Abstract PD4-04: PD4-04 A quantitative spatial analysis of microenvironmental biomarkers for breast cancer outcome
Authors: Nizzero Sara , Soni Maria Pelaez , Xu Yitian , Zhang Licheng , Zheng Junjun , Menegaz Brian A. , Jordan Lee B , Purdie Colin A , Quinlan Philip R , Nagi Chandandeep , Sepulveda Karla A , Oertle Philipp , Appenzeller Tobias A , Loparic Marko , Wang Zhihui , Chen Shu-Hsia , Cristini Vittorio , Plodinec Marija , Thompson Alastair M. 
Published: March 2023

Abstract
Background: Understanding breast cancer progression and the relationships among biomarkers in the tumor and tumor microenvironment could go beyond established prognostic markers of breast cancer. Poor response and recurrence may indeed be a consequence of the highly heterogeneous spatial distribution of biomarkers within cancers, and/or the synergistic and antagonistic relationship between co-localized biomarkers. Recently, mechanical and physical microenvironmental signatures have emerged as relevant in determining cancer aggressiveness and invasiveness. This suggests that physical structures in the tumor tissue may drive favorable immune-tumor-stroma cell patterns. However, current assessment of tumor biopsies is limited in the ability to quantify and spatially resolve several different subtypes of cells and biomarkers within tumors. Methods: We developed a 60-marker imaging mass cytometry panel to resolve high-plex spatial patterns of cells, signaling, and microenvironmental biomarkers within tumor tissues. Our panel includes 16 tumor markers, 20 immune markers and 24 microenvironmental markers. We built an innovative computational tool to identify recurring spatial patterns of these markers within the tumor microenvironment, and define the spatial scale of heterogeneity of such patterns. We correlated the results from this spatial analysis to prospectively collected long term clinical outcome variables (e.g. 10 year survival, local and distant recurrence) in primary breast cancers sampled at baseline. Our patient cohort comprised 287 samples of patients treated with surgery and a radiation, chemotherapy, endocrine therapy, or combinations of these post-surgery. Of these, 174 were from patients alive 5 years post diagnosis, and 113 from patients lost to breast cancer deaths. Living patients were evaluated for recurrence, and of these 94% were disease free at the end of the study, while 4% had local recurrence and 2% distant metastasis. Of the dead patients, 59% had local recurrence while 27% had distant metastasis. Results: We investigated first all patients independent of tumor subtype; in this analysis the presence of both endothelial (CD31+) or HLADR+ cells were consistently associated with long term survival. We further investigated the distribution of all 60 markers in Lum A, Lum B, Lum like, Her2+ and triple negative subtypes. Among the results we confirmed the prognostic role of known biomarkers such as p53+ as a biomarker for poor survival in Luminal B. We also identified complex microenvironmental patterns associated with outcome. For example, the presence of PD1+ cells in collagen rich environments were generally associated with long terms survival in Luminal B patients. With our spatial analysis tool we further investigated intra-patient and inter-patient spatial distribution and classified clusters predictive of outcome beyond heterogeneity. We found that beyond the mere positivity of each marker, the spatial distribution and co-localization of different immune, tumor and mechanical markers determines long term outcome in different subtypes. Specifically, we investigated the role of vimentin within different microenvironments and tumor subtypes. We identified the co-localization of hCa9 and vimentin as strongly associated with poor 5 year outcome, independent of tumor subtype. Moreover, in Luminal A patients while the presence of clusters exclusively positive for vimentin was associated with poor survival, vimentin co-expression with β actin and co-localization of XBP1+ cancer cells and immune cells in a collagen matrix was associated with longer survival. Conclusion: These results suggest the importance of mechanics and physics in determining spatial distribution of tumor-promoting and tumor-inhibiting immune cells, offering new avenues of physics-based therapeutic targets.
Citation Format: Sara Nizzero, Maria Pelaez Soni, Yitian Xu, Licheng Zhang, Junjun Zheng, Brian A. Menegaz, Lee B Jordan, Colin A Purdie, Philip R Quinlan, Chandandeep Nagi, Karla A Sepulveda, Philipp Oertle, Tobias A Appenzeller, Marko Loparic, Zhihui Wang, Shu-Hsia Chen, Vittorio Cristini, Marija Plodinec, Alastair M. Thompson. PD4-04 A quantitative spatial analysis of microenvironmental biomarkers for breast cancer outcome [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 PD4-04.