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Background and Aims: Through an exploratory proteomic approach based on typical hepatocellular adenomas (HCAs), we previously identified a diagnostic biomarker for a distinctive subtype of HCA with high risk of bleeding, already validated on a multicenter cohort. We hypothesized that the whole protein expression deregulation profile could deliver much more informative data for tumor characterization. Therefore, we pursued our analysis with the characterization of HCA proteomic profiles, evaluating their correspondence with the established genotype/phenotype classification and assessing whether they could provide added diagnosis and prognosis values., Approach and Results: From a collection of 260 cases, we selected 52 typical cases of all different subgroups on which we built a reference HCA proteomics database. Combining laser microdissection and mass-spectrometry-based proteomic analysis, we compared the relative protein abundances between tumoral (T) and nontumoral (NT) liver tissues from each patient and we defined a specific proteomic profile of each of the HCA subgroups. Next, we built a matching algorithm comparing the proteomic profile extracted from a patient with our reference HCA database. Proteomic profiles allowed HCA classification and made diagnosis possible, even for complex cases with immunohistological or genomic analysis that did not lead to a formal conclusion. Despite a well-established pathomolecular classification, clinical practices have not substantially changed and the HCA management link to the assessment of the malignant transformation risk remains delicate for many surgeons. That is why we also identified and validated a proteomic profile that would directly evaluate malignant transformation risk regardless of HCA subtype., Conclusions: This work proposes a proteomic-based machine learning tool, operational on fixed biopsies, that can improve diagnosis and prognosis and therefore patient management for HCAs., (© 2021 by the American Association for the Study of Liver Diseases.)

Hepatocellular adenomas (HCAs) are rare benign tumors divided into three main subgroups defined by pathomolecular features, HNF1A (H-HCA), mutated β-catenin (b-HCA), and inflammatory (IHCA). In the case of unclassified HCAs (UHCAs), which are currently identified by default, a high risk of bleeding remains a clinical issue. The objective of this study was to explore UHCA proteome with the aim to identify specific biomarkers. Following dissection of the tumoral (T) and nontumoral (NT) tissue on formalin-fixed, paraffin-embedded HCA tissue sections using laser capture methodology, we performed mass spectrometry analysis to compare T and NT protein expression levels in H-HCA, IHCA, b-HCA, UHCA, and focal nodular hyperplasia. Using this methodology, we searched for proteins which are specifically deregulated in UHCA. We demonstrate that proteomic profiles allow for discriminating known HCA subtypes through identification of classical biomarkers in each HCA subgroup. We observed specific up-regulation of the arginine synthesis pathway associated with overexpression of argininosuccinate synthase (ASS1) and arginosuccinate lyase in UHCA. ASS1 immunohistochemistry identified all the UHCA, of which 64.7% presented clinical bleeding manifestations. Interestingly, we demonstrated that the significance of ASS1 was not restricted to UHCA, but also encompassed certain hemorrhagic cases in other HCA subtypes, particularly IHCA., Conclusion: ASS1 + HCA combined with a typical hematoxylin and eosin stain aspect defined a new HCA subgroup at a high risk of bleeding. (Hepatology 2017;66:2016-2028)., (© 2017 by the American Association for the Study of Liver Diseases.)

Phenotypic identification of focal nodular hyperplasia (FNH) and hepatocellular adenoma (HCA) subtypes using immunohistochemical markers has been developed from their molecular characteristics. Our objective was to evaluate the sensitivity of these markers in the definitive diagnosis of these lesions by core needle biopsies. A total of 239 needle biopsies paired with their surgical resection specimen (group A) or without an associated resection specimen (group B) were reviewed. Using a step-by-step algorithm after standard staining, appropriate immunostaining analyses were performed to determine the certainty of diagnosis of FNH, HNF1α-inactivated HCA, inflammatory HCA, β-catenin-activated HCA, or unclassified HCA. The diagnosis of FNH was certain or probable on routine stains in 53% of needle biopsies of group A, whereas after glutamine synthetase staining, the diagnosis was certain in 86.7% as compared with 100% on the corresponding surgical specimen (P=0.04). In needle biopsies of group A, the diagnosis of HCA was certain on routine stains in 58.6% as compared with 94.3% on surgical specimens. After specific immunostaining, diagnosis was established on biopsies with 74.3% certainty, including all HCA subtypes, with similar distribution in surgical specimens. For each "certain diagnosis" paired diagnostic test (biopsy and surgical specimen), a positive correlation was observed (P<0.001). No significant difference was observed between groups A and B for FNH (P=0.714) or for HCA subtypes (P=0.750). Compared with surgical specimens, immunohistochemical analysis performed on biopsies allowed the discrimination of FNH from HCA and the identification of HCA subtypes with good performance.

Telangiectatic hepatocellular adenoma is a rare, recently recognized subtype of hepatocellular adenoma that is often underrecognized by pathologists. We report a case of hepatocellular carcinoma arising within a pigmented telangiectatic hepatocellular adenoma in a noncirrhotic man with diffuse glutamine synthetase and nuclear β-catenin positivity. This case highlights malignant transformation of telangiectatic adenomas, and describes a previously unreported association between pigment deposition and telangiectatic adenoma. Radiology, gross pathology, and histopathology are shown. Review of the literature with attention to β-catenin and glutamine synthetase staining, malignant transformation, patient characteristics, the presence of Dubin-Johnson-like pigment, and genetic characteristics of telangiectatic adenomas are discussed.

Unlabelled: We took advantage of the reported genotype/phenotype classification to analyze our surgical series of hepatocellular adenoma (HCA). The series without specific known etiologies included 128 cases (116 women). The number of nodules varies from single, <5, and >or=5 in 78, 38, and 12 cases, respectively. The resection was complete in 95 cases. We identified 46 HNF1alpha-inactivated HCAs (44 women), 63 inflammatory HCAs (IHCA, 53 women) of which nine were also beta-catenin-activated, and seven beta-catenin-activated HCAs (all women); six additional cases had no known phenotypic marker and six others could not be phenotypically analyzed. Twenty-three of 128 HCAs showed bleeding. No differences were observed in solitary or multiple tumors in terms of hemorrhagic manifestations between groups. In contrast, differences were observed between the two main groups. Steatosis (tumor), microadenomas (resected specimen), and additional benign nodules were more frequently observed in HNF1alpha-inactivated HCAs (P < 0.01) than in IHCAs. Body mass index > 25, peliosis (tumor), and steatosis in background liver were more frequent in IHCA (P < 0.01). After complete resection, new HCAs in the centimetric range were more frequently found during follow-up (>1 year) in HNF1alpha-inactivated HCA. After incomplete resection (HCA left in nonresected liver), the majority of HCA remained stable in the two main groups and even sometimes regressed. Six patients of 128 developed hepatocellular carcinoma (HCC) (all were beta-catenin-activated, whether inflammatory or not)., Conclusion: There were noticeable clinical differences between HNF1alpha-inactivated HCA and IHCA; there was no increased risk of bleeding or HCC related to the number of HCAs; beta-catenin-activated HCAs are at higher risk of HCC. As a consequence, we believe that management of HCA needs to be adapted to the phenotype of these tumors.

Agrin is a recently identified proteoglycan component of vascular and bile duct basement membranes in the liver. The selective deposition of agrin in hepatocellular carcinoma (HCC) microvessels versus sinusoidal walls prompted us to investigate the utility of agrin immunohistochemistry (IHC) in detecting malignant hepatocellular lesions. We focused on the differential diagnostic problems often presented by hepatocellular adenomas (HCAs) and dysplastic nodules. IHC for agrin was performed on 138 formalin-fixed, paraffin-embedded surgical specimens from 93 patients, including cirrhotic liver tissues (25), focal nodular hyperplasia (10), large regenerative nodules (8), low-grade (23) and high-grade (7) dysplastic nodules, small HCC (8), HCC (27), and HCA (30). Agrin immunostaining was compared with that of CD34 and, in selected cases, to glypican-3. The combination of agrin and CD34 sensitively (0.94) and specifically (0.93) identified lesions judged previously as malignant by histology. The majority of benign lesions were clearly agrin-negative, whereas the strength and extent of agrin IHC faithfully reflected dysplasia in "atypical" HCAs and in high-grade dysplastic nodules. Malignant lesions were uniformly positive. In conclusion, as agrin is highly selective for tumor blood vessels, IHC for agrin facilitates the discrimination of benign and malignant hepatocellular lesions. Moreover, whereas glypican-3 in some HCCs may appear in few scattered cells only, agrin is diffusely deposited in virtually all malignant lesions, which may prove advantageous in the evaluation of small specimens such as core biopsies.

Unlabelled: Hepatocellular carcinomas (HCCs) and bile duct carcinomas (BDCs) have a poor prognosis. Therefore, surveillance strategies including sensitive and specific serum markers for early detection are needed. Recently, Golgi Phosphoprotein 2 (GOLPH2) has been proposed as a serum marker for HCC, but GOLPH2 expression data in liver tissues was not available. Using tissue microarrays and immunohistochemistry, we semiquantitatively analyzed GOLPH2 protein expression in patients with HCC (n = 170), benign liver tumors (n = 22), BDC (n = 114) and normal liver tissue (n = 105). A newly designed sandwich enzyme-linked immunoassay (ELISA) was used to analyze GOLPH2 levels in the sera of patients with HCC (n = 62), hepatitis C virus (HCV) (n = 29), BDC (n = 10), and healthy control persons (n = 12). By immunohistochemistry 121/170 (71%) of HCC showed strong GOLPH2 expression, which was significantly associated with a higher tumor grade (P = 0.01). A total of 97/114 (85%) BDCs showed a strong GOLPH2 expression which proved to be an independent prognostic factor for overall survival (P < 0.05). Serum levels of GOLPH2 measured by ELISA were significantly elevated in patients with HCC with underlying HCV infection (median 18 mg/L, P < 0.05) and patients with BDC (median = 14.5 mg/L, P < 0.01) in comparison to healthy controls (median 4 mg/L)., Conclusion: GOLPH2 protein is highly expressed in tissues of HCC and BDC. GOLPH2 protein levels are detectable and quantifiable in sera by ELISA. In patients with hepatitis C, serial ELISA measurements in the course of the disease appear to be a promising complementary serum marker in the surveillance of HCC. GOLPH2 should be further evaluated as a serum tumor marker in BDC on a larger scale.

Unlabelled: Hepatocellular adenomas (HCAs) are a group of benign tumors forming three molecular pathological subgroups: (1) hepatocyte nuclear factor 1alpha (HNF-1alpha)-inactivated, (2) beta-catenin-activated, and (3) inflammatory. Some HCAs present both beta-catenin activation and inflammation. We analyzed magnetic resonance imaging (MRI) data for correlations between features on imaging and pathological classification of HCAs. We included 50 cases for which pathology specimens were classified into three groups based on immunohistochemical staining. Two characteristic MRI profiles were identified corresponding to HNF-1alpha-inactivated and inflammatory HCAs. Fifteen HCAs were HNF-1alpha-inactivated. The corresponding lesions showed (1) diffuse signal dropout on T1-weighted chemical shift sequence due to steatosis, (2) isosignal or slight hypersignal on T2-weighted (T2W) images, and (3) moderate enhancement in the arterial phase, with no persistent enhancement in the portal venous and delayed phases. For the diagnosis of HNF-1alpha-inactivated HCA, the positive predictive value of homogeneous signal dropout on chemical shift images was 100%, the negative predictive value was 94.7%, the sensitivity was 86.7%, and the specificity was 100%. Twenty-three HCAs were inflammatory and showed (1) an absence or only focal signal dropout on chemical shift sequence; (2) marked hypersignal on T2W sequences, with a stronger signal in the outer part of the lesions, correlating with sinusoidal dilatation areas; and (3) strong arterial enhancement, with persistent enhancement in the portal venous and delayed phases. Marked hypersignal on T2W sequences associated with delayed persistent enhancement had a positive predictive value of 88.5%, a negative predictive value of 84%, a sensitivity of 85.2%, and a specificity of 87.5% for the diagnosis of inflammatory HCA., Conclusion: HNF-1alpha-mutated HCAs and inflammatory HCAs were associated with specific MRI patterns related to diffuse fat repartition and sinusoidal dilatation, respectively.

It is often difficult to distinguish hepatocellular adenoma (HCA) from well-differentiated hepatocellular carcinoma (WDHCC) when limited tissue from a needle biopsy is evaluated. The aim of this study was to identify gene expression patterns that can distinguish HCA from WDHCC, with the ultimate goal of discovering novel diagnostic markers. Gene expression profile analysis was performed using Affymetrix U133Plus2 GeneChip microarrays on RNA isolated from frozen tissue of 6 HCA and 8 WDHCC specimens. Statistical analysis of microarray data identified 63 genes whose expression levels were significantly different between HCA and WDHCC. These included 57 genes overexpressed by HCA and 6 overexpressed by WDHCC. Eight genes were chosen for further analysis by quantitative RT-PCR on RNA derived from archived, paraffin-embedded tissue blocks of an independent validation set comprising 9 HCAs and 9 HCCs. Seven of the 8 genes demonstrated average expression differences between HCA and HCC that were concordant with the microarray findings, and their expression pattern correctly classified the 18 tumors into HCA and HCC using unsupervised clustering analysis. Furthermore, immunohistochemical staining performed on a third, independent set of 27 HCAs and 33 HCCs confirmed the expression differences at protein levels for 5 of the genes. Taken together, our data demonstrate significant molecular differences between HCA and WDHCC, despite their morphologic similarity. More importantly, we have identified a unique set of genes whose expression pattern can discriminate between these two types of hepatocellular neoplasms, suggesting the possibility of future development of ancillary molecular and immunohistochemical diagnostic methods.

Hepatitis B virus (HBV) transgenic mice (official designation, Tg [Alb-1 HBV] Bri 44) invariably develop macroscopically evident tumors within the 20th month of life. Sustained proliferative activity seems to play an important role in the development of these lesions. We previously showed that ursodeoxycholate (UDC) stimulates hepatocyte proliferation in various experimental settings. Herein, we tested the assumption that biological factors able to further increase liver cell proliferation, such as UDC, could accelerate tumor development in this animal model. For this study, 22 eight-week-old male transgenic mice were divided into 2 groups; 11 animals received a standard diet, and 11 received a UDC-enriched diet. The 2 groups were further divided into 2 subgroups of 5 and 6 animals each and were sacrificed at 3 and 15 months of age, respectively. These different times were chosen to exclude diet-related toxicity (in 3-month-old mice) and evaluate tumor growth (in 15-month-old mice). In addition, hepatocyte proliferation was assessed in all animals. In 3-month-old mice receiving UDC, cholestatic and cytolytic indices as well as liver histology were comparable to those in controls. At 15 months, all UDC-treated mice showed large multinodular tumors whereas only 33% of controls developed smaller uninodular neoplasms. Hepatocyte proliferation was increased in all animals receiving UDC compared with controls. In conclusion, the increase in serum UDC (undetectable in mice fed a standard diet), in the absence of any toxic effect on the liver, suggests the involvement of this bile salt in the stimulation of hepatocyte proliferation and tumor growth.

Hepatocellular adenoma (HA) and focal nodular hyperplasia (FNH) are 2 rare, benign liver neoplasms that often are discovered incidentally. To date, few genetic changes have been found in these 2 benign lesions. However, the 2 pathways of p53 and Wnt signaling, which may be the most common molecular targets involved in liver tumorgenesis, were studied in HA and FNH. Ten HAs and 11 FNHs were analyzed for loss of heterozygosity (LOH) and sequencing analysis of mutation hot spots in exons 5 to 8 of the p53 gene. No LOH or mutant sequences were identified, indicating that p53 was not associated with these benign lesions. Genes in the Wnt signaling pathway, including beta-catenin, axin, and adenomatous polyposis coli (APC), also were studied. Polymerase chain reaction (PCR) amplification and direct sequencing of all samples of HA and FNH displayed no mutations in exon 3 of the beta-catenin gene. However, 3 HAs (30%) contained interstitial deletions from exon 3 to exon 4. Truncated forms of beta-catenin detected by Western blot and immunohistochemical analyses showed they had accumulated in the cytoplasm and nuclei. However, for the axin and APC genes, no genetic changes, including allelic loss, interstitial deletions and point mutations, were detected in any of the HAs and FNHs. In conclusion, beta-catenin, which participates in the Wnt signaling pathway, might play a more important role in the formation of HA than in that of FNH, but p53 is not associated with the development of either HA or FNH.

Hepatic silicosis, cirrhosis, liver cell adenoma, and carcinomas developed in nude mice (NCr-Nu) given quartz by the subcutaneous and intraperitoneal routes. Syrian golden hamsters (15:16 EHS:cr) given quartz by both routes developed extensive fibrosis and cirrhosis and had higher morbidity and mortality rates after 3 months. Crystalline silica (quartz) induces fibrosis, adenomas, and carcinomas in the lungs of Fisher 344 rats, but certain strains of mice and hamsters are resistant to quartz-induced pulmonary carcinogenesis. Pulmonary fibrosis, however, is minimal in mice and absent in hamsters who received quartz intratracheally. To determine whether species differences are due to organ-specific rather than species-specific factors, susceptibility of the liver to quartz toxicity was investigated in nude mice and hamsters. The present study shows that the differential manifestations of quartz toxicity by these rodent species are dependent on factors that are organ-specific rather than host-specific. At 3 months, hepatocytes in mice were immunostained with intracellular transforming growth factor (TGF) beta 1 (LC 1-30) but not with TGF-beta 1 latency-associated peptide (LAP) protein (266-278); at 12 months, hepatocytes were immunostained with TGF-beta 1 LAP (266-278) but not with TGF-beta 1 (LC1-30). The hepatocytes of hamsters at 3 months showed immunoreactivities to TGF-beta 1 LAP (266-278) and TGF-beta 1 (LC1-30); immunostaining to TGF-beta 1 (LC1-30) was detected in nonparenchymal cells. Extracellular TGF-beta 1 (CC1-30) was detected in the silicotic granulomas and fibrous tissue in livers of both species. Quartz-induced liver carcinoma did not express TGF-beta 1 LAP (266-278) and LC (1-30) proteins, but these were detected in the cells of the adenoma in the same liver. Control animals showed no hepatic lesions nor immunoreactivity to TGF-beta 1. The spatial and temporal patterns of expression of TGF-beta 1, TGF-beta 2, TGF-beta receptor type II messenger RNAs (mRNAs), and TGF-beta 1 proteins in the different hepatic lesions suggests that TGF-beta isoforms may play a role in the pathogenesis of quartz-induced fibrosis, cirrhosis, liver cell adenoma, and carcinoma.