Comprehensive clinicopathologic study of alpha fetoprotein-expression in a large cohort of patients with hepatocellular carcinoma.

Alpha fetoprotein (AFP) is the most widely used diagnostic and prognostic serum biomarker for hepatocellular carcinoma (HCC). Despite its wide clinical use, a systematic clinicopathologic study comparing AFP expression in HCC in situ with serum AFP concentrations has not yet been conducted. To analyze AFP expression in a large cohort of patients by immunohistochemistry, we employed a comprehensive tissue microarray with 871 different HCCs of overall 561 patients. AFP immunoreactivity was detected in only about 20% of HCC core biopsies, whereas 48.9% of the patients displayed increased serum values (>12 ng/mL). Immunostaining of whole tumor slides revealed that lack of detectable immunoreactivity in core biopsies in a subgroup of patients with elevated AFP serum concentrations is due to heterogeneous intratumoral AFP expression. Serum AFP concentrations and AFP expression in situ were moderately correlated (Spearman's rank correlation coefficient .53, P = 1.2e - 13). High AFP expression detected in serum (>227.3 ng/mL) or in situ predicted unfavorable prognosis and was associated with vascular invasion, higher tumor grade and macrotrabecular-massive tumor subtype. Multivariate and ROC curve analysis demonstrated that high AFP concentrations in serum is an independent prognostic parameter and represents the more robust prognostic predictor in comparison to AFP immunostaining of core biopsies. The previously published vessels encapsulating tumor clusters (VETC) pattern turned out as an additional, statistically independent prognostic parameter. AFP-positivity was associated with increased tumor cell apoptosis, but not with increased vascular densities. Additionally, AFP-positive tumors displayed increased proliferation rates, urea cycle dysregulation and signs of genomic instability, which may constitute the basis for their increased aggressiveness.

HOXD10 expression as a prognostic factor for hepatocellular carcinoma treated with curative resection.

PURPOSE: HOXD10 downregulation resulting from epigenetic changesas well as its role as a tumor suppressor have been reported in several cancers including hepatocellular carcinomas (HCCs). However, the prognostic role of HOXD10 expression in HCC tissue samples has not been evaluated. METHODS: HOXD10 expression was investigated in 278 curatively resected HCC samples using immunohistochemistry and its effectiveness in predicting patient outcome was analyzed. RESULTS: Low expression of HOXD10 was observed in 82.7% of HCC samples, and this was associated with increased age, large tumor size and advanced stage.HOXD10 was an independent predictive factor for early tumor recurrence at less than 2 years. Patients with low HOXD10 expression showed shorter recurrence-free survival (RFS) (p=0.024) and disease-specific survival (DSS) (p=0.016) than those with high expression. Multivariate analysis confirmed that low HOXD10 expression was an independent predictor of shorter RFS (hazard ratio 1.873, p=0.006) and DSS (hazard ratio2.504, p=0.012) than high HOXD10 expression. CONCLUSIONS: The present study provides clinical evidence supporting the use of HOXD10 as a prognostic biomarker in curatively resected HCCs, and suggests that HOXD10 could also be a potential therapeutic target in HCC.

Pembrolizumab plus lenvatinib with or without hepatic arterial infusion chemotherapy in selected populations of patients with treatment-naive unresectable hepatocellular carcinoma exhibiting PD-L1 staining: a multicenter retrospective study.

BACKGROUND: Not all patients with unresectable hepatocellular carcinoma (uHCC) benefit from treatment with immune checkpoint inhibitors and molecular-targeted agents. The aim of this retrospective study was to assess the efficacy and safety of pembrolizumab plus lenvatinib plus hepatic arterial infusion chemotherapy (HAIC) versus pembrolizumab plus lenvatinib in selected populations of patients with treatment-naive uHCC exhibiting programmed cell death ligand-1 (PD-L1) staining. METHODS: Consecutive patients with treatment-naive uHCC exhibiting PD-L1 staining who were treated with pembrolizumab plus lenvatinib plus HAIC (PLH) or pembrolizumab plus lenvatinib (PL) were retrospectively identified from our medical centres from 2018 to 2021. HAIC involved oxaliplatin, fluorouracil, and leucovorin (FOLFOX). Follow-up occurred every 3 weeks for 1 year and then every 6 weeks thereafter. The primary endpoints included overall survival (OS) and progression-free survival (PFS). Secondary endpoints were the frequency of key adverse events (AEs). RESULTS: In total, 248 treatment-naive patients were retrospectively reviewed, 78 of whom were ineligible on the basis of the current criteria. Thus, 170 patients (PLH: n = 84, median age 52 years [range, 42-67]; PL: n = 86, 53 years [range, 43-69]) were eligible for the analysis. The median follow-up was 18.6 months (range, 1-26). At the final follow-up, the median OS was 17.7 months (95% confidence interval [CI], 15.2-18.3) in the PLH group versus 12.6 months (95% CI, 11.1-13.7) in the PL group (hazard ratio [HR] 0.52; 95% CI, 0.36-0.75; p = 0.001). A significant difference was also detected in the median PFS (10.9 months [95% CI, 8.7-11.4] for PLH vs. 6.8 months (95% CI, 5.2-7.4) for PL; HR 0.61, 95% CI, 0.43-0.85; p = 0.001). Significant differences in the rate of the key AEs were noted between groups (79.8% for PLH vs. 62.8% for PL, p = 0.015), but these AEs were controllable. CONCLUSIONS: Among selected populations of patients with treatment-naive uHCC exhibiting PD-L1 staining, the PLH regimen may substantially improve the survival benefits compared with the PL regimen with a controllable safety profile.

Integrated LC-MS metabolomics with dual derivatization for quantification of FFAs in fecal samples of hepatocellular carcinoma patients.

FFAs display pleiotropic functions in human diseases. Short-chain FAs (SCFAs), medium-chain FAs, and long-chain FAs are derived from different origins, and precise quantification of these FFAs is critical for revealing their roles in biological processes. However, accessing stable isotope-labeled internal standards is difficult, and different chain lengths of FFAs challenge the chromatographic coverage. Here, we developed a metabolomics strategy to analyze FFAs based on isotope-free LC-MS-multiple reaction monitoring integrated with dual derivatization. Samples and dual derivatization internal standards were synthesized using 2-dimethylaminoethylamine or dansyl hydrazine as a "light" label and N,N-diethyl ethylene diamine or N,N-diethyldansulfonyl hydrazide as a "heavy" label under mild and efficient reaction conditions. General multiple reaction monitoring parameters were designed to analyze these FFAs. The limit of detection of SCFAs varied from 0.5 to 3 nM. Furthermore, we show that this approach exhibits good linearity (R2 = 0.99374-0.99929), there is no serious substrate interference, and no quench steps are required, confirming the feasibility and reliability of the method. Using this method, we successfully quantified 15 types of SCFAs in fecal samples from hepatocellular carcinoma patients and healthy individuals; among these, propionate, butyrate, isobutyrate, and 2-methylbutyrate were significantly decreased in the hepatocellular carcinoma group compared with the healthy control group. These results indicate that the integrated LC-MS metabolomics with isotope-free and dual derivatization is an efficient approach for quantifying FFAs, which may be useful for identifying lipid biomarkers of cancer.

Efficacy of expressions of Arg-1, Hep Par-1, and CK19 in the diagnosis of the primary hepatocellular carcinoma subtypes and exclusion of the metastases.

Many conflicts arise using immunohistochemistry of Hepatocellular carcinoma (HCC), some of these conflicts arise from the biliary part within the tumor itself or from liver metastasis. The aim of this study is to investigate the extent of Arg-1, HepPar-1, and CK-19 expressions in the primary HCC subtypes as well as studying of some metastatic cases to find a distinctive immunohistochemical panel utilizing it to differentiate between these entities. MATERIAL AND METHODS: A paraffin-embedded block including 62 cases of primary HCC, and 18 cases diagnosed as metastatic tumors, were subjected for this study using Anti-liver Arginase antibody (ab125134 Cambridge, USA, polyclonal antibody, 3.75 µg/ml), HepPar-1 (polyclonal mouse antibody OCH1E5; 1:600; DAKO, CA, USA), and CK 19 Anti-Cytokeratin 19 antibody (ab15463, rabbit polyclonal antibody; 1:100; Cambridge, USA). The intensity of immunostaining was scored (0 to 3+). Nuclear and cytoplasmic staining with Arg-1 and cytoplasmic for both HepPar-1 and CK 19 are reported. RESULTS: The histopathological patterns were mainly trabecular no= (24, 38.7%), and pseudoglandular (no=14, 22.5%), mixed hepatocellular cholangiocarcinoma was observed in one case (1.6%). Arginase-1 positivity was in 55 cases (88.7%) opposite to 46 (74.19%) and 8 (12.9%) for HepPAr.1% -1 and CK 19, respectively. The intensity of expression was marked in well and moderate differentiation for Arg-1 and HepPar-1and in poorly differentiated for CK 19. Metastatic carcinoma cases revealed two cases positive for Arg-1 (11.1%), 4 cases (22.2%) positive for HepPar-1, and 13 cases (72.2%) positive for CK 19. CONCLUSION: Arg-1 and HepPar-1 are confirmative in the diagnosis of primary HCC in most cases, either separately or collectively but the priority of selection leans more towards Arg-1. Arg-1 and HepPar-1 positive with negative CK 19 expressions give more support to diagnosis of primary HCC while the reverse will support the diagnosis of tumour of biliary origin or liver metastasis.

Pharmacodynamic Biomarkers Predictive of Survival Benefit with Lenvatinib in Unresectable Hepatocellular Carcinoma: From the Phase III REFLECT Study.

PURPOSE: In REFLECT, lenvatinib demonstrated an effect on overall survival (OS) by confirmation of noninferiority to sorafenib in unresectable hepatocellular carcinoma. This analysis assessed correlations between serum or tissue biomarkers and efficacy outcomes from REFLECT. EXPERIMENTAL DESIGN: Serum biomarkers (VEGF, ANG2, FGF19, FGF21, and FGF23) were measured by ELISA. Gene expression in tumor tissues was measured by the nCounter PanCancer Pathways Panel. Pharmacodynamic changes in serum biomarker levels from baseline, and associations of clinical outcomes with baseline biomarker levels, were evaluated. RESULTS: Four hundred and seven patients were included in the serum analysis set (lenvatinib n = 279, sorafenib n = 128); 58 patients were included in the gene-expression analysis set (lenvatinib n = 34, sorafenib n = 24). Both treatments were associated with increases in VEGF; only lenvatinib was associated with increases in FGF19 and FGF23 at all time points. Lenvatinib-treated responders had greater increases in FGF19 and FGF23 versus nonresponders at cycle 4, day 1 (FGF19: 55.2% vs. 18.3%, P = 0.014; FGF23: 48.4% vs. 16.4%, P = 0.0022, respectively). Higher baseline VEGF, ANG2, and FGF21 correlated with shorter OS in both treatment groups. OS was longer for lenvatinib than sorafenib [median, 10.9 vs. 6.8 months, respectively; HR, 0.53; 95% confidence interval (CI), 0.33-0.85; P-interaction = 0.0397] with higher baseline FGF21. In tumor tissue biomarker analysis, VEGF/FGF-enriched groups showed improved OS with lenvatinib versus the intermediate VEGF/FGF group (HR, 0.39; 95% CI, 0.16-0.91; P = 0.0253). CONCLUSIONS: Higher baseline levels of VEGF, FGF21, and ANG2 may be prognostic for shorter OS. Higher baseline FGF21 may be predictive for longer OS with lenvatinib compared with sorafenib, but this needs confirmation.

The Expression of PEDF and its Putative Receptors in Hepatocellular Carcinoma and Background Liver Tissue.

BACKGROUND/AIM: Hepatocellular carcinoma (HCC) remains one of the biggest medical issues. Pigment epithelial-derived factor (PEDF) is a glycoprotein that belongs to the superfamily of serine protease inhibitors. PEDF interacts with its two receptors, adipose triglyceride lipase (ATGL) and laminin receptor (LR). MATERIALS AND METHODS: We conducted immunohistochemical staining for PEDF, LR and ATGL in 151 resected HCCs and their background liver tissues. RESULTS: High expression of LR in HCC was associated with high histological grade and portal vein invasion, while high expression of PEDF in HCC was associated with absence of portal vein invasion. High LR expression in background liver was statistically associated with low serum albumin levels and was an independent prognostic factor of worse outcomes. No cases with more than 5% fatty degeneration in the background liver tissue showed high PEDF expression. CONCLUSION: PEDF/LR/ATGL could be potential biomarkers in HCC and various chronic hepatic disorders.

Ki-67 "hot spot" digital analysis is useful in the distinction of hepatic adenomas and well-differentiated hepatocellular carcinomas.

This study aims to investigate the utility of digital protocols for Ki-67 immunohistochemistry quantitative analysis ("hot spot" method) in the setting of well-differentiated hepatocellular neoplasms. Resection cases of typical hepatic adenomas (HAs) (n = 40), atypical HAs (n = 9), and well-differentiated hepatocellular carcinomas (WD HCCs) (n = 56) were selected. HAs were further classified by immunohistochemistry using antibodies against liver fatty acid binding protein, glutamine synthetase, B-catenin, hepatic serum amyloid A, and C-reactive protein. Ki-67 proliferative index by immunohistochemistry was evaluated in all cases by digital analysis using a modified neuroendocrine tumor "hot spot" protocol. The proliferative rate of HAs (typical, median 1.2% (range 0-7.4%) and atypical, median 1.0% (range 0.3-3%)) was significantly lower than that of WD HCCs (median 4.5%, range 0-49.8%) (P < 0.0001). Only a few (7.5%) of the adenomas (all inflammatory/telangiectatic type) had proliferative rates higher than 4%, compared to most (51%) of HCCs. Ki-67 is a potentially useful adjunct marker in the evaluation of WD hepatocellular neoplasms, as "hot spot" proliferative rates are consistently very low in HAs but vary significantly in WD HCCs.

Repeated double cross-validation applied to the PCA-LDA classification of SERS spectra: a case study with serum samples from hepatocellular carcinoma patients.

Intense label-free surface-enhanced Raman scattering (SERS) spectra of serum samples were rapidly obtained on Ag plasmonic paper substrates upon 785 nm excitation. Spectra from the hepatocellular carcinoma (HCC) patients showed consistent differences with respect to those of the control group. In particular, uric acid was found to be relatively more abundant in patients, while hypoxanthine, ergothioneine, and glutathione were found as relatively more abundant in the control group. A repeated double cross-validation (RDCV) strategy was applied to optimize and validate principal component analysis-linear discriminant analysis (PCA-LDA) models. An analysis of the RDCV results indicated that a PCA-LDA model using up to the first four principal components has a good classification performance (average accuracy was 81%). The analysis also allowed confidence intervals to be calculated for the figures of merit, and the principal components used by the LDA to be interpreted in terms of metabolites, confirming that bands of uric acid, hypoxanthine, ergothioneine, and glutathione were indeed used by the PCA-LDA algorithm to classify the spectra.

Phenotype Classification using Proteome Data in a Data-Independent Acquisition Tensor Format.

A novel approach for phenotype prediction is developed for data-independent acquisition (DIA) mass spectrometric (MS) data without the need for peptide precursor identification using existing DIA software tools. The first step converts the DIA-MS data file into a new file format called DIA tensor (DIAT), which can be used for the convenient visualization of all the ions from peptide precursors and fragments. DIAT files can be fed directly into a deep neural network to predict phenotypes such as appearances of cats, dogs, and microscopic images. As a proof of principle, we applied this approach to 102 hepatocellular carcinoma samples and achieved an accuracy of 96.8% in distinguishing malignant from benign samples. We further applied a refined model to classify thyroid nodules. Deep learning based on 492 training samples achieved an accuracy of 91.7% in an independent cohort of 216 test samples. This approach surpassed the deep-learning model based on peptide and protein matrices generated by OpenSWATH. In summary, we present a new strategy for DIA data analysis based on a novel data format called DIAT, which enables facile two-dimensional visualization of DIA proteomics data. DIAT files can be directly used for deep learning for biological and clinical phenotype classification. Future research will interpret the deep-learning models emerged from DIAT analysis.

Syt-7 overexpression predicts poor prognosis and promotes cell proliferation in hepatocellular carcinoma.

Aim: To explore the prognostic significance of Syt-7 in hepatocellular carcinoma (HCC) and the potential mechanisms. Methods: Immunohistochemistry was used to examine the expression of Syt-7. Overall survival and disease-free survival were compared between Syt-7 positive and negative groups. The effects of Syt-7 knockdown on BEL-7404 cells were further evaluated. Results: Syt-7 expression was significantly higher in HCC tumorous tissues compared with paracancerous tissues. Syt-7 was closely associated with α-fetoprotein tumor size, vascular invasion, tumor node metastasis stage and tumor differentiation. Syt-7 was an independent risk factor for overall survival and disease-free survival. Additionally, Syt-7 knockdown inhibited proliferation and colony formation and induced cell cycle arrest in HCC cells. Conclusion: Syt-7 overexpression forecasts unfavorable prognosis and promotes cell proliferation in HCC.

Hepatocellular carcinomas can be Special AT-rich sequence-binding protein 2 positive: an important diagnostic pitfall.

Special AT-rich sequence-binding protein 2 (SATB2) is a sensitive and specific marker for tumors originating with the colon and appendix. It is now commonly used in surgical pathology, while working up carcinomas of unknown primary. We had anecdotally encountered occasional hepatocellular carcinomas (HCCs) that were SATB2 positive. Immunohistochemical expression of SATB2 in HCC has not yet been examined in detail. In this study, we evaluated SATB2 expression in 46 HCCs. Nineteen (41%) of 46 HCCs were positive for SATB2. SATB2 expression in HCCs was more commonly seen in poorly differentiated tumors (11 of 13 cases, 85%) than well and moderately differentiated tumors (8 of 33 cases, 24%), p value = 0.0001. No other statistically significant correlations were observed (p > 0.05). There were no other statistically significant correlations between SATB2 expression and age, gender, background liver disease, and cirrhosis (p > 0.05). Results of our study show that a significant subset (41%) of HCCs can be SATB2 positive. Awareness of this phenomenon is important as SATB2 expression in a liver tumor does not completely exclude a diagnosis of HCC.

A prognostic model composed of four long noncoding RNAs predicts the overall survival of Asian patients with hepatocellular carcinoma.

Based on accumulating evidence, long noncoding RNAs (lncRNAs) are potential biomarkers and therapeutic targets for many diseases, including tumors. In this study, we consulted The Cancer Genome Atlas (TCGA) database to explore the functions and modulatory mechanisms of lncRNAs as competing endogenous RNAs (ceRNAs) in hepatocellular carcinoma (HCC) in Asian patients and constructed a risk scoring system composed of four lncRNAs (SNHG1, STEAP3-AS1, RUSC1-AS1, and SNHG3) to predict the outcomes of Asian patients with HCC. The prognostic value of this risk model was validated in the internal validation cohort (n = 157). The stratified survival analysis revealed good performance for the risk model in stratifying clinical features. According to the Cox proportional hazard regression analysis, the four-lncRNA risk model is an independent prognostic model for Asian patients with HCC. Finally, we developed a nomogram that integrates prognostic signals and other clinical information to predict 1-, 3-, and 5-year overall survival rates. In conclusion, the prognostic lncRNAs identified in our study exerted potential biological effects on the development of HCC. The risk scoring model based on four lncRNAs may be an effective classification tool for assessing the prognosis of Asian patients with HCC.

Identification of a protein signature for predicting overall survival of hepatocellular carcinoma: a study based on data mining.

BACKGROUND: Hepatocellular carcinoma (HCC), is the fifth most common cancer in the world and the second most common cause of cancer-related deaths. Over 500,000 new HCC cases are diagnosed each year. Combining advanced genomic analysis with proteomic characterization not only has great potential in the discovery of useful biomarkers but also drives the development of new diagnostic methods. METHODS: This study obtained proteomic data from Clinical Proteomic Tumor Analysis Consortium (CPTAC) and validated in The Cancer Proteome Atlas (TCPA) and TCGA dataset to identify HCC biomarkers and the dysfunctional of proteogenomics. RESULTS: The CPTAC database contained data for 159 patients diagnosed with Hepatitis-B related HCC and 422 differentially expressed proteins (112 upregulated and 310 downregulated proteins). Restricting our analysis to the intersection in survival-related proteins between CPTAC and TCPA database revealed four coverage survival-related proteins including PCNA, MSH6, CDK1, and ASNS. CONCLUSION: This study established a novel protein signature for HCC prognosis prediction using data retrieved from online databases. However, the signatures need to be verified using independent cohorts and functional experiments.

Updates in the diagnosis of combined hepatocellular-cholangiocarcinoma.

Combined hepatocellular-cholangiocarcinoma (cHCC-CCA) is a rare primary liver carcinoma showing variable degrees of differentiation toward hepatocellular and cholangiocellular carcinoma. Its great heterogeneity in term of morphology, immunophenotype, molecular, radiological and clinical features represents a challenge still to overcome. The multidisciplinary 2018 International Consensus on the nomenclature of cHCC-CCA allowed to define key issues of this entity. Here we review the historical controversies of cHCC-CCA, resume the key elements of the 2018 consensus, now incorporated in the 2019 WHO classification, and propose a short survival guide to help surgical pathologists facing cHCC-CCA in their routine workup.

Clusterin expression in nontumor tissue in patients with resectable hepatocellular carcinoma related with postresectional survival.

BACKGROUND: Surgical resection offers an effective treatment for patients with hepatocellular carcinoma (HCC); however, it has high tumor recurrence rate. Clusterin is a highly conserved glycoprotein that enhances cell aggregation in vitro. It is upregulated in several types of cancers such as breast, ovarian, colon, prostate and kidney cancers, and HCC. Clusterin overexpression is correlated with tumor metastasis. We evaluated the significance of clusterin expression levels in serum and resected tissues of patients with HCC. METHODS: Serum, resected tumor tissue, and nontumor tissue were collected from 140 patients with HCC undergoing hepatic resection. Serum clusterin levels were determined by enzyme-linked immunosorbent assay. Clusterin expression in resected tissue was evaluated by immunohistochemistry. Median follow-up time was 57.8 months. RESULTS: Mean serum clusterin levels were found to be 130.0 ± 58.7 µg/mL (range, 10.1-366.6 µg/mL). Serum clusterin levels were independent of tumor stage and deterioration of liver function in patients. No significant difference was observed in the survival of patients with high (>130.0 ± 58.7 µg/mL) or low (≤130.0 ± 58.7 µg/mL) serum clusterin level. Clusterin was expressed in HCC tissues of 76 patients (54.3%) and nontumor liver tissues of 53 patients (37.9%). No significant difference was observed in the survival of patients with positive or negative clusterin expression in HCC tissues. In nontumor tissues, patients with positive clusterin expression were observed to have low postoperative disease-free survival rate (p = 0.001) compared to patients with negative clusterin expression. Multivariate analysis showed that tumor with macrovascular/microvascular invasion and clusterin expression in nontumor tissues are independent prognostic factors following hepatic resection. CONCLUSION: In HCC, clusterin expression in nontumor tissue shows worse prognosis after hepatic resection. Clusterin can be a prognostic marker for patients with postresection HCC.

Nanohoneycomb Surface-Enhanced Raman Spectroscopy-Active Chip for the Determination of Biomarkers of Hepatocellular Carcinoma.

Overexpression of the Lens culinaris agglutinin-reactive fraction of alpha-fetoprotein (AFP-L3) is an essential biomarker for early diagnosis of hepatocellular carcinoma (HCC). In this study, we designed a new surface-enhanced Raman spectroscopy active chip for the detection of AFP with high sensitivity and excellent repeatability. This chip was composed of a honeycomb gold nanostructure array with strong electromagnetic field coupling due to the special cavity geometric characteristics of the honeycomb structure. The honeycomb structure exhibited extraordinary performance for the specific detection of AFP in the range of 0.003-3 ng/mL and also determined the proportion of AFP-L3 with a high degree of accuracy, which has shown great potential for application in the clinical diagnosis of HCC.

Reverse capture for selectively and sensitively revealing the N-glycome of serum exosomes.

Exosomes are emerging as a promising source of disease biomarkers. However, glycans from exosomes have been less studied. Here, for the first time, the N-glycome of human serum exosomes is reported and the potential of N-glycans from exosomes as a source for biomarker discovery is revealed.

Hepatic copper and other trace mineral concentrations in dogs with hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common primary liver tumor in dogs. Abnormalities in hepatic copper, iron, zinc, and selenium concentrations increase risk for HCC development in other species, but trace mineral concentrations have not been evaluated in dogs with HCC. OBJECTIVES: To investigate hepatic trace mineral concentrations in dogs with HCC. ANIMALS: Archived liver specimens from 85 dogs with HCC and 85 control dogs. METHODS: Retrospective case-control study. A histopathology database was searched to identify dogs with HCC (test population) and an age-matched control population. Demographic information was retrieved, and H&E and rhodanine stained slides were reviewed for all cases. Copper, iron, zinc, and selenium concentrations were determined in noncancerous liver tissues (test and control population) and in HCC tissues (test population) using inductively coupled plasma mass spectrometry. RESULTS: Hepatic copper concentrations (non-neoplastic hepatic tissue) were greater in test population dogs (median, IQR; 294.9 µg/g, 233.5-475.9 µg/g) than in control dogs (202.8 µg/g, 135.0-295.3 µg/g; P < .001). Hepatic zinc concentrations in test (132.1 µg/g,108.6-163.2 µg/g) and control dogs (151.5 µg/g, 117.1-184.5 µg/g) also were different (P = .03). Within test population dogs, all trace mineral concentrations were decreased in the HCC tissue as compared to the non-neoplastic hepatic tissue (all P < .001). CONCLUSIONS AND CLINICAL IMPORTANCE: Hepatic copper accumulation and other abnormalities in hepatic trace mineral concentrations could be involved in the pathogenesis of HCC in some dogs.

Prussian blue nanoparticle-labeled aptasensing platform on graphene oxide for voltammetric detection of α-fetoprotein in hepatocellular carcinoma with target recycling.

An enzyme-free electrochemical aptasensing platform based on a graphene oxide nanosheet-modified gold-disk electrode was developed for the voltammetric detection of alpha-fetoprotein (AFP) in hepatocellular carcinoma by using a Prussian blue nanoparticle (PBNP)-labeled aptamer. The electroactive PBNP, a typical signal-generation tag, was utilized for the labeling of the aminated AFP aptamer by using covalent conjugation. The electrochemical sensing platform was prepared in a simple manner on the basis of a π-π stacking reaction between the immobilized graphene oxide and the PBNP-labeled AFP aptamer. Upon target AFP introduction, the analyte reacted with the aptamer, thus resulting in the dissociation of the PBNP from the nanosheets. In the presence of DNase I, the newly formed AFP/aptamer-PBNP complex was cleaved to release target AFP, which could react again with the aptamer on the nanosheets, thereby causing target recycling. During this process, the cleaved PBNP-aptamer was far away from the electrode to decrease the voltammetric signal. Under optimum conditions, the voltammetric peak current of the modified electrode decreased with the increment of the target AFP concentration within the linear range of 0.01-300 ng mL-1 at a low detection limit of 6.3 pg mL-1. The precision and reproducibility of the aptasensing protocol were acceptable (CV: <15% for intra-assay and inter-assay). Other possible nontarget biomarkers did not interfere significantly with the voltammetric signal of this system. Human serum samples containing target AFP were assayed with electrochemical aptasensing and a commercial human AFP ELISA kit, and gave well-matched results from these two methods. Importantly, our strategy provides a new horizon for the determination of disease-related proteins.