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Ductal carcinoma in situ (DCIS) is characterized by inter-tumor heterogeneity that poses a therapeutic challenge due to its unpredictable recurrence and progression to invasive breast cancer (IBC). Recent publications have implicated the crucial role of the tumor microenvironment particularly stromal differences between DCIS patients who progress to IBC (progressors) and those that do not (non-progressors). However, spatial regulation of the collagen proteome has yet to be investigated in the context of disease progression in DCIS. In this study, we hypothesized that the collagen proteome was significantly altered between DCIS and IBC and that differentiating collagen peptide signatures could be related to clinical outcomes. Our initial studies investigated collagen peptide signatures in lumpectomies (n=13) annotated as DCIS, DCIS and invasive ductal carcinoma (IDC), or IDC only. We leveraged our previously published method for spatial imaging of collagen proteomics on tissue to report collagen types and collagen post-translational modifications including 40 other extracellular matrix (ECM) proteins involved in the regulation of collagen fibers. Over 1000 peaks were found to be linked to annotated pathologies or adjacent regions. Initial comparison of DCIS to IDC lesions demonstrated 63 differentially expressed peaks between these regions by unpaired, two-tailed t-test (p< 0.001). Image segmentation of the 315,541 pixels demonstrated 16 high-level hierarchical groups designating unique spatially localized ECM proteomic groups. Notably, these groups overlaid with histopathological features and pathological annotations. Next, we investigated collagen peptide signatures in a subset of DCIS samples from the Resource of Archival Breast Tissue (RAHBT) (n=37). Samples were histologically diverse within the tissue microarrays, with cribriform, micropapillary, papillary, solid, and comedo necrosis architectural patterns. In our preliminary analysis, we found two peptide peaks that could distinguish the solid subtype (n=22) from comedo necrosis (n=4) and one peak that could discriminate between the cribriform (n=8) and solid subtype (n=22) by area under the receiver operating curve (AUROC)≥0.75 and Wilson/Brown t-test (p< 0.05). Evaluated per clinical outcome, four ECM peptides showed significantly different peak intensities in progressors with IBC recurrence (n=7) compared to non-progressors (n=26) (AUROC≥0.75; Wilson/Brown t-test p< 0.05). One peptide had significantly different peak intensities between progressors with contralateral IBC recurrence and DCIS recurrence (n=5) and non-progressors (n=26) (AUROC≥0.75; Wilson/Brown t-test p< 0.05). Overall, the data suggest that unique collagen signatures in DCIS could be useful for understanding recurrence and progression to IBC. Further investigation of the spatial distribution of the collagen proteome within DCIS pathologies and relative to clinical outcome is warranted. Citation Format: Taylor S. Hulahan, Elizabeth N. Wallace, Siri H. Strand, Graham A. Colditz, E Shelley Hwang, Robert West, Laura Spruill, Jeffrey Marks, Richard R. Drake, Peggi M. Angel. Unique Collagen Peptide Signatures between Ductal Carcinoma in Situ and Invasive Breast Cancer by Mass Spectrometry Tissue Imaging [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-21-03.

Ductal carcinoma in situ (DCIS) is characterized by inter-tumor heterogeneity that poses a therapeutic challenge due to its unpredictable recurrence and progression to invasive breast cancer (IBC). In recent publications, collagen stromal differences have been reported between patients that progressed to IBC (progressors) and those that did not (non-progressors). However, details on the role spatial regulation of the collagen proteome might play in this progression have yet to be studied. Here, we investigated the pathological distribution of collagen post-translational modifications in a cohort of patients classified as progressors with ipsilateral IBC recurrence compared to non-progressors. Previously published methods for collagen proteomics by targeted tissue mass spectrometry imaging were used. The method reports collagen types and post-translational modifications within the collagen triple-helical region as well as approximately 40 other extracellular matrix (ECM) proteins involved in the regulation of collagen fibers. Initial studies investigated collagen variation in lumpectomies (n=7) with DCIS, DCIS plus invasive ductal carcinoma (IDC) or IDC only. Over 590 peptides were found to be linked to annotated pathologies. A preliminary comparison of DCIS (n=2392 spectra) to IBC (n=4696 spectra) using area under the receiver operating curve (AUROC) ≥0.85 demonstrated that 47 peptides could individually discriminate between DCIS and IBC in this limited cohort. Image segmentation of the 405,652 pixels demonstrated 11 high-level hierarchical groups designating unique spatially localized ECM proteomic groups; these groups overlaid with histopathological features and pathological annotations. A total of 87 samples from the Resource of Archival Breast Tissue (RAHBT) matched with clinical characteristics were also investigated. Cores were histologically diverse within the tissue microarrays, with cribriform, micropapillary, papillary, solid, and comedo necrosis architectural patterns. Initial results suggest certain peptides may differentiate between non-progressors and progressors with ipsilateral IBC recurrence. Our current work focuses on correlating collagen signatures to mixed pathologies and the cellular content of cores. Further investigation of the collagen proteome is warranted. Overall, the data suggest unique collagen signatures in DCIS that could be useful for understanding recurrence and progression to IBC. Citation Format: Taylor S. Hulahan, Elizabeth N. Wallace, Siri H. Strand, Robert Michael Angelo, Graham Colditz, Eun-Sil Shelley Hwang, Robert West, Laura Spruill, Jeffrey R. Marks, Richard R. Drake, Peggi M. Angel. Discrete regulation of the collagen proteome among pathological features in DCIS and invasive breast cancer by mass spectrometry tissue imaging [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 B019.