f NOTCH1 in cancers in which it has an oncogenic role with antagonists such as inhibitory antibodies and gamma-secretase inhibitors . Ideally, such trials would focus on treatment of patients whose tumors show evidence of ongoing NOTCH1 activation. Given the large size of the NOTCH1 locus and the diversity of genetic aberrations that produce ligand-independent activation of NOTCH1 or stabilize NICD1, genetic screening for oncogenic NOTCH1 alterations is challenging, particularly when working with archival FPE samples. Moreover, it is suspected that ligand-mediated NOTCH1 activation also contributes to tumor cell growth and survival, and such tumors would go undetected by genetic screening. An ideal biomarker test would detect NICD1 within tumor cells directly, regardless of the underlying mechanism of NOTCH1 activation. To this end, we developed a robust, specific immunohistochemical staining method that detects NICD1 in archival samples. The test relies on a commercial rabbit monoclonal antibody, previously used only in Western blot analyses, that is specific for a neoepitope in NICD1 created by gamma-secretase-mediated proteolysis of NOTCH1, the event that triggers NOTCH1 signaling. Following optimization and validation of the test using cancer xenografts bearing diverse NOTCH1 aberrations and normal tissues, we screened a large series of human cancers of PBTZ 169 unknown NOTCH1 mutational status for activated NOTCH1. Most T-LLs and CLLs showed evidence of ongoing NOTCH1 activation, as did many peripheral T cell lymphomas and a small subset triple-negative breast cancers. Activation of NOTCH1 was also detected in a majority of angiosarcomas, in line with the observation that NICD1 is readily detectable in normal endothelial cells within tumor stroma. By contrast, little or no NOTCH1 activation was observed in mantle cell lymphoma, diffuse large B cell lymphoma, non-small cell lung cancers, and ovarian carcinoma. These findings indicate that NOTCH1 activation among B cell tumors is both more sharply restricted to and more prevalent in CLL than would be predicted from prior genotypic data, raise questions about the proposed tumor suppressive role for NOTCH1 in 10712926 vascular neoplasms, and suggest that 
IHC testing for NICD1 can be used to select patients for clinical trials of Notch pathway inhibitors, particularly those involving patients with tumors such as triplenegative breast cancer, in which NOTCH1 activation is confined to a small subset of tumors. Materials and Methods Use of Human Tissues Human tissues specimens were approved for use by Institutional Review Boards as follows. All tissues involved by lymphoid neoplasms were collected and used without informed consent under Brigham and Women’s Hospital IRB protocol 2010-P002736. A non-small cell lung carcinoma microarray was constructed with tissues obtained from patients without consent under Brigham and Women’s Hospital IRB protocol 2006-P001929. A serous ovarian cancer microarray was constructed with tissues obtained from patients without consent under Brigham and Women’s Hospital IRB protocol 2006-P000321. An angiosarcoma microarray was constructed with tissues obtained from patients without informed consent 7498254 under the University of Texas M. D. Anderson Cancer Center IRB protocol LAB04-0890. In each of these instances, the studies were granted waivers of consent on the following bases: 1) samples were gathered retrospectively from pathology archives and resulted from routine surgical procedu
