Development of a Specific, Sensitive and Selective Immunohistochemical Assay for Notch1 Intracellular Domain (N1ICD) Reveals Notch Pathway Activation in Glioblastoma Multiforme and Carcinomas of the Lung and Colon
Timothy R Holzer, Janet M Grondin, Angie D Fulford, Bharvin K Patel, Andrew E Schade, Bradley L Ackermann, Robert J Konrad, Aejaz Nasir. Eli Lilly and Company, Indianapolis, IN
Background: Aberrant Notch signaling is implicated in a number of malignancies. Pathway signaling is initiated by ligand binding to Notch1 receptor which triggers a series of catalytic cleavages, including γ-secretase cleavage which releases the intracellular domain of the Notch1 protein (N1ICD). N1ICD translocates to the nucleus and promotes transcription of genes involved in inhibition of apoptosis and promotion of cell division. Detection of N1ICD in tumor specimens may indicate an active or deregulated pathway and has the potential to identify patients who may respond to novel Notch pathway inhibitor therapeutics.
Design: We systematically characterized an anti-N1ICD rabbit monoclonal antibody (D3B8). Immunoblot profiling was performed on cell lysates. Preabsorption of the antibody with an excess of recombinant peptides designed to the intracellular forms of Notch1, 2, 3 and 4 was performed. A cell line stably transfected with a truncated murine Notch1 was treated with a γ-secretase inhibitor to assess inhibition of cleavage using a fully optimized immunohistochemical (IHC) assay. IHC was performed on tissue microarrays including a set of Glioblastoma multiforme (GBM), non-small-cell lung carcinomas (NSCLC) and colonic adenocarcinomas (CRC).
Results: Immunoblots showed clean bands at the expected molecular weight of ∼110 kDa suggesting specificity. As indicated by abolishment of staining, the primary antibody was absorbed only by the Notch1 neoepitope which demonstrated selectivity. Inhibitor treated transfectants showed a decrease in immunoreactivity which suggests assay sensitivity to pathway activation. Ten of 12 (83%) GBM, 7/16 (44%) NSCLC and 5/9 (56%) CRC showed unequivocal nuclear expression of N1ICD.
Six of 12 (50%) of the GBM expressed N1ICD protein in >35% of the neoplastic cells.
Conclusions: We developed a sensitive, specific and robust IHC assay to detect Notch pathway activation in archival tumor tissues. Based on direct histo-morphologic evidence, we underscore the potential value of the N1ICD IHC to support Notch inhibitor therapeutic trials in various histological subtypes of common and clinically aggressive human malignancies.
Wednesday, March 21, 2012 9:30 AM
Poster Session V # 258, Wednesday Morning