Dysplastic Nodules Research Articles

A novel panel of biomarkers in distinction of small well-differentiated HCC from dysplastic nodules and outcome values

BackgroundDifferential diagnosis of high-grade dysplastic nodules (HGDN) and well-differentiated hepatocellular carcinoma (WDHCC) represents a challenge to experienced hepatic clinicians, radiologists and hepatopathologists.MethodsThe expression profiles of aminoacylase-1 (ACY1), sequestosome-1 (SQSTM1) and glypican-3 (GPC3) in low-grade dysplastic nodules (LGDN), HGDN and WDHCC were assessed by immunohistochemistry. The differential diagnostic performances of these three markers alone and in combination for HGDN and WDHCC were investigated by logistic regression models (HGDN = 21; WDHCC = 32) and validated in an independent test set (HGDN, n = 21; WDHCC n = 24). Postoperative overall survival and time to recurrence were evaluated by univariate and multivariate analyses in an independent set of 500 patients.ResultsACY1, SQSTM1 and GPC3 were differentially expressed in each group. For the differential diagnosis of WDHCC from HGDN, the sensitivity and specificity of the combination of ACY1 + SQSTM1 + GPC3 for detecting WDHCC were 93.8% and 95.2% respectively in the training set, which were higher than any of the three two-marker combinations. The validities of the four diagnostic models were further confirmed in an independent test set, and corresponding good sensitivity and specificity were observed. Interestingly, GPC3 expression in HCC tissues combined with serum α-fetoprotein (AFP) was found to be an independent predictor for overall survival and time to recurrence.ConclusionsACY1 + SQSTM1 + GPC3 combination represents a potentially valuable biomarker for distinguishing between WDHCC and HGDN using immunohistochemistry. Meanwhile, low GPC3 staining combined with positive serum AFP may play a practical role in predicting poor postoperative outcome and high tumor recurrence risk.

Efficacy of early treatment on 52 patients with preneoplastic hepatitis B virus-associated hepatocellular carcinoma by compound Phyllanthus Urinaria L.

To observe the change in the number of antibodies of preneoplastic hepatocellular carcinoma (HCC) using early treatment by Compound Phyllanthus Urinaria L. (CPUL) on patients with preneoplastic hepatitis B virus (HBV)-associated HCC. A total of 102 cirrhosis patients with regenerative or dysplastic nodules whose sera were tested positive for at least one of these six proteins (five up-regulated genes URG4, URG7, URG11, URG12 and URG19, and one down-regulated gene DRG2) were assigned randomly to two groups using continual random codes by SPSS software. Fifty-two patients were in the treatment group and 50 patients were in the control group. CPUL was used in the treatment group for 3 years, while the control group did not receive any treatment. The changes in HBV-DNA level, number of antibodies, and hepatocarcinogenesis occurred were observed. Patients who did not develop HCC were followed up for another 2 years. HBV-DNA levels decreased ⩾2log in 22.2% (10/45) of patients in the treatment group in contrast to only 5.0% (2/40) of patients in the control group (P=0.0228). The number of antibodies that were tested positive in the treatment group (1.08±1.01) was significantly lower compared with the control group (2.11±1.12) after 24 months of drug treatment (P<0.01). Both the positive rates of anti-URG11 (33/52) and anti-URG19 (31/52) were over 60% at baseline in the two groups, and were decreased to 48.1% (25/52) and 46.2% (24/52) respectively at 36 months of drug treatment, while the rates increased to 68.0% (34/50) and 66.0% (33/50) respectively (P=0.0417, P=0.0436) in the control group. The positive rate of anti-DRG2 was increased to 55.8% (29/52) at 36 months of drug treatment, while in the control group was decreased to 36.0% (18/50, P=0.0452). Among the 102 patients who developed HCC, 2 were in the treatment group and 9 were in the control group, meaning that a significant difference between the two groups (P=0.0212). In 11 patients who developed HCC, anti-URG11 and anti-URG19 were always positive, while anti-DRG2 was negative. Patients newly developing HCC were 6 (20.0%) in the control group, and only one (2.5%) in the treatment group (P=0.0441) during 2-year follow-up after the end of the treatment. Anti-URG11, anti-URG19 and anti-DRG2 could be used as early markers in the prediction of the therapeutic efficacy of CPUL in treating preneoplastic HCC. CPUL is useful in preventing or delaying the development of HBV-associated cirrhosis to HCC.

Global gene expression profiling reveals SPINK1 as a potential hepatocellular carcinoma marker.

BackgroundLiver cirrhosis is the most important risk factor for hepatocellular carcinoma (HCC) but the role of liver disease aetiology in cancer development remains under-explored. We investigated global gene expression profiles from HCC arising in different liver diseases to test whether HCC development is driven by expression of common or different genes, which could provide new diagnostic markers or therapeutic targets.Methodology and Principal FindingsGlobal gene expression profiling was performed for 4 normal (control) livers as well as 8 background liver and 7 HCC from 3 patients with hereditary haemochromatosis (HH) undergoing surgery. In order to investigate different disease phenotypes causing HCC, the data were compared with public microarray repositories for gene expression in normal liver, hepatitis C virus (HCV) cirrhosis, HCV-related HCC (HCV-HCC), hepatitis B virus (HBV) cirrhosis and HBV-related HCC (HBV-HCC). Principal component analysis and differential gene expression analysis were carried out using R Bioconductor. Liver disease-specific and shared gene lists were created and genes identified as highly expressed in hereditary haemochromatosis HCC (HH-HCC) were validated using quantitative RT-PCR. Selected genes were investigated further using immunohistochemistry in 86 HCC arising in liver disorders with varied aetiology. Using a 2-fold cut-off, 9 genes were highly expressed in all HCC, 11 in HH-HCC, 270 in HBV-HCC and 9 in HCV-HCC. Six genes identified by microarray as highly expressed in HH-HCC were confirmed by RT qPCR. Serine peptidase inhibitor, Kazal type 1 (SPINK1) mRNA was very highly expressed in HH-HCC (median fold change 2291, p = 0.0072) and was detected by immunohistochemistry in 91% of HH-HCC, 0% of HH-related cirrhotic or dysplastic nodules and 79% of mixed-aetiology HCC.ConclusionHCC, arising from diverse backgrounds, uniformly over-express a small set of genes. SPINK1, a secretory trypsin inhibitor, demonstrated potential as a diagnostic HCC marker and should be evaluated in future studies.

Predictors of Dysplastic Nodule Diagnosis in Patients With Liver Cirrhosis on Unenhanced and Gadobenate Dimeglumine–Enhanced MRI With Dynamic and Hepatobiliary Phase

The purpose of this article is to assess whether unenhanced and gadobenate dimeglumine-enhanced MRI with dynamic and hepatobiliary phase may predict the diagnosis of dysplastic nodules in patients with liver cirrhosis. We retrospectively analyzed 75 cirrhotic patients (47 men and 28 women; mean [± SD] age, 55 ± 12 years) with 82 hepatocellular nodules, including histology-proven dysplastic nodules (n = 25; diameter, 1-3 cm) and hepatocellular carcinomas (n = 57; diameter, 2-3 cm) scanned by MRI before and after gadobenate dimeglumine injection during hepatic arterial phase (HAP), portal venous phase (PVP), equilibrium phase, and hepatobiliary phase. Nodule T1 and T2 intensities before contrast agent injection and nodule HAP, PVP, equilibrium phase, and hepatobiliary phase intensities were compared with the adjacent liver. Univariate and multivariate logistic regression analysis was conducted to assess how the nodule could predict dysplastic nodule diagnosis. Some imaging findings were independent predictors of dysplastic nodule diagnosis-namely, nodule T2 isohypointensity (odds ratio [OR], 12.28; 95% CI, 3.88-38.82), T1 isohyperintensity (OR, 26.74; 95% CI, 7.53-94.90), HAP isohypointensity (OR, 97.16; 95% CI, 20.06-470.49), PVP-equilibrium phase isohyperintensity (OR, 20.53; 95% CI, 5.36-78.62), and hepatobiliary phase isohyperintensity (OR, 119.6; 95% CI, 21.59-662.40). Nodule T2 and HAP isohypointensity (OR 31.47; 95% CI, 7.88-125.58), nodule T2 isohypointensity and hepatobiliary phase isohyperintensity (OR, 28.77; 95% CI, 7.79-106.19), nodule T1 isohyperintensity and HAP isohypointensity (OR, 17.22; 95% CI, 4.85-61.14), and nodule T1 and hepatobiliary phase isohyperintensity (OR, 19.39; 95% CI, 5.38-69.90) were also predictors of dysplastic nodule diagnosis. The combination of nodule appearance on T2-weighted MRI and nodule enhancement after gadobenate dimeglumine injection may predict dysplastic nodule diagnosis in patients with liver cirrhosis.

Validation of diagnostic criteria using gadoxetic acid-enhanced and diffusion-weighted MR imaging for small hepatocellular carcinoma (≤ 2.0 cm) in patients with hepatitis-induced liver cirrhosis

Gadoxetic acid and diffusion-weighted imaging (DWI) is increasingly used for the diagnosis of hepatocellular carcinoma (HCC). It is relevant to refine the diagnostic parameters for HCC, using state-of-the-art imaging techniques. To validate usefulness of magnetic resonance imaging (MRI) criteria with gadoxetic acid-enhanced MRI and DWI for diagnosis of small HCC by differentiation from dysplastic nodule (DN) or regenerative nodule (RN) in cirrhotic patients with strongly suspected small HCC. One hundred and eight patients with 102 HCCs and 29 benign nodules including 21 DNs and two large RNs (≤ 2.0 cm), and 40 patients with no HCC underwent gadoxetic acid-enhanced MRI and DWI. All patients also underwent MDCT. Index MR criteria for HCC were: (i) arterial hyperenhancement and hypointensity on hepatobiliary phase (HBP) with hyperintensity on DWI; (ii) hypovascular nodule with hyperintensity on DWI; (iii) arterial hyperenhancement and hypointensity on HBP without hyperintensity on DWI; (iv) arterial hyperenhancement and either iso- or hyperintensity on HBP, with hyperintensity on DWI; and (v) hyperintensity only on DWI. According to these criteria, MRI findings for HCCs and benign nodules were independently classified by two reviewers. On multidetector-row computed tomography, 64 HCCs (62.7%) showed typical features for HCC while 13 (12.8%) were not identified. On MRI, 84 HCCs (82.4%) showed arterial hyperenhancement and hypointensity on HBP, and hyperintensity on DWI. Eight HCCs were regarded as hypovascular HCCs with hyperintensity DWI (category 2). One HCC (0.6 cm in diameter) was demonstrated only by DWI. For each observer, 101 (99.0%) and 100 HCCs (98.0%) were discernible when applying all MRI criteria for HCC, respectively. Three DNs also fit the HCC criteria, thus the specificity was 90.9% for both observers. With the HCC criteria based on combined gadoxetic acid-enhanced MRI and DWI, it is possible to reliably diagnose small HCC including hypovascular HCCs.

Utility of an Immunohistochemical Panel Consisting of Glypican-3, Heat-shock Protein-70, and Glutamine Synthetase in the Distinction of Low-grade Hepatocellular Carcinoma From Hepatocellular Adenoma

The pathologic distinction between hepatocellular carcinoma (HCC) and hepatocellular adenoma (HCA) is sometimes problematic due to histologic overlap between the 2 entities, a problem amplified on small biopsy specimens. Several recently characterized immunohistochemical markers such as glypican-3 (GPC-3), heat-shock protein-70 (HSP-70), and glutamine synthetase (GS) help distinguish dysplastic nodules from HCC. The utility of this panel in the distinction of low-grade hepatocellular carcinoma (LG-HCC) from HCA has not been fully described. To determine whether the above markers are useful in the distinction of HCCs from HCAs. Tissue microarrays were constructed with 30 LG-HCCs and 18 HCAs. The arrays were stained with the above markers and analyzed with respect to amount and pattern of staining. GPC-3 and HSP-70 were considered positive when 10% of tumor cells showed immunoreactivity. GS was considered positive when 50% of tumor cells showed immunoreactivity. GPC-3 was positive in 13 of 30 LG-HCCs and 0 of 18 adenomas. The sensitivity was 43% and the specificity was 100%. HSP-70 was positive in 14 of 30 LG-HCCs and 0 of 18 adenomas. The sensitivity was 46% and the specificity was 100%. GS was positive in 24 of 30 LG-HCCs and 9 of 18 adenomas. The sensitivity was 80% and the specificity was 50%. GPC-3 and HSP-70 are helpful in separating carcinomas from adenomas. GS is not useful in this clinical context.

T-39 Small nodules arising in cirrhotic liver during surveillance: possibility of characterization among dysplastic nodule, early hepatocellular carcinoma and progressed hepatocellular carcinoma using CEUS

T-39 Small nodules arising in cirrhotic liver during surveillance: possibility of characterization among dysplastic nodule, early hepatocellular carcinoma and progressed hepatocellular carcinoma using CEUS

Differentiation between dysplastic nodule and early-stage hepatocellular carcinoma: The utility of conventional MR imaging

To elucidate the variety of ways early-stage hepatocellular carcinoma (HCC) can appear on magnetic resonance (MR) imaging by analyzing T1-weighted, T2-weighted, and gadolinium-enhanced dynamic studies. Seventy-three patients with well-differentiated HCC (wHCC) or dysplastic nodules were retrospectively identified from medical records, and new histological sections were prepared and reviewed. The tumor nodules were categorized into three groups: dysplastic nodule (DN), wHCC compatible with Edmondson-Steiner grade I HCC (w1-HCC), and wHCC compatible with Edmondson-Steiner grade II HCC (w2-HCC). The signal intensity on pre-contrast MR imaging and the enhancing pattern for each tumor were recorded and compared between the three tumor groups. Among the 73 patients, 14 were diagnosed as having DN, 40 were diagnosed as having w1-HCC, and 19 were diagnosed as having w2-HCC. Hyperintensity measurements on T2-weighted axial images (T2WI) were statistically significant between DNs and wHCC (P = 0.006) and between DN and w1-HCC (P = 0.02). The other imaging features revealed no significant differences between DN and wHCC or between DN and w1-HCC. Hyperintensity on both T1W out-phase imaging (P = 0.007) and arterial enhancement on dynamic study (P = 0.005) showed statistically significant differences between w1-HCC and w2-HCC. The other imaging features revealed no significant differences between w1-HCC and w2-HCC. In the follow-up for a cirrhotic nodule, increased signal intensity on T2WI may be a sign of malignant transformation. Furthermore, a noted loss of hyperintensity on T1WI and the detection of arterial enhancement might indicate further progression of the histological grade.

DNA Damage Sensor γ‐H2AX Is Increased in Preneoplastic Lesions of Hepatocellular Carcinoma

Background. Phosphorylated histone H2AX (γ-H2AX) is a potential regulator of DNA repair and is a useful tool for detecting DNA damage. To evaluate the clinical usefulness of γ-H2AX in hepatocellular carcinoma (HCC), we measured the level of γ-H2AX in HCC, dysplastic nodule, and nontumorous liver diseases. Methods. The level of γ-H2AX was measured by immunohistochemistry in fifty-eight HCC, 18 chronic hepatitis, 22 liver cirrhosis, and 19 dysplastic nodules. Appropriate cases were also examined by fluorescence analysis and western blotting. Results. All cases with chronic liver disease showed increased levels of γ-H2AX expression. In 40 (69.9%) of 58 cases with HCC, the labeling index (LI) of γ-H2AX was above 50% and was inversely correlated with the histological grade. Mean γ-H2AX LI was the highest in dysplastic nodule (74.1 ± 22.1%), which was significantly higher than HCC (P < 0.005). Moreover, γ-H2AX was significantly increased in nontumorous tissues of HCC as compared with liver cirrhosis without HCC (62.5 ± 24.7%, from 5.1 to 96.0%, P < 0.005). Conclusions. γ-H2AX was increased in the preneoplastic lesions of HCC and might be a useful biomarker for predicting the risk of HCC.

Contrast ultrasound in hepatocellular carcinoma at a tertiary liver center: First Indian experience

To assess the role of contrast enhanced ultrasonography in evaluation of hepatocellular carcinoma (HCC) at the first Indian tertiary liver center. Retrospective analysis of contrast enhanced ultrasound (CEUS) examinations over 24 mo for diagnosis, surveillance, characterization and follow up of 50 patients in the context of HCC was performed. The source and indication of referrals, change in referral rate, accuracy and usefulness of CEUS in a tertiary liver center equipped with a 64 slice dual energy computer tomography (CT) and 3 tesla magnetic resonance imaging (MRI) were studied. Sonovue (BR1, Bracco, Italy, a second generation contrast agent) was used for contrast US studies. Contrast enhanced CT/MRI or both were performed in all patients. The findings were taken as a baseline reference and correlation was done with respect to contrast US. Contrast enhanced MRI was performed using hepatocyte specific gadobenate dimeglumine (Gd-BOPTA). Iomeron (400 mg; w/v) was used for dynamic CT examinations. About 20 (40%) of the examinations were referred from clinicians for characterization of a mass from previous imaging. About 15 (30%) were performed for surveillance in chronic liver disease; 5 (10%) examinations were performed for monitoring lesions after radiofrequency ablation (RFA); 3 (6%) were post trans-arterial chemo-embolization (TACE) assessments and 3 (6%) were patients with h/o iodinated contrast allergy. About 2 (4%) were performed on hemodynamically unstable patients in the intensive care with raised alpha fetoprotein and 2 (4%) patients were claustrophobic. The number of patients referred from clinicians steadily increased from 12 in the first 12 mo of the study to 38 in the last 12 mo. CEUS was able to diagnose 88% of positive cases of HCC as per reference standards. In the surveillance group, specificity was 53.3% vs 100% by CT/MRI. Post RFA and TACE specificity of lesion characterization by CEUS was 100% in single/large mass assessment, similar to CT/MRI. For non HCC lesions such as regenerative and dysplastic nodules, the specificity was 50% vs 90% by CT/MRI. The positive role of CEUS in imaging spectrum of HCC included a provisional urgent diagnosis of an incidentally detected mass. It further led to a decrease in time for further management. A confident diagnosis on CEUS was possible in cases of characterization of an indeterminate mass, in situations where the patient was unfit for CT/MRI, was allergic to iodinated contrast or had claustrophobia, etc. CEUS was also cost effective, radiation free and an easy modality for monitoring post RFA or TACE lesions. CEUS is a valuable augmentation to the practice of ultrasonography, and an irreplaceable modality for confounding cases and interpretation of indeterminate lesions in imaging of HCC.

Accuracy and Disagreement of Computed Tomography and Magnetic Resonance Imaging for the Diagnosis of Small Hepatocellular Carcinoma and Dysplastic Nodules: Role of Biopsy

Accuracy and Disagreement of Computed Tomography and Magnetic Resonance Imaging for the Diagnosis of Small Hepatocellular Carcinoma and Dysplastic Nodules: Role of Biopsy

A serum amyloid A-positive hepatocellular neoplasm arising in alcoholic cirrhosis: a previously unrecognized type of inflammatory hepatocellular tumor

Hepatocellular adenoma usually arises in the absence of significant fibrosis. Herein, we report seven patients with serum amyloid A-positive hepatocellular neoplasm, which shares features with inflammatory hepatocellular adenoma arising in alcoholic cirrhosis. Seven patients (two women and five men, age range 41–67 years) with hypervascular hepatocellular nodules associated with alcoholic cirrhosis were retrieved from our pathological files (1997–2011). The hepatocellular nodules were multiple (>3) in all patients and 17 nodules were histologically examined. We surveyed the immunoreactivity for serum amyloid A, glutamine synthetase, and glypican-3 in the hepatocellular nodules and control lesions, including 5 focal nodular hyperplasia, 18 dysplastic nodules, and 54 hepatocellular carcinomas in various background diseases. In all, 15 of 17 nodules showed strong and distinct immunoreactivity for serum amyloid A, sharing features with inflammatory hepatocellular adenoma. The serum amyloid A-positive hepatocellular neoplasms showed increased cellular density, inflammatory infiltrate, sinusoidal dilatation, and ductular reaction to various degrees. Although about a half of dysplastic nodules and hepatocellular carcinomas showed focal immunoreactivity for serum amyloid A, the extent of serum amyloid A expression was significantly higher in serum amyloid A-positive hepatocellular neoplasms, than in control nodules. The serum amyloid A-positive hepatocellular neoplasms did not show the overexpression of glutamine synthetase or immunoreactivity for glypican-3. In contrast, most hepatocellular carcinomas showed the overexpression of glutamine synthetase and immunoreactivity for glypican-3, irrespective of background diseases. In conclusion, this study highlights a characteristic group of hepatocellular neoplasms arising in alcoholic cirrhosis, which share features with inflammatory hepatocellular adenomas. These serum amyloid A-positive hepatocellular neoplasms may be a new type of inflammatory hepatocellular tumors in alcoholic patients.

Immunostaining of glutamine synthetase is a sensitive and specific marker for diagnosing focal nodular hyperplasia in needle biopsy

Focal nodular hyperplasia (FNH) has characteristic histological features which may not be seen in needle biopsy specimens. We investigate the diagnostic role of glutamine synthetase (GS) in needle biopsy specimens. Sixty-one hepatic tumours were categorised into 20 'definite' FNHs, 13 'probable' FNHs, and 28 cases without specific diagnosis. Needle biopsy specimens of 92 non-tumourous lesions, 25 well-differentiated hepatocellular carcinomas (WDHCCs), and 4 high-grade dysplastic nodules (HGDNs) and resection specimens of 10 macroregenerative nodules were also selected for immunohistochemical stain of GS for comparison. All 20 'definite' FNHs, nine 'probable' FNHs, and five cases without specific diagnosis expressed typical map-like staining pattern of GS. The demographic data of these five cases were similar to those of FNH. All cases of chronic hepatitis B and C, cirrhosis, macroregenerative nodule and peritumourous liver tissue showed normal pericentral/periseptal pattern. Fifteen of 25 WDHCCs and one HGDN showed diffuse pattern. Ten WDHCCs and two HGDNs showed negative staining. One HGDN showed mosaic pattern. Immunohistochemical staining of GS increases the diagnostic sensitivity of FNH in needle biopsy, especially in those without typical morphology. It also helps in differentiating FNH from other tumourous and non-tumourous lesions.

Hyperintense Lesions on Gadoxetate Disodium-Enhanced Hepatobiliary Phase Imaging

The purpose of this article is to illustrate various focal hepatic lesions that may show hyperintensity on hepatobiliary phase images on gadoxetate disodium-enhanced MRI. Hyperintense lesions on gadoxetate disodium-enhanced MRI include focal nodular hyperplasia (FNH) or FNH-like nodules, hepatocellular adenoma, dysplastic nodules, and hepatocellular carcinoma. Understanding the contrast enhancement patterns on hepatobiliary phase images and other imaging findings is important to ensure correct diagnosis.

Liver cancer initiation is controlled by AP-1 through SIRT6-dependent inhibition of survivin

Understanding stage-dependent oncogenic mechanisms is critical to develop not only targeted therapies, but also diagnostic markers and preventive strategies. The mechanisms acting during cancer initiation remain elusive, largely owing to a lack of suitable animal models and limited availability of human precancerous lesions. Here we show using genetic mouse models specific for liver cancer initiation, that survival of initiated cancer cells is controlled by c-Jun, independently of p53, through suppressing c-Fos-mediated apoptosis. Mechanistically, c-Fos induces SIRT6 transcription, which represses survivin by reducing histone H3K9 acetylation and NF-κB activation. Importantly, increasing the level of SIRT6 or targeting the anti-apoptotic activity of survivin at the initiation stage markedly impairs cancer development. Moreover, in human dysplastic liver nodules, but not in malignant tumours, a specific expression pattern with increased c-Jun-survivin and attenuated c-Fos-SIRT6 levels was identified. These results reveal a regulatory network connecting stress response and histone modification in liver tumour initiation, which could be targeted to prevent liver tumorigenesis.

Molecular genetic study in hepatocellular carcinoma: microRNA deregulation in multistep hepatocarcinogenesis

Deregulation of microRNAs (miRNAs) plays an important role in human carcinogenesis. However, miRNA deregulation in the pre-malignant lesions and expression changes during multistep hepatocarcinogenesis remain elusive. We investigated the expression changes of seven cancer-related miRNAs during the early stages of HBV-related hepatocarcinogenesis, including dysplastic nodules (DN), small hepatocellular carcinomas (HCCs), and their corresponding non-tumorous livers. We found that down-regulation of miR-145 and miR-199b and up-regulation of miR-224 were frequently observed in pre-malignant DNs and these changes persisted throughout HCC development. Restoration of miR-145 in both HepG2 and Hep3B HCC cells significantly inhibited cell proliferation and reduced cell migration and cell invasion. Furthermore, these inhibitory functions of miR-145 could be substantially reduced by anti-miR-145 inhibitor. Our results showed that miRNA deregulation was an early event and accumulated throughout the various steps of HBV-associated hepatocarcinogenesis. On the other hand, we investigated the mechanisms of HCC metastasis and identified an antimetastatic miRNA, miR-139, that is down-regulated in human HCC samples. Down-regulation of miR-139 in HCC was associated with poor prognosis of patients and features of metastatic tumors, including venous invasion, microsatellite formation, absence of tumor encapsulation, and reduced differentiation. miR-139 expression was reduced in metastatic HCC tumors as compared with primary tumors. Overexpression of miR-139 in HCC cells significantly reduced cell migration and invasion in vitro and the incidence and severity of lung metastasis from orthotopic liver tumors in mice. miR-139 interacted with the 3’ untranslated region of Rho-kinase 2 (ROCK2) and reduced its expression in HCC cells. Levels of miR-139 correlated inversely with ROCK2 protein in human HCC samples. Expanding insight into the keys of miRNA dysregulation involved in HCC metastasis will yield important clues to our understanding of the complicated mechanisms underlying HCC progression and may enhance the development of new therapeutic regimens in treating advanced HCCs. Presence of tumor thrombi in the portal veins (venous metastases) is a clinicopathological feature of metastatic HCCs. By analyzing the miRNA expression profiles of non-tumorous livers, primary HCCs, and venous metastases in the same livers from 20 HCC patients with low density microarray (LDA), we identified the precise alterations of miRNA expression from non-tumorous livers to primary HCCs and venous metastases globally. Non-tumorous livers were distinctly segregated from primary HCCs and venous metastases, whereas no discernible difference in the expression pattern could be found between primary HCCs and venous metastases. However, a marked global reduction of miRNA expression levels was detected in venous metastases, as compared with primary HCCs. These data suggest that miRNA deregulation may be an early event in liver carcinogenesis and the later global miRNA down-regulation aggravates the pre-existing miRNA deregulation to further promote HCC metastasis. Our study has enriched the current understanding of the deregulation of miRNAs in HCC progression and highlighted the sequential and distinctive alterations of miRNA expression in primary HCC and venous metastasis formation.

Contrast-enhanced computed tomography and ultrasound-guided liver biopsy to diagnose dysplastic liver nodules in cirrhosis

BackgroundDysplastic nodules in cirrhosis herald a very high risk of transition to hepatocellular carcinoma. A better understanding of the relationships between dysplastic nodules and hepatocellular carcinoma development may help refining strategies of enhanced follow-up. MethodsAll consecutive cirrhotics with a histologically proven de novo dysplastic nodule, were retrospectively identified and underwent alternating abdominal ultrasound and contrast-computed tomography every 3 months. An ultrasound-guided liver biopsy was the diagnostic gold standard, whereas surveillance and recall policies were according to current guidelines. ResultsAmong 36 patients with dysplastic nodule (21 low-grade, 15 high-grade, 17.4±2.6mm), 17 (47%) showed arterial wash-in, 15 (42%) portal/venous hypodensity whereas 4 (11%) had neither pattern. During 6–128 (median 36) months, 21 patients developed a hepatocellular carcinoma at a rate of 13.8% per year, intranodular=8.7% vs extranodular=7.1% per year. Hepatocellular carcinoma occurred more frequently in high-grade than low-grade dysplastic nodules (32.2% vs 9.3% per year, p=0.0039); the maximum time to hepatocellular carcinoma transformation was 27 months for intranodular vs 67 months for extranodular tumours (p=0.025). No contrast-computed tomography pattern predicted neoplastic transformation of dysplastic nodules. ConclusionThe histological examination of liver nodules in cirrhosis lacking the imaging hallmark of hepatocellular carcinoma improves both prognostication and outcome of surveillance, since it dictates the intensity of the radiological follow-up.

Differentiating hepatocellular carcinoma from dysplastic nodules at gadobenate dimeglumine-enhanced hepatobiliary-phase magnetic resonance imaging

We evaluated whether the addition of delayed phase imaging (DPI) gadobenate dimeglumine-enhanced MRI to dynamic postcontrast imaging improves the characterization of small hepatocellular carcinoma (HCC) and the differentiation between HCC, high grade dysplastic nodules (HGDN), and low grade dysplastic nodules (LGDN). Twenty-five cirrhotic patients with 30 nodules (16 HCC, 8 HGDNs, and 6 LGDNs; maximum size of 3 cm) were included in this retrospective study. The diagnostic reference standard was histology. All the patients underwent MRI both prior to and following intravenous administration of gadobenate dimeglumine. The lesions were classified as hypointense, isointense, hyperintense on DPI for qualitative assessment. In the quantitative analysis the relative tumor-liver contrast to noise ratio (CNR) of the lesions on DPI was calculated. All HCCs were hypointense on DPI while only 8 (57.1%) of 14 DNs were hypointense and only 1 of 6 (16.6%) LGDNs was hypointense. There was a statistically significant difference in the hypointensity on DPI between HCCs and DNs (p = 0.003) in the qualitative analysis but not in the CNR values while there was a strong statistically significant difference in the hypointensity on DPI in the qualitative (p = 0.00001) and quantitative analysis (p < 0.05) between LGDNs and the group obtained by unifying HGDNs and HCCs. DPI is helpful in differentiating HCCs and HGDNs from LGDNs. Demonstration of hypointensity on DPI should raise the suspicion of HGDN or hypovascular HCC in the case of nodules with atypical dynamic pattern.

Deficiency of the Promyelocytic Leukemia Protein Fosters Hepatitis C-Associated Hepatocarcinogenesis in Mice

Overwhelming lines of epidemiological evidence have indicated that persistent infection with hepatitis C virus (HCV) is a major risk for the development of hepatocellular carcinoma (HCC). We have recently shown that HCV core protein mediates functional inactivation of the promyelocytic leukemia (PML) tumor suppressor pathway. However, the role of PML in HCC development yet remains unclear. To clarify the function of PML in liver carcinogenesis and HCV-associated pathogenesis we crossed PML-deficient mice with HCV transgene (HCV-Tg) expressing mice and treated the resulting animals with DEN/Phenobarbital, an established protocol for liver carcinogenesis. Seven months after treatment, livers were examined macroscopically and histologically. Genetic depletion of the tumor suppressor PML coincided with an increase in hepatocyte proliferation, resulting in development of multiple dysplastic nodules in 100% of the PML-deficient livers and of HCCs in 53%, establishing a tumor suppressive function of PML in the liver. In animals expressing the HCV-transgene in PML-deficient background, HCC development occurred even in 73%, while only 7% of their wildtype littermates developed HCC. The neoplastic nature of the tumors was confirmed by histology and expression of the HCC marker glutamine synthetase. Several pro- and antiapoptotic factors were tested for differential expression and liver carcinogenesis was associated with impaired expression of the proapoptotic molecule TRAIL in PML-deficient mice. In conclusion, this study provides first in vivo evidence that the tumor suppressor PML acts as an important barrier in liver carcinogenesis and HCV-dependent liver pathology.

Enhancer of zeste homologue 2 (EZH2) is a reliable immunohistochemical marker to differentiate malignant and benign hepatic tumors

BackgroundThe immunohistochemical demonstration of Enhancer of zeste homologue 2 (EZH2) proved to be a useful marker in several tumor types. It has been described to distinguish reliably hepatocellular carcinomas from liver adenomas and other benign hepatocellular lesions. However, no other types of malignant liver tumors were studied so far.MethodsTo evaluate the diagnostic value of this protein in hepatic tumors we have investigated the presence of EZH2 by immunohistochemistry in hepatocellular carcinomas and other common hepatic tumors.EZH2 expression was examined in 44 hepatocellular carcinomas, 23 cholangiocarcinomas, 31 hepatoblastomas, 16 other childhood tumor types (rhabdomyosarcoma, neuroblastoma, Wilms’ tumor and rhabdoid tumor), 17 metastatic liver tumors 24 hepatocellular adenomas, 15 high grade dysplastic nodules, 3 biliary cystadenomas, 3 biliary hamartomas and 3 Caroli’s diseases.ResultsMost of the malignant liver tumors were positive for EZH2, but neither of the adenomas, cirrhotic/dysplastic nodules, reactive and hamartomatous biliary ductules stained positively.ConclusionsOur immunostainings confirm that EZH2 is a sensitive marker of hepatocellular carcinoma, but its specificity is very low, since almost all the investigated malignant liver tumors were positive regardless of their histogenesis. Based on these results EZH2 is a sensitive marker of malignancy in hepatic tumors. In routine surgical pathology EZH2 could be most helpful to diagnose cholangiocarcinomas, because as far as we know this is the first marker to distinguish transformed and reactive biliary structures. Although hepatoblastomas also express EZH2, the diagnostic significance of this observation seems to be quite limited whereas, the structurally similar, other blastic childhood tumors are also positive.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1173195902735693