Dual-Color Immunohistochemistry Assays For Measuring Aiolos And Ikaros Proteins In Multiple Myeloma Patient Samples

IMiDs® immunomodulatory drugs Lenalidomide (LEN) and Pomalidomide (POM) are antineoplastic agents that have a significant clinical impact in the treatment of patients with multiple myeloma (MM). Recent studies indicate that the anti-proliferative activity of LEN and POM in MM cells is associated with ubiquitination and degradation of the Ikaros family zinc finger protein transcription factors, Aiolos (IKZF3) and Ikaros (IKZF1), the substrates of the Cereblon (CRBN)-dependent Cullin 4 E3-ligase complex. Measurement of Aiolos and Ikaros protein levels in MM patient samples is critical for future correlative studies. Here we describe the development and validation of dual IHC Aiolos/CD138 and Ikaros/CD138 assays using the highly specific Aiolos rabbit monoclonal antibody CGN-9-9-7, and a commercial Ikaros rabbit polyclonal antibody H-100 in combination with a commercial CD138 antibody. Aiolos and Ikaros immunoreactivity in CD138+ MM cells is scored using the semi-quantitative H-score method. H-scores range from 0 to 300 and are the sum of the products of the percentage of tumor cells (0-100%) and the intensity of staining (0-3). The assays were specific and detected high Aiolos and Ikaros expression in the positive control OCI-Ly10 lymphoma cell line, and low Aiolos and Ikaros levels in POM treated OCI-Ly10 cells. These results were confirmed via Western Blot analysis of Aiolos and Ikaros expression data in these cells lines. Assay precision was examined via staining serial sections of the same MM tumor across three different days and deemed acceptable. The dual IHC assays were used to evaluate bone marrow core biopsies or aspirate clots from 22 MM patients and H-scores were determined by a board-certified pathologist. The dual IHC assays detected a wide range of Aiolos or Ikaros immunoreactivity in the 22 MM cases, with nuclear H-scores ranging from 10 to 260 for Aiolos and 40 to 240 for Ikaros. In conclusion, the dual Aiolos/CD138 and Ikaros/CD138 IHC assays were developed to provide a specific and reliable semi-quantitative method to evaluate Aiolos and Ikaros protein levels in both bone marrow core biopsies and clots from MM patients. These assays are currently being used in clinical trials to assess the importance of Aiolos and Ikaros as biomarkers in MM patients treated with IMiDs® immunomodulatory drugs. Citation Format: Yan Ren, Maria Wang, Suzana Couto, Donna Hansel, Anita K. Gandhi, Patrick Hagner, Anjan Thakurta, Rajesh Chopra, Mike Breider. Dual-color immunohistochemistry assays for measuring Aiolos and Ikaros proteins in multiple myeloma patient samples. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5445. doi:10.1158/1538-7445.AM2015-5445