Whole-proteome phage immunoprecipitation sequencing reveals germ cell tumor–specific immunosignature

Germ cell tumors (GCTs) pose significant diagnostic challenges due to the limited performance of existing tumor markers. To address this, we utilized phage immunoprecipitation sequencing (PhIP-Seq) to develop a unique immunosignature panel with high sensitivity and specificity to improve the diagnosis and differentiation of GCTs. Using 427 serum samples (150 GCT, 277 controls), we developed and validated an immunosignature panel (GCT-iSIGN) comprising 24 peptides from 16 unique proteins. This panel achieved 93% sensitivity, 99% specificity, and an area under the curve (AUC) of 0.98, with accurate identification of all biomarker-negative GCT cases. A secondary model (Sem-iSIGN), consisting of 17 peptides from 5 proteins, differentiated seminoma from nonseminoma with 96% specificity, 65% sensitivity, and an AUC of 0.77. RNA sequencing from The Cancer Genome Atlas confirmed differential overexpression of target antigens in testicular cancer, supporting biological relevance. ELISA validation of two antigens, Endogenous Retrovirus Group K Member 7 (ERVK7), and Leucine Zipper 4 (LUZP4), demonstrated significant correlation with PhIP-Seq findings and immunohistochemical detection of ERVK7, MUC4, ZNF91 and LUZP4 in tumor tissues supported target expression. This study highlights PhIP-Seq immunosignature profiling to identify unique serum-based immunosignature panels that can serve as biomarkers for GCT. This approach addresses the shortcomings of conventional markers and offers a scalable, cost-effective tool for enhancing cancer diagnosis and management.