Abstract 6373: From genotype to phenotype: Adding phosphoproteomic analysis to molecularly-guided therapy recommendation in Cancer of Unknown Primary (CUP)

Abstract Molecularly-guided therapy can improve treatment outcomes in CUP, a heterogeneous group of cancers with very limited prognosis. Within DKFZ/NCT/DKTK MASTER, a clinically embedded prospective precision oncology registry trial, patients receive molecularly-guided treatment recommendations (TR) based on whole genome/exome sequencing (WGS/WES), transcriptome and methylome analysis from a dedicated molecular tumor board (MTB). We retrospectively added phosphoproteomic analysis (PPA) to assess its potential to provide clinically relevant predictive biomarkers. We analyzed 155 CUP patients enrolled in MASTER between May 2013 and 2023. Median overall survival was 11.8 months from enrollment, median follow-up was 25.7 months. Analyses included WES/WGS of tumor and normal tissue (n=45/n=89), as well as RNA sequencing (n=112). The MTB issued 479 TR for 134/155 patients (86%), which were applied in 45/134 patients (34%). To evaluate clinical efficacy in this highly heterogeneous cohort, we calculated progression-free survival ratios (PFSr) by comparing PFS of the first applied TR (PFS2) to that of its preceding systemic therapy (PFS1) in each patient. Mean PFSr was 2.6, median PFSr was 1.5 (range 0.2-19.0; both n=33). PFSr was >1.3 in 18/33 cases (55%). Median PFS1 was 2.5 months (range 1.0-10.0; n=36) and median PFS2 5.6 months (range 1.0-36.9; n=41). PPA was performed retrospectively in 23/45 patients (51%) with TR applications, using tumor tissue from the same biopsy as the other omic analyses. Phosphoproteomic biomarkers were identified by a standardized and partially automated workflow assessing expression and phosphorylation of a given protein, focusing on protein kinases and their substrates. In 2/23 cases (9%), PPA failed due to insufficient material. PPA led to identification of 25 biomarkers, most frequently TACSTD2 (TROP2, n=8), ERBB2 (n=4), immune checkpoints/ T cell signaling (n=5), EGFR (n=5), and MAGEA4 (n=4). For biomarkers referenced in TR, matching biomarkers from PPA were observed in 12/21 patients (57%). In 16/21 patients (76%), PPA revealed additional biomarkers. In 13/21 patients (62%), PPA predicted efficacy for 16 applied TR based on abnormal expression/phosphorylation of immune checkpoints/ T cell signaling (n=5), ERBB2 (n=4), ALK (n=2), EGFR (n=2), DNA damage repair (n=1), TACSTD2 (n=1) and SRC (n=1). PPA predicted efficacy for the first applied TR in 10/21 cases (48%) leading to a median PFSr of 2.4 (range 0.6-4.8; n=7) and a PFSr > 1.3 for 5/7 applications (71%). Conversely, PPA did not support the first applied TR in 11/21 cases (52%). Median PFSr was 1.3 (range 0.6-5.3; n=10) and PFSr was > 1.3 in 4/10 of these cases (40%). Our analysis suggests that PPA may be a valuable addition to precision oncology workflows by providing additional biomarkers, predicting meaningful PFS and improving specificity of TR. Citation Format: Maximilian Werner, Tobias Mundt, Annika Schneider, Waqar Hussain, Jonas Arnold, Cecilia Bang Jensen, Martina Fröhlich, Barbara Hutter, Jennifer Hüllein, Nagarajan Paramasivam, Amirhossein Sakhteman, Matthew The, Daniela Richter, Simon Kreutzfeldt, Christoph E. Heilig, Maria-Veronica Teleanu, Daniel B. Lipka, Annika Baude, Katja Beck, Katrin Pfütze, Maren Knödler, Damian Rieke, Johanna Falkenhorst, Ilektra Antonia Mavroeidi, Christian Brandts, Anna L. Illert, Melanie Börries, Thomas Kindler, Tobias Marcus Herold, Nicole Pfarr, Albrecht Stenzinger, Christoph Heining, Irina Kerle, Evelin Schröck, Peter Horak, Daniel Hübschmann, Lino Möhrmann, Bernhard Küster, Stefan Fröhling, Hanno Glimm. From genotype to phenotype: Adding phosphoproteomic analysis to molecularly-guided therapy recommendation in Cancer of Unknown Primary (CUP) [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 6373.