| ObjectiveTo illuminate the pathological morphology, possible molecular mechanism and relative specific microRNAs of dysplastic nodules (DNs) and early hepatocellular carcinomas (eHCCs).1. To illuminate the problems in the pathological diagnosis of DNs and eHCCs, to figure out the prognosis of patients with DNs or eHCC.2. Try to find out the probable molecular mechanisms of hepatocarcinogenesis, evaluate the prognostic value of conventional biomarkers for patients with DNs or eHCCs.3. To select the relative carcinogenesis specific miRNAs from DNs to eHCCs, and to forecast the possible target gene.Methods1. The samples diagnosed as DN or eHCC or well-differentiated HCC were collected and reassessed with new diagnostic criteria by two experienced pathologists. The final and original pathological diagnoses were compared and the correlation with the prognostic data, statistic analysis of survival was made for patients with DNs and eHCCs.2. The expression of GPC3, GS, E-cadherin, P53, Cyclin D1, CD34 were assessed by immunohistochemistry in tissue microarrays which contain paired early HCC, DN and peritumoral liver tissue from 145 patients. Statistic analysis was performed to assess the prognostic value of the biomarkers.3. Hepatocarcinogenesis specific microRNA was filtered with microRNA arrays by comparison of eHCC and DN, and the target gene was predicted.Results1. With the new criteria,16 cases were diagnosed as LGDN,50 cases were HGDN,72 cases were DN with micro invasion,7 cases were HCC. The original diagnoses of 112 cases (77.2%) were coincident with the finals. Of the 33case (22.8%) underdiagnosed,7 HCCs were diagnosed as DN or DN with microinvasion,26 eHCCs were diagnosed as HGDNs. Kaplan-Meier Analyze showed that the diagnosis of HGDN or early HCC was not associated with the overall survival (OS) (P=0.604, 0.677) or disease-free survival (DFS) (P=0.783,0.700) in all the patients and in the patients with no history of HCC. Accompany with advanced HCC or not was significantly correlated with overall survival (OS) (P=0.004), but not correlated with disease-free survival (DFS) (P=0.079) in DN and eHCC patients.2. Among the 361 samples in the tissue array,8 points were markers,83 were eHCC tissue,123 were DN tissue, and 147 were peritumoral cirrhosis liver tissue. The expression of GPC3 was significant different among eHCC (46.99%), DN (9.35%) and peritumoral liver tissue (2.85%) (P=0.000,0.000,0.026). The sensitivity and specificity of GPC3 to figure out eHCC is 47.0% and 72.2%. The expression of GS was significantly different among eHCC (72.29%), DN (22.03%) and peritumoral liver tissue (5.63%) (P=0.000,0.000,0.000). The sensitivity and specificity of GS for detection of eHCC is 72.29% and 63.83%. Combination of GPC3 and GS can increase the sensitivity and specificity for detection of eHCC to 78.3% and 85.7%. The expression of E-cadherin was significant different between eHCC (76.25%) and peritumoral liver tissue (P=0.000), DN (74.14%) and peritumoral liver tissue (P=0.000,0.000,0.026), but showed no difference between eHCC and DN (P=0.404). The expression of P53 was significantly different between eHCC and DN, eHCC and peritumoral liver tissue (P=0.000,0.000), but not between DN and peritumoral liver tissue (P=0.245). The expression of CD34 showed significant difference among the three (P=0.000,0.000, 0.000), Cyclin D1 was not significantly different among them. Kaplan-Meier Analyses showed that all the biomarkers were not significant correlated with OS or DFS.3. Compared the miRNAs profiles between DNs and eHCCs, in the process from DN to eHCC, the up-regulated microRNAs were hsa-miR-532-5p hsa-miR-1282, HS 176, hsa-miR-517a, hsa-miR-130a*, hsa-miR-154 hsa-miR-512-5p, hsa-miR-200c; and the down-regulated miRNAs included: hsa-miR-122*, solexa-603-1846, hsa-miR-93, hsa-miR-1274a, hsa-miR-1184, hsa-miR-146b-5p, hsa-miR-34a*, hsa-miR-15a, hsa-miR-25, hsa-miR-1304. hsa-miR-200c and hsa-miR-25 may play important role from DN to HCC. The target gene of hsa-miR-200c and hsa-miR-25 were ZEB2, ERRFI1, TMEFF2 and RAB23, USP28, MOAP1, separately.Conclusions1. New diagnostic criteria is useful to diagnose early HCC and HGDN, stromal invasion can differentiate eHCC from HGDN. The overall survival and disease free survival are not significant different between patients with eHCC or HGDN who undergone hepatectomy for tumor. Accompany with HCC or not is significantly correlated with OS, but not correlated with DFS in DN and eHCC patients. Patients with hepatic DN show better prognosis than patients with advanced HCC, but still suffer from high risk of HCC recurrence.2. GPC3, GS, CD34 are useful biomarkers to differentiate eHCC from HGDN. Multiple signaling pathways play roles in hepatocarcinogenesis. GS, GPC3, CD34 are up-regulated during the process from cirrhosis to DN to eHCC. P53 show in eHCC but not in HGDN, E-cad is up-regulated during DN to HCC, Cyclin D1 may be not so important in hepatocarcinogenesis. The biomarkers don't have prognostic value to DN and eHCC patients who undergo hepatectomy for tumor.3. MiRNA expression profile seems to be different among the processes of cirrhosis to DN to eHCC, cirrhosis to HCC, relatively normal liver to HCC, the molecular mechanisms of the three may be different. hsa-miR-200c and hsa-miR-25 may play important role during the process from DN to HCC, but need more studies with large samples to confirm them. |
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