Abstract 5415: Glypican-3 immunohistochemistry precision, validation, and prevalence in selected solid tumors to identify target populations for CAR T cell therapy
Authors: Weiss Glen J. , Jensen-Smith Amy , Muralidharan Sujatha 
Published: June 2022

Abstract
Background: Glypican-3 (GPC3) is an oncofetal tumor antigen that is an attractive target for chimeric antigen receptor (CAR) T cell therapy due to its highly restricted expression on normal tissue and high prevalence in several adult and pediatric solid tumors. Aberrant GPC3 expression is implicated in tumorigenesis, and GPC3+ cancers are characterized by a highly immunosuppressive landscape which induces exhaustion in tumor-resident T cells. We sought to assess the validity of a GPC3 antibody clone in immunohistochemistry (IHC) and then measure GPC3+ prevalence in selected solid tumors.
Methods: We evaluated GPC3 transcripts in human normal and tumor tissue specimens from The Cancer Genome Atlas (TCGA) and the Genome-Tissue Expression Project. GPC3 IHC was performed on formalin fixed paraffin embedded tumor tissues: hepatocellular (HCC), squamous cell lung (SCC), colorectal (CRC), serous ovarian (OC), Merkel cell carcinomas (MCC) and liposarcoma (LS) using antibody clone GC33 and scored using H-score (range 0-300), with appropriate positive and negative controls. Accuracy and precision of GPC3 antibody clone GC33 were tested in GPC3+ high prevalence tumor types compared to a validated anti-GPC3 clone (1G12). GPC3+ cutoff was set as H-score ≥ 30 (GC33) compared to &gt;20 (1G12). The prevalence of GPC3 expression measured by IHC was also used to calculate the theoretical mRNA cutoff value from the TCGA data for GPC3+ indications represented in the database.
Results: GPC3 mRNA was expressed at low levels predominantly in lung, adipose, tibial nerve, kidney and breast. GPC3 IHC of normal tissue showed predominantly faint, cytoplasmic staining in a few tissues including heart, kidney and stomach and no expression in lung or breast. HCC, SCC, LS, CRC, and OC showed high and varying levels of GPC3 mRNA expression in TCGA samples. The theoretical mRNA expression cutoff value based on IHC were HCC=1396 RSEM, SCC= 2675 RSEM, Sarcoma= 4126 RSEM; and OC 4486 RSEM; respectively. A cutoff for CRC could not be determined because the prevalence of GPC3 by IHC was &lt;10%. GPC3 IHC using GC33 showed a higher prevalence of positive cases in myxoid/round cell LS (MRCLS) relative to other LS subtypes (15/45 vs. 8/75). HCC (n=110), SCC (n=73), MRCLS (n=45), and MCC (n=20) had a prevalence of 69%, 33%, 33%, and 70%; respectively. However, &lt;10% of CRC (n=80) or OC (n=31) were GPC3+. GPC3 IHC assay using clone GC33 was fully validated for use as a lab-developed test by assessing accuracy, sensitivity, specificity, and precision in SCC, HCC, MCC and LS specimens.
Conclusions: GPC3 has a high prevalence in selected tumors with low level expression in some normal tissues, making it an attractive target for CAR T cell therapy. The GC33 clone performs similarly to 1G12 and could be used for IHC screening for CAR T cell therapy. MRCLS has higher prevalence of GPC3+ expression relative to other LS subtypes.
Citation Format: Glen J. Weiss, Amy Jensen-Smith, Sujatha Muralidharan. Glypican-3 immunohistochemistry precision, validation, and prevalence in selected solid tumors to identify target populations for CAR T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5415.