Wang, Xue

Abstract Background: Growing evidence supports the critical role of preexisting immune response in triple negative breast cancer (TNBC). However, there are limitations with current evaluation approaches: inability to functionally assess the type of immune infiltration with traditional pathologic evaluation and loss of spatial distribution in conventional high-plex gene or protein expression analyses from the whole tumor section. Here, we report the initial analysis of immune protein expression as a function of spatial distribution and clinical outcomes in TNBC samples. Methods: NanoString GeoMxTM Digital Spatial Profiling (DSP) was used to quantify 39 immune-related proteins in stromal and tumor segments from 44 TNBC samples from the FinXX trial. Samples were matched for patient characteristics, treatment arm (capecitabine vs. 5-fluorouracil), and outcome based on recurrence-free survival (RFS) with 22 samples from patients who recurred and 22 samples from patients with durable RFS. Regions of interest (ROIs) were selected based on expression of cytokeratin (tumor), CD45 (leukocytes), or CD68 (macrophages). Each ROI was segmented into tumor (pancytokeratin-positive area) and stroma (pancytokeratin-negative/nuclear SYTO13-positive area). The general linear model was used for statistical analysis of differential expression with RFS as a categorical variable (recur yes or no). Results: A total of 950 tumor and stroma segments were included in this initial analysis. In both tumor and stroma segments, over-expression of T cell activation markers (CD137, GITR) was associated with better outcome, whereas T cell markers (CD3, CD4, CD8) were not significantly associated with outcome. In tumor segments alone, improved outcome was significantly associated with increased protein expression [> 2-fold change (FC) at p<0.001] of CD56, PD-L2, HLA-DR, CD137, GITR, and CD40. In CD45-enriched stroma, improved outcome was associated with elevated expression (FC>2.0, p<0.001) of PD-L2; whereas durable RFS was associated with elevated PD-L2 and IDO1 expression in CD68-enriched stroma. In contrast, macrophage/dendritic cell markers CD68 and CD11c were not associated with outcome. In tumor cells adjacent to CD45-enriched stroma, durable RFS was associated with increased abundance (FC>2.0, p<0.001) of PD-L2, CD56, CD27, GITR, CD20, HLA-DR, and IDO1. Similarly, in tumor segment associated with CD68-enriched stroma, proteins associated with RFS included PD-L2, HLA-DR, CD56, GITR, and CD137. Among 39 immune function proteins, only elevated CTLA4 expression in CD68-enriched stroma was associated with recurrence (FC=0.55, p<0.001), whereas tumor-segment CTLA4 was associated with RFS. Conclusions: Using an in-depth analysis to precisely quantify the abundance of multiple immune function proteins in a spatially defined manner, we observed that PD-L2, IDO1, and T cell activation markers were robustly associated with durable RFS in both tumor and stromal segments. In contrast, MHC components (HLA-DR, beta-2-microglobulin), B cell markers (CD20), and NK cell markers (CD56) were strongly associated with favorable outcome in tumor but not in stromal segments. Our study highlights the conclusion that the immune landscape of TNBC is far too complex to be encompassed by any single molecular marker, and more detailed analyses of the DSP data reported here are ongoing with a view towards using quantitative multiplex analyses to refine our understanding of how therapeutic outcome is influenced by interactions among immune cells and between immune and tumor cells. Acknowledgements: Supported by the Breast Cancer Research Foundation (BCRF18-161), Bankhead Coley (6BC05 Florida Department of Health), 26.2 with Donna Foundation, US National Cancer Institute (CA15083), and the Canadian Cancer Center Citation Format: Saranya Chumsri, Douglas Hinerfeld, Jennifer M. Kachergus, Yaohua Ma, Heather A Brauer, Sarah Warren, Xue Wang, Torsten O. Nielsen, Karama Asleh, Heikki Joensuu, Edith A. Perez, E. A. Thompson. Digital spatial mapping of the immune landscape of triple negative breast cancer reveals novel features of immune-tumor cell interaction [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD5-06.

Abstract OBJECTIVE Our goal is to identify new pathways perturbed in and which may lead to GRS. METHODS We collected brain transcriptome levels from healthy controls (n=5), patients with GRS (n=9), glioma without seizures (non-GRS, n=8), and idiopathic temporal lobe epilepsy (iTLE, n=7) using Illumina-based poly-A capture high-throughput RNA sequencing approach. Differential gene expression analysis was conducted to ascertain expression differences between the four groups. We identified molecular pathways enriched for differentially expressed genes (DEG) using Ingenuity Pathway Analysis. DEGs were defined as those with log2 fold change ≥ 2 and q-value < 0.01 when compared to GRS. RESULTS Principal component analysis revealed that diagnostic grouping was the main determinant of the transcriptome variance. We identified 212 DEGs in the GRS vs. non-GRS analysis. Among these, 108 genes were up-regulated and 104 genes were down-regulated. Among the genes that were down‐regulated, there was significant enrichment for those involved in acute phase response signaling and immune cell trafficking (e.g. H19, BMP5, CXCL8). Compared to iTLE samples, there were 1015 DEGs in the GRS samples. Among these 877 genes were up-regulated and 138 genes were down-regulated. There was significant enrichment for genes involved in cell-to-cell signaling among those that were either uniquely up‐regulated (e.g. NRK, RGS10, SLC4A4, and SELPLG) or down‐regulated in the GRS samples (e.g. SLC9A3 –Na+/H+ exchanger; SLC5A5-sodium-glucose co-transporter family). Further, two of the most down-regulated genes in samples with GRS, SCLC17A6- vesicular glutamate transporter 2 and FOSB, have previously been shown to be involved in glutamatergic transmission. CONCLUSIONS Our findings suggest that inflammation-related processes and alterations in cell-to cell signaling may underlie the pathogenesis of GRS. Our results also provide support for perturbations in glutamatergic transmission in GRS. Our findings require replication in larger human cohorts and functional experiments to explore the function of the genes perturbed in GRS.

Abstract Introduction Comparative transcriptome analyses in Alzheimer's disease (AD) and other neurodegenerative proteinopathies can uncover both shared and distinct disease pathways. Methods We analyzed 940 brain transcriptomes including patients with AD, progressive supranuclear palsy (PSP; a primary tauopathy), and control subjects. Results We identified transcriptional coexpression networks implicated in myelination, which were lower in PSP temporal cortex (TCX) compared with AD. Some of these associations were retained even after adjustments for brain cell population changes. These TCX myelination network structures were preserved in cerebellum but they were not differentially expressed in cerebellum between AD and PSP. Myelination networks were downregulated in both AD and PSP, when compared with control TCX samples. Discussion Downregulation of myelination networks may underlie both PSP and AD pathophysiology, but may be more pronounced in PSP. These data also highlight conservation of transcriptional networks across brain regions and the influence of cell type changes on these networks.