Targeting anticancer immunity in oral cancer: Drugs, products, and nanoparticles.

Oral cavity carcinomas are the most frequent malignancies among head and neck malignancies. Oral tumors include not only oral cancer cells with different potency and stemness but also consist of diverse cells, containing anticancer immune cells, stromal and also immunosuppressive cells that influence the immune system reactions. The infiltrated T and natural killer (NK) cells are the substantial tumor-suppressive immune compartments in the tumor. The infiltration of these cells has substantial impacts on the response of tumors to immunotherapy, chemotherapy, and radiotherapy. Nevertheless, cancer cells, stromal cells, and some other compartments like regulatory T cells (Tregs), macrophages, and myeloid-derived suppressor cells (MDSCs) can repress the immune responses against malignant cells. Boosting anticancer immunity by inducing the immune system or repressing the tumor-promoting cells is one of the intriguing approaches for the eradication of malignant cells such as oral cancers. This review aims to concentrate on the secretions and interactions in the oral tumor immune microenvironment. We review targeting tumor stroma, immune system and immunosuppressive interactions in oral tumors. This review will also focus on therapeutic targets and therapeutic agents such as nanoparticles and products with anti-tumor potency that can boost anticancer immunity in oral tumors. We also explain possible future perspectives including delivery of various cells, natural products and drugs by nanoparticles for boosting anticancer immunity in oral tumors.

AGLR is a novel index for the prognosis of hepatocellular carcinoma patients: a retrospective study.

BACKGROUND: Most hepatocellular carcinoma (HCC) patients' liver function indexes are abnormal. We aimed to investigate the relationship between (alkaline phosphatase + gamma-glutamyl transpeptidase)/lymphocyte ratio (AGLR) and the progression as well as the prognosis of HCC. METHODS: A total of 495 HCC patients undergoing radical hepatectomy were retrospectively analyzed. We randomly divided these patients into the training cohort (n = 248) and the validation cohort (n = 247). In the training cohort, receiver operating characteristic (ROC) curve was used to determine the optimal cut-off value of AGLR for predicting postoperative survival of HCC patients, and the predictive value of AGLR was evaluated by concordance index (C-index). Further analysis of clinical and biochemical data of patients and the correlation analysis between AGLR and other clinicopathological factors were finished. Univariate and multivariate analyses were performed to identify prognostic factors for HCC patients. Survival curves were analyzed using the Kaplan-Meier method. RESULTS: According to the ROC curve analysis, the optimal predictive cut-off value of AGLR was 90. The C-index of AGLR was 0.637 in the training cohort and 0.654 in the validation cohort, respectively. Based on this value, the HCC patients were divided into the low-AGLR group (AGLR ≤ 90) and the high-AGLR group (AGLR > 90). Preoperative AGLR level was positively correlated with alpha-fetoprotein (AFP), tumor size, tumor-node-metastasis (TNM) stage, and microvascular invasion (MVI) (all p < 0.05). In the training and validation cohorts, patients with AGLR > 90 had significantly shorter OS than patients with AGLR ≤ 90 (p < 0.001). Univariate and multivariate analyses of the training cohort (HR, 1.79; 95% CI 1.21-2.69; p < 0.001) and validation cohort (HR, 1.82; 95% CI 1.35-2.57; p < 0.001) had identified AGLR as an independent prognostic factor. A new prognostic scoring model was established based on the independent predictors determined in multivariate analysis. CONCLUSIONS: The elevated preoperative AGLR level indicated poor prognosis for patients with HCC; the novel prognostic scoring model had favorable predictive capability for postoperative prognosis of HCC patients, which may bring convenience for clinical management.

Elevation of MAP17 enhances the malignant behavior of cells via the Akt/mTOR pathway in hepatocellular carcinoma.

MAP17, a small non-glycosylated membrane protein, was significantly up-regulated in hepatocellular carcinoma (HCC) tissues in our previous genome-wide microarray analysis. In this study, quantitative real-time RT-PCR and immunohistochemistry were applied to examine MAP17 mRNA and protein expression in primary HCC and matched peritumoral tissues. The disease-free survival (DFS) and overall survival (OS) was estimated using the Kaplan-Meier analysis. The expression of MAP17 was significantly higher in HCC tissues compared to the paired peritumoral tissues at both mRNA and protein levels. High MAP17 expression was positively correlated with gender, distant metastasis, early recurrence (≤ 2 year), and serum alpha-fetoprotein (all p < 0.05). Kaplan-Meier analysis showed that the DFS (p = 0.004) and OS (p = 0.013) in HCC patients with elevated expression of MAP17 were much worse than that in the low expression subgroup. High level of MAP17 was also significantly associated with a high probability of HCC early recurrence after surgical resection (p = 0.005). Cox regression analysis indicated MAP17 was an independent prognostic factor for DFS (HR, 1.710; 95% CI, 1.156-2.449, p = 0.012) and OS (HR, 1.743; 95% CI, 1.152-2.639, p = 0.009) in HCC. Silencing MAP17 significantly inhibited the proliferation, invasion and migration of HCC cells in vitro, and decreased the expression levels of Akt, p-Akt (Ser473), mTOR, p-mTOR (Ser2448) and MMP-9. Suggesting MAP17 was a novel diagnostic and prognostic biomarker for HCC patients and promoted HCC cell proliferation, invasion and migration via the Akt/mTOR pathway.

A novel prognostic index-neutrophil times γ-glutamyl transpeptidase to lymphocyte ratio (NγLR) predicts outcome for patients with hepatocellular carcinoma.

Clinical outcomes of patients with hepatocellular carcinoma (HCC) are highly variable. This study aims to identify and validate a simple, readily available, and objective prognostic index for the management of HCC. Data from 724 HCC patients undergoing curative resection were evaluated and randomly divided into two cohorts for building and validating the prognostic index. A best model, NγLR = (neutrophil count [109/L] × Î³-glutamyl transpeptidase [U/L]) /(lymphocyte count [109/L] × U/L), was selected. An optimal cut-off value of 103.6 for NγLR stratified patients into high NγLR (>103.6) and low NγLR (≤103.6) groups. NγLR > 103.6 was closely associated with HCC malignant characteristics. Elevated NγLR predicted a worse overall survival (OS) and progression-free survival (PFS) for HCC patients and remained an independent predictor for both types of survival. Moreover, early recurrence rates in patients with NγLR > 103.6 were higher than that in patients with NγLR ≤ 103.6 (P < 0.0001). NγLR was an important independent predictor of survival for HCC patients and might be a new promising method to identify patients at different risks of early recurrence and survival after curative resection.

Up-regulation of BRCA1-associated RING Domain 1 Promotes Hepatocellular Carcinoma Progression by Targeting Akt Signaling.

The present study was designed to investigate the potential clinical, pathological, prognostic value, role and mechanism of BRCA1-associated RING Domain 1 (BARD1) in Hepatocellular carcinoma (HCC). Quantitative real-time PCR and immunohistochemistry were performed to evaluate the expression of BARD1 mRNA and protein. The expression of BARD1 in the HCC tissue samples was markedly higher than that in the adjacent noncancerous liver tissues. Elevated BARD1 expression was positively correlated with tumor-node-metastasis stage, Barcelona-Clinic Liver Cancer stage, hepatitis B surface antigen, large tumor size, serum alpha-fetoprotein levels, and serum aspartate aminotransferase levels. Univariate and multivariate analyses revealed the BARD1 was an independent predictor for decreased progression-free survival and overall survival in HCC. In vitro experiments demonstrated that knocking down BARD1 significantly inhibited the proliferation, invasion and migration of HCC cells. Moreover, silencing BARD1 inhibit the signaling pathway via decreased the levels of Akt, mTOR, and MMP-9 and inhibited the phosphorylation of Akt (Ser473) and mTOR (Ser2248). Collectively, our findings suggest that BARD1 may be a novel diagnostic and prognostic biomarker of HCC, and up-regulation of BARD1 can contribute to HCC progression by targeting Akt signaling.

High level of intercellular adhesion molecule-1 affects prognosis of patients with hepatocellular carcinoma.

AIM: To determine the cut-off value of intercellular adhesion molecule-1 (ICAM-1) and assess the correlation of ICAM-1 with clinicopathological features and the prognosis of hepatocellular carcinoma (HCC) patients who underwent surgical resection. METHODS: We prospectively collected clinicopathological data from 236 HCC patients who had undergone successful hepatectomy. Receiver operating characteristic curve analysis was performed to determine the optimal cut-off value of ICAM-1. Enzyme-linked immunosorbent assay was used to measure the concentration of ICAM-1 in 236 serum samples isolated from HCC patients and the stratified analysis was used to compare the serum level of ICAM-1 in different HCC subgroups. Immunohistochemistry was performed to test the expression level of the ICAM-1 protein in 76 cases of HCC tissues and their adjacent normal liver tissues (ANLT). The survival probability of HCC patients was estimated using Kaplan-Meier plots and differences between the groups were obtained using the log-rank test. Furthermore, independent indicators of the prognosis were acquired using a stepwise Cox proportional hazard model to analyze a series of predictors that were associated with disease-free survival (DFS) and overall survival (OS) in HCC patients. RESULTS: Our findings suggested that ICAM-1 promotes HCC metastasis and high serum ICAM-1 is significantly associated with alpha-fetoprotein (AFP) (P = 0.022), clinical tumor-node-metastasis stage (P < 0.001), portal vein tumor thrombus (P = 0.005), distant metastasis (P = 0.016) and recurrence (P = 0.034). We further detected the ICAM-1 protein in HCC specimens and found that 56 of 76 (73.7%) HCC tissues had ICAM-1 positive staining while only 23 of 76 (30.3%) ANLT were positively stained (P < 0.0001). Survival analysis indicated that HCC patients with increased ICAM-1 concentrations had significantly shorter DFS and OS after resection. A multivariate analysis showed that ICAM-1 > 684 ng/mL was an independent factor for DFS (HR = 1.643; 95%CI: 1.125-2.401; P = 0.010) and OS (HR = 1.692; 95%CI: 1.152-2.486; P = 0.007). CONCLUSION: ICAM-1 may be a promising serological biomarker for HCC diagnosis and an independent predictor of DFS and OS after surgical resection and may provide a useful reference for the prediction of intra- and extrahepatic metastasis.

Preoperative Neutrophil-to-Lymphocyte Ratio as a New Prognostic Marker in Hepatocellular Carcinoma after Curative Resection.

BACKGROUND: Preoperative peripheral blood neutrophil-to-lymphocyte ratio (NLR) has been proposed to predict prognosis of hepatocellular carcinoma (HCC). However, the cutoff value of NLR in several studies is not consistent. This study aims to investigate the correlation of preoperative NLR with clinicopathologic features and the prognosis in patients who have undergone resection for HCC. METHODS: Clinical data of 256 patients with HCC who underwent radical hepatectomy were retrospectively analyzed. The patients were divided into the low-NLR group (NLR ≤ 2.31) and the high-NLR group (NLR > 2.31). A univariate analysis was performed to assess clinicopathologic characteristics that influenced disease-free survival (DFS) and overall survival (OS) in patients. The significant variables were further analyzed by a multivariate analysis using Cox regression. The Kaplan-Meier method was used to assess the DFS and OS rate. RESULTS: The value of NLR was associated with tumor size, clinical tumor-node-metastasis (TNM) stage, portal vein tumor thrombus (PVTT), distant metastasis, and aspartate aminotransferase (AST) in HCC. NLR > 2.31, size of tumor > 5 cm, number of multiple tumors, III-IV of TNM stage, PVTT, distant metastasis, and AST > 40 U/l were predictors of poorer DFS and OS. NLR > 2.31, size of tumor >5 cm, III-IV of TNM stage, and AST > 40 U/l were independent predictors of DFS and OS. CONCLUSION: Preoperative NLR > 2.31 was an adverse predictor of DFS and OS in HCC after hepatectomy. This study suggested that NLR might be a novel prognostic biomarker in HCC after curative resection.

Silencing of DLGAP5 by siRNA significantly inhibits the proliferation and invasion of hepatocellular carcinoma cells.

BACKGROUND: The dysregulation of oncogenes and tumor suppressor genes plays an important role in many cancers, including hepatocellular carcinoma (HCC), which is one of the most common cancers in the world. In a previous microarray experiment, we found that DLGAP5 is overexpressed in HCCs. However, whether the up-regulation of DLGAP5 contributes to hepatocarcinogenesis remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we showed that DLGAP5 was significantly up-regulated in 76.4% (168 of 220) of the analyzed HCC specimens when compared with adjacent liver tissue. DLGAP5 overexpression was evident in 25% (22 of 88) of the HCC specimens without AFP expression, suggesting that DLGAP5 may be a novel biomarker for HCC pathogenesis. The silencing of DLGAP5 gene expression by RNA interference significantly suppressed cell growth, migration and colony formation in vitro. The expression level of DLGAP5 was also found to be related to the methylation level of its promoter in the HCC specimens. CONCLUSIONS/SIGNIFICANCE: Taken together, these data suggest that the expression of DLGAP5 is regulated by methylation and that the up-regulation of DLGAP5 contributes to HCC tumorigenesis by promoting cell proliferation.

FOXP3 gene polymorphism is associated with hepatitis B-related hepatocellular carcinoma in China.

BACKGROUND: Previous evidence has shown that the FOXP3 gene was involved in the pathogenesis of several tumors; however, the correlation between single nucleotide polymorphisms (SNPs) in the FOXP3 gene and the susceptibility to hepatitis B-related hepatocellular carcinoma (HCC) remains unclear. METHODS: We analyzed two SNPs in the FOXP3 gene, rs2280883 and rs3761549, in 392 patients with HCC, 344 patients with chronic hepatitis B (CHB) and 372 matched healthy controls. Genotyping was performed by MALDI-TOF Mass Spectrometry for all donors. RESULTS: Compared to healthy controls, HCC patients had higher frequencies of the TT genotype (79.6%) at rs2280883 and the CC genotype (77.6%) at rs3761549 of the FOXP3 gene; CHB patients also had higher frequencies of the TT genotype (74.1%) at rs2280883 and the CC genotype (74.6%) at rs3761549. There were no significant differences in the distribution of FOXP3 genotypes between CHB donors and HCC donors. The TT genotype at rs2280883 was more frequent in patients with HCC than healthy donors (P = 0.01), but no significant difference was observed in this genotype between CHB and healthy donors (P = 0.479). C allele frequency at rs3761549 was higher in HCC patients than healthy donors (P = 0.03), but distribution of this allele was not significantly different between CHB patients and healthy donors (P = 0.11). Stratified analysis showed that the CC genotype at rs3761549 was significantly associated with a high incidence of portal vein tumor thrombus (P = 0.02) and that the TT/CT genotype at rs3761549 was significantly associated with an increased rate of tumor recurrence in HCC patients (P = 0.001). CONCLUSIONS: Our results suggested that the FOXP3 gene polymorphisms at rs2280883 and rs3761549 may be associated with hepatitis B-related HCC. At rs3761549, the CC genotype and the TT/CT genotype were associated with a high incidence of portal vein tumor thrombus and tumor recurrence, respectively.

[Correlation between interleukin-28B genetic polymorphisms and primary hepatocellular carcinoma].

OBJECTIVE: To explore the correlation between single nucleotide polymorphisms (SNPs) of interleukin-28B (IL-28B) gene and the susceptibility to primary hepatocellular carcinoma (HCC). METHODS: A total of 300 histologically confirmed HCC cases (from November 2001 to April 2010) and 310 healthy controls with no history of chronic hepatitis B or hepatocellular carcinoma (2009-2010) were selected from a hospital in Guilin and a hospital in Beijing for this case-control study.139 HCC patients in the case group had complete clinical tracking data. All the subjects were Han Chinese, with no age or gender restrictions.2 ml peripheral blood samples were drawn from each subject with informed consent. SNP of rs12972991, rs4803223, rs8099917 and rs12979860 four loci in IL-28B gene were analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF). RESULTS: The frequencies of C allele at rs12972991, G allele at rs8099917 and G allele at rs4803223 were 6.7% (40/598), 7.9% (47/598) and 10.0% (59/588) respectively in case group; all higher than the corresponding frequencies in control group, separately 2.9% (18/618), 4.1% (25/616) and 3.6% (21/608). The differences were statistically significant (χ2=9.542, 7.858, 20.736, P values all<0.05). The above alleles could increase the risk of HCC, and the OR (95%CI) values were separately 1.67 (1.13-2.46), 1.49 (1.08-2.06) and 2.91 (1.79-4.72). The genotype frequencies of AC+CC at rs12972991, GT+GG at rs8099917, GA+GG at rs4803223 were 13.0% (39/299), 14.7% (44/299) and 19.0% (56/296) respectively in case group; while the frequencies were lower in control group, separately 5.8% (18/309), 8.1% (25/308) and 6.6% (20/304). The differences were statistically significant (χ2=9.319, 6.557, 20.948, P values all<0.05). These genotypes may increase the risk of HCC, and the adjusted OR (95%CI) values were 2.24 (1.31-3.83), 1.81 (1.14-2.88) and 2.90 (1.78-4.70), respectively. The stratified analysis of the clinical data indicated that the frequency of genotype GA+GG at rs4803223 was 50.0% (13/26) in patients of tumor thrombosis in portal vein (TTPV), higher than the frequency of genotype AA (21.1%, 23/109). The difference was statistically significant (χ2=8.965, P=0.003). CONCLUSION: The results suggested that IL-28B gene polymorphisms was correlated to the susceptibility to HCC in Chinese Han ethnic population. Among them, GA + GG genotype at rs4803223 could increase the risk of TTPV in HCC patients.

[The inhibitory effect of RNA interference on STAT3 expression in liver cancer cell line SMMC7721].

AIM: To investigate the chemosensitivity small interfering RNA (siRNA) on liver cancer cell line SMMC7721. METHODS: The siRNA sequences design based on signal transducers and activators of transcription 3 (STAT3) gene, siRNA were transfected into SMMC7721 cells through liposome lipofectamine(TM); 2000. The expression inhibition of STAT3 gene in SMMC7721 cells was measured by real-time relative quantitative PCR. The cells growth inhibition rate were measured by MTT colorimetry after 10 µmol/L 5-fluorouracil (5-Fu) action. RESULTS: The siRNA expression vector to aim directly at STAT3 gene was constructed successfully. The result of real-time PCR revealed that specificity siRNA were transfected into SMMC7721 cells could inhibit the expression of STAT3 gene. STAT3 gene in SMMC7721 cells were specialty and effectually inhibit the expression by RNA interference (RNAi). MTT colorimetry detection result revealed that SMMC7721 cells inhibition rate significantly increasing after siRNA action. CONCLUSION: The siRNA expression vector can active inhibit expression of STAT3 gene in SMMC7721 cells, enhance its sensitivity to chemotherapeutics 5-Fu, to provide experiment based on for the biological therapy of tumor.

Regulatory T cell depletion enhances tumor specific CD8 T-cell responses, elicited by tumor antigen NY-ESO-1b in hepatocellular carcinoma patients, in vitro.

Immunotherapy in hepatocellular carcinoma based on one or a few tumor specific antigens have shown limited antitumor efficacy. As a major suppressive factor in tumor immune response, better understanding of the role of regulatory T cells (Tregs) in hepatocellular carcinoma might be important for design of future immunotherapy-based clinical protocols. Tregs from 49 HCC patients and 40 controls were identified by flow cytometric analysis for the phenotype. Functional studies were performed by analyzing their inhibition to immune responses. Finally investigating whether elimination of Tregs was capable of enhancing the immunostimulatory efficacy of NY-ESO-1b peptides. In HCC peripheral blood and tumor-infiltrating lymphocytes, we found increased numbers of Tregs, which expressed high levels of HLA-DR, GITR and CD103. The prevalence of Tregs increased with during progressive stages in HCC patients. Moreover, the elimination of Treg cells followed by stimulating with NY-ESO-1b peptide significantly improved the anti-tumor cytotoxic T lymphocytes responses in HCC patients compared with stimulating with NY-ESO-1b peptide alone. The immune response efficiency increased from 37.5 to 62.5%. In conclusion, the increase in frequency of Treg cells might play a role in suppression of the immune response against HCC and for the design of immunotherapy the incorporation of the Treg cell depletion strategy will achieve potent anti-tumor immunity with therapeutic impact.

Expression of CIITA-related MHCII molecules in tumors linked to prognosis in hepatocellular carcinoma.

The prognosis of hepatocellular carcinoma (HCC) after surgery is poor due to its high recurrence rate. In order to unfold the mechanism of different recurrent-free survival (RFS) times following resection, expression profiling of tumor tissues from 32 HCC patients with different RFS time were used to identify differential expression of individual genes and signaling pathway components correlated with RFS time. Quantitative RT-PCR, Western blotting, and immunohistochemistry were used to validate the expression of selected genes. Up-regulation of several immune related genes and pathways, especially HLA II-related antigen presenting pathways, significantly correlated with longer RFS time. The expression of MHCII molecules were found to be mainly located in either CD68+ cells or CD45+ cells, and their expression significantly correlated with the expression of CIITA (HLA II genes transactivator) in the tumor. The results suggest that the high expression level of CIITA and MHCII molecules in hepatocellular carcinoma tissue is an effective prognostic marker for longer RFS time in HCC.

Specific CD8(+ )T cell responses to HLA-A2 restricted MAGE-A3 p271-279 peptide in hepatocellular carcinoma patients without vaccination.

The MAGE-A3 protein, one of the promising tumor antigens for immunotherapy, is highly expressed in human hepatocellular carcinoma (HCC). In this study, we estimated the specific CD8(+) T cell immune response to MAGE-A3 p271-279 peptide (M3(271)) in the peripheral blood of HCC patients without antigen vaccination in order to evaluate its immunotherapeutic potential in these patients. After expansion in vitro, the functional IFN-gamma producing M3(271) specific CD8(+) T cells were detected in 30.8% (8/26) of HLA-A2(+)MAGE-A3(+) HCC patients. The effector CD8(+ )T cells could release cytotoxic molecules of granzyme B and perforin after restimulation with natural HLA-A2(+)MAGE-A3(+) HCC cell lines in the samples tested. The functional supertype of HLA-A2 in the presentation of HLA-A*0201 restricted M3(271) peptide has been identified in the Chinese HCC patients of Han ethnicity, that widely expanded the applicability of this tumor peptide vaccine in Chinese HCC patients. Thus, the functionally detectable pre-existence of M3(271)-specific CD8(+) T cells in HCC patients makes M3(271) a potential target for immunotherapy in these patients. The responsive CD8(+ )T cells to both NY-ESO-1 and MAGE-A3 antigens provide a rationale for the application of a bivalent vaccine in HCC patients with tumors expressing both antigens.

[The frequency, phenotypes and functions of CD4+ CD25+ regulatory T cells in hepatocellular carcinoma patients].

OBJECTIVES: (1) To evaluate the prevalence, phenotypes and suppressive function of CD4+CD25+ regulatory T cells (Tregs) among the in peripheral blood mononuclear cells (PBMCs) and tumor-infiltration lymphocytes (TILs) from hepatocellular carcinoma (HCC) patients and patients with chronic hepatitis B. (2) To investigate the correlation between the frequency of CD4+CD25+ Tregs and clinical characteristics of HCC patients. METHODS: PBMCs and TILs in 18 HCC patients, 10 chronic hepatitis B (CHB) patients and 15 healthy donors were evaluated for the phenotypes of CD4+CD25+ Tregs and the proportion of CD4+CD25+ Tregs as a percentage of the total CD4+ cells, by flow cytometric analysis with three or four color staining. The relationship between the frequency of CD4+CD25+ Tregs and tumor TNM stages was analyzed. The CD4+CD25+ Tregs and CD4+CD25- T cells were isolated from PBMC of HCC patients and donors. The suppressive function of CD4+CD25+ Tregs was analyzed. RESULTS: The percentages of CD4+CD25+ Tregs of the HCC patients (6.38% +/- 6.30%) and CHB patients (4.29% +/- 1.82%) were significantly higher than those of the healthy donors (1.58% +/- 0.55%, P less than 0.01). Among the TILs, the percentage of CD4+CD25+ Tregs was higher (t = 4.39, P < 0.01). There were significant differences in the prevalence of CD4+CD25+ Tregs in early and advanced stage HCCs (stage II vs. III, P less than 0.05; stage II vs. IV P < 0.01). The proliferative capacity of CD4+CD25- T cells was inhibited by the presence of CD4+CD25+ T cells in a dose-dependent manner where the level of suppression was correlated to the ratio of the two-cell populations. CONCLUSION: These results suggest that the increase in frequency of CD4+CD25+ Tregs might play a role in the suppression of the immune response against HCC, which may contribute to the HCC cells that escaped from immunological surveillance.

The spontaneous CD8+ T-cell response to HLA-A2-restricted NY-ESO-1b peptide in hepatocellular carcinoma patients.

PURPOSE: Hepatocellular carcinoma (HCC) can express various cancer-testis antigens including NY-ESO-1, members of the SSX family, members of the MAGE family, SCP-1, and CTP11. Immunotherapy directed against these antigens is a potential alternative treatment for HCC. To date, it remains unclear whether HCC patients have spontaneous immune responses to these tumor antigens. The objectives of this study were to measure immune responses to NY-ESO-1, a promising cancer vaccine candidate, in HCC patients using the HLA-A2-restricted NY-ESO-1b peptide (p157-165) to measure cellular responses and whole protein to measure antibody responses. EXPERIMENTAL DESIGN: In HLA-A2(+) patients with NY-ESO-1(+) HCC, we analyzed T-cell antigen-dependent interferon (IFN)-gamma and/or Granzyme B release by enzyme-linked immunospot (ELISPOT) assay and IFN-gamma-producing intracellular cytokine flow cytometry (CytoSpot). As an assay independent of T-cell function, we performed tetramer staining. Antibodies to whole NY-ESO-1 were assayed by enzyme-linked immunosorbent assay. RESULTS: The frequency of specific CD8(+) T-cell responses to NY-ESO-1b in 28 NY-ESO-1 mRNA(+)HLA-A2(+) HCC patients was 35.7% (10 of 28). The average magnitude of effector CD8(+) T cells was 0.3% (89 +/- 59 per 2.5 x 10(4) CD8(+) cells) and 1.2% as measured by IFN-gamma release ELISPOT and CytoSpot assays, respectively. These in vitro induced NY-ESO-1b-specific CD8(+) T cells can also recognize HepG2 cells transfected with pcDNA3.1-NY-ESO-1 in both IFN-gamma and Granzyme B ELISPOT assays. Frequencies of NY-ESO-1b-specific T cells in several patients were confirmed by tetramer staining. Nonfunctional tetramer(+)CD8(+) T cells were also present. The CD8(+) T-cell response was apparently increased in patients with late-stage HCC. A discordance between antibody and CD8(+) T-cell responses in HCC patients was observed. CONCLUSIONS: The elevated frequency of specific CD8(+) T-cell responses to NY-ESO-1b in NY-ESO-1 mRNA(+)HLA-A2(+) HCC patients suggests that NY-ESO-1 is appropriate for use in the immunotherapy of HCC patients.

[The genetic polymorphism of melanoma-associated antigen 1 in Chinese normal donors and hepatoma patients].

OBJECTIVES: To investigate the expression of melanoma-associated antigen 1 (MAGE-1) in Chinese hepatocellular carcinoma (HCC) patients and to determine the existence and distribution of single nucleotide polymorphisms (SNP) of MAGE-1 gene. METHODS: Total RNA was extracted from cancer tissues and adjacent tissues from 19 HCC patients and the expression of MAGE-1 mRNA was examined by using RT-PCR. The PCR products were sequenced to analysis the gene variation. Genomic DNA was extracted from cancer tissues, adjacent tissues and peripheral blood cells of 19 HCC patients, as well as from peripheral blood cells of 23 healthy donors. The PCR product of MAGE-1 DNA was sequenced to determine the existence and distribution of SNPs of MAGE-1 gene. RESULTS: 9 of 19 (47.4%) tumor tissues and none of adjacent tissue from HCC patients expressed MAGE-1 mRNA. There were three kinds of gene variations of cDNA in 9 MAGE-1 mRNA positive patients. One type was named type I including 1 patient, which sequence is as same as that of the GenBank M77481. The other was named TGA type including 5 patients, which involved three nucleotide changes (C159T, A272G and G393A) and result in two amino acid changes (T32A and R72Q). Another one was named GTG type including 3 patients, which involved three nucleotide changes (A272G, C991T, A1125G) and result in only one amino acid changes (T32A). According to the analysis of genomic DNA, above three types were not specific mutations of tumor tissue, but were SNPs. These SNPs types were distributed in HCC patients and normal donors with the frequencies of 26.3% (5/19) and 60.9% (14/23) for type I, 57.9% (11/19) and 47.8% (11/23) for TGA type, and 21.1% (4/19) and 21.7% (5/23) for GTG type, respectively. The sequences of two new SNPs had been deposited in GenBank with the accession numbers of AF463515 and AY148486. In male population, the distributions of SNPs were not correlated to HCC suffering or MAGE-1 expression. Several new HLA-restricted epitopes were probably resulted from SNPs existing. The three-dimensional models of MAGE-1 proteins of type I and TGA type was established by using computer software. CONCLUSION: The expression rate of MAGE-1 gene in Chinese HCC patients is high. Three SNP types of MAGE-1 gene exist in Chinese population. The three-dimensional models of MAGE-1 proteins were obtained by computer processing. These results will be helpful to developing MAGE-1 peptide-vaccine for HCC immunotherapy.