Glycoproteomics as a powerful liquid biopsy-based screening tool for non-small cell lung cancer.

Abstract

e21148 Background: Protein glycosylation is the most abundant and complex form of post-translational protein modification. Glycosylation profoundly affects protein structure, conformation, and function. The elucidation of the potential role of differential protein glycosylation as biomarkers has so far been limited by the technical complexity of generating and interpreting this information. We have recently established a novel, powerful platform that combines liquid chromatography/mass spectrometry with a proprietary artificial-intelligence-based data processing engine that allows, for the first time, highly scalable interrogation of the glycoproteome. Methods: Using this platform, we interrogated 694 glycopeptide (GP) and non-glycosylated peptide transitions derived from 74 serum proteins in pre-treatment peripheral blood samples from a cohort of 316 individuals with non-small-cell lung cancer (NSCLC) (128 females, 187 males, 1 with unknown sex, median age 66 years, age range 31-89 years, stage 0-4 N’s: 1 / 99 / 80 / 84 / 49, 3 missing) and a comparison cohort of 194 healthy control samples (102 females, 92 males, median age 52 years, age range 30-63 years). Age- and sex-adjusted differential expression analysis for 596 normalized biomarkers were performed to evaluate statistically significant differential abundances using an FDR-adjusted q-value of 0.05 as a cutoff. Repeated five-fold cross-validated LASSO-regularized logistic regression was performed to create a multivariable classifier that predicts whether a serum sample belongs to the healthy or NSCLC cohort. Results: We identified 432 biomarkers with significant abundance differences at FDR ≤ 0.05 between samples with NSCLC and healthy controls. Using 70% of the complete cohort (balanced by case/control membership, NSCLC stage, sex, and age quartile) as a training set, we selected a total of 375 glycopeptide and non-glycosylated peptide biomarker features that remained differentially expressed at FDR-adjusted q-value ≤ 0.05 as input into a LASSO-regularized multivariable classifier. This resulting in a 19-biomarker model exhibiting an accuracy of 94.8% (96.9% sensitivity, 91.2% specificity) and AUC of 0.989. This classifier was validated in an independent test set comprising the remaining 30% of subjects, yielding an accuracy of 94.5% (95.5% sensitivity, 93.0% specificity) and AUC of 0.975. Sensitivity in the test set was 100% / 96% / 99% / 96% / 94% / 10%, in stages 0-4 and missing, respectively. Conclusions: Our results indicate that glycoproteomic biomarkers can be leveraged as a strong liquid biopsy-based screening tool for patients at high risk of NSCLC, as an alternative to imaging modalities.

Medical Relevance, State-of-the-Art and Perspectives of “Sweet Metacode” in Liquid Biopsy Approaches

“…Another important aspect of liquid biopsy is to provide information about the stage of the disease and a prognosis for the patient's survival, possibly a design for a personalised therapy plan [21]. Herein we present the current state-of-the-art for glycoprofiling of novel glycoproteomic biomarkers, mainly focusing on sialic acids (N-acetylneuraminic acid-Neu5Ac-and N-glycolylneuraminic acid-Neu5Gc-respectively) in early cancer diagnostics as a possible multi-omics liquid biopsy approach for the analysis of prevalence and also for more aggressive cancer types, such as non-small-cell lung cancer [22,23]. The importance of these methods is further illustrated by the fact that the global incidence and mortality for different cancer diseases is predicted to grow in the upcoming years.…”

Negative Charge-Carrying Glycans Attached to Exosomes as Novel Liquid Biopsy Marker

“…It could provide diagnostic, prognostic and/or therapeutic information and it could be used for monitoring metastases, identifying changes in genetic or epigenetic codes, determining optimal treatments for individual patients and assessing treatment efficacy [ 17 ]. Aberrant glycoform profiling may also be used as a novel liquid biopsy approach in combination with tissue-specific glycoproteins (as in the case of the free prostate-specific antigen (free PSA for prostate cancer) or extracellular vesicles, such as exosomes [ 18 ].…”