Glutamine Synthetase Staining Research Articles

Dynamics of glutamine synthetase expression in hepatic ischemia-reperfusion injury: Implications for therapeutic interventions.

Hepatic ischemia-reperfusion injury (IRI) poses a great challenge in liver surgery and transplantation because of oxidative stress and inflammatory responses. The changes in glutamine synthetase (GS) expression during hepatic IRI remain unclear. To investigate the dynamic expression of GS during hepatic IRI. Following hepatic ischemia for 1 h and reperfusion, liver tissue samples were collected at 0.5, 6, and 24 hours postreperfusion for fixation, embedding, sectioning. Hematoxylin and eosin staining and GS staining were performed. GS expression rapidly decreases in hepatocytes around the central vein after IRI, reaching its lowest point at 6 hours postreperfusion, and then gradually recovers. GS is highly sensitive to IRI, highlighting its potential role as an indicator of liver injury states and a target for therapeutic intervention.

Evaluation of the histologic and immunohistochemical (CD34, glutamine synthetase) findings in idiopathic non-cirrhotic portal hypertension (INCPH)

AimIdiopathic non-cirrhotic portal hypertension (INCPH) is a vascular disorder of uncertain origin. Diagnosis can be challenging on liver biopsy. Despite diverse histomorphologic findings documented in literature, studies on the frequency of these findings are lacking. This study aims to assess both the histomorphologic features and the immunoexpression patterns of CD34 and glutamine synthetase (GS) in liver biopsies and searched for their contribution to the pathologic diagnosis of INCPH.Materials and methodsHematoxylin–eosin, CD34, and GS-stained liver needle biopsy sections of 16 patients clinically diagnosed with INCPH were retrospectively analyzed. Histologic findings such as portal vein narrowing, obliteration, or loss were grouped as major findings, while portal vein herniation, hypervascularized portal tracts, and periportal abnormal vessels were grouped as minor findings, and their frequency were evaluated. Periportal endothelial CD34 stained areas were measured via ocular micrometer. The distribution of GS immunoexpression was evaluated. Eighteen healthy liver donor biopsies were evaluated as controls.ResultsIn INCPH cases, 58% of portal tracts showed major findings, compared to 15% in the control group (p < 0.001). Minor findings were observed in 16% of INCPH cases and 7% of controls (p = 0.014). The number of portal tracts with histologic findings is significantly higher in INCPH than in control liver biopsies. Abnormal portal tract distribution, like being close to each other, was seen in 75% of INCPH cases but not in controls (p < 0.001). Nodular regenerative hyperplasia (NRH) was present in 31% of cases. Periportal CD34 expression was higher in INCPH, and affected areas were larger than in controls (p < 0.001). Irregular GS staining, i.e. GS staining with patchy distribution in zone 3, and/or periportal and zone 2 hepatocytes, was found in 62% of INCPH cases, while controls showed the usual pattern (p < 0.001).ConclusionIn the biopsy diagnosis of INCPH, in addition to the presence of major histologic findings and the amount of portal tracts displaying these features, the expression of endothelial CD34 in periportal areas, and irregular hepatocellular GS expression can also be considered as supporting feature.

MiR-26a-5p Attenuates Oxidative Stress and Inflammation in Diabetic Retinopathy through the USP14/NF-κB Signaling Pathway.

Diabetic retinopathy (DR) is an ocular disease caused by diabetes and may lead to vision impairment and even blindness. Oxidative stress and inflammation are two key pathogenic factors of DR. Recently, regulatory roles of different microRNAs (miRNAs) in DR have been widely verified. miR-26a-5p has been confirmed to be a potential biomarker of DR. Nevertheless, the specific functions of miR-26a-5p in DR are still unclear. Primary cultured mouse retinal Müller cells in exposure to high glucose (HG) were used to establish an in vitro DR model. Müller cells were identified via morphology observation under phase contrast microscope and fluorescence staining for glutamine synthetase. The in vivo animal models for DR were constructed using streptozotocin-induced diabetic C57BL/6 mice. Western blotting was performed to quantify cytochrome c protein level in the cytoplasm and mitochondria of Müller cells and to measure protein levels of glial fibrillary acidic protein (GFAP), ubiquitin-specific peptidase 14 (USP14), as well as factors associated with NF-κB signaling (p-IκBα, IκBα, p-p65, and p65) in Müller cells or murine retinal tissues. ROS production was detected by CM-H2DCFDA staining, and the concentration of oxidative stress markers (MDA, SOD, and CAT) was estimated by using corresponding commercial kits. Quantification of mRNA expression was conducted by RT-qPCR analysis. The concentration of proinflammatory factors (TNF-α, IL-1β, and IL-6) was evaluated by ELISA. Hematoxylin-eosin staining for murine retinal tissues was performed for histopathological analysis. Immunofluorescence staining was conducted to determine NF-κB p65 nuclear translocation in Müller cells. Furthermore, the interaction between miR-26a-5p and USP14 was verified via the luciferase reporter assays. HG stimulation contributed to Müller cell dysfunction by inducing inflammation, oxidative injury, and mitochondrial damage to Müller cells. miR-26a-5p was downregulated in Müller cells under HG condition, and overexpression of miR-26a-5p relieved HG-induced Müller cell dysfunction. Moreover, miR-26a-5p targeted USP14 and inversely regulated USP14 expression. Additionally, HG-evoked activation of NF-κB signaling was suppressed by USP14 knockdown or miR-26a-5p upregulation. Rescue assays showed that the protective impact of miR-26a-5p upregulation against HG-induced Müller cell dysfunction was reversed by USP14 overexpression. Furthermore, USP14 upregulation and activation of NF-κB signaling in the retinas of DR mice were detected in animal experiments. Injection with miR-26a-5p agomir improved retinal histopathological injury and weakened the concentration of proinflammatory cytokines and oxidative stress markers in the retinas of DR mice. miR-26a-5p inhibits oxidative stress and inflammation in DR progression by targeting USP14 and inactivating the NF-κB signaling pathway.

Focal nodular hyperplasia–like nodules arising in the setting of hepatic vascular disorders with portosystemic shunting show β-catenin activation

Hepatocellular nodules can develop in the setting of chronic hepatic vascular disorders including those characterized by portosystemic shunts such as Abernethy malformation and post-Fontan procedure. The nodules can range from benign lesions such as regenerative nodules, focal nodular hyperplasia (FNH), and hepatocellular adenoma (HCA) to malignant neoplasms such as hepatocellular carcinoma (HCC). In many instances, these nodules are difficult to place into well-defined categories based on radiologic or histologic features. Nodular lesions that resemble FNH are common in this context and have been described as FNH-like nodules, the nature of which is not well-established. This study examines 6 liver resections from patients with vascular disease characterized by portosystemic shunts. A wide range of nodules were present in these cases, including regenerative nodules (n=2), FNH and FNH-like (n=30), HCA (n=10), HCA-like (n=13), and HCC (n=2). Six nodules from 3 patients were categorized as FNH-like due to one or more features such as nodular architecture, fibrous septa, and ductular reaction, but lack of typical map-like glutamine synthetase (GS) staining. Further characterization of these 6 FNH-like nodules showed diffuse GS staining in all nodules (3 diffuse homogeneous, 3 diffuse heterogeneous). Targeted next-generation sequencing identified CTNNB1 alterations in all tested FNH-like nodules (n=4). These results indicate that FNH-like nodules in the setting of chronic hepatic vascular disorders can be neoplastic. Since the presence of β-catenin activation portends a potential risk for malignant progression, GS and β-catenin immunohistochemistry should be obtained in all cases showing FNH-like morphology, with molecular analysis performed in cases with indeterminate staining pattern.

Lipid Droplet-Associated Proteins Perilipin 1 and 2: Molecular Markers of Steatosis and Microvesicular Steatotic Foci in Chronic Hepatitis C

Chronic infection with hepatitis C (HCV) is a major risk factor in the development of cirrhosis and hepatocellular carcinoma. Lipid metabolism plays a major role in the replication and deposition of HCV at lipid droplets (LDs). We have demonstrated the importance of LD-associated proteins of the perilipin family in steatotic liver diseases. Using a large collection of 231 human liver biopsies with HCV, perilipins 1 and 2 have been localized to LDs of hepatocytes that correlate with the degree of steatosis and specific HCV genotypes, but not significantly with the HCV viral load. Perilipin 1- and 2-positive microvesicular steatotic foci were observed in 36% of HCV liver biopsies, and also in chronic hepatitis B, autoimmune hepatitis and mildly steatotic or normal livers, but less or none were observed in normal livers of younger patients. Microvesicular steatotic foci did not frequently overlap with glycogenotic/clear cell foci as determined by PAS stain in serial sections. Steatotic foci were detected in all liver zones with slight architectural disarrays, as demonstrated by immunohistochemical glutamine synthetase staining of zone three, but without elevated Ki67-proliferation rates. In conclusion, microvesicular steatotic foci are frequently found in chronic viral hepatitis, but the clinical significance of these foci is so far not clear.

Spontaneous Occurrence of Various Types of Hepatocellular Adenoma in the Livers of Metabolic Syndrome-Associated Steatohepatitis Model TSOD Mice.

Male Tsumura-Suzuki Obese Diabetes (TSOD) mice, a spontaneous metabolic syndrome model, develop non-alcoholic steatohepatitis and liver tumors by feeding on a standard mouse diet. Nearly 70% of liver tumors express glutamine synthetase (GS), a marker of hepatocellular carcinoma. In contrast, approximately 30% are GS-negative without prominent nuclear or structural atypia. In this study, we examined the characteristics of the GS-negative tumors of TSOD mice. Twenty male TSOD mice were sacrificed at 40 weeks and a total of 21 tumors were analyzed by HE staining and immunostaining of GS, liver fatty acid-binding protein (L-FABP), serum amyloid A (SAA), and beta-catenin. With immunostaining for GS, six (29%) tumors were negative. Based on the histological and immunohistological characteristics, six GS-negative tumors were classified into several subtypes of human hepatocellular adenoma (HCA). One large tumor showed generally similar findings to inflammatory HCA, but contained small atypical foci with GS staining and partial nuclear beta-catenin expression suggesting malignant transformation. GS-negative tumors of TSOD mice contained features similar to various subtypes of HCA. Different HCA subtypes occurring in the same liver have been reported in humans; however, the diversity of patient backgrounds limits the ability to conduct a detailed, multifaceted analysis. TSOD mice may share similar mechanisms of HCA development as in humans. It is timely to review the pathogenesis of HCA from both genetic and environmental perspectives, and it is expected that TSOD mice will make further contributions in this regard.

Hepatocellular neoplasms with loss of liver fatty acid binding protein: Clinicopathologic features and molecular profiling

HNF1A-inactivated hepatocellular adenomas (H-HCA) show steatosis, no atypia and loss of liver fatty acid binding protein (LFABP). LFABP loss also occurs in hepatocellular carcinoma (HCC). This study examines 68 LFABP-negative tumors: 33 typical H-HCA, 10 atypical hepatocellular neoplasms (AHN), 7 well-differentiated (WD) HCC, 18 moderately or poorly differentiated (MD/PD) HCC. Capture based sequencing was performed in 13 cases (8 AHN, 5 WD-HCC). Patients with HCA, AHN and WD-HCC were nearly all women. AHN and WD-HCC resembled H-HCA but had higher degree of atypia and/or reticulin loss. Variant features like inconspicuous fat (59% vs. 12%, p=0.03), predominance of eosinophilic cells (59% vs. 21%, p=0.01) and pseudoacini were more common in AHN and WD-HCC. Myxoid change and prominent lipofuscin were more common in WD-HCC (29% each) than H-HCA and AHN combined (2% and 7% respectively). Compared to WD-HCC, LFABP-negative MD/PD HCC were more commonly associated with male gender, viral hepatitis and cirrhosis. Biallelic HNF1A alterations were seen in all 13 (100%) sequenced cases. Additional mutations and/or copy number alterations were observed in 38% of AHN and 100% of WD-HCC. Diffuse glutamine synthetase (GS) staining was seen in 13% of cases, with no nuclear β-catenin or Wnt signaling alterations. In conclusion, variant features such as lack of fat, peliosis, myxoid change, pseudoacini and abundant lipofuscin are more common in AHN and/or WD-HCC. LFABP-negative MD/PD HCC have different clinicopathologic features compared to WD-HCC. The significance of diffuse GS in a subset of these cases is unclear.

Diagnostic challenges of focal nodular hyperplasia: interobserver variability, accuracy, and the utility of glutamine synthetase immunohistochemistry.

The diagnosis of focal nodular hyperplasia (FNH) and the interpretation of glutamine synthetase (GS) staining can be challenging on biopsies. We aimed to evaluate the reproducibility of needle biopsy diagnosis of FNH, the effect of GS immunohistochemistry on FNH diagnosis, and which histological features are most useful for the diagnosis of FNH. The study included virtual needle biopsies generated from 75 resection specimens (30 FNHs, 15 hepatocellular adenomas, 15 hepatocellular carcinomas, and 15 non-lesional liver specimens). Pathologists were reasonably accurate (83.1%) in the diagnosis of FNH with haematoxylin and eosin alone. Ductular reaction and nodularity had the highest sensitivity for a diagnosis of FNH (88.1% and 82.2%, respectively), whereas central scar was the most specific feature (90.6%). The presence of two or more of the classic histological features had 89.6% sensitivity and 86.2% specificity for a diagnosis of FNH. Diagnostic accuracy was significantly higher with the addition of a GS stain. A map-like GS staining pattern was highly specific (99.3%) for FNH. However, GS staining was interpreted as non-map-like in 14.4% of reviews of true FNH cases, and overall interobserver agreement for interpretation of the GS staining pattern was only moderate (kappa=0.42). Pathologists are reasonably accurate in the diagnosis of FNH on virtual biopsies, and GS staining improves accuracy. However, a subset of FNH cases remain challenging. Steatosis and a pseudo-map-like GS staining pattern were associated with increased difficulty. Therefore, although a map-like GS staining pattern is useful for confirmation of a diagnosis, the lack of a map-like GS staining pattern on needle biopsy does not necessarily exclude a diagnosis of FNH.

Application of Immunohistochemistry in the Pathological Diagnosis of Liver Tumors

Although radiological diagnostics have been progressing, pathological diagnosis remains the most reliable method for diagnosing liver tumors. In some cases, definite pathological diagnosis cannot be obtained by histological evaluation alone, especially when the sample is a small biopsy; in such cases, immunohistochemical staining is very useful. Immunohistochemistry is the most frequently used technique for molecular pathological diagnosis due to its broad application, ease of performance and evaluation, and reasonable cost. The results occasionally reflect specific genetic mutations. The immunohistochemical markers of hepatocellular carcinoma include those of hepatocellular differentiation—such as hepatocyte paraffin 1 and arginase-1—and those of malignant hepatocytes—such as glypican-3, heat shock protein 70, and glutamine synthetase (GS). To classify the subtypes of hepatocellular adenoma, examination of several immunohistochemical markers, such as liver fatty acid-binding protein, GS, and serum amyloid A, is indispensable. Immunohistochemical staining for GS is also important for the diagnosis of focal nodular hyperplasia. The representative immunohistochemical markers of intrahepatic cholangiocarcinoma include cytokeratin (CK) 7 and CK19. In this article, we provide an overview of the application of immunohistochemistry in the pathological diagnosis of liver tumors referring to the association with genetic alterations. Furthermore, we aimed to explain the practical points in the differential diagnosis of liver tumors by immunohistochemical staining.

Cytoarchitectural analysis of the neuron-to-glia association in the dorsal root ganglia of normal and diabetic mice.

Dorsal root ganglia (DRGs) host the somata of sensory neurons which convey information from the periphery to the central nervous system. These neurons have heterogeneous size and neurochemistry, and those of small-to-medium size, which play an important role in nociception, form two distinct subpopulations based on the presence (peptidergic) or absence (non-peptidergic) of transmitter neuropeptides. Few investigations have so far addressed the spatial relationship between neurochemically different subpopulations of DRG neurons and glia. We used a whole-mount mouse lumbar DRG preparation, confocal microscopy and computer-aided 3D analysis to unveil that IB4+ non-peptidergic neurons form small clusters of 4.7±0.26 cells, differently from CGRP+ peptidergic neurons that are, for the most, isolated (1.89±0.11 cells). Both subpopulations of neurons are ensheathed by a thin layer of satellite glial cells (SGCs) that can be observed after immunolabeling with the specific marker glutamine synthetase (GS). Notably, at the ultrastructural level we observed that this glial layer was discontinuous, as there were patches of direct contact between the membranes of two adjacent IB4+ neurons. To test whether this cytoarchitectonic organization was modified in the diabetic neuropathy, one of the most devastating sensory pathologies, mice were made diabetic by streptozotocin (STZ). In diabetic animals, cluster organization of the IB4+ non-peptidergic neurons was maintained, but the neuro-glial relationship was altered, as STZ treatment caused a statistically significant increase of GS staining around CGRP+ neurons but a reduction around IB4+ neurons. Ultrastructural analysis unveiled that SGC coverage was increased at the interface between IB4+ cluster-forming neurons in diabetic mice, with a 50% reduction in the points of direct contacts between cells. These observations demonstrate the existence of a structural plasticity of the DRG cytoarchitecture in response to STZ.

Glutamine Synthetase and Cytochrome P‐4502E1 Characterize Metabolic Zonation in Human Liver: Immunohistochemical Study

Hepatic metabolic zonation endows metabolic homeostasis and proper functioning of the liver under various physiological conditions. The zonation reflects the heterogeneity of gene expression and functions of hepatocytes along the centro‐portal axis of the liver lobule. The Wnt/ß‐catenin signaling pathway is the master regulator of hepatic zonation. The pathway maintains the metabolic zonation of glutamine synthetase (GS) and cytochrome P‐4502E1 (2E1) —among others—in the centrilobular parenchyma. GS detoxifies ammonia entering the liver as well as serves as an immunohistological marker of perivenous hepatocytes that make up the first three cell layers around the central vein. 2E1 mediates metabolism of xenobiotics and has a broader centrilobular expression than that of GS. While the zonation of GS and 2E1 has been documented in animal models, the phenomenon remains to be evaluated in human liver. This study assessed expression of GS and 2E1 in human cadaveric livers by immunohistochemistry. Ten livers of cadavers (mean age, 79.9 years) with minimal fibrotic changes were studied. Consecutive paraffin liver sections were stained with a polyclonal GS antibody or 2E1 antibody using an immunoperoxidase method. The central vein was defined as a hepatic vein located at the center of the classic liver lobule and showed a cross‐sectional diameter not exceeding 150 μm. In each liver, five to six veins were examined for the distribution of GS+ hepatocytes in the centrilobular area. In most cases, GS+ hepatocytes were arranged in a band of 1 to 3 cells wide in the perivenous area around the central veins (perivenous cells). In some instances, GS staining was observed in hepatocytes beyond the perivenous area (recorded as non‐perivenous cells). Midlobular and periportal hepatocytes lacked GS staining. Expressed as per central vein, counts of perivenous GS+ cells were 14.9 ± 2.2 and non‐perivenous cells were 5.6 ± 1.8. The mean 2E1 zonal score was 1.35 ± 0.35. Total GS+ cell numbers (perivenous plus non‐perivenous) were found to correlate with 2E1 zonal scores (P &gt; 0.05, N = 10).ConclusionsExpression of GS and 2E1 characterizes the metabolic zonation in the liver of aged cadavers, in accordance with the findings of animal studies. The correlation of GS with 2E1 expression suggests a link between the hepatic zonation of GS and 2E1, which is consistent with the notion that the zonation is mediated by the Wnt/ß‐catenin signaling pathway.

Hepatocellular adenoma in Taiwan: Distinct ensemble of male predominance, overweight/obesity, and inflammatory subtype.

The clinicopathologic features of hepatocellular adenoma in Asian populations have been poorly defined. The study aimed to characterize this rare entity in a single institution in Taiwan. In total, 45 hepatocellular adenomas from 1995 to 2018 were included and sent for pathologic review and molecular subtyping. The numbers of patients with hepatocellular adenoma has doubled in the recent decade. Surprisingly, men outnumbered women in our cohort (n=26, 58% vs N=19, 42%). A collection of clinical information revealed that overweight/obesity accounts for most of the associated conditions of hepatocellular adenoma. Only three women took oral contraceptives. There were 34 inflammatory (75%), three LFABP-negative (7%), four β-catenin activated (9%), and four unclassified (9%) hepatocellular adenomas. Ten inflammatory hepatocellular adenomas demonstrated strong and homogeneous glutamine synthetase staining and were thus also β-catenin activated. Notably, overweight and obesity were significantly associated with inflammatory hepatocellular adenoma than other subtypes (P=.029 and .056, respectively) and were strongly correlated with steatosis in background liver (P=.028 and.007, respectively). Malignant transformation (four borderline tumors and two hepatocellular carcinomas) was identified in six adenomas (two women and four men). All six hepatocellular adenomas with malignancy were β-catenin activated; β-catenin activation could serve as a biomarker for malignant progression. The clinicopathologic features of hepatocellular adenoma in Taiwan are distinct from those reported in Western countries. Rare oral contraceptive usage and an emerging epidemic of overweight/obesity in Taiwan provides new insights into the pathogenesis of hepatocellular adenoma.

Genomic profiling of well-differentiated hepatocellular neoplasms with diffuse glutamine synthetase staining reveals similar genetics across the adenoma to carcinoma spectrum

Well-differentiated hepatocellular neoplasms are currently classified in the World Health Organization scheme as hepatocellular adenoma or hepatocellular carcinoma. There is no recognized diagnostic category for atypical cases with borderline features, and we have designated these as atypical hepatocellular neoplasms. Diffuse glutamine synthetase staining is used as a surrogate marker to detect β-catenin activation, a well-recognized high risk feature in hepatocellular tumors. This study examined 27 well-differentiated hepatocellular neoplasms with diffuse glutamine synthetase staining, including 7 atypical hepatocellular neoplasms with no cytoarchitectural atypia, 6 atypical hepatocellular neoplasms with focal cytoarchitectural atypia, and 14 well-differentiated hepatocellular carcinomas. Capture-based next-generation sequencing was performed, and alterations in WNT pathway genes (CTNNB1, APC, AXIN1) were seen in 81% of cases (10/13 atypical hepatocellular neoplasms and 12/14 of hepatocellular carcinomas), while the molecular basis of diffuse glutamine synthetase staining was unclear in the remaining 19% of cases. Additional non-WNT pathway mutations (TP53, TSC1, DNMT3A, CREBBP) or copy number alterations were present in 56% of atypical hepatocellular neoplasms, with no significant difference in cases with or without focal cytoarchitectural atypia, supporting that all cases with β-catenin activation should be classified as atypical irrespective of atypia. Atypical hepatocellular neoplasm and hepatocellular carcinoma also demonstrated largely similar genomic profiles, but TERT promoter mutations were restricted to hepatocellular carcinoma (21%) and copy number alterations were more common in hepatocellular carcinoma (64 vs 31%). Mutational and copy number analysis may be helpful in characterization and risk stratification of atypical hepatocellular neoplasms when morphology and glutamine synthetase staining yield ambiguous results.

Nodular Regenerative Hyperplasia: Expression Pattern of Glutamine Synthetase and a Potential Role for Hepatic Progenitor Cells.

Nodular regenerative hyperplasia (NRH) is one of the most frequent causes of noncirrhotic intrahepatic hypertension, but is a difficult histologic diagnosis. The expression of glutamine synthetase (GS) and cytokeratin 7 (CK7) has been reported to be increased in other regenerative/vascular conditions, while CK7 and BerEP4 are also markers of hepatic progenitor cells. The aims of this study were to investigate the use of GS, CK7, and BerEP4 as the potential markers for NRH. This is a retrospective case series of NRH at Centre Hospitalier de l'Universite de Montreal between 1993 and 2013. Normal liver from partial hepatectomies for tumors were used as controls. GS, CK7, CK19, and BerEP4 immunohistochemical stains were performed on all specimens. Immunohistochemical staining patterns were scored from 0 to 3+. NRH was identified in 46 samples obtained from 26 patients. Liver chemistry profile was cholestatic in 45% of the patients. In 93% of the NRH cases, there was abnormal zone 2 expression of GS. Weak panacinar GS staining was seen in all the NRH cases. Aberrant CK7 expression was present in all cases of NRH, but were not associated with cholestasis. BerEP4 was overexpressed in 47% of the NRH cases (P<0.05); all cases with diffuse BerEP4 staining also showed extensive CK7 expression (P<0.01). NRH showed increased immunohistochemical GS staining that may support its morphologic diagnosis. Our findings suggest that there is an activation of hepatic progenitor cells in NRH.

Immunohistochemical Classification of Hepatic Adenoma, A Single Center Experience

Objectives: Hepatocellular adenoma (HCA) is a tumor with heterogenous molecular pathogenesis and varying malignant potential. Subclassification of HCAs is one of the important issues for the management decision to perform surgery or to follow up the patient. Iran is in intermediate status regarding the incidence of hepatocellular carcinoma, and on the other hand, incidence of viral precursors especially hepatitis B is decreasing, so it can be important to know the frequency of premalignant subtypes of liver cell adenomas in Iran. Methods: During the study period (10 years from 2008 to 2018) 40 cases of HCA (35 female and 5 males) were retrieved from the archives of the pathology departments of the affiliated hospitals of Shiraz University of Medical Sciences. The diagnosis was confirmed and the best paraffin block was used for IHC staining for liver fatty acid binding protein (LFABP), glutamine synthetase (GS), β-catenin and serum amyloid A (SAA). Histologic findings were also recorded. In the mean time, clinical charts of the patients were reviewed and clinicopathologic findings were compared. Results: The most common subtype in our cases was hepatocyte nuclear factor-1α inactivated and the least common was β-catenin activated subtype. We didn’t find any cases of mixed subtype. Inflammatory subtype was seen in 10% of the cases. Another 10% of our cases were unclassified because all of the IHC markers were negative. Conclusions: Our results confirmed that immunohistochemistry should be routinely performed to subclassify HCAs. We also showed that frequencies of subtypes differ according to the studied population. For our population (Middle East or west Asia) HCAs with malignant potential are the least common.

A Common Presentation of Right Upper Quadrant Pain in an Uncommon Disease

The presentation of right upper quadrant abdominal pain is common, non—specific and challenging to diagnose. Right upper quadrant (RUQ) pain can be benign but may also signal serious pathology. Early diagnosis is key to preventing possible complications. This case aims to highlight a presentation of RUQ pain in a young male which heralded a more sinister pathology. A previously healthy 36year—old male presented with one—week of intermittent fevers and progressive sharp RUQ pain with chills, night sweats, early satiety and fatigue. Initial assessment was significant for HR 116, RUQ tenderness with guarding, no rebound or organomegaly. Laboratory findings were only significant for lactate 11.08mg/dL. Cultures were negative, AFP was normal. HIV, HBV and HCV were negative.Abdominal ultrasound revealed heterogeneous large right hepatic lobe mass suggestive of hemorrhage, hepatic adenoma, metastasis, fibrolamellar hepatocellular carcinoma (HCC) or hepatic abscess. MRI with contrast had similar findings with large non—enhancing geographic regions, T2—hyperintense and T1—hypointense, consistent with necrosis or blood products. MRI was suggestive of atypical HCC, fibrolamellar HCC, angiosarcoma or metastases; hepatic adenoma unlikely in the absence of risk factors. CT—guided biopsy revealed hepatic neoplasm favoring adenoma with atypical findings evidenced by immunostaining, diffuse strong glutamine synthetase staining and no nuclear beta—catenin staining, positive serum amyloid A, and retained LFABP. Glutamine synthetase staining was concerning for beta—catenin invasion. He underwent a right hepatic lobectomy, with pathology finding of hepatic adenoma, and no recurrence at 1—year follow—up. Hepatocellular adenomas (HCA) are rare, benign tumors which occur mostly in women and are strongly associated with the use of estrogens, anabolic and androgenic steroids. There are no specific diagnostic serologic or imaging studies. The new Bordeaux HCA subtype classification is based on genetic and phenotypic characteristics and provides insight into the pathological and radiological diagnosis and clinical management. Complications such as rupture, hemorrhage and malignant transformation to HCC increase mortality markedly. Mortality is low when recognized and treated early. It is, therefore, an important differential to consider in a patient with RUQ pain in the presence of a liver mass with no underlying cirrhosis. The absence of risk factors does not exclude the diagnosis.3020_A.tif Figure 1: Abdominal ultrasound: Large heterogeneous right hepatic lobe mass with suggestion of internal hemorrhage.3020_B.tif Figure 2: Contrast—MRI, coronal and axial views: marked enlargement of the right hepatic lobe secondary to a large mass with intrahepatic blood products suggesting rupture/hemorrhage3020_C.tif Figure 3: Liver biopsy histology: Hepatocellular neoplasm, favor adenoma with atypical findings. Diffuse strong glutamine synthetase staining, serum amyloid A positive, retained LFABP, no nuclear beta—catenin staining and noncontributory reticulin special stain

Hepatic angiomyolipoma: mutation analysis and immunohistochemical pitfalls in diagnosis.

Hepatic angiomyolipoma (AML) often shows epithelioid morphology with inconspicuous fat. Epithelioid component can mimic hepatocellular adenoma (HCA) or carcinoma (HCC). The aims of this study were to examine the expression of commonly used markers for HCA or HCC in hepatic AML and highlight pitfalls in diagnosis. Resected hepatic AMLs (n = 16) were reviewed; reticulin stain, immunohistochemistry for glutamine synthetase (GS), β-catenin and liver fatty acid binding protein (LFABP) were performed along with Sanger sequencing of exon 3 of CTNNB1 and next-generation sequencing (NGS). Predominant epithelioid component (≥50%) was seen in 80% of cases. Foamy macrophage was present in 33% of cases. High-risk histological features were often present in tumours with benign outcome: marked atypia (19%), mitoses (20%) and necrosis (33%). GS staining (≥10% of tumour) was seen in epithelioid components in 13 (87%) cases, and was diffuse (>50% of tumour) in six (40%) cases. LFABP staining or nuclear β-catenin staining was not seen in any case. Sanger sequencing and NGS did not reveal CTNNB1 mutation in any tested case. NGS demonstrated TSC2 mutations in all five cases tested. The predominance of epithelioid component resembling HCA or HCC is common in hepatic AML. Absence of LFABP and presence of fat can be mistaken for HNF1α-inactivated HCA. Diffuse GS staining can be mistaken for β-catenin-activated HCA or HCC. Diffuse GS expression is not related to CTNNB1 mutation. All tested cases showed TSC2 mutation, supporting this as the driving genetic event for hepatic AML.

Peri-tumoral hyperintensity on hepatobiliary phase of gadoxetic acid-enhanced MRI in hepatocellular carcinomas: correlation with peri-tumoral hyperplasia and its pathological features

Peri-tumoral hyperintensity (P-hyperintensity) is occasionally seen in hepatocellular carcinoma (HCC) on the hepatobiliary (HB) phase of gadoxetic acid-enhanced MRI (EOB-MRI). A recent study reported peri-tumoral hyperplasia (P-hyperplasia) associated with over-expression of glutamine synthetase (GS) in HCC or metastatic carcinoma. The aim of this study was to analyze the correlation between P-hyperintensity on the HB phase and GS expression indicating P-hyperplasia and reveal its pathological features. Seventy-seven surgically resected HCCs from 68 patients were analyzed. The grade of P-hyperintensity on HB phase was divided according to the degree of the peri-tumoral hyperintense signal: grade 0 (no P-hyperintensity), grade 1 (less than 50% of the tumor border), grade 2 (50%-80%), grade 3 (80%-100%). Immunohistochemical staining for GS and organic anion transporter polypeptides (OATP)1B3 was performed. The relationships among P-hyperplasia (peri-tumoral GS expression) and OATP1B3 expression, P-hyperintensity, and pathological features of the tumor were analyzed. Thirty-four HCCs were classified as P-hyperintensity grade 0, 29 HCCs as grade 1,10 nodules as grade 2, and 4 HCCs as grade 3. P-hyperplasia was observed in 3/34 (8.8%) P-hyperintensity grade 0, 16/29 (55.2%) grade 1, 9/10 (90%) grade 2, and 4/4 (100%) grade 3. The incidence of P-hyperplasia was significantly increased in P-hyperintensity grades 1-3 compared with grade 0 (p<0.0001). Hepatocytes in all P-hyperplasia sites demonstrated definite OATP1B3 expression. Microscopic hepatic venous invasion was significantly increased in P-hyperintensity-positive HCCs compared with negative HCCs (p=0.0017). P-hyperintensity on HB phase in HCC may indicate p-hyperplasia with GS and OATP1B3 expression and a higher incidence of microscopic hepatic venous invasion.

Focal β-catenin mutation identified on formalin-fixed and paraffin-embedded inflammatory hepatocellular adenomas.

The identification of hepatocellular adenoma (HCA) with mutation in exon 3 of the CTNNB1 gene encoding for β-catenin is clinically relevant due to a higher risk of malignant transformation. Inflammatory HCA (IHCA) can exhibit β-catenin activation (β-IHCA). We report two cases with multiple IHCA in which focal β-catenin activation has been found in one of the IHCA. In both cases, the diagnosis of IHCA was confirmed on the resected nodules by routine stains, immunohistochemical detection of C-reactive protein (CRP) and molecular biology on frozen material. An additional molecular analysis was performed on formalin-fixed paraffin-embedded (FFPE) material that showed focal glutamine synthetase (GS) staining, the surrogate marker of β-catenin activation. In case 1, it was a 1.8-cm area within the 7.5 cm IHCA, and in case 2 a small 0.3-cm area within a 1.8 cm resected IHCA located close to a larger IHCA, negative for GS. In both cases, nuclear β-catenin expression and decreased reticulin network were observed in the GS expressing foci, together with cholestasis and diffuse CD34 expression in case 1. Molecular analysis by pyrosequencing on FFPE material using the GS-stained slides as reference to select areas with/without positive staining revealed a CTNNB1 exon 3 mutation restricted to the areas exhibiting both positive GS and CRP expression, whereas wild-type CTNNB1 was found in areas showing only CRP staining. These two cases illustrate focal β-catenin activation that can occur within IHCAs. Additional data are needed to determine if β-catenin mutation is a secondary event in IHCA.

Amburana cearensis seed extract protects brain mitochondria from oxidative stress and cerebellar cells from excitotoxicity induced by glutamate

Ethnopharmacological relevanceAmburana cearensis (Allemao) A.C.Sm. is a medicinal plant of the Brazilian Caatinga reported to present antioxidant and anti-inflammatory activity. This study aimed to evaluate the neuroprotective effect of the extracts obtained from the seeds of A. cearensis in primary cultures of cerebellar cells subjected to excitotoxicity induced by glutamate and brain mitochondria submitted to oxidative stress. Materialsand methods: Primary cultures of cerebellar cells were treated with the ethanol (ETAC), hexane (EHAC), dichloromethane (EDAC) and ethyl acetate (EAAC) extracts of the seeds of A.cearensis and subjected to excitotoxicity induced by glutamate (10µM). Mitochondria isolated from rat brains were submitted to oxidative stress and treated with ETAC. ResultsOnly the EHAC extract reduced cell viability by 30% after 72h of treatment. Morphological analyses by Immunofluorescence showed positive staining for glutamine synthetase, β-III tubulin, GFAP and IBA1 similar to control cultures, indicating a better preservation of astrocytes, neurons and microglia, after excitotoxic damage induced by glutamate in cerebellar cultures treated with the extracts. The ETAC extract also protected mitochondria isolated from rat brains from oxidative stress, reducing the swelling, dissipation of the membrane potential, ROS production and calcium influx. ConclusionThus, this study suggests that the seed extracts from A. Cearensis exhibit neuroprotective potential against oxidative stress and excitotoxicity induced by glutamate and can be considered a potential therapeutic agent in the treatment of neurodegenerative diseases.