Impact Of Microscopically Margin-positive Resection On Survival In Children With Hepatoblastoma & ITRAQ-based Proteomics Analysis Of Small Cell Undifferentiated Hepatoblastoma

Part 1 Impact of microscopically margin-positive resection on survival in children with hepatoblastomaBackground Hepatoblastoma(HB)is the most common primary malignant neoplasm of the liver in children.Surgery remains the mainstay of therapy for HB.In children with large lesions,aggressive hepatic resection can compromise the remaining liver volume.Besides,a large tumor might also approach or even encase vital vessels that should be preserved.In such cases,cautious resection may remove macroscopic residuals,but microscopic residuals might still be detected by histological examination.The impact of R1(microscopically margin-positive)resection on the survival of patients with HB remains debatable.This study aimed to compare the long-term outcomes of R0(microscopically margin-negative)and R1 resection for HB in children after hepatectomy.Methods We retrospectively reviewed files of children with HB who underwent resection at our institution between September 1,2005,and November 30,2017.Survival analyses and prognostic factors were evaluated using Kaplan-Meier curves and Cox regression models.Results Of 259 patients,218(84.2%)underwent R0 and 41(15.8%)R1 resection.After adjusting for confounding factors(sex,age,staging,metastasis,macrovascular involvement,tumor multifocality,initial alpha fetoprotein[AFP]level,postoperatively histologic subtype,chemotherapy regimen,neoadjuvant chemotherapy responses,and total chemotherapeutic cycles),R1 resection demonstrated a non-significantly lower overall survival(OS:hazard ratio[HR]=0.75;95%confidence interval[CI]0.34-1.64)and shorter event-free survival(EFS:HR=0.97;95%CI 0.53-1.78)rates than R0 resection.However,stratified analysis showed significantly increased risk of poor OS(HR=4.59;95%CI 1.87-11.30;P=0.048)and EFS(HR=8.33;95%CI 3.29-21.10;P=0.001)in children with metastasis.In children with postoperatively mixed pathologic subtype,R1 resection also significantly increased the risk of poor OS(HR=3.15;95%CI 1.48-6.71;P=0.004)and EFS(HR=3.12;95%CI 1.64-5.95;P=0.001).After further analysis of the relapse sites in children with metastasis and/or postoperatively mixed pathologic subtype,we found that there was no significant difference between R0 resection with metastasis and R1 resection with metastasis in the incidence of local recurrence(P=0.494);however,a significant difference in the incidence of local recurrence was seen between RO and R1 resection for subgroups with mixed pathologic subtypes(P=0.035).In the R1 resection group,the univariate analysis revealed that initial AFP<100ng/ml,mixed pathologic subtype,metastasis,macrovascular involvement,age 3 or older had poor prognosis(P<0.05).PRETEXT IV stage significantly reduced the 5-year EFS rate(P=0.003),but did not significantly reduce the 5-year OS rate of HB children(P=0.050).Conclusions With effective chemotherapy,microscopic margin status may not be associated with survival outcomes in children with HB undergoing hepatectomy.However,the stratified analysis showed that R1 resection might be associated with decreased survival in children with mixed epithelial/mesenchymal HB,compared with R0 resection,and not affect survival outcomes in those with an epithelial subtype and without metastasis.Conversely,it appears that children with an epithelial subtype and R1 resection achieve similar survival outcomes compared to those with an epithelial subtype and R0 resection,additional chemotherapy regimens or second look surgery was not necessary for these children if AFP levels and imaging showed normal results.Part 2 iTRAQ-based proteomics analysis of small cell undifferentiated hepatoblastomaBackground Hepatoblastoma(HB)is the most common childhood liver malignancy.Serum alpha fetoprotein(AFP)is commonly regarded as a tumor marker for early diagnosis and prognosis assessment of children with HB.However,the sensitivity and specificity of AFP is still inadequate for HB diagnosis.Elevated AFP can not only be detected in other diseases such as hepatocellular carcinoma and hepatic hemangioma,but also in healthy fetuses and neonates.Besides,children with small cell undifferentiated HB are always associated with extremely aggressive clinical behavior,and this type of HB sometimes showed nonsignificantly elevated or even low AFP levels.Therefore,novel diagnostic markers with satisfactory sensitivity and specificity to small cell undifferentiated HB for early diagnosis and prognosis judgment remain to be discovered.Proteomics offers great promise for unveiling the complex molecular events of tumorigenesis and the identification of tumor biomarkers.In this study,we performed quantitative proteomic analysis to identify proteins that are dysregulated in small cell undifferentiated HB compared to normal liver tissue.Methods Paired small cell undifferentiated HB(n=3)and pericarcinous tissues(control,n=3)were analyzed using isobaric tags for relative and absolute quantitation(iTRAQ).The screening criteria for differentially expressed proteins were as follows:1)Fold change(FC)≥ 1.5 and P<0.05;or 2)FC<2/3 and P<0.05.And these proteins were further analyzed by bioinformatics.Results Among a total of 749 dysregulated proteins,510 were upregulated and 239 were downregulated.The top 20 upregulated proteins included Creatine kinase B-type at number one,followed by Keratin(type I cytoskeletal 19),Fatty acid-binding protein(brain),Histone H1.5,Nuclear autoantigenic sperm protein,Solute carrier family 2 facilitated glucose transporter member 1,Stathmin,Immunoglobulin heavy constant delta(Fragment),Laminin subunit beta-1,Histone H1.2,Periostin,Macrophage-capping protein,Serpin H1,Laminin subunit gamma-1,ATP-dependent 6-phosphofructokinase(platelet type),D-3-phosphoglycerate dehydrogenase,Insulin-like growth factor 2 mRNA-binding protein 1,Coagulation factor XIII A chain,Nucleolin,and Versican core protein.Gene ontology(GO)analysis showed that there were 3710,541 and 817 entries were annotated in terms of biological process,cellular composition and molecular function,respectively.Based on Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis,a total of 52 pathways were significantly related to the occurrence of small cell undifferentiated HB,among which the ribosome pathway was the most significant.Conclusions Compared with the normal liver tissue,the expression of proteins was significantly altered in small cell undifferentiated HB.The value of some of the upregulated proteins as potential tumor markers has been demonstrated in other malignancies.GO analysis revealed that dysregulated proteins may be active in cell secretion.The results of this study may provide a basis and experimental direction for further researches on potential tumor markers of small cell undifferentiated HB.