance of iron deposition in two patients. No side effect of treatment was noted at any time during the study. In conclusion, the present data raise the possibility that an angiotensin II receptor antagonist may be therapeutically efficacious for NASH.

High, but not low, molecular weight hyaluronan prevents T-cell-mediated liver injury by reducing proinflammatory cytokines in mice.

BACKGROUND: The extracellular matrix component hyaluronan (HA) modulates the production of various cytokines and chemokines by activated inflammatory cells. In this study, we investigated whether exogenous administration of HA influences T-cell-mediated liver injury and cytokine production. METHODS: Liver injury was induced by administration of concanavalin A (Con A) or D-galactosamine/lipopolysaccharide (GalN/LPS), and 0.05%-0.35% (v/v) HA (MW 250, 470, 780, 900, and 1200 kDa) was administered intravenously 18 h before Con A or GalN/LPS injection. Plasma ALT level was determined enzymatically and plasma cytokine levels were determined by ELISA. RESULTS: The elevated plasma levels of ALT at 8 h after Con A and at 7 h after GalN/LPS injection were significantly decreased by pretreatment with high molecular weight HAs (780, 900, and 1200 kDa) but not low molecular weight HAs (250 and 470 kDa). High molecular weight HA (900 kDa) significantly reduced plasma tumor necrosis factor-alpha, interferon gamma, macrophage inflammatory protein 2, and interleukin 4 levels after Con A injection. However, this inhibitory effect on plasma cytokines was not observed with low molecular weight HA (250 kDa) pretreatment. CONCLUSIONS: The present results suggest that high molecular weight but not low molecular weight HA prevents liver injury by reducing proinflammatory cytokines in a T-cell-mediated liver injury model. The extracellular matrix component hyaluronan (HA) modulates the production of various cytokines and chemokines by activated inflammatory cells. In this study, we investigated whether exogenous administration of HA influences T-cell-mediated liver injury and cytokine production.

Antithrombin III prevents concanavalin A-induced liver injury through inhibition of macrophage inflammatory protein-2 release and production of prostacyclin in mice.

BACKGROUND/AIMS: Recently, we have reported that macrophage inflammatory protein-2 (MIP-2) plays a pivotal role in concanavalin A (Con A)-induced liver injury. In this study, we investigated the effect of antithrombin III (AT-III) on liver damage, and production of MIP-2 and prostacyclin in this model. METHODS: Liver injury was induced by intravenous injection of Con A (15 mg/kg) and AT-III was administered (50, 250 and 500 units/kg, iv) 30 mm before Con A injection. Plasma levels of alanine aminotransferase (ALT), MIP-2 and 6-keto-prostaglandin F1alpha (6k-PG-F1alpha), stable metabolite of prostaglandin I(2) (prostacyclin), were determined. RESULTS: The elevated plasma ALT levels 8, 16, 24 h after Con A injection were inhibited by AT-III pretreatment. The elevated plasma MIP-2 levels were significantly inhibited by AT-III pretreatment compared with vehicle treatment. The inhibitory effect of AT-III on plasma ALT and MIP-2 in Con A-induced liver injury was attenuated by indomethacin (5 mg/kg, ip). Plasma concentration of 6k-PG-F1alpha at 2 h after AT-III injection was significantly elevated compared with baseline and vehicle pretreatment. CONCLUSIONS: These findings suggest that AT-III prevents Con A-induced liver injury through an inhibition of MIP-2 release and a production of prostacyclin.