Prognostic factors affecting postoperative survival of patients with solitary small hepatocellular carcinoma

Background: Small hepatocellular carcinoma(sHCC) is a unique variant of HCC that is characterized by small tumor size(maximum tumor diameter predic≤3 cm) and favorable long?term outcomes. The present study aimed to define clin?icopathologic factors that t survival in patients with s HCC.Methods: The study population consisted of 335 patients who underwent hepatectomy for solitary s HCC between December 1998 and 2010. Prognostic factors were evaluated using Kaplan–Meier curves and Cox proportional hazard models.Results: The 5?year overall survival(OS) and recurrence?free survival(RFS) rates were 77.7% and 59.9%, respectively. Kaplan–Meier curves showed that tumor size and vascular invasion had prognostic significance within this relatively selected cohort(P < 0.05). Multivariate analysis confirmed that increased tumor size and vascular invasion were independent prognostic factors for short OS(hazard ratio [HR] = 2.367, 95% confidence interval [CI] 1.406–3.985; HR = 2.954, 95% CI 1.781–4.900) and RFS(HR = 1.779, 95% CI 1.259–2.514; HR < 0.05). Importantly, a proposed prognostic scoring model was deri= 1.699, 95% CI 1.165–2.477) in s HCC patients(Pved according to the two variables; tumor size and extent of vascular invasion were significantly associated with OS and RFS in patients with s HCC(P < 0.001).Conclusions: Tumor size and vascular invasion are feasible and useful prognostic factors for s HCC. The proposed prognostic model, based on tumor size and vascular invasion, is informative in predicting survival in s HCC patients undergoing hepatectomy.

Knockdown of nucleophosmin induces S-phase arrest in HepG2 cells

Nucleophosmin/B23 (NPM) is a universally expressed nucleolar phosphoprotein that participates in proliferation, apoptosis, ribosome assembly, and centrosome duplication; however, the role of NPM in cell cycle regulation is not well characterized. We investigated the mechanism by which NPM is involved in cell cycle regulation. NPM was knocked down using siRNA in HepG2 hepatoblastoma cells. NPM translocation following actinomycin D (ActD) treatment was investigated using immunofluorescent staining. Expression of NPM and other factors involved in cell cycle regulation was examined by Western blotting. Cell cycle distribution was measured using flow cytometry to detect 5-ethynyl-2′-deoxyuridine (EdU) incorporation. Cell proliferation was quantified by the MTT assay. Knockdown of NPM increased the percentage of HepG2 cells in S phase and led to decreased expression of P53 and P21Cip1/WAF1. S-phase arrest in HepG2 cells was significantly enhanced by ActD treatment. Furthermore, knockdown of NPM abrogated ActD-induced G2/M phase cell cycle arrest. Taken together, these data demonstrate that inhibition of NPM has a significant effect on the cell cycle.

The propensity for tumorigenesis in human induced pluripotent stem cells is related with genomic instability

The discovery of induced pluripotent stem cells (iPSCs) is a promising advancement in the field of regenerative medicine. Previous studies have indicated that the teratoma-forming propensity of iPSCs is variable; however, the relationship between tumorigenic potential and genomic instability in human iPSCs (HiPSCs) remains to be fully elucidated. Here, we evaluated the malignant potential of HiPSCs by using both colony formation assays and tumorigenicity tests. We demonstrated that HiPSCs formed tumorigenic colonies when grown in cancer cell culture medium and produced malignancies in immunodeficient mice. Furthermore, we analyzed genomic instability in HiPSCs using whole-genome copy number variation analysis and determined that the extent of genomic instability was related with both the cells′ propensity to form colonies and their potential for tumorigenesis. These findings indicate a risk for potential malignancy of HiPSCs derived from genomic instability and suggest that quality control tests, including comprehensive tumorigenicity assays and genomic integrity validation, should be rigorously executed before the clinical application of HiPSCs. In addition, HiPSCs should be generated through the use of combined factors or other approaches that decrease the likelihood of genomic instability.

NHE7 upregulation potentiates the uptake of small extracellular vesicles by enhancing maturation of macropinosomes in hepatocellular carcinoma

Background:Small extracellular vesicles(sEVs)mediate intercellular commu-nication that contributes to hepatocellular carcinoma(HCC)progression via multifaceted pathways.The success of cell entry determines the effect of sEV on recipient cells.Here,we aimed to delineate the mechanisms underlying the uptake of sEV in HCC.Abbreviations:AF,Alexa Fluor;ANOVA,analysis of variance;ATP9A,ATPase Phospholipid Transporting 9A;BCECF-AM,2’,7’-bis-(2-barboxyethyl)-5-(and-6)-carboxyfluorescein,acetoxymethyl ester;BSA,bovine serum albumin;CCMR,Centre for Comparative Medicine Research;CRISPR,clustered regularly interspaced short palindromic repeats;CTL,ctrl;CXCR4,C-X-C Chemokine Receptor Type 4;DAPI,4′,6-diamidino-2-phenylindole;DFS,disease-free survival;DMEM,Dulbecco’s Modified Eagle Medium;DMSO,dimethyl sulfoxide;Dox,doxycycline;EEA1,early endosome antigen 1;EIPA,5-(N-ethyl-N-isopropyl)-amiloride;FBS,fetal bovine serum;FITC,fluorescein isothiocyanate;GAPDH,glyceraldehyde-3-phosphate dehydrogenase;GM130,Golgi matrix protein 130;HCC,hepatocellular carcinoma;HEPES,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;HPRT1,hypoxanthine phosphoribosyltransferase 1;H-score,histoscore;IAA,indole-3-acetic acid;KD,knockdown;KO,knockout;mAID,mini-auxin-inducible degron;MTT,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;NHE7,Na(+)/H(+)exchanger 7;ns,non-significant;OD,optical density;OS,overall survival;PBS,phosphate-buffered saline;PCR,polymerase chain reaction;pHe,endosomal pH;pHi,intracellular pH;PKH67,Paul Karl Horan 67;Rab21,Ras-associated binding protein 21;RIPA,radioimmunoprecipitation assay;SAM,synergistic activation mediator;SEMs,standard error of the means;sEVs,small extracellular vesicles;sgRNA,single-guide RNA;shRNA,short-hairpin RNA;SLC9,solute carrier gene 9;SLiCE,Seamless Ligation Cloning Extract;TCGA,The Cancer Genome Atlas;TCL,total cell lysates;TGN,trans-Golgi network;TMA,tissue microarray;TMR,tetramethyl rhodamine;TSG101,tumor susceptibility gene 101.Yue Yao and Yi Xu contributed equally to this work.Methods:Macropinocytosis was examined by the ability of cells to internalize dextran and sEV.Macropinocytosis was analyzed in Na(+)/H(+)exchanger 7(NHE7)-knockdown and-overexpressing cells.The properties of cells were stud-ied using functional assays.pH biosensor was used to evaluate the intracellular and endosomal pH.The expression of NHE7 in patients’liver tissues was exam-ined by immunofluorescent staining.Inducible silencing of NHE7 in established tumors was performed to reveal the therapeutic potential of targeting NHE7.Results:The data revealed that macropinocytosis controlled the internaliza-tion of sEVs and their oncogenic effect on recipient cells.It was found that metastatic HCC cells exhibited the highest efficiency of sEV uptake relative to normal liver cells and non-metastatic HCC cells.Attenuation of macropinocytic activity by 5-(N-ethyl-N-isopropyl)-amiloride(EIPA)limited the entry of sEVs and compromised cell aggressiveness.Mechanistically,we delineated that high level of NHE7,a sodium-hydrogen exchanger,alkalized intracellular pH and acidized endosomal pH,leading to the maturation of macropinosomes.Inducible inhibition of NHE7 in established tumors developed in mice delayed tumor development and suppressed lung metastasis.Clinically,NHE7 expression was upregulated and linked to dismal prognosis of HCC.Conclusions:This study advances the understanding that NHE7 enhances sEV uptake by macropinocytosis to promote the malignant properties of HCC cells.Inhibition of sEV uptake via macropinocytosis can be exploited as a treatment alone or in combination with conventional therapeutic approaches for HCC.

Integrated genetic and epigenetic analysis reveals DNA repair alterations in multifocal hepatocellular carcinoma

Dear Editor,Hepatocellular carcinoma(HCC)ranks the fourth most lethal cancer worldwide and over 50%of cases are diagnosed as multifocal HCC(mHCC)with dismal prognosis.1 mHCC displays more complicated intratumor heterogeneity(ITH)and clonal evolution course which decreases the efficacy of clinical treatments.

Intra-operative molecular diagnosis of sentinel lymph node and prediction of non-sentinel lymph node metastasis in breast cancer patients

To the Editor:Sentinel lymph node biopsy has become a universal procedure performed in node-negative breast cancer patients.The accurate intra-operative assessment of the sentinel lymph node(SLN)is prominent in enabling axillary lymph node dissection(ALND)synchronously performed during the breast surgery,in case of the morbidity,inconvenience,and extra cost resulting from a second operation.One-step nucleic acid amplification(OSNA)assay(Sysmex,Kobe,Japan)is a moderately sensitive molecular technique combining node tissue homogenization and subsequent reverse-transcription loop-mediated isothermal amplification of cytokeratin-19(CK-19)mRNA in a single reaction.The assay could accurately discern nodal metastases of over 0.2 mm intraoperatively.