Masaki Anraku

Video-Assisted Mediastinoscopy Compared With Conventional Mediastinoscopy: Are We Doing Better?

BACKGROUNDnConventional mediastinoscopy (CM) is recently being replaced by video-assisted mediastinoscopy (VAM), with potentially better yield and better safety profile for VAM.nnnMETHODSnAll 645 mediastinoscopies (505 CM, 140 VAM) performed between May 2004 and May 2008 were reviewed. Numbers of stations biopsied, total number of lymph nodes dissected, pathology results, and complications were recorded. Patients were divided into two groups: staging for lung cancer group (n = 500) and diagnostic group (n = 145). The staging group was further analyzed, using 304 patients who eventually underwent thoracotomy to evaluate accuracy and negative predictive value of mediastinoscopy, comparing between the two methods (233 CM, 71 VAM).nnnRESULTSnAverage age was 65 years (range, 26 to 91), and 382 were male. There was no mortality. Eight complications (1.2%) occurred, more in the VAM group (3.8%) than in the CM group (0.8%; p = 0.04). The total number of dissected nodes was higher in the VAM group than in the CM group (7.0 +/- 3.2 versus 5.0 +/- 2.8, p < 0.001), and so was the number of stations sampled (3.6 versus 2.6, p < 0.01). Sensitivity was higher for VAM (95% versus 92.2%, p = not significant), and so was the negative predictive value (98.6% versus 95.7%, p = not significant). Most false negative biopsies (8 of 11, 73 %) occurred in station 7.nnnCONCLUSIONSnBoth methods are safe. More lymph nodes and stations were evaluated by VAM, with trend toward higher negative predictive value. The higher rate of minor complications seen with VAM might be related to a more aggressive and thorough dissection.

Gene Expression Profiling in the Lungs of Patients With Pulmonary Hypertension Associated With Pulmonary Fibrosis

BACKGROUNDnPulmonary hypertension (PH) associated with pulmonary fibrosis (PF) is a severe condition with poor outcome. It is unknown whether patients with PF with associated PH (APH) represent a distinct phenotype of the disease. We hypothesized that the lung tissue gene expression pattern of patients with APH has a characteristic profile when compared with patients with PF without APH. We sought to determine if different gene expression signatures in PF could be determined based on pulmonary arterial pressures (PAPs) and to provide new insights into the pathobiology of APH.nnnMETHODSnMicroarray analysis (Affymetrix) was performed after RNA was extracted from explanted lungs in 116 consecutive patients with PF (development set, n = 84; validation set, n = 32) and seven subjects with idiopathic pulmonary arterial hypertension undergoing lung transplant (LTx). PAP were recorded intraoperatively immediately before starting LTx. The development set was divided into three groups according to mean PAP (mPAP): severe PH group (mPAP ≥ 40 mm Hg, n = 17); intermediate PH group (mPAP 21-39 mm Hg, n = 45); NoPH group (mPAP ≤ 20 mm Hg, n = 22).nnnRESULTSnDistinct gene signatures were observed. Patients in the severe PH group showed increased expression of genes, gene sets, and networks related to myofibroblast proliferation and vascular remodeling, whereas patients in the NoPH group strongly expressed proinflammatory genes. Two-dimensional hierarchic clustering based on 222 differentially expressed genes (severe PH vs no PH) dichotomized subjects into two phenotypes in the intermediate PH group and in the validation set. Real-time polymerase chain reaction confirmed the differential expression of selected genes.nnnCONCLUSIONSnGene expression profiles distinguish PF phenotypes with and without APH. This observation can have important implications for future trials.

Synergistic Antitumor Effects of Regulatory T Cell Blockade Combined with Pemetrexed in Murine Malignant Mesothelioma

CD4+CD25+ regulatory T cells (Tregs) can promote the growth of some tumors, but it is unknown whether this is true for all tumors, including malignant pleural mesothelioma. We have previously shown that the existence of Tregs was associated with poor survival in patients with malignant pleural mesothelioma. In this study, using an intrathoracic murine model of malignant mesothelioma (MM), we provide evidence suggesting that Treg blockade could enhance survival when combined with pemetrexed in established tumor. AC29 murine MM cells were injected into the right pleural cavity of CBA mice for tumor development. Four days after the tumor injection, tumor-bearing mice were then treated with pemetrexed alone, Treg blockade alone, or a combination of pemetrexed and Treg blockade. We observed a synergistic antitumor effect of Treg blockade combined with pemetrexed resulting in prolonged survival. The combination of Treg blockade and pemetrexed was associated with decreased tumor-infiltrating Tregs, increased IL-2 production, dendritic cell maturation, and increased CD3+CD8+IFN-γ+ tumor-infiltrating T cells when compared with mice treated with pemetrexed alone or Treg blockade alone. The survival benefit was abrogated if anti-CD8 mAb was administered simultaneously. Likewise, the survival benefit resulting from the combined Treg blockade with pemetrexed was not observed when immunodeficient mice were used. Therefore, this study suggests that Treg blockade combined with pemetrexed can suppress mesothelioma growth in established tumor in vivo through an immune-mediated process. This study also validates a new intrathoracic tumor model of pleural effusion to explore the role of antitumor immunity in murine MM.

Regression of Allograft Airway Fibrosis: The Role of MMP-Dependent Tissue Remodeling in Obliterative Bronchiolitis after Lung Transplantation

Obliterative bronchiolitis after lung transplantation is a chronic inflammatory and fibrotic condition of small airways. The fibrosis associated with obliterative bronchiolitis might be reversible. Matrix metalloproteinases (MMPs) participate in inflammation and tissue remodeling. MMP-2 localized to myofibroblasts in post-transplant human obliterative bronchiolitis lesions and to allograft fibrosis in a rat intrapulmonary tracheal transplant model. Small numbers of infiltrating T cells were also observed within the fibrosis. To modulate inflammation and tissue remodeling, the broad-spectrum MMP inhibitor SC080 was administered after the allograft was obliterated, starting at post-transplant day 21. The allograft lumen remained obliterated after treatment. Only low-dose (2.5 mg/kg per day) SC080 significantly reduced collagen deposition, reduced the number of myofibroblasts and the infiltration of T cells in association with increased collagenolytic activity, increased MMP-2 gene expression, and decreased MMP-8, MMP-9, and MMP-13 gene expression. In in vitro experiments using cultured myofibroblasts, a relatively low concentration of SC080 increased MMP-2 activity and degradation of type I collagen. Moreover, coculture with T cells facilitated persistence of myofibroblasts, suggesting a role for T-cell infiltration in myofibroblast persistence in fibrosis. By combining low-dose SC080 with cyclosporine in vivo at post-transplant day 28, partial reversal of obliterative fibrosis was observed at day 42. Thus, modulating MMP activity might reverse established allograft airway fibrosis by regulating inflammation and tissue remodeling.

Antitumor impact of interferon-γ producing CD1d-restricted NKT cells in murine malignant mesothelioma

CD1d-restricted natural killer T (iNKT) cells have been shown to provide adjuvant activity against cancer by producing interferon (IFN)-&ggr;. However, the role of invariant NKT (iNKT) cells in the tumor microenvironment has not yet been fully addressed. Our aim is to elucidate the antitumor effect of iNKT cells in the tumor microenvironment by using an intrathoracic murine malignant pleural mesothelioma model that we had previously developed and to provide pleural effusion as a good surrogate of the tumor microenvironment. We found that the number of iNKT cells increased dramatically in the pleural effusion after intrathoracic tumor cell injection at an earlier phase compared with accumulation of CD8+ T cells. These iNKT cells showed increased expression of CD25 and increased ratio of cells positive for IFN-&ggr; intracellular staining. iNKT cells sorted from pleural effusion of tumor burden mice produced larger amount of IFN-&ggr; compared with naive mice. Mice pretreated in vivo with anti-CD1d-blocking Ab showed increased amount of pleural effusion and decreased ratio of total and effector-type CD8+ T cells as well as decreased intracellular IFN-&ggr; expression of CD8+T-cell in the pleural effusion. In vivo administration of &agr;-galactosylceramide (&agr;-GalCer) showed prolonged survival associated with less pleural effusion and increased ratio of IFN-&ggr;-positive iNKT cells and CD8+ T cells in the pleural effusion. Therefore, this study suggests that iNKT cells accumulating in the tumor microenvironment play an antitumor effect by producing IFN-&ggr; and enhancing subsequent CD8+ T-cell response. Furthermore, in vivo administration of &agr;-GalCer could suppress mesothelioma growth by activating iNKT cells.

Abstract 777: Antitumor role of early infiltrating interferon-gamma producing NKT cells in murine malignant mesothelioma

Introduction: NKT cells can provide adjuvant activity against cancer by producing large amounts of IFN-gamma which activate other immune cells, and orchestrate protective anti-tumor immunity. Recently, induction of the NKT cell-dependent anti-tumor immune response using its ligand alpha-galactosylceramide has been attempted in several tumor types. However, the role of NKT cells in the tumor microenvironment has not yet been fully addressed. Our aim is to elucidate the role of NKT cells in the thoracic cavity by using a murine malignant mesothelioma model. Methods: Half a million of AB12 murine malignant mesothelioma cells were injected into the right pleural cavity of female Balb/c mice for tumor development. The control mice were injected with the same amount of PBS. In this model, thoracic cavity was filled with tumors and pleural effusion by day 14. On days 0 (before tumor cell injection), 3, 6, 10 and 14 after tumor cell injection, mice were sacrificed and the pleural effusion and tumors were collected. Lymphocytes were isolated by performing a Ficoll gradient centrifugation. Cell phenotype was identified by cell surface marker staining and cytokine expression was analyzed by intracellular cytokine staining using flow cytometry. Cytokine concentrations in the pleural effusion were measured by ELISA. Resluts: The absolute number of CD3 positive cells in the pleural cavity did not increase between day 0 (4.7±0.9 x105), day 3 (5.5±0.7 x105) and day 6 (7.1x±1.6 x105) but did start to increase from day 10 (1.2±0.2 x106) after tumor cell injection. In the control mice, the absolute number of CD3 positive cells was not different throughout the experiment. Although the absolute number of CD8 positive cells and CD4 positive cells were not different between day 0 (CD8: 1.5±0.2 x105, CD4: 2.8±0.2 x105, respectively), day 3 (CD8: 1.8±0.2 x105, CD4: 2.6±0.2 x105, respectively) and day 6 (CD8: 1.9±0.4 x105, CD4: 4.3±0.2 x105, respectively) after tumor cell injection, the absolute number of NKT cells (CD3 and DX5 double positive cells) increased dramatically at relatively earlier phase from 1.6±0.1 x104 on day 0, to 6.4±0.3 x104 on day 3 and 12.1±1.0 x104 on day 14. Intracellular IFN-gamma staining of NKT and CD8 T cells showed that the ratio of IFN-gamma positive NKT to whole NKT cells started increasing from day 3 (day0: 7.0±1.3%, day3: 31±3%, respectively) and peaked on day 6 (59±11%) whereas the ratio of IFN-gamma positive to whole CD8 T cells started increasing from day 6 (day0: 12.1±3.5%, day6: 36±6.5%) and peaked on day 10 (39±3.4%). Conclusion: These results indicate that the NKT cells are recruited to the pleural cavity at a relatively early phase of tumor formation and could potentially affect the subsequent CD8 T cell response by the production of IFN-gamma. Further experiments are on going to see the role of NKT cells and to test the impact of chemotherapy on these cells in this model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 777. doi:10.1158/1538-7445.AM2011-777