Josiah Brown Poster Abstract


Kara Reitz
Robert Prins
Joey Orpilla
Evaluation of tumor-infiltrating cell function via multiplex immunohistochemistry of Ki-67 and pSTAT1 in patients treated with pembrolizumab for recurrence/progression of glioblastoma.


Glioblastoma multiforme (GBM) is a lethal primary brain tumor in adults that is exceptionally difficult to treat. Patients diagnosed with GBM typically have a median survival of  <1 year following initial diagnosis; thus, there is great interest in both identifying new and improving current treatment modalities. More recently, there has been notable success in patients with advanced cancers using immunotherapeutic agents, including those that target T-cell checkpoint receptors such as PD-1 (i.e. pembrolizumab). The goal of this project is to establish a standardized protocol for evaluating lymphocyte activation in GBM for use as a predictive biomarker of survival and therapeutic effect. Brain tumor samples from “A pilot surgical trial to evaluate early immunologic pharmacodynamic parameters for the PD-1 checkpoint inhibitor, pembrolizumab (MK-3475), in patients with surgically accessible recurrent/progressive glioblastoma” will be stained for the expression of lymphocytes, CD4+ and CD8+, and the following activation/proliferation markers: Ki67 (a marker of cell proliferation) and pSTAT1 (a marker of cell activation that is initiated by IFN-g).



An immunohistochemistry (IHC) protocol was established for staining Ki67 and pSTAT1 in healthy human tonsil tissue. Slides were stained using a multiplex IHC assay for CD8, CD4, and Ki67; pSTAT1 was stained on its own. DAPI was used as a nuclear counterstain. HALO was used to quantify and qualify expression of each marker, particularly in terms of observed level of expression and localization in tissue.



Ki67 was expressed in the nuclei of cells within the germinal centers of tonsil follicles, as expected. The protocol for pSTAT1 was not found to stain in tonsil tissue and will further be evaluated using other types of tumor tissue (i.e. melanoma).



The establishment of these IHC protocols are critical to eventually delineating the expression and localization of Ki67 and pSTAT1 in brain tumor tissue. This will help to characterize the degree of immune and lymphocyte activation in the setting of an immune checkpoint blockade, which we believe may be predictive of therapeutic efficacy.