Formalin-fixed Paraffin-embedded Liver Research Articles

Intrahepatic Transcriptomics Differentiate Advanced Fibrosis and Clinical Outcomes in Adults With Fontan Circulation

BackgroundThe molecular mechanisms underlying Fontan-associated liver disease (FALD) remain largely unknown. ObjectivesThis study aimed to assess intrahepatic transcriptomic differences among patients with FALD according to the degree of liver fibrosis and clinical outcomes. MethodsThis retrospective cohort study included adults with the Fontan circulation. Baseline clinical, laboratory, imaging, and hemodynamic data as well as a composite clinical outcome (CCO) were extracted from medical records. Patients were classified into early or advanced fibrosis. RNA was isolated from formalin-fixed paraffin-embedded liver biopsy samples; RNA libraries were constructed with the use of an rRNA depletion method and sequenced on an Illumina Novaseq 6000. Differential gene expression and gene ontology analyses were performed with the use of DESeq2 and Metascape. ResultsA total of 106 patients (48% male, median age 31 years [IQR: 11.3 years]) were included. Those with advanced fibrosis had higher B-type natriuretic peptide levels and Fontan, mean pulmonary artery, and capillary wedge pressures. The CCO was present in 23 patients (22%) and was not predicted by advanced liver fibrosis, right ventricular morphology, presence of aortopulmonary collaterals, or Fontan pressures on multivariable analysis. Samples with advanced fibrosis had 228 upregulated genes compared with early fibrosis. Samples with the CCO had 894 upregulated genes compared with those without the CCO. A total of 136 upregulated genes were identified in both comparisons and were enriched in cellular response to cytokine stimulus or oxidative stress, VEGFA-VEGFR2 signaling pathway, TGF-β signaling pathway, and vasculature development. ConclusionsPatients with FALD and advanced fibrosis or the CCO exhibited upregulated genes related to inflammation, congestion, and angiogenesis.

Molecular confirmation of alpha 1-antitrypsin deficiency in liver transplant setting: A province-wide experience.

Patients suspected of Alpha 1-Antitrypsin (A1AT) abnormality based on low serum concentration are routinely confirmed through polymerase chain reaction (PCR) testing of peripheral blood. Genotyping formalin-fixed paraffin-embedded (FFPE) tissue is a novel approach that could aid in detecting variant A1AT. We performed qPCR on FFPE liver explants with Periodic Acid Schiff after Diastase (PASD)- and A1AT-positive globules to confirm and estimate the frequency of A1AT deficiency in transplant cases. Eighteen (12.68%) of 142 patients with end-stage liver disease showed PASD/A1AT positive globules. FFPE of the explants was tested through qPCR to detect S and Z alleles. A second age- and sex-matched control group consisting of five liver transplant patients with negative globules was included in the study. qPCR assay was successful with all the samples meeting QC parameters. All patients included in the study elucidated Z allele variants; 2 homozygous (11.1%) and 16 heterozygous (88.9%). The control group demonstrated normal wild-type MM allele. Screening for A1AT deficiency using serum levels is not sufficiently sensitive to detect deficiency, especially in carriers. If A1AT testing was not performed preoperatively and the risk is high based on the PASD/A1AT-positive globules in the explants, then molecular testing of FFPE tissue can be a viable method for confirming the diagnosis.

The Intensity and Pattern of Syndecan-1 (CD138) Expression in Normal and Diseased Livers.

Introduction Heparan sulfate proteoglycans (HSPGs) belongto the syndecan family, and syndecan-1 (CD138) is a heparan sulfate proteoglycan. Syndecan-1 has a potential role in cell-matrix and cell-cell communications as they are present in cell epithelium. Its expression is different in an extensive range of benign, inflammatory, and neoplastic diseases. In routine histopathology, it is used as a marker for plasma cells. However, it is expressed in a large variety of normal and neoplastic epithelia including squamous epithelium and gastric glandular epithelium expressed in other tissues, i.e., the liver. In the liver, variable expression is seen in cirrhosis, hepatitis, and carcinoma. The objective of this study was to investigatethe expression of this marker in normal, inflammatory, and neoplastic lesions of the liver. This in turn may help clinicians to select patients whomay benefit from anti-CD138 therapy. It is currently used in the diagnosis and management of plasma cell proliferations. Material and methods This is a retrospective study in which weretrieved 53 formalin-fixed paraffin-embedded (FFPE) liver specimen blocks and selected one block from each case by reviewing the hematoxylin and eosin (H&E) slides of each case. Syndecan-1 (CD138), pancytokeratin, and CD68 expression were analyzed immunohistochemically (IHC) to evaluate the percentage and intensity of CD138 expression in various hepatic entities and identify those entities where syndecan-1 can be consistently used to make a definitive diagnosis. Results The expression of pancytokeratin and CD68 was analyzed in hepatocytes and Kupffer cells, respectively. For syndecan-1 (CD138), 15.4% of cases showed basolateral membranous positivity, 44.6% of cases showed complete membranous positivity, and 40% of cases showed no positivity in hepatocytes. Cytokeratin (CK) was positive as expected in hepatocytes, and CD68 was expressed in Kupffer cells. Conclusion CD138 does not appear to be a reliablesurrogate marker for liver disease. However, it may be included with other ancillary markers as a predictor of the stage of chronic liver disease and metastatic potential. The response to anti-CD138 therapy needs to be further studied.

Growth differentiation factor 7 autocrine signaling promotes hepatic progenitor cell expansion in liver fibrosis

Background and aimLiver fibrosis is prevalent among chronic diseases of the liver and represents a major health burden worldwide. Growth differentiation factor 7 (GDF7), a member of the TGFβ protein superfamily, has been recently investigated for its role in repair of injured organs, but its role in chronic liver diseases remains unclear. Here, we examined hepatic GDF7 expression and its association with development and progression of human liver fibrosis. Moreover, we determined the source and target cells of GDF7 in the human liver.MethodsGDF7 expression was analyzed in fibrotic and healthy human liver tissues by immunohistochemistry and qPCR. Cell-specific accumulation of GDF7 was examined by immunofluorescence through co-staining of cell type-specific markers on formalin-fixed paraffin-embedded human liver tissues. Public single cell RNA sequence databases were analyzed for cell type-specific expression of GDF7. In vitro, human liver organoids and LX-2 hepatic stellate cells (LX-2) were treated with recombinant human GDF7. Human liver organoids were co-cultured with activated LX-2 cells to induce an autocrine signaling circuit of GDF7 in liver organoids.ResultsGDF7 protein levels were elevated in fibrotic liver tissue, mainly detected in hepatocytes and cholangiocytes. In line, GDF7 mRNA was mainly detected in liver parenchymal cells. Expressions of BMPR1A and BMPR2, encoding GDF7 receptors, were readily detected in hepatocytes, cholangiocytes and stellate cells in vivo and in vitro. In vitro, recombinant GDF7 promoted liver organoid growth and enhanced expression of the progenitor cell markers (LGR5, AXIN2), but failed to activate LX-2 cells. Still, activated LX-2 cells induced GDF7 and LGR5 expression in co-cultured human liver organoids.ConclusionsCollectively, this study reveals a role of GDF7 in liver fibrosis and suggests a potential pro-regenerative function that can be utilized for amelioration of hepatic fibrosis caused by chronic liver disease.

Association between the Expression Patterns of Hepatitis B Surface Antigen and Hepatitis B Core Antigen with Clinicopathological Parameters and Antiviral Therapy in Liver Biopsies Obtained from Chronically Infected Hepatitis B Positive Omani Patients

Background: Hepatitis B virus (HBV) infection is considered a major global health problem. The main objectives of the current study is to determine the patterns of expression of the hepatitis B surface antigen (HBsAg) and HBV core antigen (HBcAg) in liver tissue samples obtained from hepatitis B virus-infected Omani patients and to associate between the pattern of the expression of HBsAg and HBcAg with the other clinical parameters and anti-viral therapy. Methods : The expression patterns of HBsAg and HBV core antigen HBcAg were determined by immunohistochemistry (IHC), in 58 formalin-fixed paraffin-embedded liver tissue biopsies obtained from chronic hepatitis B Omani patients. The association between positivity for HBV antigens with gender, age group, histological appearances, and antiviral therapy was determined. Results: IHC-positive staining of HBsAg was demonstrated in 28 patients (48.3%) patients, of whom 4 (6.9%) also showed HBcAg expression. The expression pattern of HBsAg was predominantly cytoplasmic and was seen in 25 (89.3%) of patients, whereas expression of HBcAg was nuclear in 3 (75%) of patients. HBsAg and HBcAg IHC positivity were more common among males than females and among those aged 39–58 years (P = 0.130 and 0.569, respectively). The presence of lymphocytic infiltration in the majority of liver biopsies examined in the present study indicates that the liver damage could be attributed to immunologically mediated events, especially because HBV is a non-cytopathogenic virus. No significant statistical association was found between positivity for HBsAg/HBcAg by IHC and antiviral treatment. Conclusions: Determination of the expression patterns of HBV antigens in liver biopsies obtained from chronic hepatitis B Omani patients and the association between these expression patterns with other clinical histopathological parameters and anti-viral therapy, will contribute greatly to a better understanding of the pathogenesis of HBV in this unique cohort group of infected individuals.

The comparative performance of a custom Canine NanoString® panel on FFPE and snap frozen liver biopsies

Formalin-Fixed Paraffin Embedded (FFPE) biopsies would provide a critical mass of cases to allow investigation of canine liver disease, however their use is often limited by challenges typically associated with transcriptomic analysis. This study evaluates the capability of NanoString® to measure the expression of a broad panel of genes in FFPE liver samples. RNA was isolated from matched histopathologically normal liver samples using FFPE (n = 6) and snap frozen in liquid nitrogen (n = 6) and measured using a custom NanoString® panel. Out of the 40 targets on the panel, 27 and 23 targets were above threshold for non-diseased snap frozen and FFPE tissue respectively. The binding density and total counts were significantly reduced in the FFPE samples relative to the snap frozen samples (p = 0.005, p = 0.01, respectively), confirming a reduction in sensitivity. The concordance between the snap frozen and FFPE samples was high, with correlations (R) ranging between 0.88 and 0.99 between the paired samples. An additional 14 immune-related targets, undetectable the non-diseased FFPE liver, were above threshold when the technique was applied to a series of diseased samples, further supporting their inclusion on this panel. This use of NanoString® based analysis opens up huge opportunity for retrospective evaluation of gene signatures in larger caseloads through harnessing the capacity of archived FFPE samples This information used alongside clinical and histological data will not only afford a way to explore disease etiopathogenesis, it may also offer insight into sub-types of liver disease in dogs, which cannot be discerned using more traditional diagnostic methods.

Visualizing in situ viral replication across the natural history of chronic HBV infection.

Chronic HBV infection evolves through different phases. Interactions between viral replication and the host immune response in the liver underlie the pathogenesis of this disease. The aim of this study was to directly visualize the HBV replication intermediates at a single-cell resolution inscribed on morphological changes corresponding to disease activity. A set of archived formalin-fixed paraffin-embedded liver needle biopsies from treatment-naïve patients were collected and categorized into phases according to the American Association for the Study of the Liver Diseases (AASLD) guidelines. HBV RNA and DNA were detected using in situ hybridization assays. The hepatocytes were ubiquitously infected in subjects with immune tolerance, and their percentage was gradually decreased in immune-active and inactive chronic hepatitis B phases. HBV-infected hepatocytes were prone to localize close to fibrous septa. The subcellular distribution of signals was able to distinguish hepatocytes with productive infection from those harboring HBV integrants and transcriptionally inactive covalently closed circular DNAs. A smaller number of hepatocytes with productive infection and more harboring transcriptionally inactive covalently closed circular DNA or HBV integrants became apparent in the inactive chronic hepatitis B phase. An atlas of in situ characteristics of viral-host interactions for each phase is described, which sheds light on the nature of viral replication and disease pathogenesis among the phases of chronic HBV infection.

Pigment epithelium-derived factor downregulation in oestrogen receptor positive breast cancer bone metastases is associated with menopause

Pigment epithelium-derived factor (PEDF) has a critical role in bone development and anti-tumour function in breast cancer (BC). As the expression and role of PEDF in BC bone metastases is unknown, we aimed to characterise PEDF in primary and metastatic BC. Subcellular PEDF localisation was semi-quantitatively analysed via immunohistochemistry in patient-matched, archived formalin-fixed paraffin-embedded primary BC and liver, lung, and decalcified bone metastases specimens. PEDF localisation was evaluated in 23 metastatic BC patients diagnosed with ER+, human epidermal growth factor receptor-2 (HER2) negative BC or TNBC. Cytoplasmic (p = 0.019) and membrane (p = 0.048) PEDF was lower in bone metastases compared to primary ER+/HER2- BC. In contrast, nuclear PEDF scores were higher in metastases compared to primary TNBC (p = 0.027), and increased membrane PEDF in metastatic tissue had improved disease-free interval (p = 0.016). Nuclear PEDF was decreased in bone metastases compared to primary ER+//HER2- BC in post-menopausal patients (p = 0.029). These novel findings indicate PEDF plays a role in clinical BC metastasis. Significantly lower PEDF levels in the post-menopausal compared to pre-menopausal setting suggests future PEDF research may have greater clinical importance in the post-menopausal ER+/HER2- BC population.

Expression of HLA and Autoimmune Pathway Genes in Liver Biopsies of Young Subjects With Autoimmune Hepatitis Type 1.

Objectives:To test the hypothesis that autoimmune hepatitis (AIH type I) in young subjects is due to genetic differences in proinflammatory genes responding to viral triggers in patients and controls.Methods:Intrahepatic gene expression was compared between AIH type I (n = 24, age 9–30 years) patients (hereafter referred to as the AIH group) and controls (n = 21, age 4–25 years). RNA sequencing was performed on complementary DNA (cDNA) libraries made from total RNA extracted from formalin-fixed paraffin-embedded (FFPE) liver biopsy samples. Gene expression levels were quantified, and differentially expressed genes were functionally analyzed. Pathway analysis was performed using the databases Kyoto Encyclopedia of Genes and Genomes (KEGG) and PANTHER. The remaining sequences were mapped to the RefSeq complete set of viral genomes.Results:Differential gene analysis identified 181 genes that were significantly differentially expressed (136 upregulated in the AIH group). Autoimmune pathway genes such as CD19 and CD20 which are important in B cell regulation and maturation as well as, CD8 and LY9, which are T-cell related, were upregulated in our AIH group. Genes implicated in AIH pathogenesis including CXCL10, which is thought to be associated with AIH severity and progression, complement genes (C1QA, C1QB, and C1QC), and human leucocyte antigen (HLA) genes (HLA-DRB1, HLA-DRA, HLA-B, and HLA-C) were upregulated in samples from the AIH group. Specific viral etiologies were not found.Conclusions:Unbiased next-generation sequencing and differential gene expression analysis of the AIH group has not only added support for the role of B cells in the pathogenesis and treatment of AIH but also has introduced potential new therapeutic targets: CXCL10 (anti-CXCL10) and several complement system–related genes.

Abstract 5291: Characterizing the interplay between angiogenic and immunoactive factors of hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related death globally. Patients typically present at an advanced stage and less than 50% reach the maximum 1-year survival rate, when given as first-line treatment, Sorafenib. This highlights the need for early detection and novel therapeutic targets crucial to increase overall survival (OS) for patients with HCC. Given the important role of angiogenesis in HCC from its early stage and its rich immune composition, anti-angiogenic and immune checkpoint inhibitors (ICI), are two therapeutic approaches when combined marked the first treatment in more than a decade to significantly improve the overall survival and progression-free survival in patients with advanced HCC compared to Sorafenib. While the combination of agents inhibiting angiogenesis and ICI have recently entered the clinic, the interplay between angiogenic factors and immunity in the context of this approach remains poorly understood. Here we focus on understanding the interplay between the vascular state of the tumor and the immune response in HCC. As a first step, we focus on defining the immune and vasculature landscape of the central tumor, peripheral tumor, adjacent liver to the tumor, and distal liver regions of each lesion by immunohistochemistry (IHC). Forty (40) formalin-fixed paraffin-embedded (FFPE) human liver tissue samples containing untreated and non-viral HCC tumors, obtained from the Liver Disease Biobank of the RI-MUHC were used to perform IHC. Images were viewed and scored using the Aperio ImageScope software. With respect to the vasculature, we observe that all tumors have a combination of both angiogenic (CD34/Ki67+ve) and co-optioning (CD31 +ve) features, with no uniform distribution. Our immune markers demonstrate that both adaptive and innate immune cells are present at the interface and different tumors demonstrate different levels of infiltration. Next, to identify the immune subtype populations (ie macrophage M1 vs M2, Treg, etc) present, we will use the NanoString Whole Transcriptome Atlas spatial profiler technology. We will also then link the vasculature to any specific immune profile. For example, it has been shown in other cancer types that macrophages are associated with co-option. This project presents preliminary evidence for the interaction of vascular factors with immune cells, thus providing insight into the biological rationale of why 30% of patients responded to combined angiogenic and immunotherapy treatment. Citation Format: Audrey Kapelanski-Lamoureux, Flemming Kondrup, Lucyna Krzywon, Stephanie K. Petrillo, Anthoula Lazaris, Peter Metrakos. Characterizing the interplay between angiogenic and immunoactive factors of hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5291.

Immunohistochemical Expression of Oxidative Stress and Apoptosis Markers in Archived Liver Specimens from Dogs with Chronic Hepatitis

Chronic hepatitis (CH) in dogs is histologically characterized by an inflammatory infiltration of the liver accompanied by hepatocellular apoptosis or necrosis, varying degrees of fibrosis and regeneration. Oxidative stress has been suggested to play a role in the pathogenesis of various liver diseases, including CH. This study assessed the immunohistochemical expression of markers of oxidative stress (4-hydroxynonenal [4-HNE] and malondialdehyde [MDA]) and apoptosis (active caspase-3 [casp-3]) in 35 surplus archived formalin-fixed paraffin-embedded liver biopsies from 25 dogs with CH and 10 control dogs that had no significant hepatic changes. Correlations between immunohistochemical markers and necroinflammatory, fibrosis and histological copper scores, and hepatic copper concentrations were also determined. There were no significant differences in 4-HNE expression between the two groups. Control dogs had lower hepatic MDA scores than dogs with CH. MDA scores were positively correlated with copper scores as well as hepatic copper concentrations. There was no significant difference in casp-3-positive hepatocytes between groups. However, a positive correlation between casp-3 immunoreactivity and copper scores, as well as hepatic copper concentrations, was identified. Necroinflammatory and fibrosis scores were positively correlated with immunoreactivity for MDA and casp-3. MDA and casp-3 are expressed in canine liver and both markers are correlated with necroinflammatory scores, fibrosis scores and hepatic copper accumulation. Our results suggest the utility of immunolabelling for MDA and casp-3 for assessment of hepatic oxidative stress and apoptosis, respectively, in dogs with CH.

Histological paraffin-embedded block: a good alternative specimen to detect the use of opiates at least 20 years ago

PurposeSince the solely certain remnants of a performed autopsy are formalin-fixed paraffin-embedded (FFPE) samples, stored in the archives of every institute of legal medicine, we managed to extract molecules of toxicological interest from these specimens.MethodsWe assessed the analysis of ten fresh liver samples collected from heroin-related deaths and then histologically processed the same samples. The embedded blocks were then extracted by means of a new extracting method and the eluates were measured. We also selected five toxicological cases of heroin-related fatalities that were examined 20 years ago, collected the toxicological result documents of the analysis that were carried out at the time and then processed the corresponding FFPE liver samples that were stored in the archives.ResultsWe managed to isolate heroine-related metabolites from 20-year-old paraffin-embedded blocks and calculated ratios to evaluate the performance of our new extraction.ConclusionsAccording to our study, it is feasible to carry out a toxicological examination on old histological samples and, therefore, this matrix can be considered as a new alternative specimen for chemical-analytical evaluations of past cases or when fresh samples are not available anymore. The new extractive method was evaluated as efficient in treating these complex, paraffin-embedded samples. It was surprising that the target compounds could be quantitated from FFPE bocks created as long as 20 years ago.

Case study: Targeted RNA-sequencing of aged formalin-fixed paraffin-embedded samples for understanding chemical mode of action

Formalin-fixed paraffin-embedded (FFPE) samples are the only remaining biological archive for many toxicological and clinical studies, yet their use in genomics has been limited due to nucleic acid damage from formalin fixation. Older FFPE samples with highly degraded RNA pose a particularly difficult technical challenge. Probe-based targeted sequencing technologies show promise in addressing this issue but have not been directly compared to standard whole-genome RNA-Sequencing (RNA-Seq) methods. In this study, we evaluated dose-dependent transcriptional changes from paired frozen (FROZ) and FFPE liver samples stored for over 20 years using targeted resequencing (TempO-Seq) and whole-genome RNA-Seq methods. Samples were originally collected from male mice exposed to a reference chemical (dichloroacetic acid, DCA) at 0, 198, 313, and 427 mg/kg-day (n = 6/dose) by drinking water for 6 days. TempO-Seq showed high overlap in differentially expressed genes (DEGs) between matched FFPE and FROZ samples and high concordance in fold-change values across the two highest dose levels of DCA vs. control (R2 ≥ 0.94). Similarly, high concordance in fold-change values was observed between TempO-Seq FFPE and RNA-Seq FROZ results (R2 ≥ 0.92). In contrast, RNA-Seq FFPE samples showed few overlapping DEGs compared to FROZ RNA-Seq (≤5 for all dose groups). Modeling of DCA-dependent changes in gene sets identified benchmark doses from TempO-Seq FROZ and FFPE samples within 1.4-fold of RNA-Seq FROZ samples (93.9 mg/kg-d), whereas RNA-Seq FFPE samples were 3.3-fold higher (310.3 mg/kg-d). This work demonstrates that targeted sequencing may provide a more robust method for quantifying gene expression profiles from aged archival FFPE samples.

Celiac disease associated liver dysfunction: Study of the histological features and co-localisation patterns of IgA and IgG anti-tissue transglutaminase antibody (TTG-2) in liver biopsies

Celiac disease (CeD) associated liver dysfunctions (CALD) have wide range of manifestation ranging from milder symptoms alleviating on gluten free diet (GFD), to pathological conditions such as autoimmune hepatitis, cirrhosis etc. However, characteristic histopathological signatures diagnostic of this entity have not been described yet. Diagnosis relies solely on clinical suspicion due to co-occurrence of two pathologies. The aim of this study is to elucidate histological features of CALD and demonstrate IgA/IgG anti-tTG colocalization patterns as a hypothetic evidence towards etiology. 28 formalin fixed paraffin embedded (FFPE) core liver biopsies from patients with treatment naïve CeD with associated liver dysfunction and another 5 FFPE liver core biopsies from patients with other proven pathologies and normal serum anti-tTG titres, were retrieved and IgA/IgG anti-tTG colocalization was carried out both by dual immunohistochemistry (IHC) and dual confocal immunofluorescence (IF) techniques. After a period of GFD, clinically indicated liver biopsies from 5 of these 28 patients were obtained and subjected to repeat testing via the same techniques. Histopathological examination in CALD revealed the following spectrum: steatohepatitis 4 (14.2%), autoimmune hepatitis 4 (14.2%), non-cirrhotic portal venopathy 1 (3.5%), secondary hemochromatosis 1 (3.5%), steatohepatitis with necrotising granuloma 1 (3.5%), cirrhosis 3 (10.7%), irregular sinusoidal dilatation 14 (50%). In CALD, IgA/IgG anti-tTG colocalization was noted in 85.6% cases by dual IHC technique and in 92.8% cases by dual IF technique. Follow up biopsies in 5 CeD cases showed no co-localisation. Deposition of the circulating IgA/IgG anti-tTG antibodies in suspected patients of CALD seems to be a sensitive and specific technique for diagnosing their celiac association, thereby opening up newer insights into management protocols.

Profiling of transcriptional biomarkers in FFPE liver samples: PLS-DA applications for detection of illicit administration of sex steroids and clenbuterol in veal calves

Administering growth promoters to meat-producing animals is strictly regulated within the European Community to preserve consumers’ safety. Nevertheless, illicit misuse of steroids and other veterinary drugs to increase animal production has still been found by National Residues Control Plans (NRCPs), mainly based on expensive targeted multi-class/multi-residue analytical methods, used for confirming quantification analysis of these residues in tissues and biological fluids. The setup of complementary novel diagnostic methods, based on indirect biomarkers of exposure to anabolic substances is, therefore, needed to update current tests available in NRCPs at the screening level, where cheaper, sensitive and untargeted methods are required. Biological effects of illicit treatment transcriptomic analysis has reached a good grade of standardization, identified by microarray and RNAseq technologies of large panels of differentially expressed gene targets (DEG) related to abuse of different classes of anabolic molecules (e.g. sex steroids, thyreostats, β2-agonists, glucocorticoids, etc.). The aim of the work was to identify a minimum set of molecular markers able to detect illicit administration of two of the most recurrent classes of growth promoters (sex steroids and B2-agonists) on formalin-fixed paraffin-embedded (FFPE) tissues, being a potentially straightforward sampling strategy at the slaughterhouse level. A PCR array to allow quantitative profiling of 48 genetic targets was, therefore, developed on FFPE liver samples from veal calves. Conventional univariate and alternative approaches based on multivariate classification methods were considered for gene expression profiling of 92 samples collected from different animal trials, where effects of nandrolone, estradiol, their combination, and clenbuterol were studied.The developed Partial Least Squares – Discriminant Analysis (PLS-DA) models show the successful setup of a novel screening strategy based on multiple transcriptional biomarkers, able to expand tested sample sizes, and consequently strengthen current NRCPs.

MALDI-TOF mass spectrometry imaging for N-glycans on FFPE tissue sections of mouse NASH liver through Sialic acid Benzylamidation.

Glycans play an important physiological role and are drawing attention as biomarkers that capture pathophysiological changes. Glycans can be detected by mass spectrometry, but recently matrix-assisted laser desorption/ionization- mass spectrometry imaging (MALDI-MSI) has enabled the visualization of glycans distribution on tissues. In this study, focusing on sialylated glycan (sialoglycans), we investigated the amidation reaction used to visualize glycans distribution, and developed a method of sialic acid derivatization by benzylamidation which is more sensitive than conventional amidation. Furthermore, we adapted this method for visualizing glycans in formalin-fixed paraffin-embedded (FFPE) liver tissue from normal mice and non-alcoholic steatohepatitis (NASH) model mice using MALDI-MSI. As a result, an increase in the distribution of glycan N-Acetylneuraminic acid-(NeuAc) ions was observed in the NASH mouse liver, and the change in glycan structure in the NASH model was clarified.

High-risk gene expression profiles for colorectal liver metastases

Presenter: Michael Turgeon MD | Emory University Background: Over half of patients with colorectal cancer develop liver metastases with a minority of patients considered resectable at presentation. While there has been significant progress in the development of new biologics for the treatment of colorectal liver metastases (CRLM), only a small proportion of patients harbor actionable mutations. Furthermore, gene expression patterns of liver-specific metastases remain unclear. Our aim was to identify high-risk gene expression profiles for patients with CRLM to better inform prognosis and the development of novel targeted therapies. Methods: We examined 53 formalin-fixed paraffin-embedded (FFPE) colorectal liver metastasis samples from patients who underwent complete metastasectomy from 2009-2017 at a single institution. Immune cell infiltration was confirmed with CD45 antibody staining via immunohistochemistry (IHC). Targeted mRNA expression profiling was performed with extracted RNA using the NanoString Immune Oncology (IO360) 750-gene panel. Expression counts were normalized by the geometric mean of the positive-control and the most stable housekeeping genes were selected using the geNorm algorithm followed by log2 transformation. The differential expression analysis with fold changes and p-values were calculated using the NanoString methodology. Groups of interest were dichotomized based on synchronous versus metachronous disease and risk profiles. Risk profiles were based on median and quartile overall survival (OS) cut-points. “High-risk” was considered OS below the median or 25th percentile, while “low-risk” was OS above the median, above the 75th percentile, or no death due to disease. Results: Of 53 patients, median overall survival was 58 months. Median follow-up was 36 months. Using the NanoString IO360 panel, we detected no differentially expressed genes between synchronous (n=28) and metachronous (n=25) CRLM. Considering risk profiles, four differentially expressed genes were identified comparing tumors from high-risk (n=4) versus low-risk (n=18) patients based on a median OS cut-point of 45 months. Pathway analysis indicated these gene expression signatures were associated with antigen presentation, DNA repair, and collagen synthesis. When comparing the 25th percentile (high-risk n=2) to the 75th percentile (low-risk n=16), a total of 68 down-regulated genes were detected that were primarily involved in the antigen presentation pathways. Conclusion: Patients with colorectal liver metastases with poor overall survival may have a distinct, “high-risk” gene expression profile that manifests in alterations in antigen presentation, DNA repair, and collagen synthesis pathways. The identified genes serve as promising molecular markers for prognostication and for the development of novel targeted therapies. Next steps include NanoString analysis with an expanded cohort, immunohistochemistry to validate protein expression, and modulation of gene expression in cell lines.

Development of the C12Im-Cl-assisted method for rapid sample preparation in proteomic application.

Chromatography and mass spectrometry (MS) techniques have greatly improved the power of proteomic analyses. However, sample processing methods used prior to MS, including protein extraction and digestion, remain bottlenecks in the large-scale clinical application of proteomics. Ionic liquids, composed entirely of ions, have high solubility in various solvents. In this study, the effects of the cationic surfactant 1-dodecyl-3-methylimidazolium chloride (C12Im-Cl) on protein digestion were evaluated for clinical proteomic applications. C12Im-Cl was compatible with trypsin and reduced the protein digestion time from 16 h to 1 h. Residual C12Im-Cl was easily removed with a strong anion exchange membrane before MS. We evaluated the performance of C12Im-Cl extraction and rapid protein digestion using formalin-fixed paraffin-embedded liver cancer tissues. The number of proteins and peptides identified was nearly equal to that identified by the traditional filter-aided sample preparation method (2705 vs. 2739 and 16 682 vs. 17 214). In general, the C12Im-Cl-aided rapid sample preparation method is promising for proteomic applications.

Prostate-specific membrane antigen expression in hepatocellular carcinoma, cholangiocarcinoma, and liver cirrhosis.

BACKGROUNDPrimary liver cancer includes three subtypes: Hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (CCA), and combined hepatocellular carcinoma. Patients with primary liver cancer experienced poor prognosis and high mortality, so early detection of liver cancer and improved management of metastases are both key strategies to reduce the death toll from liver cancer. Prostate-specific membrane antigen (PSMA) expression in the tumor-associated neovasculature of nonprostate malignancies including liver cancer has been reported recently, but conclusive evidence of PSMA expression based on the pathological type of liver cancer remains limited.AIMTo study the expression of PSMA in HCC, CCA, and liver cirrhosis.METHODSA total of 446 formalin-fixed paraffin-embedded (FFPE) liver tumor and liver cirrhosis tissue samples were obtained retrospectively from the Pathology Department of Tongji Hospital. Immunohistochemistry was used to detect PSMA expression in these 446 FFPE liver biopsy specimens (213 HCC, 203 CCA, and 30 liver cirrhosis). The tumor compartment and the associated neovascular endothelium were separately analyzed. PSMA expression was examined by two certified pathologists, and the final results were presented in a 4-point scoring system (0-3 points). Correlation between PSMA expression and clinicopathological information was also assessed.RESULTSPSMA was expressed primarily in the neovascular endothelium associated with tumors. The positive rate of PSMA staining in HCC was significantly higher than that in CCA (86.8% vs 79.3%; P = 0.001) but was only 6.6% in liver cirrhosis (P = 0.000). HCC cases had more 3-score PSMA staining than CCA had (89/213, 41.8% vs 35/203, 17.2%; P = 0.001). PSMA expression correlated positively with the stage and grade of HCC and CCA. In both liver cancer subtypes, there were more PSMA+ cases in stages III–V diseases than in stages I and II. High staining intensity of PSMA was more frequently observed in liver cancers at high grade and advanced stage. There was no significant association of PSMA expression with sex, age, region, α-fetoprotein, hepatitis B surface antigen, or tumor size in both tumor subtypes.CONCLUSIONNeovascular PSMA may be a promising marker to differentiate HCC from liver cirrhosis and a prognostic marker for anti-tumor angiogenesis therapy for HCC.

Transcriptome Profiling Reveals New Insights into the Immune Microenvironment and Upregulation of Novel Biomarkers in Metastatic Uveal Melanoma.

Simple SummaryUveal melanoma (UM) is a rare aggressive eye cancer. Although treatment of the eye tumour is successful, about 50% of UM patients develop a relapse of their cancer in the liver. At present, such advanced disease is not curable. A better understanding of the metastatic UM (mUM) in the liver is essential to improve patient survival. This study examines both the response of immune cells within the liver to the UM secondaries (metastases), as well as the expression of various proteins by the UM cells. Our study demonstrates that there is a limited immune response to the mUM, but reveals that a certain type of reactive immune cell: a protumourigenic subset of macrophage is dominant within the mUM. Our research also reveals novel proteins within the mUM, which are specific to these cells and therefore may be targetable in future therapies.Metastatic uveal melanoma (mUM) to the liver is incurable. Transcriptome profiling of 40 formalin-fixed paraffin-embedded mUM liver resections and 6 control liver specimens was undertaken. mUMs were assessed for morphology, nuclear BAP1 (nBAP1) expression, and their tumour microenvironments (TME) using an “immunoscore” (absent/altered/high) for tumour-infiltrating lymphocytes (TILs) and macrophages (TAMs). Transcriptomes were compared between mUM and control liver; intersegmental and intratumoural analyses were also undertaken. Most mUM were epithelioid cell-type (75%), amelanotic (55%), and nBAP1-ve (70%). They had intermediate (68%) or absent (15%) immunoscores for TILs and intermediate (53%) or high (45%) immunoscores for TAMs. M2-TAMs were dominant in the mUM-TME, with upregulated expression of ANXA1, CD74, CXCR4, MIF, STAT3, PLA2G6, and TGFB1. Compared to control liver, mUM showed significant (p < 0.01) upregulation of 10 genes: DUSP4, PRAME, CD44, IRF4/MUM1, BCL2, CD146/MCAM/MUC18, IGF1R, PNMA1, MFGE8/lactadherin, and LGALS3/Galectin-3. Protein expression of DUSP4, CD44, IRF4, BCL-2, CD146, and IGF1R was validated in all mUMs, whereas protein expression of PRAME was validated in 10% cases; LGALS3 stained TAMs, and MFGEF8 highlighted bile ducts only. Intersegmental mUMs show differing transcriptomes, whereas those within a single mUM were similar. Our results show that M2-TAMs dominate mUM-TME with upregulation of genes contributing to immunosuppression. mUM significantly overexpress genes with targetable signalling pathways, and yet these may differ between intersegmental lesions.