Serum LECT2 levels in mice with β-catenin gene mutated HCC.

<p>A. Representative picture of β-catenin (left panel) and GS (right panel) immunohistochemistry of liver of a tumor bearing mouse at 8 months after DEN/PB treatment. Magnification, 100×. B. Using frozen tissue from a representative tumor, β-catenin gene exon-3 mutation affecting codon 33 (red box) was confirmed by direct sequencing. C. Serum LECT2 levels were significantly (*) increased in tumor bearing versus non-tumor bearing DEN/PB treated mice as analyzed by ELISA. (* <i>p</i><0.01). D. Representative pictures of frozen sections from which tumors (T1-T3) were scraped for direct sequencing. E. Sequence analysis from three tumor lesions (T1-T3) show S33Y-β-catenin gene mutations in codon 33 (red boxes) by direct sequencing. F. <i>Glutamine Synthetase (Glul)</i> and <i>Lect2</i> expression in three tumor lesions (T1-T3) were assessed by qRT-PCR. Gene expression of background liver tissues surrounding tumor are shown as N.</p

Clinical characteristics of control patients with cirrhosis but no HCC.

†<p>HCV, hepatitis type C virus; ETOH, alcoholic; NASH, non-alcoholic steatohepatitis; AIH, autoimmune hepatitis.</p><p>*serum LECT2 level value was very high (103.9 ng/mL).</p

Candidate genes encoding secreted protein.

<p>Candidate genes encoding secreted protein.</p

The role of serum LECT2 level as a diagnostic biomarker in HCC.

<p>A. Serum LECT2 levels in all HCC patients as compared to patients with chronic liver fibrosis (CH/LC), and healthy volunteer (HV) as assessed by ELISA. (*<i>p</i><0.01). B. ROC analysis for the utility of LECT2 as a diagnostic marker of HCC with AUC = 0.82. C. Fisher's Exact test shows that based on the cut-off value of serum LECT2 level at 50 ng/mL, sensitivity, specificity, positive predictive value, negative predictive value for the diagnosis of HCC were 59.3%, 96.1%, 97.0%, and 53.2%, respectively.</p

No correlation of β-catenin mutations or β-catenin activation to serum LECT2 levels in patients.

<p>A. Serum LECT2 levels in patients with HCC with <i>CTNNB1</i> mutations, absent <i>CTNNB1</i> mutations, patients with chronic liver fibrosis (CH/LC), and healthy volunteer (HV) as assessed by ELISA. (*<i>p</i><0.05). B. No correlation observed between <i>LECT2</i> expression in tumor and serum levels of LECT2 in HCC patients (n = 28). C. No correlation observed between <i>LECT2</i> expression in β-catenin mutated tumors and serum levels of LECT2 in these HCC patients (n = 4). D. No correlation observed between <i>LECT2</i> expression in non-β-catenin mutated tumors and serum levels of LECT2 in these HCC patients (n = 24). E. Heat map shows expression of β-catenin target genes in β-catenin-mutated (MT) and non-mutated wild-type (WT) HCC patients (n = 28). Genes assessed included <i>AXIN2</i>, <i>REGUCALCIN</i>, <i>LECT2</i>, and <i>GLUL</i>. (+) indicates β-catenin activity as seen by increased expression of at least 2 target genes whereas (−) indicates absent β-catenin activation reflected by lack of target gene expression. F. Serum LECT2 levels showed insignificant difference in HCC patients that lacked β-catenin gene mutations but showed high expression of β-catenin target genes versus patients who have neither β-catenin gene mutations nor any increase in β-catenin target gene expression. G. β-Catenin target gene expression shown by qRT-PCR. Steel-Dwass test was performed to compare the values among three groups. *, <i>p</i><0.05. (+) indicates β-catenin activity as seen by increased expression of at least 2 target genes whereas (−) indicates lack of β-catenin activity due lack of target gene expression.</p

Regulation of LECT2 expression by β-catenin.

<p>A. Strategy to identify biomarker for β-catenin activation. Microarray analysis was performed using liver tissue from hepatocyte-specific β-catenin knockout (KO) and wild-type (WT) mice, which identified 14 secreted targets. <i>Lect</i>2 expression was 117-fold lower in KO livers. B. β-Catenin expression in Hep3B cells and stable cell lines established with wild-type β-catenin (Hep3B WT)- or mutated β-catenin (Hep3B S33Y)-transfected cells. C. Representative Western blot shows increased LECT2 protein levels in Hep3B S33Y cells as compared to Hep3B WT. D. Hep3B S33Y cells transfected with either <i>β-catenin</i> or <i>control</i> siRNA showed decreased β-catenin and LECT2 protein levels in a representative Western blot. E. Increased LECT2 protein levels were observed in culture media collected from Hep3B S33Y cells as compared to Hep3B WT as analyzed by ELISA. Basal media was used as a negative control. F. Occupation of <i>Lect2</i> promoter by TCF4 especially in Hep3B S33Y cells was as assessed by ChIP. Albumin promoter is not regulated by β-catenin but by HNF1α, which is used as quality control for chromatin.</p

Clinical characteristics of patients with HCC.

<p>Clinical characteristics of patients with HCC.</p