Abstract PR010: Characterizing N-glycan profiles of DCIS progression using tissue imaging MALDI mass spectrometry

Abstract

Identifying distinct biomarkers of DCIS and determining the invasive potential of DCIS are of great clinical importance. N-linked glycans present on the cell surface of DCIS lesions and surrounding stroma are potential biomarkers of disease and tissue, but remain largely unexplored. An N-glycan targeted imaging mass spectrometry (IMS) approach was initiated to identify N-glycans associated with DCIS and progression in clinical FFPE tissues. A cohort of pathologist annotated DCIS and IDC tissues with a range of disease severity as well as the RAHBT TMA DCIS cohort were evaluated. The RAHBT cohort contains primary DCIS lesions with known long-term outcomes and serves as a good sample set to identify early markers indicating progressive potential, while the DCIS and IDC large tissue biopsies provide information on distinct glycan profiles when DCIS has progressed to IDC. Initially, tissue samples containing DCIS alone or both DCIS and IDC were processed for N-glycan MALDI-IMS analysis, the goal being to establish a consensus panel of each analyte and determine distinct profiles of DCIS when alone vs. when IDC was present. For N-glycans, a peak list of 54 N-glycans was selected for evaluation of each DCIS only tissue, as well as the mixed DCIS/IDC tissues. By highest intensity, the major DCIS-associated glycans were in the high-mannose and paucimannose structure categories. Additionally, a series of tri- and tetra-antennary multi-fucosylated glycans were also detected specifically in the DCIS lesions. In tissues containing both IDC and DCIS lesions, the high mannose glycans were also detected in the IDC regions, and GlcNAc-bisected glycans were seen elevated in these as compared to DCIS-only tissues. For samples in the RAHBT TMA cohort (progressors N=43; non-progressors N=78), the bisected N-glycans seen elevated with IDC were also seen to be significantly increased in the RAHBT samples that would eventually progress to IDC. Additional analyses to evaluate N-glycan isomer distributions for fucosylated and sialylated glycan species are also ongoing. The cumulative N-glycan data will be assessed with the extensive spatial genomic, transcriptomic and immunohistological data already generated for the same samples in the RAHBT cohort. This novel combination of multi-enzymatic digests with histopathology annotations represents an extensive multi-dimensional profile of DCIS and a novel tissue biomarker discovery approach. Citation Format: Elizabeth N. Wallace, Grace Grimsley, Siri H. Strand, Robert Michael Angelo, Graham Colditz, E. Shelley Hwang, Robert West, Jeffrey R. Marks, Peggi M. Angel, Richard R. Drake. Characterizing N-glycan profiles of DCIS progression using tissue imaging MALDI mass spectrometry [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr PR010.

Non-targeted isomer-sensitive N-glycome analysis reveals new layers of organ-specific diversity in mice

“…While the CFG's analytical activities have come to an end, their seminal glycan profiling datasets still represent a vital and fundamental resource to the field of mammalian glycobiology. More recently, new glyco-analytical developments and the paucity in modern, comprehensive N-glycome datasets prompted several studies aiming at the systemic profiling of tissue-specific N-glycosylation patterns using MALDI-TOF MS [19][20][21][22][23][24] . Despite the remarkable compositional variations that were detected by such ("single-stage") MS approaches, these methods intrinsically fall short in capturing the unique level of N-glycan structure micro-heterogeneity.…”

“…We used PGC-LC to chromatographically separate closely related N-glycan structure isomers and an Orbitrap mass-analyzer for high resolution MS/MS data-acquisition. Conventionally, the analysis of such LC-MS(/MS)-Nglycome datasets is based on the targeted, selective extraction for known or anticipated glycancompositions and -masses 20,22,25 . Subsequent glycan-structure assignment incorporates additional information on pre-established, relative chromatographic retention times 9,26,27 , complementary MS/MS data and expert knowledge on glycan biosynthetic pathways.…”