Abstract 2032: Comprehensive spatial transcriptomic analysis of an integrated, diverse cohort reveals distinct molecular topographic patterns in triple negative breast cancer
Authors: Bassiouni Rania , Idowu Michael , Gibbs Lee D. , Grizzard Pamela J. , Webb Michelle G. , Noriega Ashley , Robila Valentina , Craig David W. , Carpten John D. 
Published: June 2022

Abstract
Background: Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer that disproportionately affects African-American and black (AAB) women, who have higher rates of incidence and mortality. To understand this disparity, we must apply advanced molecular profiling to racially diverse cohorts. We propose the use of spatial transcriptomics to map whole transcriptome data to several thousand coordinates within tissue sections, for comprehensive and unbiased characterization of TNBC tumors and their microenvironment.
Methods: We applied the Visium Spatial Gene Expression method (10x Genomics) to 28 fresh frozen TNBC tissue sections obtained from 14 patients (7 AAB and 7 Non-Hispanic White). Sections were stained with H&E, and regions of tumor and stroma were annotated by a pathologist. The assay yielded whole transcriptome data from over 36,000 spatially-defined features, averaging 1,380 features per section. Expression data from each sample was normalized and subject to dimensionality reduction and clustering analysis. All sections were also integrated into a single dataset, from which integrated clusters (IC) were defined. Several bioinformatics tools were used to annotate the data at the feature, cluster, and IC level. Join count statistical analysis was employed to quantify patterns of spatial aggregation or dispersion of ICs across samples.
Results: Clustering analysis revealed that all samples exhibited a great deal of heterogeneity; most contained several transcriptionally distinct regions of both tumor and stroma. Application of the ESTIMATE gene sets at the feature level accurately defined tumor and non-malignant regions when compared to histopathological annotation. Gene set enrichment analysis at the cluster level allowed further classification of biological processes within each sample. Following integration of all 28 samples, 9 ICs were defined from transcriptional data. Although all ICs were present in all samples, IC5 - characterized by a strong hypoxic signature - was overrepresented in AAB samples. ICs were then mapped back to individual samples and subject to join count statistics. This revealed strong spatial autocorrelation within each IC, as well as significant spatial pairings of ICs. The fibrotic clusters IC3 and IC7 were strongly contiguous in all samples (z-score &gt; 5), as were tumor clusters IC1 and IC4. Conversely, we found spatial exclusion between IC3 and tumor clusters IC5 and IC2.
Conclusion: Our study provides novel evidence of spatially related populations across diverse TNBC samples. Our findings suggest that, while TNBC tumors are highly heterogeneous, they exhibit elements of a common spatio-transcriptional architecture. Moreover, this provides a new framework in which to evaluate transcriptional differences between racial groups within a broader spatial context.
Citation Format: Rania Bassiouni, Michael Idowu, Lee D. Gibbs, Pamela J. Grizzard, Michelle G. Webb, Ashley Noriega, Valentina Robila, David W. Craig, John D. Carpten. Comprehensive spatial transcriptomic analysis of an integrated, diverse cohort reveals distinct molecular topographic patterns in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2032.