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Role of leukocyte cell-derived chemotaxin 2 as a biomarker in hepatocellular carcinoma.

Okabe H, Delgado E, Lee JM, Yang J, Kinoshita H, Hayashi H, Tsung A, Behari J, Beppu T, Baba H, Monga SP - PLoS ONE (2014)

Bottom Line: Leukocyte cell-derived chemotaxin-2 (LECT2) expression was decreased in KO livers.Hep3BS33Y expressed and secreted more LECT2 in media as compared to Hep3BWT.Intriguingly, patients without β-catenin mutations showed significantly higher serum LECT2 levels (54.26 ± 22.25 ng/mL; n = 46).

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America; Department of Gastroenterological Surgery, Graduate School of Life Sciences, Kumamoto University, Kumamoto, Japan.

ABSTRACT
We sought to identify a secreted biomarker for β-catenin activation commonly seen in hepatocellular carcinoma (HCC). By examination of our previously published genearray of hepatocyte-specific β-catenin knockout (KO) livers, we identified secreted factors whose expression may be β-catenin-dependent. We verified expression and secretion of the leading factor in HCC cells transfected with mutated (Hep3BS33Y)-β-catenin. Serum levels of biomarker were next investigated in a mouse model of HCC with β-catenin gene (Ctnnb1) mutations and eventually in HCC patients. Leukocyte cell-derived chemotaxin-2 (LECT2) expression was decreased in KO livers. Hep3BS33Y expressed and secreted more LECT2 in media as compared to Hep3BWT. Mice developing HCC with Ctnnb1 mutations showed significantly higher serum LECT2 levels. However patients with CTNNB1 mutations showed LECT2 levels of 54.28 ± 22.32 ng/mL (Mean ± SD; n = 8) that were insignificantly different from patients with non-neoplastic chronic liver disease (32.8 ± 21.1 ng/mL; n = 15) or healthy volunteers (33.2 ± 7.2 ng/mL; n = 11). Intriguingly, patients without β-catenin mutations showed significantly higher serum LECT2 levels (54.26 ± 22.25 ng/mL; n = 46). While β-catenin activation was evident in a subset of non-mutant β-catenin HCC group with high LECT2 expression, serum LECT2 was unequivocally similar between β-catenin-active and -normal group. Further analysis showed that LECT2 levels greater than 50 ng/ml diagnosed HCC in patients irrespective of β-catenin mutations with specificity of 96.1% and positive predictive value of 97.0%. Thus, LECT2 is regulated by β-catenin in HCC in both mice and men, but serum LECT2 reflects β-catenin activity only in mice. Serum LECT2 could be a potential biomarker of HCC in patients.

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No correlation of β-catenin mutations or β-catenin activation to serum LECT2 levels in patients.A. Serum LECT2 levels in patients with HCC with CTNNB1 mutations, absent CTNNB1 mutations, patients with chronic liver fibrosis (CH/LC), and healthy volunteer (HV) as assessed by ELISA. (*p<0.05). B. No correlation observed between LECT2 expression in tumor and serum levels of LECT2 in HCC patients (n = 28). C. No correlation observed between LECT2 expression in β-catenin mutated tumors and serum levels of LECT2 in these HCC patients (n = 4). D. No correlation observed between LECT2 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 AXIN2, REGUCALCIN, LECT2, and GLUL. (+) 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. *, p<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.
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pone-0098817-g003: No correlation of β-catenin mutations or β-catenin activation to serum LECT2 levels in patients.A. Serum LECT2 levels in patients with HCC with CTNNB1 mutations, absent CTNNB1 mutations, patients with chronic liver fibrosis (CH/LC), and healthy volunteer (HV) as assessed by ELISA. (*p<0.05). B. No correlation observed between LECT2 expression in tumor and serum levels of LECT2 in HCC patients (n = 28). C. No correlation observed between LECT2 expression in β-catenin mutated tumors and serum levels of LECT2 in these HCC patients (n = 4). D. No correlation observed between LECT2 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 AXIN2, REGUCALCIN, LECT2, and GLUL. (+) 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. *, p<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.

Mentions: Sera were available from 54 HCC patients through appropriate IRB approvals. Eight of the 54 patients showed β-catenin gene (CTNNB1) alterations in the form of missense mutations in exon-3 (Table 2). Remaining 46 patients lacked any genetic alterations in exon-3 of CTNNB1. Additionally, we enrolled healthy volunteers and also patients with cirrhosis due to chronic liver disease (Table 3) to determine serum LECT2 levels and address its efficacy as a tumor marker. Based on Tukey-Kramer post hoc test, serum LECT2 levels in patients with mutated β-Catenin (54.28±22.32 ng/mL; n = 8) were not statistically different from either patients with cirrhosis (32.8±21.1 ng/mL, p = 0.091; n = 15) or healthy volunteers (33.2±7.2 ng/mL, p = 0.137; n = 11). On the other hand, patients who did not harbor CTNNB1 mutations showed significantly higher LECT2 level (54.26±22.25 ng/mL; n = 46) than those with cirrhosis and from healthy volunteers (p = 0.0044 and 0.0176, respectively) (Figure 3A).


Role of leukocyte cell-derived chemotaxin 2 as a biomarker in hepatocellular carcinoma.

Okabe H, Delgado E, Lee JM, Yang J, Kinoshita H, Hayashi H, Tsung A, Behari J, Beppu T, Baba H, Monga SP - PLoS ONE (2014)

No correlation of β-catenin mutations or β-catenin activation to serum LECT2 levels in patients.A. Serum LECT2 levels in patients with HCC with CTNNB1 mutations, absent CTNNB1 mutations, patients with chronic liver fibrosis (CH/LC), and healthy volunteer (HV) as assessed by ELISA. (*p<0.05). B. No correlation observed between LECT2 expression in tumor and serum levels of LECT2 in HCC patients (n = 28). C. No correlation observed between LECT2 expression in β-catenin mutated tumors and serum levels of LECT2 in these HCC patients (n = 4). D. No correlation observed between LECT2 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 AXIN2, REGUCALCIN, LECT2, and GLUL. (+) 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. *, p<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.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4043833&req=5

pone-0098817-g003: No correlation of β-catenin mutations or β-catenin activation to serum LECT2 levels in patients.A. Serum LECT2 levels in patients with HCC with CTNNB1 mutations, absent CTNNB1 mutations, patients with chronic liver fibrosis (CH/LC), and healthy volunteer (HV) as assessed by ELISA. (*p<0.05). B. No correlation observed between LECT2 expression in tumor and serum levels of LECT2 in HCC patients (n = 28). C. No correlation observed between LECT2 expression in β-catenin mutated tumors and serum levels of LECT2 in these HCC patients (n = 4). D. No correlation observed between LECT2 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 AXIN2, REGUCALCIN, LECT2, and GLUL. (+) 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. *, p<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.
Mentions: Sera were available from 54 HCC patients through appropriate IRB approvals. Eight of the 54 patients showed β-catenin gene (CTNNB1) alterations in the form of missense mutations in exon-3 (Table 2). Remaining 46 patients lacked any genetic alterations in exon-3 of CTNNB1. Additionally, we enrolled healthy volunteers and also patients with cirrhosis due to chronic liver disease (Table 3) to determine serum LECT2 levels and address its efficacy as a tumor marker. Based on Tukey-Kramer post hoc test, serum LECT2 levels in patients with mutated β-Catenin (54.28±22.32 ng/mL; n = 8) were not statistically different from either patients with cirrhosis (32.8±21.1 ng/mL, p = 0.091; n = 15) or healthy volunteers (33.2±7.2 ng/mL, p = 0.137; n = 11). On the other hand, patients who did not harbor CTNNB1 mutations showed significantly higher LECT2 level (54.26±22.25 ng/mL; n = 46) than those with cirrhosis and from healthy volunteers (p = 0.0044 and 0.0176, respectively) (Figure 3A).

Bottom Line: Leukocyte cell-derived chemotaxin-2 (LECT2) expression was decreased in KO livers.Hep3BS33Y expressed and secreted more LECT2 in media as compared to Hep3BWT.Intriguingly, patients without β-catenin mutations showed significantly higher serum LECT2 levels (54.26 ± 22.25 ng/mL; n = 46).

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America; Department of Gastroenterological Surgery, Graduate School of Life Sciences, Kumamoto University, Kumamoto, Japan.

ABSTRACT
We sought to identify a secreted biomarker for β-catenin activation commonly seen in hepatocellular carcinoma (HCC). By examination of our previously published genearray of hepatocyte-specific β-catenin knockout (KO) livers, we identified secreted factors whose expression may be β-catenin-dependent. We verified expression and secretion of the leading factor in HCC cells transfected with mutated (Hep3BS33Y)-β-catenin. Serum levels of biomarker were next investigated in a mouse model of HCC with β-catenin gene (Ctnnb1) mutations and eventually in HCC patients. Leukocyte cell-derived chemotaxin-2 (LECT2) expression was decreased in KO livers. Hep3BS33Y expressed and secreted more LECT2 in media as compared to Hep3BWT. Mice developing HCC with Ctnnb1 mutations showed significantly higher serum LECT2 levels. However patients with CTNNB1 mutations showed LECT2 levels of 54.28 ± 22.32 ng/mL (Mean ± SD; n = 8) that were insignificantly different from patients with non-neoplastic chronic liver disease (32.8 ± 21.1 ng/mL; n = 15) or healthy volunteers (33.2 ± 7.2 ng/mL; n = 11). Intriguingly, patients without β-catenin mutations showed significantly higher serum LECT2 levels (54.26 ± 22.25 ng/mL; n = 46). While β-catenin activation was evident in a subset of non-mutant β-catenin HCC group with high LECT2 expression, serum LECT2 was unequivocally similar between β-catenin-active and -normal group. Further analysis showed that LECT2 levels greater than 50 ng/ml diagnosed HCC in patients irrespective of β-catenin mutations with specificity of 96.1% and positive predictive value of 97.0%. Thus, LECT2 is regulated by β-catenin in HCC in both mice and men, but serum LECT2 reflects β-catenin activity only in mice. Serum LECT2 could be a potential biomarker of HCC in patients.

Show MeSH
Related in: MedlinePlus