Hepatic sonic hedgehog protein expression measured by computer assisted morphometry significantly correlates with features of non-alcoholic steatohepatitis.

BACKGROUND: Hepatic expression of Sonic Hedgehog (SHH) is associated with Non-alcoholic fatty liver disease (NAFLD) and development of Non-alcoholic steatohepatitis (NASH). Hepatic SHH detection increases with the diagnosis of NASH. This pilot study was designed to confirm that staining for SHH is useful in NASH diagnosis and determine whether quantification of staining by computer assisted morphometry (CAM) can be used to assess severity of ballooning degeneration. METHODS: SHH was detected by immunohistochemistry (IHC) on paraffin-embedded liver sections in subjects (N = 69) with biopsy proven NAFLD and no liver disease (control). Serum samples were also available for these subjects. Post-staining, a digitized image of the section was acquired and an area quantification algorithm was used to quantify the degree of SHH expression. Additionally, circulating M30, M65, and SHH were measured by ELISA. RESULTS: Notably, hepatic SHH expression correlated with histologic ballooning degeneration (rho = 0.62, p < 0.0001), steatosis grade (rho = 0.554, P < 0.001), Mallory-Denk bodies (rho = 0.54, P < 0.001), pericellular fibrosis (rho = 0.527, P < 0.001), and lymphocytic infiltration (rho = 0.435, P < 0.0002). Additionally, hepatic SHH expression correlated with circulating M65 (rho = 0.588, p < 0.0001), and circulating M30 (rho = 0.375, p = 0.001), as well as AST and ALT (rho = 0.43, p = 0.0004, and rho = 0.27, p = 0.03, respectively). Further, serum M30 was almost twice as high in NASH patients compared to non-NASH (539.1 ± 290.8 U/L vs. 287.6 ± 190.5 U/L; p = 0.0002), while M65 was almost three times higher in NASH patients compared to non-NASH (441.2 ± 464.2 U/L vs. 162.8 ± 353.1 U/L, P = 0.0006). Logistic modeling indicates hepatic SHH expression and presence of type 2 diabetes as independent predictors of advanced fibrosis (defined as portal and pericellular fibrosis > 2: OR = 1.986, p = 0.01, and OR = 3.280, p = 0.03, respectively). CONCLUSION: Thus, our findings show quantitation of SHH expression by CAM can provide a tool for quantifying changes in hepatocyte injury and assist in unambiguous staging/grading of NASH. Our study showed minimal interobserver variability using CAM based quantification. Once validated, CAM assessment of hepatic SHH could benefit clinical trials or long term outcomes studies of NASH subjects.

An exploratory study examining how nano-liquid chromatography-mass spectrometry and phosphoproteomics can differentiate patients with advanced fibrosis and higher percentage collagen in non-alcoholic fatty liver disease.

BACKGROUND: Non-alcoholic steatohepatitis (NASH) is among the leading causes of liver disease worldwide. It is increasingly recognized that the phenotype of NASH may involve a number of different pathways, of which each could become important therapeutic targets. The aim of this study is to use high resolution mass spectrometry (MS) and phosphoproteomics techniques to assess the serum proteome and hepatic phosphoproteome in subjects with NASH-related fibrosis. METHODS: Sixty-seven biopsy-proven NAFLD subjects with frozen sera and liver tissue were included. Reverse phase protein microarray was used to quantify the phosphorylation of key signaling proteins in liver and nano-liquid chromatography (LC)-MS was used to sequence target biomarkers in the serum. An image analysis algorithm was used to quantify the percentage of collagen (% collagen) using computer-assisted morphometry. Using multiple regression models, serum proteomes and phosphorylated hepatic proteins that were independently (p ≤ 0.05) associated with advanced fibrosis (stage ≥ 2) and higher % collagen were assessed. RESULTS: Phosphorylated signaling pathways in the liver revealed that apoptosis signal-regulating kinase 1, mitogen-activated protein kinase (ASK1-MAPK pathway involving ASK1 S38 (p < 0.02) and p38 MAPK (p = 0.0002)) activated by the inflammatory cytokine interleukin (IL-10) (p < 0.001), were independently associated with higher % collagen. LC-MS data revealed that serum alpha-2 macroglobulin (α2M) (p = 0.0004) and coagulation factor V (p = 0.0127) were independently associated with higher % hepatic collagen. CONCLUSIONS: Simultaneous profiling of serum proteome and hepatic phosphoproteome reveals that the activation of ASK1 S38, p38 MAPK in the liver, and serum α2M and coagulation factor V are independently associated with hepatic collagen deposition in patients with NASH. These data suggest the role of these pathways in the pathogenesis of NASH-related fibrosis as a potential therapeutic target.

Anti-adipocyte antibody response in patients with non-alcoholic fatty liver disease.

BACKGROUND AND AIM: A significant number of autoantibodies have been reported in patients with non-alcoholic fatty liver disease (NAFLD) patients. In the present study, our aim was to assess the role of disease and cell-specific antibodies, namely anti-adipocyte antibodies (anti-AdAb) in patients with NAFLD and non-alcoholic steatohepatitis (NASH). METHODS: Flow cytometry was used to detect the presence of anti-AdAb (immunoglobulin M [IgM] and immunoglobulin G [IgG]) in sera from patients with biopsy-proven NAFLD (n = 98) and in controls (n = 49) without liver disease. Univariate and multivariate analysis was performed to draw associations between anti-AdAb IgM and IgG levels and the different clinical variables. RESULTS: Patients with NAFLD had significantly higher levels of anti-AdAb IgM and significantly lower levels of AdAb IgG when compared with controls (P = 0.002 and P < 0.001, respectively). Patients with NASH had significantly higher levels of anti-AdAb IgM when compared with non-NASH NAFLD patients, P = 0.04. In multivariate analysis, anti-AdAb IgM was independently associated with a higher risk for NASH (odds ratio[OR]: 2.90 [confidence interval (CI) 1.18-7.16], P = 0.02). Anti-AdAb IgM was also found to be independently associated with portal inflammation in patients with NAFLD (OR: 3.01 [CI 1.15-7.90 P = 0.02]). CONCLUSIONS: Anti-AdAb IgM was independently associated with NAFLD and NASH while anti-AdAb IgG was found to be protective against NAFLD. Anti-AdAb IgM was found specifically to be associated with the inflammatory processes in NAFLD. These findings indicate that the anti-AdAb IgM and IgG may play an immunomodulatory role in the pathogenesis of NAFLD and NASH.

Major Histocompatibility Complex Class I-Related Chain A Alleles and Histology of Nonalcoholic Fatty Liver Disease.

Major histocompatibility complex class I-related chain A (MICA) is a highly polymorphic gene that modulates immune surveillance by binding to its receptor on natural killer cells, and its genetic polymorphisms have been associated with chronic immune-mediated diseases. The progressive form of nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), is characterized by accumulation of fat and inflammatory cells in the hepatic parenchyma, potentially leading to liver cell injury and fibrosis. To date, there are no data describing the potential role of MICA in the pathogenesis of NAFLD. Therefore, our aim was to assess the association between MICA polymorphism and NASH and its histologic features. A total of 134 subjects were included. DNA from patients with biopsy-proven NAFLD were genotyped using polymerase chain reaction-sequence-specific oligonucleotide for MICA alleles. Liver biopsies were assessed for histologic diagnosis of NASH and specific pathologic features, including stage of fibrosis and grade of inflammation. Multivariate analysis was performed to draw associations between MICA alleles and the different variables; P ≤ 0.05 was considered significant. Univariate analysis showed that MICA*011 (odds ratio [OR], 7.14; 95% confidence interval [CI], 1.24-41.0; P = 0.04) was associated with a higher risk for histologic NASH. Multivariate analysis showed that MICA*002 was independently associated with a lower risk for focal hepatocyte necrosis (OR, 0.24; 95% CI, 0.08-0.74; P = 0.013) and advanced fibrosis (OR, 0.11; 95% CI, 0.02-0.70; P = 0.019). MICA*017 was independently associated with a higher risk for lymphocyte-mediated inflammation (OR, 5.12; 95% CI, 1.12-23.5; P = 0.035). Conclusion: MICA alleles may be associated with NASH and its histologic features of inflammation and fibrosis. Additional research is required to investigate the potential role of MICA in increased risk or protection against NAFLD.

Morphometry Confirms Fibrosis Regression From Sustained Virologic Response to Direct-Acting Antivirals for Hepatitis C.

Sustained virologic response (SVR) after direct-acting antiviral (DAA) therapy for chronic hepatitis C results in significant decreases in liver stiffness measured by transient elastography (TE). The aim of this study was to clarify if TE can guide post-SVR management in patients with advanced fibrosis or cirrhosis prior to treatment as current guidelines are unclear on the role of TE after SVR. In total, 84 patients with hepatitis C virus and advanced fibrosis or cirrhosis and from a single center underwent DAA treatment and achieved SVR. Overall, 62% had improved liver stiffness that was consistent with regression of at least one stage of fibrosis. In the cirrhosis group, 48% showed fibrosis regression by at least two stages by TE (<9.5 kPa). In the F3 fibrosis group, 39% regressed by at least two stages (<7 kPa). The median time from SVR to regression by TE was 1 year. Fifteen patients with liver biopsies prior to SVR underwent a biopsy after SVR; 13 of these patients had improved liver stiffness (to <9.5 kPa). The post-SVR liver biopsies of only 4 patients showed F1-F2 while 11 patients showed F3-F4; however, morphometry of the first 11 biopsied patients revealed that 10 patients had an average 46% decrease in collagen content. Conclusion: This is the first DAA study that also has paired liver biopsies showing fibrosis regression. After SVR is achieved, improvements in liver stiffness measured by TE are seen in a majority of patients with advanced fibrosis/cirrhosis within 2 years. TE improvements are overstated when compared to histologic staging but confirmed with morphometric analysis. It is unclear whether TE following SVR can reliably predict when patients no longer require advanced fibrosis/cirrhosis monitoring after SVR.

Utilization of fluorescence in situ hybridization with cytokeratin discriminators in TOP2A assessment of chemotherapy-treated patients with breast cancer.

Tumor biomarkers increasingly provide information for predicting outcomes with chemotherapeutic regimens (personalized medicine). Topo2A is a DNA helicase targeted by anthracyclines, cytotoxic therapeutics used in both adjuvant and palliative treatments of breast cancer. TOP2A gene amplification/deletion is implicated in response to anthracycline-based chemotherapy. We describe an approach for analyzing formalin-fixed, paraffin-embedded breast tumors on tissue microarrays with TOP2A fluorescence in situ hybridization coupled with cytokeratin immunofluorescence to target tumor cells. Stained tissue from patient specimens was imaged and analyzed using Metafer/Metacyte (Metasystems, Waltham, MA, USA), including customized image classifiers. TOP2A/CEN17 ratios of 2.0 or greater (amplified) and 0.8 or less (deleted) were observed for 10.0% and 6.1% of the patients, respectively. Patient outcomes for adjuvant chemotherapy (cyclophosphamide-epirubicin-fluorouracil, cyclophosphamide-methotrexate-fluorouracil, no chemotherapy) were evaluated. No statistical significance was achieved for clinical end points regarding TOP2A status in anthracycline-treated patients. However, patients with TOP2A aberrations receiving methotrexate-based therapy exhibited a significant decrease in 5-year distant disease-free survival and breast cancer-specific overall survival, especially for patients with TOP2A deletions (disease-free survival: hazard ratio, 5.31 [P = .001], and breast cancer-specific overall survival: hazard ratio, 6.45 [P ≤ .001]). No significant differences were seen in patients included in the no-chemotherapy group. Topo2A protein levels were assessed by immunohistochemistry with no correlative statistical relevance to immunofluorescence/fluorescence in situ hybridization-based prognosis for cyclophosphamide-epirubicin-fluorouracil or cyclophosphamide-methotrexate-fluorouracil groups. Interestingly, aberrant (under)expressing patients in the no-chemotherapy group exhibited better 5-year distant disease-free survival (hazard ratio, 0.39; P = .004), trending toward more favorable breast cancer-specific overall survival (hazard ratio, 0.61; P = .11). Our results indicate a strategy by which fluorescence in situ hybridization scoring targeted to cytokeratin-positive tumor cells may provide a tool for added precision and efficiency in TOP2A evaluation from tumor tissue.