Targeted cancer immunotherapy with irradiated, granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting, allogeneic cancer cell lines has been an effective approach to reduce tumor burden in several patients. It is generally assumed that to be effective, these cell lines need to express immunogenic antigens coexpressed in patient tumor cells, and antigen-presenting cells need to take up such antigens then present them to patient T cells. We have previously reported that, in a phase I pilot study (ClinicalTrials.gov NCT00095862), a subject with stage IV breast cancer experienced substantial regression of breast, lung, and brain lesions following inoculation with clinical formulations of SV-BR-1-GM, a GM-CSF-secreting breast tumor cell line. To identify diagnostic features permitting the prospective identification of patients likely to benefit from SV-BR-1-GM, we conducted a molecular analysis of the SV-BR-1-GM cell line and of patient-derived blood, as well as a tumor specimen. Compared to normal human breast cells, SV-BR-1-GM cells overexpress genes encoding tumor-associated antigens (TAAs) such as PRAME, a cancer/testis antigen. Curiously, despite its presumptive breast epithelial origin, the cell line expresses major histocompatibility complex (MHC) class II genes (

Background: This study aimed to identify potential crucial genes and construction of microRNA-mRNA negative regulatory networks in osteosarcoma by comprehensive bioinformatics analysis.
Methods: Data of gene expression profiles (GSE28424) and miRNA expression profiles (GSE28423) were downloaded from GEO database. The differentially expressed genes (DEGs) and miRNAs (DEMIs) were obtained by R Bioconductor packages. Functional and enrichment analyses of selected genes were performed using DAVID database. Protein-protein interaction (PPI) network was constructed by STRING and visualized in Cytoscape. The relationships among the DEGs and module in PPI network were analyzed by plug-in NetworkAnalyzer and MCODE seperately. Through the TargetScan and comparing target genes with DEGs, the miRNA-mRNA regulation network was established.
Results: Totally 346
Conclusion: Our study indicated possible differentially expressed genes and miRNA, and microRNA-mRNA negative regulatory networks in osteosarcoma by bioinformatics analysis, which may provide novel insights for unraveling pathogenesis of osteosarcoma.

BACKGROUND: Tremelimumab is an antibody that blocks CTLA-4 and demonstrates clinical efficacy in a subset of advanced melanoma patients. An unmet clinical need exists for blood-based response-predictive gene signatures to facilitate clinically effective and cost-efficient use of such immunotherapeutic interventions.
METHODS: Peripheral blood samples were collected in PAXgene® tubes from 210 treatment-naïve melanoma patients receiving tremelimumab in a worldwide, multicenter phase III study (discovery dataset). A central panel of radiologists determined objective response using RECIST criteria. Gene expression for 169 mRNA transcripts was measured using quantitative PCR. A 15-gene pre-treatment response-predictive classifier model was identified. An independent population (N = 150) of refractory melanoma patients receiving tremelimumab after chemotherapy enrolled in a worldwide phase II study (validation dataset). The classifier model, using the same genes, coefficients and constants for objective response and one-year survival after treatment, was applied to the validation dataset.
RESULTS: A 15-gene pre-treatment classifier model (containing ADAM17, CDK2, CDKN2A, DPP4, ERBB2, HLA-DRA, ICOS, ITGA4, LARGE, MYC, NAB2, NRAS, RHOC, TGFB1, and TIMP1) achieved an area under the curve (AUC) of 0.86 (95% confidence interval 0.81 to 0.91, p < 0.0001) for objective response and 0.6 (95% confidence interval 0.54 to 0.67, p = 0.0066) for one-year survival in the discovery set. This model was validated in the validation set with AUCs of 0.62 (95% confidence interval 0.54 to 0.70 p = 0.0455) for objective response and 0.68 for one-year survival (95% confidence interval 0.59 to 0.75 p = 0.0002).
CONCLUSIONS: To our knowledge, this is the largest blood-based biomarker study of a checkpoint inhibitor, tremelimumab, which demonstrates a validated pre-treatment mRNA classifier model that predicts clinical response. The data suggest that the model captures a biological signature representative of genes needed for a robust anti-cancer immune response. It also identifies non-responders to tremelimumab at baseline prior to treatment.

BACKGROUND: Secreted frizzled-related protein 1 (SFRP1) expression is down-regulated in a multitude of cancers, including breast cancer. Loss of Sfrp1 also exacerbates weight gain as well as inflammation. Additionally, loss of SFRP1 enhances TGF-β signaling and the downstream MAPK pathway. TGF-β has been shown to increase the expression of Early Growth Response 2 (EGR2), a transcription factor implicated in immune function in a wide variety of cell types. The work described here was initiated to determine whether SFRP1 modulation affects TGF-β mediated EGR2 expression in mammary tissues as well as macrophage polarization.
METHODS: Real-time PCR analysis was performed to examine EGR2 expression in human and murine mammary epithelial cells and tissues in response to SFRP1 modulation. Chemical inhibition was employed to investigate the roles TGF-β and MAPK signaling play in the control of EGR2 expression in response to SFRP1 loss. Primary murine macrophages were isolated from Sfrp1
RESULTS: Knockdown of SFRP1 expression increases the expression of EGR2 mRNA in human mammary epithelial cells and addition of rSFRP1 decreases the expression of EGR2 when added to explant mammary gland tissues. Chemical inhibition of both TGF-β and MAPK signaling in Sfrp1
CONCLUSIONS: Loss of SFRP1 likely contributes to tumor progression by altering the expression of a critical transcription factor in both the epithelium and the immune system.

OBJECTIVE: Inflammatory bowel diseases (IBDs) including ulcerative colitis (UC) and Crohn's disease (CD) increased the risk for developing colorectal cancer. However, there is no effective therapy for IBDs. The aim of this study was to identify potential therapeutic targets for inflammatory bowel disease (IBD) and explore the possible mechanism underlying this disease.
MATERIALS AND METHODS: Gene expression profile GSE6731 was downloaded from Gene Expression Omnibus database, which included 9 UC samples and 19 CD samples. Differentially expressed genes (DEGs) between affected colon tissues and non-affected tissues were identified in UC and CD group. Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis of DEGs were performed. Modules in the protein-protein interaction (PPI) network were identified, and significant node genes were selected.
RESULTS: Total 619 DEGs including 285 up-regulated genes and 334 down-regulated genes were identified in UC group and total 1159 DEGs of CD including 585 up-regulated genes and 574 down-regulated genes were selected. Module was selected from PPI network. From the PPI network and module, DEGs of mitogen-activated protein kinase 3 (MAPK3), N-myc downstream regulated 1 (NDRG1) and major histocompatibility complex, class II, DR alpha (HLA-DRA) have high degree.
CONCLUSIONS: MAPK3, NDRG1 and HLA-DRA may play key roles in the progression and development of IBD. They may be used as specific therapeutic targets in the treatment of IBD. However, further experiments are still needed to confirm our results.

The FOXP1 (forkhead box P1) transcription factor is a marker of poor prognosis in diffuse large B-cell lymphoma (DLBCL). Here microarray analysis of FOXP1-silenced DLBCL cell lines identified differential regulation of immune response signatures and major histocompatibility complex class II (MHC II) genes as some of the most significant differences between germinal center B-cell (GCB)-like DLBCL with full-length FOXP1 protein expression versus activated B-cell (ABC)-like DLBCL expressing predominantly short FOXP1 isoforms. In an independent primary DLBCL microarray data set, multiple MHC II genes, including human leukocyte antigen DR alpha chain (HLA-DRA), were inversely correlated with FOXP1 transcript expression (P<0.05). FOXP1 knockdown in ABC-DLBCL cells led to increased cell-surface expression of HLA-DRA and CD74. In R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone)-treated DLBCL patients (n=150), reduced HLA-DRA (<90% frequency) expression correlated with inferior overall survival (P=0.0003) and progression-free survival (P=0.0012) and with non-GCB subtype stratified by the Hans, Choi or Visco-Young algorithms (all P<0.01). In non-GCB DLBCL cases with <90% HLA-DRA, there was an inverse correlation with the frequency (P=0.0456) and intensity (P=0.0349) of FOXP1 expression. We propose that FOXP1 represents a novel regulator of genes targeted by the class II MHC transactivator CIITA (MHC II and CD74) and therapeutically targeting the FOXP1 pathway may improve antigen presentation and immune surveillance in high-risk DLBCL patients.

INTRODUCTION: Colorectal cancer is the third most common cancer in the world, a small fraction of which is represented by locally advanced rectal cancer (LARC). If not medically contraindicated, preoperative chemoradiotherapy, represent the standard of care for LARC patients. Unfortunately, patients shows a wide range of response rates in which approximately 20% has a complete pathological response, whereas in 20 to 40% the response is poor or absent.
RESULTS: The following specific gene signature, able to discriminate responders' patients from non-responders, were founded: AKR1C3, CXCL11, CXCL10, IDO1, CXCL9, MMP12 and HLA-DRA. These genes are mainly involved in immune system pathways and interact with drugs traditionally used in the adjuvant treatment of rectal cancer.
DISCUSSION: The present study suggests that new ideas for therapy could be found not only limited to studying genes differentially expressed between the two groups of patients but deepening the mechanisms, associated to response, in which they are involved.
METHODS: Gene expression studies performed by: Agostini et al., Rimkus et al. and Kim et al. have been merged through a meta-analysis of the raw data. Gene expression data-sets have been processed using A-MADMAN. Common differentially expressed gene (DEG) were identified through SAM analysis. To further characterize the identified DEG we deeply investigated its biological role using an integrative computational biology approach.

Acute myeloid leukemia (AML) is a heterogeneous group of malignancies that may be sensitive to the NK cell antitumor response. However, NK cells are frequently defective in AML. In this study, we found in an exploratory cohort (n = 46) that NK cell status at diagnosis of AML separated patients in two groups with a different clinical outcome. Patients with a deficient NK cell profile, including reduced expression of some activating NK receptors (e.g., DNAX accessory molecule-1, NKp46, and NKG2D) and decreased IFN-γ production, had a significantly higher risk of relapse (p = 0.03) independently of cytogenetic classification in multivariate analysis. Patients with defective NK cells showed a profound gene expression decrease in AML blasts for cytokine and chemokine signaling (e.g., IL15, IFNGR1, IFNGR2, and CXCR4), Ag processing (e.g., HLA-DRA, HLA-DRB1, and CD74) and adhesion molecule pathways (e.g., PVR and ICAM1). A set of 388 leukemic classifier genes defined in the exploratory cohort was independently validated in a multicentric cohort of 194 AML patients. In total, these data evidenced the interplay between NK cells and AML blasts at diagnosis allowing an immune-based stratification of AML patients independently of clinical classifications.

αE(CD103)β7 is a TGFβ-regulated integrin that mediates retention of lymphocytes in peripheral tissues by binding to E-cadherin expressed on epithelial cells. We recently reported that αE(CD103)β7 specifically demarcates intraepithelial CD8(+) tumor-infiltrating lymphocytes (CD8 TIL) in ovarian cancer and that CD103(+) TIL have a surface profile consistent with an active effector phenotype (HLA-DR(+), Ki67(+), and CD127(lo)). These findings led us to hypothesize that, over time, CD103-mediated retention of CD8 TIL within the tumor epithelium might result in chronic stimulation by tumor antigen, which in turn might lead to an exhausted phenotype. To investigate this possibility, we evaluated PD-1 expression in a large cohort of ovarian tumors (N = 489) with known CD103(+) TIL content. PD-1(+) cells were present in 38.5% of high-grade serous carcinomas (HGSC), but were less prevalent in other histologic subtypes. PD-1(+) TIL were strongly associated with increased disease-specific survival in HGSC (HR, 0.4864; P = 0.0007). Multicolor immunohistochemistry and flow cytometry revealed a high degree of PD-1 and CD103 coexpression, specifically within the CD8 TIL compartment. PD-1(+)CD103(+) CD8 TIL were quiescent when assessed directly ex vivo yet were capable of robust cytokine production after pharmacologic stimulation. Moreover, they showed negligible expression of additional exhaustion-associated markers, including TIM-3, CTLA-4, and LAG-3. Thus, as hypothesized, CD103(+) CD8 TIL express PD-1 and appear quiescent in the tumor microenvironment. However, these cells retain functional competence and demonstrate strong prognostic significance. We speculate that, after standard treatment, PD-1(+)CD103(+) CD8 TIL might regain functional antitumor activity, an effect that potentially could be augmented by immune modulation.

Glioblastoma multiforme (GBM) is the most malignant type of human glioma, and has a poor prognosis. Screening differentially expressed genes (DEGs) in brain tumor samples and normal brain samples is of importance for identifying GBM and to design specific-targeting drugs. The transcriptional profile of GSE30563, containing three genechips of brain tumor samples and three genechips of normal brain samples, was downloaded from Gene Expression Omnibus to identify the DEGs. The differences in the expression of the DEGs in the two different samples were compared through hierarchical biclustering. The co-expression coefficient of the DEGs was calculated using the information from COXPRESdb, the network of the DEGs was constructed and functional enrichment and pathway analysis were performed. Finally, the transcription factors of important DEGs were predicted. A total of 1,006 DEGs, including 368 upregulated and 638 downregulated DEGs, were identified. A close correlation was demonstrated between six important genes, associated with immune response, HLA-DQB1, HLA-DRB1, HLA-DPA1, HLA-B, HLA-DMA and HLA-DRA, and the immune response. Allograft rejection was selected as the most significant pathway. A total of 17 transcription factors, including nuclear factor (NF)-κB and NF-κB1, and their binding sites containing these six DEGs, were also identified. The DEGs, including major histocompatibility complex (MHC) class II, DQβ1, MHC class II, DRβ1, MHC class IB, MHC class II, DMα, MHC class II, DPα1, MHC class II, DRα, may provide novel targets for the diagnosis and treatment of GBM. The transcription factors of these six genes and their binding sites may also provide evidence and direction for identifying target-specific drugs.

BACKGROUND: Low expression of HLA class II antigens has been associated with more aggressive disease in several human malignancies including adult adrenocortical tumors (ACT), but their clinical relevance in pediatric ACT needs to be investigated.
PROCEDURE: This study analyzed the expression profile of three class II histocompatibility genes (HLA-DRA, HLA-DPA1, and HLA-DPB1) in 58 consecutive pediatric ACT (13 adenomas and 45 carcinomas) by quantitative real time PCR and their association with clinical and biological features. HLA-DPA1 protein level was determined by immunohistochemistry.
RESULTS: A significant association (P < 0.01) was observed between lower expression levels of the three genes analyzed and poor prognostic factors such as age ≥ 4 years, tumor size ≥ 200 cm(3), tumor weight ≥ 100 g, and metastatic disease; the presence of an unfavorable event and death. Underexpression of the HLA-DRA, HLA-DPA1, and HLA-DPB1 genes were associated with lower 5-year event-free survival (EFS) (P = 0.017, P < 0.001, and P = 0.017, respectively). Cox multivariate analysis showed that HLA-DPA1 was an independent prognostic factor (P = 0.029) when analyzed in association with stage IV, age and tumor size. Significantly lower EFS was also observed in patients with negative/weak immunostaining for HLA-DPA1 (P = 0.002). Similar results were observed when only patients classified as having carcinomas were analyzed.
CONCLUSION: Our results suggest that lower expression of HLA-DRA, HLA-DPA1, and HLA-DPB1 genes may contribute to more aggressive disease in pediatric ACT. HLA-DPA1 immunostaining may represent potential aggressiveness marker in this tumor.

Primary effusion lymphoma (PEL) presents as a serous lymphomatous effusion without tumor masses exclusively in body cavities and mainly occurs in human immunodeficiency virus-1 (HIV-1)-infected patients. We established a new PEL cell line, designated GTO, from the pericardial effusion of a 39-year-old Japanese patient with acquired immunodeficiency syndrome-related PEL. This cell line was infected with human herpesvirus-8, but not with Epstein-Barr virus. Southern blot hybridization demonstrated that GTO cells display monoclonal rearrangement of the IgH gene, suggesting clonal B cell proliferation. GTO cells weakly express or lack T cell-associated markers (CD3, CD5, CD8), the majority of B cell-associated markers (CD19, CD20, CD21, CD79a), the α chains of β 2 integrins (CD11a, CD11b, CD11c), HLA-DR, CD30, and surface immunoglobulin (sIgM, sIgG sIgκ, sIgλ), TCR (α/β, γδ), but express CD45, and post-germinal center B cell/plasma cell-associated antigens (CD38, CD138). They also express a high level of cell-surface CD4 and can be infected by HIV-1. Immunodeficient mice intraperitoneally xenografted with GTO cells developed ascites containing lymphoma cells. The establishment of GTO and a GTO xenograft mouse model may help to provide insights toward a better understanding of the pathogenesis of PEL and the relationship between HIV-1 and HHV-8.

BACKGROUND: Malignant cells in tumours of B-cell origin account for 0.1% to 98% of the total cell content, depending on disease entity. Recently, gene expression profiles (GEPs) of B-cell lymphomas based on microarray technologies have contributed significantly to improved sub-classification and diagnostics. However, the varying degrees of malignant B-cell frequencies in analysed samples influence the interpretation of the GEPs. Based on emerging next-generation sequencing technologies (NGS) like tag sequencing (tag-seq) for GEP, it is expected that the detection of mRNA transcripts from malignant B-cells can be supplemented. This study provides a quantitative assessment and comparison of the ability of microarrays and tag-seq to detect mRNA transcripts from malignant B-cells. A model system was established by eight serial dilutions of the malignant B-cell lymphoma cell line, OCI-Ly8, into the embryonic kidney cell line, HEK293, prior to parallel analysis by exon microarrays and tag-seq.
RESULTS: We identified 123 and 117 differentially expressed genes between pure OCI-Ly8 and HEK293 cells by exon microarray and tag-seq, respectively. There were thirty genes in common, and of those, most were B-cell specific. Hierarchical clustering from all dilutions based on the differentially expressed genes showed that neither technology could distinguish between samples with less than 1% malignant B-cells from non-B-cells. A novel statistical concept was developed to assess the ability to detect single genes for both technologies, and used to demonstrate an inverse proportional relationship with the sample purity. Of the 30 common genes, the detection capability of a representative set of three B-cell specific genes--CD74, HLA-DRA, and BCL6 - was analysed. It was noticed that at least 5%, 13% and 22% sample purity respectively was required for detection of the three genes by exon microarray whereas at least 2%, 4% and 51% percent sample purity of malignant B-cells were required for tag-seq detection.
CONCLUSION: A sample purity-dependent loss of the ability to detect genes for both technologies was demonstrated. Taq-seq, in comparison to exon microarray, required slightly less malignant B-cells in the samples analysed in order to detect the two most abundantly expressed of the selected genes. The results show that malignant cell frequency is an important variable, with fundamental impact when interpreting GEPs from both technologies.

The induction of the major histocompatibility (MHC), antigen-presenting class II molecules by interferon-gamma, in solid tumor cells, requires the retinoblastoma tumor suppressor protein (Rb). In the absence of Rb, a repressosome blocks the access of positive-acting, promoter binding proteins to the MHC class II promoter. However, a complete molecular linkage between Rb expression and the disassembly of the MHC class II repressosome has been lacking. By treating A549 lung carcinoma cells with a novel small molecule that prevents phosphorylation-mediated, Rb inactivation, we demonstrate that Rb represses the synthesis of an MHC class II repressosome component, YY1. The reduction in YY1 synthesis correlates with the advent of MHC class II inducibility; with loss of YY1 binding to the promoter of the HLA-DRA gene, the canonical human MHC class II gene; and with increased Rb binding to the YY1 promoter. These results support the concept that the Rb gene regulatory network (GRN) subcircuit that regulates cell proliferation is linked to a GRN subcircuit regulating a tumor cell immune function.

Cytokine expression in a tumor microenvironment can impact both host defense against the tumor and tumor cell survival. In this study, we sought to clarify whether the cytokine gene expression profile could have clinical associations with ovarian cancer. We analyzed the expression of 16 cytokine genes (IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12p35, IL-12p40, IL-15, IFN-γ, TNF-α, IL-6, HLA-DRA, HLA-DPA1 and CSF1) in 50 ovarian carcinomas. Hierarchical clustering analysis of these tumors was carried out using Cluster software and differentially expressed genes were examined between clear cell carcinoma (CCC) and other subtypes. Following this examination we evaluated the biological significance of IL-6 knockdown in CCC. Unsupervised hierarchical clustering analysis of cytokine gene expression revealed two distinct clusters. The relationship between the two clusters and clinical parameters showed statistically significant differences in CCC compared to other histologies. CCC showed a dominant Th-2 cytokine expression pattern driven largely by IL-6 expression. Inhibition of IL-6 in CCC cells suppressed Stat3 signaling and rendered cells sensitive to cytotoxic agents. The unique cytokine expression pattern found in CCC may be involved in the pathogenesis of this subtype. In particular, high IL-6 expression appears likely to be driven by the tumor cells, fueling an autocrine pathway involving IL-6 expression and Stat3 activation and may influence survival when exposed to cytotoxic chemotherapy. Modulation of IL-6 expression or its related signaling pathway may be a promising strategy of treatment for CCC.

One mechanism frequently utilized by tumor cells to escape immune system recognition and elimination is suppression of cell surface expression of Major Histocompatibility Class II (MHC II) molecules. Expression of MHC II is regulated primarily at the level of transcription by the Class II Transactivator, CIITA, and decreased CIITA expression is observed in multiple tumor types. We investigate here contributions of epigenetic modifications to transcriptional silencing of CIITA in variants of the human breast cancer cell line MDA MB 435. Significant increases in histone H3 lysine 27 trimethylation upon IFN-γ stimulation correlate with reductions in transcription factor recruitment to the interferon-γ inducible CIITA promoter, CIITApIV, and with significantly increased CIITApIV occupancy by the histone methyltransferase enhancer of zeste homolog 2 (EZH2). Most compelling is evidence that decreased expression of EZH2 in MDA MB 435 variants results in significant increases in CIITA and HLA-DRA mRNA expression, even in the absence of interferon-γ stimulation, as well as increased cell surface expression of MHC II. Together, these data add mechanistic insight to prior observations of increased EZH2 expression and decreased CIITA expression in multiple tumor types.

The underlying etiological cause of non-hereditary colorectal cancer has yet to be determined. The adenoma-carcinoma sequence is widely accepted, however, a sole trigger has not been specified. Therefore, we sought to further define genotypic and phenotypic parameters that could be involved in promoting a possible infection, inflammation and hyper-proliferation, followed by the adenoma-carcinoma sequence. Expression of phenotype-related parameters for MHC class I (HLA-A N-20 and β2 microglobulin) and class II (HLA-DRα and HLA-DR) as well as CD45 and carcinoembryonic antigen (CEA) were investigated immunohistochemically in a series of 93 colorectal cancers. Additionally, in 49 of the tumours the MHC class II genotype was analysed. MHC class II genotype analyses revealed a tendency towards DRB1*08 and DQB1*04. A significant association among the MHC class I markers or the MHC class II markers was found. No difference in marker expression could be detected between tumour and stromal tissue, however a significant inverse expression existed for markers of the functionally different class I or II systems. With the exception of CEA, there was no correlation between expression of any marker and tumour grade. Only 2% of tumours expressed no markers for MHC class I and II. Further studies on MHC class I and II genotype and phenotype relation in colorectal cancer may help to identify trigger mechanisms for tumourigenesis, involved markers and possible mechanisms of subsequent immune escape.

Histone deacetylase (HDAC) is strongly associated with epigenetic regulation and carcinogenesis, and its inhibitors induce the differentiation or apoptosis of cancer cells. Valproic acid (VPA) is one of the clinically available HDAC inhibitors. We investigated the anticancer effects of VPA in combination with gemcitabine (GEM) in the human cholangiocarcinoma cell line HuCCT1, and explored the mechanisms of the anticancer effects using microarray analysis. The anticancer effects of VPA or gemcitabine (GEM), and the effects of VPA combined with GEM, were studied by a cell proliferation assay. A microarray analysis was performed and the genes were picked up using GeneSpring GX11.5, followed by Ingenuity Pathways Analysis (IPA) and determination of gene expression by RT-PCR. GEM (5 nM) and VPA (0.5 mM) reduced proliferation by 23%, which significantly augmented the anticancer effect of GEM alone or VPA alone (P<0.01). Using microarray analysis, 43 genes were identified with the comparison between the GEM group and the GEM plus VPA combination group. Interactions were identified between genes of the 'Cellular Development' network relevant to the differentiation of cancer cells using IPA. Furthermore, GEM combined with VPA up-regulated the HLA-DRA expression compared to the single agents (P<0.01). VPA augmented the effects of GEM by enhancing the gene network mainly including HLA-DRA, possibly through the modification of cancer cell differentiation.

Absent in Melanoma 2 (AIM2) is a member of the HIN-200 family of hematopoietic, IFN-inducible, nuclear proteins, associated with both, infection defense and tumor pathology. Recently, AIM2 was found to act as a DNA sensor in innate immunity. In addition, we and others have previously demonstrated a high frequency of AIM2-alterations in microsatellite unstable (MSI-H) tumors. To further elucidate AIM2 function in colorectal tumors, we here addressed AIM2-responsive target genes by microarray based gene expression profiling of 22 244 human genes. A total of 111 transcripts were significantly upregulated, whereas 80 transcripts turned out to be significantly downregulated in HCT116 cells, constitutively expressing AIM2, compared with AIM2-negative cells. Among the upregulated genes that were validated by quantitative PCR and western blotting we recognized several interferon-stimulated genes (ISGs: IFIT1, IFIT2, IFIT3, IFI6, IRF7, ISG15, HLA-DRA, HLA-DRB, TLR3 and CIITA), as well as genes involved in intercellular adhesion and matrix remodeling. Expression of ISGs correlated with expression of AIM2 in 10 different IFN-γ treated colorectal cancer cell lines. Moreover, small interfering RNA-mediated knock-down of AIM2 resulted in reduced expression of HLA-DRA, HLA-DRB and CIITA in IFN-γ-treated cells. IFN-γ independent induction of HLA-DR genes and their encoded proteins was also demonstrated upon doxycyclin-regulated transient induction of AIM2. Luciferase reporter assays revealed induction of the HLA-DR promoter upon AIM2 transfection in different cell lines. STAT-signaling was not involved in IFN-γ independent induction of ISGs, arguing against participation of cytokines released in an autostimulating manner. Our data indicate that AIM2 mediates both IFN-γ dependent and independent induction of several ISGs, including genes encoding the major histocompatibility complex (MHC) class II antigens HLA-DR-α and -β. This suggests a novel role of the IFN/AIM2/ISG cascade likewise in cancer cells.

Prior genome-wide association (GWA) studies have identified 10 susceptibility loci for risk of chronic lymphocytic leukemia (CLL). To identify additional loci, we performed a GWA study in 407 CLL cases (of which 102 had a family history of CLL) and 296 controls. Moreover, given the strong familial risk of CLL, we further subset our GWA analysis to the CLL cases with a family history of CLL to identify loci specific to these familial CLL cases. Our top hits from these analyses were evaluated in an additional sample of 252 familial CLL cases and 965 controls. Using all available data, we identified and confirmed an independent association of 4 single-nucleotide polymorphisms (SNPs) that met genome-wide statistical significance within the IRF8 (interferon regulatory factor 8) gene (combined P values ≤ 3.37 × 10(-8)), located in the previously identified 16q24.1 locus. Subsetting to familial CLL cases, we identified and confirmed a new locus on chromosome 6p21.3 (combined P value = 6.92 × 10(-9)). This novel region harbors the HLA-DQA1 and HLA-DRB5 genes. Finally, we evaluated the 10 previously reported SNPs in the overall sample and replicated 8 of them. Our findings support the hypothesis that familial CLL cases have additional genetic variants not seen in sporadic CLL. Additional loci among familial CLL cases may be identified through larger studies.

Previous epidemiologic studies showed four times increased risk of acute lymphoblastic leukemia (ALL) in children of women with multiple sclerosis (MS). MS shows a risk association with Human leukocyte antigens (HLA)-DRA single nucleotide polymorphism (SNP) rs3135388, which is a proxy marker for DRB1*1501. We examined the relevance of rs3135388 in childhood ALL risk along with two other HLA-DRA SNPs in two case-control groups: 114 cases and 388 controls from South Wales (UK) and 100 Mexican Mestizo cases and 253 controls. We first confirmed the correlation between rs3135388 and DRB1*1501 in HLA-typed reference cell lines. We noted a female-specific risk association in childhood ALL (pooled odds ratio (OR) = 2.6, 95% confidence interval (CI) = 1.5-4.5, Mantel-Haenszel P = 0.0009) similar to the stronger association of DRB1*1501 in females with MS. Examination of an HLA-C 5' flanking region SNP rs9264942, known to correlate with HLA-C expression, showed a protective association in girls (OR = 0.4, 95% CI = 0.2-0.7, Mantel-Haenszel P = 0.0003) similar to the protective HLA-Cw*05 association in MS. In a reference cell line panel, HLA-Cw5 homozygous samples (n = 8) were also homozygous for the minor allele of the SNP. Likewise, the male-specific protective association of interferon-gamma (IFNG) SNP rs2069727 in MS was replicated with the same sex specificity in childhood ALL (OR = 0.6, 95% CI = 0.4-1.0, Mantel-Haenszel P = 0.03). Two other SNPs in superkiller viralicidic activity 2-like and tenascin XB that are markers for systemic lupus erythematosus susceptibility showed female-specific associations but due to linkage disequilibrium with HLA-DRB1*15. Our observations supported the epidemiologic link between MS and childhood ALL and added the sex effect to this connection. It appears that only girls born to mothers with MS may have an increased risk of ALL. Investigating the mechanism of these sex-specific associations may help understand the pathogenesis of MS and ALL.

PURPOSE: Inflammatory genes and microRNAs have roles in colon carcinogenesis; therefore, they may provide useful biomarkers for colon cancer. This study examines the potential clinical utility of an inflammatory gene expression signature as a prognostic biomarker for colon cancer in addition to previously examined miR-21 expression.
EXPERIMENTAL DESIGN: Quantitative reverse transcriptase-PCR. was used to measure the expression of 23 inflammatory genes in colon adenocarcinomas and adjacent noncancerous tissues from 196 patients. These data were used to develop models for cancer-specific mortality on a training cohort (n = 57), and this model was tested in both a test (n = 56) and a validation (n = 83) cohort. Expression data for miR-21 were available for these patients and were compared and combined with inflammatory gene expression.
RESULTS: PRG1, IL-10, CD68, IL-23a, and IL-12a expression in noncancerous tissue, and PRG1, ANXA1, IL-23a, IL-17a, FOXP3, and HLA-DRA expression in tumor tissues were associated with poor prognosis based on Cox regression (/Z-score/ >1.5) and were used to generate the inflammatory risk score (IRS). IRS was associated with cancer-specific mortality in the training, test (P = 0.01), and validation (P = 0.02) cohorts. This association was strong for stage II cases (P = 0.002). Expression of miR-21 was associated with IL-6, IL-8, IL-10, IL-12a, and NOS2a, providing evidence that the function of this microRNA and these inflammatory genes are linked. Both IRS and miR-21 expression were independently associated with cancer-specific mortality, including stage II patients alone.
CONCLUSION: IRS and miR-21 expression are independent predictors of colon cancer prognosis and may provide a clinically useful tool to identify high-risk patients.

In human B cells, effective major histocompatibility complex (MHC) class II-antigen presentation depends not only on MHC class II, but also on the invariant chain (CD74 or Ii), HLA-DM (DM) and HLA-DO (DO), the chaperones regulating the antigen loading process of MHC class II molecules. We analysed immediate ex vivo expression of HLA-DR (DR), CD74, DM and DO in B cell chronic lymphocytic leukaemia (B-CLL). Real-time reverse transcription polymerase chain reaction demonstrated a highly significant upregulation of DRA, CD74, DMB, DOA and DOB mRNA in purified malignant cells compared to B cells from healthy donors. The increased mRNA levels were not translated into enhanced protein levels but could reflect aberrant transcriptional regulation. Indeed, upregulation of DRA, DMB, DOA and DOB mRNA correlated with enhanced expression of class II transactivator (CIITA). In-depth analysis of the various CIITA transcripts demonstrated a significant increased activity of the interferon-gamma-inducible promoter CIITA-PIV in B-CLL. Comparison of the aberrant mRNA levels with clinical outcome identified DOA mRNA as a prognostic indicator for survival. Multivariate analysis revealed that the prognostic value of DOA mRNA was independent of the mutational status of the IGHV genes. Thus, aberrant transcription of DOA forms a novel and additional prognostic indicator for survival in B-CLL.

Direct comparison and ranking of vaccine formulations in pre-clinical studies will expedite the identification of cancer vaccines for clinical trials. Two human ErbB-2 (Her-2) vaccines, naked DNA and whole cell vaccine, were tested side-by-side in wild type and Her-2 transgenic mice. Both vaccines can induce humoral and cellular immunity to the entire repertoire of Her-2 epitopes. Mice were electro-vaccinated i.m. with a mixture of pGM-CSF and pE2TM, the latter encodes Her-2 extracellular and transmembrane domains. Alternatively, mice were injected i.p. with human ovarian cancer SKOV3 cells that have amplified Her-2. In wild type mice, comparable levels of Her-2 antibodies (Ab) were induced by these two vaccines. However, T cell immunity and protection against Her-2(+) tumors were superior in DNA vaccinated mice. In BALB Her-2 transgenic (Tg) mice, which were tolerant to Her-2, DNA and cell vaccines were administered after regulatory T cells (Treg) were removed by anti-CD25 mAb. Again, comparable levels of Her-2 Ab were induced, but DNA vaccines rendered greater anti-tumor activity. In B6xDR3 Her-2 Tg mice that expressed the autoimmune prone HLA-DR3 allele, higher levels of Her-2 Ab were induced by SKOV3 cell than by Her-2 DNA. But anti-tumor activity was still more profound in DNA vaccinated mice. Therefore, Her-2 DNA vaccine induced greater anti-tumor immunity than cell vaccine, whether mice were tolerant to Her-2 or susceptible to autoimmunity. Through such side-by-side comparisons in appropriate pre-clinical test systems, the more effective vaccine formulations will emerge as candidates for clinical trials.

BACKGROUND AND PURPOSE: Nasopharyngeal carcinoma (NPC) represents a seldom malignancy in most developed countries. Nevertheless, NPC receives an endemic form in concrete racial entities. The aims of this study were to detect the presence of Epstein-Barr virus DNA (EBV-DNA) in peripheral blood of NPC patients, to molecularly define human leukocyte antigens (HLA) DRB1*, DQA1* and DQB1* allele frequencies, and, finally, to determine whether the genetic predisposition of an individual to NPC depends on the liability to EBV infection.
PATIENTS AND METHODS: A total of 101 patients of Hellenic origin and nationality, with histologically proven NPC, participated in this study. EBV-DNA detection was also applied in 66 patients with EBV-related malignancies (Hodgkin's [HL] and non-Hodgkin's lymphoma [NHL]) and infectious mononucleosis (IM), as well as in 80 healthy EBV-seropositive controls.
RESULTS: 81% of the NPC patients, 77.8% with HL, 72.2% with NHL, and 66.7% with IM were EBV-DNA positive, whereas the EBV genome was detected only in 15% of the healthy controls. These differences were statistically significant in all cases. Analysis of HLA class II antigens showed decreased frequency of the DRB1*07 (p = 0.003), DQA1*0103 (p = 0.002), and DQA1*0201 (p = 0.003) alleles among NPC patients. A significant association between the HLA-DR/DQ alleles and the presence of EBV-DNA in peripheral whole blood was not established.
CONCLUSION: Circulating EBV-DNA and specific HLA class II alleles may predispose to or protect from NPC. However, the results of this study suggest that the genetic predisposition of an individual to NPC is independent of the liability to EBV infection.

We, for the first time, directly compared gene expression profiles in human non-small cell lung carcinomas (NSCLCs) and in human fetal lung development. Previously reported correlations of gene expression profiles between lung cancer and lung development, deduced from matching data on mouse development and human cancer, have brought important information, but suffered from different timing of mouse and human gene expression during fetal development and fundamental differences in tumorigenesis in mice and humans. We used the suppression subtractive hybridization technique to subtract cDNAs prepared from human fetal lung samples at weeks 10-12 and 22-24 and obtained a cDNA library enriched in the transcripts more abundant at the later stage. cDNAs sequencing and RT-PCR analysis of RNAs from human fetal and adult lungs revealed 12 differentially transcribed genes: ADH1B, AQP1, FOLR1, SLC34A2, CAV1, INMT, TXNIP, TPM4, ICAM-1, HLA-DRA, EFNA1 and HLA-E. Most of these genes were found up-regulated in mice and rats at later stages than in human lung development. In surgical samples of NSCLC, these genes were down-regulated as compared to surrounding normal tissues and normal lungs, thus demonstrating opposite expression profiles for the genes up-regulated during fetal lung development.

BACKGROUND: Melanoma progression occurs through three major stages: radial growth phase (RGP), confined to the epidermis; vertical growth phase (VGP), when the tumor has invaded into the dermis; and metastasis. In this work, we used suppression subtractive hybridization (SSH) to investigate the molecular signature of melanoma progression, by comparing a group of metastatic cell lines with an RGP-like cell line showing characteristics of early neoplastic lesions including expression of the metastasis suppressor KISS1, lack of alphavbeta3-integrin and low levels of RHOC.
METHODS: Two subtracted cDNA collections were obtained, one (RGP library) by subtracting the RGP cell line (WM1552C) cDNA from a cDNA pool from four metastatic cell lines (WM9, WM852, 1205Lu and WM1617), and the other (Met library) by the reverse subtraction. Clones were sequenced and annotated, and expression validation was done by Northern blot and RT-PCR. Gene Ontology annotation and searches in large-scale melanoma expression studies were done for the genes identified.
RESULTS: We identified 367 clones from the RGP library and 386 from the Met library, of which 351 and 368, respectively, match human mRNA sequences, representing 288 and 217 annotated genes. We confirmed the differential expression of all genes selected for validation. In the Met library, we found an enrichment of genes in the growth factors/receptor, adhesion and motility categories whereas in the RGP library, enriched categories were nucleotide biosynthesis, DNA packing/repair, and macromolecular/vesicular trafficking. Interestingly, 19% of the genes from the RGP library map to chromosome 1 against 4% of the ones from Met library.
CONCLUSION: This study identifies two populations of genes differentially expressed between melanoma cell lines from two tumor stages and suggests that these sets of genes represent profiles of less aggressive versus metastatic melanomas. A search for expression profiles of melanoma in available expression study databases allowed us to point to a great potential of involvement in tumor progression for several of the genes identified here. A few sequences obtained here may also contribute to extend annotated mRNAs or to the identification of novel transcripts.

Chronic myelogenous leukemia (CML) is characterized by the presence of Philadelphia chromosome resulting from bcr/abl translocation. To clarify the association between HLA class II allele and haplotype frequencies in CML, 50 patients referred to Hematology Oncology and Bone Marrow Transplantation (BMT) center, Shariaty Hospital, Tehran, Iran, were randomly selected and compared with a group of 80 unrelated healthy blood donor subjects. HLA class II alleles were determined by PCR-SSP method. The results showed that the frequencies of DQB1*03011 (P=0.01) and DQA1*0505 (P=0.05) were higher, while that of DQB1*03032 (P=0.04) was lower in patients than in the controls. Regarding age-at-onset, the frequency of HLA-DRB1*07 (P=0.03) and -DQA1*0201 (P=0.03) alleles were higher in patients younger than 35 years. The most frequent haplotypes in our CML patients were HLA-DRB1*11/-DQB1*03011/-DQA1*0505 (P=0.01) and HLA-DRB1*04/-DQB1*0302/-DQA1*03011 (P=0.02). In conclusion, it is suggested that positive and negative association in certain HLA alleles and haplotypes exist in Iranian patients with CML.

Previous studies have demonstrated some significant differences in HLA allele frequencies in leukemic patients and normal subjects. We have analyzed HLA class II alleles and haplotypes in 60 Iranian patients with acute myelogenous leukemia (AML) and 180 unrelated normal subjects. Blood samples were collected after obtaining informed consents. From the patients and control DNA extraction and HLA typing were performed using PCR-SSP method. Significant positive association with the disease was found for HLA-DRB1*11 allele (35% vs. 24.7%, p=0.033). Two alleles including HLA-DRB4 and -DQB1*0303 were found to be significantly decreased in patients compared to controls. Regarding haplotype analysis, no significant association was found between case and control groups. It is suggested that HLA-DRB1*11 allele plays as a presumptive predisposing factor while the HLA-DRB4 and -DQB1*0303 alleles are suggested as protective genetic factors against acute myelogenous leukemia. Larger studies are needed to confirm and establish the role of these associations with acute myelogenous leukemia.

The development of cancer is a multistep process that is characterized by the accumulation of genetic alterations in cells and changed cellular interactions with the surrounding healthy tissues. The human immune system is believed to be intrinsically involved in this process. The correlation of certain human leukocyte antigen (HLA)-class I and II haplotypes with tumorigenesis is documented in a variety of tumors. However, few data exist on the possible association of specific HLA-class II alleles or haplotypes with ovarian cancer. In our sample of 52 Caucasian patients with primary ovarian carcinoma and 239 female healthy local controls, we observed a significantly increased incidence of the HLA-class II haplotypes DRB1*0301 - DQA1*0501 - DQB1*0201 (p < 0.001) and DRB1*1001 - DQA1*0101 - DQB1*0501 (p < 0.001) in the patients. Our data suggest that HLA-class II loci or individual HLA-class II haplotypes may be involved in the pathogenesis of ovarian cancer.