Metabolomic-Based Noninvasive Serum Test to Diagnose Nonalcoholic Steatohepatitis: Results From Discovery and Validation Cohorts

Nonalcoholic fatty liver disease (NAFLD) is the most common type of chronic liver disease worldwide and includes a broad spectrum of histologic phenotypes, ranging from simple hepatic steatosis or nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH). While liver biopsy is the reference gold standard for NAFLD diagnosis and staging, it has limitations due to its sampling variability, invasive nature, and high cost. Thus, there is a need for noninvasive biomarkers that are robust, reliable, and cost effective. In this study, we measured 540 lipids and amino acids in serum samples from biopsy-proven subjects with normal liver (NL), NAFL, and NASH. Using logistic regression analysis, we identified two panels of triglycerides that could first discriminate between NAFLD and NL and second between NASH and NAFL. These noninvasive tests were compared to blinded histology as a reference standard. We performed these tests in an original cohort of 467 patients with NAFLD (90 NL, 246 NAFL, and 131 NASH) that was subsequently validated in a separate cohort of 192 patients (7 NL, 109 NAFL, 76 NASH). The diagnostic performances of the validated tests showed an area under the receiver operating characteristic curve, sensitivity, and specificity of 0.88 +/- 0.05, 0.94, and 0.57, respectively, for the discrimination between NAFLD and NL and 0.79 +/- 0.04, 0.70, and 0.81, respectively, for the discrimination between NASH and NAFL. When the analysis was performed excluding patients with glucose levels >136 mg/dL, the area under the receiver operating characteristic curve for the discrimination between NASH and NAFL increased to 0.81 +/- 0.04 with sensitivity and specificity of 0.73 and 0.80, respectively. Conclusion: The assessed noninvasive lipidomic serum tests distinguish between NAFLD and NL and between NASH and NAFL with high accuracy.Supported by the National Institutes of Health Blueprint for Neuroscience Research (R01AT001576 to S.C.L., J.M.M.), Agencia Estatal de Investigacion of the Ministerio de Economia, Industria y Competitividad (SAF2014-52097R to J.M.M.), CIBER Hepatic and Digestive Diseases and Instituto de Salud Carlos III (PIE14/0003 to J.M.M.), Etorgai 2015-Gobierno Vasco (ER-2015/00015 to R.M., I.M.A., C.A., A.C.), Plan de Promocion de la Innovacion 2015-Diputacion Foral de Bizkaia (6/12/IN/2015/00131 to A.C., C.A.), National Institute of Diabetes and Digestive and Kidney Diseases (RO1DK81410 to A.J.S.), and Czech Ministry of Health (RVO VFN64165 to L.V.)

Histopatologicka diagnostika chronickych hepatitid.

Liver biopsy interpretation changed substantionally with the change of chronic hepatitis classification. Grading and staging of chronic hepatitis appears to be one of the most important tasks for liver biopsy. Conventional verbal approach does not seem to fulfill demands of precise and reproducible methods of evaluation mild forms of chronic hepatitis, mostly hepatitis C. There are many semiquantitative scoring systems for numeric grading and staging. These systems are used to initiate treatment and to compare different liver samples in an individual patient to follow and to manage the development of his/her disease as well as in large cohorts of patients in clinical trials. There is no consensus on the use of these systems. Scientific papers evaluating individual scoring systems are rare and papers evaluating and comparing different systems are extremely rare. Intraobserver reproducibility of Knodell HAI (Desmet modification, 25 cases), Ishak HAI (45 cases) and Scheuer system (25 cases) were studied with the use of kappa statistics. Total activity score reproducibility is fair in both modifications of HAI (kappa 0,23 and 0,25) while it is good in Scheuer system (kappa 0,65). For interface hepatitis moderate reproducibility (kappa 0,52) and moderate/good (kappa 0,95) were reached in Desmet HAI and Ishak HAI modifications respectively. It was very good (kappa 0,65) in Scheuer system. Reproducibility of lobular activity was fair in both modifications of HAI (kappa 0,56 and 0,46) and good in Scheuer system (kappa 0,66). Portal inflammation showed minimal reproducibility in Desmet HAI modification (kappa 0,17) and fair agreement in Ishak HAI modification. The construction of Scheuer system does not allow to evaluate portal inflammation grading. The reproducibility of staging score was good/very good in Desmet HAI modification (kappa 0,71) and very good in Ishak HAI modification (kappa 0,71) as well as in Scheuer system (kappa 0,87 and 0,95 respectively). Suggestions to improve definitions in Ishak system, to standardize microscopic field for local lobular activity grading and to standardize liver biopsy format are done. CD45RO method is a suitable method to evaluate focal lobular activity and piecemeal necrosis in difficult cases.Available from STL Prague, CZ / NTK - National Technical LibrarySIGLECZCzech Republi

Aberrantly elevated suprabasin in the bone marrow as a candidate biomarker of advanced disease state in myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are preleukemic disorders characterized by clonal growth of mutant hematopoietic stem and progenitor cells. MDS are associated with proinflammatory signaling, dysregulated immune response, and cell death in the bone marrow (BM). Aging, autoinflammation and autoimmunity are crucial features of disease progression, concordant with promoting growth of malignant clones and accumulation of mutations. Suprabasin (SBSN), a recently proposed proto‐oncogene of unknown function, physiologically expressed in stratified epithelia, is associated with poor prognosis of several human malignancies. Here, we showed that SBSN is expressed in the BM by myeloid cell subpopulations, including myeloid‐derived suppressor cells, and is secreted into BM plasma and peripheral blood of MDS patients. The highest expression of SBSN was present in a patient group with poor prognosis. SBSN levels in the BM correlated positively with blast percentage and negatively with CCL2 chemokine levels and lymphocyte count. In vitro treatment of leukemic cells with interferon‐gamma and demethylating agent 5‐azacytidine (5‐AC) induced SBSN expression. This indicated that aberrant cytokine levels in the BM and epigenetic landscape modifications in MDS patients may underlie ectopic expression of SBSN. Our findings suggest SBSN as a candidate biomarker of high‐risk MDS with a possible role in disease progression and therapy resistance

Low Plasma Citrate Levels and Specific Transcriptional Signatures Associated with Quiescence of CD34+ Progenitors Predict Azacitidine Therapy Failure in MDS/AML Patients

To better understand the molecular basis of resistance to azacitidine (AZA) therapy in myelodysplastic syndromes (MDS) and acute myeloid leukemia with myelodysplasia-related changes (AML-MRC), we performed RNA sequencing on pre-treatment CD34+ hematopoietic stem/progenitor cells (HSPCs) isolated from 25 MDS/AML-MRC patients of the discovery cohort (10 AZA responders (RD), six stable disease, nine progressive disease (PD) during AZA therapy) and from eight controls. Eleven MDS/AML-MRC samples were also available for analysis of selected metabolites, along with 17 additional samples from an independent validation cohort. Except for two patients, the others did not carry isocitrate dehydrogenase (IDH)1/2 mutations. Transcriptional landscapes of the patients’ HSPCs were comparable to those published previously, including decreased signatures of active cell cycling and DNA damage response in PD compared to RD and controls. In addition, PD-derived HSPCs revealed repressed markers of the tricarboxylic acid cycle, with IDH2 among the top 50 downregulated genes in PD compared to RD. Decreased citrate plasma levels, downregulated expression of the (ATP)-citrate lyase and other transcriptional/metabolic networks indicate metabolism-driven histone modifications in PD HSPCs. Observed histone deacetylation is consistent with transcription-nonpermissive chromatin configuration and quiescence of PD HSPCs. This study highlights the complexity of the molecular network underlying response/resistance to hypomethylating agents

Use of Non-Invasive Parameters of Non-Alcoholic Steatohepatitis and Liver Fibrosis in Daily Practice - An Exploratory Case-Control Study

<div><p>Background</p><p>Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of a metabolic syndrome. To date, liver biopsy has been the gold standard used to differentiate between simple steatosis and steatohepatitis/fibrosis. Our aim was to compare the relevance of serum non-invasive parameters and scoring systems in the staging of liver fibrosis and non-alcoholic steatohepatitis (NASH) in patients with NAFLD.</p><p>Methods and Findings</p><p>A total of 112 consecutive patients diagnosed with NAFLD were included. A liver biopsy was performed on 56 patients. The Kleiner score was used for the staging and grading of the histology. Non-invasive parameters for fibrosis (hyaluronic acid; AST/ALT; fibrosis scoring indexes OELF, ELF, BARD score, APRI, NAFLD fibrosis score); and inflammation (M30 and M65 cytokeratin-18 fragments) were measured and calculated. The same analyses were performed in 56 patients diagnosed with NAFLD, who were not indicated for liver biopsy. Based on the liver histology, NASH was diagnosed in 38 patients; simple steatosis in 18 patients. A cut-off value of 750 U/L of serum M65 discriminated patients with and without NASH with a 80% sensitivity and 82% specificity (95% CI:57–95). Fibrosis stage F0–F2 was present in 39 patients; F3–F4 in 17 patients. Serum concentrations of hyaluronic acid were higher in patients with advanced fibrosis (p<0.01); a cut-off value of 25 µg/l discriminated patients with F3–F4 with a 90% sensitivity and 84% specificity from those with F0–F2 (95% CI:59–99). When applying the non-invasive criteria to those patients without a liver biopsy, NASH could only be diagnosed in 16%; however, advanced fibrosis could be diagnosed in 35% of them.</p><p>Conclusions</p><p>In patients with NAFLD, non-invasive serum parameters with a high accuracy can differentiate those patients with NASH and/or advanced fibrosis from those with simple steatosis. A substantial portion of those patients not indicated for liver biopsy might have undiagnosed advanced fibrosis.</p></div

Sensitivity and specificity of different parameters for differentiation between those patients with significant fibrosis (F3,4) and those with none to moderate fibrosis (F0–F2).

<p>HA: hyaluronic acid, CI: confidence interval.</p><p>Sensitivity and specificity of different parameters for differentiation between those patients with significant fibrosis (F3,4) and those with none to moderate fibrosis (F0–F2).</p

Sensitivity and specificity of different parameters for differentiation between the patients with and without histological sings of NASH.

<p>M30, M65: fragments of cytokeratin-18, GGT: γ-Glutamyltransferase, CI: confidence interval.</p><p>Sensitivity and specificity of different parameters for differentiation between the patients with and without histological sings of NASH.</p

Clinical and laboratory data in patients with NAFLD, and the presence or absence of histological signs of NASH.

<p>BMI: body mass index, GGT: γ-Glutamyltransferase, PIIINP: aminoterminal peptide of pro-collagen III, TIMP-1: tissue inhibitor of matrix metalloproteinase 1, IL 2: interleukin 2, IL 6: interleukin 6, TNFα: tumor necrosis factor alpha, M30, M65: fragments of cytokeratin-18, hsCRP: high sensitive C-reactive protein, HA: hyaluronic acid, ns: non-significant. The results are given as mean ±standard deviation.</p><p>Clinical and laboratory data in patients with NAFLD, and the presence or absence of histological signs of NASH.</p

Comparison of clinical and laboratory parameters between NAFLD patients and control subjects.

<p>IL 2: interleukin 2, IL 6: interleukin 6, TNFα: tumor necrosis factor alpha, M30, M65: fragments of cytokeratin-18, hsCRP: high sensitive C-reactive protein, HA: hyaluronic acid, ns: non-significant. The results are given as mean ±standard deviation.</p><p>Comparison of clinical and laboratory parameters between NAFLD patients and control subjects.</p

Correlation between stage of fibrosis and clinical parameters, laboratory parameters, and fibrosis indexes.

<p>BMI: body mass index, TIMP-1: tissue inhibitor of matrix metalloproteinase 1, IL 6: interleukin 6, HA: hyaluronic acid.</p><p>Correlation between stage of fibrosis and clinical parameters, laboratory parameters, and fibrosis indexes.</p