Proteomic analysis of human plasma and serum for identifying and validating disease-specific marker proteins and peptides has one major drawback besides its unique advantage as a readily available sample source for diagnostic assays. This disadvantage is represented by the predominance of several high- and middle-abundant proteins, which clearly hamper identification and quantification approaches of potential and validated protein and peptide biomarkers, which are often of very low abundance. During the last decades, a significant number of depletion and enrichment techniques evolved to address these two issues. We present here a cost-effective and easy-to-use strategy for protein depletion comprising a thermal precipitation protocol followed by a two-step liquid/liquid precipitation as well as using an immunoaffinity chromatography method for the specific enrichment and isolation of the low-abundance polypeptide N-terminal pro-B-type natriuretic peptide and its precursor proBNP clinically used as biomarkers for the detection of severe human heart failure and related diseases. The applicability of this approach is shown by SDS -CGE, SDS-PAGE, electrochemiluminescence immunoassay and nano-LC ESI-MS/MS. Our thermal precipitation protocol followed by a two-step liquid/liquid precipitation could also serve as a potential depletion technique for the characterization of other low-abundance peptides and proteins.

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The prevalence of early-stage non-small cell lung cancer (NSCLC) with curative treatment options is expected to increase with recent implementation of annual screening programs. Predictors and molecular drivers of disease relapse, especially the role of intra-tumoral immune dysfunction, remains unclear but critical for the refinement of therapeutic decisions. By leveraging a comprehensive individual portrait of each patient's immune system potential novel mechanisms associated with tumor relapse in early-stage NSCLC may be identified. We profiled 11 non-relapsed (at least 2 year FU) lung adenocarcinoma patients and 11 covariate-matched (gender, age, stage) relapsed patients, who underwent curative treatment in stage IA-IIIB disease. We used NeXT SummitTM for variant and CNA calling, gene expression quantification, neoantigen prediction, HLA profiling (typing, mutation, and loss of heterozygosity), T-cell receptor and tumor microenvironment (TME) profiling. Neoantigen peptide sequences were subjected to further filtering and clustering based on between-patient similarity scores, with the goal of identifying shared clusters of relapse-associated neoantigens in each possible pair of patients. Differential network analyses were applied to the TME composition estimates to investigate relapse-associated patterns of cellular co-occurrence and interaction. When considering neoantigens selected on the basis of similarity, we found that those belonging to non-relapsed patients had significantly lower HLA binding rank (17.8 points) compared to that of relapsed patients (P=0.02), indicating weaker binding for relapsed cases. Clustering of both the most similar and frequently shared neoantigens correlated with relapse (P < 0.002). In the TME, we observed differential immune cell co-occurrence associated with relapse status, such as Tregs are positively correlated with B and CD4 T cells only in relapsed patients (Pearson’s R=0.7 and 0.74, both P<0.02 vs. R=0.18 and 0.35, both P>0.2 in non-relapsed patients), indicating suppressive anti-tumor immunity. Relapsed patients did not share significant enrichment of mutations in any biological pathway. Surprisingly, mutation purity (less mutations than expected by chance) was observed in relapsed patients, suggesting selective killing and escape. In this pilot cohort, we used an integrated platform to broadly characterize both the tumor and immune system, enabling identification of relapse-associated neoantigens that may share universal features which enhance HLA binding. Relapses in early-stage LUAD patients were associated with neoantigens with lower immunogenicity and an immunosuppressive TME. These findings demonstrate that deeper profiling of shared neoantigen features has the potential to become an early biomarker of relapse, informing patient therapy selection and surveillance. Citation Format: Martina M. Sykora, Jason Pugh, Bailiang Li, Finn O. Mildner, Hubert Hackl, Arno Amann, Fabienne I. Nocera, Rachel M. Pyke, Lee McDaniel, Charles W. Abbott, Sean M. Boyle, Richard O. Chen, Dominik Wolf, Sieghart Sopper, Gabriele Gamerith. Immune infiltrate co-occurrence and neoantigen similarity are prognostic factors in early stage NSCLC [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 6668.