Gauging NOTCH1 activation in cancer using immunohistochemistry

Michael J. Kluk, Todd Ashworth, Hongfang Wang, Birgit Knoechel, Emily F. Mason, Elizabeth A. Morgan, David Dorfman, Geraldine Pinkus, Oliver Weigert, Jason L. Hornick, Lucian R. Chirieac, Michelle Hirsch, David J. Oh, Andrew P. South, Irene M. Leigh, Celine Pourreyron, Andrew J. Cassidy, Daniel J. DeAngelo, David M. Weinstock, Ian E. KropDeborah Dillon, Jane E. Brock, Alexander J. F. Lazar, Myron Peto, Raymond J. Cho, Alexander Stoeck, Brian B. Haines, Sriram Sathayanrayanan, Scott Rodig, Jon C. Aster

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    67 Citations (Scopus)

    Abstract

    Fixed, paraffin-embedded (FPE) tissues are a potentially rich resource for studying the role of NOTCH1 in cancer and other pathologies, but tests that reliably detect activated NOTCH1 (NICD1) in FPE samples have been lacking. Here, we bridge this gap by developing an immunohistochemical (IHC) stain that detects a neoepitope created by the proteolytic cleavage event that activates NOTCH1. Following validation using xenografted cancers and normal tissues with known patterns of NOTCH1 activation, we applied this test to tumors linked to dysregulated Notch signaling by mutational studies. As expected, frequent NICD1 staining was observed in T lymphoblastic leukemia/lymphoma, a tumor in which activating NOTCH1 mutations are common. However, when IHC was used to gauge NOTCH1 activation in other human cancers, several unexpected findings emerged. Among B cell tumors, NICD1 staining was much more frequent in chronic lymphocytic leukemia than would be predicted based on the frequency of NOTCH1 mutations, while mantle cell lymphoma and diffuse large B cell lymphoma showed no evidence of NOTCH1 activation. NICD1 was also detected in 38% of peripheral T cell lymphomas. Of interest, NICD1 staining in chronic lymphocytic leukemia cells and in angioimmunoblastic lymphoma was consistently more pronounced in lymph nodes than in surrounding soft tissues, implicating factors in the nodal microenvironment in NOTCH1 activation in these diseases. Among carcinomas, diffuse strong NICD1 staining was observed in 3.8% of cases of triple negative breast cancer (3 of 78 tumors), but was absent from 151 non-small cell lung carcinomas and 147 ovarian carcinomas. Frequent staining of normal endothelium was also observed; in line with this observation, strong NICD1 staining was also seen in 77% of angiosarcomas. These findings complement insights from genomic sequencing studies and suggest that IHC staining is a valuable experimental tool that may be useful in selection of patients for clinical trials.

    Original languageEnglish
    Article numbere67306
    Number of pages14
    JournalPLoS ONE
    Volume8
    Issue number6
    DOIs
    Publication statusPublished - 2013

    Keywords

    • OVARIAN-CANCER
    • BREAST-CANCER
    • GENE AMPLIFICATION
    • CHRONIC LYMPHOCYTIC-LEUKEMIA
    • B-CLL
    • CODING GENOME
    • MARGINAL ZONE LYMPHOMA
    • SQUAMOUS-CELL CARCINOMA
    • STEM-CELLS
    • T-ALL

    Cite this

    Kluk, M. J., Ashworth, T., Wang, H., Knoechel, B., Mason, E. F., Morgan, E. A., Dorfman, D., Pinkus, G., Weigert, O., Hornick, J. L., Chirieac, L. R., Hirsch, M., Oh, D. J., South, A. P., Leigh, I. M., Pourreyron, C., Cassidy, A. J., DeAngelo, D. J., Weinstock, D. M., ... Aster, J. C. (2013). Gauging NOTCH1 activation in cancer using immunohistochemistry. PLoS ONE, 8(6), [e67306]. https://doi.org/10.1371/journal.pone.0067306