Histomorphology and immunohistochemistry are the most common ways of cancer classification in routine cancer diagnostics, but often reach their limits in determining the organ origin in metastasis. These cancers of unknown primary, which are mostly adenocarcinomas or squamous cell carcinomas, therefore require more sophisticated methodologies of classification. Here, we report a multiplex protein profiling-based approach for the classification of fresh frozen and formalin-fixed paraffin-embedded (FFPE) cancer tissue samples using the digital western blot technique DigiWest. A DigiWest-compatible FFPE extraction protocol was developed, and a total of 634 antibodies were tested in an initial set of 16 FFPE samples covering tumors from different origins. Of the 303 detected antibodies, 102 yielded significant correlation of signals in 25 pairs of fresh frozen and FFPE primary tumor samples, including head and neck squamous cell carcinomas (HNSC), lung squamous cell carcinomas (LUSC), lung adenocarcinomas (LUAD), colorectal adenocarcinomas (COAD), and pancreatic adenocarcinomas (PAAD). For this signature of 102 analytes (covering 88 total proteins and 14 phosphoproteins), a support vector machine (SVM) algorithm was developed. This allowed for the classification of the tissue of origin for all five tumor types studied here with high overall accuracies in both fresh frozen (90.4%) and FFPE (77.6%) samples. In addition, the SVM classifier reached an overall accuracy of 88% in an independent validation cohort of 25 FFPE tumor samples. Our results indicate that DigiWest-based protein profiling represents a valuable method for cancer classification, yielding conclusive and decisive data not only from fresh frozen specimens but also FFPE samples, thus making this approach attractive for routine clinical applications.

Precision medicine's goal of achieving a better response rate by avoiding ineffective therapies has sparked new approaches, including the testing of patient-derived 3d (PD3Ds) tumor cultures for modeling individual patient response, and the use of various molecular pathology techniques for advanced tumor profiling. An intrinsic limitation of well-established genomics methodologies is, however, that they cannot directly assess cell-signaling activity within the tumor cells defined by the phosphorylation status of cellular signaling pathway networks. Here we present the development of a robust protein profiling strategy utilizing the DigiWest immuno-assay platform, to obtain data on the activation status of key cellular signaling networks implicated in cancer, and on proteins targeted by FDA-approved drugs. This includes targeted cancer therapies for e.g. EGFR, HER2, PI3K, mTOR, ALK and AKT. Accordingly, we compiled a list of relevant pathway nodes and their phosphorylation sites that yield activity information on RAS/RAF/ERK, PI3K/AKT and mTOR signaling pathways. Based on this, we validated 242 total and phospho antibodies in a pre-set oncoproteomic DigiWest panel that yields information on the activity of all major signaling pathways from the receptor level down to transcription factors, apoptosis and proliferation. This oncoproteomic panel can be utilized for elucidating drug response in pre-clinical cell models, in PD3D organoid models or in clinical tumor samples. Exemplary, we show differential activation states in cancer cell lines representing the most prevalent cancer types and the impact of pathway activation in different models. Finally, we tested this panel in PD3D organoids that were subjected to screening against common targeted therapies. While the initial results are promising, further work on evaluating how such an oncoproteomic panel profiling might contribute to the rationale for personalized therapy decisions is required. Disclosure Statement: NMI TT Pharmaservices offers DigiWest protein profiling as a service. CELLphenomics offers PD3D cell cultures and drug testing as a service. Citation Format: Gerrit Erdmann, Przemylaw Dudys, Anja Arndt, Christian Regenbrecht, Markus Templin, Christoph Sachse. Beyond genomics - A precision oncology approach utilizing multiplex protein profiling for tumor and tumor organoid samples [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 810.

Physiologically relevant in vitro tumor models are crucial in any research setting from drug development to individualized treatment predictions. Patient-derived 3D cell culture models (PD3D®) are validated cancer models which recapitulate the biology of the donor tissue from genotype to phenotype. A multitude of PD3D® replicates can be generated and these are suitable for high-throughput-screening. However, the influence of media conditions on specific drug sensitivity profiles widely remains elusive and thus the predictive value of screenings is obscured. Here we have investigated pathway activities in dependence of different media compositions and mapped out differences in response to targeted therapies and distinct growth media. We assessed the response of PD3D® models derived from colorectal carcinoma (CRC) patients towards two targeted drugs aiming at different levels of the EGFR/Ras/Raf/Mek/Erk axis in defined, serum-free culture media. One media composition contained growth factors in concentration prevalently used in published cell culture protocols, while the other composition is geared to better match physiological levels of relevant supplements. We observed a significant impact of media conditions on sensitivity towards targeted cancer drugs. However, these differences were as individual as the models and the sensitivity against the treatment. To uncover the underlying mechanisms in the observed discrepancy we used DigiWest - a targeted proteomics technology - to assess pathways activity in the different models. We applied a panel of over 250 (phospho-)antibodies against proteins in the MAPK/ERK/RAS, PI3K/AKT and mTOR pathways as well as cell cycle and proliferation. Using functional pathway analysis, we mapped out the difference in response to targeted therapies and distinct growth media. In conclusion, media composition has great impact on the response to targeted drugs in vitro and therefore needs to be harmonized to ensure relevant results. Combination of PD3D® models and DigiWest functional pathway analysis represents a powerful tool to identify the molecular mechanisms underlying differential drug response. Further development of this collaborative approach will lead to a better understanding of the drug resistance or sensitivity and potentially even identification of signatures related to them. Citation Format: Jürgen Loskutov, Gerrit Erdmann, Anja Arndt, Quirin Graf Adelmann, Przemyslaw Dudys, Marie Flechner, Madeleine Nadolny, Katja Osman, Ulrike Pfohl, Christoph Reinhard, Markus Templin, Katja Uhlig, Lena Wedeken, Christian Regenbrecht. Let’s get physiological! Impact of media conditions on drug response to targeted therapies in CRC organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 187.