Abstract
Germ cell tumors (GCTs) pose significant diagnostic challenges because of the limited performance of existing tumor markers. Here, we used phage immunoprecipitation sequencing (PhIP-Seq) to develop a unique immunosignature panel to improve diagnosing and differentiating GCT. 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, identifying 23/24 biomarker-negative GCT cases. A secondary model (Sem-iSIGN), consisting of 17 peptides from five proteins, differentiated seminoma from nonseminoma with 96% specificity, 65% sensitivity, and AUC of 0.77. RNA sequencing data from The Cancer Genome Atlas confirmed differential overexpression of target antigens in testicular cancer. ELISA validation of ERVK7 and LUZP4 and immunohistochemical detection of ERVK7, MUC4, ZNF91, and LUZP4 in tumor tissues supported target expression. This study highlights PhIP-Seq immunoprofiling to identify serum-based immunosignature panels that can serve as biomarkers for GCTs. This approach addresses the shortcomings of conventional markers and offers a scalable, cost-effective tool for improving cancer diagnosis and management.
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Data availability
The raw PhIP-Seq sequencing files (FASTQ format) generated and analyzed during this study are available through Zenodo at https://doi.org/10.5281/zenodo.17478533. Processed enrichment matrices and associated metadata are included within the repository. De-identified clinical information is included in supplementary data 1. Source data are provided with this paper.
Code availability
The complete bioinformatics pipeline used for PhIP-Seq data processing and modeling, including scripts for sequence alignment, enrichment calculation, and classifier development, is publicly available at https://doi.org/10.5281/zenodo.17478533.
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Acknowledgements
We thank Sara Vinje for administrative assistance. We thank Gerard Hebert for editorial assistance. We are grateful to the patients and their families for their participation in this study. This work is supported by Department of Defense (DOD) grant #CA210208 awarded to D.D. (PI), B.A.C., and S.D. (Co-I).
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Conception and design of the study: A.M.K., S.D., and D.D. Acquisition of data: M.B.H., A.M.K., H.K., B.A.C., S.B.D.L., B.C.L., J.C.C., Y.G., R.G.L.-C., N.K.P., S.R.H., J.S., J.E.O., A.A.-S., S.J.P., J.R.M., S.D., and D.D. Data analysis: M.B.H, A.M.K., S.B.D.L., and S.D. Drafting the manuscript or figures: M.B.H., A.M.K., S.B.D.L., Y.G., J.C.C., N.K.P., and D.D. Study supervision: D.D. All authors reviewed and edited the manuscript and approved the final version of the manuscript for submission.
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A.A.S. has participated on advisory boards for Roche Diagnostics and Fujirebio Diagnostics. She has received speaker honoraria from Roche Diagnostics. S.J.P. reports receiving grants, personal fees paid to Mayo Clinic, and nonfinancial support from Alexion Pharmaceuticals, Inc. and MedImmune, Inc./Viela Bio; receiving personal fees from Genentech/Roche, UCB, and Astellas, outside the submitted work; holding patent 8,889,102 (application 12–678350) issued and patent 9,891,219B2 (application 12–573942) issued. D.D. consulted for UCB, Immunovant, Argenx, Arialys, and Astellas Pharmaceuticals. All compensation for consulting activities is paid directly to Mayo Clinic. He is a named inventor on a filed patent that relates to KLHL11 as a marker of autoimmunity and germ cell tumors. He has patents pending for LUZP4-IgG, cavin-4-IgG, and SKOR2-IgG as markers of neurological autoimmunity. He has received funding from the DOD (CA210208 and PR220430), David J. Tomassoni ALS Research Grant Program, and UCB. The rest of the authors have no competing interests.
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Hammami, M.B., Knight, A.M., Kherbek, H. et al. Whole-proteome phage immunoprecipitation sequencing reveals germ cell tumor–specific immunosignature. Nat Commun (2026). https://doi.org/10.1038/s41467-026-71174-9
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DOI: https://doi.org/10.1038/s41467-026-71174-9
