Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (EBV+ DLBCL) is typically an aggressive tumor in elderly patients. However, in a subset of young patients, EBV+ DLBCL follows a relatively indolent clinical course and exhibits a good response to chemotherapy. This lymphoma comprises polymorphous lymphoma and large cell lymphomas subtypes, with the latter subtype showing a significantly poorer prognosis. It is unknown whether the genetic background differs between age groups and histopathological subtypes. To investigate the genetic basis, heterogeneity, and recurrently mutated genes in EBV+ DLBCL, we performed whole-exome sequencing of DNA from 11 tissue samples of this lymphoma. Sequencing revealed that the most common substitution was the transition C>T/G>A. Genetic features-including the numbers of mutated genes in exonic region, single-nucleotide variants (SNV), and indels-did not significantly differ between age groups or histological subtypes. Matching with the COSMIC database revealed that the main mutational signature was signature 3, which is associated with failure of DNA double-strand break-repair by homologous recombination. Mutant-Allele Tumor Heterogeneity (MATH) scores showed that EBV+ DLBCL exhibited broad intratumor heterogeneity, and were positively correlated with Ann Arbor Stage and ≥2 extranodal lesion sites. We identified 57 selected recurrently mutated genes. The most commonly mutated five genes-LNP1 (11/11), PRSS3 (10/11), MUC3A (9/11), FADS6 (9/11), and TRAK1 (8/11)-were validated by Sanger sequencing. These mutated genes have not previously been identified. Overall, our present results demonstrate the tremendous genetic heterogeneity underlying EBV+ DLBCLs, and highlight the need for personalized therapeutic approaches to treating these patients.

BACKGROUND: T-cell immunoglobulin mucin 3 (TIM3) is a negative immune checkpoint and plays a crucial part in tumor-induced immune suppression. However, the mechanism of TIM3 in regulating immunosuppression in head and neck squamous cell carcinoma (HNSCC) was still not quite clear.
METHODS: We carried out the immunohistochemistry staining of HNSCC tissue microarrays. Through quantification of the histoscore, we performed the correlation analysis among the TIM3, Galectin-9, Foxp3, CD68 and CD163. The effects of TIM3 on regulatory T cells (Tregs) and macrophages were detected by utilizing the Tgfbr1/Pten 2cKO HNSCC mouse model. Flow cytometry were used to analysis the percent of Tregs, macrophages and IFN-γ.
RESULTS: We demonstrated the close association among TIM3/Galectin-9 pathway, regulatory T cell marker (Foxp3) and macrophage marker (CD68, CD163) in human HNSCC. In the transgenic HNSCC mouse model, blockade of TIM3 by the anti-TIM3 monoclonal antibody induced a reduction of CD4
CONCLUSIONS: The present study demonstrated that TIM3 was associated with the immunosuppression in HNSCC. And targeting TIM3 can enhance anti-tumor immune response by decreasing Tregs in HNSCC.

The treatment of advanced or refractory non-small cell lung cancer has been historically difficult owing to the lack of studies on effective systemic cure. The progress in lung cancer treatment has plateaued, necessitating new options for additional benefits. Immune checkpoint proteins are co-inhibitory factors that can diminish the antigen-specific immune responses by attenuating the regulatory role of cytotoxic T-lymphocyte-associated protein 4, programmed cell death-1, lymphocyte-activation gene 3, and T-cell immunoglobulin mucin-3. The therapeutic strategies targeting immune checkpoints mainly focus on the monoclonal antibody of these regulatory factors, which may facilitate clinical decision making. An enhanced understanding of the drug-resistance mechanisms and the therapeutic efficacy regulation will provide opportunities to improve the clinical outcomes of non-small cell lung cancer patients. Preclinical and clinical trials on these key immune-regulatory agents, which has heralded a new era in immuno-oncology in non-small cell lung cancer treatment, are currently in development.

BACKGROUND: The purpose of the present study was to explore novel biomarkers that can predict the clinical outcome of patients before treatment or during vaccination. These would be useful for the selection of appropriate patients who would be expected to exhibit better treatment outcomes from vaccination, and for facilitating the development of cancer vaccine treatments.
METHODS: From a single-arm, non-randomized, human leukocyte antigen (HLA)-A-status-blind phase II trial of a vaccine treatment using three HLA-A*2402-restricted peptides for advanced pancreatic cancer (PC), we obtained peripheral blood samples from 36 patients of an HLA-A*2402-matched group and 27 patients of an HLA-A*2402-unmatched group.
RESULTS: Multivariate analysis (HR = 2.546; 95% CI = 1.138 to 5.765; p = 0.0231) and log-rank test (p = 0.0036) showed that a high expression level of programmed death-1 (PD-1) on CD4+ T cells was a negative predictive biomarker of overall survival in the HLA-A*2402-matched group . Moreover, a high expression level of PD-1 on CD4+ T cells was a negative predictor for the induction of cytotoxic T lymphocytes (p = 0.0007). After treatment, we found that the upregulation of PD-1 and T cell immunoglobulin mucin-3 (Tim-3) expression on CD4+ and CD8+ T cells was significantly associated with a poor clinical outcome in the HLA-A*2402-matched group (p = 0.0330, 0.0282, 0.0046, and 0.0068, respectively). In contrast, there was no significant difference for these factors in the HLA-A*2402-unmatched group.
CONCLUSIONS: Our results indicate that the upregulation of PD-1 and Tim-3 expression on CD4+ and CD8+ T cells may restrict T cell responses in advanced PC patients; therefore, combination immunotherapy with blockade of PD-1 and Tim-3 to restore T cell responses may be a potential therapeutic approach for advanced PC patients.
TRIAL REGISTRATION: Clinical-Trail-Registration: UMIN000008082 .

Inhibition of immune checkpoint proteins (checkpoints) has become a promising anti-esophageal cancer strategy. We here tested expressions of immune checkpoints in human esophageal cancers. Our results showed the expressions of many immune checkpoints, including CD28, CD27, CD137L, programmed death 1 (PD-1), T cell immunoglobulin mucin-3 (TIM-3), T cell Ig and ITIM domain (TIGIT), CD160, cytotoxic T lymphocyte antigen 4 (CTLA-4), CD200, CD137 and CD158, were dysregulated in peripheral T cells of esophageal cancer patients. Further, the expressions of PD-1, TIM-3 and TIGIT were upregulated in tumor infiltrating lymphocytes (TILs), which might be associated with TILs exhaustion. Meanwhile, the expressions of PD-1 and TIM-3 on CD4+ T cells were closely associated with clinic pathological features of esophageal cancer patients. These results indicate that co-inhibitory receptors PD-1, TIM-3 and TIGIT may be potential therapeutic oncotargets for esophageal cancer.

PURPOSE: Tyrosine kinase inhibitors are effective in gastrointestinal stromal tumors (GISTs) but often are of transient benefit as resistance commonly develops. Immunotherapy, particularly blockade of the inhibitory receptor programmed death 1 (PD-1) or the ligand programmed death ligand 1 (PD-L1), has shown effectiveness in a variety of cancers. The functional effects of PD-1/PD-L1 blockade are unknown in GISTs.
EXPERIMENTAL DESIGN: We analyzed tumor and matched blood samples from 85 patients with GISTs and determined the expression of immune checkpoint molecules using flow cytometry. We investigated the combination of imatinib with PD-1/PD-L1 blockade in Kit
RESULTS: The inhibitory receptors PD-1, lymphocyte activation gene 3, and T-cell immunoglobulin mucin-3 were upregulated on tumor-infiltrating T cells compared with T cells from matched blood. PD-1 expression on T cells was highest in imatinib-treated human GISTs. Meanwhile, intratumoral PD-L1 expression was variable. In human GIST cell lines, treatment with imatinib abrogated the IFNγ-induced upregulation of PD-L1 via STAT1 inhibition. In Kit
CONCLUSIONS: PD-1/PD-L1 blockade is a promising strategy to improve the effects of targeted therapy in GISTs. Collectively, our results provide the rationale to combine these agents in human GISTs. Clin Cancer Res; 23(2); 454-65. ©2016 AACR.

MUC3A is a membrane-associated mucin that recent evidence reveals the role of MUC3A in pathogenesis and progression of cancers. To evaluate the association between MUC3A expression with overall survival (OS) and recurrence-free survival (RFS) in patients with localized clear-cell renal cell carcinoma (ccRCC), we retrospectively detected MUC3A expression in samples of 384 postoperative localized ccRCC patients by immunohistochemistry. Median follow-up was 73 months (range: 42 - 74 mo). Overall, 41 patients died, 47 experienced recurrence. High MUC3A expression occurred in 45.8% of localized ccRCC cases, which was significantly associated with high pT-stage, high Fuhrman grade, high frequency of necrosis and LVI, and increased risk of recurrence and death (Logrank test P < 0.001 and P < 0.001, respectively). By multivariate analysis, MUC3A expression was confirmed as an adverse independent prognostic factor for OS and RFS. The prognostic accuracy of UISS, SSIGN, Leibovich models was significantly increased when MUC3A expression was integrated. Meanwhile, MUC3A was enrolled into a newly built nomogram with other factors selected by multivariate analysis. Calibration curves revealed optimal consistency between observations and prognosis. In conclusion, high MUC3A expression is an adverse prognostic biomarker for OS and RFS in postoperative localized ccRCC patients.

Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate therapeutic target in AML. Eradication of LSCs should be a critical and efficient therapeutic approach for the cure of AML. T-cell immunoglobulin mucin-3 (TIM-3) is expressed in most types of AML LSCs, but not in normal hematopoietic stem cells (HSCs); therefore, TIM-3 would be one of the promising therapeutic targets to specifically kill AML LSCs, sparing normal HSCs. In xenograft models reconstituted with human AML LSCs or human normal HSCs, an anti-human TIM-3 mouse antibody with cytotoxic activities exerts a potent anti-leukemic effect by targeting AML LSCs but does not affect normal human hematopoiesis in vivo. Here, we would like to introduce the recent studies on TIM-3 in normal and malignant hematopoiesis.

Gastric cancer is one of the most prevalent and aggressive cancers worldwide, and its molecular mechanism remains largely elusive. Here we report the genomic landscape in primary gastric adenocarcinoma of human, based on the complete genome sequences of five pairs of cancer and matching normal samples. In total, 103,464 somatic point mutations, including 407 nonsynonymous ones, were identified and the most recurrent mutations were harbored by Mucins (MUC3A and MUC12) and transcription factors (ZNF717, ZNF595 and TP53). 679 genomic rearrangements were detected, which affect 355 protein-coding genes; and 76 genes show copy number changes. Through mapping the boundaries of the rearranged regions to the folded three-dimensional structure of human chromosomes, we determined that 79.6% of the chromosomal rearrangements happen among DNA fragments in close spatial proximity, especially when two endpoints stay in a similar replication phase. We demonstrated evidences that microhomology-mediated break-induced replication was utilized as a mechanism in inducing ∼40.9% of the identified genomic changes in gastric tumor. Our data analyses revealed potential integrations of Helicobacter pylori DNA into the gastric cancer genomes. Overall a large set of novel genomic variations were detected in these gastric cancer genomes, which may be essential to the study of the genetic basis and molecular mechanism of the gastric tumorigenesis.

In chronic infectious diseases and cancer, CD8⁺ T cells specific for viral and/or tumor antigens undergo repeated T cell receptor (TCR) stimulation due to the persistence of pathogens or cancer cells, gradually losing their ability to secrete interleukin 2 (IL-2), tumor necrosis factor-a (TNF-a), and interferon-g (IFN-g). These CD8⁺ T cells are finally eliminated by apoptosis, a process referred to as immune exhaustion. The worsening immune function is accompanied by phenotypic changes in CD8⁺ T cells, for example, by changes in the expression of exhaustion markers such as programmed cell death protein 1(PD-1), cytotoxic T-lymphocyte antigen 4 (CTLA-4), T cell immunoglobulin mucin 3 (TIM-3), and lymphocyte activation gene 3 (LAG-3). The exhaustion molecules on CD8⁺ T cells interact with their respective ligands and induce negative signals in activated T cells, leading to CD8⁺ T cell dysfunction. In 2013, the combined use of anti-PD-1 and anti- CTLA-4 antibodies was shown to induce extraordinary anti-tumor effects in patients with advanced melanoma. The regulation of exhausted CD8⁺ T cells has now emerged as a promising therapy to treat cancer.

Despite initial response to therapy, most acute myeloid leukemia (AML) patients relapse. To eliminate relapse-causing leukemic stem/progenitor cells (LPCs), patient-specific immune therapies may be required. In vitro cellular engineering may require increasing the "stemness" or immunogenicity of tumor cells and activating or restoring cancer-impaired immune-effector and antigen-presenting cells. Leukapheresis samples provide the cells needed to engineer therapies: LPCs to be targeted, normal hematopoietic stem cells to be spared, and cancer-impaired immune cells to be repaired and activated. This study sought to advance development of LPC-targeted therapies by exploring nongenetic ways to slow the decay and to increase the immunogenicity of primary CD34(+) AML cells. CD34(+) AML cells generally displayed more colony-forming and aldehyde dehydrogenase activity than CD34(-) AML cells. Along with exposure to bone marrow stromal cells and low (1%-5%) oxygen, culture with RepSox (a reprogramming tool and inhibitor of transforming growth factor-β receptor 1) consistently slowed decline of CD34(+) AML and myelodysplastic syndrome (MDS) cells. RepSox-treated AML cells displayed higher CD34, CXCL12, and MYC mRNA levels than dimethyl sulfoxide-treated controls. RepSox also accelerated loss of T cell immunoglobulin mucin-3 (Tim-3), an immune checkpoint receptor that impairs antitumor immunity, from the surface of AML and MDS cells. Our results suggest RepSox may reduce Tim-3 expression by inhibiting transforming growth factor-β signaling and slow decay of CD34(+) AML cells by increasing CXCL12 and MYC, two factors that inhibit AML cell differentiation. By prolonging survival of CD34(+) AML cells and reducing Tim-3, RepSox may promote in vitro immune cell activation and advance development of LPC-targeted therapies.

BACKGROUND: T-cell immunoglobulin mucin-3 (TIM3) has recently been described as an acute myeloid leukemia (AML) stem cell antigen expressed on leukemic myeloblasts, but not on normal hematopoietic stem cells. TIM3 is also expressed by monocytes, natural killer cells, and several T cell subsets; however, normal myeloblasts have not been well-characterized or compared to AML. A specific flow cytometric marker capable of separating leukemic myeloblasts from non-neoplastic myeloblasts would be diagnostically useful, especially in the post-chemotherapy setting.
METHODS: TIM3 myeloblast expression was assessed in 69 bone marrow and/or peripheral blood specimens, including 27 AML and 42 non-neoplastic cases (20 with a recent history of chemotherapy). TIM3 median fluorescence intensity (MFI) was evaluated within myeloblast, monocyte, T cell, and natural killer cell populations.
RESULTS: The median percentage of myeloblasts positive for TIM3 was lower in non-neoplastic specimens without a history of recent chemotherapy (50.3%) as compared to AML (71.4%), but not significantly different as compared to non-leukemic myeloblasts in the post-chemotherapy setting (72.4%). Mean myeloblast TIM3 MFI was higher in AML myeloblasts and non-leukemic myeloblasts in the post-chemotherapy setting as compared to non-neoplastic myeloblasts in cases lacking a history of chemotherapy. Mean monocyte, natural killer cell, and T-cell TIM3 MFI remained relatively constant in varied clinical settings.
CONCLUSIONS: We confirm that leukemic myeloblasts overexpress TIM3 as compared to non-neoplastic controls; however, high levels of expression may also be seen among non-leukemic myeloblasts in the post-chemotherapy setting. This overlap limits the diagnostic utility of TIM3 as a specific marker of neoplasia.

BACKGROUND: T cell immunoglobulin mucin-3 (Tim-3) has been identified as a negative regulator of anti-tumor immunity. Recent studies highlight the important role of Tim-3 in the CD8(+) T cell exhaustion that takes place in both human and animal cancer models. However, the nature of Tim-3 expression in the tumor cell and the mechanism by which it inhibits anti-tumor immunity are unclear. This present study aims to determine Tim-3 is expressed in cervical cancer cells and to evaluate the role of Tim-3 in cervical cancer progression.
METHODOLOGY: A total of 85 cervical tissue specimens including 43 human cervical cancer, 22 cervical intraepithelial neoplasia (CIN) and 20 chronic cervicitis were involved. Tim-3 expression in tumor cells was detected and was found to correlate with clinicopathological parameters. Meanwhile, expression of Tim-3 was assessed by RT-PCR, Western Blot and confocal microscopy in cervical cancer cell lines, HeLa and SiHa. The migration and invasion potential of Hela cells was evaluated after inhibiting Tim-3 expression by ADV-antisense Tim-3.
CONCLUSIONS: We found that Tim-3 was expressed at a higher level in the clinical cervical cancer cells compared to the CIN and chronic cervicitis controls. We supported this finding by confirming the presence of Tim-3 mRNA and protein in the cervical cell lines. Tim-3 expression in tumor cells correlated with clinicopathological parameters. Patients with high expression of Tim-3 had a significant metastatic potential, advanced cancer grades and shorter overall survival than those with lower expression. Multivariate analysis showed that Tim-3 expression was an independent factor for predicting the prognosis of cervical cancer. Significantly, down-regulating the expression of Tim-3 protein inhibited migration and invasion of Hela cells. Our study suggests that the expression of Tim-3 in tumor cells may be an independent prognostic factor for patients with cervical cancer. Moreover, Tim-3 expression may promote metastatic potential in cervical cancers.

Gestational choriocarcinoma is a malignant trophoblastic tumor that usually occurs in the uterus after pregnancy. The tumor is curable with advanced chemotherapy, but the molecular mechanism of choriocarcinoma tumorigenesis remains unclear. This is partly because the low incidence makes it difficult to obtain clinical samples for investigation and because an appropriate choriocarcinoma cell model to study the tumorigenesis has not been developed. We have established a new choriocarcinoma cell line, induced choriocarcinoma cell-1 (iC(3)-1), that possesses unique characteristics compared to other choriocarcinoma cell lines, including production of tumors that consist of the two types of cells commonly found in choriocarcinoma and mimicking of the clinical pathology. Existing trophoblast cell lines utilized in previous choriocarcinoma studies have had significantly dissimilar gene expression profiles. Therefore, it is important to choose an appropriate cell line for a particular study based on the characteristics of the cell line. In this study, to clarify the genetic characteristics of iC(3)-1 and to explore the tumorigenesis mechanism, we examined the gene profile of iC(3)-1 compared to those of existing cell lines and normal placental tissue. Bioinformatics analysis showed that several characteristic genes, IGF1R, CHFR, MUC3A, TAF7, PARK7, CDC123 and PSMD8, were significantly upregulated in iC(3)-1 compared to BeWo and JEG3 cells. Interestingly, HAS2, CD44 and S100P were significantly upregulated in iC(3)-1 compared to parental HTR8/SVneo cells and normal third trimester placenta. Choriocarcinoma samples also showed immunoreactivity to HAS2, CD44 and S100. In summary, the gene expression profile of iC(3)-1 suggests that studies using this cell line can make an important contribution to improved understanding of choriocarcinoma tumorigenesis.

Foxp3(+) regulatory T (T(reg)) cells are dominant suppressor cells which regulate conventional T (T(conv)) cells. Inside tumor microenvironment, T(reg) cells have been known to become potent in suppressing T(conv) cell responses, thereby enabling tumor cells to circumvent immune response. However, the underlying mechanism by which tumor-infiltrating T(reg) cells display enhanced suppressive function is still unresolved. To understand characteristics and function of tumor-infiltrating T(reg) cells as well as T(conv) cells in the tumor site, we analyzed their phenotypes either within tumor burden or at distant site of tumor using both heterotopic and orthotopic mouse cancer models. Compared to CD8(+) T cells at distant site of tumor, tumor-infiltrating CD8(+) T cells dramatically upregulated programmed death 1 (PD-1) and other inhibitory receptors, thereby being more exhausted functionally. Tumor-infiltrating CD4(+) T cells also expressed higher level of PD-1 than CD4(+) T cells at distant site of tumor but very surprisingly, upregulation of PD-1 occurred in CD4(+)Foxp3(+) T(reg) as well as CD4(+)Foxp3(-) T(conv) cells. Moreover, tumor infiltrating T(reg) cells upregulated other inhibitory receptors such as T cell immunoglobulin mucin 3 (TIM-3), cytotoxic T lymphocyte antigen-4 (CTLA-4), glucocorticoid-induced tumor necrosis factor receptor (GITR), and lymphocyte activation gene-3 (LAG-3). These results suggest that upregulation of PD-1 and other inhibitory receptors on tumor-infiltrating T(reg) cells is related with their enhanced suppressive function.

MUC3A is a membrane-bound glycoprotein that is aberrantly expressed in carcinomas and is a risk factor for a poor prognosis. However, the exact mechanism of MUC3A expression has yet to be clarified. Here, we provide the first evidence that MUC3A gene expression is controlled by the CpG methylation status of the proximal promoter region. We show that the DNA methylation pattern is intimately correlated with MUC3A expression in breast, lung, pancreas and colon cancer cell lines. The DNA methylation status of 30CpG sites from -660 to +273 was mapped using MassARRAY analysis. MUC3A-negative cancer cell lines and those with low MUC3A expression (e.g., MCF-7) were highly methylated in the proximal promoter region, corresponding to 9CpG sites (-345 to -75bp), whereas MUC3A-positive cell lines (e.g., LS174T) had low methylation levels. Moreover, 5-aza-2'-deoxycytidine and trichostatin A treatment of MUC3A-negative cells or those with low MUC3A expression caused elevation of MUC3A mRNA. Our results suggest that DNA hypomethylation in the 5'-flanking region of the MUC3A gene plays an important role in MUC3A expression in carcinomas of various organs. An understanding of epigenetic changes in MUC3A may contribute to the diagnosis of carcinogenic risk and to prediction of outcome in patients with cancer.

The aim was to establish an in vitro model for studies of innate defence mechanisms of human intestinal epithelium. Ultrastructural characterization and determination of mRNA expression levels for apical glycocalyx and mucous components showed that polarized, tight monolayers of the colon carcinoma cell lines T84 and Caco2 acquire the features of mature- and immature columnar epithelial cells, respectively. Polarized monolayers were challenged with non-pathogenic Gram+ and Gram- bacteria from the apical side and the proinflammatory cytokines interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) from the basolateral side. Immune responses were estimated as changes in mRNA expression levels for the mucous component mucin-2 (MUC2), the glycocalyx components carcinoembryonic antigen (CEA), CEA-related cell adhesion molecule-1 (CEACAM1), CEACAM6, CEACAM7 and MUC3, the antimicrobial factors human beta-defensin-1 (hBD1), hBD2, hBD3 and lysozyme, the chemokine IL-8 and the cytokines IL-6 and TNF-alpha. Tight monolayer cells were generally unresponsive to bacterial challenge, but increased their hBD2 levels when challenged with Bacillus megaterium. T84 cells also increased their TNF-alpha levels upon bacterial challenge. Tight monolayer cells responded to cytokine challenge suggesting awareness of basolateral attack. TNF-alpha induced significantly increased levels of IL-8 and TNF-alpha itself in both cell lines suggesting recruitment and activation of immune cells in the underlying mucosa in vivo. Cytokine challenge also increased levels of CEACAM1, which includes two functionally different forms, CEACAM1-L and CEACAM1-S. In T84 cells, IFN-gamma was selective for CEACAM1-L while TNF-alpha upregulated both forms. Increased CEACAM1 expression may influence epithelial function and communication between epithelial cells and intraepithelial lymphocytes.

BACKGROUND: MUC1 and MUC3 are from a large family of glycoproteins with an aberrant expression profile in various malignancies. Much interest has been focused on the role of these proteins in the development and progression of colorectal cancer; however, no previous studies have included the highly confounding variable of vascular invasion in their survival analysis. Using high throughput tissue microarray technology we assessed the prognostic value of MUC1 and MUC3 expression in the largest cohort of colorectal cancer patients to date. We propose that tumours lacking expression of MUC1 and MUC3 will be more likely to metastasise, due to previously observed loss of cell-cell adhesion, and this will therefore lead to more aggressive cancers with poorer prognosis.
METHODS: A tissue micro-array was prepared from tumour samples of 462 consecutive patients undergoing resection of a primary colorectal cancer. A comprehensive prospectively recorded data base with mean follow up of 75 months was collected and included common clinicopathological variables and disease specific survival. Immunohistochemical analysis of MUC1 and MUC3 expression was performed using antibodies NCL-MUC1 and 1143/B7 respectively, results were correlated with the variables within the database.
RESULTS: Positive expression of MUC1 and MUC3 was seen in 32% and 74% of tumours respectively. On univariate analysis no correlation was seen with either MUC1 or MUC3 and any of the clinicopathological variables including tumour grade and stage, vascular invasion and tumour type. Kaplan-Meier analysis demonstrated a significant reduction in disease specific survival with MUC1 positive tumours (p = 0.038), this was not seen with MUC3 (p = 0.552). On multivariate analysis, using Cox proportional hazards model, MUC1 expression was shown to be an independent marker of prognosis (HR 1.339, 95%CI 1.002-1.790, p = 0.048).
CONCLUSION: MUC1 expression in colorectal cancer is an independent marker of poor prognosis, even when vascular invasion is included in the analysis. These results support previous studies suggesting a role for MUC1 in colorectal cancer development possibly through its effects on cell adhesion.

T cell Ig mucin 3 (Tim-3) has been found to play an important role in Th1-mediated auto- and alloimmune responses, but the function of soluble form of Tim-3 (sTim-3) remains to be elucidated. In this study, we report the inhibitory effect of sTim-3 on T cell-mediated immune response. In this study, sTim-3 mRNA was found, among different tissues and organs, only in splenic cells, and the activation of splenocytes resulted in up-regulated production of both sTim-3 mRNA and protein. We constructed a eukaryotic expression plasmid, psTim-3, which expresses functional murine sTim-3. In C57BL/6 mice inoculated with B16F1 melanoma cells, the growth of tumor was facilitated by the expression of this plasmid in vivo. Furthermore, sTim-3 inhibited the responses of T cells to Ag-specific stimulation or anti-CD3 mAb plus anti-CD28 mAb costimulation and the production of cytokines IL-2 and IFN-gamma in vitro. In tumor rejection model, sTim-3 significantly impaired T cell antitumor immunity, evidenced by decreased antitumor CTL activity and reduced amount of tumor-infiltrating lymphocytes in tumor. Real-time PCR analysis of gene expression in tumor microenvironment revealed the decreased expression of Th1 cytokine genes and the unchanged profile of the genes related to T regulatory cell function, suggesting that the inhibitory effect of sTim-3 on the generation of Ag-specific T cells in vivo is dominated by T effector cells rather than T regulatory cells. Our studies thus define sTim-3 as an immunoregulatory molecule that may be involved in the negative regulation of T cell-mediated immune response.

Mucins are high-molecular weight epithelial glycoproteins with a high content of clustered oligosaccharides O-glycosidically linked to tandem repeat peptides rich in threonine, serine, and proline. There are two structurally and functionally distinct classes of mucins: secreted gel-forming mucins (MUC2, MUC5AC, MUC5B, and MUC6) and transmembrane mucins (MUC1, MUC3A, MUC3B, MUC4, MUC12, MUC17), although the products of some MUC genes do not fit well into either class (MUC7, MUC8, MUC9, MUC13, MUC15, MUC16). MUC1 mucin, as detected immunologically, is increased in expression in colon cancers, which correlates with a worse prognosis. Expression of MUC2 secreted gel-forming mucin is generally decreased in colorectal adenocarcinoma, but preserved in mucinous carcinomas, a distinct subtype of colon cancer associated with microsatellite instability. Another secreted gel-forming mucin, MUC5AC, a product of normal gastric mucosa, is absent from normal colon, but frequently present in colorectal adenomas and colon cancers. The O-glycosidically linked oligosaccharides of mucins can be described in terms of core type, backbone type, and peripheral structures. Colon cancer mucins have differences in both core carbohydrates and in peripheral carbohydrate structures that are being investigated as diagnostic and prognostic markers, and also as targets for cancer vaccines. Colon cancer mucins typically have increases in three core structures: Tn antigen (GalNAcalphaThr/Ser), TF antigen (Galbeta3GalNAc) and sialyl Tn (NeuAcalpha6GalNAc). The type 3 core (GlcNAcbeta3Ga1NAc) predominant in normal colonic mucin is lacking in colon cancer mucins. There are cancer-associated alterations in the peripheral carbohydrates of colonic mucins including a decrease in O-acetyl-sialic acid and a decrease in sulfation. There are, however, cancer-associated increases in sialyl LeX and related structures on mucins and other glycoproteins that can serve as ligands for selectins, increasing the metastatic capacity of colon cancer cells. The endogenous galactoside-binding protein galectin-3, which is expressed at higher levels in colon cancers than normal colon, binds to colon cancer mucin as well as other glycoproteins. Interference of the binding of selectins and galectin-3 to mucin may show therapeutic or preventative promise for colon cancer.

OBJECTIVES: To investigate, by real-time quantitative reverse transcriptase-polymerase chain reaction, the expression of MUC3 and vascular endothelial growth factor (VEGF) to correlate them with histologic parameters and with prognosis. Human mucins are large O-glycoproteins expressed in epithelial tissues. Deregulation of mucin genes has been demonstrated in several epithelial neoplasms. In the kidney, MUC3 is expressed in normal convoluted tubules and in renal clear cell carcinoma.
METHODS: Twenty-six renal clear cell carcinoma specimens were studied. For all tumors, samples of normal and tumor kidney were frozen. After RNA extraction, using ultracentrifugation through a cesium chloride cushion, VEGF and MUC3 mRNA were analyzed by real-time quantitative reverse transcriptase-polymerase chain reaction. The pathologic parameters included Fuhrman nuclear grade and TNM stage. All follow-up data were available.
RESULTS: The median level of MUC3 and VEGF expression was greater in tumor areas compared with normal areas (P < 0.002 and P < 0.001, respectively). The MUC3 tumor/normal tissue expression ratio was greater in nuclear grade 3 tumor than in low grades (grade 1-2; P < 0.005). No statistically significant relationship was found with the prognosis for MUC3 and VEGF in our study.
CONCLUSIONS: The results of this study demonstrate that MUC3 and VEGF are overexpressed in renal clear cell carcinoma, and the MUC3 expression ratio is greater in nuclear grade 3 than in grades 1 and 2 (low grades) tumor. These findings suggest the implication of MUC3 in renal carcinogenesis.

The mouse Muc3 mucin is a membrane-bound glycoprotein highly expressed in the intestinal tract. We have characterized the mouse Muc3 5' structure and regulation of its promoter by cytokines and growth factors. The first two exons of Muc3 are separated by an intron of over 8 kb. Exon 3 contains the tandem repeat domain. Ten exons reside 3' to the tandem repeat domain. The 5' nonrepetitive sequence contains 104 amino acids characterized by a putative signal sequence, a single cysteine and 28% serine/threonine. No TATA box is found near the transcription start site. The promoter has consensus binding sites for AP1, CREB, SP1, NF kappa B, GATA binding protein and Cdx. Muc3 promoter constructs demonstrate that IL4, IL6, EGF or PMA increased promoter activity to 35-58% of control. TNF alpha and IFN gamma showed lesser stimulation. These data indicate that cytokines and growth factors are capable of regulating Muc3 gene expression, suggesting that this protein may play an active role in intestinal mucosal defense.

INTRODUCTION: Ductal adenocarcinoma of the pancreas has recently been suggested to arise from histologically identifiable ductal lesions known as pancreatic intraepithelial neoplasia (PanINs). Altered levels and patterns of mucin gene expression have been reported to occur in epithelial cancers.
AIM: To examine the pattern of expression of membrane-associated mucins, MUC3 and MUC4, and a mucin-associated carbohydrate tumor antigen, sialyl Le(x), in these precursor lesions and ductal adenocarcinoma of the pancreas.
METHODOLOGY: A total of 144 PanIN lesions and 85 cases of ductal adenocarcinoma of the pancreas were examined by using immunohistochemistry and in situ hybridization methods.
RESULTS: MUC3 showed a progressive increase in expression in PanINs of increasing dysplasia and was also highly expressed in ductal adenocarcinoma. In contrast, neoexpression of MUC4 and sialyl Le(x) antigen was observed, mainly in PanIN-3 and ductal adenocarcinoma. In addition, a decrease in the expression of MUC3 and MUC4 was correlated with the degree of de-differentiation of the tumor.
CONCLUSION: Aberrant expression of membrane mucins MUC3 and MUC4 and of a mucin-associated carbohydrate tumor antigen Sialyl Le(x) in PanINs and adenocarcinoma further supports the progression model for pancreatic adenocarcinoma.

Membrane mucins have several functions in epithelial cells including cytoprotection, extravasation during metastases, maintenance of luminal structure, and signal transduction. In this paper we describe a large membrane mucin expressed in the normal intestine. This novel mucin, designated MUC17, contains an extended, repetitive extracellular glycosylation domain and a carboxyl terminus with two EGF-like domains, a SEA module domain, a transmembrane domain, and a cytoplasmic domain with potential serine and tyrosine phosphorylation sites. RNA blot analysis and in situ hybridization indicates that MUC17 is expressed in select pancreatic and colon cancer cell lines and in intestinal absorptive cells. Radiation hybrid mapping localized MUC17 to chromosome 7q22 where it resides in close proximity with three other membrane mucin genes, MUC3A, MUC3B, and MUC12. Thus, these membrane mucins reside together in a gene cluster, but are expressed in different tissues and are likely to have different functions as well.

OBJECTIVES: To explore the clinicopathological significance of the expression of MUC2 (mucin 2) and MUC3 (mucin 3) genes in tissues of the normal gastric mucosa, intestinal metaplasia (IM) and gastric carcinoma.
METHODS: MUC2 and MUC3 apomucins were detected by immunohistochemistry; MUC2 and MUC3 mRNAs were detected by in situ hybridization.
RESULTS: In the normal stomach, antibody detecting MUC2 apomucin and oligonucleotide probe detecting MUC2 mRNA were not reactive with mucus-producing cells in the superficial epithelium and neck, which were weakly reactive with antibody and probe detecting MUC3. In the duodenum, MUC2 apomucin and mRNA were found at the peri- and supranuclear area of goblet cells, but MUC3 apomucin and mRNA were at the cytoplasm of goblet cells and columnar cells. MUC2 and MUC3 apomucins and its mRNAs were found in 85.2%, 88.9% and 31.6%; 57.1% of specimens of IM, 67.4%, 66.7% and 57.9%, 43.6% of specimens of gastric carcinoma. There were no associations between expressions of MUC2 and MUC3 genes and different types of IM. Patients with moderate/well differentiation had higher proportion of MUC2 apomucin expression than those with poor differentiation (P < 0.05), and patients with positive staining of MUC3 apomucin in the cancerous tissues had higher proportions of metastasis of lymph nodes (P < 0.01), serosal invasion (P < 0.05) and clinical stages III-IV (P < 0.05).
CONCLUSIONS: MUC2 and MUC3 genes are mainly expressed in the mucosa of the duodenum, and marked expression is seen during the neoplastic transformation of stomach mucosa. MUC3 apomucin might have a poor prognostic significance.

Neoplastic transformation of epithelial cells is commonly associated with alterations in the synthesis and structures of mucin. Mucin protein epitopes and mRNA levels were frequently altered in adenocarcinomas compared to corresponding normal tissues. Clinically, hepatolithiasis has been regarded as a risk factor for cholangiocarcinoma. The aims of this study were to determine the possible alteration of mucin gene expression in stone-containing intrahepatic bile ducts and cholangiocarcinomas and to try to predict whether or not hepatolithiasis has a predisposition to development of cholangiocarcinoma. In situ hybridization with DIG-tailed oligonucleotides was performed on sections of paraffin-embedded tissues of stone-containing intrahepatic bile ducts, cholangiocarcinomas, and normal controls to identify the expression of MUC2, MUC3, MUC4, MUC5B, and MUC5AC in nonneoplastic and neoplastic biliary epithelium. The findings showed that (1) while multiple diverse mucin genes were expressed in the biliary epithelium, MUC3 and MUC5B mRNA were the main mucin genes expressed in the biliary epithelium of stone-containing intrahepatic bile ducts and normal controls; (2) absent or decreased expression of MUC2, MUC3, and MUC5B of mRNA was found in cholangiocarcinomas in contrast to nonneoplastic biliary epithelium; and (3) increased expression of MUC4 and MU5AC of mRNA was found in cholangiocarcinomas and the biliary epithelium, especially for dysplastic cells of stone-containing intrahepatic bile ducts compared with normal controls. In this study, using in situ hybridization we demonstrated that neoplastic transformation of the biliary epithelium is accompained by alterations in mucin gene expression, the altered mucin gene expression in dysplastic cells of stone-containing intrahepatic bile ducts may reflect a higher potential for malignant transformation in these cells, and it could be a precursor of cholangiocarcinoma in the presence of hepatolithiasis.

Altered expression of mucin gene products has been described in many epithelial cancers including colorectal cancer. However, mucins are heavily O-glycosylated making the study of apomucin expression difficult. In this study, we describe a novel method of chemical deglycosylation of mucin gene products on paraffin embedded formalin-fixed tissue sections. In the normal and cancerous colorectum, our results suggest that alkali-catalyzed beta-elimination of periodate oxidized glycan method of chemical deglycosylation modifies the structure of carbohydrates sensitive to mild periodate oxidation resulting in less steric hindrance and selectively removes Tn and sialyl-Tn structures, partially exposing the underlying apomucin epitopes. Using this method, we have demonstrated that the MUC1 tandem repeat epitope recognized by MAb 139H2 is masked predominantly due to steric hindrance by carbohydrate structures whereas the MUC2 tandem repeat epitope recognized by MAb CCP58 and pAb MRP and the MUC3 tandem repeat epitope recognized by pAb M3P are masked by the presence of carbohydrate side chains O-linked to Ser/Thr residues within the epitope. Considerable differences in the level and pattern of expression of the epitopes in the tandem repeat region of apomucins of MUC1, MUC2, and MUC3 were observed between normal and cancerous colorectal cancer tissues. We conclude that this novel chemical deglycosylation method that causes selective cleavage of distinct glycans will be useful in unmasking various mucin gene products and glycoproteins containing similar O-glycosidic linkages in the tissue sections of formalin-fixed paraffin embedded normal and pathological tissues.

Epithelial mucins are glycoproteins secreted by epithelial cells and their carcinomas. At least nine mucin genes have been identified, and their products (MUC1-MUC9) are expressed in various epithelia. MUC1 is a mucin expressed in breast epithelial cells, whereas MUC2 and MUC3 are primarily intestinal mucins. Although MUC1 and MUC2 expression has been documented in invasive ductal carcinoma of the breast, mucin expression in pure ductal carcinoma in situ (DCIS) has not been investigated. Sixty-one of 105 cases of DCIS without coexisting infiltrating carcinoma diagnosed during a 30-month period were selected as having sufficient tissue for study. Paraffin-embedded tissue sections were stained using immunohistochemical techniques with mouse monoclonal anti-MUC1, anti-MUC2, and rabbit-specific polyclonal anti-MUC3 antibodies. Immunoreactive epitopes of MUC1, MUC2, and MUC3 were expressed in DCIS in 61, 19, and 16 of 61 cases, respectively. MUC2 and MUC3 staining intensity in DCIS was markedly less than that observed for MUC1. Luminal and/or cytoplasmic patterns of staining were observed for MUC1. MUC2 and MUC3 showed only cytoplasmic staining. Cytoplasmic-only staining of MUC1 was associated with a higher grade of DCIS. Any MUC2 staining was also associated with a higher grade of DCIS. Coexpression of MUC2 and MUC3 was present in only 6 of 61 cases, and MUC3 staining was unrelated to the grade of DCIS. Cytoplasmic expression of MUC1 and MUC2 appears to be associated with a higher grade of DCIS. MUC3 expression appears to be independent of grade and expression of MUC1 and MUC2. The relationship of mucin expression and grade warrants further study.

Phorbol esters such as phorbol 12-myristate 13-acetate (PMA) have been reported to modulate diverse cellular responses through signal transduction pathways including the protein kinase C (PKC) pathway. In the present study, we sought to determine the effect of PMA on mucin gene expression and on the biological properties of a human colon cancer cell line, HM3. The cells were treated for 8 and 24 h with various concentrations of PMA and total RNA was extracted and Northern and slot blot analyses were carried out using MUC2, MUC3 and MUC5AC mucin cDNA probes to assess the steady state levels of mRNA. Spent media were collected and the level of cancer associated carbohydrate antigens (T, Tn, sialyl Tn, sialyl Lex, and sialyl Lea) and matrix-degrading metalloproteinase (MMPs) activity were examined. Trypsinized cells were used for assessing in vitro invasion, motility and adhesion to matrigel. Our results showed that PMA caused upregulation of steady state mRNA levels of MUC2, MUC3 and MUC5AC which was inhibited after treatment with protein synthesis inhibitors. Calphostin C, a highly specific inhibitor of protein kinase C significantly inhibited the PMA induced induction of mRNA levels of MUC2, MUC3, and MUC5AC. The levels of all cancer-associated mucin carbohydrate antigens examined in the media were increased by PMA treatment. PMA also caused an increase in MMPs activity and in in vitro invasion and motility properties, but did not affect adhesion of HM3 cells to matrigel. Thus, PMA caused a significant increase in the expression of all three mucin genes through signaling pathways involving protein kinase C and increased secretion of mucin associated carbohydrate antigens. These changes were associated with increases in MMP activity as well as by increases in the invasive and motility properties of HM3 colon cancer cells. These data suggest that protein kinase C signaling pathways may be involved in mucin gene regulation and in modulating the invasive and metastatic properties of colon cancer cells.

Epithelial mucins are a family of secreted and cell surface glycoproteins expressed by epithelial tissues and implicated in epithelial cell protection, adhesion modulation and signaling. The gene encoding human MUC3 (hMUC3), localised to chromosome 7q22, is most highly expressed in the small intestine. It has previously been reported to be a non-transmembrane mucin with minimal homology to its suggested orthologues from rat (rMuc3) and mouse (mMuc3). RT-PCR was performed to investigate the carboxyl terminus of the published sequence of hMUC3 from normal colon and small intestine tissues and also from a series of 10 colorectal cancer cell lines. Two distinct PCR products were identified. In contrast to the previously published hMUC3 sequence, which terminates shortly after a single cysteine-rich EGF-like domain, conceptual protein translation of the dominant and largest PCR product identified two extracellular cysteine-rich EGF-like domains separated by an N-glycosylation-rich domain and a potential coiled-coil region, followed by a putative transmembrane region and a 75 amino acid cytoplasmic tail. The smaller of the two PCR products was found to be an alternative splice variant of MUC3 including the first EGF-like domain but lacking part of the second EGF-like domain and the transmembrane region. Nine out of 10 colorectal cancer cell lines were found to express MUC3. Interestingly, one of the cell lines, LoVo, expressed predominantly the alternative splice form lacking a transmembrane domain. Structural homology of the new protein sequence of hMUC3 with rMuc3 and mMuc3 indicates it is closely related to the rodent proteins and is likely to be involved in ligand-binding and intracellular signaling. The new finding that MUC3 encodes a transmembrane molecule presents a new paradigm for the structure of this mucin and the manner in which it may function.