Diagnostic Methods Of Dual-Phenotype Hepatocellular Carcinoma And Its Co Rrelation With Early Recurrence

Objective To explore a new method of double-phenotype hepatocellular carcinoma(DPHCC)with immunofluorescence double-staining,and to investigate the correlation between early postoperative recurrence and stem cell marker expression in patients with DPHCC.Methods1.Establish methods for diagnosing DPHCC: paraffin specimens of 80 patients with HCC in 2015 were collected.Hep par-1 and CK19 antibodies were used to mark 80 paraffin specimens of HCC patients.Hep par-1 and CK19 were respectively fluorescently labeled by immunofluorescence staining.Hep par-1 was labeled green with FITC-conjugated secondary antibody,CK19 was labeled red with TRITC-conjugated secondary antibody,the overlap(yellow)of red and green fluorescence was observed,it was diagnosed as DPHCC if the overlap rate on the cell was more than 15%.2.The accuracy of fluorescent staining in diagnosing DPHCC was verified by the gold standard of immunohistochemical method of DPHCC:paraffin specimens of 80 patients were serially sectioned and the adjacent two sections were respectively treated with Hep par-1 and CK19.Searcheing for the parts of Hep par-1 and CK19 are both positive,then DPHCC could be diagnosed when the both positive sites exceeded 15%of the visual field.3.The clinicopathologic data of 80 patients with HCC in 2015 were collected.Chi-square test was used to compare baseline differences between the two groups.Kaplan-Meier method was used to compare the disease-free survival rates of the two groups.COX regression model was used to evaluate risk factors affecting early recurrence of HCC;4.From the frozen tissue samples of 80 patients with HCC above,47 specimens were selected.According to the rusult of staining,47 specimens were divided into DPHCC group and Non-DPHCC group,the expression of cancer stem cell markers(CD90,CD133 and EPCAM)in two groups were compared by real-time quantitative PCR(q PCR)and Western blot(WB).Results1.The immunofluorescence double staining effect was better,red and green coloration was clear,and the overlapping part was yellow.The three colors had higher staining contrast in the same slice.In 80 cases of HCC,19 cases were diagnosed by immunofluorescence double staining method.For DPHCC,19 cases were diagnosed as CK19+ HCC,42 cases were diagnosed as CK19-HCC,and those who did not meet the DPHCC diagnostic criteria were included in the non-DPHCC group.2.Immunohistochemical staining of the serial sections was clearly performed.The diagnostic results of DPHCC were the same as those of immunofluorescence double-staining.3.The baseline characteristics did not differ between the two groups(all variables P > 0.05).The median recurrence-free survival was 7 and 16 months in DPHCC group and Non-DPHCC group,respectively,and the difference was statistically significant(P = 0.001).The recurrence-free survival of the DHHCC group and the Non-DPHCC group.The recurrence-free survival rates were 47.4%,36.8%,5.3% and 80.3%,63.6% and 38.3% respectively in the 6-month,12-month and 24-month periods of the DPHCC and Non-DPHCC groups(P=0.001).Univariate analysis showed that CA199 > 37 ng/ml(P = 0.043),DPHCC(P = 0.002)and CK19 positive(P = 0.005)were the risk factors for the recurrence of HCC.Multivariate analysis showed that AFP > 400ng/ml(P = 0.013)and multiple nodules(P = 0.011)were independent risk factors for HCC recurrence. 4.The results of q PCR showed that the expressions of CD133(p =0.013)and Ep CAM(P = 0.006)in m RNA level were higher than that of Non-DPHCC group in DPHCC group.Western blotting showed that the expression of Ep CAM(P < 0.001)and CD133(P = 0.031)in DPHCC group were higher than that in Non-DPHCC group.The results of q PCR showed that the expression of Ep CAM(P =0.006)and CD133(P = 0.013)in DPHCC group were higher than that in Non-DPHCC group.There was no significant difference in CD90 expression between the two groups at protein or m RNA levels.Conclution1.The immunofluorescence double staining has high contrast and accurate diagnosis and can be used as one of the methods for diagnosing DPHCC.2.DPHCC is a subtype of HCC that has a high early recurrence rate.3.Liver CSCs of CD133 and EPCAM are highly expressed in DPHCC and low in Non-DPHCC.