Identifying Peptide Spectra In Wilms' Tumor That Associate With Adverse Events

Abstract

Introduction: MALDI-TOF-IMS (matrix assisted laser desorption ionization time of flight imaging mass spectrometry) is a recently validated method for proteomic analysis of formalin-fixed, paraffin-embedded (FFPE) archived tissue specimens. We hypothesized that high-throughput proteomic analysis of Wilms' Tumor (WT) would yield biomarkers predictive of advanced disease stage and disease relapse. Methods: Two tissue microarrays (TMA) were assembled that contain 156 punches from FFPE WT tissue blocks stored in our embryonal tumor tissue repository. Multiple histologically selected tissue punches were taken from each block and included 30 WT, one renal rhabdomyosarcoma, five different gestational age human fetal kidneys, one hepatic, one pulmonary, and one nodal WT metastases. Coordinates of specimens within the TMA were uploaded to an acoustic robotic microspotter for deposition of trypsin and matrix formation. MALDI-TOF-IMS was performed, segregating peptide spectra based on differential mass-to-charge (m/z) ratios. Post-acquisition processing of this information enabled display of these m/z peaks as an ion-density map, where the relative abundance of the selected ion across the tissue is displayed on a color-intensity scale at each coordinate location, or pixel. Peptide spectra were compared across the two TMAs with respect to histology, similarity to expression patterns in fetal kidneys, stage of disease, disease relapse, and vital status. Results: Unique peptide spectral patterns were identified that corresponded with metastasis, treatment effect, and tumor necrosis. For example, increased expression of peptides with m/z of 1515.6, 1790.6, and 2215.5 corresponded with tumor-specific histologic regions of four separate pulmonary metastases in the same patient. In contrast, within these same pulmonary metastases, and in an additional pulmonary metastasis from a different patient, peptides with m/z of 1032.8, 1326.1, and 2190.0 showed decreased expression relative to other samples on the TMA. High peptide expression at m/z of 1529.3 and 1274.8 correlated with tumor necrosis and histologic post-treatment effect. Conclusions: MALDI-TOF-IMS has the ability to reveal unique peptide spectral patterns that associate with adverse events occurring in WT. More samples are necessary to draw such conclusions from the current study. Sequencing of these individual peptides will enable the search for biomarkers of disease that can clarify our understanding of WT pathogenesis, disease relapse, and adverse events. Identifying protein markers of epithelial and stromal differentiation may give insight into tumor differentiation.