Author of

• 11842

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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
  • Moderators:
  • Coordinates: 10/29/2013, 09:30 - 16:30, Exhibit Hall, Ground Level

  • 11845

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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 - 09:30  |  Author(s): M. Dean
    • 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.