The Preliminary Clinical And Proteomics Study Of Early Recurrence/Metastasis Of Huge Hepatocellular Carcinoma (HHCC) Following Radical Resection

ObjectiveBy using iTRAQ-label-based quantitative proteomic technique, the present studyaims to screen and identify the differential protein expressions associated with earlyrecurrence/metastasis of huge hepatocellular carcinoma（HHCC） following radicalresection,in order to screen new predicting biomarkers and potential therapeutictargets.Methods1. A total of166patients with primary HCC larger than10cm were recruitedfrom January2006to December2010. These patients underwent radical resection andthe clinicopathology was retrospectively analyzed.Univariable and multivariableLogistic regression analyses were performed and a mathematic model predicting therecurrence/metastasis of HHCC at different time points was constructed.2. Proteomics study:the resection tissue samples of HHCC were applied to thisexperiment. The specimens were divided into3groups according to clinical follow-uprecurrence/metastasis or not（follow-up time are24months） after receiving HHCCresection:a recurrence/metastasis time≤6months group （n=20）,a recurrence/metastasistime between6and12months group （n=20） and a non-recurrence/metastasis>12months group （n=20）.After quantification and enzymolysis of the protein extract fromthe specimens of the3groups, Protein expression profile was assessed by using thetechnique of iTRAQ （reagent117,119and121were used to label the recurrence time≤6months group, the recurrence time between6and12months group and thenon-recurrence>12months group, respectively）-2DLC-MS/MS.The MS/MS peptideswere analyzed by using ProteinPilot software （version4.2）, proteins were identifiedand quantitatived by using the Intemational Swissprot database （20111015, human）.For117:121or119:121differentially expressed proteins, the fold change cut-offratio>2or <0.5was considered as the existence of significant differential expression（P<0.05）. The bio-informative analysis software Ingenuity Pathways Analysis （IPA）was used to conduct bio-informative analysis, including GO function assessment, signaling pathway analysis and transcription regulation networks analysis of thedifferentially expressed protein.3. Application of proteomics experimental part of the tissue samples andgrouping criteria,3（Galectin-4,S100A12and Alpha-methylacyl-CoA racemase） ofthe differentially expressed proteins identified in Part II were selected to verify theirexpressions.Western Blot and immunohistochemical assessment were used to detectthe expression of the protein. Real time-quantitative PCR （Q-PCR） technique wasused to detect the mRNA expression of the differentially-expressed protein.4. The Q-PCR technique was applied to examine the mRNA expression ofGalectin-4in40HHCC compared with paired adjacent tissues and6healthy livertissues.The techniques of tissue microarray and immunohistochemical staining wereemployed to examine the expression profiles of Galectin-4in specimens （bothcarcinoma tissues and peri-carcinoma tissues） collected from the73patients withrecurrence/metastasis （≤12months） HHCC and73patients with non-recurrent/metastasis HHCC,as well as the expression profiles in10healthy liver tissues.Inaddition,the implications of Galectin-4and its correlations with other clinical patholo-gical parameters and recurrence/metastasis were also studied.Results1. The recurrence/metastasis rate was36.7%（61/166） within6months，at6¹2months after surgery, the recurrence/metastasis rate was27.1%（45/166）. Univariableanalysis showed pre-operative AFP of≥1210ng/ml （X₄）,tumor capsule （X₇）,post-operative pathological grade (X₁₁) and microscopic tumor thrombus (X₁₀) wererelated to the recurrence/metastasis within6months after surgery. Pre-operativeserum AFP of≥1210ng/ml （X₄） and microscopic tumor thrombus (X₁₀) wereassociated with the recurrence/metastasis within6¹2months after surgery.Multivariable analysis revealed the risk factors of recurrence within6months aftersurgery included pre-operative AFP of≥1210ng/ml （X₄）,without tumor capsule （X₇）and microscopic tumor thrombus (X₁₀),while those within6¹2months after surgerywere pre-operative serum AFP of≥1210ng/ml （X₄） and microscopic tumor thrombus(X₁₀). The equation for the prediction of recurrence/metastasis within6months was P=Y/（1+Y）,Y=EXP(-0.852+0.753X₄-1.273X₇+2.524X₁₀) and that within6¹2months was P=Y/（1+Y）, Y=EXP(-0.804-0.897X₄+0.923X₁₀).2. A total of1407proteins were identified through the searching ofInternational Swissprot （20111015,human） database. Based on the conditions ofdifferential expression protein identification, a total of87proteins were found. Incontrast to the non-recurrence/metastasis group, there were46proteins expresseddifferentially proteeins （15up-regulated expressionand31down-regulated expression）in the recurrence/metastasis time≤6months group.Compare with the non-recurrence/metastasisgroup, there were49proteins expressed differentially （33up-regulatedexpression and16down-regulated expression） in the recurrence/metastasis timebetween6and12months group.In relative to the non-recurrence/metastasis group,theother two groups （recurrence/metastasis time≤6months+the recurrence/metastasistime between6and12months） had8differentially-expressed proteins in common,namely, Galectin-4, Lactotransferrin, Eukaryotic translation initiation factor3subunitB,Biliverdin reductase A,60kDa SS-A/Ro ribonucleoprotein,Apolipoprotein A-II,Protein S100A12and Alpha-methylacyl-CoA racemase（AMACR）.3. The87differentially-expressed proteins were analysised by GO database,suggesting that most of the differentially expressed proteins were localized in theextracellular domain; they played roles in the functions of protein binding,they weremainly involved in metabonomics and regulation. Accoding to IPA software analysis,the most important biological pathway is EIF2signaling pathway,the highest proteinconcentration degree of protein network is regulated and controlled by ERK1/2,a toalof14proteins were involved in.4. Part of differentially expressed proteins validation results show that, Whencompared with the non-recurrence/metastasis group, the expressions of bothGalectin-4mRNA and protein in the other two groups （recurrence/metastasis time≤6months+the recurrence/metastasis time between6and12months） were alldown-regulated,the expressions of both S100A12and AMACR mRNA and protein inthe recurrence/metastasis time≤6months group were down-regulated while those inthe recurrence/metastasis time between6and12months group were up-regulated.The results were accordance with the results of proteomic study.5. Galectin-4mRNA expression levels in HHCC were higer than para-carcinoma tissues and normal tissues rsepectively with statistical significance（P<0.05）. Expanding clinical samples using tissue chips combined with immunohisto-chemical method to detect Galectin-4expressed in HHCC.The positive rate ofGalectin-4in the carcinoma tissues was39.0%（57/146）, which was significantlylower than those in the peri-carcinoma tissues （71.9%,105/146） and the healthy livertissues （80%,8/10）; and differences were statistically significant （P were <0.05）.Galectin-4protein was expressed in19.1%（14/73） of the carcinoma tissues from therecurrence （≤12months） group, while this ratio in the non-recurrence group （58.9%,43/73） was much higher. The difference between these two groups was statisticallysignificant （P=0.000）.The negative expression of Galectin-4was independent ofpatient’s age,gender,complication of cirrhosis,HBV infection,pathological grading.andhepatic capsule （P were>0.05）, but associated with pre-operative serum AFP value,and microscopic tumor thrombus （P were <0.05）.Kaplan-Meier survival curveanalysis showed that the3-year survival ratio in the negative expression of Galectin-424.0%was lower than the positive group34.8%（χ²=6.589,P=0.010）,the1-yeardisease free survival rate in the negative expression of Galectin-4（53.1%） was muchlower than the positive group83.6%,（χ²=5.8369, P=0.016）.Conclusions1. The recurrence/metastasis rate is high within12month after resection ofHHCC. Pre-operative AFP levels,tumor capsule and microvascular invasion are riskfactors of early recurrence/metastasis of huge HCC after surgery.2. The effect factors at different time points of early recurrence/metastasis ofhuge HCC after surgery are different,the equation for prediction of HHCCrecurrence/metastasis at different time points varies.3. The differentially expressed proteins include Galectin-4,S100A12,AMACRwhich have been confirmded were expected to be potential warning protein predictingearly recurrence/metastasis of HHCC after resection.4. Galectin-4mRNA expression was up-regulated in HHCC comparing with para-carcinoma tissues and normal tissues.While the protein expression wasdown-regulated in the huge HCC tissue.Galectin-4is associated with earlyrecurrence/metastasis of HHCC,Galectin-4lower expression suggests lower3yearsurvival rates and1year disease-free survival rate,Galectin–4can provide effectivebasis for HHCC prognosis judgment.