Mass Spectrometry Imaging Reveals Neutrophil Defensins as Additional Biomarkers for Anti-PD-(L)1 Immunotherapy Response in NSCLC Patients

Abstract

(1) Background: Therapeutic blocking of the interaction between programmed death-1 (PD-1) with its ligand PD-L1, an immune checkpoint, is a promising approach to restore the antitumor immune response. Improved clinical outcomes have been shown in different human cancers, including non-small cell lung cancer (NSCLC). Unfortunately, still a high number of NSCLC patients are treated with immunotherapy without obtaining any clinical benefit, due to the limitations of PD-L1 protein expression as the currently sole predictive biomarker for clinical use; (2) Methods: In this study, we applied mass spectrometry imaging (MSI) to discover new protein biomarkers, and to assess the possible correlation between candidate biomarkers and a positive immunotherapy response by matrix-assisted laser desorption/ionization (MALDI) MSI in 25 formalin-fixed paraffin-embedded (FFPE) pretreatment tumor biopsies (Biobank@UZA); (3) Results: Using MALDI MSI, we revealed that the addition of neutrophil defensin 1, 2 and 3 as pretreatment biomarkers may more accurately predict the outcome of immunotherapy treatment in NSCLC. These results were verified and confirmed with immunohistochemical analyses. In addition, we provide in-vitro evidence of the immune stimulatory effect of neutrophil defensins towards cancer cells; and (4) Conclusions: With proteomic approaches, we have discovered neutrophil defensins as additional prospective biomarkers for an anti-PD-(L)1 immunotherapy response. Thereby, we also demonstrated that the neutrophil defensins contribute in the activation of the immune response towards cancer cells, which could provide a new lead towards an anticancer therapy.

Reproducible Lipid Alterations in Patient-Derived Breast Cancer Xenograft FFPE Tissue Identified with MALDI MSI for Pre-Clinical and Clinical Application

“…Matrix-assisted laser desorption and ionization (MALDI) MSI can be used to determine both low-mass molecules and proteins in FFPE tissue [10,11]. However, analysis of lipids has proven difficult in this type of tissue due to their depletion during initial processing with organic solvents, which is further compounded by the remaining lipids being implicated in cross-binding with other structures in the tissue [12][13][14].…”

Mass Spectrometry (Imaging) for Detection and Identification of Cyclic AMPs: Focus on Human Neutrophil Peptides (HNPs)

“…Using this approach, three peptides m/z 3369.5, m/z 3440.6 and m/z 3484.6 have found to be discriminative between a responding and a nonresponding NSCLC patient towards anti-PD-(L)1 immunotherapy, shown in Figure 1 [21].…”

“…Based upon this, the corresponding amino acid sequence can be determined and the precursor ion can be identified [17]. Even if the AMPs have a cyclic nature, due to their cysteine bond formation, mass spectrometry can be used as an identification tool, although a specific approach is needed [18][19][20][21]. The added benefit of using proteomic approaches to study AMPs is the fact that the majority of AMPs are post-or co-translational proteolytically processed from their large polyprotein precursor, resulting in the release of the active AMP.…”

Implementation of MALDI Mass Spectrometry Imaging in Cancer Proteomics Research: Applications and Challenges

“…The correct identification of the tumor origin is an important aspect in the personalized medicine field [ 38 ]. Recently, our research group reported the use of MALDI mass spectrometry imaging to measure therapeutic response in NSCLC patients who received immunotherapy [ 39 ]. MSI analysis was performed on pre-treatment FFPE biopsies and differential analysis revealed neutrophil defensin 1, neutrophil defensin 2 and neutrophil defensin 3 as predictive biomarkers for the response to immune-checkpoint-based immunotherapy in NSCLC patients.…”

“…These outcomes were verified with immunohistochemical analyses specific for these neutrophil defensins and after pathological scoring of neutrophil defensin expression on different cell types, statistical analysis exposed a significant association between neutrophil defensin expression and a positive immunotherapy response. This is of major importance for immunotherapy decision, for a better quality of life for the NSCLC patient, to avoid treatment of patients who are not likely to respond and to reduce unnecessary treatment costs [ 39 ].…”