| Part I Prognostic nomograms for the pre- and post-operative predictions of long-term survival for patients who underwent liver resection for huge hepatocellular carcinomaObjective Liver resection is an effective treatment for selected patients with huge hepatocellular carcinoma(HCC, diameter ≥ 10 cm). This study aimed to develop nomograms for the pre- and post-operative predictions of prognosis for these patients.Methods 464 consecutive patients who underwent liver resection for huge HCC at the EHBH between January 2008 and December 2009 were collected and divided into a training cohort(n = 310) and an internal validation cohort(n = 154). Another 90 patients who were operated at the FMU between January 2008 and April 2010 served as an external validation cohort. The surgical morbidity, mortality, time to recurrence and OS were observed. Two prognostic nomograms were developed based separately on the data obtained before and after surgery. Discrimination and predictive accuracy of the models were measured using concordance index(C-index), calibration curves and validation study.Results The post-operative 4-year tumor recurrence and OS rates were 79.0%, 41.2% and 78.8%, 37.6% in the patients from the EHBH and the FMU, respectively. Independent predictors of OS on multivariable analysis using the pre- and post-operative data were respectively incorporated into the two nomograms. In the training cohort, the calibration curves for the probability of 4-year post-operative survival fitted well. The C-indexes of the pre- and post-operative nomograms in predicting OS were 0.75(95% confidence interval, 0.72 to 0.78) and 0.78(0.75 to 0.81). The internal and external validation studies optimally supported these results. The post-operative 1-, 3- and 5-year tumor recurrence and OS rates of EHBH were 43.8%, 74.1% and 82.1%; 75.1%, 49% and 33.3%, respectively. The post-operative 1-, 3- and 5-year tumor recurrence and OS rates of FMU were 38.3%, 69.2% and 84.1%; 77.7%, 44.9% and 21.5%, respectively. The calibration curves showed good agreements between the prediction by the models and the actual observation for the probability of 3- and 5- year post-resectional survival. The C-indexes of the pre- and post-operative nomograms in predicting OS were 0.75(95% confidence interval, 0.72 to 0.78) and 0.78(0.75 to 0.81) in the training cohort, and 0.70(0.67 to 0.73) and 0.75(0.72 to 0.78)in the validation cohort, respectively. The independent risk factors of OS based on the pre-operative data were serum AFP level >200 μg/L, high HBV-DNA level, multiple tumor, larger tumor diameter and presence of PVTT. The independent risk factors of tumor recurrence were AFP level > 200 μg/L, high HBV-DNA level, multiple tumors, large tumor diameter, and presence of PVTT. The independent risk factors of OS based on the post-operative data were high HBV-DNA level, multiple tumors, large tumor diameter, vascular invasion, incomplete tumor capsule and poorly differentiated tumor. The independent risk factors of tumor recurrence were high HBV-DNA level, multiple tumors, large tumor diameter, vascular invasionand incomplete tumor capsule.Conclusions The two nomograms achieved accurate pre- or post-operative predictions of long-term survival for patients with huge HCC after liver resection.Part II A differential proteomic study on post-operative recurrence of huge hepatocellular carcinoma(HCC) following radical resectionObjective By using i TRAQ-label-based quantitative proteomic technique, the present study aims to screen the differential protein expressions associated with post-operative recurrence of HCC.Methods The fresh HCC tissue specimens were grouped according to the patients’ time to recurrent(TTR) after liver resection. Protein expression profile was assessed by using the technique of i TRAQ-2DLC-MS/MS, and differential expression proteins were analysed by the bioinformatics.Results Based on the conditions of protein identification, 409 proteins were found to have significant differential expressions between <6 months group and >2 years group, 518 proteins were found to have significant differential expressions between 6-12 months group and >2 years group. In our study, we screened 10 proteins by the expressions with the different time of recurrence. Up-regulation proteins were S100A9, MPO, APOA4, TMEM97, NR5A2, and down-regulation proteins were DHRS2, TTC4, CHD2, HPD, FTCD. With reference to GO database, the differentially-expressed proteins were related to Steroid metabolism, oxidation reduction and protein metabolism process. From the results of IPA analysis, the highest degree of differentially-expressed protein enrichment pathways were regulated by NR5A2、IL1、ERK1/2、NFk B and IFNα.Conclusions i TRAQ quantitative proteomic technique is a powful tool for acquiring a more comprehensive view of the protein expression in the post-operative recurrence of HCC. The bioinformatics can further discovery the function of differentially expressed proteins and its regulatory network.Part III Verifications of the differentially expressed proteins associated with post-operative recurrence of huge HCCObjective To verify the differentially expressed proteins, from both protein and m RNA levels, by Q-PCR and Western blot esperiment.Methods Select S100A9, DHRS2, TMEM97 and NR5A2 to verify their expressions. Collect fresh HCC tissues to detect the m RNA expression and the protein expression by Q-PCR and Western blot esperiment, respectively. Collect paraffin-embedded tissues to perform immunohistochemical experiment.Results The Q-PCR and Western blot esperiment’s results showed that the high level of TMEM97 was the risk factors of tumor recurrence. The immunohistochemical experiment showed that the high level of(P=0.005,HR1.534), serum AFP level >200 μg/L( 0.003,1.595), multiple tumor(0.000,2.422)、larger tumor diameter(0.018,1.042), PVTT(0.002,1.776), MVI(0.019,1.504), incomplete tumor capsule(0.004,0.595)、transfusions(0.028,1.590) were the independent risk factors of tumor recurrence.Conclusions The expression of TMEM97 was up-regulated in shorter TTR HCC tissues. higher expression of TMEM97 suggested lower disease-free survival, and thus might be used as a potential alternative marker for recurrence. |
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