Tomonobu Fujita

Immune Suppression and Resistance Mediated by Constitutive Activation of Wnt/β-Catenin Signaling in Human Melanoma Cells

Cancer-induced immunosuppression is a major problem reducing antitumor effects of immunotherapies, but its molecular mechanism has not been well understood. We evaluated immunosuppressive roles of activated Wnt/β-catenin pathways in human melanoma for dendritic cells (DCs) and CTLs. IL-10 expression was associated with β-catenin accumulation in human melanoma cell lines and tissues and was induced by direct β-catenin/TCF binding to the IL-10 promoter. Culture supernatants from β-catenin–accumulated melanoma have activities to impair DC maturation and to induce possible regulatory DCs. Those immunosuppressive culture supernatant activities were reduced by knocking down β-catenin in melanoma cells, partly owing to downregulation of IL-10. Murine splenic and tumor-infiltrating DCs obtained from nude mice implanted with human mutant β-catenin–overexpressed melanoma cells had less ability to activate T cells than did DCs from mice with control melanoma cells, showing in vivo suppression of DCs by activated Wnt/β-catenin signaling in human melanoma. This in vivo DC suppression was restored by the administration of a β-catenin inhibitor, PKF115-584. β-catenin–overexpressed melanoma inhibited IFN-γ production by melanoma-specific CTLs in an IL-10–independent manner and is more resistant to CTL lysis in vitro and in vivo. These results indicate that Wnt/β-catenin pathways in human melanoma may be involved in immunosuppression and immunoresistance in both induction and effector phases of antitumor immunoresponses partly through IL-10 production, and they may be attractive targets for restoring immunocompetence in patients with Wnt/β-catenin–activated melanoma.

Autocrine and paracrine loops between cancer cells and macrophages promote lymph node metastasis via CCR4/CCL22 in head and neck squamous cell carcinoma

Lymph node metastasis is a poor prognostic factor for patients with head and neck squamous cell carcinoma (HNSCC). However, its molecular mechanism has not yet been fully understood. In our study, we investigated the expression of CCR4 and its ligand CCL22 in the HNSCC tumor microenvironment and found that the CCR4/CCL22 axis was involved in lymph node metastasis of HNSCC. CCR4 was expressed in 20 of 31 (64.5%) human tongue cancer tissues, and its expression was significantly correlated with lymph node metastasis (p < 0.01) and lymphatic invasion (p < 0.05). CCR4 was expressed in three of five human HNSCC cell lines tested. CCR4+ HNSCC cells, but not CCR4− cells, showed enhanced migration toward CCL22, indicating that functional CCR4 was expressed in HNSCC cell lines. CCL22 was also expressed in cancer cells (48.4% of tongue cancer tissues) or CD206+ M2‐like macrophages infiltrated in tumors and draining lymph nodes. CCL22 produced by cancer cells or CD206high M2‐like macrophages increased the cell motility of CCR4+ HNSCC cells in vitro in an autocrine or paracrine manner. In the mouse SCCVII in vivo model, CCR4+ cancer cells, but not CCR4− cells, metastasized to lymph nodes which contained CCL22 producing M2‐like macrophages. These results demonstrate that lymph node metastasis of CCR4+ HNSCC is promoted by CCL22 in an autocrine or M2‐like macrophage‐dependent paracrine manner. Therefore, the CCR4/CCL22 axis may be an attractive target for the development of diagnostic and therapeutic strategies for patients with HNSCC.

Phase I pilot study of Wilms tumor gene 1 peptide‐pulsed dendritic cell vaccination combined with gemcitabine in pancreatic cancer

This study aimed to evaluate the feasibility of and immune response to Wilms tumor gene 1 (WT1) peptide‐pulsed dendritic cell vaccination combined with gemcitabine (DCGEM) as a first‐line therapy among patients with advanced pancreatic cancer. Ten HLA‐A*2402 patients were treated with WT1 peptide‐pulsed DC vaccination (1 × 107 cells) on days 8 and 22 and gemcitabine (1000 mg/m2) on days 1, 8 and 15. Induction of a WT1‐specific immune response was evaluated using the delayed‐type hypersensitivity (DTH) skin test, interferon‐γ enzyme‐linked immunospot and HLA tetramer assays, along with assays for various immunological factors. DCGEM was well‐tolerated, and the relative dose intensity of gemcitabine was 87%. Disease control associated with a low neutrophil/lymphocyte ratio was observed in all three patients with DTH positivity; it was also correlated with a low percentage of granulocytic myeloid derived suppressor cells in the pretreatment peripheral blood (P = 0.017). Patients with liver metastases and high levels of inflammatory markers such as C‐reactive protein and interleukin‐8 (IL‐8) showed poor survival even though a WT1‐specific immune response was induced in them. WT1 peptide‐pulsed DCGEM is feasible and effective for inducing anti‐tumor T‐cell responses. Our results support future investigations for pancreatic cancer patients with non‐liver metastases and favorable immunological conditions. This trial was registered with the University hospital Medical Information Network (UMIN) Clinical Trials Registry (http://www.umin.ac.jp/ctr/ number: UMIN‐000004855).

International validation of the consensus Immunoscore for the classification of colon cancer: a prognostic and accuracy study

BACKGROUND The estimation of risk of recurrence for patients with colon carcinoma must be improved. A robust immune score quantification is needed to introduce immune parameters into cancer classification. The aim of the study was to assess the prognostic value of total tumour-infiltrating T-cell counts and cytotoxic tumour-infiltrating T-cells counts with the consensus Immunoscore assay in patients with stage I-III colon cancer. METHODS An international consortium of 14 centres in 13 countries, led by the Society for Immunotherapy of Cancer, assessed the Immunoscore assay in patients with TNM stage I-III colon cancer. Patients were randomly assigned to a training set, an internal validation set, or an external validation set. Paraffin sections of the colon tumour and invasive margin from each patient were processed by immunohistochemistry, and the densities of CD3+ and cytotoxic CD8+ T cells in the tumour and in the invasive margin were quantified by digital pathology. An Immunoscore for each patient was derived from the mean of four density percentiles. The primary endpoint was to evaluate the prognostic value of the Immunoscore for time to recurrence, defined as time from surgery to disease recurrence. Stratified multivariable Cox models were used to assess the associations between Immunoscore and outcomes, adjusting for potential confounders. Harrells C-statistics was used to assess model performance. FINDINGS Tissue samples from 3539 patients were processed, and samples from 2681 patients were included in the analyses after quality controls (700 patients in the training set, 636 patients in the internal validation set, and 1345 patients in the external validation set). The Immunoscore assay showed a high level of reproducibility between observers and centres (r=0·97 for colon tumour; r=0·97 for invasive margin; p<0·0001). In the training set, patients with a high Immunoscore had the lowest risk of recurrence at 5 years (14 [8%] patients with a high Immunoscore vs 65 (19%) patients with an intermediate Immunoscore vs 51 (32%) patients with a low Immunoscore; hazard ratio [HR] for high vs low Immunoscore 0·20, 95% CI 0·10-0·38; p<0·0001). The findings were confirmed in the two validation sets (n=1981). In the stratified Cox multivariable analysis, the Immunoscore association with time to recurrence was independent of patient age, sex, T stage, N stage, microsatellite instability, and existing prognostic factors (p<0·0001). Of 1434 patients with stage II cancer, the difference in risk of recurrence at 5 years was significant (HR for high vs low Immunoscore 0·33, 95% CI 0·21-0·52; p<0·0001), including in Cox multivariable analysis (p<0·0001). Immunoscore had the highest relative contribution to the risk of all clinical parameters, including the American Joint Committee on Cancer and Union for International Cancer Control TNM classification system. INTERPRETATION The Immunoscore provides a reliable estimate of the risk of recurrence in patients with colon cancer. These results support the implementation of the consensus Immunoscore as a new component of a TNM-Immune classification of cancer. FUNDING French National Institute of Health and Medical Research, the LabEx Immuno-oncology, the Transcan ERAnet Immunoscore European project, Association pour la Recherche contre le Cancer, CARPEM, AP-HP, Institut National du Cancer, Italian Association for Cancer Research, national grants and the Society for Immunotherapy of Cancer.

Prognostic significance of interleukin-8 and CD163-positive cell-infiltration in tumor tissues in patients with oral squamous cell carcinoma

Purpose We investigated whether serum interleukin (IL)-8 reflects the tumor microenvironment and has prognostic value in patients with oral squamous cell carcinoma (OSCC). Experimental Design Fifty OSCC patients who received radical resection of their tumor(s) were enrolled. Preoperative sera were measured for IL-8 by ELISA. Expression of IL-8 and the infiltration of immune cells in tumor tissues were analyzed by an immunohistochemical staining of surgical specimens. Results We found that disease-free survival (DFS) was significantly longer in the Stage I/II OSCC patients with low serum IL-8 levels compared to those with high levels (p = 0.001). The tumor expression of IL-8, i.e., IL-8(T) and the density of CD163-positive cells in the tumor invasive front, i.e., CD163(IF) were correlated with the serum IL-8 level (p = 0.033 and p = 0.038, respectively), and they were associated with poor clinical outcome (p = 0.007 and p = 0.002, respectively, in DFS) in all patients. A multivariate analysis revealed that N status, IL-8(T) and CD163(IF) significantly affected the DFS of the patients. Further analysis suggested that combination of N status with serum IL-8, IL-8(T) or CD163(IF) may be a new criterion for discriminating between OSCC patients at high and low risk for tumor relapse. Interestingly, the in vitro experiments demonstrated that IL-8 enhanced generation of CD163-positive M2 macrophages from peripheral blood monocytes, and that the cells produced IL-10. Conclusions These findings indicate that IL-8 may be involved in poor clinical outcomes via generation of CD163-positive M2 macrophages, and that these factors in addition to N status may have prognostic value in patients with resectable OSCSS.

Clinical significance of serum p53 antibodies in patients with ulcerative colitis and its carcinogenesis

Background: For early detection of ulcerative colitis (UC)‐associated colorectal cancer (CRC), surveillance colonoscopy is recommended in UC patients at high risk. However, poor acceptability deteriorates its effectiveness and a suitable marker for selecting patients at high risk is needed. Here we evaluated clinical usefulness of the measurement of anti‐p53 antibodies (Abs) by enzyme‐linked immunosorbent assay (ELISA) using sera samples from UC patients. Methods: Sera from 286 patients with UC, 82 patients with sporadic CRC, and 63 healthy controls (HC) were obtained. Serum anti‐p53 antibodies were detected with ELISA. Immunohistochemical detection was also performed in patients who developed dysplasia or CRC. Results: Serum p53 Ab was positive in 15.0% of UC, while it was positive only in 1.6% of HCs. In sporadic CRCs, 52.4% of 82 patients were positive. In UC patients with disease duration equal to or longer than 8 years, positivity of serum p53 Ab was significantly higher than those in patients with shorter duration. Eight of 13 (61.5%) UC patients with CRC or dysplasia were positive for serum p53 Abs, which was significantly higher than that in patients without neoplasia. All UC patients with CRC were positive for p53 staining, while 2 were negative for serum p53 Ab. Finally, levels of serum p53 Ab had fallen in 4 patients with CRC we could monitor after surgery. Conclusions: This study revealed that p53 Ab developed in the progression of UC‐associated CRC but not in all patients with neoplasia, suggesting that serological detection of p53 Abs by ELISA is not suitable in primarily selecting patients at high risk; however, it is helpful in salvaging patients who drop from a surveillance program. (Inflamm Bowel Dis 2007)

Frequent immune response to a melanocyte-specific protein KU-MEL-1 in patients with Vogt-Koyanagi-Harada disease

Aim: To isolate autoantigens possibly involved in the pathogenesis of Vogt-Koyanagi-Harada (VKH) disease. Methods: Autoantigens recognised by immunoglobulin G antibodies (IgG Ab) in sera from VKH patients were isolated by screening the lambda phage cDNA libraries made from melanocytes and a highly pigmented melanoma cell line with the patients’ sera. Presence of IgG specific for the autoantigens in sera from patients with various panuveitis and healthy individuals was evaluated. Relation between the specific IgG and various clinicopathological features was examined. Results: KU-MEL-1 was found to be one of the 81 isolated positive clones representing 35 distinct genes, which is a previously isolated melanoma antigen preferentially expressed in melanocytes. The IgG Ab specific for KU-MEL-1 was detected in sera from patients with VKH in significantly higher amounts than in sera from patients with Behçet’s disease, sarcoidosis, and from healthy individuals. Positive serum KU-MEL-1 Ab was significantly associated with HLA-DRB1*0405 and male VKH patients. Conclusion: KU-MEL-1 was identified as a new autoantigen for VKH. The highly frequent induction of IgG Ab for KU-MEL-1 in HLA-DRB1*0405 positive VKH patients may suggest the possible involvement of KU-MEL-1 specific CD4+ T cells in the pathogenesis of VKH, suggesting the possible use in the development of diagnostic and therapeutic treatments for VKH patients.

Cancer-testis antigen BORIS is a novel prognostic marker for patients with esophageal cancer

Esophageal squamous cell cancer (ESCC) is one of the most common lethal tumors in the world, and development of new diagnostic and therapeutic methods is needed. In this study, cancer‐testis antigen, BORIS, was isolated by functional cDNA expression cloning using screening technique with serum IgG Abs from ESCC patients. BORIS was previously reported to show cancer‐testis antigen like expression, but its immunogenicity has remained unclear in cancer patients. BORIS was considered to be an immunogenic antigen capable of inducing IgG Abs in patients with various cancers, including four of 11 ESCC patients. Immunohistochemical study showed that the BORIS protein was expressed in 28 of 50 (56%) ESCC tissues. The BORIS expression was significantly associated with lymph node metastasis in ESCC patients with pT1 disease (P = 0.036). Furthermore, the patients with BORIS‐positive tumors had a poor overall survival (5‐year survival rate: BORIS‐negative 70.0% vs BORIS‐positive 29.9%, log‐rank P = 0.028) in Kaplan–Meier survival analysis and log‐rank test. Multivariate Cox proportional hazard model demonstrated that BORIS expression was an independent poor prognostic factor (hazard ratio = 4.158 [95% confidence interval 1.494–11.57], P = 0.006). Downregulation of BORIS with specific siRNAs resulted in decreased cell proliferation and invasion ability of ESCC cell lines. BORIS may be a useful biomarker for prognostic diagnosis of ESCC patients and a potential target for treatment including by BORIS‐specific immunotherapy and molecular target therapy.

Studies on the N-[(trans-4-Isopropylcyclohexyl)-carbonyl]-d-phenylalanine (A-4166) receptor in HIT T-15 cells: Displacement of [3H]glibenclamide

A-4166 is a new type of oral hypoglycemic agent that does not contain a sulfonylurea moiety. To clarify the mechanism of insulin secretion by A-4166, a specific receptor for A-4166 was investigated in a hamster pancreatic beta cell line (HIT T-15), using [3H]A-4166 or [3H]glibenclamide as a ligand. The saturation binding of [3H]A-4166 to HIT cell membranes was not observed up to 10 microM. In the displacement study, unlabeled A-4166 inhibited [3H]A-4166 binding to HIT cell membranes, but glibenclamide did not. On the other hand, A-4166 inhibited [3H]glibenclamide binding to the sulfonylurea receptor (Ki = 248 nM). A-4166 inhibited 86Rb efflux from HIT cells (IC50 = 350 nM). The EC50 for insulin secretion by A-4166 was 20 microM in HIT cells when they were incubated for 30 min in Krebs-Ringer bicarbonate buffer containing 16 mM HEPES supplemented with 5 mg/mL BSA in the absence of glucose. These data demonstrate the possibility of the presence of two kinds of binding sites for A-4166: one of them is the sulfonylurea receptor, and the other might be a binding site specific for A-4166.

Stimulating Activity of A-4166 on Insulin Release in in situ Hamster Pancreatic Perfusion

Using the in situ hamster pancreatic perfusion system, the stimulating action of A-4166 on insulin release was examined in comparison with that of glibenclamide. Both antidiabetic agents stimulated insulin release, but its onset by A-4166 was faster than that by glibenclamide. In the presence of a basal glucose concentration (3 mmol/l), insulin releases induced by A-4166 and glibenclamide were inhibited by preexisting diazoxide. At higher glucose concentrations (5-16.7 mmol/l), however, A-4166 was able to reverse the inhibitory effect of diazoxide on the first and second phases of insulin release, while glibenclamide did not reverse the first-phase release. On the other hand, in the presence of 16.7 mmol/l of glucose A-4166 completely reversed the inhibitory action of diazoxide added simultaneously, but glibenclamide reversed it only partially. In the presence of 8 mmol/l of glucose, the stimulating action of A-4166 and glibenclamide on insulin release was hardly affected by inhibitors of ATP production. These results indicate that the stimulating action of A-4166 on insulin release is different from glibenclamide in response to the inhibitory action of diazoxide. These results also suggest that A-4166 is an effective agent for release of insulin by acting on the KATP channel, especially under an impaired function of pancreatic B cells.