Abstract A046 Expression of novel immune checkpoints HHLA2 and B7x on circulating tumor cells (CTCs) of pediatric, adolescent and young adult solid tumors
Authors: Yuan Ziqiang , Libutti Steven K. , Cole Peter , Moerdler Scott 
Published: September 2024

Abstract
Introduction: The poor outcomes observed in high-risk pediatric solid tumors often stem from the burden of metastatic disease. The utility of liquid biopsies from peripheral blood has emerged as a promising avenue of comprehending the intricate process of metastasis and an area of active investigation. In addition, novel immunotherapies are under development for cancer treatment, particularly for pediatric high-risk and metastatic cases. HHLA2 and B7x are the newest members of the B7/CD28 family immune checkpoint regulators, which typically facilitate tumor immune escape by suppressing the host immune system’s ability to recognize and eliminate tumors. These checkpoints are widely expressed across various adult cancers. Clinical trials in adult cancer patients have shown efficacy with antibodies to B7/CD28 family member immune checkpoints. However, the expression of HHLA2 and B7x on Circulating Tumor Cells (CTCs) of pediatric solid tumors remains poorly understood. Here we describe the initial investigations of HHLA2 and B7x expression on circulating tumor cells of pediatric solid tumors, in effort to begin to understand their role in the metastatic cascade. Methods: We assessed immune checkpoints HHLA2 and B7x on CTCs using the CellSieve size-based microfilitration and our developed HHLA2/B7x multiple cocktailed assay kits. The blood specimens were pre-filtered through CellSieve microfilters. Captured cells were further analyzed by immunofluorescence staining with the fluorescently labeled antibody cocktail containing anti-Vimentin antibody (a biomarker for CTC), anti-CD45 antibody (a biomarker for lymphocyte), and DAPI, plus our checkpoint antibodies (HHLA2 and B7x). To validate the HHLA2/B7x multiple cocktailed assay kits, we used known cultured cell lines expressing these checkpoints as positive controls: A204 for HHLA2, SK-BR-3 for B7x. H460 served as a negative control for HHLA2, and MD-MB-231 as a negative control for B7x. Results: Blood specimens were collected from ten patients with pediatric cancers, including osteosarcoma, Ewing sarcoma, chordoma, rhabdomyosarcoma, lung carcinoma, hepatocellular carcinoma, neuroblastoma, and ovarian germ cell tumor. The CTCs were identified in all ten patients with pediatric cancers. Our findings revealed HHLA2 expression on CTCs in 40% of patients (4/10) and B7x expression in 33.3% of patients (1/3). Normal control blood samples were also tested to confirm that the positive signaling was not due to background noise. Conclusion: These results demonstrate an exciting proof of concept with the novel finding of HHLA2 and B7x checkpoint detection on CTCs of pediatric solid tumors. Further investigation is needed to understand the role these checkpoint expressing CTCs play in contributing to the metastatic cascade. Moreover, these findings hold promise for identifying biomarkers which may be able to help guide treatment decision for progressive disease before it becomes detectable through conventional radiographic methods. Acknowledgements: We are grateful for support from NJ PHORCE.
Citation Format: Ziqiang Yuan, Steven K. Libutti, Peter Cole, Scott Moerdler. Expression of novel immune checkpoints HHLA2 and B7x on circulating tumor cells (CTCs) of pediatric, adolescent and young adult solid tumors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr A046.