Mutanome-guided immunopeptidomics of blood plasma for neoepitope detection in solid tumors is constrained by cfDNA variant calling sensitivity and MS detection limits
- 1German Cancer Research Center (DKFZ)
Abstract
Introduction: Neoepitopes form the basis of tumor-specific immune responses. Tissue biopsy, the primary source for neoepitope detection, is limited and invasive. Therefore, we aimed to identify neoepitopes by mutanome-guided immunopeptidomics from plasma of cancer patients. Methods: Mass spectrometry (MS) data analysis of HLA ligands from plasma (n = 4) was guided by patient-specific mutanomes of cell-free DNA (cfDNA) from plasma or tumor genomic DNA (tgDNA) from tissue. Matched tumor tissue and healthy donor plasma served as controls. Neoepitopes were validated with synthetic peptides, and immunogenicity was assessed using IFN-gamma ELISpot and intracellular cytokine staining. Results: Wild-type immunopeptidomes from tissue and plasma overlapped by 58%, with 91% of plasma HLA ligands rediscovered in tissue. 13 out of 15 tumor-associated HLA ligands detected in plasma were rediscovered in the matching tissue. However, no neoepitopes in plasma were identified by immunopeptidomics guided by cfDNA mutanomes, likely reflecting the limited overlap between cfDNA and tgDNA mutanomes (15%). Using the tgDNA mutanome as a complementary reference, two neoepitopes were detected in one patient's plasma, albeit at the MS detection limit. Both neoepitopes were also discovered in tissue, along with three tissue-exclusive neoepitopes. Two tissue-exclusive neoepitopes induced antigen-specific T cell responses in healthy donor PBMCs. Conclusion: In summary, plasma immunopeptidomics enables profiling of HLA ligands from wild-type proteins, including TAAs. In principle, neoepitope detection from plasma at the peptide level is feasible, but tissue remains the gold standard for variant calling and neoepitope identification. Improved detection methods may enable minimally invasive approaches in the future.
Related
- MSIPep: A pipeline for immunogenic peptide identification and prioritization integrating immunopeptidomics and transcriptomics data
- Multi-Task Bacterial Colony Detection and Classification Using YOLOv8 with Edge Optimization for Resource-Constrained Deployment
- Higher rewards lead to more accurate flower detection and increased contrast sensitivity in the bumblebee Bombus terrestris
- Cost-efficient filtration and replication strategies match or exceed detection sensitivity for marine eDNA diversity at an intertidal site under a standardized laboratory workflow
- Achieving near single-molecule sensitivity in separation-free protein detection using engineered kinetic proofreading
BibTeX
@misc{kienzle2026mutanomeguided,
title = {{Mutanome-guided immunopeptidomics of blood plasma for neoepitope detection in solid tumors is constrained by cfDNA variant calling sensitivity and MS detection limits}},
author = {A. Kienzle and J. P. Becker and B. Fendl and E. Tomasich and G. Heller and K. Wellach and K. Samimi and B. Eiz-Vesper and A. M. Starzer and J. M. Berger and V. Sunder-Plassmann and B. Kiesel and C. Bock and G. Widhalm and M. Preusser and A. S. Berghoff and A. B. Riemer},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.21.753080},
url = {https://www.biorxiv.org/content/10.64898/2026.09.21.753080v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Mutanome-guided immunopeptidomics of blood plasma for neoepitope detection in solid tumors is constrained by cfDNA variant calling sensitivity and MS detection limits
AU - A. Kienzle
AU - J. P. Becker
AU - B. Fendl
AU - E. Tomasich
AU - G. Heller
AU - K. Wellach
AU - K. Samimi
AU - B. Eiz-Vesper
AU - A. M. Starzer
AU - J. M. Berger
AU - V. Sunder-Plassmann
AU - B. Kiesel
AU - C. Bock
AU - G. Widhalm
AU - M. Preusser
AU - A. S. Berghoff
AU - A. B. Riemer
PY - 2026
DA - 2026/09/21
PB - bioRxiv
DO - 10.64898/2026.09.21.753080
UR - https://www.biorxiv.org/content/10.64898/2026.09.21.753080v1
AB - Introduction: Neoepitopes form the basis of tumor-specific immune responses. Tissue biopsy, the primary source for neoepitope detection, is limited and invasive. Therefore, we aimed to identify neoepitopes by mutanome-guided immunopeptidomics from plasma of cancer patients. Methods: Mass spectrometry (MS) data analysis of HLA ligands from plasma (n = 4) was guided by patient-specific mutanomes of cell-free DNA (cfDNA) from plasma or tumor genomic DNA (tgDNA) from tissue. Matched tumor tissue and healthy donor plasma served as controls. Neoepitopes were validated with synthetic peptides, and immunogenicity was assessed using IFN-gamma ELISpot and intracellular cytokine staining. Results: Wild-type immunopeptidomes from tissue and plasma overlapped by 58%, with 91% of plasma HLA ligands rediscovered in tissue. 13 out of 15 tumor-associated HLA ligands detected in plasma were rediscovered in the matching tissue. However, no neoepitopes in plasma were identified by immunopeptidomics guided by cfDNA mutanomes, likely reflecting the limited overlap between cfDNA and tgDNA mutanomes (15%). Using the tgDNA mutanome as a complementary reference, two neoepitopes were detected in one patient's plasma, albeit at the MS detection limit. Both neoepitopes were also discovered in tissue, along with three tissue-exclusive neoepitopes. Two tissue-exclusive neoepitopes induced antigen-specific T cell responses in healthy donor PBMCs. Conclusion: In summary, plasma immunopeptidomics enables profiling of HLA ligands from wild-type proteins, including TAAs. In principle, neoepitope detection from plasma at the peptide level is feasible, but tissue remains the gold standard for variant calling and neoepitope identification. Improved detection methods may enable minimally invasive approaches in the future.
ER -