Abstract 1027: Immunohistochemistry (IHC) to assess tumor cell-associated OX40 expression in lymphoma PDXs
Authors: Lin Jie , Tu Xiaolong , Zhang Likun , Yu Hongjiang , JIang Cheng , Yang Tao , Li Henry Qixiang 
Published: March 2024

Abstract
Background. Tumor necrosis factor receptor superfamily member 4, or OX40, is an immunostimulatory receptor usually expressed in activated T-cells. The OX40-OX40L (ligand of OX40) interaction plays a crucial role in the enhancement of T-cell proliferation, survival, and cytokine production, including those in tumor microenvironment (TME) leading to increased anti-tumor immunity1. OX40 agonist antibodies have been widely tested for immunotherapy for solid tumors2,3. Interestingly, in the treatment of lymphoma, the situation may become more complicate, where OX40 could also be found to be expressed on lymphoma cells, in addition to TME. This phenomenon might or might not impact OX40 agonist therapy. This report aims at developing an immunohistochemistry (IHC) assay to assess tumor-associated OX40 by utilizing morphology approach to understand OX40 expression in lymphoma cells, as opposed on tumor-infiltrate T-lymphocytes (TILs) present in TME. FFPE samples from patient-derived xenografts (PDXs), recapitulating the main pathological features of the original tumors, but exclusive of human TILs, were used as the first stage of the assay development, avoiding TIL interference prior to human sample testing.
Methods. Ninety leukemia and lymphoma PDXs are comprehensively annotated, including RNAseq and histopathology (HuBaseTM: https://hubase.crownbio.com). IHC staining of PDX FFPE slides or TMA (tissue microarray) of various cancers was performed using a commercial OX40 antibody (CST, #61637) and Bond RX Automated IHC/ISH Staining System (Leica Biosystems), followed by whole slide imaging by NanoZoomer NDP2.0-HT Digital Slide System (Hamamatsu) and quantitative analysis by HALOTM image analysis software (Indica labs). OX40 IHC scores were also compared to the RNA expression determined by RNAseq.
Results. A total of 90 leukemia and lymphoma PDXs were comprehensively evaluated. RNASeq data (human mRNA) indeed revealed OX40 gene expression (cutoff as Log2 (FPKM)&gt;3) in 8.9% of models (8/90 models); on the other hand, IHC data also confirmed these OX40 protein expression; and the IHC scores largely correlated with the RNA data. Together, our data firmly confirmed the expression of OX40 in certain hematological cancers, beyond just in TME. The ongoing studies is investigating the characteristics of the OX40 positive lymphoma/leukemia, and the preliminary data will be presented in the annual conference. Furthermore, we are also using the same assay to explore other cancer types beyond hematological cancers.
Conclusion. A tumor associated OX40 expression IHC workflow was preliminarily established and validated to facility the OX40 immunotherapy development.
Citation Format: Jie Lin, Xiaolong Tu, Likun Zhang, Hongjiang Yu, Cheng JIang, Tao Yang, Henry Qixiang Li. Immunohistochemistry (IHC) to assess tumor cell-associated OX40 expression in lymphoma PDXs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1027.