Protein expression profiling of vascular endothelial growth factor and its receptors identifies subclasses of hepatocellular carcinoma and predicts survival

Purpose  To examine expression profile and prognostic significance of vascular endothelial growth factor (VEGF) and its receptors in hepatocellular carcinoma (HCC) and peritumoral tissue. Methods  Expression of VEGF-A, VEGF-C, and VEGF receptor 1(VEGFR-1), VEGFR-2, and VEGFR-3 in tumor and peritumoral liver tissue was studied by immunohistochemistry in a tissue microarray from 107 patients with HCC. Unsupervised hierarchical cluster analyses were conducted to identify relevant clusters. Results  Staining of VEGF-A, VEGF-C, VEGFR-2, and VEGFR-3 was mostly found on the tumor cells and peritumoral hepatocytes, but VEGFR-1 was mostly expressed in stromal cells. In most of the cases, the expression of VEGF-A, VEGFR-1, VEGFR-2, and VEGFR-3 in was higher in peritumoral liver tissue, while VEGF-C expression was higher in tumor. Unsupervised hierarchical clustering analysis identified four prognostically different clusters, of which cluster A was classified into the “poor prognosis group,” and the other three clusters were classified into the “good prognosis group” (P = 0.047). Further analysis with a set of seven markers reproduced the same four cluster groups with significantly different recurrence free probability (RFP) (P = 0.018), and the low RFP group was associated with more intrahepatic satellite lesions. Multivariate analysis showed that classification defined by seven biomarkers was of prognostic significance (P = 0.000). Conclusions  Expression of VEGF and its receptors was higher in peritumoral tissue than in tumor in HCC. Seven biomarkers predicted patients’ RFP, which consisted of tumoral expression of VEGF-A, VEGFR-1, and VEGF-C as well as peritumoral expression of VEGF-A, VEGFR-1, VEGFR-2, and VEGFR-3. J.-B. Jia and P.-Y. Zhuang contributed equally to this work.     

肝细胞癌癌组织和癌旁肝组织Ep-CAM和C-Kit表达及其意义探讨*

目的 探讨肝细胞癌（HCC）患者癌组织和癌旁肝组织C-Kit蛋白和肿瘤干细胞标志上皮细胞黏附分子（EpCAM）蛋白表达的变化。方法 2014年3月~2017年1月我院经手术切除治疗的HCC患者癌组织和癌旁肝组织标本90份,采用免疫组化染色法检测癌组织和癌旁肝组织Ep-CAM蛋白和C-Kit蛋白表达情况,并比较不同分化、有无包膜、不同病灶大小、术前不同血清甲胎蛋白（AFP）水平癌组织Ep-CAM蛋白和C-Kit蛋白表达阳性率的差异。结果 在本组90例HCC患者肝组织中,癌组织Ep-CAM蛋白和C-Kit蛋白表达阳性率分别为65.6%和74.4%,均显著高于癌旁组织的11.1%和4.4%,差异具有统计学意义（P<0.05）;50例Ⅲ级和Ⅳ级组织学分化、49例术前血清AFP>400 ng/ml、28例发生血管浸润的癌组织Ep-CAM蛋白表达阳性率分别为76.0%、79.6%和82.1%,显著高于40例Ⅰ级和Ⅱ级组织学分化、41例血清AFP≤400 ng/ml和62例未发生肿瘤血管浸润的癌组织（分别为52.5%、48.8%和58.1%）;Ⅲ级和Ⅳ级组织学分化、术前血清AFP>400 ng/ml、发生血管浸润的癌组织C-Kit蛋白表达阳性率分别为86.0%、87.8%和89.3%,也显著高于Ⅰ级和Ⅱ级组织学分化、血清AFP≤400 ng/ml和未发生肿瘤血管浸润的癌组织（分别为60.0%、58.5%和67.7%）,差异均具有统计学意义（P<0.05）。结论 HCC患者癌组织Ep-CAM蛋白和C-Kit蛋白表达阳性率显著增高,并与肿瘤组织学分级、术前血清AFP水平和是否发生肿瘤血管浸润有关,其临床意义还有待进一步探讨。     

原发性肝癌患者血清leptin,VEGF和AFP表达的意义

目的:探讨HCC患者血清Lep,VEGF和AFP表达的临床意义.方法:用ELISA检测30例健康对照、31例肝硬化和146例HCC患者血清Lep,VEGF和AFP含量,分析Lep,VEGF和AFP及其联合检测对HCC的诊断、临床分期和预后的意义.结果:HCC患者血清Lep水平与BMI显著相关(r=0.64,P<0.01),且显著低于肝硬化组/健康对照组(19μg/Lvs35,27μg/L,P<0.05).随TNM分期增加和转移、复发,HCC血清Lep水平明显降低(P<0.05).HCC患者血清VEGF水平显著高于肝硬化组/健康对照组(398ng/Lvs179,167ng/L,P<0.01),VEGF和AFP间无相关性,二者对HCC诊断的敏感性和特异性分别为71.2%,73.3%和80.6%,83.9%,联合检测可提高诊断的敏感性(91.8%)和准确度(90.4%).HCC患者VEGF水平与肿瘤大小呈正相关(<3cm:237±96ng/L,>3cm:398±124ng/L,P<0.01),Ⅲ,Ⅳ期患者血清VEGF水平也明显高于Ⅰ,Ⅱ期患者(346±131,401±152ng/Lvs228±89,259±102ng/L,P<0.01),术后转移和复发患者血清VEGF水平明显升高(P<0.05).结论:血清Lep测定可判断HCC患者营养状况和预后,血清VEGF与AFP联合测定可作为诊断HCC和判断其预后的重要指标.     

Expression of vascular endothelial growth factor in surgical specimens of hepatocellular carcinoma

Purpose: Vascular endothelial growth factor (VEGF) has been reported to play an important role in angiogenesis in hepatocellular carcinoma (HCC). However, there is great variation in reports on the distribution of VEGF expression, especially in non-carcinoma liver cells. Furthermore, some reports have mentioned that endothelial cells were positive for VEGF antibody but have not evaluated its significance. In this study, we focused our attention to these problems and try to solve them. We also analyzed the factors influencing VEGF expression and evaluated the prognostic potential of VEGF protein in HCC. Methods: We examined the VEGF expression in specimens surgically removed from 46 HCC patients and 3 patients with liver cancer metastatic from the colon, and in 4 specimens of liver tissue with benign disease, by immunohistochemical methods. Results/conclusions: VEGF was expressed in HCC cells and hepatocytes and on vascular endothelial cells. Our finding that about seven times more endothelial cells were positive for VEGF antibody in carcinoma areas than in non-carcinoma areas (P < 0.001) suggests that VEGF is a very important angiogenesis factor for HCC growth. VEGF expression in HCC cells and non-carcinoma liver cells and on endothelial cells did not closely correlate with the disease recurrence rate (P > 0.05), suggesting that VEGF expression may not be useful as an individual factor for estimating the prognosis of HCC. A statistical analysis of the relationships between VEGF expression and clinicopathological variables revealed the following: preoperative transcatheter arterial embolization enhanced VEGF expression in both HCC cells and non-carcinoma liver cells. The histological grade of HCC and the level of alanine aminotransferase was related to VEGF expression in non-carcinoma liver cells and on endothelial cells in HCC areas. Tumor size and the histological status of the accompanying chronic hepatitis also influenced the VEGF expression on endothelial cells. Our findings concerning not only HCC but also the surrounding liver and endothelial cells may provide useful information for further research on the role of VEGF expression in HCC patients. Received: 29 July 1999 / Accepted: 11 October 1999     

大肠癌组织中P16蛋白和血管内皮生长因子的表达及其临床意义

目的：探讨大肠癌组织中P16蛋白和血管内皮生长因子（VEGF）表达及其临床意认。方法：用S－P免疫组织化学方法测定66例大肠癌组织和20例正常大肠组织中P16蛋白和VEGF的表达。结果：大肠癌中P16蛋白阳性率为48．5％（32／66）明显低于对照组的70．0％（14／20）（P＜0．01），VEGF阳性率为72．7％（48／66）则明显高于对照组的15．0％（3／20）（P＜0．01）：P16蛋白和VEGF在大肠癌中表达具有明显负相关性；P16蛋白和VEGF表达与大肠癌组织学类型、肿瘤直径、肿瘤部位无关（P＞0．05），而与淋巴结转移、Duke's分期五年生存率有明显的关系（P＜0．01）。结论：大肠癌中存在P16蛋白下调和VEGF上调，P16蛋白和VEGF表达可作为反映大肠癌生物学行为的指标之一。     

NHE1 mRNA在肝癌组织的表达及意义

目的 探讨钠氢交换蛋白1（NHE1）mRNA在原发性肝细胞肝癌组织及癌旁组织的表达及意义。方法采用RT—PCR检测诊断为HCC患者34例的肝癌及癌旁组织中NHE1mRNA的表达情况。结果所有肝癌组织和癌旁组织NHE1均有表达,相对表达量分别为2．892±0．882和0．802±0．206,两者存在显著性差异（P〈0．001）。作为对照的21例肝组织NHE1的相对表达量为0．872±0．186。肝癌组织与对照肝组织比较存在显著性差异（P〈0．001）;癌旁组织与对照肝组织比较差异无统计学意义（P〉0．10）。将从同一患者取材的肝癌组织和癌旁组织检测到的NHE1mRNA的表达量进行配对t检验,两者间存在显著性差异（P〈0．001）。结论NHE1mRNA在肝癌组织的表达增高,可能在肝癌的发生中起一定的作用。     

血管内皮生长因子受体与肝细胞癌血管生成关系的研究进展

新生血管是肿瘤生长和转移的基础.肝细胞肝癌是典型的多血管肿瘤,其发生、发展、转移、侵袭都和血管生成密切相关.血管的生成主要依靠血管生长因子和血管生长抑制因子的调控,其中研究最多也是最重要的是血管内皮生长因子(VEGF)及其受体(VEGFR).VEGFR在机体内作用不同,参与肝癌血管生长的主要是VEGFR-1(flt-1)和VEGFR-2(flk-1).针对VEGFR的抗肿瘤血管治疗在实验室取得了不错的疗效,部分已经进入了临床试验.     

Clinical significance of cylindromatosis expression in primary hepatocellular carcinoma

Background and study aimThere is currently a lack of sensitive biomarkers for the diagnosis of hepatocellular carcinoma (HCC). Low expression of cylindromatosis (CYLD), a tumor suppressor gene that encodes a deubiquitinase, is associated with the development of HCC. The present study, therefore, aimed to determine the clinical utility of measuring CYLD expression in the early diagnosis of HCC.Patients and methodsThe present study comprised 257 patients from the Affiliated Hospital of Qingdao University including 90 patients with HCC, 41 patients with liver cirrhosis (LC), 46 patients with hepatitis B (HB), and 80 healthy controls. qPCR was used to measure the amounts of CYLD mRNA in stored blood samples. The sensitivity and specificity of CYLD mRNA in diagnosing HCC was analyzed using receiver operator characteristic (ROC) curves. We also obtained HCC data from the Oncomine database to further verify our results.ResultsThe relative levels of CYLD mRNA in peripheral blood from patients with HCC (median, 0.060; interquartile range [IQR], 0.019–0.260) was significantly lower than in blood from patients with LC (median, 3.732; IQR, 0.648–14.573), HB (median, 0.419; IQR, 0.255–1.809) and healthy controls (median, 1.262; IQR, 0.279–3.537; P < 0.05). CYLD mRNA levels in peripheral blood were significantly higher in patients with LC compared to healthy controls and patients with HB. Oncomine data demonstrated that CYLD mRNA expression levels in HCC tissues were significantly lower than in normal liver tissues. ROC analysis demonstrated that the combined use of peripheral blood levels of CYLD and AFP had the greatest diagnostic accuracy for HCC (area under the curve (AUC), 0.897; 95 % confidence interval [CI], 0.853–0.942). CYLD had utility as a supplementary marker to AFP for diagnosing HCC.ConclusionCirculating levels of CYLD mRNA are significantly decreased in patients with HCC, indicating CYLD may have utility as a biomarker of HCC. Combined measurement of CYLD mRNA and AFP protein had the greatest diagnostic accuracy.     

Clinical significance of cylindromatosis expression in primary hepatocellular carcinoma

Background and study aimThere is currently a lack of sensitive biomarkers for the diagnosis of hepatocellular carcinoma (HCC). Low expression of cylindromatosis (CYLD), a tumor suppressor gene that encodes a deubiquitinase, is associated with the development of HCC. The present study, therefore, aimed to determine the clinical utility of measuring CYLD expression in the early diagnosis of HCC.Patients and methodsThe present study comprised 257 patients from the Affiliated Hospital of Qingdao University including 90 patients with HCC, 41 patients with liver cirrhosis (LC), 46 patients with hepatitis B (HB), and 80 healthy controls. qPCR was used to measure the amounts of CYLD mRNA in stored blood samples. The sensitivity and specificity of CYLD mRNA in diagnosing HCC was analyzed using receiver operator characteristic (ROC) curves. We also obtained HCC data from the Oncomine database to further verify our results.ResultsThe relative levels of CYLD mRNA in peripheral blood from patients with HCC (median, 0.060; interquartile range [IQR], 0.019–0.260) was significantly lower than in blood from patients with LC (median, 3.732; IQR, 0.648–14.573), HB (median, 0.419; IQR, 0.255–1.809) and healthy controls (median, 1.262; IQR, 0.279–3.537; P < 0.05). CYLD mRNA levels in peripheral blood were significantly higher in patients with LC compared to healthy controls and patients with HB. Oncomine data demonstrated that CYLD mRNA expression levels in HCC tissues were significantly lower than in normal liver tissues. ROC analysis demonstrated that the combined use of peripheral blood levels of CYLD and AFP had the greatest diagnostic accuracy for HCC (area under the curve (AUC), 0.897; 95 % confidence interval [CI], 0.853–0.942). CYLD had utility as a supplementary marker to AFP for diagnosing HCC.ConclusionCirculating levels of CYLD mRNA are significantly decreased in patients with HCC, indicating CYLD may have utility as a biomarker of HCC. Combined measurement of CYLD mRNA and AFP protein had the greatest diagnostic accuracy.     

Inhibitory effect of antisense vascular endothelial growth factor RNA on the profile of hepatocellular carcinoma cell line in vitro and in vivo

AIM:To evaluate the effect of antisense vascularendothelial growth factor(VEGF)RNA(PCMV-FGEV)transfection on the profile of hepatocellular carcinoma(HCC)SMMC-7721 cells in vitro and in vivo.METHODS:SMMC-7721 cells were transfectedwith PCMV-FGEV antisense,PCMV-VEGF sense andempty vector plasmid encapsulated by lipofectamineas antisense group,sense group and control grouprespectively.The positive cell clones were selectedwith G418.The stable transfection and expressionof VEGF in the cells were determined by RT-PCR andimmunohistochemistry.Cell proliferation was observedby MTT assay.FACS analysis was used to determine theeffect of PCMV-FGEV transfection on cell apoptosis.Thegrowth of transfected cells in Wvo was also observed innude mice.RESULTS:VEGF expression was reduced in SMMC-7721transfected with PCMV-FGEV,which was confirmed byRT-PCR and immunohistochemistry.No effect of PCMV-FGEV transfection was found on cell proliferation andcell apoptosis of SMMC-7721 in vitro.The growth of cellstransfected with PCMV-FGEV was slow in nude miceand accompanied with obvious apoptosis.The latenttime of tumors in the antisense group was 25.0±1.8d,which was longer than that in sense and controlgroups(F=19.455,P<0.01).The average tumor weightin antisense group(0.96 g±0.28 g)was the smallestamong the three groups(F=21.501,P<0.01).CONCLUSION:The expression of VEGF can be inhibitedby antisense PCMV-FGEV.Antisense PCMV-FGEV has no effect on cell proliferation and apoptosis of SMMC-7721in vitro but can inhibit tumor growth and induce cellapoptosis in vivo.     

小窝蛋白1和血管内皮生长因子在结直肠癌组织中的表达及临床意义

目的:探讨小窝蛋白1(Cav-1)和血管内皮生长因子(VEGF)在结直肠癌组织中的表达,分析其与结直肠癌临床病理因素的关系及意义.方法:收集辽宁省肿瘤医院2007-01/2009-06肿瘤外科手术切除的83例结直肠癌标本及其配对正常结直肠组织（距癌灶边缘＞5 cm）.应用免疫组织化学SP法检测Cav-1和VEGF蛋白在...     

MMP-3和VEGF在胰腺癌中的表达及临床意义

目的:探讨MMP-3和VEGF蛋白在胰腺癌组织中的表达及其与胰腺癌临床病理特征、预后及胰腺癌组织中血管新生的关系.方法:用免疫组织化学检测了56例手术切除的胰腺癌组织和正常胰腺组织中MMP-3、VEGF的蛋白表达和微血管密度,并请相关专业人员进行微血管密度计数分析.结果:在56例胰腺癌标本中有42例MMP-3(75.0...     

肝癌组织血管内皮生长因子表达水平的免疫组化研究

目的　旨在研究血管内皮生长因子 (VEGF)与肝癌微血管形成、生长和转移诸方面的关系。方法　对临床 3 6例肝癌术后癌组织 ,以免疫组织化学法研究VEGF在肿瘤组织的胞内分布及其表达 ;并以ELISA法测定癌灶、癌旁及远癌组织中的VEGF蛋白的表达水平。结果　癌组织中VEGF阳性表达率为 63 .9% ;无包膜或包膜不完整组VEGF阳性表达率与有包膜组存在显著差异 ;肝癌伴有远处转移组VEGF阳性表达水平显著高于无转移组 (P <0 .0 1) ,癌灶组织中VEGF的表达水平明显高于癌旁、远癌组织(P <0 .0 1)。结论资料提示VEGF在肝癌组织中高度表达 ,它在HCC的血管形成、肿瘤发展和转移过程中起重要作用 ,提示癌组织中VEGF过度表达是反映肿瘤侵袭生长及转移潜能的有效指标     

High expressions of vascular endothelial growth factor and platelet-derived endothelial cell growth factor predict poor prognosis in alpha-fetoprotein-negative hepatocellular carcinoma patients after curative resection

Purpose  To evaluate the prognosis value of vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF) in alpha-fetoprotein (AFP)-negative hepatocellular carcinoma (HCC) patients after curative resection. Methods  Tumor tissue microarrays (TMAs) were used to detect the expressions of VEGF and PD-ECGF in consecutive 162 AFP-negative HCC patients undergoing curative resection between 1997 and 2000 in our institute. Clinicopathologic data for these patients were evaluated. The prognostic significance was assessed using Kaplan–Meier survival estimates and log-rank tests. Multivariate study with Cox’s proportional hazard model was used to evaluate the prognosis-related aspects. Results  The positive rates of VEGF and PD-ECGF in tumor tissues were 59.9% (97/162) and 62.3% (101/162), respectively. Univariate analysis showed that VEGF and PD-ECGF were prognostic factors for relapse-free survival (P = 0.034 and P = 0.033, respectively). Multivariate analyses demonstrated that the co-index (VEGF/PD-ECGF) was an independent prognostic factor for overall survival and relapse-free survival (P = 0.002 and P = 0.000, respectively). Conclusion  The co-index of VEGF and PD-ECGF is a promising independent predictor for recurrence and survival of AFP-negative HCC patients after curative resection. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. J. Hu, Y. Xu and Z.-Z. Shen have contributed equally to this work.     

Expression of platelet-derived endothelial cell growth factor and vascular endothelial growth factor in hepatocellular carcinoma and portal vein tumor thrombus

Purpose: Both platelet-derived endothelial cell growth factor (PD-ECGF) and vascular endothelial growth factor (VEGF) are known to promote the development of new blood vessels, which are fundamental to tumor growth and metastasis. We aimed at evaluating the gene expression of PD-ECGF and VEGF in hepatocellular carcinoma (HCC) and portal vein tumor thrombus (PVTT). Patients and methods: Surgical specimens (28 HCC, 28 nontumorous liver tissues and 18 PVTT) were studied by Northern blot analysis. The levels of PD-ECGF mRNA and VEGF mRNA expression were measured by densitometric scanning of the autoradiographs, and they were normalized to the level of expression of an internal control (glyceraldehyde-phosphate dehydrogenase) mRNA. Results: The expression rates of PD-ECGF mRNA in PVTT, HCC and nontumorous liver tissues were 77.8% (14/18), 67.9% (19/28) and 35.7% (10/28), being 88.9% (16/18), 75.0% (21/28) and 17.9% (5/28) respectively for VEGF mRNA. The expressions of PD-ECGF mRNA and VEGF mRNA were higher in HCC with PVTT than when PVTT was absent (P < 0.05). The PVTT was more often seen in patients with positive expression of both PD-ECGF mRNA and VEGF mRNA in HCC than in patients who were positive for only one of these factors or negative for both (P < 0.05). Conclusion: Both PD-ECGF and VEGF correlated well with the formation of PVTT of HCC. Received: 20 June 1999 / Accepted: 20 July 1999     

Targeting vascular endothelial growth factor with the monoclonal antibody bevacizumab inhibits human hepatocellular carcinoma cells growing in an orthotopic mouse model

Background: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer related death worldwide. Liver resection or transplantation is curative for a subset of patients with localized disease, but treatments for advanced disease are generally toxic and ineffective. Aberrant expression of the vascular endothelial growth factor (VEGF) has been implicated in the progression of HCC and represents a valid target for anticancer therapy. Bevacizumab, a humanized anti‐VEGF monoclonal antibody, is currently being evaluated in the treatment of HCC. In addition, other novel anti‐angiogenesis agents are being developed in HCC. Aim: This study examines the effect of bevacizumab in a newly characterized orthotopic model of the disease using the human HCC cell line, Hep 3B, and provides preclinical evidence that an anti‐angiogenic approach holds promise in HCC. Results: Administration of bevacizumab 5 mg/kg intraperitoneal twice a week significantly decreased microvessel density in tumours, decreased human serum α‐fetoprotein measurements and prolonged the time to progression for treatment mice compared with control mice. Conclusions: Our findings suggest that targeting VEGF with bevacizumab may be an effective approach to the treatment of HCC and further study of other novel anti‐angiogenic agents in HCC is warranted.     

Clinicopathological features of hepatocellular carcinoma evaluated by vascular endothelial growth factor expression

AIM: To evaluate the significance of the expression of vascular endothelial growth factor (VEGF), its correlation with clinicopathological variables were studied in the tissue of hepatocellular carcinoma (HCC) and surrounding liver. METHODS: In 56 samples (tumor and non-tumor liver tissue) collected from 28 patients, VEGF expression was examined by immunohistochemistry and western blot analysis. RESULTS: The value of VEGF expression by western blotting was correlated with immunohistochemical staining grade. In tumor tissue, the value of VEGF expression correlated with tumor size (P = 0.034), á-fetoprotein (P = 0.036) and protein induced by vitamin K absence-II by simple regression, and histological grade (P = 0.0132) by the unpaired t-test. The level of VEGF expression in non-tumor liver was found to correlate with the value of serum albumin (P = 0.008), cholinesterase (P = 0.012) and prothrombin activity (P = 0.046). The frequency of simple nodular type in gross appearance decreased in cases with high tumor/non-tumor (T/N) ratio (P = 0.022), and the degree of portal vein invasion progressed with an increase in the T/N ratio (P = 0.008). The T/N ratio was significantly higher in early recurrence cases (P = 0.0081). CONCLUSION: This study on the expression of VEGF might be useful to estimate the liver condition and the clinicopathological features of HCC.