Abstract 4646: Technical, intratumoral and interpatient variability of a novel assay that quantifies fractional occupancy of PD1 by PDL1 in clinical biospecimens

Abstract Background: New biomarkers are needed to predict clinical response to anti-PD1 therapy. We have been evaluating whether quantifying fractional occupancy of PD1 by PDL1 (PD1 FO) within a tumor may be of value as a predictive biomarker. The Ligand-Receptor Complex-binding Aptamer (LIRECAP) assay used to quantify PD1 FO is based on the preferential binding of one RNA aptamer to unoccupied PD1 (P aptamer) and the other to the PD1/PDL1 complex (C aptamer). Here, we report the technical, intratumoral, and inter-patient variability of the PD1 LIRECAP assay using clinical biospecimens. Methods: Biospecimens: Formalin Fixed Paraffin Embedded (FFPE) sarcoma biospecimens were selected based on their availability with varying expression of PDL1. All samples had significant T cell infiltration. Ten-μm-thick scrolls were cut, deparaffinized, rehydrated and used for analysis. To assess technical variability, scrolls were macerated, aliquoted, and repeat LIRECAP assays were performed on the same sample. To assess intratumoral variability, multiple scrolls were cut from the same biospecimen. To assess inter-patient variability, biospecimens from different patients with high (tumors 1, 2 and 3) or low PDL1 (tumors 4, 5 and 6) were evaluated. PD1-PDL1 LIRECAP assay: An equimolar mix of a “P aptamer” and a “C aptamer was added to a macerated scroll. After extensive washing, bound P and C aptamers were quantified using a TaqMan-based RT-qPCR with a set of common PCR primers (against their constant regions) and two unique TaqMan probes (against their unique central regions). The ratio of bound aptamers (C:P ratio) reflected PD1 FO. Results: Technical variability. The PD1 LIRECAP assay showed high technical reproducibility, with low coefficient of variation (CV) for C:P ratio between the aliquots from the same scroll (e.g. Tumor #1: 10%; Tumor #5: 7%). Intratumoral variability. Intratumoral variability was greater than technical variability demonstrating regional variations in PD1 FO within an individual tumor (CV: Tumors #1 - 71%; #2=84%; #3=44%; #4=45%; #5=76%; #6=79%). Interpatient variability. PDL1high tumors showed a higher C:P ratio, indicating higher PD1 FO, when compared to PDL1low tumors (p=0.017) with higher interpatient variability in PDL1low patients (CV: PDL1high=15%; PDL1low=32%). Conclusions and Significance: Evaluation of clinical FFPE biospecimens using the PD1 LIRECAP assay is technically feasible and reproducible. There is intratumoral variability in PD1 FO indicating regional differences within a tumor. Nevertheless, PD1 FO was highest in the PDL1high biospecimens. Studies assessing the value of PD1 FO as a predictive biomarker as determined by the PD1 LIRECAP assay are ongoing with the understanding that further evaluation of PD1 FO as a biomarker for response to anti-PD1 therapy will need to take intratumoral variability into account. Citation Format: Suresh Veeramani, Chaobo Yin, Kristen L. Coleman, George J. Weiner. Technical, intratumoral and interpatient variability of a novel assay that quantifies fractional occupancy of PD1 by PDL1 in clinical biospecimens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4646.