High preoperative levels of serum glypican-3 containing N-terminal subunit are associated with poor prognosis in patients with hepatocellular carcinoma after partial hepatectomy.

Glypican-3 (GPC3) is a glycosylphosphatidylinositol-anchored cell surface glycoprotein overexpressed in hepatocellular carcinoma (HCC) cells and may serve as a potential molecular target for therapeutic intervention. This study evaluated the prognostic significance of serum GPC3 in HCC patients receiving curative surgery. A novel sandwich enzyme-linked immunosorbent assay for the quantitative and sensitive determination of serum GPC3 N-terminal subunit antigen (sGPC3N) was developed and used to measure sGPC3N levels in 25 healthy volunteers and 115 HCC patients who underwent curative partial hepatectomy. The relationships between sGPC3N and clinicopathologic features were analyzed and the prognostic impact on overall survival (OS) or disease-free survival (DFS) was also investigated. Mean and median levels of sGPC3N in healthy controls were 110.12 and 115.95 pg mL(-1) , respectively, with 185.52 pg mL(-1) (mean + 2 SD) being set as the upper limit of the normal range. In HCC patients, sGPC3N levels were significantly increased (mean/median, 405.16/236.19 pg mL(-1) ) compared to healthy controls (p < 0.0001), and 60% of HCC cases (69/115) showed sGPC3N levels that were higher than the upper normal limit. High sGPC3N levels were significantly associated with serum AFP level, high Child-Pugh score and positive HCV. Kaplan-Meier analysis indicated that elevated pre-operative sGPC3N was associated with shorter OS and DFS after hepatectomy (p ≤ 0.01). Multivariate analysis revealed elevated sGPC3N as an independent poor prognostic marker for OS (p < 0.05) and DFS (p < 0.01). The pre-operative sGPC3N level serves as an independent prognostic biomarker in HCC patients.

[Evaluation of serum PIVKA-II by Lumipulse PrestoII assay].

Measurements of serum concentrations of Des-gamma-carboxy Prothrombin (PIVKA-II) are widely used for diagnosing hepatocellular carcinoma (HCC). Recently, in Lumipulsef assay, it was reported that antibodies against alkaline phosphatase (ALP) derived from anti bleeding sheets led false high values of PIVKA-II in the patients with HCC resection. To improve the previous issue, newly developed Lumipulse PrestoII assay was examined. (1) The assay was reliable and positively correlated with the previous assays (Lumipulse f and Picolumi, R = 0.997 and 0.994 (n=115), respectively). (2) Eleven cases, which had false high values of PIVKA-II by the Lumipulsef assay, were examined by the PrestoII assay with excess of inactive ALP. The false high values of 10 cases were improved, but only one was still high. False reactivity of this case was stronger than other cases, more effective adsorption was required. (3) Comparing the absorbent activity of inactive ALP among 6 different kinds, we found inactive ALP with much higher adsorbent activity. When this inactive ALP was applied to assay, false high values of PIVKA-II were improved in all 11 cases. In conclusion, the PrestoII assay, which applies the inactive ALP with high activity, is reliable and useful for clinical screening.

Correction: Serum Wisteria floribunda agglutinin-positive Mac-2-binding protein levels predict the presence of fibrotic nonalcoholic steatohepatitis (NASH) and NASH cirrhosis.

[This corrects the article DOI: 10.1371/journal.pone.0202226.].

Serum Wisteria floribunda agglutinin-positive Mac-2-binding protein levels predict the presence of fibrotic nonalcoholic steatohepatitis (NASH) and NASH cirrhosis.

OBJECTIVE: The race for finding effective treatments for nonalcoholic fatty liver disease (NAFLD) has been slowed down by the high screen-failure rate for including patients in trials due to the lack of a noninvasive biomarker that can identify patients with significant disease. Recently, Wisteria floribunda agglutinin-positive Mac-2-binding protein (WFA+ -M2BP) has shown promise in predicting liver fibrosis. The aims of this study were to evaluate the utility of WFA+ -M2BP as a biomarker to sub-classify patients with NAFLD according to their disease severity and to assess its correlation with histologic features of NAFLD. METHODS: Patients undergoing biopsy for clinical suspicion of NAFLD and healthy controls were included. Patients with NAFLD were classified into: NAFL, early NASH (F0-F1), fibrotic NASH (F2-F3), and NASH cirrhosis (F4). Levels of WFA+ -M2BP in sera was measured by a HISCL™ M2BPGi™ assay kit using an automated immunoanalyzer (HISCL™-800; Sysmex, Kobe, Japan). Analysis of covariance was used to assess difference in WFA+ -M2BP between the groups and Spearman's correlation coefficients were used to assess correlation with histological features. RESULTS: Our cohort consisted of 20 healthy controls and 198 patients with biopsy-proven NAFLD divided as follows: 52 with NAFL, 62 with early NASH, 52 with fibrotic NASH, and 32 with NASH cirrhosis. WFA+ -M2BP level was found to be significantly increased in the fibrotic NASH and NASH cirrhosis groups compared to healthy controls and those with early NAFLD after adjusting for age, gender and BMI. Furthermore, patients with NASH cirrhosis had significantly higher WFA+ -M2BP levels (2.4[1.5, 4.2] C.O.I (Cut-off Index)) than those with fibrotic NASH (1.2[0.79, 1.9]), p < 0.001. WFA+ -M2BP level had moderate correlation with inflammation, ballooning and NAFLD activity score and strong correlation with fibrosis stage. Additionally, ROC curve analysis demonstrated that WFA+ -M2BP accurately differentiated F2-4 from F0-F1. CONCLUSION: In a large cohort of patients with the full spectrum of NAFLD, WFA+ -M2BP levels predicted the presence of advanced disease and correlated strongly with fibrosis stage.

[IgG heterophile antibody causes false positivity for CA19-9, which is overcome with bovine immunoglobulin].

Immunoassay using antibodies is widely applied to measure various clinical parameters such as tumor markers. However, many mechanisms of interference for this method have been reported. We studied serum sample showing false positive CA19-9 values in the range of 24 to approximately 286U/ml depending on the reagent lots used. PEG-pretreated serum did not show false positivity. Using high performance liquid chromatography (HPLC), the CA19-9 false positive peak was obtained around 160kD, which is the same as IgG. Antibody-coated microbeads, labeled antibody and buffer were used in different combinations between different lots, and the buffer was found to be the cause of false positive findings. The amount of bovine IgG contaminated in bovine serum albumin(BSA) in the reagent buffer markedly differed between lots of BSA, and reagent with a low amount of bovine IgG showed false positive results. Bovine immunoglobulin was superior to mouse IgG in the attempt to avoid a false positive reaction. We concluded that the cause of false positive CA19-9 findings in this serum sample was IgG heterophile antibody, which reacts with both of mouse and bovine IgG. The heterophile antibody had greater affinity to bovine IgG than to mouse IgG. The difference between CA19-9 values obtained by different reagent lots was due to different amounts of bovine IgG contaminated in BSA used in the reagent. Bovine immunoglobulin was superior to mouse IgG in the attempt to absorb the heterophile antibody and avoid false positive reaction in this case.