Role of immunosuppression and tumor differentiation in predicting recurrence after liver transplantation for hepatocellular carcinoma: A multicenter study of 412 patients

AIM： To assess pre-orthotopic liver transplantation （OLT） factors that could be evaluated pre-operatively or controlled post-operatively associated with hepatocellular carcinoma （HCC） recurrence and disease-free survival after liver transplantation （LT）.METHODS： Four hundred and twelve patients transplanted for HCC between 1988 and 1998 in 14 French centers, who survived the postoperative period were studied. Kaplan Meier estimates were calculated for 24 variables potentially associated with recurrence of HCC. Uni- and multivariate analyses were conducted to identify independent predictors of recurrence. RESULTS： Overall 5-year disease-free survival was 57.1%. By univariate analysis, variables associated with disease-free survival were： presence of cirrhosis （P = 0.001）, etiology of liver disease （P = 0.03）, α fetoprotein level （〈 200, 200 to 2000, or 〉 2000; P 〈 0.0001）, y-GT activity （N, N to 2N or 〉 2N; P = 0.02）, the number of nodules （1, 2-3 or ≥ 4; P = 0.02）, maximal diameter of the largest nodule （〈 3 cm, 3 to 5 cm or 〉 5 cm; P 〈 0.0001）, the sum of the diameter of the nodules （〈 3 cm, 3 to 5 cm, 5 to 10 cm or 〉10 cm; P 〈 0.0001）, bilobar location （P = 0.01）, preoperative portal thrombosis （P 〈 0.0001）, peri-operative treatment of the tumor （P = 0.002） and chemoembolization （P = 0.03）, tumor differentiation （P = 0.01）, initial type of calcineurin inhibitor （P = 0.003）, the use of antilymphocyte antibodies （P = 0.02）, rejection episodes （P = 0.003） and period of LT （P 〈 0.0001）. By multivariate analysis, 6 variables were independently associated with HCC recurrence： maximal diameter of the largest nodule （P 〈 0.0001）, time of LT （P 〈 0.0001）, tumor differentiation （P 〈 0.0001）, use of anti-lymphocyte antibody （ATG） or anti-CD3 antibody （OKT3） （P = 0.005）, preoperative portal thrombosis （P = 0.06） and the number of nodules （P = 0.06）. CONCLUSION： This study identifies immunosuppression, through the use of ATG or OKT3, as a predictive factor of tumor recurrence, and confirms the prognostic value of tumor differentiation.

Tumor differentiation phenotype in gastric differentiated-type tumors and its relation to tumor invasion and genetic alterations

AIM： To clarify the relations between tumor differentiation phenotype and tumor invasion or genetic alterations in gastric differentiated-type tumors. METHODS： We examined the tumor differentiation phenotype, the presence of mutations in APC and p53, and the microsatellite instability （MSI） status in 48 gastric adenomas and 171 differentiated-type carcinomas, The tumor differentiation phenotype was determined by examining the expression of human gastric mucin （HGM）, NUC6, MUC2 and CD10, The tumors were then classified into gastric- （G-）, gastric and intestinal mixed （GI-）, or intestinal- （I-） phenotypes, according to the immunopositivity of the above markers, The presence of mutations in APC and p53 and the MSI status were also investigated in all the tumors, RESULTS： Gastric adenomas were significantly associated with CDIO expression, I-phenotype tumors and the presence of APC mutations, compared with carcinomas （66.7% vs 25.1%, P 〈 0.0001; 56.3% vs 14.6%, P 〈 0.0001; 39.6% vs 14.0%, P 〈 0.0001, respectively） and inversely associated with expressions of HGM and MUC6 and the presence of p53 mutations （10.4% vs 62.6%, P 〈 0.0001; 39.6% vs 64.3%, P = 0.003; 2.0% vs 26.3%, P = 0.001, respectively）. The frequency of APC mutations was significantly higher in HGM-negative tumors, MUC6-negative tumors, CD10-positive tumors and I-phenotype tumors than in HGM-positive tumors, MUC6- positive tumors, CD10-negative tumors and G-phenotype tumors （32.7% vs 7.1%, P 〈 0.0001; 27.8% vs 14.0%, P = 0.0182; 37.3% vs 10.4%, P 〈 0.0001; and 38.5% vs 9.5%, P = 0.0017, respectively）. The frequency of MSI was significantly higher in MUC6-positive tumors, CD10- negative tumors and G-phenotype tumors than in MUC6- negative tumors, CD10-positive tumors and I-phenotype tumors （24.8% vs 6.7%, P = 0.0009; 22.2% vs 8.0%, P = 0.0143; and 28.6% vs 9.6%, P = 0.0353, respectively）. CONCLUSION： The tumor differentiation phenotype is closely related to tumor invasion and genetic alterations in gastric differentiated-type tumors.

Pilot Study of Molecular Mechanism on Vasculogenic Mimicry in Bi-directional Differentiated Malignant Tumors

Objective: To investigate the role of collagen IV and PAS positive substancesecreted by tumor cells in vasculogenic mimicry (VM) and the effects of VM on tumor cells expressingVEGF. Methods: 158 cases of bi-direction differential malignant tumor specimens withparaffin-embedded were enrolled into our study and made tissue microarray which were dual-stainedwith CD31-PAS and stained with collagen IV. The difference of the areas and distribution withpattern surrounded by between CD31 and PAS positive respectively were identified via grid-counting,as well as the difference of VEGF expression with VE absent and present. Results: The basementmembrane of VM was both PAS and collagen IV positive. VEGF expression in the bi-directiondifferential malignant tumor was higher VM-absent than VM-present and the difference wasstatistically significance in malignant melanoma and alveolar rhabdomyosarcoma (P 【 0.05).Conclusion: PAS positive substance and collagen IV compose the wall of VE and VE could provide theoxygen and nutrition for tumor growth and progression.

All-trans retinoic acid(ATRA)inhibits insufficient radiofrequency ablation(IRFA)-induced enrichment of tumor-initiating cells in hepatocellular carcinoma

Objective:Local recurrence of hepatocellular carcinoma(HCC)after radiofrequency ablation(RFA)treatment remains a serious problem.Tumor-initiating cells(TICs)are thought to be responsible for tumor relapse.Here,we investigated the effect of the TIC differentiation inducer,all-trans retinoic acid(ATRA),on RFA and explored the potential molecular mechanisms.Methods:The proportions of CD133+and epithelial cell adhesion molecule(Ep CAM);TICs in recurrent HCC after RFA and primary HCC were first determined in clinic.Then,the effect of heat intervention or insufficient RFA(IRFA)on the malignant potential of HCC cells,including cell migration,sphere formation ability,tumor growth,the proportion of CD133+and Ep CAM+TICs and expression of stem cell-related genes,was evaluated in vitro and in vivo.Finally,the effect of ATRA on the tumor growth and the proportion of TICs was evaluated.Results:In clinical data,a higher proportion of CD133+and Ep CAM+TICs was found in recurrent tumors than in primary tumors.In vitro heat intervention promoted the cell migration and sphere formation ability.Additionally,it increased the proportion of CD133+and Ep CAM+TICs and the expression of stem cell-related genes.In addition,after IRFA the residual tumors in xenografts grew faster and had more TICs than untreated tumors.ATRA remarkably inhibited residual tumor growth after IRFA by elimination of TICs though the PI3 K/AKT pathway.Combination treatment with ATRA resulted in longer survival outcomes in mouse xenografts than RFA alone.Conclusions:ATRA,as a TIC differentiation inducer,could help to improve the effect of RFA treatment,which was partially attributed to its effect against TICs.The data indicated its potential as an alternative drug in the development of better therapeutic strategies for use in combination with RFA.

p21 and p27 immunoexpression in gastric well differentiated endocrine tumors(ECL-cell carcinoids)

AIM： To investigate the expression of cyclin-dependent kinase inhibitors p21 and p27 in gastric well differentiated endocrine tumors （GWDET） （ECLocell carcinoids）.METHODS： The expressions of p21 and p27 were examined immunhistochemically in endoscopic biopsy specimens from 16 patients matching the diagnostic criteria of GWDET. Percentage of positive nuclear staining either weak or strong was noted. The association of immunoexpressions with age, gender, tumor localization, multifocality and accompanying chronic atrophic gastritis, neuroendocrine cell hyperplasia （NEH）, neuroendocrine dysplasia （NED）, intestinal metaplasia （IM）, Ki-67 proliferation index and clinical outcome were also evaluated.RESULTS： All cases expressed p27 with a mean expression score of 43.6%, while 31.3% of the cases showed any p21 expression, p21 and p27 immunoexpressions were significantly correlated with each other （P 〈 0.01）, and the p21-expressing group had higher p27 expression scores （68% vs 22%）. p21 and p27 expressions were lower in women, in non-atrophic mucosa and cases whose tumors were located somewhere other than fundus without submucosal extension. On contrary, p21 and p27 expressions were higher in males and the patients with submucosal extension and atrophic gastritis. Cases presenting lower p27 scores had solitary tumors showing neither NEH-NED nor IM. Despite, cases with lower p21 expression presented multifocal tumors accompanied by NEH-NED. However, no correlation of p21 and p27 expressions was found with age and Ki-67 expression.CONCLUSION： p27 is widely expressed in GWDETs, while p21 expression is sparse and observed in two thirds of the cases. Loss of p21 and p27 expressions may be correlated with different carcinoid tumor subtypes; however,more studies are needed to assess the role of these prospective markers in gastrointestinal endocrine tumors.

Silence of HIN-1 expression through methylation of its gene promoter in gastric cancer

AIM: To clarify the role of high in normal-1 (HIN-1) gene promoter methylation during gastric cancer development. METHODS: Gastric cancer cell lines and tissue specimens were analyzed for expression of HIN-1 mRNA and protein using the semi-quantitative reverse transcription polymerase chain reaction and immunohistochemistry. The methylation of the HIN-1 gene promoter was detected in gastric carcinoma cells and tissues using methylation-specific polymerase chain reaction. The 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium cell viability assay and flow cytometry were used to assess the changes in behaviors of gastric cancer cells with or without 5-aza-2’-deoxycytidine treatment. RESULTS: HIN-1 was not expressed in 4 of 5 gastric cancer cell lines. The demethylation reagent 5-aza-2’-deoxycytidine was able to induce or upregulate HIN-1 expression in gastric cancer cell lines, which is associated with reduction of tumor cell viability. Furthermore, methylation of the HIN-1 gene promoter was shown in 57.8% (26/45) of the primary gastric cancer and 42.1% (17/38) of adjacent tissue samples, but was not shown in normal gastric mucosa (0/10). From the clinicopathological data of the patients, methylation of the HIN-1 gene promoter was found to be associated with tumor differentiation (P = 0.000). CONCLUSION: High methylation of HIN-1 gene promoter results in silence of HIN-1 expression in gastric cancer. 5-aza-2’-deoxycytidine reverses HIN-1 methylation and reduces viability of gastric cancer cells.

Molecular pathogenesis and therapeutic strategies of human osteosarcoma

Osteosarcoma（OS）is a devastating illness with rapid rates of dissemination and a poor overall prognosis,despite aggressive standard-of-care surgical techniques and combination chemotherapy regimens.Identifying the molecular mechanisms involved in disease pathogenesis and progression may offer insight into new therapeutic targets.Defects in mesenchymal stem cell differentiation,abnormal expression of oncogenes and tumor suppressors,and dysregulation within various important signaling pathways have all been implicated in development of various disease phenotypes.As such,a variety of basic science and translational studies have shown promise in identifying novel markers and modulators of these disease-specific aberrancies.Born out of these and similar investigations,a variety of emerging therapies are now undergoing various phases of OS clinical testing.They broadly include angiogenesis inhibitors,drugs that act on the bone microenvironment,receptor tyrosine kinase inhibitors,immune system modulators,and other radio-or chemo-sensitizing agents.As new forms of drug delivery are being developed simultaneously,the possibility of targeting tumors locally while minimizing systemic toxicityis is seemingly more achievable now than ever.In this review,we not only summarize our current understanding of OS disease processes,but also shed light on the multitude of potential therapeutic strategies the scientific community can use to make long-term improvements in patient prognosis.

STUDY ON EFFECTS OF ARSENIC TRIOXIDE ON GASTRIC CANCER CELL LINES

Objective To evaluate the effects of arsenic trioxide (As-2O-3) on apoptosis and differentiation of gastric cancer cell lines (GCCL). Methods MKN45 and SGC7901 cells were treated with As-2O-3 at different concentrations, then the apoptosis rates and cell cycle were determined by flow cytometry assays, the morphologic changes were observed under fluorescence microscopy and electronic microscopy, and the gene expressions were tested with immunohistologic staining. Results Higher apoptosis rates of GCCL were seen in the As-2O-3-treated group at concentrations of 5μmol and 10μmol, as compared with those in the 5-Fu-treated group. Cell-nuclear pyknosis and chromosomal condensation were observed. The As-2O-3 at a concentration of 0.5 μmol could induce the cell cycle changes of GCCL, revealing an increase in the proportion of G1/G0 phase cells and a decrease in the proportion of S phase cells. From the fifth day after treatment of SGC7901 with As-2O-3 at a low concentration, P53 and bcl-XL genes expression rates were reduced, Bax gene expression rate increased, and bcl-2 gene expression showed little change. Conclusion As-2O-3 could induce GCCL apoptosis at a high concentration and differentiation at a low concentration, but it could not completely reverse the malignant biological behaviours of cancer cells.

Water–soluble and polarity–sensitive near–infrared fluorescent probe for long–time specific cancer cell membranes imaging and C. Elegans label

A novel D–π –A structure and near–infrared fluorescent probe(DCITT) with high polarity sensitivity and membrane targeting was reported. The fluorescent spectra of DCITT were polarity dependent and Stokes shift was greater than 300 nm. Due to its high fluorescence quantum yield, low cytotoxicity and photostability, DCITT could be used as a labeling probe in multicellular organisms. In particular, DCITT effectively distinguished tumor cells from normal cells because it could specifically light up the cancer cells membrane based on strong red fluorescence for a long time. On this basis, a polar–sensitive cell membrane probe is developed to differentiate tumor cells from normal cells, which provides an idea and method for the early diagnosis of tumor at cellular level.

A Hybrid Mathematical Model of Tumor-Induced Angiogenesis with Blood Perfusion

Angiogenesis, the growth of new blood vessel from existing ones, is a pivotal stage in cancer development,and is an important target for cancer therapy. We develop a hybrid mathematical model to understand the mechanisms behind tumor-induced angiogenesis. This model describes uptake of Tumor Angiogenic Factor（TAF）at extracellular level, uses partial differential equation to describe the evolution of endothelial cell density including TAF induced proliferation, chemotaxis to TAF, and haptotaxis to extracellular matrix. In addition we also consider the phenomenon of blood perfusion in the micro-vessels. The model produces sprout formation with realistic morphological and dynamical features, including the so-called brush border effect, the dendritic branching and fusing of the capillary sprouts forming a vessel network. The model also demonstrates the effects of individual mechanisms in tumor angiogenesis： Chemotaxis to TAF is the key driving mechanisms for the extension of sprout cell; endothelial proliferation is not absolutely necessary for sprout extension; haptotaxis to Extra Cellular Matrix（ECM） gradient provides additional guidance to sprout extension, suggesting potential targets for anti-angiogenic therapies.