Serum N-glycan profiling as a diagnostic biomarker for the identification of hepatitis B virus-associated hepatocellular carcinoma

BackgroundChanges in N-glycosylation of proteins are thought to play a key role in cancer. This study aims to investigate the changes in the serum N-glycan profiles of patients with hepatitis B virus (HBV)-related liver disease, and to evaluate the role of N-glycan markers in the noninvasive diagnosis of hepatocellular carcinoma (HCC).MethodsSerum samples were available for 21 patients with HCC, 20 patients with liver cirrhosis (LC), 20 patients with chronic hepatitis B (CHB), and 20 healthy subjects. Serum N-glycans were released and analyzed using DNA sequencer-assisted fluorophore-assisted carbohydrate electrophoresis (DSA-FACE). Serum AFP was determined by electrochemiluminescence (ECL) (AFP reference value range: <10 ng/mL).ResultsThere were characteristic changes in the serum N-glycan profiles of patients with HBV-related liver disease, including NA2FB, NA3, and NA3Fb. NA2FB was the most abundant in LC patients, while NA3Fb abundance was the highest in HCC patients. For HCC screening in patients, especially in patients with LC, the sensitive of Log peak 9 (94.4%) and Log (peak 9/peak 7) (88.9%) were better than alpha-fetoprotein (AFP) (33.3–61.1%), and their specificity was similar to that of AFP. The receiver operating characteristic (ROC) curve showed that the accuracy of Log peak 9 (AUC: 0.81±0.07) and Log (peak 9/peak 7) (AUC: 0.87±0.06) was better than that of AFP (AUC: 0.72±0.09), while the accuracy of AFP combined with the above 2 indexes was better than that of a single index. Moreover, Log (peak 9/peak 7) combined with AFP (AUC: 0.89±0.06) had the best accuracy in the diagnosis of HCC.ConclusionsOur research indicates that N-glycan may serve a new, valuable, and noninvasive alternative method for diagnosing HCC, and it may be a supplement to AFP in the diagnosis of HCC in patients with HBV-related liver disease.     

血浆外泌体中的CD48蛋白作为潜在的肝细胞癌诊断标志物的研究

背景与目的：约60%的肝细胞癌（hepatocellular carcinoma,HCC）患者在晚期才被确诊,因而无法得到及时有效的治疗,目前临床上常用于HCC诊断的血清标志物的灵敏度均偏低。本研究通过分析血浆外泌体蛋白质组的表达差异,鉴定可能用于HCC诊断的候选外泌体蛋白质标志物。方法：首先,采用鸟枪法蛋白质组质谱分析方法,对发掘人群[包括4名健康对照者（healthy control,HC）组和4例HCC患者组]的血浆外泌体进行全蛋白质组的定性和定量分析,并筛选差异表达蛋白质。然后,采用酶联免疫吸附试验（enzyme-linked immunosorbent assay,ELISA）在独立的验证人群[包括56例HCC患者组、20例肝硬化（liver cirrhosis,LC）患者组和48名HC组]中进行验证实验,并评估候选蛋白质分子在HCC诊断中的潜在价值。结果：在发掘人群中,共鉴定到1 434种血浆外泌体蛋白质,其中的CD48蛋白在HCC患者中的表达水平显著高于HC组个体。进一步采用ELISA在独立验证人群中证实CD48蛋白在HCC患者中的表达水平显著高于LC和HC个体,受试者工作特征（receiver operating characteristic,ROC）曲线的曲线下面积（area under curve,AUC）为0.886。当联合检测血浆外泌体中的CD48蛋白和血浆中的甲胎蛋白（alpha-fetoprotein,AFP）时,AUC可提升至0.970。结论：血浆外泌体中的CD48蛋白可能作为HCC的候选诊断标志物,当其与AFP联合应用时可能进一步提高HCC的临床诊断能力。     

Kidney-type glutaminase (GLS1) is a biomarker for pathologic diagnosis and prognosis of hepatocellular carcinoma

The lack of sensitive and specific biomarkers hinders pathological diagnosis and prognosis for hepatocellular carcinoma (HCC). Since glutaminolysis plays a crucial role in carcinogenesis and progression, we sought to determine if the expression of kidney-type and liver-type glutaminases (GLS1 and GLS2) were informative for pathological diagnosis and prognosis of HCC. We compared the expression of GLS1 and GLS2 in a large set of clinical samples including HCC, normal liver, and other liver diseases. We found that GLS1 was highly expressed in HCC; whereas, expression of GLS2 was mainly confined to non-tumor hepatocytes. The sensitivity and specificity of GLS1 for HCC were 96.51% and 75.21%, respectively. A metabolic switch from GLS2 to GLS1 was observed in a series of tissues representing progressive pathologic states mimicking HCC oncogenic transformation, including normal liver, fibrotic liver, dysplasia nodule, and HCC. We found that high expression of GLS1 and low expression of GLS2 in HCC correlated with survival time of HCC patients. Expression of GLS1 and GLS2 were independent indexes for survival time; however, prognosis was predominantly determined by the level of GLS1 expression. These findings indicate that GLS1 expression is a sensitive and specific biomarker for pathological diagnosis and prognosis of HCC.     

Cortactin overexpression correlates with poor prognosis in hepatocellular carcinoma

Objective: To investigate cortactin expression in hepatocellular carcinoma (HCC) and explore its significance in the prognosis of HCC patients.Methods: Immunohistochemistry was performed for paraffin samples of 119 pairs of HCC tissues (HCCs) and paratumorous liver tissues (PTLTs) to evaluate cortactin expression. The cortactin expression difference in HCCs and PTLTs were analyzed by the McNemar's test. The relationship of cortactin expressions in HCCs and clinicopathologic factors was analyzed with Mann-Whitney U test. The Kaplan-Meier method and log-rank test were employed to compare the overall survival between Cortactin negative expression group, weak expression group and strong expression group. Expression of cortactin was further determined in 19 pairs of fresh HCCs and PTLTs specimens with Western blotting.Results: Cortactin expression rate was significantly higher in HCCs (53/119, 44.5%) than that in PTLTs (2/119, 1.7%) (P<0.001). The upregulated cortactin expression in HCCs was significantly correlated to absence of capsule formation (P=0.012), vascular invasion (P=0.037) and high Edmondson-Steiner grade (P=0.020), and predicted shorter overall survival. Western blotting demonstrated that cortactin expression was upregulated in 9 out of 19 HCCs (47.4%) compared to corresponding PTLTs.Conclusion: Cortactin expression is upregulated in HCC and is related to shorter overall survival of patients, suggesting that cortactin might play roles in the metastasis of HCC and predict a poor prognosis of HCC patients.     

Emerging roles of heterogeneous nuclear ribonucleoprotein K (hnRNP K) in cancer progression

The heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a nucleic acid-binding protein that serves as a docking platform integrating transduction pathways to nucleic acid -directed processes. Recently, this protein has emerged as an important player in carcinogenesis process. HnRNP K is overexpressed in several human cancers and its aberrant cytoplasmic localization has been associated with a worse prognosis for patients, suggesting that it has a role in cancer progression. Herein, we provide a brief overview of the multifunctional roles of hnRNP K and discuss clinical studies that have demonstrated its involvement in cancer development and progression.     

Heterogeneous nuclear ribonucleoprotein K: altered pattern of expression associated with diagnosis and prognosis of prostate cancer

Using proteomic analysis of the nuclear matrix (NM), we found that heterogeneous nuclear ribonucleoprotein K (hnRNP K), a member of the hnRNP family with pleiotropic functions, was differentially expressed in prostate cancer (PCa) tissues. This study aimed to characterise the expression of hnRNP K and its subcellular localisation in PCa, utilising immunohistochemical and quantitative western blot techniques. Furthermore, the hnRNP K expression was studied in human PCa cell lines in order to determine its modulation by bicalutamide, the anti-androgen widely used in PCa therapy. Immunohistochemical staining of paraffin-embedded tissues showed that hnRNP K was overexpressed in PCa, where it was localised both in the cytoplasm and in the nucleus. Staining of non-tumour tissues showed exclusively nuclear localisation and a less intense or absent signal. Immunoblot analysis demonstrated that the hnRNP K level within the NM was higher in PCa compared with non-tumour tissues and closely correlated with Gleason score (P=0.008). Higher expression within the NM was significantly (P=0.032) associated with poor prognosis. In two-dimensional western blot analysis hnRNP K presented several isoforms; the one with pI 5.1 was the most differently expressed between non-tumour and PCa tissues. Preliminary results indicate that hnRNP K can be modulated in vitro by a non-steroidal anti-androgen. Taken together, our findings suggest that hnRNP K has potential implications at the diagnostic, prognostic and therapeutic levels in PCa.     

胞核雄激素受体测定对评价前列腺癌、肝癌受体状态的意义

背景与目的:雄激素受体(androgenreceptor,AR)与前列腺癌(prostatecarcinoma,PC)、原发性肝癌(hepatocellularcacinoma,HCC)的发生、发展有关,对临床治疗方案选择及预后的评估等有一定影响,准确判断肿瘤组织的AR状态有重要的临床意义。本研究旨在通过对胞核雄激素受体(nuclearAR,AnR)进行分析,探讨AnR对评估前列腺癌、原发性肝癌雄激素受体状态的意义。方法:取94例PC和192例HCC患者的肿瘤组织及癌周组织,采用受体放射配基结合分析法(radioligandbindingassay,RBA)对组织中胞浆雄激素受体(cytosolAR,AcR)、胞核雄激素受体的亲和力(affinity,KD)、最大结合容量(maximumconcentrationofreceptor,Bmax)进行分析。结果:PC患者中肿瘤组织AcR、AnR的Bmax值(58.82±34.73)、(543.70±249.44)fmol/mgprotein明显高于癌周组织的(21.63±14.89)、(89.20±47.32)fmol/mgprotein(P<0.001);其KD值(0.84±0.52)、(2.15±0.79)nmol/L与癌周组织的(0.78±0.49)、(2.24±0.84)nmol/L之间的差异无统计学意义(P>0.50)。HCC患者中,肿瘤组织AcR、AnR的Bmax值(18.09±16.87)、(59.93±34.12)fmol/mgprotein亦明显高于癌周组织的(10.87±7.60)、(25.54±20.10)fmol/mgprotein(P<0.001);其KD值(0.76±0.57)、(1.89±0.74)nmol/L与     

Molecular phenotypes reveal heterogeneous engraftments of patient-derived hepatocellular carcinoma xenografts

ObjectivePatient-derived xenograft (PDX) models provide a promising preclinical platform for hepatocellular carcinoma (HCC). However, the molecular features associated with successful engraftment of PDX models have not been revealed.MethodsHCC tumor samples from 76 patients were implanted in immunodeficient mice. The molecular expression was evaluated by immunohistochemistry. Patient and tumor characteristics as well as tumor molecular expressions were compared for PDX engraftment using the Chi-square test. The independent prediction parameters were identified by logistic regression analyses.ResultsThe engraftment rate for PDX models from patients with HCC was 39.47% (30/76). Tumors from younger patients and patients with elevated preoperative alpha-fetoprotein level had higher engraftment rates. Tumors with poor differentiation and vascular invasion were related to engraftment success. The positive expression of CK19, CD133, glypican-3 (GPC3), and Ki67 in tumor samples was associated with engraftment success. Logistic regression analyses indicated that GPC3 and Ki67 were two of the strongest predictors of PDX engraftment. Tumors with GPC3/Ki67 phenotypes showed heterogeneous engraftment rates, with 71.9% in GPC3⁺/Ki67⁺ tumors, 30.8% in GPC3⁻/Ki67⁺ tumors, 15.0% in GPC3⁺/Ki67⁻ tumors, and 0 in GPC3⁻/Ki67⁻ tumors. ConclusionsSuccessful engraftment of HCC PDXs was significantly related to molecular features. Tumors with the GPC3⁺/Ki67⁺ phenotype were the most likely to successfully establish HCC PDXs.     

Galectin‐4 serves as a prognostic biomarker for the early recurrence / metastasis of hepatocellular carcinoma

Galectin‐4 is a multifunctional lectin found at both intracellular and extracellular sites. It could serve as a tumor suppressor intracellularly and promote tumor metastases extracellularly during colorectal cancer development. However, galectin‐4 expression and its prognostic value for patients with hepatocellular carcinoma (HCC) have not been well investigated. Here we report that galectin‐4 was significantly downregulated in early recurrent/metastatic HCC patients, when compared to non‐recurrent/metastatic HCC patients. Low expression of gelectin‐4 was well associated with larger tumor size, microvascular invasion, malignant differentiation, more advanced TNM stage, and poor prognosis. Cancer cell migration and invasion could be significantly reduced through overexpression of galectin‐4, but upregulated by knocking down of galectin‐4 in vitro. Moreover, the serum galectin‐4 level could be significantly elevated solely by hepatitis B virus infection. Combined with clinicopathological features, the higher serologic level of galectin‐4 was well associated with more aggressive characteristics of HCC. Taken together, galectin‐4 expression closely associates with HCC progression and might have potential use as a prognostic biomarker for HCC patients.     

Identification of potential prognostic biomarkers for hepatocellular carcinoma

BackgroundThe incidence of liver cancer is increasing every year. Hepatocellular carcinoma (HCC) accounts for nearly 90% of liver cancer, and the overall 5-year survival rate of become of Hepatocellular carcinoma patients less than 20%. However, the molecular mechanism of HCC progression and prognosis still requires further exploration.MethodsIn this study, we downloaded the gene expression data from the Cancer Genome Atlas (TCGA) Genomic Data and the official website of GEO database. Weighted gene co-expression network analysis (WGCNA) and Pearson’s correlation coefficient were utilized to detect the gene modules. The shared differentially-expressed genes (DEGs) were screened out by a Venn diagram, and the hub genes were identified through protein-protein interaction (PPI) network analyses. GO and KEGG enrichment analyses were constructed for these hub genes. Overall survival (OS) and correlation analysis were conducted to investigate the relationship between the hub genes and clinical features.ResultsWe screened out 27 shared DEGs, and the mainly enriched GO terms were mitotic nuclear division, chromosomal region, and tubulin binding. Furthermore, the top three enriched KEGG pathways were “cell cycle”, “oocyte meiosis”, and “p53 signaling pathway”. According to the Maximal Clique Centrality (MCC) algorithm, the top 10 candidate hub genes were MYC, MCM3, CDC20, CCNB1, BIRC5, UBE2C, TOP2A, RRM2, TK1, and PTTG1, among which BIRC5, CDC20, and UBE2C showed a strong correlation with the OS.ConclusionsThree hub genes (BIRC5, CDC20, and UBE2C) were identified and found to be correlated to the progression and prognosis of HCC. These may become potential targets for HCC therapy.     

Methylation of multiple genes as molecular markers for diagnosis of a small,well‐differentiated hepatocellular carcinoma

The current study was conducted to identify robust methylation markers and their combinations that may prove useful for the diagnosis of early hepatocellular carcinoma (HCC). To achieve this, we performed in silico CpG mapping, direct sequencing and pyrosequencing after bisulfite treatment, and quantitative methylation‐specific PCR (MSP) in HCC and non‐HCC liver tissues. In the filtering group (25 HCCs), our direct sequencing analysis showed that, among the 12 methylation genes listed by in silico CpG mapping, 7 genes (RASSF1A, CCND2, SPINT2, RUNX3, GSTP1, APC and CFTR) were aberrantly methylated in stages I and II HCCs. In the validation group (20 pairs of HCCs and the corresponding non‐tumor liver tissues), pyrosequencing analysis confirmed that the 7 genes were aberrantly and strongly methylated in early HCCs, but not in any of the corresponding non‐ tumor liver tissues (p < 0.00001). The results obtained using our novel quantitative MSP assay correlated well with those observed using the pyrosequencing analysis. Notably, in MSP assay, RASSF1A showed the most robust performance for the discrimination of HCC and non‐HCC liver tissues. Furthermore, a combination of RASSF1A, CCND2 and SPINT2 showed 89–95% sensitivity, 91–100% specificity and 89–97% accuracy in discriminating between HCC and non‐HCC tissues, and correctly diagnosed all early HCCs. These results indicate that the combination of these 3 genes may aid in the accurate diagnosis of early HCC. © 2009 UICC     

A novel classification in predicting prognosis and guiding postoperative management after R0 liver resection for patients with hepatocellular carcinoma and microvascular invasion

BackgroundMicrovascular invasion (MVI) is a significant risk factor affecting survival outcomes of patients after R0 liver resection (LR) for hepatocellular carcinoma (HCC). The current classification of MVI is not refined enough to prognosticate long-term survival of these patients, and a new MVI classification is needed.MethodsPatients with HCC who underwent R0 LR at the Eastern Hepatobiliary Surgery Hospital from January 2013 to December 2013 and with resected specimens showing MVI were included in this study with an aim to establish a novel MVI classification. The classification which was developed using multivariate cox regression analysis was externally validated.ResultsThere were 180 patients in the derivation cohort and 131 patients in the external validation cohort. The following factors were used for scoring: α-fetoprotein level (AFP), liver cirrhosis, tumor number, tumor diameter, MVI number, and distance between MVI and HCC. Three classes of patients could be distinguished by using the total score: class A, ≤3 points; class B, 3.5–5 points and class C, >5 points with distinct long-term survival outcomes (median recurrence free survival (mRFS), 22.6, 10.2, and 1.9 months, P < 0.001). The predictive accuracy of this classification was more accurate than the other commonly used classifications for HCC patients with MVI. In addition, the mRFS of class C patients was significantly prolonged (1.9 months vs. 6.2 months, P < 0.001) after adjuvant transcatheter arterial chemoembolization (TACE).ConclusionsA novel MVI classification was established in predicting prognosis of HCC patients with MVI after R0 LR. Adjuvant TACE was useful for class C patients.     

Relationship of ethnicity and overall survival in patients treated with sorafenib for advanced hepatocellular carcinoma

Background

Although both the SHARP and the Asian-Pacific trials showed improved overall survival (OS) for sorafenib, the magnitude of benefit was substantially less for Asians, who have a higher prevalence of hepatitis B viral (HBV) infection. Whether the worse prognosis is related to ethnicity or to the etiology of hepatocellular carcinoma (HCC) remains unclear. The aim of this study was to identify prognostic factors among patients with HCC who received sorafenib in British Columbia (BC), which has a sizeable Asian population.

Methods

A total of 255 consecutive patients with advanced HCC who initiated sorafenib from January 2008 to February 2013 were identified using our pharmacy database. Clinicopathological variables and outcomes were retrospectively collected. Prognostic factors were assessed by univariate and multivariate analyses.

Results

Median age was 63 years, 80.2% were men, and 38% were Asian. Among them, 34.5% had HBV and 29.8% had hepatitis C viral (HCV) infection. In addition, 68.6% had cirrhosis and 45.9% had liver-limited disease. Median progression-free and OS were 3.7 [95% confidence interval (CI): 3.3-4.2] and 7.5 months (95% CI: 5.7-9.2), respectively. On multivariate analysis, Eastern Cooperative Oncology Group performance status (ECOG PS) and HCV positivity correlated with better OS (P<0.001 and 0.04, respectively), but ethnicity did not (P=0.622).

Conclusions

When treated with sorafenib at the same institution, Asians and Caucasians with advanced HCC had similar OS. ECOG PS and HCV were the only significant prognostic factors. A higher proportion of HCV positivity might explain why the SHARP trial achieved better OS when compared to the Asian-Pacific trial.     

奥沙利铂治疗肝细胞癌耐药的相关机制研究进展

奥沙利铂在我国已被批准用于晚期肝细胞癌（HCC）的治疗,以奥沙利铂为基础的FOLFOX 4方案已成为晚期HCC的标准治疗之一,显著延长了患者的生存期,展现出安全有效、耐受性良好等特点。尽管如此,奥沙利铂治疗HCC的临床疗效仍十分有限,其治疗失败与HCC产生耐药密切相关。本文就奥沙利铂治疗HCC的耐药机制研究进展作一综述,希望能帮助临床和科研工作者了解最新动态,并为他们的工作拓宽思路。     

Radiofrequency ablation after transarterial embolization as therapy for patients with unresectable hepatocellular carcinoma

Purpose

To evaluate the usefulness of transcatheter arterial embolization (TAE) followed by radiofrequency ablation (RFA) as combined treatment for unresectable hepatocellular carcinoma (HCC).

Patients and methods

Thirty-six consecutive patients (cirrhosis, Child-Pugh class A or B) with solitary or oligonodular HCC were treated (41 lesions; mean size, 58.9 mm; range, 30–120 mm). RFA was performed after one TAE treatment. Local efficacy was evaluated with multiphasic computed tomography (CT) performed an average of two months after RFA and once during later follow-up.

Results

The mean follow-up period was 16 months (range, 2–45 months). Technical success (namely, complete tumor devascularization during the arterial phase) was achieved for 59% of lesions at the first CT evaluation and for 46% at the second evaluation. Among prognostic factors included in the analysis, only lesion diameter (<50 mm versus ≥50 mm) was statistically significant in terms of predicting local success (Fisher's exact test: 85% versus 43% at first CT, p < 0.01; 70% versus 36% during follow-up, p = 0.05). There were no major periprocedural complications. Kaplan–Meier analysis showed survival rates of 84% at 12 months and 57% at 24 months.

Conclusions

Combined therapy – TAE then RFA – for unresectable HCC lesions in patients with cirrhosis produces a relatively high complete local response rate compared with TAE or RFA alone. Our results, considered with those from other case series, may help design prospective, randomized clinical trials to test combination therapy versus single-modality therapy in terms of risks and benefits.