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R. West



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    MO03 - Thymic Malignancies (ID 123)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Medical Oncology
    • Presentations: 1
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      MO03.08 - Increased Galectin-1 Expression in a Thymic Epithelial Tumor Tissue Microarray (TMA) and Galectin-1 Knockdown Studies in a Thymoma Cell Line (ID 1238)

      10:30 - 12:00  |  Author(s): R. West

      • Abstract
      • Presentation
      • Slides

      Background
      Thymoma is a rare malignancy with a paucity of data on biology. Thymic epithelial tumors are often admixed with developing T-lymphocytes in the microenvironment. Galectin-1 (gal-1) is a beta-galactosidase binding protein involved in T-cell development via thymic stromal and thymocyte interaction as well as thymocyte development through negative selection. Gal-1 also induces apoptosis of effector T-lymphocytes, promotes angiogenesis, and is a poor prognostic indicator when overexpressed in several tumor types. To our knowledge expression of gal-1 has not been examined in thymic epithelial tumors.

      Methods
      A TMA was constructed from 68 patients with thymic malignancies and 8 benign thymic controls at Stanford University School of Medicine (Stanford, CA). Immunohistochemical stains for galectin-1 (1:200 dilution; citrate pre-treatment; mouse monoclonal; Novocastra) were performed on 4 µM-thick TMA sections. Galectin-1 cytoplasmic staining of the epithelial cell component was scored as negative (0), focal positive (1+), or strong positive (2+) by a Stanford pathologist, who was blinded to the clinical data. Gal-1 expression was averaged for each patient sample. Statistical analysis was performed using SAS Enterprise Guide v5.0 (Cary, NC). Non-parametric statistical analyses were used to compare average patient gal-1 expression between thymic tumors and benign thymic controls. Stable gal-1 knockdown was achieved in IU-TAB1, a human thymoma WHO type AB cell line, using the pLKo.1 vector with gal-1 shRNA (Open Biosystems). Lentivirus was produced using the Trans-Lentiviral Packaging System (Thermo Scientific). In vitro proliferation cell counts were performed by hemocytometer. After hypoxia exposure (0.5% O~2~), apoptotic cells were labeled using the APO-Direct kit and quantified by flow cytometry (BD Biosciences).

      Results
      Demographics for 68 patients: M:F (53%/47%), Mean age at diagnosis: 55 years, WHO Histology: A (10%), B (57%), AB (24%), C (4%), unclassified (4%), Pathologic Maseoka Stage: I (46%), IIa (18%), IIb (4%), III (18%), IVa (9%) IVb (6%). Gal-1 expression was increased among thymic tumor tissue compared to unpaired controls (mean avg gal-1 expression 1.5 vs. 0.125, p=0.0012, Kruskal-Wallis test). Logistic regression of tumor vs. control thymus by gal-1 generated a C-statistic of 0.845. A significant increase in gal-1 expression was noted across all WHO thymoma subtypes except thymic carcinoma (type C) (p < 0.05, non-parametric ANOVA with post-hoc ranked Dunnett’s t-test). Among 11 thymic tumors analyzed with paired adjacent resected benign thymus tissue from the same patient, a significant increase was noted in gal-1 expression among tumor compared with adjacent resected normal benign thymus (mean avg gal-1 1.82 vs. 0.35, p=0.004, sign-rank test). In vitro, gal-1 knockdown did not affect IU-TAB1 proliferation. Preliminary results showed gal-1 knockdown increased apoptosis under hypoxia compared to scramble control.

      Conclusion
      Gal1 expression was increased among thymoma compared with benign thymus controls and paired adjacent resected benign thymus. A robust C-statistic of 0.845 indicates that gal-1 expression may discriminate tumor from benign thymus. Increased gal-1 expression was conserved across WHO histologic subtype except for thymic carcinoma—whose analysis was limited due to small sample size. Gal-1 knockdown might increase apoptosis under hypoxia. We are continuing to investigate the biologic and clinical significance of increased gal-1 expression in thymoma.

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    P2.15 - Poster Session 2 - Thymoma (ID 191)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Thymoma & Other Thoracic Malignancies
    • Presentations: 1
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      P2.15-003 - Gli1, Notch1 and CTNNB1 Expression by Automated Quantitative Immunofluorescence (AQUA) in a Thymic Malignancy Tissue Microarray (TMA) (ID 778)

      09:30 - 16:30  |  Author(s): R. West

      • Abstract

      Background
      Thymoma is a rare malignancy, with a paucity of data on its biology and on the role of targeted therapeutics. Wnt, notch and sonic hedgehog pathway interactions between thymocytes and thymic stroma are important to both thymus and T-cell development. AQUAnalysis[®] is a digital image analysis software that continuously measures multiplexed protein expression and has the potential to overcome limitations of small sample sizes and tissue heterogeneity in the tumor microenvironment. We analyzed a thymoma TMA for gli1, notch1 and CTNNB1 (β-Catenin) expression by AQUA[®] as surrogate markers of activity of the sonic hedgehog, notch and wnt pathways, respectively. We hypothesized this preclinical screen may provide rationale for attacking these pathways with targeted therapeutics in thymoma.

      Methods
      A TMA was constructed from 68 patients with thymic malignancies and 8 benign thymic controls at Stanford University School of Medicine (Stanford, CA). Gli1, notch1 and CTNNB1 expression were assayed using quantitative fluorescent immunohistochemistry at the Tom Baker Cancer Center (Alberta, Canada). The TMA was stained with anti-gli1 rabbit mAb (monoclonal antibody), clone EPR4523 (Epitomics, Burlingame, CA, USA); anti-Notch1 rabbit mAb, clone EP1238Y (Epitomics, Burlingame, CA, USA); and anti-beta-catenin mouse mAb, clone β-Catenin-1 (Dako Mississauga, ON, Canada) using a Dako autostainer. To isolate expression of these stem-cell pathway proteins separately in the tumor and the lymphocytes, the TMA was also stained with anti-pan-cytokeratin guinea pig mAb (Acris, San Diego, CA, USA); anti-vimentin rat mAb, clone 280618 (R&D Systems, Minneapolis, MN, USA); and anti-CD45 rabbit mAb, clone EP322Y (Epitomics, Burlingame, CA, USA). Automated image acquisition was performed using an Aperio Scanscope FL (Aperio Inc., Vista, CA, USA). Images were then analyzed using the AQUAnalysis® program, version 2.3.4.1. A tumor-specific mask and a tumor cytoplasmic mask were generated to distinguish thymoma cells from surrounding stromal tissue by thresholding the pan-cytokeratin images to identify pan-cytokeratin positive cells as tumor cells and define the tumor cytoplasm. Statistical analysis was performed using SAS Enterprise Guide v5.0 (Cary, NC). Two-tailed t-tests were used to compare the differences between thymic tumor and benign control tissue. ANOVA and Dunnett’s t-test was used to compare differences in gli1, notch1, and CTNNB1 expression by WHO histology.

      Results
      Demographics for 68 patients: M:F (53%/47%), Mean age at diagnosis: 55 years, WHO Histology: A (10%), B (57%), AB (24%), C (4%), unclassified (4%), Pathologic Masaoka Stage: I (46%), IIa (18%), IIb (4%), III (18%), IVa (9%) IVb (6%). No difference in gli1 (mean 201 vs. 211, p=0.31), CTNNB1 (mean 396 vs. 418, p=0.66) or notch1 expression (mean 317 vs. 325, p=0.82) was noted between thymic tumors and controls. In a subset analysis, we found no significant differences by WHO histology compared to controls.

      Conclusion
      AQUA® was used to help overcome limitations of analyzing protein expression in histologically heterogeneous thymic tumors with small sample sizes. We found no clinically or statistically significant increased expression of gli1, notch1, and CTNNB1 in thymoma compared to benign thymic tissue. Thus, this study provides no evidence for upregulation of the sonic hedgehog, notch or wnt pathways in thymic tumors.