GeoMx Digital Spatial Profiling Research Articles

Abstract PR004: Characterizing the effects of neoadjuvant therapy in PDAC

Abstract Pancreatic ductal adenocarcinoma lethality can be attributed to a combination of rapid metastatic dissemination and intrinsic resistance to conventional therapies. Our prior studies using single cell RNA-seq in pancreatic circulating tumor cells (CTCs) revealed that these “seeds of metastasis” had a biphenotypic state of both epithelial and mesenchymal features. This suggested that CTCs are highly plastic cells that exist in an intermediate state along epithelial (E) to mesenchymal (M) transition (EMT). Analysis of CTC markers in primary PDAC tumors revealed that this subpopulation of cancer cells was concentrated at the interface of tumor cells and stromal cancer associated fibroblasts (CAFs). To evaluate the contribution of CAFs in PDAC EMT plasticity, we utilized patient derived PDAC-CAF co-culture preclinical models demonstrating that PDAC EMT heterogeneity was modulated by the density of CAFs and partially driven by TGF-b secretion. Moreover, analysis of patient derived PDAC tumor spheres treated with FOLFIRINOX (FFX) demonstrated selection or induction of EMT changes that were also found in patient tumors that were resected after neoadjuvant FFX. Altogether, our collective work along with others demonstrates the importance of EMT plasticity in PDAC cell metastatic propensity and the ability to resist cytotoxic chemotherapy. A multi-institutional randomized Phase II trial supported by SU2C-Lustgarten Foundation evaluating the role of the TGF-b modulating activities of losartan on locally advanced PDAC response to chemotherapy and suppression of metastatic dissemination has neared completion. This 3 arm (n=40 per arm) trial of neoadjuvant FFX, FFX + losartan, or FFX+ losartan + nivolumab (anti-PD1). Here, we have performed EMT RNA in situ hybridization in all post-treatment resection specimens to determine if there is a difference in EMT proportions between the different arms of the study. As an orthogonal unbiased approach, we have utilized the NanoString GeoMx Digital Spatial Profiler whole transcriptome assay (18,000+ protein coding genes) on each of the resection specimens with analysis of the tumor cells, CAFs, and immune cell separately for multiple regions of interest in these specimens. This provides deeper molecular insight of the PDAC tumor cells that resisted neoadjuvant therapy, the changes in the surrounding CAFs, and the modulation of immune infiltrates that might differ between each arm of the study. We anticipate the results of the trial will be completed before the conference and initial correlative analysis of spatial transcriptomics and RNA-ISH will be presented. Citation Format: Amaya Pankaj, Michael J. Raabe, Bidish Patel, Evan R. Lang, Joshua Kocher, Katherine H. Xu, Linda T. Nieman, Alec C. Kimmelman, David P. Ryan, Theodore S. Hong, William L. Hwang, Martin Aryee, David T. Ting. Characterizing the effects of neoadjuvant therapy in PDAC [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR004.

TMIC-55. IMMUNE DIGITAL-SPATIAL PROFILING AND CHARACTERIZATION OF GLIOMA MICROENVIRONMENT AFTER ADOPTIVE CELLULAR THERAPY

Abstract INTRODUCTION Our group has previously shown that Adoptive Cellular Therapy (ACT) is efficacious against CNS malignancies including medulloblastoma, brain stem glioma, and glioblastoma. In this study, we characterized the spatial distribution of immune cells within the tumor microenvironment after ACT and conducted regional genomic analysis in situ to obtain insights into the underlying cellular dynamics. This method enabled us to define specific differences in gene expression within the T cells in the treated versus untreated tumors including changes in gene expression of regulatory T cell (Treg) associated genes, such as Runx1 and TGF-β. We also found a significant increase in Batf3 expression associated with therapy. METHODS We performed in situ GeoMx Digital Spatial Profiling (DSP) and whole genome transcriptomics (RNA) of murine glioma KR-158B-luciferase tumors after adoptive cellular therapy. ACT was conducted in orthotopic KR158B tumor-bearing mice. Histological slides were sent to be stained for CD45, CD3, GFP (to mark hematopoietic stem cell-derived cells) and nuclei, and processed following GeoMx DSP workflow for RNA. Overall gene expression clustering profiles were conducted using a Principal Component Analysis (PCA), and relative gene expression differences were analyzed by unpaired t-tests. RESULTS/CONCLUSIONS We observed differential expression of a substantial number of genes in the ACT-treated group compared to control. Our findings also indicate that tumors treated with ACT cluster separately from control tumors in PCA analysis. Furthermore, we found downregulation of genes associated with the suppressive properties of regulatory T cells (Runx1 and TGF-β), and upregulation of dendritic cell characteristic genes (Batf3) and antigen presentation genes (H2-Ab1) in ACT-treated tumors vs control. These findings provide insights into the immune cell dynamics and specific genes that are differentially expressed within the tumor microenvironment. This could help improve current adoptive cellular therapies to treat brain tumors.

Abstract 15810: Cerebral Thrombus Transcriptome in Acute Ischemic Stroke

Establishment of mechanical thrombectomy as the standard of care provides a unique opportunity to refine stroke subtype mechanism by studying thrombus retrieved from stroke patients. Using traditional pathologic methods, researchers have described the form and structure of cerebral thrombi. These methods do not spatially resolve tissues and cell populations with functional segmentation. We applied next generation genomic tools to determine the spatially resolved transcriptome within cerebral thrombus. Methods: Cerebral thrombus was retrieved under sterile conditions. Tissue was formalin-fixed and paraffin embedded in standard fashion. Using the GeoMx Digital Spatial Profiling platform, morphology marker antibodies and WTA RNA probes coupled to photocleavable oligonucleotide tags were applied to the slide. After hybridization of the probes to tissue sections, 24 regions of interest (ROIs) were selected, and oligonucleotide tags were released from the discrete regions of the tissue via UV exposure. Released tags were quantitated using next generation sequencing and counts were mapped back to tissue location, yielding a spatially-resolved digital profile of analyte abundance. Results: Sequencing quality was inspected for sufficient saturation and data normalized to the third quartile to account for differences in cellularity. 18,676 genes were assayed across the 24 ROI. Of these, 4,590 genes were expressed in 10% of ROI and 1,127 genes were expressed in 50% of ROI. Conclusion: Significant transcriptional activity is seen within cerebral thrombus and preliminary analysis shows feasibility for a novel application of spatial transcriptomics. Further evaluation of expressed genes provides information about the microenvironment of thrombus origin and mechanistic insight to stroke subtype etiology. Figure. Number of genes detected across increasing % of ROIs.

Transcriptional and Immune Landscape of Cardiac Sarcoidosis.

Background:Cardiac involvement is an important determinant of mortality among sarcoidosis patients. Although granulomatous inflammation is a hallmark finding in cardiac sarcoidosis, the precise immune cell populations that comprise the granuloma remain unresolved. Furthermore, it is unclear how the cellular and transcriptomic landscape of cardiac sarcoidosis differs from other inflammatory heart diseases.Methods:We leveraged spatial transcriptomics (GeoMx digital spatial profiler) and single-nucleus RNA sequencing to elucidate the cellular and transcriptional landscape of cardiac sarcoidosis. Using GeoMX digital spatial profiler technology, we compared the transcriptomal profile of CD68+ rich immune cell infiltrates in human cardiac sarcoidosis, giant cell myocarditis, and lymphocytic myocarditis. We performed single-nucleus RNA sequencing of human cardiac sarcoidosis to identify immune cell types and examined their transcriptomic landscape and regulation. Using multichannel immunofluorescence staining, we validated immune cell populations identified by single-nucleus RNA sequencing, determined their spatial relationship, and devised an immunostaining approach to distinguish cardiac sarcoidosis from other inflammatory heart diseases.Results:Despite overlapping histological features, spatial transcriptomics identified transcriptional signatures and associated pathways that robustly differentiated cardiac sarcoidosis from giant cell myocarditis and lymphocytic myocarditis. Single-nucleus RNA sequencing revealed the presence of diverse populations of myeloid cells in cardiac sarcoidosis with distinct molecular features. We identified GPNMB (transmembrane glycoprotein NMB) as a novel marker of multinucleated giant cells and predicted that the MITF (microphthalmia-associated transcription factor) family of transcription factors regulated this cell type. We also detected additional macrophage populations in cardiac sarcoidosis including HLA-DR (human leukocyte antigen-DR)+ macrophages, SYTL3 (synaptotagmin-like protein 3)+ macrophages and CD163+ resident macrophages. HLA-DR+ macrophages were found immediately adjacent to GPMMB+ giant cells, a distinct feature compared with other inflammatory cardiac diseases. SYTL3+ macrophages were located scattered throughout the granuloma and CD163+ macrophages, CD1c+ dendritic cells, nonclassical monocytes, and T cells were located at the periphery and outside of the granuloma. Finally, we demonstrate mTOR (mammalian target of rapamycin) pathway activation is associated with proliferation and is selectively found in HLA-DR+ and SYLT3+ macrophages.Conclusions:In this study, we identified diverse populations of immune cells with distinct molecular signatures that comprise the sarcoid granuloma. These findings provide new insights into the pathology of cardiac sarcoidosis and highlight opportunities to improve diagnostic testing.

216.13: Spatial Protein Profiling of Leukocyte Infiltrates in Renal Biopsies After Cellular Therapy

Background: Regulatory T cells (Tregs) are powerful modulators of immune function, and are of increasing interest as an adoptive cellular therapy for the treatment of transplant rejection. Elucidate the impact of Treg therapeutics in vivo is crucial to identify the therapeutic efficacy. This study aimed to examine the infiltrated leukocytes to provide a clearer picture of the dynamic process that occurs in the transplanted tissue after cellular therapy compared to rejection. Methods: Cellular infiltrates were assessed by GeoMx digital spatial profiling (DSP) platform in tissue biopsies from three transplant patients who received Treg therapy and two control patients with confirmed rejection, with biopsies taken at similar time points after transplantation (6-12 months). Various regions of interest (ROIs) were selected (9 -12 regions) from each patient with diverse infiltration types (Figure 1). In addition, FOXP3hi CD4+ and FOXP3low/neg CD4+ regions were selected from both groups to assess the protein signatures of these cellular infiltrates. Results: The FOXP3 signal was highly enriched in regions from cell therapy compared to rejection patients, suggesting that infused Tregs are likely homing to the allograft. The B cell marker CD20 was highly expressed in cell therapy ROIs than in rejection ROIs, suggesting a link between infused Tregs and allograft infiltration by B cells. Conversely, the rejection ROIs have a high expression of Ki67, indicating the presence of highly proliferative cells. The monocyte/macrophage markers CD14, CD68, and CD163 were also enriched in the rejection ROIs, suggesting that macrophages may be associated with graft rejection in these patients. Further analysis revealed interesting differences between the cellular infiltrates in FOXP3hi segments from cell therapy patients compared with rejection group such as enrichment in proteins related to signalling, including STAT3 and p-STAT3, and coinciding with enrichment in regulatory molecules, including CTLA-4 and PD-1. VISTA protein was found to be highly enriched in FOXP3hi segments from cell therapy patients, suggesting that VISTA might play a regulatory role in suppressing the alloimmune response. Our analysis suggests that FOXP3hi cells that are present in regions collected from rejecting patients are likely suppressive Tregs, since FOXP3hi cells coincide with enrichment of Tregs-associated markers. Nevertheless, these Tregs failed to control the alloresponse, as demonstrated by the enrichment of CD8 T cells, which were notably represented in FOXP3hi regions in rejection tissues. Conclusion: The GeoMx DSP allowed for an in-depth analysis of leukocyte infiltrates and provided comprehensive information about the molecular processes associated with stable renal allograft in patients receiving Treg therapy, which in turn may assist in understanding the mechanisms of therapeutic immune regulation and optimal timing for immunosuppression minimisation.

Spatially resolved proteomic profiling identifies tumor cell CD44 as a biomarker associated with sensitivity to PD-1 axis blockade in advanced non-small-cell lung cancer

BackgroundMost patients with advanced non-small-cell lung cancer (NSCLC) fail to derive significant benefit from programmed cell death protein-1 (PD-1) axis blockade, and new biomarkers of response are needed. In this...

824 Spatial transcriptomic analysis of HS skin lesions reveals that tunnels are immunologically active and activity correlates with disease severity

Hidradenitis suppurativa (HS) is an inflammatory skin disease characterized by neutrophil-rich inflammatory nodules, abscesses or tunnels (sinus tracts). Tunnels, unique to HS, are deep dermal cavities lined with squamous epithelium that are immunologically active, contributing to disease pathology. The role of tunnels in driving ongoing disease is poorly understood. We used the GeoMX Digital Spatial Profiler to analyze a moderate (Hurley stage II) compared to a severe (Hurley stage III) HS skin lesion to explore biological differences between and within lesions. Four distinct regions per sample were analyzed: tunnel epithelium, immune infiltrate adjacent to a tunnel, immune infiltrate not associated with a tunnel, and sub-epidermis. We observed higher expression of keratinization genes (e.g. K16, S100A7) in the more differentiated tunnel epithelium of the severe lesion compared to the moderate lesion tunnel. Genes for proinflammatory cytokines (e.g. IL36, IL1B), chemokines (e.g. CXCL1, CCL20), neutrophil activation (e.g. ELANE, MPO) and B cells (CD79A, SDC1) had higher expression in the severe compared to moderate HS lesion. We found a spatial gradient in gene expression for IL36 and neutrophil-attracting chemokines (CXCL1, CXCL8), from high expression in the tunnel epithelium to lower expression in the sub-epidermis. Both lesions displayed these gradients, but those in the severe HS lesion were more pronounced. Conversely, spatial gradients of fibroblast-related genes (e.g. PDGFR, FAP) had higher expression in the sub-epidermis and lower expression adjacent to tunnel epithelium. These data suggest a marked increase in dermal inflammation in more severe HS lesions. The spatial gradients of cytokine and chemokine gene expression support the idea that more differentiated tunnels are more immunologically active, potentially contributing to ongoing severe disease, rather than representing remnants of prior disease.

Abstract 5676: Digital spatial profiling for determination of pancreatic stromal components and correlation with survival

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related death in the United States. Recently, intensive research has revealed the remarkable heterogeneity and complexity of the different components in the pancreatic cancer tumor microenvironment. However, our knowledge regarding active stroma (dominated with activated cancer-associated fibroblasts), infiltrated immune cells and actual tumor cells interactions in the tumor microenvironment, and how these interactions impact patient outcomes, remains limited. Digital spatial profiling (DSP) technology now allows us to quantitatively analyze transcript and protein expression on intact tissue, offering an opportunity to study the intra-tumor cell-to-cell communication. Formalin-fixed paraffin-embedded (FFPE) pancreatic tumors from patients with shorter survival (DFS < 12 months, n=2), and longer survival (DFS > 36 months, n=2) were recruited in this study. Immunofluorescent histochemistry staining of tissue morphology identification uses antibodies to pan-cytokeratin (panCK+) for PDAC cells, alpha-smooth muscle actin (aSMA+) for cancer-associated fibroblasts (CAFs). Six replicated regions of interest (ROI) of tumor, CAFs adjacent and distal to tumor area were annotated from each tissue sample and a panel of 78 transcripts (73 target genes and 5 reference genes) were profiled using GeoMx DSP (Nanostring). Person’s correlation (R2) was computed for regression of GeoMx DSP data. All statistical analyses were performed using the GraphPad Prism software. p-values were calculated on two-sided t-tests and p < 0.05 was considered statistically significant. As expected, the morphologic selection successfully separates panCK+ PDAC cells from CAFs demonstrated by the correspondent expression of epithelial genes of EpCam and KRT, allowing single cell profiling. Within the panel of 78 genes, 9 genes (approximal 12%) were detected with significantly higher expression levels in PDAC cells than CAFs. Five genes (BATF3, IL12b, ITGB8, CD4 and IFNAR1) were found increased expression levels in CAFs-adj comparing to CAFs-dis, regulating molecular functions of Organismal Death (p value = 3.88E-03), Maturation of Cells (p value = 2.94E-06), and Migration of Cells (p value = 2.37E-03). When comparing the differential transcriptomes in PDAC cells between the longer and shorter survival groups, higher expression of antigen presentation gene HLA-E and integrin alpha- and beta- family genes (ITGAV, ITGAX, ITGB2, and ITGB8) was seen in longer-lived patients. Proliferation genes (CCND1 and MKI67) were also found to be increased in CAFs in patients with longer survival. Our results suggest that further exploration of gene and protein expression and interactions within the pancreatic tumor microenvironment may improve our understanding of tumor and stromal communication and how it impacts on patient outcomes. Citation Format: Gerik W Tushoski, Dongtao Ann Fu, Lingsong Meng, Kelly Herremans, Andrea N Riner, Christopher E Forsmark, Zhiguang Huo, Steven J Hughes, Song Han. Digital spatial profiling for determination of pancreatic stromal components and correlation with survival [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 5676.

Abstract 989: Spatial profiling of androgen receptor splice variant 7 transcriptional activity in prostate cancer metastases

Abstract Introduction: Androgen receptor splice variant 7 (AR-V7) is expressed in metastases from patients with castration resistant prostate cancer (CRPC) and shows a high level of inter- and intra-tumoral variability. However, the downstream activity generated by AR-V7 in tissue has not been shown. Whether AR-V7 is active in tissue or whether AR-V7 is a non-functioning biomarker with full-length AR is not known. To address this question, we constructed a tissue microarray (TMA) of 56 metastases from 27 patients with CRPC to analyze spatial gene expression using the GeoMx Digital Spatial Profiler. Immunostaining was performed to define epithelial, vascular, and stromal compartments. The stained tissues were then hybridized with barcoded tagged oligonucleotides targeting 2093 unique genes, which included those representing AR, AR-V cryptic exons, AR and neuroendocrine activity, and immune cell markers. One 500 um region of interest (ROI) was assessed per tissue core (approximately 1200 cells). A sequential section from the same TMA was then stained with AR-V7 and AR-C-terminal specific antibodies. ROIs for RNA and protein were selected to be similar between slides. In addition to DSP, each metastasis was assessed by RNA-seq on the bulk tissue. Results: The most differentially expressed genes (FDR<0.05) based on association with AR-V7 staining were known downstream AR regulated genes including KLK2 and 3, FKBP5, NKX3.1, TMPRSS2, FASN, and TARP. Additionally, genes associated with proliferation and stemness, e.g., POLB, KRT1, SOX2, were significantly expressed. Since 93% of patients were on ADT at time of tissue collection and over 80% also had been treated with either abiraterone or enzalutamide, the increase in AR downstream genes would not be expected to occur from ligand activation of AR-FL. We also have previously shown that knock-down of AR-V7 in LNCaP95 cells results in loss of AR binding to AREs. In these metastases, then, activation of AR downstream genes would be a result of AR-V7 nuclear transport of AR-FL through AR-V7/AR-FL heterodimers or transcriptional activation by AR-V7 homodimers. Of further note, AR cryptic exons 1, 2, and 5 were also significantly expressed in AR-V7 positive ROIs (p< 0.0001). RNA-seq intron/exon junction reads were used to demonstrate that additional AR-Vs, most commonly AR-V9, were also expressed in tissues positive for AR-V7, suggesting that AR splicing is a common event in CRPC. Finally, expression of neuroendocrine (NE) genes INSM1 and TUBB2 were not expressed in AR-V7 positive ROIs (negatively correlated, p<0.001), indicating that AR-V7 and NE phenotypes cannot co-exist in the same cell. Conclusion: AR-V7 continues to drive prostate cancer through activation of the AR-cistrome. Its expression is heterogenous in metastases along with NE cells, suggesting that in the presence of AR-V7 and NE markers, therapy needs to be directed at the N-terminus of AR and NE components. Citation Format: Cynthia C. Sprenger, Ilsa Coleman, Michelle Kriner, Lauren Brady, Margaret Hoang, Martine Roudier, Mamatha Damodarasamy, Radhika Patel, Lawrence True, Peter Nelson, Michael Haffner, Stephen Plymate. Spatial profiling of androgen receptor splice variant 7 transcriptional activity in prostate cancer metastases [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 989.

Abstract 1715: Phenotypic characterization of spatial immune infiltration niches in non-small cell lung cancer

Abstract Introduction: Prolonged response to checkpoint inhibitors is still rare in patients with non-small cell lung cancer (NSCLC) and the performance of current predictive biomarkers is limited. Thus, more accurate biomarkers and alternative treatment targets are needed to optimize immunotherapy. The objective of this study was to advance the understanding of cancer immunity by characterization of spatially resolved compartments of immune infiltration. Material and methods: Using the GeoMx Digital Spatial Profiler (NanoString), 49 proteins were measured in 183 regions of interest (ROIs) from FFPE tissue sections of 33 NSCLC patients. The pan-leukocyte marker CD45 was used to define region of interests (ROIs) of three spatial phenotypes: (1) tumor infiltrating leukocytes (n=39), (2) tertiary lymphoid structures (TLS, n=19) and (3) tumor stroma without specific leukocyte aggregation (n=125). Differential protein expression (FDR<0.05) was determined using generalized linear mixed models with patient as random effect, to account for multiple samples per patient. Results: Normalized levels of lymphocyte phenotypic markers (CD3, CD8, CD4, and CD20) did not differ between stroma and tumor infiltrates, however the infiltrates had higher expression of markers of immune suppression (PD-L1, B7-H3, CTLA-4, IDO-1, PD-L2, 4-1BB, LAG3), regulatory T-cells (FOXP3), macrophages (CD68), and antigen presentation (CD40, OX40L, CD11c). The stromal ROIs were characterized by markers of fibroblast activation (Fibronectin, SMA, FAP alpha), and higher levels of VISTA and CD27 compared to infiltrating regions, and VISTA, ARG1 and CD66b compared to TLS. In comparison, TLS had higher levels of lymphocyte phenotypic markers (CD20, CD3, CD4, CD8), markers of activation (CD45RO, CD40, ICOS), proliferation (Ki67), and antigen presentation (HLA-DR, B2M, CD11c). In general, markers for macrophages (CD68), neutrophils (CD66b), regulatory T-cells (FOXP3) and immune suppression (B7-H3, IDO1, PD-L1, CTLA4) were more abundant in the vicinity of tumor cells, while CD27, ICOS, SMA and CD3 increased with longer distance to tumor cells. PD-L1 and IDO1 protein expression was higher in tumors that were immunohistochemical positive for PD-L1. Conclusions: Spatially resolved protein profiling showed that NSCLC TLS are characterized by activated, proliferating lymphocytes and antigen presentation. We identified a general immunosuppressive microenvironment and indication of T cell exhaustion in the tumor cell compartment and its close vicinity. The clear discrimination of different immune infiltration niches independent of the overall level of leukocytes in the sampled regions illustrates the value of spatial profiling for deconvoluting how immune cells can dictate tumors, and to identify predictive immune signatures in the context of immunomodulatory treatment of NSCLC patients. Citation Format: Anna Sandström Gerdtsson, Artur Mezheyeuski, Patrick Micke, Sara Ek. Phenotypic characterization of spatial immune infiltration niches in non-small cell lung 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 1715.

Abstract CT154: B-cell infiltration in lung cancer predicts response to neoadjuvant pembrolizumab

Abstract Background Neoadjuvant immune checkpoint inhibitor treatment is a promising approach for resectable cancer, including non-small cell lung cancer (NSCLC). The characteristics of potential responders to such treatments and the molecular underlying events are not known. Methods We have conducted a phase I, investigator-initiated single-center study (MK3457-223), to examine the safety of neoadjuvant pembrolizumab for stage I-II resectable NSCLC and to determine the recommended phase II dose/schedule (RP2D/S). FFPE biopsies and surgical specimens were subjected to correlative studies. NanoString’s GeoMx Digital Spatial Profiler (DSP) analysis was conducted on pre-treatment samples and post-treatment responder samples. Protein (72 proteins) and mRNA expression data (73 genes) analysis was conducted on regions of interest (ROIs), defined as mostly CD8 positive or mostly pan-cytokeratin positive (presumed cancer cells). Pathology assessment was done on the surgical specimen to identify major pathologic response (MPR; ≤10% remaining viable cells). Statistical analysis was done to compare responders (MPR+) to non-responders (MPR-) by Mann Whitney with false discovery rate correction. Immunohistochemistry (IHC) was conducted on post-treatment samples. Results Twenty-six patients initiated treatment on the study. Two patients (8%, 95% C.I 0-18%) had adverse events that precluded surgery, 1 patient refused surgery after treatment. 7 patients (27%, 95% C.I 10-44%) achieved a major pathologic response (MPR; responders), 3 patients (12%, 95% C.I 0-24%) achieved complete pathologic response. Responders had a longer interval from treatment to surgery (43 days vs. 36 days, univariate analysis, p-value 0.043). RP2D/S was determined as 2 treatments of 200mg pembrolizumab at 3 week interval, followed by surgery at least 2 weeks later. The expression of several proteins and genes differed between responders and non-responders. Pre-treatment, CD20 protein was the most differentially expressed protein both in in CD8+ (4.7 fold, p=0.002) and in cancer cells (4.8 fold, p=0.001) ROIs, in both cases higher in the responders compared to the non-responders. Comparing pre to post-treatment expression in responding tumors, the protein found to be upregulated to the highest extent following pembrolizumab treatment was CD20 protein (6.2-fold, p=0.001), as was its encoding gene, MS4A1 (2.4-fold, p=0.006). CD20 IHC of post-treatment samples demonstrated tertiary lymphoid structures (TLS) to be more prevalent in responders compared to non-responders (3.2-fold, p<0.05). Conclusions Longer interval from treatment to surgery was associated with higher rate of MPR. Presence of tumor-infiltrating B-cells and evolvement of TLSs was strongly correlated with pathologic response to neoadjuvant pembrolizumab in early stage NSCLC. Citation Format: Jair Bar, Iris Kamer, Oranit Zadok, Damien Urban, Marina Perelman, Ilanit Redinsky, Aliza Ackerstein, Sameh Daher, Efrat Ofek, Amir Onn, Nona Zeitlin, Alon Ben-Nun, Ran Kremer, Inbal Daniel, Yossef Glantzspiegel, Irit Gat-Viks. B-cell infiltration in lung cancer predicts response to neoadjuvant pembrolizumab [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 CT154.

Abstract 2578: High regulatory T cells infiltrate within tertiary lymphoid structure restricts response to immune checkpoint blockers in sarcomas

Abstract Background: While composition of the tumor microenvironment (TME) is a prerequisite for an effective antitumor immunity, infiltration of organized B- and T-cells aggregates called tertiary lymphoid structures (TLS) has been recently demonstrated to predict response to immune checkpoint blockers (ICB) in sarcomas. However, only a minority of patient derive benefit, suggesting the implication of additional key determinants of ICB-mediated response in TLS-positive sarcomas, such as TLS composition. Using high-throughput spatial transcriptomics and multiplex immunofluorescence (IHF), we aimed at investigating the association between TLS composition and clinical outcome to ICB. Methods: In an exploratory cohort, we spatially profiled the expression of more than 18000-protein encoding genes from responders (R) and non-responders (NR) using Nanostring’s GeoMx Digital Spatial Profiler (DSP) Whole Transcriptome Atlas (WTA) assay. A first set of regions of interest (ROI) was selected in the TLS and further segmented in “B-cells” vs “no B-cells” areas according to CD20+ staining; a second set of ROI was selected in the tumor tissue and further segmented into “tumor” vs “stroma” areas according to CD45+ staining. Deconvolution of data was performed using SpatialDecon algorithm to estimate cell population within TLS. We then evaluated the association between immune cell composition and response to ICB in each segment. In a validation cohort, we performed multiplexed-IHF assay enabling detection of T cells (CD8/GzmA/CD4/FoxP3/CD56) and B cells. These panel was applied to whole sections baseline sarcoma samples. We investigated the association between immune cell composition and clinical benefit in term of progression-free survival (PFS) and overall survival (OS). Results: Six patients were selected for the exploratory cohort, including 3 R and 3 NR. Among the top immune cell infiltrate within TLS segment, NR demonstrated higher Treg infiltrate versus R in “no B-cells” compartment (3.4% vs 2.0%, respectively; p=0.010), whereas no association was observed between Treg infiltration and response to ICB in both stromal (p=0.67) or tumor cells (p=0.36) compartments from tumor area. In the validation cohort (N=16), we observed that Treg density within TLS was higher in NR versus R (p=0.0059). Patients with Treg-enriched TLS had shorter PFS (2.6 vs 11.1 months, p=0.042) and OS (9.0 vs 18.3 months, p=0.12) compared to those with Treg-low TLS infiltration. Concordantly, the CTLA-4 key Treg regulator gene was upregulated in the TLS regions from NR. Conclusions: Altogether, our findings suggest that the presence of Treg within TLS may exert a negative influence on the capacity of TLS to generate an effective antitumor immune response in sarcoma patients treated with ICB, providing new insights in understanding role of TLS in antitumor immunotherapy. Citation Format: Florent Peyraud, Jean-Philippe Guegan, Christophe Rey, Marina Pulido, Emmanuelle Bompas, Sophie Piperno-Neumann, Christine Chevreau, Nicolas Penel, François Bertucci, Maud Toulmonde, Carine Bellera, Catherine Sautès-Fridman, Antoine Bougoüin, Coralie Cantarel, Michèle Kind, Mariella Spalato-Ceruso, Bérengère Dadone-Montaudie, Jean-Yves Blay, Wolf Herman Fridman, François Le Loarer, Alban Bessede, Antoine Italiano. High regulatory T cells infiltrate within tertiary lymphoid structure restricts response to immune checkpoint blockers in sarcomas [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 2578.

Abstract 2028: Spatially resolved proteomic profiling identifies tumor cell CD44 as a novel indicative biomarker of sensitivity to PD-1 axis blockade in advanced non-small cell lung cancer (NSCLC)

Abstract Introduction: Most patients with advanced NSCLC fail to develop durable responses from PD-1 axis blockade, and more robust predictive biomarkers are needed. In this study, we aimed to discover and validate spatially resolved protein markers associated with sensitivity to PD-1 axis inhibition across multiple NSCLC cohorts. Methods: We first used the GeoMx Digital Spatial Profiling (DSP) system in a discovery cohort of 56 NSCLC patients treated with PD-1 axis inhibitors at Yale (USA). Pre-treatment tumors were represented in a tissue microarray (YTMA471) and analyzed using a 71-plex primary antibody panel. Proteins were measured from three molecularly defined tissue compartments: tumor (CK+), leukocytes (CD45+/CD68-), and macrophages (CD68+). We used quantitative immunofluorescence (QIF) to orthogonally validate candidate biomarkers. For external validation of identified predictors, we assessed pre-treatment whole tissue sections from a cohort of 128 NSCLC patients treated with single-agent PD-1 axis inhibitors at the Hospital 12 de Octubre (Madrid, Spain) using DSP (39-plex protein panel, measured from CK+ and CD45+ compartments). In addition, we analyzed two immunotherapy untreated cohorts to address prognostic significance: YTMA423 (Yale, USA; n = 252) and CIMA-CUN (UNAV, Spain; n = 124), using QIF and DSP respectively. Results: Using continuous log-scaled data, we found 5 markers independently associated with progression-free survival (PFS) in the tumor compartment (including PD-L1, p = 0.002). Among the novel candidate predictors, tumor cell CD44, a marker of pluripotency and stemness, was associated with longer PFS (multivariate HR = 0.63, p= 0.002). Using QIF, we orthogonally validated that CD44 expression in the tumor compartment was associated with longer PFS (p <0.001) and overall survival (OS) (p = 0.03). In the external validation cohort, CD44 expressed in the tumor compartment, but not in the immune compartment, was predictive of clinical benefit (OR 1.22, p = 0.018), and was significantly associated with longer PFS (first tertile cutpoint: HR 0.62, p = 0.03). In contrast, high CD44 expression was not associated with survival in the two untreated cohorts. Using DSP data from two cohorts, regions of interest with elevated expression of CD44 in tumor cells consistently showed prominent (Fold Change>1.5, p<0.05) upregulation of TIM-3, and PD-L1 and multiple co-stimulatory molecules (including CD40, ICOS, CD27). Conclusion: This work highlights CD44 as a novel indicative biomarker of sensitivity to PD-1 axis blockade in NSCLC. NSCLCs with high CD44 expression in epithelial cells may be associated with an immune contexture primed for higher response to immune checkpoint blockade. Further studies are needed to understand the interplay between CD44+ cancer stem cell phenotype and mechanisms of immune evasion. Citation Format: Myrto Moutafi, Magdalena Molero, Sandra Martinez-Morilla, Javier Baena, Ioannis Vathiotis, Niki Gavrielatou, Laura Castro, Gorka Ruiz de Garibay, Vera Adradas, Daniel Orive, Karmele Valencia, Alfonso Galvo, Luis M. Montuenga, Santiago Ponce, Kurt Schalper, Luis Paz-Ares, David L. Rimm, Jon Zugazagoitia. Spatially resolved proteomic profiling identifies tumor cell CD44 as a novel indicative biomarker of sensitivity to PD-1 axis blockade in advanced non-small cell lung cancer (NSCLC) [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 2028.

Abstract CT139: Intratumoral (IT) microdosing of the investigational SUMOylation Inhibitor TAK-981 in a phase 0 CIVO trial demonstrates the reactivation of type I Interferon (IFN1) signaling in head and neck squamous cell carcinoma (HNSCC)

Abstract Purpose/Objectives: The human tumor microenvironment (TME) has a dramatic impact on cancer prognosis and therapeutic response, but accurate models of the native TME do not exist. The Comparative In Vivo Oncology (CIVO) platform was developed as a means to assess the effect of investigational agents on the native TME in a Phase 0 microdosing study. CIVO was clinically validated using approved agents and is used for the first time here to assess the impact of an investigational agent - the SUMOylation inhibitor TAK-981 - on the native human TME in HNSCC. Materials/Methods: Eligible subjects have a confirmed HNSCC diagnosis, ECOG 0-2, and planned surgical resection. Injectable tumors were at the primary site or within cervical lymph nodes but had to be surface-accessible and ≥ 2cm. TAK-981 or control microdoses were simultaneously administered via a CIVO device and co-injected with a fluorescent tracking marker for injection site identification and visualization. Tumors were resected 24 or 72 hours after injection, processed, and then analyzed at a central site. Multiplexed biomarker staining and molecular profiling via GeoMx Digital Spatial Profiling were performed to capture pharmacodynamic responses in the native TME. Results: As of January 2022, 8 subjects provided informed consent and were enrolled, and no adverse events associated with the injection procedure or microdoses have been reported. Biomarker analysis demonstrated TAK-981 distribution around the injection site accompanied by reduction of SUMOylation. Dose-dependent elevation of IFN1 signaling was also observed in TAK-981-exposed areas within the TME. Elevated IFN1 signaling was accompanied by TME reconfiguration, with increased macrophage M1 polarization and activation of dendritic cells, NK cells, and CD8+ T cells. TAK-981 exposure was also associated with upregulation of CXCL10, PD-L1, and an IFNγ gene expression signature predictive of response to immune checkpoint blockade. Conclusion: IT microdosing with CIVO provided early insights into complex functional responses induced by the investigational agent TAK-981 that can only be accurately evaluated in the intact, native TME of a patient’s tumor. SUMO pathway inhibition in HNSCC tumors following TAK-981 exposure led to functional activation of multiple immune cell types, effectively shifting the local TME toward an inflamed “hot” state, highlighting TAK-981’s potential as an immune stimulating agent for treating patients with solid tumors. These data were generated while TAK-981 was still in Phase I dose escalation trials (via IV administration), highlighting CIVO’s ability to safely study investigational agents. Further evaluation of TAK-981 alone and in combination with other agents is ongoing in this Phase 0 CIVO microdosing trial. Citation Format: Jeffrey Houlton, Harrison Cash, Haodong Xu, Paul L. Swiecicki, Keith Casper, Steven B. Chinn, Daniel R. Clayburgh, Ryan J. Li, Robert J. Christian, Aaron Halfpenny, Annemieke van Zante, Beryl A. Hatton, Kimberly Sottero, Marc O. Grenley, Connor Burns, Jason Frazier, Jonathan Derry, Gloria Kung, Emily Beirne, Nathan J. Schauer, Atticus Turner, Wendy Jenkins, Kirsten Anderson, Richard A. Klinghoffer, Dennis Huszar, Allison Berger, Karuppiah Kannan. Intratumoral (IT) microdosing of the investigational SUMOylation Inhibitor TAK-981 in a phase 0 CIVO trial demonstrates the reactivation of type I Interferon (IFN1) signaling in head and neck squamous cell carcinoma (HNSCC) [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 CT139.

Abstract 2027: Spatial omics and multiplexed imaging to discover new biomarkers of response or resistance to immune checkpoint inhibitors (ICI) in advanced non-small cell lung cancer (NSCLC)

Abstract Introduction: Despite the impressive outcomes with ICI in NSCLC, only a minority of the patients show long-term benefit from ICI. While PD-L1 immunohistochemistry is an approved companion diagnostic test, it is neither sensitive nor specific. Here, we describe the use of spatial transcriptomics using the GeoMx Digital Spatial Profiler (DSP) as a discovery platform to find biomarkers for ICI response or resistance. Methods: Pre-treatment samples from 56 patients with NSCLC treated with ICI were collected and analyzed in a tissue microarray (TMA) format in two-fold redundancy. The human whole transcriptome, represented by 18000 genes assessed by oligonucleotide-tagged in situ hybridization, was sequenced on the NovaSeq platform to quantify the mRNAs present in each region of interest. Three molecularly defined tissue compartments, defined by fluorescence co-localization (tumor [panCK+], leukocytes [CD45+/CD68-], macrophages [CD68+]) were generated to assess mRNA. All statistical testing was performed using a two-sided significance level of α=0.05. Result: 54000 gene variables were generated per case, from them about 27000 were analyzed after removing targets with expression lower than a prespecified frequency. Expression distribution of EPCAM in tumor, PTPRC in leukocytes and CD68 in macrophages confirmed that DSP successfully profiled the molecular compartments. Differential gene expression (DEG) analysis was performed for 6-month clinical benefit across two distinct tumor TMA spots per patient. This resulted in a DEG list of about 250, 125 and 122 genes in CK, CD45 and CD68 compartment respectively. When we used the intersection of significantly associated with benefit genes the candidate list was reduced dramatically to 5, 10 and 10 distinct genes in CK (CYBA, ESD, SH3BGRL3, TOP2B, UFM1), CD45 (ATRN, DGAT1, DNAAF9, GMPS, IFT20, MPHOSPH6, PKP4, SOCS2, TFAP4, TGFB3) and CD68 (ANKS3, ARFRP1, CTSD, DPYD, GTF3C5, HS2ST1, KIAA0895L, PIGR, SFTPA2, YEATS4) compartment respectively. From them, using the Cox Proportional-Hazards Model we further identified CYBA, ESD and UFM1 in CK, DGAT1 and IFT20 in CD45 and ANKS3 and DPYD in CD68 to predict survival as well. Conclusions: Using DSP technology allows rapid, patient-specific assessment of the transcriptome in TMAs by in situ hybridization in spatially defined molecular compartments. Here we show a small set of candidate genes that are associated with outcome in this ICI-treated cohort. By intersecting two non-adjacent cores of the same patient tissue sample, we have begun to dissect tumor heterogeneity and pilot biomarker candidate information with unique molecularly defined compartments for tumor cells, lymphocytes, and macrophages. Results from this cohort may lead to a novel, spatially defined, transcriptomic approach for developing new biomarkers for immunotherapy. Citation Format: Myrto Moutafi, Sandra Martinez-Morilla, Rolando Garcia-Milian, Thazin Nwe Aung, Ioannis Vathiotis, Niki Gavrielatou, Vasiliki Xirou, Leonidas Salichos, David L. Rimm. Spatial omics and multiplexed imaging to discover new biomarkers of response or resistance to immune checkpoint inhibitors (ICI) in advanced non-small cell lung cancer (NSCLC) [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 2027.

Abstract 6327: Profiling T-cell subpopulations based on spatial proteome in mantle cell lymphoma

Abstract Background: In precision medicine era, we need to develop tools that better predict treatment response with, for e.g. immuno-oncology drugs. One hypothesis is that diverse treatment response is governed by differences in the tumor microenvironment (TME) both within the tumor and surrounding areas. Thus, novel ways to measure the heterogeneity within defined immune-cell populations is the key and now possible by using spatially-resolved multiplexed platforms. Here, we have investigated the phenotype of four T-cell subtypes both within and excluded from the tumor-rich regions in a large cohort of primary mantle cell lymphoma (MCL) patients. We propose that profiling different cell populations could potentially provide insight into patient prognosis and identify clinically actionable targets, but also in understanding whether tumor cells locally re-educate T-cells through cell-to-cell interaction or by soluble factors Method: We analyzed archival FFPE presented as tissue microarrays of 196 cores from 104 MCL patients, collected from the Biobank of Lymphoma of Southern Sweden, by using the, GeoMX-digital spatial profiler (DSP, Nanostring Inc.). Cellular regions of interest were selected by immunofluorescense staining of CD8, CD3 and CD57 to identify four subpopulations of T cells - early cytotoxic (CD3+ CD8+ CD57-), early non-cytotoxic (CD3+ CD8- CD57-), late cytotoxic (CD3+ CD8+ CD57+) and late non-cytotoxic (CD3+ CD8- CD57+). 601 regions distributed among the four T-cell subtypes were collected from both within the tumor-rich and tumor deficient zones and probed for 69 proteins for sub-characterization. Result: Our results show that the distribution and expression profile of all subpopulations differ considerably based on their position relative to the tumor. Using linear mixed effects model to account for effect of multiple sampling per patient, we observed higher cytolytic activity (upregulation of granzyme A and B) (FDR cut-off < 0.05) in early cytotoxic and late non-cytotoxic cells, within the tumor-rich zone compared to the tumor-deficient area. In contrast, proteins such as STING, VISTA, neurofibromin and fibronectin were consecutively upregulated in all subtypes in tumor-deficient zones. We also observe differential expression profiles for proteins involved in for example cell death regulation and apoptotic signaling pathways. Our analysis provide insight into effector functions important for response to treatment in MCL. Conclusion: This study reveals the phenotypic differences of T-cell subsets in MCL with a higher resolution compared to previous studies. The spatially-resolved analysis shows that the proximity to tumor cells highly affect the profile of different T-cell subsets and that further analysis of re-education mechanisms should be pursued. This study advance the understanding of the MCL tissue microenvironment and for the basis for connecting such differences to clinical outcome. Citation Format: Lavanya Lokhande, Joana de Matos Rodrigues, Anna Sandstrom Gerdtsson, Anna Porwit, Mats Jerkeman, Sara Ek. Profiling T-cell subpopulations based on spatial proteome in mantle cell lymphoma [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 6327.

Abstract 2031: High plex proteomic prognostic marker discovery for patients with pancreatic adenocarcinoma using digital spatial profiling

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) long-term outcomes following surgical resection remain poor, with only 20% of patients surviving 5 years post pancreatectomy. With its immune-privileged nature, starting from the early pre-neoplastic state, it appears to easily evade the antitumor immune response. Although there is rationale for targeting PDAC immune pathways, little benefit has been observed to date. The aim of the study was to interrogate the immune landscape of PDAC utilising the Nanostring GeoMx Digital Spatial Profiler (DSP), enabling high-plex proteomic characterisation whilst maintaining tumor microenvironment (TME) topographical features. We assessed tumor samples from 28 treatment-naive PDAC cases represented in a multiregional FFPE tissue microarray (TMA) for which extensive IHC, molecular, genomic characterisation and clinicopathological follow-up data is available. Multiplex immunofluorescent staining for nuclei, panCK, aSMA and CD3 was used to guide region selection using the GeoMx DSP system in multiple TMA cores. 60 immune markers were quantified simultaneously in different tissue compartments defined by immunofluorescence co-localization including tumor (panCK+), immune stromal (PanCK-) regions. The spatially informed assessment by DSP was validated by both regression and variable prognostication compared with IHC for stromal CD3, CD8 and CD68 in near serial TMA PDAC sections. Unsupervised analysis of proteome data (DSP) in the panCK-negative regions identified an immune poor group associated with shorter OS (13.0 versus 31.1mths, P=0.005). When transcriptomic subtype was considered, the checkpoint inhibitor B7-H3 was significantly upregulated in the squamous subtype tumours versus the classical group (Log2: 1.63, P = 0.001). Patients with high B7-H3 expression, using a median expression cut-off, were associated with shorter OS on multivariate analysis (HR: 4.16, P=0.01) a finding that was validated in an external cohort at the bulk transcriptome level. This pilot scale discovery study shows the potential of the Nanostring DSP technology to identify spatially-informed biomarkers with prognostic relevance in biopsy sized samples from treatment-naive PDAC. A number of relevant candidate immune predictors were identified in a spatial context, which are currently undergoing validation in a larger independent cohort and within the neoadjuvant setting. Future studies will apply this technology to pre and post treatment biopsy samples. Citation Format: Assya Legrini, Holly Leslie, Stephan Dreyer, Joanne Edwards, Andrew Biankin, David Chang, Nigel Jamieson. High plex proteomic prognostic marker discovery for patients with pancreatic adenocarcinoma using digital spatial profiling [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 2031.

Abstract 66: A multi-institution examination of concordance in spatial transcriptomics using the GeoMx Cancer Transcriptome Atlas

Abstract Multiplexed spatial profiling can enable biological insights by characterizing gene expression within discrete physical locations of a tissue. However, these advanced techniques can be confounded by variability of sample collection, storage, or profiling protocols, making it difficult to accurately compare data generated by different laboratories. Further, as more spatial profiling datasets become publicly available, having methods to enable meta-analysis of samples collected on different studies will maximize the learnings to support the advancement of treatments or identifying patient segments. To quantify the variability of spatial profiling data at different laboratories and to advance data normalization methodologies, 4 independent laboratories used the NanoString® GeoMx® Digital Spatial Profiling (DSP) to profile serial 5 µm sections of tissue and cell pellet arrays (CPAs). GeoMx DSP enables high throughput, spatially resolved analysis of gene or protein expression from fresh or archival human tissues. In this study, the GeoMx Cancer Transcriptome Atlas was used to profile >1800 genes simultaneously. We examined the concordance of GeoMx data generated in the different laboratories when controlling for methodical variation (e.g., reagents, tissue source) and experimentally varying region of interest (ROI) size, collection site, and sample preservation methods. Sections of tonsil, colon, and 2 CPAs were profiled separately at the 4 laboratories. Each analyzed fresh cut (FC) tissues and two sites examined sample stability by analyzing the impact of storing slides at -80°C for 1 month prior to spatial profiling. Concordance analysis was performed using the Horn-Morisita Index on raw data comparing paired and unpaired ROIs across each set of slides. In CPA samples where each pellet was a different tumor type (e.g., NSCLC, melanoma), we observed strong clustering by cell line. While data initially showed varying degrees of clustering by slide, factoring out this variable removed the association of slide, allowing integration of the data across profiling locations without affecting concordance within slides. In tonsil, ROIs with increasing area were profiled. Comparing expression between pairs of samples for a given area, concordance increased with ROI size (R = -0.40, p<6e-06). Finally, we observed little impact of preservation method (FC vs -80°C) in these data. In this study, we quantify slide-specific variation observed in high-plex RNA profiling by the DSP platform and detail methods for accounting for this variation. We note that many downstream analyses (e.g., differential expression) already model slide effects during the analysis, but modeling it explicitly allows for direct comparison of concordance with other approaches (e.g., clustering, PCA). These methods support the use of multi-institution studies leveraging the GeoMx platform. Citation Format: Tyler Hether, Tim Howes, David Scoville, Charlie Glaser, Yanyun Li, Rami Vanguri, Neeman Mohibullah, Wan-Jung Chang, Todd Yoder, Minnal Gupta, Kathy Ton, Yan Liang, Ying Huang, Zach Herbert, Jason Reeves, Elizabeth Mittendorf, Simon Lacey, Travis Hollmann, Sarah Warren, Theresa LaVallee. A multi-institution examination of concordance in spatial transcriptomics using the GeoMx Cancer Transcriptome Atlas [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 66.

Abstract 2029: Multiomic analysis of whole transcriptome and high plex protein assays on a single FFPE slide

Abstract The GeoMx࣪ Digital Spatial Profiler (DSP) enables high-plex, high-throughput spatial profiling and quantification from a single slide for either protein or RNA. To fully understand the interplay between RNA and protein in tissue, we have developed a novel multi analyte assay that allows for the profiling of both analytes from the area of interest (AOI) on a single slide. Here we describe the development and performance of the multi analyte assay on cell pellet array (CPA) and various tissues including tonsil and colorectal cancer (CRC) using a high plex GeoMx Protein Panel and the GeoMx Human Whole Transcriptome Atlas (WTA). We successfully identified conditions to detect RNA and protein simultaneously, with comparable sensitivity and specificity to the single analyte conditions. The data accurately matched cell lines in an unbiased correlation analysis between GeoMx WTA and the entire CCLE RNAseq database. Furthermore, the assay performance was maintained in tissue samples. Tonsil and colorectal cancer biopsies were profiled for RNA (whole transcriptome) and protein from the same AOI, allowing simultaneous measurements of transcriptional and translational regulation of key biological pathways. This multi-analyte profiling enables deeper characterization of precious biological samples that are available in limited quantities. Citation Format: Shilah Bonnett, Nicholas Confuorto, Kit Fuhrman, Giang Ong, Alyssa Rosenbloom, John Lyssand, Gary Geiss, Joseph Beechem. Multiomic analysis of whole transcriptome and high plex protein assays on a single FFPE slide [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 2029.

Abstract 2023: Spatial transcriptomic evidence for an inflammatory, pro tumorigenic microenvironment in normal human dense breast tissue

Abstract Breast cancer affects over 2,000,000 women worldwide, with more than 680,000 deaths per year. Increased breast density is one of the risk factors for breast cancer, albeit less than the risk associated with increased age and genetic mutation. It has been suggested that high breast density accounts for 15% of the breast cancers diagnosed. On radiographic examination, breast density appears as opaque regions associated with increased cellularity and matrix, which in some circumstances contribute to difficulty observing small tumors. Dense regions in the breast have been associated with both morphological changes as well as molecular changes in the tissue. How these changes may play a role in increased risk for tumorigenesis are not well described. In this study, we examine the partial spatial transcriptomes of specific regions in normal breast tissue containing dense regions, including regions of normal density, the interface of dense and non-dense regions and the dense regions themselves using the GeoMx Digital Spatial Profiling system (DSP). Multi-label immunofluorescence was used to distinguish tissue morphology in FFPE samples from two health patients. We selected 24 regions of interest (ROIs) from each tissue section included dense, interface and non-dense breast. Using barcoded DNA oligos attached in situ hybridization probes (for RNA) via ultraviolet (UV)- photocleavable linkers, we screened over 1800 genes from Cancer Transcriptome Atlas panel. Our results indicated an elevated expression of CD68 and CD33, markers for macrophages and myeloid-derived suppressor cells (MDSCs) in dense breast regions as compared to the non-dense areas. Identifying markers for myeloid lineage cells was followed by an increased expression of the genes EOMES, TIGIT and RORA, which are engaged in the immunosuppressive phenotype. This could be hypothesized to support a pro-tumorigenesis microenvironment preventing T cells from recognizing newly arisen tumor cells. In addition, we also observed a significant expression of cytokines (IL12A and IL26) and chemokines (CCL19 and CXCL10) in the dense breast, which are involved in the regulation of inflammatory response and chemotaxis of monocytes and T-lymphocytes cells. Both CCL19 and IL26 have been demonstrated to play a role in cell proliferation. Thus, these initial studies suggest that the dense breast microenvironment is potentially a pro-tumorigenic environment that, with sufficient factors, gives rise to tumorigenesis. These data also suggest potential nodes of therapeutic intervention that may lower such risk. Citation Format: Ana Karina de Oliveira, Patcharin Pramoonjago, Christopher A. Moskaluk, Jay W. Fox. Spatial transcriptomic evidence for an inflammatory, pro tumorigenic microenvironment in normal human dense breast tissue [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 2023.