Non-neoplastic Liver Tissue Research Articles

Metallothioneins Gene Regulation Through Promoter DNA Methylation According to Zn and Cu Trace Elements Status in Human Hepatocellular Carcinoma (P05-002-19)

ObjectivesHepatocellular carcinoma (HCC) is the most frequent primary liver cancer, yet mechanisms of hepatocarcinogenesis are largely unknown. A particular interest was recently dedicated to the role of trace elements and metallothioneins (MTs), a group of proteins involved in metal ions homeostasis and detoxification, have been suggested as possible tumor suppressor genes. The study of MTs transcriptional regulation by promoter DNA methylation is the object of study as a possible mechanism responsible for gene silencing through epigenetics. MethodsTwenty-seven HCC patients undergoing surgery intervention were enrolled and clinically characterized. MT1G and MT1H gene expression was performed by Real Time qPCR. DNA methylation analysis in 23 HCC and homologous non-neoplastic liver tissue (N) was performed by Bisulfite-Amplicon Sequencing (BSAS) in an overlapping region (∼400 bp) of the promoters of the two genes. Cu and Zn concentrations were measured in serum and liver tissues (HCC and N) by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Kaplan-Meier analysis of survival was performed according to serum trace elements. ResultsMT1G and MT1H were transcriptionally repressed in HCC tissue as compared to N. A correlation was observed between the mRNA levels of the two MTs, in particular MT1G was repressed in 23 out of 27 HCC tissue (P = 0.0366) and MT1H was repressed in 24 out of 27 HCC tissue (P = 0.0077). The promoter region resulted hypermethylated in 9 out of 19 HCC that showed MT1G and MT1H down-regulation. Serum Zn and Cu levels were within the normal range while HCC tissue exhibited significantly reduced Zn levels as compared to N (P < 0.0001). Tissue Cu levels did not show significant differences. Serum trace elements levels were also analyzed according to patients clinical features and those with Cu levels higher than the 75th percentile had a significantly poorer prognosis than those within the lowest Cu levels quartile (P < 0.05). ConclusionsMT1G and MT1H are repressed in HCC tissue. In a subset of patients the downregulation was associated to promoter hypermethylation, supporting the hypothesis of MT1G and MT1H as possible tumor suppressor genes in HCC. Evidence of a correlation between serum Cu levels and survival rate provide new insights for the role of this microelement in liver carcinogenesis. Funding SourcesNo funding sources.

Diagnosis of Liver Neoplasms by Computational and Statistical Image Analysis.

BackgroundDistinguishing well-differentiated hepatocellular carcinoma (WD-HCC), hepatocellular adenoma (HA) and non-neoplastic liver tissue (NNLT) solely on morphology is often challenging. The purpose of this study was to evaluate the use of computational image analysis to distinguish WD-HCC, HA and NNLT.MethodsSeventy-seven cases comprising of WD-HCC (n = 26), HA (n = 23) and NNLT (n = 28) were retrieved and reviewed. A total of 485 hematoxylin and eosin (H&E) photomicrographs (× 400, 0.09 µm2) of WD-HCC (n = 183), HA (n = 173), NNLT (n = 129) and nine whole-slide scans (three of each diagnosis) were obtained, color deconvoluted and digitally transformed. Quantitative data including nuclear density, nuclear sphericity, nuclear perimeter, and nuclear eccentricity from each image were acquired. The data were analyzed by one-way analysis of variance (ANOVA) with Tukey post hoc test, followed by unsupervised and supervised (Chi-square automatic interaction detection (CHAID)) cluster analysis.ResultsUnsupervised cluster analysis identified three well defined clusters of WD-HCC, HA and NNLT. Employing the four most discriminating nuclear features, supervised analysis was performed on a training set of 383 images, and validated on the remaining 102 test images. The analysis identified WD-HCC (sensitivity 100%, specificity 98%), HA (sensitivity 71%, specificity 85%) and NNLT (sensitivity 70%, specificity 86%). An analysis of whole-slide images identified WD-HCC with sensitivity and specificity of 100%.ConclusionsWe have successfully demonstrated that computational image analysis of nuclear features can differentiate WD-HCC from non-malignant liver with high accuracy, and can be used to assist in the histopathological diagnosis of hepatocellular carcinoma.

Single-stranded DNA binding protein 2 expression is associated with patient survival in hepatocellular carcinoma

BackgroundSSBP2, single-stranded DNA binding protein 2, is a subunit of the ssDNA-binding complex that is involved in the maintenance of genome stability. The majority of previous studies have suggested a tumor-suppressive role of SSBP2, which is silenced by promoter hypermethylation in several human malignancies, such as hematologic malignancies, prostate cancer, esophageal squamous cell carcinoma, ovarian cancer, and gallbladder cancer. However, an oncogenic role of SSBP2 has been suggested in glioblastoma patients. We investigated the clinicopathologic significance of SSBP2 expression in hepatocellular carcinoma.MethodsWe constructed tissue microarrays consisting of 21 normal liver parenchyma and 213 hepatocellular carcinoma tissues with corresponding adjacent non-neoplastic tissues. SSBP2 expression was investigated by immunohistochemistry, and positive expression was defined as more than 10% of the tumor cells to show nuclear staining. We then analyzed the correlations between SSBP2 expression and various clinicopathologic characteristics, and further studied the role of SSBP2 in cell growth and migration.ResultsHepatocytes were negative for SSBP2 immunohistochemistry in all normal liver samples, whereas the nuclei of normal bile duct epithelium and sinusoidal endothelium were immunoreactive. Positive immunoreactivity was found in one (0.6%) out of 180 non-neoplastic liver tissue samples adjacent to the tumor and in 16 (8.5%) out of 189 hepatocellular carcinomas. Positive SSBP2 expression was significantly correlated with tumor multifocality (P = 0.027, chi-square test), high histologic grade (P = 0.003, chi-square test), and frequent vascular invasion (P = 0.001, chi-square test). Kaplan-Meier survival curves revealed that patients with SSBP2 expression had poor prognosis in both disease-free and overall survival (P = 0.004 and P = 0.026, respectively, log-rank test). SSBP2-positive tumors also had a higher Ki-67 proliferation index (P < 0.001, t-test). Furthermore, downregulation of SSBP2 in the Huh7 cell line inhibited cell migration (P = 0.022, t-test) with altered expression of epithelial-mesenchymal transition markers.ConclusionsThe minority of hepatocellular carcinomas expressed SSBP2 by immunohistochemistry, whereas normal hepatocytes were negative. SSBP2-positive hepatocellular carcinomas were significantly associated with aggressive phenotypes and poor clinical outcome.

Evaluation of the role of Notch1 expression in hepatic carcinogenesis with clinico-pathological correlation

The role of Notch pathway in hepatocarcinogenesis is unclear with conflicting results reported from different researchers. This study aimed to investigate the exact role of Notch1 in hepatocarcinogenesis and its influence on survival and to determine the possibility of it being a target therapy. Differential immunohistochemical expression of Notch1 in 100 cases of hepatocellular carcinoma (HCC) and adjacent non-neoplastic liver tissue was performed. The results showed that expression of Notch1 was significantly higher in the non-neoplastic hepatic tissues than in HCC tissues (p<0.001), but there was no significant difference in Notch1 expression between cirrhotic and non-cirrhotic liver tissue (p=0.197). Notch1 expression was higher in low grade than in high grade HCC (p=0.036). Notch1 expression showed reverse correlation with mitotic count (p=0.008), and necrosis (p=0.005). The disease free survival was shorter in patients displaying low levels of Notch1 expression (p=0.045). The overall survival showed no significant difference between high and low levels of Notch1 expression; however, it was somewhat longer in patients with high Notch1 expression (p=0.220). In conclusion, the tumour suppressor role of Notch1 was supported and the use of Notch1 agonists may have a role in improving the prognosis of HCC.

Abstract B37: Assessing DNA damage response pathway proteins ATM and p53 in fibrolamellar hepatocellular carcinoma with PRKACA-DNAJB1 fusion protein

Abstract Introduction: Fibrolamellar hepatocellular carcinoma (FL-HCC) is a rare variant of liver cancer that is prevalent in younger patients between 10-35 years of age and is usually not associated with underlying cirrhosis. Overall outcomes tend to be poor due to lack of effective chemotherapy. Recently, a gene fusion transcript has been identified in a significant proportion of FL-HCC whereby PRKACA, a cAMP-associated protein, and heat shock protein DNAJB1 fuse following a deletion of 400kb region. In our previous study, immunoblot analysis of p53, a tumor-suppressor protein, was significantly upregulated in FL-HCC samples in comparison to normal liver tissues. p53 is a downstream target of ATM, which is a key regulator of DNA damage response (DDR) pathway. Therefore, we hypothesized that increased p53 protein expression may be associated with ATM activation in FL-HCC. Methods: We analyzed 3 tumor and non-neoplastic FL-HCC patient samples containing DNAJB1-PRKACA fusion protein using immunohistochemistry (IHC) on paraffin-embedded sections with anti-ATM, anti-phosphorylated ATM (S1981), anti-p53, and anti-phospho-p53 (S15) antibodies. Immunoblot analysis was performed for cleaved PARP protein to assess the apoptotic cells in FL-HCC samples. ImageJ software was utilized to quantify the nuclear, cytoplasmic/mitochondrial localization of each protein. Results: Nuclear ATM expression was significantly increased in tumor samples compared to normal liver tissues in 3/3 FL-HCC patients (p=1.43E-04, p=1.51E-07 and 1.68E-05). Nuclear expression of activated (phosphorylated) ATM (p-ATM) was significantly upregulated in 2/3 tumor compared to the normal samples (p=9.47E-09 and p=0.00). Interestingly, the tumor sample lacking nuclear phospho-ATM was the only tumor sample with a significant expression of p-ATM in the mitochondrial/cytoplasmic compartment. In assessing the expression pattern of p-53, 2/3 patients revealed statistically significant increase in total p-53 compared to normal liver sample. While nuclear phospho-p53 expression was not significantly altered in any of the three tumor samples compared to non-neoplastic liver tissue, there was a statistically significant increase in the activated form of p53 (phospho-p53) expression in the cytoplasmic region in 2/3 FLHCC samples compared to normal. Coincidentally, the same 2 patients who demonstrated increased total nuclear ATM and phospho-ATM showed increased cytoplasmic p53 and phosphorylated p53. Immunoblot analysis for cleaved-PARP revealed no PARP activation in FL-HCC tumor samples. Conclusion: The expression pattern of total ATM and phospho-ATM in FL-HCC patients with PRKACA-DNAJB1 suggests that the DDR pathway may be activated in these tumors. However, increases in p53 protein expression and phosphorylated (S15) p53 are not associated with apoptosis given lack of increase in PARP cleavage. Further studies aimed at understanding the mechanisms preventing p53-induced apoptosis or DNA repair are therefore necessary. Citation Format: Anju Karki, Khashayar Vakili, Antonio R. Perez-Atayde. Assessing DNA damage response pathway proteins ATM and p53 in fibrolamellar hepatocellular carcinoma with PRKACA-DNAJB1 fusion protein [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B37.

Cytochrome P450 4A11 expression in tumor cells: A favorable prognostic factor for hepatocellular carcinoma patients.

Elevated cytochrome p450 (CYP) 4A gene expression has been linked to the aggravation of various cancers and affects various regulated metabolites. In hepatocellular carcinoma (HCC), the clinicopathological value of CYP4A has not yet been explored, although CYP4A is expressed at high levels in the liver. The goal of this study was to evaluate the clinicopathological value of CYP4A11 expression in HCC. We performed immunohistochemical analysis of CYP4A11 and correlated the results with clinicopathological features of HCC (n=155). Western blotting and reverse transcription-polymerase chain reaction against CYP4A11 and CYP4A22 were also performed for 15 and 20 pairs of fresh-frozen primary HCC and non-neoplastic liver tissue, respectively. Moreover, we analyzed the underlying mechanism by comparing the high and low CYP4A11 mRNA expression groups using gene set enrichment analysis. CYP4A11 expression level was higher in non-neoplastic hepatocytes than those in HCC cells (P<0.001), and CYP4A11 expression positively correlated with favorable prognostic factors, including tumor size, histological grade, and pathological tumor stage (P=0.007, P=0.005, and P=0.007). Multivariate analysis revealed that CYP4A11 expression was an independent prognostic factor of overall and disease-free survival (P=0.002 and P=0.033). Based on gene set enrichment analysis, high CYP4A11 mRNA expression negatively correlated with the expression of cell cycle-related genes. These findings support the notion that CYP4A11 expression is a favorable prognostic factor of HCC and suggest potential predictive diagnostic and prognostic roles of CYP4A11 expression in HCC.

C-reactive Protein Overexpression in the Background Liver of Hepatitis B Virus-Associated Hepatocellular Carcinoma Is a Prognostic Biomarker.

BackgroundChronic hepatitis B virus (HBV) infection is a leading cause of hepatocellular carcinoma (HCC). Peripheral blood C-reactive protein (CRP) concentration and CRP overexpression in HCC cells are proven to be prognostic markers for HCC, but the significance of CRP expression in non-neoplastic hepatocytes, which are the primary origin of CRP, has not been studied. This study was conducted to determine the clinicopathologic significance of CRP immunoreactivity in the background liver of HBV-associated HCC.MethodsCRP immunostaining was done on tissue microarrays of non-neoplastic liver tissues obtained from surgically resected, treatment-naïve HBV-associated HCCs (n = 156). The relationship between CRP immunoreactivity and other clinicopathologic parameters including cancer-specific survival was analyzed. CRP immunoreactivity was determined using a 4-tier grading system: grades 0, 1, 2, and 3.ResultsCRP was positive in 139 of 156 cases (89.1%) of non-neoplastic liver in patients with HCCs: grade 1 in 83 cases (53.2%); grade 2 in 50 cases (32.1%); and grade 3 in six cases (3.8%). The patients with diffuse CRP immunoreactivity (grade 3) had decreased cancer-specific survival (p = .031) and a tendency for shorter interval before early recurrence (p = .050). The degree of CRP immunoreactivity correlated with serum CRP concentration (p < .001).ConclusionsCRP immunoreactivity in non-neoplastic liver is a novel biomarker for poor cancer-specific survival of HBV-associated HCC and correlates with serum CRP concentration.

Abstract 4150: MDM4 expression is increased in fibrolamellar hepatocellular carcinoma with PRKACA-DNAJB1 fusion protein

Abstract Introduction: Fibrolamellar hepatocellular carcinoma (FL-HCC) is a rare variant of hepatocellular carcinoma (HCC) that preferentially affects young adults with no underlying liver disease. Overall patient outcome is poor due to the lack of effective treatment options. Recent studies have shown a 400 kb pair deletion resulting in DNJAB1-PRKACA fusion transcript and protein in majority of FL-HCC tumors. The exact oncogenic mechanism of this fusion protein is yet to be elucidated. In our previous studies we had noted upregulation of p53 and phosphorylated p53 in tumor samples compared to normal liver tissue without expected p53-induced apoptosis. Since MDM4 (a.k.a. MDMX or HDMX), a known negative regulator of p53, is overexpressed in various human cancers, we sought to examine its role in dysregulation of p53 signaling pathway in FL-HCC containing PRKACA-DNAJB1 fusion. Methods: We analyzed 7 FL-HCC tumors containing DNAJB1-PRKACA fusion protein and 5 non-neoplastic samples. Immunohistochemistry (IHC) on formalin-fixed and paraffin-embedded sections was performed using primary anti-MDM4 antibody. Diaminobenzidine (DAB) substrate kit was utilized to detect the signal and the slides were counterstained with hematoxylin. The nuclear MDM4 expression was assessed using immune reactive score (IRS) in a blinded manner. The IRS gives a range of 0-12 as a product of multiplication between positive cells proportion score (0-4) and staining intensity score (0-3). We also analyzed transcript levels of MDM4 from tumor and non-neoplastic liver samples on 4/7 patients. Reactions were performed in duplicates and MDM4 was normalized to GAPDH. Results: We found that the percentage of positive MDM4 cells ranged from 0 to 80 in normal liver samples (5/7 patients) and 10 to 100 in tumor samples (7/7 patients). The IRS score of MDM4 ranged from 0 to 6 in non-neoplastic liver tissue and 2 to 12 in tumor tissue. Statistically, the IRS score of MDM4 protein expression was significantly upregulated in tumor samples in comparison to non-neoplastic samples (P=0.0098). Additionally, the MDM4 transcript levels in 2/4 tumors examined were increased by 2- and 180-fold, respectively, compared to non-neoplastic liver tissue. Conclusion: The overexpression of MDM4 protein in FL-HCC patients with PRKACA-DNAJB1 suggests that the p53 effector function of DNA repair or induction of apoptosis may be dysregulated by increased MDM4 expression. Further studies targeted at blocking MDM4 inhibition of p53 activity in FL-HCC will be important in determining its contribution to tumor cell survival and proliferation. Citation Format: Anju Karki, Juan Putra, Michael LaQuaglia, Antonio Perez-Atayde, Khashayar Vakili. MDM4 expression is increased in fibrolamellar hepatocellular carcinoma with PRKACA-DNAJB1 fusion protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4150.

Higher CYP2E1 Activity Correlates with Hepatocarcinogenesis Induced by Diethylnitrosamine.

Hepatocellular carcinoma (HCC) is one of the most common types of primary liver cancer and the third most frequent cause of cancer death worldwide. Diethylnitrosamine (DEN) is one of the recognized risk factors for hepatocarcinogenesis likely due to CYP2E1-mediated metabolic activation. However, CYP2E1-mediated DEN metabolic activity in non-neoplastic liver tissue from HCC patients has not been determined; the role of CYP2E1 activity, in particular CYP2E1 constitutive activity and CYP2E1 inhibited activity, with respect to the hepatocarcinogenesis induced by DEN is not yet clear. Herein, we determined CYP2E1-mediated DEN metabolic activity in non-neoplastic liver tissue from HCC patients and found that CYP2E1-mediated DEN metabolic activity was significantly elevated with a 43.3% positive rate, and clinicopathologic parameters did not affect the activity. Then, using a Sprague-Dawley rat liver tumor model induced by DEN, the relationship between CYP2E1 constitutive/inhibited activity and hepatocarcinogenesis was explored. The results showed that the CYP2E1 constitutive activity was strongly correlated with tumor incidence and severity of liver tumorigenesis (nodule numbers and size), whereas inhibition of CYP2E1 activity decreased hepatocyte proliferation, liver injury, and liver carcinogenesis in the presence of DEN. In conclusion, the higher CYP2E1 activity would lead to an increased incidence of HCC as a result of CYP2E1-mediated DEN activation. Therefore, higher CYP2E1 activity might be a risk factor for HCC induced by DEN.

Hepcidin and DNA promoter methylation in hepatocellular carcinoma.

The liver hormone hepcidin regulates iron homoeostasis that is often altered in hepatocellular carcinoma (HCC). Epigenetic phenomena control gene expression through a dynamic fashion; therefore, considering the plasticity of both iron homoeostasis and epigenetic mechanisms and their role in liver carcinogenesis, we investigated whether hepcidin gene (HAMP) expression is modulated by DNA methylation, thus affecting iron status in human HCC. Thirty-two patients affected by nonviral HCC were enrolled, and their main clinical and biochemical characteristics were obtained. Neoplastic and homologous non-neoplastic liver tissues were analysed for HAMP promoter DNA methylation, for HAMP gene expression and for iron content. An invitro demethylation assay with a human hepatocarcinoma cell line was performed to evaluate the role of DNA methylation on HAMP transcriptional repression. Gene expression and DNA methylation analyses on tissues showed that HAMP was transcriptionally repressed in HCC tissues consensually with a promoter hypermethylation. Furthermore, patients with HCC had low serum hepcidin concentrations, and HCC tissues had relative iron depletion as compared to non-neoplastic liver tissues. The cell culture model showed the functional role of DNA hypermethylation by downregulating HAMP gene expression. Through a quantitative methylation analysis on HCC tissues, we then proved that methylation at definite CpG sites within consensus sequences for specific transcription factors is possibly the mechanism underlying HAMP repression. This study highlights a novel role for HAMP downregulation through DNA promoter hypermethylation and emphasises the significance of epigenetics in the regulation of iron metabolism in HCC.

A pilot systematic genomic comparison of recurrence risks of hepatitis B virus-associated hepatocellular carcinoma with low- and high-degree liver fibrosis

BackgroundChronic hepatitis B virus (HBV) infection leads to liver fibrosis, which is a major risk factor in hepatocellular carcinoma (HCC) and an independent risk factor of recurrence after HCC tumor resection. The HBV genome can be inserted into the human genome, and chronic inflammation may trigger somatic mutations. However, how HBV integration and other genomic changes contribute to the risk of tumor recurrence with regards to the different degree of liver fibrosis is not clearly understood.MethodsWe sequenced mRNAs of 21 pairs of tumor and distant non-neoplastic liver tissues of HBV-HCC patients and performed comprehensive genomic analyses of our RNAseq data and public available HBV-HCC sequencing data.ResultsWe developed a robust pipeline for sensitively identifying HBV integration sites based on sequencing data. Simulations showed that our method outperformed existing methods. Applying it to our data, 374 and 106 HBV host genes were identified in non-neoplastic liver and tumor tissues, respectively. When applying it to other RNA sequencing datasets, consistently more HBV integrations were identified in non-neoplastic liver than in tumor tissues. HBV host genes identified in non-neoplastic liver samples significantly overlapped with known tumor suppressor genes. More significant enrichment of tumor suppressor genes was observed among HBV host genes identified from patients with tumor recurrence, indicating the potential risk of tumor recurrence driven by HBV integration in non-neoplastic liver tissues. We also compared SNPs of each sample with SNPs in a cancer census database and inferred samples’ pathogenic SNP loads. Pathogenic SNP loads in non-neoplastic liver tissues were consistently higher than those in normal liver tissues. Additionally, HBV host genes identified in non-neoplastic liver tissues significantly overlapped with pathogenic somatic mutations, suggesting that HBV integration and somatic mutations targeting the same set of genes are important to tumorigenesis. HBV integrations and pathogenic mutations showed distinct patterns between low and high liver fibrosis patients with regards to tumor recurrence.ConclusionsThe results suggest that HBV integrations and pathogenic SNPs in non-neoplastic tissues are important for tumorigenesis and different recurrence risk models are needed for patients with low and high degrees of liver fibrosis.

Abstract 3514: Assessing the role of CREB in fibrolamellar hepatocellular carcinoma with PRKACA-DNAJB1 fusion protein

Abstract Introduction: Fibrolamellar Hepatocellular Carcinoma (FLHCC) is a rare form of liver cancer that occurs predominantly in adolescents or young adults. Curing or prolonging the lives of patients with FLHCC is extremely challenging. Despite aggressive surgical resections of primary tumors as well as metastatic lesions, outcomes tend to be poor due to lack of effective chemotherapy. Previous studies have demonstrated the presence of PRKACA-DNAJB1 fusion protein in the majority of FLHCC patients. DNAJB1 is a heat shock protein and PKA is a kinase that phosphorylates numerous substrates including cAMP-regulatory element-binding protein (CREB), a proto-oncogenic transcription factor. Therefore, we sought to examine whether the presence of the fusion protein in FLHCC is associated with increased CREB phosphorylation. Methods: We analyzed tumor and matched non-neoplastic liver tissue from 7 pediatric patients with a pathologic diagnosis of FLHCC. We assessed the presence of the PRKACA-DNAJB1 fusion protein using PKA antibody, as well as total CREB and phosphorylated (p-CREB) using immunoblot analyses with specific antibodies. Results: Immunoblot analysis using anti-PKA antibody confirmed the occurrence of PRKACA-DNAJB1 fusion transcripts in 6/7 (86%) FLHCC tumor samples. Immunoblot analyses of the 6 patients with the fusion protein revealed a trend towards a decrease in p-CREB expression in tumor samples compared to non-neoplastic tissue controls (p=0.15). Conclusions: The pattern of p-CREB in FLHCC with PRKACA-DNAJB1 fusion protein suggests that the fusion protein does not exert its proto-oncogenic effects through the transcriptional activity of CREB. Citation Format: Anju Karki, Michael J. LaQuaglia, Amanda M. Dios, Ghazaleh Sadri-Vakili, Khashayar Vakili. Assessing the role of CREB in fibrolamellar hepatocellular carcinoma with PRKACA-DNAJB1 fusion protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3514. doi:10.1158/1538-7445.AM2017-3514

Comparison of Subtypes of Hepatocellular Adenoma to Hepatocellular Carcinoma and Non-Neoplastic Liver Tissue in Terms of PTEN Expression

PTEN is a tumour suppressor gene whose loss of function has been found to be present in a variety of neoplasms, both benign and malignant. In hepatocellular carcinoma (HCC), loss of PTEN is associated with poorly differentiated cancer, advanced clinical stage and tendency to recur. The extent and meaning of PTEN loss in hepatocellular adenoma (HA), one of the precursor lesions for HCC, has not yet been analysed. The aim of the present study was to evaluate the possible loss of PTEN expression in HA in the wider context of hepatocarcinogenesis. Immunohistochemical analysis of PTEN expression was performed in non-neoplastic liver tissue, HAs and HCCs. It has been found that the loss of PTEN was markedly present in poorly differentiated HCC, whereas well to moderately differentiated HCC showed similar levels of PTEN expression to nonneoplastic liver. HAs presented as a heterogeneous group, with loss of PTEN observed in the inflammatory and HNF1A-mutated subtype and relatively intact PTEN expression in HA with nuclear β-catenin overexpression. This suggests that the loss of PTEN might occur both in HA and HCC, constituting different outcomes of the same molecular lesion in the various contexts of malignant or benign neoplasms.

Distinction between inflammatory hepatocellular adenoma and mass effect on liver sampling

Inflammatory hepatocellular adenoma (IHA) is characterized by sinusoidal dilation, inflammation, and bile ductular reaction. However, these changes can also be seen in nonneoplastic liver tissue adjacent to a mass lesion. This differential may arise in biopsy tissue attempting to sample a liver mass. Serum amyloid A (SAA) immunostaining is useful for the diagnosis of IHA but is not entirely specific. In addition, the histologic pattern of mass effect (ME) has received little formal scrutiny. We compared the clinical, morphologic, and immunohistochemical findings in 18 IHA and 36 livers with ME. Several histologic findings were evaluated in all cases. SAA and CD34 staining were also performed. Patients with IHA were younger (P<.0001) and more often female (P=.0044) than patients with specimens showing ME, but lesions were multifocal on imaging in two-thirds of patients in each category. Unpaired arteries were only seen in IHA (P<.0001), whereas ductular reaction was more common in ME (P=.012). Strong SAA immunostaining was observed in 100% of IHAs and 56% of ME cases (P=.0004), and CD34 staining was seen in 95% of IHAs and 6% of ME cases (P<.0001). Unpaired arteries and staining for SAA and CD34 best distinguish IHA from ME. However, unpaired arteries may be absent because of sampling, and SAA is not available in all laboratories. Ductular reaction can occur in IHA but is more common in ME.

Clinicopathological features of 11 Epstein-Barr virus-associated intrahepatic cholangiocarcinoma at a single center in China.

To date, only 20 cases of Epstein–Barr virus (EBV)-associated intrahepatic cholangiocarcinomas (IHCCs) have been reported in the literature.Pathology records of IHCC from January 1, 2007 to December 31, 2013 were retrieved from our hospital. Clinical information related to EBV-associated IHCC were also obtained, including gender, age at initial diagnosis, tumor size, tumor–node–metastasis stage, and follow-up duration. Surgically resected stage-matched EBV-negative IHCCs with full follow-up were selected for comparison. All liver specimens were fixed in 10% neutral-buffered formalin and paraffin-embedded tissue blocks containing cholangiocarcinoma and nonneoplastic liver tissue. Hematoxylin and eosin-stained sections were present in all cases.Among 329 primary IHCC patients, intranuclear expression of EBV was only found in 11 patients (3.3%), with an age range of 30 to 67 years (mean, 53.2 years; median, 54 years). The group consisted of 4 male and 7 female patients (M:F ratio 1:1.8). Histopathological analysis showed 1 case (9.1%) belonged to the typical lymphoepithelioma-like carcinoma (LELC), primarily composed of undifferentiated tumor cells intimately admixed with abundant lymphoplasmacytic cells. Two cases (18.2%) belonged to the conventional-type IHCCs, showing irregularly shaped neoplastic glands and scattered lymphoplasmacytic infiltration. The remaining 8 cases (72.7%) belonged to the lymphoepithelioma-like cholangiocarcinomas (LELCCs), showing varied glandular differentiation and dense lymphoplasmacytic infiltration. The overall survival of EBV-positive IHCCs was not significantly different from that of EBV-negative IHCCs (P = 0.512).Our data demonstrate that EBV-associated IHCC is very rare and may be subclassified into 3 different pathological types including LELC, conventional-type IHCC and LELCC on the basis of the tumor cellular differentiation, and host cellular immune responses in the tumors. The etiological, clinical, pathological, and molecular features are needed to be future studied by multicentric efforts in recruiting more EBV-associated IHCC patients.

YAP Subcellular Localization and Hippo Pathway Transcriptome Analysis in Pediatric Hepatocellular Carcinoma.

Pediatric hepatocellular carcinoma (HCC) is a rare tumor which is associated with an extremely high mortality rate due to lack of effective chemotherapy. Recently, the Hippo pathway and its transcriptional co-activator Yes-associated protein (YAP) have been shown to play a role in hepatocyte proliferation and development of HCC in animal models. Therefore, we sought to examine the activity of YAP and the expression of Hippo pathway components in tumor and non-neoplastic liver tissue from 7 pediatric patients with moderately differentiated HCC. None of the patients had underlying cirrhosis or viral hepatitis, which is commonly seen in adults with HCC. This highlights a major difference in the pathogenesis of HCC between children and adults. We found a statistically significant increase in YAP nuclear localization in 100% of tumors. YAP target gene (CCNE1, CTGF, Cyr61) mRNA expression was also increased in the tumors that had the most significant increase in YAP nuclear localization. Based on Ki67 co-localization studies YAP nuclear localization was not simply a marker of proliferation. Our results demonstrate a clear increase in YAP activity in moderately differentiated pediatric HCC, providing evidence that it may play an important role in tumor survival and propagation.

Human growth hormone and human prolactin function as autocrine/paracrine promoters of progression of hepatocellular carcinoma

The death rates of hepatocellular carcinoma (HCC) are extremely high due to the paucity of therapeutic options. Animal models and anecdotal clinical evidence indicate a potential role of hGH and hPRL in HCC. However, the prognostic relevance and the functional role of tumor expression of these hormones in human HCC are not defined. Herein, we analyzed the mRNA and protein expression of hGH and hPRL in histopathological samples of non-neoplastic liver and HCC by in situ hybridization, PCR and immunohistochemistry techniques. Increased mRNA and protein expression of both hormones was observed in HCC compared with non-neoplastic liver tissues. hGH expression was significantly associated with tumor size and tumor grade. No significant association was observed between the expression of hPRL and any histopathological features. Amplification of both hGH and hPRL genes in HCC was observed when compared to non-neoplastic tissue. Expression of both hGH and hPRL was associated with worse relapse-free and overall survival in HCC patients. In vitro and in vivo functional assays performed with HCC cell lines demonstrated that autocrine expression of hGH or hPRL in HCC cells increased STAT3 activation, oncogenicity and tumor growth while functional antagonism with hGH-G120R significantly reduced these parameters. Hence, tumor expression of hGH/hPRL is associated with a worse survival outcome for patients with HCC and hGH/hPRL function as autocrine/paracrine promoters of HCC progression.

Distinct set of chromosomal aberrations in childhood hepatocellular carcinoma is correlated to hepatitis B virus infection

Hepatocellular carcinoma (HCC) is rarely observed in children and adolescents, but it is reported to be correlated with hepatitis B virus (HBV+) infections. This correlation is not easily explained, because in adults, HBV infections lead to the development of HCC only after decades, not within a few years. In HBV+ adulthood HCC, distinct chromosomal imbalances have been observed. Similar analyses have not been reported for childhood HCC. Here, we investigated whether chromosomal changes were associated with childhood HCC. We analysed formalin-fixed paraffin-embedded (FFPE) samples derived from 17 patients, 0-18 years old, who underwent partial hepatectomies due to HBV+ or HBV- associated HCC. In parallel, in 15 cases, we also analysed non-neoplastic liver tissues adjacent to the HCC. All samples were analysed with high resolution, microarray-based, comparative genomic hybridisation (aCGH). Overall, genomic aberrations in childhood HCC resembled those reported for adulthood HCC. In HBV+ HCC samples, chromosomes 1, 6, 7, 9, 17, 19, and 22 were significantly changed compared to those in HBV- HCC samples. Most interestingly, aberrations for chromosomes 7, 8, 9, 11, and 19 were also observed in corresponding non-neoplastic samples. A specific set of chromosomal abnormalities, including gains in chromosomes 8q, 9q, 11q, and 19, was significantly enriched in HBV+ compared to HBV- non-neoplastic tissues. In childhood HCC, HBV+ was correlated to increased chromosomal instability and specific chromosomal imbalances. A subset of aberrations might be essential in HCC carcinogenesis because they occurred in adjacent, non-neoplastic tissues. In particular, the gain in chromosome 19 appeared to be highly important.

Expression of Raf kinase inhibitor protein in human hepatoma tissues by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight methods.

Hepatocellular carcinoma (HCC) is the most common malignant liver tumor. To reduce the mortality and improve the effectiveness of therapy, it is important to search for changes in tumor-specific biomarkers whose function may involve in disease progression and which may be useful as potential therapeutic targets. Materials and Mehtods: In this study, we use two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to observe proteome alterations of 12 tissue pairs isolated from HCC patients: Normal and tumorous tissue. Comparing the tissue types with each other, 40 protein spots corresponding to fifteen differentially expressed between normal and cancer part of HCC patients. Raf kinase inhibitor protein (RKIP), an inhibitor of Raf-mediated activation of mitogen-activated protein kinase/extracellular signal-regulated kinase, may play an important role in cancer metastasis and cell proliferation and migration of human hepatoma cells. RKIP may be considered as a marker for HCC, because its expression level changes considerably in HCC compared with normal tissue. In addition, we used the methods of Western blotting and real time-polymerase chain reaction to analysis the protein expression and gene expression of RKIP. The result showed RKIP protein and gene expression in tumor part liver tissues of HCC patient is lower than peritumorous non-neoplastic liver tissue of the corresponding HCC samples. These results strongly suggest that RKIP may be considered to be a marker for HCC and RKIP are down-regulated in liver cancer cell.

Analysis of wedge resection of gallbladder bed and lymphadenectomy on adequate oncologic clearance for gallbladder cancer.

Surgery (R0 resection) is the mainstay of treatment of gallbladder cancer (GBC) as GBC is relatively resistant to currently known chemotherapy and radiotherapy regimens. to assess if wedge resection of the gallbladder bed achieves an adequate oncological clearance in GBC (namely T1 and T2) and some T3 GBC with minimal liver infiltration. Patients with GBC who underwent radical cholecystectomy (en bloc cholecystectomy, wedge resection of the gallbladder fossa with a ≥2 cm rim of nonneoplastic liver tissue, and regional lymph node dissection) between October 2012 and June 2015 after obtaining informed consent. Of thirty patients, mean age of 52 years, 5 had T1b, 13 T2, and 12 T3 GBC. R0 resection was achieved in all thirty GBC patients. Hepatic invasion was found in seven patients. The depth of hepatic invasion ranged from 0 to 9 mm. Follow-up ranged from a minimum of 12 to 43 months. Nineteen (63%) patients had N0 and 11 (37%) had N1 GBC. Total lymph node (TLND) count ranged from 1 to 12/patient with a median of 3. There was no local recurrence or systemic relapse of the disease. Wedge resection of the gallbladder bed achieves an adequate oncological clearance in early GBC. TLND counts remain poor even after a thorough standard lymph node dissection for resectable GBC.